JP2009062351A - Method for inhibiting cell proliferation and inhibitor for cell proliferation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a chemical capable of readily and inexpensively inhibiting proliferation of cells of human and animals. <P>SOLUTION: The method for inhibiting cell proliferation includes holding a cell in the presence of at least one substance selected from 5-aminolevulinic acids and their salts and nanoparticles containing at least one substance selected from metals and metal compounds. The inhibitor for cell proliferation includes at least one substance selected from 5-aminolevulinic acids and their salts, and nanoparticles containing at least one substance selected from metals and metal compounds. Inhibition of cell proliferation can be carried out readily and inexpensively than conventional method without requiring irradiation with light and an expensive light-emitting apparatus, because inhibition of cell proliferation is performed even when keeping the cell in a dark place and inhibition of cell proliferation can be carried out without light irradiation. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for inhibiting the growth of human or animal cells using a molecule selected from 5-aminolevulinic acids and salts thereof, and nanoparticles containing at least one substance selected from metals and metal compounds, and its Regarding drugs.

5-Aminolevulinic acid (5-ALA) is a biological material for biosynthesis of protoporphyrin IX (PpIX), which is a precursor of vitamin B12, heme, and chlorophyll, in human and animal cells. It is. 5-aminolevulinic acid hydrochloride (5-Aminolevulinic acid hydrochloride), 5-aminolevulinic acid methyl ester hydrochloride (5-aminolevulinic acid methyl ester hydrochloride) and the like are widely known as substances selected from such 5-aminolevulinic acids and salts thereof. ing. Since 5-aminolevulinic acid is often expressed as “5-ALA”, in the following description, when referring to molecules selected from 5-aminolevulinic acids and salts thereof without particularly distinguishing the types, It will be expressed as “5-ALA group molecule”. In addition, 5-aminolevulinic acid hydrochloride and 5-aminolevulinic acid methyl ester hydrochloride are expressed as “5-ALA-HCl” and “5-ALA-Me”, respectively.
On the other hand, nanoparticles including at least one kind of substance selected from metals and metal compounds are also referred to as “metal-containing nanoparticles” when they are called without distinction. In addition, nanoparticles consisting only of metals are called “metal” nanoparticles (the metal name is assigned to “metal”), and nanoparticles consisting only of metal compounds are referred to as “metal compound” nanoparticles (“metal compound” We will call the name of the compound). For example, when it is made of gold, it will be called gold nanoparticles.

Now, it is known that by administering a 5-ALA family molecule to a cell, the cell takes in those substances into the cell and biosynthesizes PpIX, and particularly in cancer cells, the biosynthesized PpIX is It is widely known that it accumulates in cells in large quantities (examples of literature: Gauller JM et al., 1997 and Casas A. et al., 1999). It is known that PpIX works as a photosensitizer and produces cytotoxicity (phototoxicity) by irradiation with light having high light intensity. An application of this phenomenon to the treatment of cancer is a photodynamic treatment method, for example, applied to the treatment of skin cancer (reference examples: Dougherty, TJ et al., 1998, Donnelly, R.F. et al. 2005, and Fotinos, N. et al., 2006, etc.).
In recent years, photodynamic therapy using a drug containing nanoparticles composed of egg lecithin and 5-ALA-HCl has been reported (Hurlimann et al., 1998, and US Pat. No. 6,559,183). Egg lecithin is a kind of lipid, and nanoparticles are formed in the form of oil-in-water emulsions (oil-in-water emulsions). In this photodynamic therapy, a drug containing nanoparticles composed of egg lecithin and 5-ALA-HCl is administered to a tumor, and the administration site of the drug is irradiated with white light having a light intensity of 200 mW / cm ^2. It is said that a significant effect of photocytotoxicity was obtained. Hurlimann et al. Also reported that strong fluorescence was observed from the tumor site 6 hours after administration of a drug containing nanoparticles containing egg lecithin and 5-ALA-HCl to the tumor. However, it has not been reported that cell growth is suppressed without light irradiation. In addition, the drugs actually used are assumed to contain nanoparticles having various particle sizes of 10 to 200 nanometers (nm), and any particle size of the nanoparticles is effective in suppressing cell proliferation. And does not disclose the utility of nanoparticles having a particle size of less than 10 nm.

On the other hand, using human breast cancer cell-derived cultured cells (MCF-7), PpIX biosynthesized in the cells when the cells are incubated for 1 to 4 hours in the presence of an oil-in-water emulsion and 5-ALA-HCl. There is also a research report on the amount of (Nielsen et al., 2004). Nielsen et al. Reported that in this study, the amount of PpIX biosynthesized from 5-ALA-HCl incorporated into cells was promoted, but as to whether cell growth is suppressed or not. Not reported.
On the other hand, Zeisser-Laboubu et al. Discuss photodynamic therapy using metal nanoparticles, giving examples of reports that gold nanoparticles and iron oxide nanoparticles do not cause cytotoxicity to living bodies (Zeisser). -Laboubu, M. et al., 2006). Examples of reports introduced in this document are as follows. When gold nanoparticles coated with molecules that are derivatized to phthalocyanine are prepared, and the gold nanoparticles are administered to perform photodynamic therapy, singlet oxygen that is considered to be greatly involved in photocytotoxicity It has been reported that it can be produced with a high quantum yield (Wieder, ME et al., 2006). In addition, when iron oxide nanoparticles coated with molecules belonging to porphyrin are prepared and the HeLa cells are incubated for 5 hours in the presence of the nanoparticles, the HeLa cells are cytotoxic in the dark (dark). (hereinafter referred to as dark toxicity) has been reported (Gu, H. et al., 2005). Zeisser-Laboubu et al. Also discussed nanoparticles without photosensitizers, for example gold nanoparticles with a particle size of 1.9 nm were intravenously administered at a concentration of 7 mg / g against mouse tumors. Introduced a report (Hainfeld J.F. et al., 2004) that most gold nanoparticles were excreted through urine without letting the specimen become lethal when administered. is doing.

On the other hand, there are several recent reports on the cytotoxicity of gold nanoparticles without photosensitizers, in addition to this report. There are reports that cytotoxicity was shown when gold nanoparticles (Au55 clusters) with a particle size of 1.4 nm were administered to cancer cells or cultured cells of normal cells (Tsoli, M. et al., 2005). ). Further, gold nanoparticles having a particle size of 14 nm and stabilized with acetic acid are prepared, and the gold nanoparticles are added to human fibroblasts (0.2 to 0.8 mg / ml). There is a report that cytotoxicity was also shown when administered at a concentration of ca. 1-4 mM) and held for 2-6 days (Pernodet, N. et al., 2006). However, as shown in the following reported examples, it has been reported that administration of gold nanoparticles at a relatively low concentration does not show cytotoxicity. For example, gold nanoparticles having a particle size of 18 nm and stabilized with acetic acid or biotin are prepared, and each of these gold nanoparticles is 250 mM with respect to a type of leukemia cancer cell (K562 leukemia cells). There has been a report that when administered at concentrations up to 5%, gold nanoparticles were incorporated into cells and no cytotoxicity was shown (Connor, EE et al, 2005). Colloidal hydrophilic gold nanoparticles having a particle size of 14 to 100 nm and stabilized with acetic acid are prepared, and the gold nanoparticles are administered to HeLa cells at a relatively low concentration (14 nm, 50 nm). And each gold nanoparticle having a particle size of 74 nm is administered at a concentration of 1.0 mM, 20.0 mM, and 16.0 mM, respectively, even if the gold nanoparticle is incorporated into the cell. There are reports that no toxicity has been shown (Chithrani, BD et al., 2006). Furthermore, colloidal hydrophilic gold nanoparticles having a particle size of 3.5 nm and reduced with borohydride are prepared, and the gold nanoparticles are 10 to 100 mM with respect to one type of macrophage cells. There has been a report that no significant cytotoxicity was shown even when administered at a concentration of and maintained for 24-72 hours (Shukla, R. et al., 2005).
The documents cited above are listed in the next section together with the documents cited in the following description.

Cited references

1. Nano-emulsion of 5-aminolevulinic acid. U. S. Patent 6,559,183
2. Chithrani, B.M. D. Ghazani, A .; A. Chan, W .; C. W. Determinating the Size and Shape Dependence of Gold Nanoparticle Uptake into Mammalian Cells. Nano Lett. 6: 662-668; 2006.
3. Connor, E .; E. Mwamuka, J .; Gole, A .; Murphy, C .; J. et al. Wyatt, M .; D. Gold Nanoparticles Are Take Up By Human Cells but Do Not Cause Accutity. small 1: 325-327; 2005.
4). Dougherty, T .; J. et al. Gomer, C .; J. et al. Henderson, B .; W. Jori, G .; Kessel, D .; Korbelik, M .; Moan, J .; Peng, Q .; REVIEW Photodynamic Therapy. Journal of the National Cancer Institute, 90: 889-905, 1998.
5). Donnelly, R.D. F. McCarron P .; A. Woolson, D .; Invited review, Drug delivery of aminolevulinic acid from topical formations intended for photodynamic therapy. Photochem. Photobiol. 81: 750-767;
6). Fiedler, D .; M.M. Eckl, P .; M.M. Krammer, B .; Does δ-aminolevulinic acid induce genotoxic effects? Photochcm. PhotobIol. B: Biology, 33: 39-44; 1996.
7). Photonos, N.M. Campo, M .; A. Popowycz, F .; Gurny, R .; Lange, N .; 5-Aminolevulinic Acids Derivatives in Photomedicine: Characteristics, Applications and Perspectives. Photochem Photobiol. 82: 994-1015; 2006.
8). Gu, H .; Xu, K .; Yang, Z .; Changa, C .; K. Xu, B .; Synthesis and cellular uptake of porphyrin detected iron oxide nanoparticulates-a potential candidate for bimodal antibiotic therapy. Chem. Commun. 4270-4272, 2005.
9. Hainfeld, J. et al. F. Slatkin, D .; N. Smileowitz, H .; M.M. The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol. 49: N309-N315, 2004.
10. Hainfeld, J. et al. F. Slatkin, D .; N. Focella, T .; M.M. Smileowitz, H .; M.M. Gold nanoparticles: a new X-ray contrast agent. The British Journal of Radiology, 79: 248-253, 2006.
11. Hurlimann, A .; F. Hanggi, G .; And Panizzon, R .; G. Photodynamic Therapeutic Super Basal Cell Carcinoma Using Topical 5-Aminolevulinic Acid in a Nanocolloid Lotion. Dermatology, 197: 248-254; 1998.
12 Kneipp, J. et al. Kneipp, H .; McLaughlin, M .; Brown, D .; Kneipp, K .; In Vivo Molecular Probing of Cellular Components with Gold Nanoparticulates and Nanoaggregates. Nano Lett. 6: 2225-2231;
13. Nielsen, H .; M.M. Aemisegger, C .; Burmeister, G .; Schuchter, U .; Gander, B .; Effect of Oil-in-Water Emulsions on 5-Aminolevulinic Acid Uptake and Metabolism to PIX in Cultured MCF-7 Cells. Pharm. Res. 21: 2253-260; 2004.
14 Pernodet, N .; Fang, X .; Sun, Y .; Bakhtina, A .; Ramakrishnan, A .; Sokolov, J .; Ulman, A .; Rafailovich, M .; Adverse Effects of Citrate / Gold Nanoparticles on Human Dermal Fibroblasts. small, 2: 766-773; 2006.
15. Rosenthal, I .; Sostaric, J .; Z. Riesz, P .; Sonodynamic therapy-a review of the synergistic effects of drugs and ultrasound. Ultrasou. Sonochem. 11: 349-363; 2004.
16. Shukla, R.A. Bansal, V .; Chaudhary, M .; Basu, A .; Bhonde, R .; R. Sastry, M .; Biocompatibility of gold nanoparticulates and the endotoxicate inside the cellular compensation: A microscopic overview. Langmuir 21: 10644-10654; 2005.
17. Tsoli, M.M. Kuhn, H .; Brandau, W .; Esche, H .; Schmid, G .; Cellular Uptake and Toxicity of Au55 Clusters. small 1: 841-844; 2005.
18. Wieder, M .; E. Hone, D .; C. Cook, M .; J. et al. Handsley, M .; M.M. Gavrilovic, J .; Russell, D .; A. Intracellular photodynamic therapy with photosensitizer-nanoparticle conjugates: cancer therapy using a 'Trojan horse'. Photochem. Photobiol. Sci. 5: 727-734; 2006.
19. Zeisser-Laboubu, M .; Vargas, A .; Delie, F .; Nonparticulars for Photodynamic Therapy of Cancer. in Nanomaterials for Cancer Therapy, ed. Kumar, C.I. Wiley-VCH Verlag GmbH & Co. kGaA, Weinheim, 2006, pp. 40-86.

