JP2010280594A - Biocompatible preparation containing composite particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biocompatible preparation exhibiting improved retentivity in the blood. <P>SOLUTION: The biocompatible preparation includes a composite particles. The composite particle has macromolecular graft chains bonded to the surface of a fine particle in such an ultrahigh density that the steric repulsion between the graft chains is caused. The fine particles are preferably silica, a metal, a metal oxide or a metal sulfide. The biocompatible preparation is preferably used for delivering a medicament to a target diseased part. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a biocompatible formulation comprising composite particles.

  In order to administer a drug to animals including humans, it is necessary to administer the drug into the blood. Moreover, in order to diagnose animals including humans, it is necessary to administer diagnostic agents into the blood. In either case, the drug or diagnostic agent needs to stay in the blood for the required amount of time and in the required amount.

  In order to administer a drug or diagnostic agent into the blood of an animal, there are methods in which the drug or diagnostic agent is conjugated with an administration carrier, or is combined with or complexed with a carrier. As such a carrier, a polymer or the like is known. On the other hand, since the drug or the diagnostic agent is a foreign substance for the animal, the foreign substance exclusion system is activated and is normally quickly removed from the blood. For example, the polymer and the like tend to have low biocompatibility. Specifically, when a serum antibody protein called opsonin adheres to a carrier, it is phagocytosed (supplemented / decomposed) by phagocytic cells present in the liver, spleen, etc. with the aid of complement (Non-patent Documents 1 and 2). reference). Alternatively, the carrier tends to be quickly removed from the blood because it is degraded by degrading enzymes in the blood.

“Advanced Materials”, 2004, Vol. 16, p. 1959-1966. “Analytical Chemistry”, 2008, volume 80, p. 9597-9603.

  Accordingly, an object of the present invention is to provide a biocompatible preparation with improved blood retention.

  The present invention is a biocompatible preparation containing composite particles, wherein the composite particles are composite particles in which polymer graft chains are bonded to the surface of the fine particles at a very high density before steric repulsion occurs between the graft chains.

  The present invention has the advantage that biocompatibility is improved and, as a result, blood retention is improved.

FIG. 1 is a transmission electron microscope (TEM) photograph of the composite particles (Table 1, Entry 9). FIG. 2 is a graph showing the relationship between the molecular weight of polymer graft chains of composite particles (fine particles are silica particles) and pharmacokinetics (Example 2). FIG. 3 is a graph showing the relationship between the core particle size of the fine particles of composite particles (fine particles are silica particles) and pharmacokinetics (Example 2). FIG. 4 is a graph showing the change over time in the residual ratio of blood of composite particles (fine particles are silica particles) (Example 2). FIG. 5 is a graph showing in-vivo distribution of composite particles (fine particles are silica particles) (Example 2). FIG. 6 is an image of fluorescence imaging in a mouse of composite particles (fine particles are silica particles) (Example 3). FIG. 7 is a transmission electron microscope (TEM) photograph of composite particles (fine particles are iron oxide nanoparticles) (Example 4).

  The inventors of the present invention show that a composite particle in which a polymer graft chain is bonded to the surface of a fine particle at an ultra-high density until a steric repulsion occurs between graft chains has a high blood residual rate 1 hour after intravenous administration to cancer-bearing mice. And newly found to accumulate in cancer tissues at a high rate in cancer-bearing mice. Based on this finding, the present inventors have solved the above problems.

  That is, as described above, the present invention is a biocompatible preparation containing composite particles, wherein the composite particles are super-high-density before the steric repulsion occurs between the graft chains, and the polymer graft chains are bonded to the surface of the fine particles. Composite particles.

  In the present invention, “polymer graft chain” means a polymer chain formed by extending two or more monomers from the surface of fine particles by a polymerization reaction. The term “ultra high density” means the density of graft chains when the graft chains are closely packed to such an extent that steric repulsion occurs between the graft chains. In this case, the graft chains extend substantially in the direction perpendicular to the surface. Takes the perfect form. The “bond” means a bond formed by a general chemical reaction, and examples thereof include a covalent bond and an ionic bond.