As described above, a method for suppressing cell proliferation using a drug containing nanoparticles composed of 5-ALA-HCl and one kind of lipid and irradiation with light having a high light intensity has been conventionally known. Yes. However, irradiating light with a high light intensity of 200 mW / cm ² is a cumbersome operation, and many devices for performing such high-intensity light irradiation are expensive, so this method is It was not always easy to implement. In addition, since nanoparticles composed of a kind of lipid are organic substances of living organisms, they may be corroded or deteriorated depending on, for example, high-temperature and high-humidity environmental conditions or long-term storage. Therefore, with regard to the conventionally known drugs containing nanoparticles composed of 5-ALA-HCl and one kind of lipid, for example, transportation and storage require great care and cost for maintaining the quality. There is a possibility, and it cannot be said that it can be used easily anywhere.

On the other hand, even in the method of suppressing cell growth using nanoparticles made of metal, there are few methods that can be sufficiently suppressed in the past, and it is necessary to administer a large amount of nanoparticles even in the method that can be suppressed, and There is a problem that it takes too much time, for example, it takes 2 to 6 days for the suppression effect to appear.
An object of the present invention is to provide a method and a drug capable of suppressing the proliferation of human and animal cells without causing the above-mentioned problems.

The method for inhibiting cell proliferation of the present invention that solves the above problems is roughly divided into the following three.
The first method for inhibiting cell proliferation of the present invention that solves the above-mentioned problems is a substance selected from at least one molecule selected from 5-aminolevulinic acids and salts thereof (5-ALA group molecules), metals and metal compounds. The cells are retained in the presence of nanoparticles (metal-containing nanoparticles) containing at least one of (Claim 1).
Although the mechanism by which cell growth is suppressed by the present invention has not been clarified, as shown in the examples below, since cell growth is suppressed even when cells are held in the dark, photocytotoxicity is observed. Does not seem to have been brought by. One way of thinking is that 5-ALA group molecules can be easily taken into cells by coexistence with metal-containing nanoparticles, and 5-ALA group molecules taken up in cells in large quantities are directly or indirectly, or alternatively It may be possible to change to other molecules such as PpIX to induce cytotoxicity. Another way of thinking is that metal-containing nanoparticles can be easily taken into cells by coexistence with 5-ALA group molecules, and metal-containing nanoparticles taken in large amounts into cells can induce cytotoxicity. Sex is also mentioned. Alternatively, there is a possibility that the 5-ALA group molecule and the metal-containing nanoparticle are bonded or attached to each other to be integrated, and induce cytotoxicity. In any case, it is conceivable that dark toxicity is induced in cells by some cooperative action of 5-ALA group molecules and metal-containing nanoparticles. This dark toxicity is thought to inhibit cell activity and suppress cell proliferation even without light irradiation.

According to the method for suppressing cell growth of the present invention, cell growth can be suppressed without light irradiation, so that no complicated light irradiation or expensive light irradiation device is required, and it is easier and less expensive than the conventional method. In addition, cell growth can be suppressed.
Moreover, according to the method for inhibiting cell growth of the present invention, it is possible to inhibit the growth of cells, whether normal cells or abnormal cells. The abnormal cells mentioned here refer to cells that cause disease, such as cancer cells of malignant tumors, and also include cells of benign tumors that may turn into malignant tumors. When aiming at the treatment and prevention of diseases by suppressing the growth of abnormal cells in the living body, it is necessary to carry out so as not to suppress the growth of normal cells.

  Furthermore, if the metal-containing nanoparticles sufficiently contain at least one substance selected from metals and metal compounds, for example, environmental conditions where bioorganic substances such as high temperature and humidity and drying are likely to corrode or deteriorate, and long-term storage However, the metal-containing nanoparticles are less likely to corrode or deteriorate. Therefore, according to the method for inhibiting cell proliferation of the present invention, nanoparticles can be easily used in more environments.

  On the other hand, the second method for inhibiting cell growth of the present invention that solves the above-mentioned problem is that at least one molecule (5-ALA group molecule) selected from 5-aminolevulinic acids and salts thereof has a particle size of less than 10 nm. It is characterized by retaining cells in the presence of nanoparticles (not claimed). Although the mechanism by which cell growth is suppressed by this method has not been clarified, there is a possibility that the size effect of the nanoparticles is involved because the nanoparticles have a particle size of less than 10 nm.

  Furthermore, the third method for inhibiting cell growth of the present invention that solves the above-described problems is the presence of at least one molecule selected from 5-aminolevulinic acids and salts thereof (5-ALA group molecule) and nanoparticles. The cells are retained for 12 hours or longer (not claimed). Although the mechanism by which cell growth is suppressed by this method has not been clarified, there is a possibility that the time effect of the retention time due to the retention for 12 hours or more is involved.

On the other hand, the cell growth inhibitor of the present invention that solves the above problems is roughly divided into two as follows.
The first cell growth inhibitor of the present invention that solves the above problems is a substance selected from at least one molecule selected from 5-aminolevulinic acids and salts thereof (5-ALA group molecule), a metal and a metal compound. And a nanoparticle (metal-containing nanoparticle) containing at least one of (Claim 6).

The cell growth inhibitor of the present invention can be used in any of the cell growth suppression methods of the present invention described above, and is particularly suitable for the cell growth suppression method of claim 1.
Moreover, as long as the metal-containing nanoparticle sufficiently contains at least one substance selected from metals and metal compounds, the bioorganic substance such as high-temperature and high-humidity is easily corroded or deteriorated as described above. It is less likely to corrode or deteriorate. Therefore, the cell growth inhibitor of the present invention does not need to pay great attention and cost to quality maintenance in transportation and storage, and can be used easily anywhere.

The second cell growth inhibitor of the present invention that solves the above problem is a nano having a particle size of less than 10 nm and at least one molecule (5-ALA group molecule) selected from 5-aminolevulinic acids and salts thereof. Particles (not claimed).
The cell growth inhibitor of the present invention can be used in the second and third methods of inhibiting cell growth of the present invention, and is particularly suitable for the second method of inhibiting cell growth of the present invention. Is.

  According to the method for inhibiting cell growth and the inhibitor of the present invention, it is possible to suppress the growth of human or animal cancer cells, and therefore, there is a possibility that cancer treatment of humans or animals can be performed.

Embodiments of the three cell growth inhibiting methods and two cell growth inhibiting agents of the present invention shown in the means for solving the above problems will be sequentially described below.
In the first method for inhibiting cell growth according to the present invention (Claim 1), at least one molecule selected from 5-aminolevulinic acids and salts thereof (5-ALA group molecule), a substance selected from metals and metal compounds A cell is hold | maintained in presence with the nanoparticle (metal containing nanoparticle) containing at least 1 type. This method of inhibiting cell growth can be carried out by administering a 5-ALA group molecule and metal-containing nanoparticles to cells and then holding the cells for a predetermined time in the presence of these substances. Here, it is not unimaginable to use 5-ALA biosynthesized by cells, but it is difficult to obtain an amount of 5-ALA sufficient to obtain an inhibitory effect on cell proliferation by this method. It is desirable to administer a 5-ALA group molecule to the above because it can be easily carried out.

{Molecules selected from 5-aminolevulinic acids and salts thereof (5-ALA group molecules)}
In the present invention, the 5-ALA group molecule is not particularly limited as long as it is pharmaceutically acceptable. Further, the salt of 5-aminolevulinic acid is preferably an acid addition salt of an organic acid or an inorganic acid, and examples thereof include hydrochloride. As this hydrochloride, 5-aminolevulinic acid hydrochloride (5-ALA-HCl) and / or 5-aminolevulinic acid methyl ester hydrochloride (5-ALA-Me) can be used. Since these hydrochlorides are harmless to living organisms unless used at high concentrations, they are easy to handle. Further, 5-ALA-Me has an advantage that the pH value of the solute is not greatly changed compared to 5-ALA-HCl. In the present invention, only one type of 5-ALA group molecule may be used, or two or more types may be used. When two or more kinds of 5-ALA group molecules are used, they may be used at the same time or may be used separately at different times.

  The 5-ALA group molecule used in the present invention may be used at any concentration. For example, when 5-ALA-HCl and / or 5-ALA-Me is used, it may be used at a concentration of 2.5 to 10 mM. desirable. If the concentration is less than 2.5 mM, cell proliferation may not be sufficiently suppressed. On the other hand, if the concentration exceeds 10 mM, the cells may be toxic alone, and handling may be difficult.

{Metal-containing nanoparticles}
The substance selected from a metal and a metal compound can be selected from at least one of transition metals, transition metal oxides, transition metal nitrides, and transition metal sulfides. In particular, it is preferable to use metal-containing nanoparticles containing a noble metal. Examples of the noble metal include gold (Au), silver (Ag), platinum (Pt), rhodium (Rh), ruthenium (Ru), palladium (Pd), and iridium (Ir). Since noble metals are extremely excellent in chemical stability, metal-containing nanoparticles containing sufficient noble metals can be heated at high temperature to sterilize harmful bacteria, or exposed to strong acids such as hydrochloric acid and 5-ALA-HCl. Even if it is exposed to an enzyme that degrades biological organic molecules, or stored for a long time, its chemical stability is unlikely to be greatly reduced.

  According to the above-mentioned conventional research reports, since gold nanoparticles do not give significant cytotoxicity to a living body unless they are administered in a large amount, if gold nanoparticles are used in the method for inhibiting cell proliferation of the present invention, human or It may be applicable to animal cancer treatment. The cancer treatment candidates include skin cancer, breast cancer and leukemia. In addition, since the present invention can suppress cell proliferation even in a dark place, it may be applicable to subcutaneous cancer treatment where light irradiation is difficult. In particular, gold nanoparticles are not exposed to strong acidity or exposed to enzymes that degrade biological organic molecules. May also be applicable.

  Furthermore, there has recently been a report showing that gold nanoparticles can be used as an X-ray contrast agent (Hainfeld JF et al., 2006). Since the nanoparticles used in the method for inhibiting cell growth of the present invention may be visually detected using X-rays or the like in this way, after the nanoparticles are administered to the affected area for the purpose of treating cancer or the like. Can enable tracking of metal-containing nanoparticles.

Examples of the transition metal oxide include aluminum oxide (Al ₂ O ₃ ), iron oxide (Fe ₂ O ₃ ), zinc oxide (ZnO), copper oxide (CuO), and titanium oxide (TiO ₂ ). Furthermore, silicon oxide (SiO ₂ ) may be used. Further, the nanoparticles may be made of a metal compound semiconductor such as CdSe, CdS, or CdTe, or may be so-called quantum dots.

  By the way, the metal-containing nanoparticle used in the present invention can have a substance selected from a metal and a metal compound on at least the surface of the nanoparticle. Furthermore, the nanoparticles may be those in which a substance selected from metals and metal compounds covers the surface of the nanoparticles in layers. In addition, the metal-containing nanoparticles used in the present invention may be complete from a substance selected from metals and metal compounds. In this case, the paraphrasing of metal-containing nanoparticles as “metal” nanoparticles or “metal compound” nanoparticles was mentioned earlier. The metal-containing nanoparticles used in the present invention may contain other inorganic substances or organic substances in addition to a substance selected from metals and metal compounds.

The metal-containing nanoparticles used in the present invention may be either a hydrophilic colloid or a hydrophobic colloid, but is preferably a hydrophilic colloid. Or a cluster may be sufficient.
The metal-containing nanoparticles used in the present invention may have any shape. For example, spherical, polygonal, columnar and rod-shaped ones can be mentioned, and spherical nanoparticles are particularly preferable.

The metal-containing nanoparticles used in the present invention may have any size. For example, in the case of using spherical metal-containing nanoparticles, it is preferable to have a particle size of 100 nm or less, and more preferable to have a particle size of 17 nm or less.
The metal-containing nanoparticles used in the present invention may be used at any concentration, but when spherical gold nanoparticles having a particle diameter of 17 to 100 nm are used, it is preferably used at a concentration of 2.2 μM or more. When spherical gold nanoparticles having a particle diameter of 5 nm are used, it is preferable to use them at a concentration of 1.4 μM or more.

The metal-containing nanoparticles used in the present invention may contain a target substance that can bind to, attach to, or be taken up by specific cells. Examples of the specific cells include cells that cause illness and injury such as cancer cells. On the other hand, the target substance is preferably a biological substance such as an amino acid, protein, or lipid, and particularly includes an antibody.
The metal-containing nanoparticles preferably have such a target substance bonded or attached to the particle surface. In particular, when using antibodies that can specifically bind to cancer cells or metal-containing nanoparticles that have a target substance that is specifically taken up by cancer cells on the particle surface, it can be administered to tissues where normal cells and cancer cells are mixed. However, the metal-containing nanoparticles can be collected toward cancer cells and not from normal cells. The metal-containing nanoparticles gathered toward the cancer cell can selectively kill the cancer cell by cooperating with at least one of 5-aminolevulinic acid and its salt, thereby suppressing the growth of the cancer cell. it can.

  The metal-containing nanoparticles may contain the target substance indirectly via an intervening substance. The intervening substance may be a linker-like substance that links the metal-containing nanoparticles and the target substance, or may be a coating substance that is coated in a layer on the surface of the metal-containing nanoparticles. The coating material can include the above-described target, and examples thereof include those bonded or attached to the surface of the coating material. If these mediators are those that can be decomposed or washed away after the target substance is bound or taken up by the cell, the metal-containing nanoparticles once bound or taken up by the target substance into the cell will be reintroduced into the cell. Can be released from the metal, and the degree of freedom of movement of the metal-containing nanoparticles can be increased.

  For example, for tissues where normal cells and cancer cells are mixed, metal-containing nanoparticles that have antibodies that can specifically bind to cancer cells or target substances that are specifically taken up by cancer cells on the particle surface. And the 5-ALA group molecule is administered in anticipation of gathering of the metal-containing nanoparticles toward the cancer cells. This places cancer cells in the presence of both 5-ALA family molecules and metal-containing nanoparticles. By maintaining this state for a predetermined time, the cancer cells can be selectively killed, and the proliferation of the cancer cells can be suppressed. At this time, since the metal-containing nanoparticles are rare in normal cells, even if 5-ALA group molecules invade, they do not receive the effect of inhibiting cell proliferation.

The metal-containing nanoparticles used in the present invention may contain at least one 5-ALA group molecule. In particular, it is preferable that a 5-ALA group molecule is bonded or attached to the surface of the metal-containing nanoparticle.
The metal-containing nanoparticles may include at least one 5-ALA group molecule indirectly with an intervening substance interposed. This intervening substance may be a linker-like substance that links metal-containing nanoparticles and 5-ALA group molecules, or may be a coating substance that is coated on the surface of metal-containing nanoparticles in layers. . The coating material can contain 5-ALA group molecules, such as those bonded or attached to the surface of the coating material.

  Furthermore, the metal-containing nanoparticles used in the present invention preferably further contain the above-mentioned target substance together with a 5-ALA group molecule, and both of these substances are directly or indirectly bound to the particle surface as described above. Alternatively, it is desirable that it be adhered.

  Further, the metal-containing nanoparticles used in the present invention are composed of nanoparticles (main body particles) containing a substance selected from metals and metal compounds, and particles (sub-particles) inside and / or on the surface of the main body particles. It may be. The sub-particles may include a substance selected from metals and metal compounds, or may not include a substance selected from metals and metal compounds. The former subparticles may contain any substance other than metals and metal compounds, and the latter subparticles may comprise any substance. The secondary particles may have any shape, and may have any particle size. Further, when the secondary particles are provided on the surface of the main body particles, the secondary particles may be bonded and / or attached to the main body particles.

{cell}
The present invention is applicable to any human or animal cell. As already described above, the cells may be normal or abnormal, but are preferably malignant tumor cancer cells.
Moreover, the method for inhibiting cell growth of the present invention can also be applied to cultured cells used in academic tests or research. Examples of such cultured cells include human leukemia cancer cells such as HL-60 and HL-525, human breast cancer cells such as MCF-7, and human normal fibroblasts such as 1522. The present invention is useful when suppressing the growth of such cultured cells or discarding them.

{Method of administering 5-ALA group molecule and metal-containing nanoparticles}
A method for administering 5-ALA group molecules and metal-containing nanoparticles to cells will be described. When a 5-ALA group molecule is administered to a cell that suppresses growth, the 5-ALA group molecule may be in any state chemically, such as a liquid, a viscous material, or a solid (powder). . The administration of the 5-ALA group molecule may be performed by any method. For example, when a solution containing the 5-ALA group molecule is administered, the solution can be administered by pipetting, injection, application, or the like. Moreover, when administering the viscous substance containing a 5-ALA group molecule | numerator, it can administer by application | coating.

  On the other hand, when a metal-containing nanoparticle is administered to a cell that suppresses proliferation, the metal-containing nanoparticle may be in any state chemically, such as a solution, a viscous substance, or a solid (powder). It is done. The metal-containing nanoparticles may be administered by any method. For example, when a solution containing metal-containing nanoparticles is administered, it can be administered by pipetting, injection, application, and the like. Moreover, when administering the viscous material containing a metal containing nanoparticle, it can administer by application | coating. Also, the metal-containing nanoparticles may be stabilized with any chemical when administered. For example, when gold nanoparticles are administered, the gold nanoparticles are preferably stabilized with acetic acid.

The 5-ALA group molecule and the metal-containing nanoparticles may be administered simultaneously or separately. Further, the 5-ALA group molecule may be administered prior to the metal-containing nanoparticle, or the metal-containing nanoparticle may be administered prior to the 5-ALA group molecule.
In the present invention, when a 5-ALA group molecule and metal-containing nanoparticles are administered to a cell, the cell may be in any condition. For example, the temperature of the cell may be any temperature, and the pH value may be any value. In addition, the cells may be in an aerobic or anaerobic atmosphere, or in an atmosphere rich in carbon dioxide. Furthermore, the cells may be under light or in the dark.

{Cell retention method}
In the method for inhibiting cell growth of the present invention, when cells are held in the presence of 5-ALA group molecules and metal-containing nanoparticles, the cells may be held under any conditions.
At this time, the cell may be held at any temperature, and can be maintained at a temperature at which the cells can grow well in the absence of 5-ALA group molecules and / or metal-containing nanoparticles. This holding temperature can be appropriately selected according to the cell type. For example, in the case of human cells, the temperature can be 37 ° C. and the surrounding temperature.

Further, the cell may be retained at any pH value. In addition, the cells may be retained under either an aerobic or anaerobic atmosphere, or under an atmosphere rich in carbon dioxide. Furthermore, the retention of the cells may be performed under light or in the dark.
The retention time of the cells may be any time, and can be maintained for a time during which the cells can proliferate well in the absence of 5-ALA group molecules and / or metal-containing nanoparticles. This holding time can be appropriately selected according to the cell type.