In biocompatible formulations of the present invention, the graft density of said polymeric graft chain is preferably from 0.1 to 1.2 stranded / nm ^2, at 0.3 to 1.2 strands / nm ² More preferably. By controlling this graft density, it is possible to control the blood retention of the composite particles in the biocompatible preparation of the present invention. In the present invention, “graft density” or “density” means the number of graft chains bonded to the surface per unit area (nm ² ) of the fine particle surface. Graft density (σ) is determined by the amount of polymer graft chains (graft amount, w) formed by extending from the fine particle surface by elemental analysis, and the value and the surface area of core fine particles (S, nm ² ), graft polymer The number average molecular weight Mn and the Avogadro number (Av) are used as follows.
Calculation method: σ = (w / Mn) Av / S
In the biocompatible preparation of the present invention, the number average molecular weight (Mn) of the polymer graft chain is preferably 10,000 to 320,000, and more preferably 13,000 to 290,000. By controlling the number average molecular weight of the graft chain, it is possible to control the blood retention of the biocompatible preparation of the present invention. Specifically, when the number average molecular weight of the graft chain is large, the blood retention of the biocompatible preparation is improved. The degree of polymerization of the polymer graft chain is, for example, 10 to 100,000.

  In the biocompatible preparation of the present invention, the polymer graft chain can be obtained, for example, by living radical polymerization of a monomer based on a polymerization initiating group on the surface of fine particles. The type of the monomer is preferably one or more selected from the group consisting of acrylic acid derivatives, methacrylic acid derivatives, acrylamide derivatives, methacrylamide derivatives, and styrene derivatives, but is not limited thereto, and is within the scope recognized by those skilled in the art. You may select suitably. This polymer graft chain may be bonded to another polymer graft chain. Specifically, in living radical polymerization, a block copolymer may be grafted using a crosslinkable monomer as one kind of monomer, and the introduced crosslinkable group may be reacted. Living radical polymerization may be random polymerization, block polymerization, composition gradient type, or the like. In the polymer graft chain, a specific ligand may be introduced at the terminal and in the vicinity of the terminal. A biocompatible preparation containing composite particles having such a ligand is preferable because the ligand can be selectively delivered to the affected area. The specific ligand is not particularly limited as long as it is a specific ligand known in the art.

  In the composite particle of the biocompatible preparation of the present invention, the polymer graft chain in the region close to the fine particle is a drug-supporting layer, and the polymer graft chain in a region far from the fine particle is a polymer that responds to external stimulation. It may be a composite particle having a certain two-layer structure. With such particles, physicochemical properties of the polymer graft chain in the region far from the fine particle are changed by external stimulation, and as a result, drug release from the polymer graft chain in the region near the fine particle is controlled. . The drug carrying layer may bind the polymer graft chain and the drug by an action such as hydrophobic interaction or complex formation. This is because the bond due to such action is broken by an external stimulus (for example, pH, heat, light). Examples of the drug include therapeutic drugs or preventive drugs. In addition, cosmetics or the like may be used in place of the drug.

  In the biocompatible preparation of the present invention, examples of the fine particles include silicon oxide such as silica; metal; metal oxide; metal sulfide and the like, and silicon oxide; metal; metal oxide is preferable. Examples of the metal include noble metals such as Au, Ag, Pt, and Pd; Ti, Zr, Ta, Sn, Zn, Cu, V, Sb, In, Hf, Y, Ce, Sc, La, Eu, Ni, Examples include transition metals such as Co and Fe. Depending on the type of microparticles, the biocompatible formulation of the present invention can be used for various applications. Specifically, for example, if the fine particles are a metal or a metal oxide, the biocompatible preparation of the present invention can be used for diagnosis. The fine particles are preferably monodisperse particles.

  In the biocompatible preparation of the present invention, the fine particles are preferably fine particles having a particle size of 3 nm to 5 μm, more preferably fine particles having a particle size of 10 nm to 3 μm. By controlling the particle size of the fine particles, it is possible to control the blood retention of the biocompatible preparation of the present invention. Specifically, when the particle size of the fine particles is small, the retention in the blood of the biocompatible preparation is improved. The size of the biocompatible preparation of the present invention can be controlled by the particle size of the fine particles and the length of the polymer graft chain (number average molecular weight). That is, when the particle size of the fine particles is increased, the size of the biocompatible preparation is increased even if the length of the polymer graft chain is the same. In the biocompatible preparation of the present invention, the particle size of the fine particles may be one type or a mixture of two or more types. When the particle size of the fine particles is one, the size of the biocompatible preparation is uniform, which is preferable for DDS applications.