{Combination with photodynamic therapy or ultrasonic dynamic therapy}
In the method for inhibiting cell growth of the present invention, while the cells are held in the presence of the 5-ALA group molecule and the metal-containing nanoparticles, the administration is performed regardless of the presence or absence of the action of the metal-containing nanoparticles. It is considered that a part of the ALA group molecule is taken into the cell and PpIX is biosynthesized. As described in the section of the prior art, if the target cell is a cancer cell, the biosynthesized PpIX is considered to be accumulated in the cell. PpIX is also known to be a photosensitizer that induces phototoxicity in cancer cells by irradiation with visible light or ultraviolet light, and it has already been described that these effects are applied in photodynamic therapy.
Therefore, in the present invention, visible light and / or ultraviolet light may be irradiated while and / or after holding cancer cells in the presence of 5-ALA group molecules and metal-containing nanoparticles. Phototoxicity can be induced in cancer cells by irradiation with visible light and / or ultraviolet light. According to this means, dark toxicity is induced in cells by some kind of cooperative action of 5-ALA group molecules and metal-containing nanoparticles, but by adding phototoxicity by irradiation with visible light and / or ultraviolet rays. , Cell proliferation is further suppressed.

  At least one of visible light and ultraviolet light used in this means may have any wavelength and may have any light intensity (photon number). In particular, it is known that red light easily reaches a place where the subcutaneous light of a living body is difficult to reach. Therefore, if red light is used as the visible light, it is possible to effectively suppress the growth of cancer cells in the affected area where the subcutaneous light of a living body is difficult to reach. Moreover, it is known that PpIX can efficiently absorb red light having a wavelength in the range of 630 to 640 nm. Therefore, if red light having a wavelength in the range of 630 to 640 nm is used in this means, the proliferation of cancer cells can be more effectively suppressed.

  Furthermore, PpIX is also known to be a sensitizer that induces certain toxicities in cancer cells by ultrasonic irradiation, and attempts have been made to apply such effects to sonodynamic therapy (Rosenthal, I. et al., 2004). Therefore, in the present invention, ultrasonic waves may be irradiated during and / or after holding cancer cells in the presence of 5-ALA group molecules and metal-containing nanoparticles. According to the present means, dark toxicity is induced in the cell by some cooperative action of the 5-ALA group molecule and the metal-containing nanoparticle, and the proliferation of the cell is caused by the addition of the toxicity induced by ultrasound. Is further suppressed. The ultrasonic wave used in this means may have any frequency and may have any ultrasonic intensity.

{Second method for inhibiting cell proliferation of the present invention}
In the present invention, cells are retained in the presence of at least one 5-ALA group molecule and nanoparticles having a particle size of less than 10 nm. In this method for inhibiting cell growth, at least one 5-ALA group molecule and nanoparticles having a particle size of less than 10 nm are administered to cells, and then the cells are held for a predetermined time in the presence of these substances. Can be implemented. The nanoparticles used in the present invention preferably have a particle size of 5 nm.

  As will be described in the following Examples, it has been found that cell proliferation is remarkably suppressed when nanoparticles having a particle size of 5 nm are used in the present invention. In addition, when nanoparticles having a particle size of 17 nm were used, it was also found that cell proliferation was remarkably suppressed even when compared with nanoparticles having a particle size of 5 nm. From these facts, it can be predicted with high probability that cell proliferation is remarkably suppressed when nanoparticles having a particle size of less than 10 nm are used.

The nanoparticles used in the present invention may be made of any substance regardless of whether they are inorganic or organic. For example, the same metal-containing nanoparticles as described in the first method for inhibiting cell growth of the present invention can be used. However, if gold nanoparticles are used, there is a possibility that they can be applied to cancer treatment of humans and animals. There is. The nanoparticles used in the present invention may be diamond, fullerene, or carbon nanotubes.
The present invention can be carried out in the same manner as in the first method for inhibiting cell growth of the present invention, except that nanoparticles having such a material and having a particle size of less than 10 nm are used.

{The third method of inhibiting cell proliferation of the present invention}
In the present invention, cells are held for 12 hours or more in the presence of at least one 5-ALA group molecule and nanoparticles. This method for inhibiting cell growth can be carried out by administering at least one 5-ALA group molecule and nanoparticles to cells and then holding the cells in the presence of these substances for 12 hours or more.

The nanoparticles used in the present invention may be made of any substance regardless of whether they are inorganic or organic. For example, the nanoparticles described in the first or second method for inhibiting cell growth according to the present invention can be used. In particular, if nanoparticles containing gold are used, they can be applied to human or animal cancer treatment. there is a possibility.
The present invention can be carried out in the same manner as the first method for inhibiting cell growth of the present invention, except that nanoparticles having such a material are used and the cells are held for 12 hours or more.

{First cell growth inhibitor of the present invention (Claim 6)}
The cell growth inhibitor of the present invention contains at least one 5-ALA group molecule and nanoparticles (metal-containing nanoparticles) containing at least one substance selected from metals and metal compounds. At least one 5-ALA group molecule can be the same as that used in the first method for inhibiting cell proliferation of the present invention. Also, the metal-containing nanoparticles can be the same as the metal-containing nanoparticles used in the first method for inhibiting cell growth of the present invention.
The cell growth inhibitor of the present invention may be composed only of 5-ALA group molecules and metal-containing nanoparticles, or may contain other substances in addition to these substances.

  The cell growth inhibitor of the present invention may be any of a liquid, a viscous material, and a solid material. As long as it is a liquid, it may be either an aqueous liquid or an oily liquid. In this solution, the 5-ALA group molecules may have any concentration, and the metal-containing nanoparticles may have any concentration. When 5-ALA-HCl or 5-ALA-Me is used, these salts are water-soluble and can be prepared as an aqueous solution. On the other hand, as long as it is a viscous material, it may be an aqueous viscous material or an oily viscous material. Further, the viscous material may have any viscosity. On the other hand, as long as it is a solid, it may be in a block form or a powder form.

The cell growth inhibitor of the present invention may be prepared by any method. For example, as a method for preparing the cell growth inhibitor of the present invention as an aqueous liquid, an aqueous solution containing a 5-ALA group molecule and an aqueous liquid containing at least one substance selected from metals and metal compounds are mixed. A method is mentioned.
The present invention has the possibility of being used as a drug for the treatment of human or animal cancer. The drug may be any type of liquid medicine, pill, tablet, capsule, and ointment.

{Second cell growth inhibitor of the present invention}
The cell growth inhibitor of the present invention contains at least one 5-ALA group molecule and nanoparticles having a particle size of less than 10 nm. At least one 5-ALA group molecule can be the same as that used in the first method for inhibiting cell proliferation of the present invention. On the other hand, the nanoparticles having a particle size of less than 10 nm can be the same as the nanoparticles used in the second method for inhibiting cell growth of the present invention.
The cell growth inhibitor of the present invention may consist only of 5-ALA group molecules and nanoparticles having a particle size of less than 10 nm, or may contain other substances in addition to these substances. There may be.
The present invention can be carried out in the same manner as the first cell growth inhibitor of the present invention except that it contains nanoparticles having such a material and having a particle size of less than 10 nm.

The embodiment of the present invention will be described in more detail with reference to examples.
{Example 1-1}
In this example, cultured cells derived from human leukemia cancer cells (HL) in the presence of 5-ALA-HCl and five types of gold nanoparticles (5 nm, 17 nm, 41 nm, 82 nm, and 103 nm) having different particle sizes. -60) was held for 23 hours.
Powdered 5-ALA-HCl (Sigma-Aldrich) was dissolved in water to prepare an aqueous solution of 5-ALA-HCl having a concentration of 1M. On the other hand, the gold nanoparticles are made entirely of gold and are spherical. Here, a gold citrate colloid solution (Tanaka Kikinzoku Kogyo Co., Ltd.) in which gold nanoparticles were stabilized with a citrate buffer and dispersed as a hydrophilic colloid was used. As shown in Table 1, the gold citrate colloid solution used in this example contains 5 types of gold nanoparticles having a particle size of 5 nm, 17 nm, 41 nm, 82 nm, and 103 nm with a narrow particle size distribution. It is. Table 1 also shows the pH of each gold citrate colloid solution and the concentration of gold nanoparticles contained in each gold citrate colloid solution.

HL-60 cells (American Type Culture Collection) were passaged and cultured in RPMI 1640 liquid medium containing 10% fetal calf serum. The number of HL-60 cells used here doubles every 23 ± 1 hour (time ± SEM) in an atmosphere in which 37 ° C. and 5% CO ₂ concentration are maintained. Cells were harvested and resuspended in fresh RPMI 1640 liquid medium containing 10% fetal calf serum. A 24-well cell culture plate (Corning Incorporated Corning, model 3524) was prepared, and 990 μl of cell suspension was dispensed into each well. The above 5 types of gold citrate with different gold nanoparticle diameters are added immediately after adding 5.5 μl of 1M 5-ALA-HCl aqueous solution to the cell suspension dispensed in 5 of them. 110 μl of colloid solution was added to each cell suspension. These cell samples contain 5 mM 5-ALA-HCl. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. .

Also, to prepare a comparative cell sample containing 5 mM 5-ALA-HCl but no gold nanoparticles, 5.5 μl of 1M 5-ALA was added to another 2-well cell suspension. -Immediately after adding aqueous HCl, 110 μl of citrate buffer (pH 3.3) was added to one, and 110 μl of water was added to the other. In addition, when 5.5 μl of 1M 5-ALA-HCl aqueous solution was added to 990 μl of cell suspension, the pH of the cell suspension was changed from 7.8 to 7.0. Further, when a gold citrate colloid solution or a citrate buffer was added to the cell suspension containing 5-ALA-HCl, the pH of the cell suspension hardly changed.
On the other hand, to prepare a comparative cell sample having the same pH as the cell sample containing 5-ALA-HCl, 5.3 μl of about 1 M HCl aqueous solution was added to the other 7-well cell suspension. Immediately thereafter, the above-mentioned five kinds of gold citrate colloidal solutions having different gold nanoparticle diameters, citrate buffer (pH 3.3), and water were added to each cell suspension in an amount of 110 μl. Here, when 5.3 μl of about 1 M HCl aqueous solution was added to 990 μl of cell suspension, the pH of the cell suspension changed from 7.8 to 7.0. When the gold citrate colloid solution or citrate buffer was added to the cell suspension added with HCl, the pH of the cell suspension hardly changed.

On the other hand, in order to prepare a comparative cell sample containing neither 5-ALA-HCl nor HCl, the above five types of gold nanoparticles having different particle diameters from the other 7-well cell suspensions were prepared. 110 μl of gold citrate colloid solution, citrate buffer (pH 3.3), and water were added to each cell suspension. When a gold citrate colloid solution or a citrate buffer was added to a cell suspension containing neither 5-ALA-HCl nor HCl, the pH of the cell suspension hardly changed.
Note that the concentration of the number of cells in each of the above cell samples was set to approximately 5 × 10 ⁵ cells / ml. This cell concentration is a value measured with a cell number measuring device (Coulter multisizer, model Z2). The same applies to the following embodiments. Here, in the preparation of a cell sample containing 5-ALA-HCl, the operation from the start of adding 5-ALA-HCl to the cell suspension until the cell sample is placed in the dark place in the following incubator is extremely difficult. Performed within 10 minutes under dim white light. In particular, since a cell sample containing 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm was prepared last, the time of exposure to dim white light was about a few minutes. The intensity of the dim white light was 3.0 ± 0.3 × 10 ⁻³ μW when measured with a light intensity measuring device (EG & G, model 550-1) equipped with a multi-probe (EG & G, model 550-2). / Cm ² . Cell samples containing HCl and cell samples containing neither 5-ALA-HCl nor HCl were prepared under the same conditions as those for preparing cell samples containing 5-ALA-HCl.

The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 23 hours. Thus, after 23 hours incubation of the cell sample and the comparative cell sample was completed, all cell samples were collected from the cell culture plate. The cell recovery operation was performed within 30 minutes under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ).

  The cell concentration of the cell sample collected in this way was measured with a cell counter (Coulter multisizer, model Z2). In addition, since this cell number measuring device measures what keeps the shape of the cell, it measures not only living cells but also dead cells that keep the shape of the cells. As shown in FIG. 1A, each of the comparative cell samples that did not use 5-ALA-HCl and / or gold nanoparticles showed a 2-fold increase in cell concentration, but 5-ALA-HCl And cell samples held in the presence of gold nanoparticles having a particle size of 5 to 103 nm, the rate of increase in cell concentration was less than twice. Moreover, when the cell concentration was compared between these cell samples, it was found that the rate of increase in the cell concentration was lower as the particle size of the gold nanoparticles was smaller. Among these cell samples, in particular, a cell sample using gold nanoparticles having a particle size of 17 nm contributes to an increase in cell number of a comparative cell sample that does not use at least one of 5-ALA-HCl and gold nanoparticles. In contrast, the cell concentration increased by about 15%. Also, cell samples using gold nanoparticles with a particle size of 5 nm showed no increase in cell concentration.

  Next, the concentration of viable cells was measured by trypan blue staining for the cell samples retained in the presence of 5-ALA-HCl and gold nanoparticles. As shown in FIG. 1B, many dead cells were not observed in the comparative cell sample that did not use 5-ALA-HCl and / or gold nanoparticles, but 5-ALA-HCl and 5-41 nm Dead cells were noticeably observed in the cell samples retained in the presence of gold nanoparticles with particle sizes. On the other hand, in the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 82 to 103 nm, not many dead cells were observed. In addition, when the concentration of living cells was compared between cell samples held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 41 nm, the smaller the gold nanoparticles, the smaller the number of living cells. It was found that the concentration was lowered. Among these cell samples, the cell sample using gold nanoparticles having a particle size of 17 nm in particular showed a live cell concentration of about 30% lower than that of the comparative cell sample. In addition, the cell sample using gold nanoparticles having a particle size of 5 nm showed a viable cell concentration of about 70% less than the viable cell concentration of the comparative cell sample.

  Furthermore, for cytotoxicity induced in cells by holding in the presence of 5-ALA-HCl and gold nanoparticles, cytotoxicity detection reagent (Cell Counting Kit F, Dojindo) and fluorescent microplate reader (HTS7000, Perkin Elmer) ). This cytotoxicity detection reagent contains Calcein-AM, and an assay using this reagent (hereinafter referred to as CCK-F assay) is excited at a wavelength of 485 nm when Calcein-AM reacts with a living cell. This utilizes the property of changing to a fluorescent substance that emits fluorescence at 535 nm with respect to light. In addition, the fluorescent microplate reader used here uses a filter that passes excitation light of 485 nm wavelength and a filter that passes light emission of 535 nm in combination with a cell sample prepared in a 96-well cell culture plate. It is an apparatus capable of measuring emission of 535 nm wavelength by irradiating with excitation light of wavelength.

In the above CCK-F assay, the cell sample medium is replaced with phosphate buffer (Dulbecco's Phosphate-Buffered Saline; DPBS), and then the cell sample is transferred to a 96-well cell culture plate. It carried out according to. Here, the operation of replacing the medium of the cell sample with DPBS was performed by repeating the operation of precipitating the cells by centrifugation (1000 rpm, 5 minutes), adding the DPBS after removing the supernatant. In this CCK-F assay, the work from the start of replacing the medium of the cell sample to DPBS until the addition of Calcein-AM to the cell sample is performed with extremely dim white light (9.5 × 10 ⁻³ μW / cm ² ) and performed within 1-2 hours. After adding Calcein-AM to the cell sample and leaving it in a completely dark place at room temperature for 1 hour, the fluorescence intensity of 535 nm emitted from the cell sample by the excitation light having a wavelength of 485 nm is changed to the above fluorescence microplate. Measurement was performed using a reader. In addition, when this fluorescence measurement was performed simultaneously also about the cell sample which has not added Calcein-AM, the fluorescence was not measured at all.

  As shown in FIG. 1C, the fluorescence intensity obtained with the cell sample retained in the presence of 5-ALA-HCl and gold nanoparticles with a particle size of 5-41 nm is It can be seen that it is clearly lower than the fluorescence intensity obtained with a comparative cell sample that did not use at least one of the particles. On the other hand, the fluorescence intensity obtained in the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 82 to 103 nm is compared with the fluorescence intensity obtained in the cell sample for comparison. It turns out that it is not so low. This result indicates that dark toxicity was remarkably induced in the cells by maintaining the cells in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 41 nm. . In addition, when the fluorescence intensity was compared between cell samples held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 41 nm, the smaller the particle size of the gold nanoparticles, the more the fluorescence It can also be seen that the strength decreases. Among these cell samples, the cell sample using gold nanoparticles having a particle size of 5 nm in particular exhibits a fluorescence intensity that is about 50% less than the fluorescence intensity of the comparative cell sample, and shows the lowest value. It was.

  From the above results, it is understood that by maintaining HL-60 cells for 23 hours in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 103 nm, the proliferation of the cells is suppressed. It was. It was also found that cell proliferation was most suppressed by using gold nanoparticles having a particle size of 5 nm.

{Example 1-2}
In this example, 4-ALA-HCl was different from 4-ALA-HCl in the same manner as in Example 1-1 except that the concentration of 5-ALA-HCl was 2.5 mM, 5 mM, and 10 mM ( HL-60 cells were retained in the presence of 5 nm, 17 nm, 41 nm, and 82 nm) gold nanoparticles.
A 24-well cell culture plate (Corning Incorporated Corning, model 3524) was prepared, and 990 μl of cell suspension was dispensed into each well. Immediately after adding 2.75 μl of 1M 5-ALA-HCl aqueous solution to the cell suspension dispensed in 6 wells, 4 types (5 nm, 17 nm, 41 nm and 82 nm) gold citrate colloid solution (Table 1), citrate buffer (pH 3.3), and water were added in an amount of 110 μl to each cell suspension. In addition, immediately after adding 5.5 μl of 1M 5-ALA-HCl aqueous solution to the cell suspension dispensed in another 6 holes, the above-mentioned four kinds of gold citrate colloid solutions having different particle diameters are used. , Citrate buffer (pH 3.3), and water were added to each cell suspension in an amount of 110 μl. Further, 11 μl of 1M 5-ALA-HCl aqueous solution was added to the cell suspension dispensed in another 6 holes, and immediately after the above four kinds of colloidal gold citrate solutions having different particle diameters, 110 μl of acid buffer (pH 3.3) and water were added to each cell suspension. For the cell suspension dispensed in the remaining 6 wells, the above-mentioned four kinds of gold citrate colloid solutions having different particle diameters, citrate buffer (pH 3.3) were added without adding 5-ALA-HCl aqueous solution. ), And water were added in an amount of 110 μl to each cell suspension.

These cell samples contain 5-ALA-HCl at respective concentrations of 0 mM, 2.5 mM, 5 mM, and 10 mM. In addition, a cell sample containing gold nanoparticles with a particle size of 5 nm contains gold nanoparticles at a concentration of 22 μM, and a cell sample containing gold nanoparticles with a particle size of 17 nm, 41 nm, and 82 nm contains a concentration of 36 μM. .
When 2.75 μl of 1M 5-ALA-HCl aqueous solution was added to 990 μl of cell suspension, the pH of the cell suspension changed from 7.8 to 7.2. In addition, when 11 μl of 1M 5-ALA-HCl aqueous solution was added to 990 μl of cell suspension, the pH of the cell suspension was changed from 7.8 to 6.6. Further, when a gold citrate colloid solution or a citrate buffer was added to the cell suspension containing these 5-ALA-HCl, the pH of the cell suspension hardly changed.
For any of the above cell samples, the concentration of the number of cells before holding for 23 hours was set to approximately 5 × 10 ⁵ cells / ml. Here, in the preparation of a cell sample containing 5-ALA-HCl, the operation from the start of adding 5-ALA-HCl to the cell suspension until the cell sample is placed in a dark place in the following incubator is extremely dim. It was performed within 15 minutes under white light (3.0 × 10 ⁻³ μW / cm ² ).

The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 23 hours. Thus, after 23 hours incubation of the cell samples was completed, all cell samples were collected from the cell culture plate. The cell recovery operation was performed within 30 minutes under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ).
The cell concentration of the cell sample collected in this way was measured with a cell counter (Coulter multisizer, model Z2). As shown in FIG. 2, when the concentration of 5-ALA-HCl is 2.5 mM or 10 mM, the rate of increase in cell concentration of a cell sample containing gold nanoparticles having a particle size of 5 to 82 nm is 5 -It was found that the cell proliferation was effectively suppressed, as was the case with the cell sample in which the concentration of ALA-HCl was 5 mM, as compared with the cell sample for comparison without gold nanoparticles. Further, it was found that when the concentration of 5-ALA-HCl was 5 mM, the increase in the cell concentration of the cell sample containing gold nanoparticles decreased with the greatest degree. From this result, it was found that the concentration of 5-ALA-HCl is preferably in the range of 2.5 to 10 mM, and cell proliferation is most effectively suppressed by setting the concentration to 5 mM in particular.