  In the biocompatible preparation of the present invention, the molecular weight distribution of the polymer graft chain is preferably 1 to 1.5, more preferably 1 to 1.3. This is because if the molecular weight distribution is in the range of 1 to 1.5, the biocompatible preparation has a homogeneous graft surface.

  The biocompatible preparation of the present invention is preferably used, for example, for drug delivery to a target affected part (organ, tissue, cell, pathogen, etc.), preferably cancer cells. The biocompatible preparation of the present invention is characterized in that the adsorption of proteins acting for removal from the blood is suppressed, and as a result, the residual ratio in blood is high. Therefore, the biocompatible preparation of the present invention has a high accumulation in cancer tissue. As this reason, EPR effect (Enhanced Permeability and Retention Effect) can be considered. Substances with a molecular size of 50-250 nm are known to accumulate depending on the anatomical characteristics of the blood vessels of cancer tissue (“Cancer Research”, 1986, 46, p. 6387- 6392). The biocompatible preparation of the present invention has high blood retention, and as a result, the probability of passing through cancer tissue through the bloodstream increases. As a result, it is thought that it accumulates more in cancer.

  Moreover, the biocompatible preparation of the present invention in which the fine particles of the composite particles are gold nanoparticles can be used as an X-ray sensitizer. This is because gold nanoparticles have X-ray absorption characteristics. In addition, the biocompatible preparation of the present invention in which the fine particles of the composite particles are gold nanorods can be used as an agent in near infrared thermotherapy. This is because gold nanorods can absorb heat and then generate heat. The biocompatible preparation of the present invention in which the fine particles of the composite particles are iron oxide particles can be used as a magnetic agent, an MRI contrast agent, an agent in thermotherapy, and the like. Moreover, the biocompatible preparation of the present invention in which the fine particles of the composite particles are quantum dots (for example, CdSe, CdS) can be used as an agent in a fluorescent probe. The biocompatible preparation of the present invention in which the fine particles of the composite particles are hollow fine particles can be used as gas-containing particles, ultrasonic contrast agents, and the like. It is also possible to combine fine particles. For example, the biocompatible preparation of the present invention in which a mixture of X-ray absorbing Au particles and MRI probe iron oxide particles is a fine particle of composite particles can be used as a diagnostic molecular probe having multimodality.

  In the biocompatible preparation of the present invention, the composite particles can be produced, for example, as follows. First, a compound as a polymerization initiating group-containing coupling agent is fixed to the surface of the fine particles by a chemical adsorption method. Then, one or more types of monomers are supplied to perform living radical graft polymerization. By fixing the polymerization initiating group-containing coupling agent on the surface of the fine particles in advance, the graft polymerization can be advanced while keeping the graft density constant on the surface of the fine particles. In other words, the graft amount can be increased in proportion to the Mn (number average molecular weight) of the graft chain, the polymerization can proceed in a living manner, and almost all the graft chains on the surface of the fine particles can be grown almost uniformly. . That is, in the composite particle of the present invention, steric hindrance between adjacent graft chains on the surface of the fine particle is reduced. During the living radical / graft polymerization, a polymerization initiator not fixed on the surface of the fine particles may be allowed to coexist.

The compound as the polymerization initiating group-containing coupling agent can be selected in consideration of the affinity with the fine particles. Specifically, for example, when the fine particles are a metal such as gold, silver, or platinum, as the polymerization initiating group-containing coupling agent, for example, a disulfide compound represented by the following formula (I) is preferable.

_{(Br (CH 3) 2 CCOO} (CH 2) 11 S) 2 - (I)

When the fine particles are a metal such as gold, silver, or platinum, examples of the coupling agent that does not contain the polymerization initiating group include alkyl disulfide compounds.

  When the fine particles are silicon oxide, metal oxide, or metal sulfide particles, the polymerization initiation group-containing coupling agent is preferably a silane compound represented by the following formula (II).

In said formula, n is an integer, 3-10 are preferable and 4-8 are more preferable. R ¹ is a C ₁ -C ₃ alkyl group, preferably a C ₁ or C ₂ alkyl group. R ² is a C ₁ or C ₂ alkyl group. X is a halogen atom, preferably a bromine or chlorine atom. Examples of the compound represented by the formula (II) include (2-bromo-2-methyl) propionyloxypropyltriethoxysilane (BPE), (2-bromo-2-methyl) propionyloxyhexyltriethoxysilane (BHE) and the like. Can be mentioned.

  When the fine particles are silicon oxide, metal oxide, or metal sulfide, examples of the coupling agent that does not contain the polymerization initiating group include alkylsilane coupling agents.

The graft density on the surface of the fine particles can be changed by adjusting the ratio of the polymerization initiator-containing coupling agent to the coupling agent not containing the polymerization initiator group. Specifically, when the fine particles are silicon oxide particles, if the ratio of the polymerization initiator-containing coupling agent to the coupling agent not containing a polymerization initiator group is 1: 0, the graft density is 0.1. This is a chain / nm ² or more.

  After completion of the polymerization, the target composite particles can be used alone or in combination with methods usually used in the art from the reaction solution, such as extraction, distillation, washing, concentration, precipitation, filtration, drying, adsorption, precipitation, chromatography, and the like. Can be isolated.

  The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited by the following examples.

The following abbreviations are used in the description of the present specification.
THF: Tetrahydrofuran DMF: Dimethylformamide DMSO: Dimethyl sulfoxide Cy5.5: 1H-Benza [e] Indolium, 2- [5- [3- [6-[(2,5-Dioxo-1-pyrrolidinyl) oxy]- 6-oxohexyl] -1,3-dihydro-1,1-dimethyl-6,8-disulfo-2H-benz [e] indole-2-ylidene] -1,3-pentadien-1-yl] -3- Ethyl-1,1-dimethyl-6,8-disulfo-, inner salt, sodium salt (1: 3); 1H-Benz [e] indolium, 2- [5- [3- [6-[(2, 5-dioxo-1-pyrrolidinyl) oxy] -6-oxohexyl] -1,3-dihydro-1,1-dimethyl-6,8-disulfo-2H-benz [e] indol-2-ylidene] -1,3 -pentadien-1-yl] -3-ethyl-1,1-dimethyl-6,8-disulfo-, inner salt, sodium salt (1: 3)

Production example of composite particles (fine particles are silica particles) (1) Synthesis of polymerization initiator-containing coupling agent (2-bromo-2-methyl) propionyloxypropyltriethoxysilane (BPE)

  The synthesis of BPE was performed by a two-step reaction. As a first step, a mixed solution of allyl alcohol (170 g), triethylamine (237 g) and dichloromethane (2 L) was ice-cooled, and 2-bromoisobutyryl bromide (450 g) was added dropwise thereto. Thereafter, the reaction solution was stirred at 0 ° C. for 3 hours, and further stirred at room temperature for 10 hours. The reaction solution was concentrated, THF was added to precipitate a salt, and the mixture was filtered. The filtrate was concentrated, and the resulting solution was diluted with chloroform (1 L), which was diluted with 1N hydrochloric acid aqueous solution (2 × 1 L), saturated bicarbonate. A sodium aqueous solution (2 × 1 L) and pure water (2 × 1 L) were washed in this order. The organic phase was dried, concentrated, and purified by distillation under reduced pressure to obtain 1- (2-bromo-2-methyl) propionyloxy-2-propene (BPP) in a yield of 90%.

  As a second step, BPP (100 g), triethoxysilane (170 g) and calsted catalyst (600 μL) were sequentially added into the flask, and the mixture was stirred at room temperature for 12 hours in an argon atmosphere. After the reaction, it was purified by distillation under reduced pressure, and BPE was synthesized with a yield of 70%.