{Example 1-3}
In this example, gold nanoparticles with a particle size of 5 nm have various concentrations ranging from 1.4 μM to 22 μM, and gold nanoparticles with a particle size of 17 nm, 41 nm or 82 nm have a concentration of 2.2 μM to 36 μM. Except for various concentrations in the range, in the same manner as in Example 1-1, 5-ALA-HCl and four types of gold nanoparticles having different particle diameters (5 nm, 17 nm, 41 nm, and 82 nm) HL-60 cells were retained in the presence.
First, with respect to four types (5 nm, 17 nm, 41 nm, and 82 nm) of gold citrate colloid liquids (Table 1) having different particle diameters of gold nanoparticles, the concentration of gold nanoparticles is adjusted with a citrate buffer (pH 3.3). Those diluted to 1/2, 1/4, 1/8, and 1/16 were prepared. Then, a total of 24-well cell culture plates (Corning Incorporated Corning, model 3524) were prepared, and 990 μl of cell suspension was dispensed into each well. Immediately after adding 5.5 μl of 1M 5-ALA-HCl aqueous solution to each of the 20-well cell suspensions, the above four kinds of gold citrate colloid solutions and their diluted solutions (gold nanoparticles) Was diluted to 1/2, 1/4, 1/8, and 1/16) in an amount of 110 μl to each cell suspension. In addition, immediately after adding 5.5 μl of 1M 5-ALA-HCl aqueous solution to the cell suspension dispensed in the other two holes, two kinds of citrate buffers (pH 3.1) having different pHs were immediately added. And pH 3.3) was added to each cell suspension in an amount of 110 μl. These cell samples contain 5-ALA-HCl at a concentration of 5 mM.

For both the cell sample and the comparative cell sample, the concentration of the number of cells before holding for 23 hours was set to approximately 5 × 10 ⁵ cells / ml. Here, in the preparation of a cell sample containing 5-ALA-HCl, the operation from the start of adding 5-ALA-HCl to the cell suspension until the cell sample is placed in the dark place in the following incubator is extremely difficult. It was performed within 30 minutes under dim white light (3.0 × 10 ⁻³ μW / cm ² ).
The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 23 hours. Thus, after 23 hours incubation of the cell samples was completed, all cell samples and comparative cell samples were collected from the cell culture plate. The cell recovery operation was performed within 30 minutes under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ).

The cell concentration of the cell sample collected in this way was measured with a cell counter (Coulter multisizer, model Z2). As shown in FIG. 3, for gold nanoparticles having a particle size of 5 nm, the cell concentration was compared to a comparative cell sample containing no gold nanoparticles when the concentration was in the range of 1.4 μM to 22 μM. It was found that the rate of increase was significantly lower and the higher the concentration of gold nanoparticles, the lower the rate of increase in cell concentration. It was also found that the rate of increase in cell concentration was the lowest at a concentration of 22 μM, and cell proliferation was most effectively suppressed.
On the other hand, when gold nanoparticles having a particle diameter of 17 nm are used, when the gold nanoparticle concentration is in the range of 2.2 μM to 36 μM, the cell concentration increases compared to the comparative cell sample containing no gold nanoparticles. It has been found that the rate of increase in cell concentration decreases as the rate is significantly lower and the concentration of gold nanoparticles increases. It was also found that the rate of increase in cell concentration was the lowest at a concentration of 36 μM, and cell proliferation was most effectively suppressed.
On the other hand, even when gold nanoparticles having a particle size of 41 nm and 82 nm are used, the cells are compared with the comparative cell sample containing no gold nanoparticles when the concentration of the gold nanoparticles is in the range of 2.2 μM to 36 μM. The rate of increase in cell concentration decreased as the rate of increase in concentration decreased and the concentration of gold nanoparticles increased. It was also found that the rate of increase in cell concentration was the lowest at a concentration of 36 μM, and cell proliferation was most effectively suppressed.

{Example 1-4}
In this example, 5-ALA-HCl and granules were obtained in the same manner as in Example 1-1 except that the retention time was selected from 0 hour, 4 hours, 8 hours, 12 hours, 18 hours, and 23 hours. HL-60 cells were maintained in the presence of three types of gold nanoparticles with different diameters (5 nm, 17 nm, and 41 nm).
A 24-well cell culture plate (Corning Incorporated Corning, model 3524) was prepared, and 990 μl of cell suspension was dispensed into each well. Immediately after adding 5.5 μl of 1M 5-ALA-HCl aqueous solution to each of the five-hole cell suspensions, three types (5 nm, 17 nm, and 41 nm) with different gold nanoparticle diameters were obtained. Gold citrate colloid solution (Table 1), citrate buffer (pH 3.3), and water were added in an amount of 110 μl to each cell suspension. These cell samples contain 5-ALA-HCl at a concentration of 5 mM. In addition, 110 μl of water was added to the cell suspension dispensed into the remaining one hole. This cell sample was prepared on 6 cell culture plates.
In each of the above cell samples, the concentration of the number of cells before holding was set to approximately 5 × 10 ⁵ cells / ml. Here, in the preparation of a cell sample containing 5-ALA-HCl, the operation from the start of adding 5-ALA-HCl to the cell suspension until the cell sample is placed in a dark place in the following incubator is extremely dim. It was performed within 10 minutes per cell culture plate under white light (3.0 × 10 ⁻³ μW / cm ² ).

Five of the six cell culture plates having the cell samples prepared as described above were transferred into a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration. Samples were placed in the incubator for 4 hours, 8 hours, 12 hours, 18 hours, and 23 hours, respectively. Thus, after each incubation of the cell sample was completed, all cell samples were collected from the cell culture plate. For the remaining one cell culture plate, all cell samples were immediately collected from the cell culture plate without being transferred into the incubator. This was used as a cell sample that was held for 0 hour. The cell recovery operation was performed within 20 minutes for each cell culture plate under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ) in any cell culture plate. .
The cell concentration of the cell sample collected in this way was measured with a cell counter (Coulter multisizer, model Z2). As shown in FIG. 4A, in the cell samples that were held for 0 hours, 4 hours, and 8 hours, there was no significant decrease in cell concentration compared to the comparative cell samples that did not contain gold nanoparticles. However, in the cell samples that were held for 12 hours, 18 hours, and 23 hours, a significant decrease in cell concentration was seen relative to the comparative cell sample that did not contain gold nanoparticles. From this result, it can be seen that a retention time of 12 hours or more is preferable to suppress cell proliferation by retaining HL-60 cells in the presence of 5-ALA-HCl and gold nanoparticles.

Apart from this experiment, 5-ALA-HCl and five different particle sizes (5 nm, 17 nm, 41 nm, 82 nm, and 103 nm) were used in the same manner as in Example 1-1 except that the retention time was 4 hours. ) Was kept for 4 hours in the presence of each gold nanoparticle.
A 24-well cell culture plate (Corning Incorporated Corning, model 3524) was prepared, and 990 μl of cell suspension was dispensed into each well. Immediately after adding 5.5 μl of 1M 5-ALA-HCl aqueous solution to the 15-well cell suspension, 5 types (5 nm, 17 nm, 41 nm, 82 nm, And 103 nm) colloidal gold citrate solution (Table 1) was added in an amount of 110 μl to each of the 3-well cell suspensions. To another 4-well cell suspension, 110 μl of 3 kinds of citrate buffers (pH 3.1, 3.3, or 6.5) and water having different pHs were added to each cell suspension. . These cell samples contain 5-ALA-HCl at a concentration of 5 mM. In addition, 110 μl of water was added to the cell suspension dispensed in another hole.

In each of the above cell samples, the concentration of the number of cells before holding was set to approximately 5 × 10 ⁵ cells / ml. This cell concentration is a value measured with a cell number measuring device (Coulter multisizer, model Z2). Here, in the preparation of a cell sample containing 5-ALA-HCl, the operation from the start of adding 5-ALA-HCl to the cell suspension until the cell sample is placed in a dark place in the following incubator is extremely dim. It was performed within 30 minutes under white light (3.0 × 10 ⁻³ μW / cm ² ).
The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator kept at 37 ° C. and 5% CO ₂ concentration and left for 4 hours. After the cell sample incubation was thus completed, all cell samples were collected from the cell culture plate. The cell collection operation was performed within 30 minutes under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ) in any cell culture plate.

The cell sample thus collected was subjected to CCK-F assay using a cytotoxicity detection reagent (Cell Counting Kit F, Dojindo) and a fluorescent microplate reader (HTS 7000, Perkin Elmer). This CCK-F assay was carried out according to the instruction manual after replacing the cell sample medium with DPBS and then transferring the cell sample to a 96-well cell culture plate. Here, the operation of replacing the medium of the cell sample with DPBS was performed by repeating the operation of precipitating the cells by centrifugation (1000 rpm, 5 minutes), adding the DPBS after removing the supernatant. In this CCK-F assay, the work from the start of replacing the medium of the cell sample to DPBS until the addition of Calcein-AM to the cell sample is performed with extremely dim white light (9.5 × 10 ⁻³ μW / cm ² ) and performed within 1-2 hours. After adding Calcein-AM to the cell sample and leaving it in a completely dark place at room temperature for 1 hour, the fluorescence intensity of 535 nm emitted from the cell sample by the excitation light having a wavelength of 485 nm is changed to the above fluorescence microplate. Measurement was performed using a reader. In addition, when this fluorescence measurement was performed simultaneously also about the cell sample which has not added Calcein-AM, the fluorescence was not measured at all.

  As shown in FIG. 4B, the fluorescence intensities obtained with cell samples retained in the presence of 5-ALA-HCl and gold nanoparticles with a particle size of 17-103 nm are comparable to those without gold nanoparticles. It was found to be almost the same as the fluorescence intensity obtained with the cell sample for use. On the other hand, the fluorescence intensity obtained in the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm is evident in comparison with the fluorescence intensity obtained in the comparative cell sample. It can be seen that it is low. This result shows that when cells are held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 17 to 103 nm, the cell growth is not sufficiently suppressed in the holding time of 4 hours. ing. On the other hand, when cells are held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm, cell growth is suppressed even with a holding time of 4 hours.

{Example 1-5}
In this example, 5-ALA-HCl and three kinds (5 nm, 17 nm, and 41 nm) of gold nanoparticles having different particle diameters were mixed and then administered to the cells, except for Example 1-1. Similarly, HL-60 cells were maintained in the presence of 5-ALA-HCl and gold nanoparticles.
Volume ratio of 1M 5-ALA-HCl aqueous solution and three types of gold citrate colloid solutions (5 nm, 17 nm, and 41 nm) having different particle diameters of gold nanoparticles (Table 1) at a volume ratio of 1:20 And left at room temperature for 1 hour. A mixed solution of the gold citrate colloid solution and the 5-ALA-HCl aqueous solution is a cell growth inhibitor containing 5-ALA-HCl and gold nanoparticles. Further, 1M 5-ALA-HCl aqueous solution and citrate buffer (pH 3.3) were mixed at a volume ratio of 1:20 and left at room temperature for 1 hour. Further, 1M 5-ALA-HCl aqueous solution and water were mixed at a volume ratio of 1:20 and left at room temperature for 1 hour.
A 24-well cell culture plate (Corning Incorporated Corning, model 3524) was prepared, and 990 μl of cell suspension was dispensed into each well. Of the five wells of the cell suspension, a mixture of the above three types of 5-ALA-HCl aqueous solution and gold citrate colloid solution, a mixture of 5-ALA-HCl aqueous solution and citrate buffer, and 110 μl of a mixed solution of 5-ALA-HCl aqueous solution and water was added to each cell suspension.