(2) Introduction of initiating group on the surface of silica fine particles As silica fine particles, silica fine particles having an average particle size of 130, 290, 740 and 1550 nm (manufactured by Nippon Shokubai Co., Ltd., product name “Seahoster KE-E10”) (Average particle size 130 nm), “Seahoster (registered trademark) KE-E30” (average particle size 290 nm), “Seahoster (registered trademark) KE-E70” (average particle size 740 nm), “Seahoster (registered trademark) KE-E150 (Average particle size 1550 nm)), silica fine particles having an average particle size of 15 nm prepared by the lysine addition method, and silica fine particles having an average particle size of 60 nm prepared by the reverse micelle method were used.

  A typical example of initiating group introduction to the surface of silica fine particles is shown below. A dispersion of silica fine particles in ethanol (7.7 wt%, 30 mL) was added to a mixed solution of 28% aqueous ammonia (13.9 g) and ethanol (200 mL). The mixture was stirred at 40 ° C. for 2 hours, BPE (2 g) dissolved in ethanol (10 mL) was added dropwise, and the mixture was stirred at 40 ° C. for 18 hours. Thereafter, the silica fine particles were collected by a centrifuge and washed with ethanol and then anisole to obtain silica fine particles having an initiating group. Silica fine particles having this initiating group were stored in anisole.

  (3) Surface-initiated living radical polymerization using silica fine particles

Pyrex glass with silica fine particles having initiating groups, polyethylene glycol methacrylate (PEGMA), (4-hydroxyphenyl) ethyl methacrylate (PEMA), Cu (I) Cl, dinonylbipyridine and ethyl 2-bromoisobutyrate (EBIB) The tube was put in a tube, deaerated by a freeze-thaw method, sealed under vacuum, and then polymerized at 70 ° C. for a predetermined time to obtain composite particles in which polymer graft chains were bonded to the surface of silica fine particles. The monomer conversion rate in the polymer graft chain was determined by NMR, and the molecular weight and molecular weight distribution of the free polymer produced from EBIB were determined by gel filtration chromatography (GPC). The purification of the composite particles (fine particles are silica particles) was performed by repeating centrifugation and redispersion in acetone and pure water. The composite particles were dispersed in pure water and stored at room temperature. By treating the composite particles with hydrogen fluoride, the polymer graft chains were cut from the silica fine particles, and the molecular weight and molecular weight distribution of the polymer graft chains were determined by GPC. The graft density was calculated from the graft amount determined by thermogravimetric analysis (TGA), and it was confirmed that all samples had a high graft density of 0.2 chain / nm ² or more. The polymerization conditions and polymerization results used here are summarized in Table 1 and used as a sample list. FIG. 1 shows a transmission electron microscope (TEM) photograph of the composite particle (Entry 9).

(4) Labeling of composite particles (fine particles are silica particles) with radioisotope (RI) Dispersion liquid (concentration = 1 wt%, solvent = pure water) of composite particles (fine particles are silica particles, produced in Example 1 (3)) , 100 μL) into a microcentrifuge tube, followed by Na ¹²⁵ I (5 μL, Perkinelmer NEZ033), then Chloramine T solution (concentration = 0.2 mg / ml, solvent = 0.5 M phosphate buffer aqueous solution (pH 7.5, 0.00). 5M NaCl, 100 μL) was added, and the mixture was stirred with a vortex mixer for 2 minutes. Thereto was further added a sodium metalsulfite solution (concentration = 4 mg / mL, solvent = pure water, 100 μL), and the mixture was stirred with a vortex mixer for 2 minutes. The obtained mixture was separated and purified with a PD-10 column (manufactured by GE Healthcare) (eluent: physiological saline), and the distillate containing fine particles ( ¹²⁵ I-labeled composite particles) was recovered as a ¹²⁵ I-labeled composite particle dispersion. did.

Pharmacokinetic evaluation of RI-labeled composite particles
^{A 125} I-labeled composite particle dispersion (produced in Example 1 (4), 100 μL) was intravenously administered from the tails of healthy and cancer-bearing mice (6 weeks old). Blood was collected from the mouse eye and the radioactivity was measured with a gamma counter (ARC-301B, Aloka Co., Ltd.) to determine the blood concentration of the composite particles. In addition, the mice were sacrificed after a predetermined time, the radioactivity of each tissue was measured, and the distribution of the composite particles in the body was evaluated.

  The retention in blood of conventional microparticle preparations (silica microparticles themselves, microparticles in which polyethylene glycol is bonded to the surface of silica microparticles, etc.) is described in “Advanced Materials”, 2004, Volume 16. , Pp. 1959-1966 and “Analytical Chemistry”, 2008, 80, p. 9597-9603.

  FIG. 2 shows the graft chain length dependence in the pharmacokinetics of composite particles (fine particles are silica particles). The average particle size of the silica fine particles of the composite particles used here is 130 nm, and the molecular weight of the graft chain is 13000 (entry 1 in table 1), 49000 (entry 2 in table 1), 95000 (entry 3 in table 1) and 290000 (entry 5 in Table 1). FIG. 2 shows the residual ratio in blood 1 hour after administration. From FIG. 2, it can be confirmed that the biocompatible preparation of the present invention has a high blood survival rate of about 50 to 70%, and the blood survival rate tends to increase as the graft chain length increases. It was.

  FIG. 3 shows the core (fine particle) particle size dependence in the pharmacokinetics of composite particles (fine particles are silica particles). The average particle size of the silica fine particles (core) of the composite particles used here is 15 nm (entry 9 in Table 1), 60 nm (entry 10 in Table 1), 130 nm (entry 3 in Table 1), 290 nm (in Table 1). Entry 6), 740 nm (entry 7 in Table 1) and 1550 nm (entry 8 in Table 1). FIG. 3 shows the residual ratio of the composite particles in the blood 1 hour after administration. It can be confirmed that the biocompatible preparation of the present invention shows a high residual ratio in blood of 50% or more when the core particle diameter is 130 nm or less, although the residual ratio in blood decreases as the core particle diameter increases. It was.

  FIG. 4 shows the change over time in the residual ratio of the composite particles (fine particles are silica particles) in blood. Here, healthy and cancer-bearing mice were used. The average particle diameter of the silica fine particles of the composite particles used is 15 nm (entry 9 in Table 1) and 290 nm (entry 6 in Table 1), and the molecular weight of the graft chain is 98000 (entry 9 in Table 1) and 113000, respectively. (Entry 6 in Table 1). In FIG. 4, “15 nm SiP_healthy” is a composite particle of Table 9 as a composite particle using a healthy mouse, and “15 nm SiP_cancer-bearing” is a composite particle of Table 9 as a composite particle using a tumor-bearing mouse. "290nmSiP_healthy" uses healthy mice as composite particles in Table 1 for entry 6, and "290nmSiP_cancer-bearing" uses tumor-bearing mice as composite particles in Table 1 for entry 6 composite particles. The result is shown. Similar to the results in FIG. 3, the biocompatible preparation of the present invention retains the blood for a long period of time when the particle size (core) is small (15 nm), and remains in the blood as high as about 40% even after 24 hours. The rate was confirmed. Moreover, it has confirmed that the blood residual rate in the cancer bearing mouse | mouth of the biocompatible formulation of this invention has the tendency which becomes low compared with the case of a healthy mouse | mouth.

  FIG. 5 shows the distribution in the body of the composite particles (fine particles are silica particles). The measurement sample was the same as that used in FIG. 4, and the distribution in the body after the end of the experiment of FIG. 4 (48 hours after administration) was evaluated. In the biocompatible preparation of the present invention, it was confirmed that a large amount of composite particles having an average particle diameter of silica fine particles (core) of 290 nm accumulated in the liver, spleen and the like. In the case of composite particles having excellent blood retention and silica fine particles (core) having an average particle size of 15 nm, the biocompatible preparation of the present invention has relatively little accumulation in the liver, spleen, etc., and 5 in tumor tissue. It was confirmed that about 10% was accumulated.