On the other hand, immediately after adding 5.5 μl of 1M 5-ALA-HCl aqueous solution to another 5-well cell suspension, the above three kinds of gold citrate colloid solution, citrate buffer (pH 3.3) were added. ) And water were added in an amount of 110 μl to each cell suspension.
For any of the above cell samples, the concentration of the number of cells before holding was set to approximately 5 × 10 ⁵ cells / ml. Here, in the preparation of a cell sample containing 5-ALA-HCl, the operation from the start of adding 5-ALA-HCl to the cell suspension until the cell sample is placed in a dark place in the following incubator is extremely dim. It was performed within 30 minutes under white light (3.0 × 10 ⁻³ μW / cm ² ).
The cell culture plate with the cell sample prepared as described above and the comparative cell sample was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration, and left for 23 hours. After the cell sample incubation was thus completed, all cell samples were collected from the cell culture plate. The cell collection operation was performed within 30 minutes under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ) in any cell culture plate.

The cell concentration of the cell sample collected in this way was measured with a cell counter (Coulter multisizer, model Z2). As shown in FIG. 5, in the cell sample in which 5-ALA-HCl aqueous solution and citrate buffer (pH 3.3) or water were mixed and added to the cell suspension, they were added without mixing. Similar to the cell sample, a 2-fold increase in cell concentration was observed.
On the other hand, when gold nanoparticles with a particle size of 5 nm are used, in a cell sample in which a 5-ALA-HCl aqueous solution and its gold citrate colloid solution are mixed and added to the cell suspension, the increase in cell concentration is It was hardly seen. Moreover, the rate of increase in the cell concentration was slightly higher than that of the cell sample added without mixing the 5-ALA-HCl aqueous solution and the gold citrate colloid solution. In addition, when the viable cell concentrations obtained by trypan blue staining were compared for these cell samples, the cell samples obtained by mixing and adding a 5-ALA-HCl aqueous solution and a gold citrate colloid solution were more suitable. It was found that the viable cell concentration was higher than the cell sample added without mixing.

In addition, when gold nanoparticles having a particle size of 17 nm are used, the cell concentration of a cell sample in which a 5-ALA-HCl aqueous solution and its gold citrate colloid solution are mixed and added to the cell suspension is also increased. The percentage of was higher than that of the cell samples added without mixing them. In addition, when the viable cell concentrations obtained by trypan blue staining were compared for these cell samples, the cell samples obtained by mixing and adding a 5-ALA-HCl aqueous solution and a gold citrate colloid solution were more suitable. It was found that the viable cell concentration was higher than the cell sample added without mixing.
Further, when gold nanoparticles having a particle size of 41 nm are used, the cell concentration of the cell sample added to the cell suspension after mixing the 5-ALA-HCl aqueous solution and the gold citrate colloid solution is also increased. The percentage was higher than that of the cell sample added without mixing them. Moreover, when the viable cell concentration obtained by the trypan blue staining method was compared for each of these cell samples, the cell sample in which the 5-ALA-HCl aqueous solution and the gold citrate colloid solution were mixed and added was compared. It was found that the viable cell concentration was higher than the cell sample added without mixing.

  These results indicate that adding the 5-ALA-HCl aqueous solution to the cell suspension and then adding the gold citrate colloid solution has a higher effect of killing the cells than adding them by mixing them. . However, even when a 5-ALA-HCl aqueous solution and a gold citrate colloid solution are mixed and added, cell growth is sufficiently suppressed when a gold citrate colloid solution containing gold nanoparticles having a particle size of 5 nm is used. I found out.

{Example 2-1}
In this example, HL-60 cells were maintained for 24 hours in the presence of 5-ALA-Me and five types of gold nanoparticles having different particle sizes (5 nm, 17 nm, 41 nm, 82 nm, and 103 nm).
Powdered 5-ALA-Me (Methyl δ-aminolevulinate hydrochloride, Sigma-Aldrich) was dissolved in water to prepare an aqueous solution of 5-ALA-Me having a concentration of 1M. A 24-well cell culture plate (Corning Incorporated Corning, model 3524) was prepared, and 990 μl of cell suspension was dispensed into each well. The above 5 types of gold citrate with different gold nanoparticle diameters are immediately added to each cell suspension dispensed in 5 wells by adding 5.5 μl of 1M 5-ALA-Me aqueous solution. 110 μl of colloidal solution (Table 1) was added to each cell suspension. These cell samples contain 5 mM 5-ALA-Me. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. .

Also, to prepare a comparative cell sample containing 5 mM 5-ALA-Me but no gold nanoparticles, 5.5 μl of 1M 5-ALA was added to another 2-well cell suspension. -Immediately after adding the aqueous Me solution, 110 μl citrate buffer (pH 3.3) was added to one, and 110 μl water was added to the other. In addition, when 5.5 μl of 1M 5-ALA-Me aqueous solution was added to 990 μl of cell suspension, the pH of the cell suspension was changed from 7.8 to 7.0. Further, when a gold citrate colloid solution or a citrate buffer was added to the cell suspension containing 5-ALA-HCl, the pH of the cell suspension hardly changed.
On the other hand, in order to prepare a comparative cell sample containing 5-ALA-HCl, 5.5 μl of 1M 5-ALA-HCl aqueous solution was added to each of the other 8 cell suspensions. Immediately afterwards, the above-mentioned six kinds of gold citrate colloid solutions having different gold nanoparticle diameters, citrate buffer (pH 3.3), and water were added to each cell suspension in an amount of 110 μl.

Note that the concentration of the number of cells in each of the above cell samples was set to approximately 5 × 10 ⁵ cells / ml. This cell concentration is a value measured with a cell number measuring device (Coulter multisizer, model Z2). Here, the operation from the start of adding 5-ALA-Me to the cell suspension until the cell sample was placed in a dark place in the following incubator was performed within 10 minutes under extremely dim white light. In particular, since a cell sample containing 5-ALA-Me and gold nanoparticles having a particle size of 5 nm was prepared last, the time of exposure to dim white light was about a few minutes. The comparison cells containing 5-ALA-HCl were also prepared under the same conditions as those for preparing the cell samples containing 5-ALA-Me.
The cell culture plate with the cell sample prepared as described above and the comparative cell sample was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 24 hours. Thus, after the 24-hour incubation of the cell samples was completed, all cell samples were collected from the cell culture plate. The cell recovery operation was performed within 30 minutes under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ).

  The cell concentration of the cell sample collected in this way was measured with a cell counter (Coulter multisizer, model Z2). As shown in FIG. 6, in the cell sample to which 5-ALA-Me and citrate buffer (pH 3.3) or water were added, the cell concentration was increased 1.6 times. In contrast to these cell samples, cell samples held in the presence of 5-ALA-Me and gold nanoparticles having a particle size of 5 to 103 nm both showed an increase in cell concentration lower than 1.6 times. It was. From these facts, by holding cells in the presence of 5-ALA-Me and gold nanoparticles having a particle size of 5 to 103 nm, gold nanoparticles are not present even in the presence of 5-ALA-Me. It was found that the cell growth was further suppressed with respect to the cell sample retained in (1). Moreover, when the cell concentration was compared between these cell samples, it was found that the cell proliferation was further suppressed as the particle size of the gold nanoparticles became smaller. Among these cell samples, especially cell samples using gold nanoparticles with a particle size of 5 nm did not show any increase in cell concentration.

{Example 2-2}
In this example, 4-ALA-Me was different from 5-ALA-Me in the same manner as in Example 2-1, except that the concentration of 5-ALA-Me was 2.5 mM, 3.75 mM, and 5 mM. HL-60 cells were retained in the presence of each type of gold nanoparticles (5 nm, 17 nm, 41 nm, and 82 nm).
A 24-well cell culture plate (Corning Incorporated Corning, model 3524) was prepared, and 990 μl of cell suspension was dispensed into each well. Four kinds of gold citrate with different gold nanoparticle diameters are immediately added to each of the cell suspensions dispensed in the six wells by adding 2.75 μl of 1M 5-ALA-Me aqueous solution. Colloid solution (Table 1), citrate buffer (pH 3.3), and water were added in an amount of 110 μl to each cell suspension. In addition, immediately after adding 4.1 μl of 1M 5-ALA-Me aqueous solution to the cell suspension dispensed in another 6 holes, four types (5 nm, 110 μl of 17 nm, 41 nm, and 82 nm) gold citrate colloid solution (Table 1), citrate buffer (pH 3.3), and water were added to each cell suspension. Further, 5.5 μl of 1M 5-ALA-Me aqueous solution was added to the cell suspension dispensed in another 6 holes, and the above four types of gold citrate colloid solution and citrate buffer were immediately added. (PH 3.3) and water were added in an amount of 110 μl to each cell suspension. For the cell suspension dispensed in the remaining 6 wells, the above four types of gold citrate colloid solution, citrate buffer (pH 3.3), and water were added without adding 5-ALA-Me aqueous solution. 110 μl was added to each cell suspension.

These cell samples contain 5-ALA-Me at respective concentrations of 0 mM, 2.5 mM, 3.75 mM, and 5.5 mM. In addition, a cell sample containing gold nanoparticles with a particle size of 5 nm contains gold nanoparticles at a concentration of 22 μM, and a cell sample containing gold nanoparticles with a particle size of 17 nm, 41 nm, and 82 nm contains a concentration of 36 μM. .
When 2.75 μl of 1M 5-ALA-Me aqueous solution was added to 990 μl of cell suspension, the pH of the cell suspension changed from 7.9 to 7.8. When 4.1 μl of 1M 5-ALA-Me aqueous solution was added to 990 μl of cell suspension, the pH of the cell suspension changed from 7.9 to 7.7. Further, when a gold citrate colloid solution or a citrate buffer was added to the cell suspension containing these 5-ALA-Me, the pH of the cell suspension hardly changed.
For any of the above cell samples, the concentration of the number of cells prior to 24-hour retention was set at approximately 5 × 10 ⁵ cells / ml. Here, in the preparation of a cell sample containing 5-ALA-Me and a cell sample for comparison, the cell sample is placed in the dark place in the following incubator after adding 5-ALA-Me to the cell suspension. The above operations were performed within 15 minutes under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ).