Targeting of composite particles to tumor tissue and fluorescence imaging (1) Preparation of fluorescent dye-labeled composite particles Silica fine particles having an initiation group (produced in Example 1 (2)), polyethylene glycol methacrylate (PEGMA), vinylbenzyl phthalimide (VBP) ), Cu (I) Cl, dinonylbipyridine and ethyl 2-bromoisobutyrate (EBIB) were placed in a Pyrex glass tube, degassed by freeze-thawing, sealed under vacuum, and polymerized at 70 ° C. for a predetermined time. Thus, composite particles in which the polymer graft chains were bonded to the surface of the silica fine particles were obtained (see entry 13 in Table 1 for the ratio of starting materials). The purification of the composite particles (fine particles are silica particles) was performed by repeating centrifugation and redispersion in acetone and pure water. After purification, hydrazine monohydrate (0.51 mL) was added to the composite particle ethanol dispersion (35 mL), and the mixture was refluxed at 90 ° C. for 1 hour. Thereafter, centrifugation and redispersion were repeated twice each with ethanol, acetone, pure water, and carbonate buffer (0.1 M, pH = 9.3) to wash the composite particles. The composite particles dispersed in a carbonate buffer solution and a reactive fluorescent dye, Cy5.5 (registered trademark, manufactured by GE Healthcare (# Q15408), one) were reacted at room temperature for 3 days. Purification was carried out by repeating centrifugation and pure water dispersion to prepare a fluorescently labeled composite particle dispersion (concentration = 1.3 wt%).

(2) Preparation of fluorescent labeled silica fine particles having no polymer grafted chain Aminopropyltriethoxysilane (20 μg), dimethylaminopyridine (1 μg), Cy5.5 (registered trademark, manufactured by GE Healthcare (# Q15408), 1 This) and DMSO (100 mg) were mixed at 80 ° C. for 3 days to prepare a DMSO solution. Pure water (960 mg), L-lysine (1 mg) and octane (50 mg) were mixed at 60 ° C., then the above DMSO solution and tetraethoxysilane (72 mg) were added, and the mixture was stirred for 24 hours. Stir for hours. The reaction solution was purified by dialysis against pure water. The final concentration of the fine particle dispersion was 1.1 wt%.

(3) Fluorescence imaging measurement A fluorescence labeled composite particle dispersion (manufactured in Example 3 (1), 100 μL) was intravenously administered from the tail of a tumor-bearing mouse (6 weeks old). In FIG. 6 (see the upper part of FIG. 6, “PPEGMA-SiP”), 1 hour (t = 1 h), 6 hours (t = 6 h), 24 hours (t = 24 h) and 48 hours after administration of the composite particle dispersion. The fluorescence intensity when (t = 48 h) has elapsed, that is, the distribution state of the composite particles is shown. The fluorescence image was obtained with a LAS4000 imaging device (manufactured by FUJIFILM Corporation). It can be seen that the tumor tissue is near the base of the left limb of the mouse, and the area around it is darkly colored over time. That is, it was revealed that the composite particles accumulate at a high rate in the tumor tissue. This is achieved by the EPR effect (Enhanced Permeability and Retention Effect. Cancer cells have increased blood vessel wall permeability compared to normal cells, and substances up to a certain size can easily accumulate). This is because the composite particles are excellent in blood retention. On the other hand, in the case of fluorescently labeled silica fine particles having no polymer graft chain (produced in Example 3 (2)), the fluorescence intensity after 1 hour (t = 1h) and 24 hours (t = 24h) has passed. It was confirmed that it was slightly accumulated in the tumor tissue (see “SiP without brush” in the lower part of FIG. 6). This indicates that binding of the polymer graft chain to the surface of the fine particles is extremely effective for long-term blood retention and accumulation in tumor tissue.

Production Example of Composite Particles (Fine Particles are Iron Oxide Nanoparticles) Instead of silica fine particles, magnetite (Fe ₃ O ₄ ) nanoparticles with an average particle diameter of 16 nm (prepared by the iron-oleic acid complex method, average particle diameter is transmission type) Composite particles (fine particles were iron oxide nanoparticles) were synthesized in the same manner as in Example 1 (entry 11 in Table 1) except that the projected area equivalent circle diameter was measured by electron microscopy. The electron micrograph is shown in FIG. The molecular weight and molecular weight distribution index of the polymer graft chain of the obtained composite particles were 12000 and 1.18, respectively.

Example of production of composite particles (fine particles are gold nanoparticles) (1) Production of gold nanoparticles having living radical polymerization initiator on the surface Chloro dissolved in pure water (20 mL) in boiling pure water (1500 mL) Gold acid (HAuCl ₄ , 220 mg) was added and stirred vigorously. Thereto was added sodium citrate (750 mg) dissolved in pure water (20 mL). After vigorously stirring for 5 minutes, the reaction solution was cooled with ice water, and a disulfide initiator of the formula (I) (1.70 mg) dissolved in tetrahydrofuran (500 mL) was added thereto, followed by stirring for 12 hours at room temperature. The generated gold nanoparticles were collected by a centrifuge and washed with a mixed solution of methanol and water (methanol: water = 1: 1) and a mixed solution of hexane and ethyl acetate (hexane: ethyl acetate = 1: 1). Then, it was air-dried to obtain gold nanoparticles having a living radical polymerization initiator on the surface (yield: 100 mg, average particle diameter: 12 nm, average particle diameter was measured by a transmission electron microscope to determine the equivalent diameter of the projected area).

_{(Br (CH 3) 2 CCOO} (CH 2) 11 S) 2 - (I)

(2) Living radical polymerization in the presence of gold nanoparticles Gold nanoparticles having an initiator on the surface (manufactured in Example 5 (1), 100 mg), polymerization initiator not bonded to the surface of fine particles 2-bromoisobutyrate ( 64 mg), methyl methacrylate (MMA, 10 g), sparteine (240 mg) and dimethylformamide (9.4 g) were added into a glass tube containing CuBr (71 mg), and the solution was freeze-thawed. Degassed and sealed. The polymerization was carried out at 40 ° C. for 3 hours or 15 hours.

  After the completion of the polymerization, composite particles (microparticles are gold nanoparticles) having a polymer graft chain of poly (methyl methacrylate) (PMMA) on the surface of the microparticles were collected by a precipitation fractionation method using an acetone / water system.

(3) Analysis of polymer graft chain on the surface of gold nanoparticle Composite particles having a polymer graft chain of PMMA on the surface of microparticles (fine particles are gold nanoparticles, manufactured in Example 5 (2), 20 mg) were dissolved in iodine dichloromethane. The polymer graft chain was cleaved from the surface of the fine particles (gold nanoparticles) by dissolving in a solution (1 mg / mL, 2 mL) and stirring for 12 hours. Analysis of the cleaved polymer graft chain was performed by gel filtration chromatography. As a result, the following results were obtained.

Composite particles (fine particles are gold nanoparticles) 1 (polymerization time, 3 hours)
Number average molecular weight of polymer graft chain = 12,000
Molecular weight distribution index of polymer graft chain = 1.24.

Composite particles (fine particles are gold nanoparticles) 2 (polymerization time, 15 hours)
Number average molecular weight of polymer graft chain = 22,000
Molecular weight distribution index of polymer graft chain = 1.30.

Moreover, the graft density was estimated to be about 0.3 chain / nm ² from the graft amount measured by Mn and elemental analysis.

  The biocompatible preparation of the present invention can also be applied for use as a drug nanocarrier, imaging reagent and the like.

Claims (8)

A biocompatible formulation comprising composite particles,
A preparation in which the composite particle is a composite particle in which a polymer graft chain is bonded to the surface of the fine particle at a very high density until steric repulsion occurs between the graft chains. The grafting density of the polymer graft chain, formulation of claim 1 which is 0.1 to 1.2-stranded / nm ^2.   The formulation according to claim 1 or 2, wherein the polymer graft chain has a number average molecular weight (Mn) of 10,000 to 320,000.   The polymer graft chain is obtained by one or more living radical polymerizations selected from the group consisting of acrylic acid derivatives, methacrylic acid derivatives, acrylamide derivatives, methacrylamide derivatives and styrene derivatives based on polymerization initiation groups on the surface of the fine particles. The preparation according to any one of claims 1 to 3.   The preparation according to any one of claims 1 to 4, wherein the fine particles are silicon oxide, metal, metal oxide, or metal sulfide.   The formulation according to any one of claims 1 to 5, wherein the molecular weight distribution of the polymer graft chain is 1 to 1.5.   The preparation according to any one of claims 1 to 6, wherein the fine particles are fine particles having a particle diameter of 3 nm to 5 µm.   The preparation according to any one of claims 1 to 7, which is used for transporting a drug to a target affected area.

Images