The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 24 hours. Thus, after the 24-hour incubation of the cell samples was completed, all cell samples were collected from the cell culture plate. The cell recovery operation was performed within 30 minutes under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ).
The cell concentration of the cell sample collected in this way was measured with a cell counter (Coulter multisizer, model Z2). As shown in FIG. 7, in a cell sample containing 5-ALA-Me at a concentration of 2.5 mM or 3.75 mM and containing gold nanoparticles with a particle size of 5-82 nm, 5-ALA-Me is Similar to the cell sample containing 5 mM, the increase in the cell concentration was reduced and the cell proliferation was effectively suppressed compared to the comparative cell sample that did not use gold nanoparticles. . Further, it was found that when the concentration of 5-ALA-Me was 5 mM, the increase in the cell concentration of the cell sample containing the gold nanoparticles decreased with the greatest degree.
From these results, it was found that the concentration of 5-ALA-Me is preferably in the range of 2.5 to 5 mM, and cell proliferation is most effectively suppressed by setting the concentration to 5 mM in particular.

{Example 2-3}
In this example, 5-ALA-Me and three types (5 nm, 17 nm, and 41 nm) of gold nanoparticles having different particle sizes were mixed and then administered to the cells, except for Example 1-1. Similarly, HL-60 cells were maintained in the presence of 5-ALA-Me and gold nanoparticles.
1M 5-ALA-Me aqueous solution and three kinds (5 nm, 17 nm, and 41 nm) of gold citrate colloid solutions (Table 1) having different gold nanoparticle diameters are mixed at a ratio of 1:20. What was left to stand at room temperature for 1 hour, 2 hours, or 3 hours was prepared. This mixed solution of 5-ALA-Me aqueous solution and gold citrate colloid solution is a cell growth inhibitor containing 5-ALA-Me and gold nanoparticles. Further, a 1M 5-ALA-Me aqueous solution and a citrate buffer (pH 6.5) were mixed at a ratio of 1:20, and the mixture was allowed to stand at room temperature for 1 hour, 2 hours, or 3 hours. Furthermore, 1M 5-ALA-HCl aqueous solution and each of the three kinds of gold citrate colloid solutions were mixed at a ratio of 1:20, and the mixture was allowed to stand at room temperature for 1 hour, 2 hours, or 3 hours.
A 24-well cell culture plate (Corning Incorporated Corning, model 3524) was prepared, and 990 μl of cell suspension was dispensed into each well. Of the five-hole cell suspension, a mixed solution of the above-mentioned 5-ALA-Me aqueous solution and gold citrate colloid solution, a mixed solution of 5-ALA-Me aqueous solution and citrate buffer, and 5- 110 μl of a mixture of an ALA-HCl aqueous solution and a gold citrate colloid solution was added to each cell suspension.

On the other hand, 1M5-ALA-Me aqueous solution and the above-mentioned three kinds of gold citrate colloid solutions were mixed at a ratio of 1:20, and immediately after that, this mixed solution was added to a cell suspension in another three wells of 110 μl. Added one by one. Further, immediately after mixing the 1M 5-ALA-Me aqueous solution and the citrate buffer (pH 6.5) at a ratio of 1:20, 110 μl of this mixed solution was added to another one-well cell suspension. . Further, 1M 5-ALA-HCl aqueous solution and gold citrate colloidal solution containing gold nanoparticles having a particle size of 17 nm were mixed at a ratio of 1:20, and this mixture was immediately added to another 1 hole. 110 μl was added to the cell suspension.
On the other hand, immediately after adding 5.5 μl of 1M 5-ALA-Me aqueous solution to another 5-well cell suspension, the above three kinds of gold citrate colloid solution, citrate buffer (pH 6.5) were added. 110 μl was added to each cell suspension. Further, add 5.5 μl of 1M 5-ALA-HCl aqueous solution to another one-well cell suspension, and immediately add 110 μl of gold citrate colloidal solution containing gold nanoparticles having a particle size of 17 nm. It was.

For any of the above cell samples, the concentration of the number of cells before holding for 23 hours was set to approximately 5 × 10 ⁵ cells / ml. Here, the operation from the start of adding the mixed solution of the 5-ALA-Me aqueous solution and the gold citrate colloid solution to the cell suspension until the cell sample is placed in the dark place in the following incubator is very dim white light. It was performed within 30 minutes under (3.0 × 10 ⁻³ μW / cm ² ).
The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 23 hours. Thus, after 23 hours incubation of the cell sample and the comparative cell sample was completed, all cell samples were collected from the cell culture plate. The cell recovery operation was performed within 30 minutes under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ).

  The cell concentration of the cell sample collected in this way was measured with a cell counter (Coulter multisizer, model Z2). As shown in FIG. 8, the increase in the cell concentration of the cell sample added to the cell suspension by mixing the gold citrate colloid solution containing gold nanoparticles having a particle size of 5 nm and the 5-ALA-Me aqueous solution. The proportions were found to be slightly higher than those of cell samples added without mixing them. It was also found that the standing time within 3 hours was not affected by the standing time of the mixed solution. Moreover, the rate of increase in the cell concentration of the cell sample added to the cell suspension by mixing the gold citrate colloidal solution containing gold nanoparticles with a particle size of 41 nm and the 5-ALA-Me aqueous solution is mixed with them. There was no significant difference compared to that of the cell sample added without. It was also found that the standing time within 3 hours was not affected by the standing time of the mixed solution.

However, for a cell sample in which a mixed solution of a 5-ALA-Me aqueous solution and a gold citrate colloid solution containing gold nanoparticles having a particle size of 17 nm is added to the cell suspension, The rate of increase in cell concentration is significantly lower than that of a cell sample obtained by adding a 5-ALA-Me aqueous solution to a cell suspension and then adding a gold citrate colloid solution containing gold nanoparticles having a particle size of 17 nm. I understood.
On the other hand, the rate of increase in the cell concentration of the cell sample mixed with the gold citrate colloid solution containing gold nanoparticles having a particle size of 17 nm and the 5-ALA-HCl aqueous solution and added to the cell suspension is Was larger than that of the cell sample added without mixing. It was also found that the standing time within 3 hours was not affected by the standing time of the mixed solution.

In addition, the rate of increase in the cell concentration of the cell sample mixed with the citrate buffer (pH 6.5) and the 5-ALA-Me aqueous solution and added to the cell suspension is the cell sample added without mixing them. There was no big difference compared to the one. It was also found that the standing time within 3 hours was not affected by the standing time of the mixed solution.
From these results, by mixing a 5-ALA-Me aqueous solution and a gold citrate colloidal solution containing gold nanoparticles with a particle size of 17 nm, and then adding what was left for 1 hour or more to the cell suspension, It was found that cell proliferation can be further suppressed.

{Example 3}
In this example, cultured cells derived from human leukemia cancer cells (HL) in the presence of 5-ALA-HCl and five types of gold nanoparticles (5 nm, 17 nm, 41 nm, 82 nm, and 103 nm) having different particle sizes. -525) was held for 22 hours.
HL-525 cells (American Type Culture Collection) were cultured in RPMI 1640 liquid medium containing 10% fetal calf serum. The number of HL-525 cells used here doubles every 22 ± 1 hour (time ± SEM) in an atmosphere maintained at 37 ° C. and 5% CO ₂ concentration. Harvested cells were resuspended in fresh RPMI 1640 liquid medium containing 10% fetal calf serum.

Powdered 5-ALA-HCl (Sigma-Aldrich) was dissolved in water to prepare an aqueous solution of 5-ALA-HCl having a concentration of 1M. A 24-well cell culture plate (Corning Incorporated Corning, model 3524) was prepared, and 960 μl of cell suspension was dispensed into each well. Immediately after adding 6 μl of 1M 5-ALA-HCl aqueous solution to the cell suspension dispensed in 5 wells, 5 kinds of gold citrate colloid solutions with different gold nanoparticle diameters ( 240 μl of Table 1) was added to each cell suspension. These cell samples contain 5 mM 5-ALA-HCl. In addition, gold nanoparticles having a particle size of 5 nm contain gold nanoparticles at a concentration of 44 μM, and gold nanoparticles having a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 72 μM. .
Also, to prepare a comparative cell sample containing 5 mM 5-ALA-HCl but no gold nanoparticles, 6 μl of 1M 5-ALA-HCl was added to another two-well cell suspension. Immediately after adding the aqueous solution, 240 μl of citrate buffer (pH 3.3) was added to one, and 240 μl of water was added to the other.

Note that the concentration of the number of cells in each of the above cell samples was set to about 5 × 10 ⁵ cells / ml. This cell concentration is a value measured with a cell number measuring device (Coulter multisizer, model Z2). Here, the operation from the start of adding 5-ALA-HCl to the cell suspension until the cell sample was placed in a dark place in the following incubator was performed within 10 minutes under extremely dim white light. In particular, since a cell sample containing 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm was prepared last, the time of exposure to dim white light was about a few minutes.
The cell culture plate having the cell sample prepared as described above and the comparative cell sample was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ and left for 22 hours. After the 22 hour incubation of the cell samples was thus completed, all cell samples were collected from the cell culture plate. The cell recovery operation was performed within 30 minutes under extremely dim white light (3.0 × 10 ⁻³ μW / cm ² ).

  The cell concentration of the cell sample collected in this way was measured with a cell counter (Coulter multisizer, model Z2). As shown in FIG. 9, in the cell sample to which 5-ALA-HCl and citrate buffer (pH 3.3) or water were added, the cell concentration was increased 1.8 times. In contrast to these cell samples, cell samples held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 103 nm all showed an increase in cell concentration lower than 1.8 times. It was. From these facts, by holding the cells in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 103 nm, gold nanoparticles are not present even in the presence of 5-ALA-HCl. It was found that the cell growth was further suppressed with respect to the cell sample retained in (1). Moreover, when the cell concentration was compared between these cell samples, it was found that the rate of increase in the cell concentration was lower as the particle size of the gold nanoparticles was smaller. Among these cell samples, especially the cell sample using gold nanoparticles having a particle size of 17 nm increases the cell concentration by about 30% compared to the increase in cell concentration of the cell sample without using gold nanoparticles. showed that. In addition, the cell sample using gold nanoparticles having a particle size of 5 nm showed an increase in cell concentration of about 50% less than the increase in cell concentration of the cell sample without using gold nanoparticles.

{Example 4-1}
In this example, cultured cells derived from human breast cancer cells (MCF-) in the presence of 5-ALA-HCl and five types of gold nanoparticles having different particle sizes (5 nm, 17 nm, 41 nm, 82 nm, and 103 nm). 7) was held for 19 hours.
Powdered 5-ALA-HCl (Sigma-Aldrich) was dissolved in water to prepare an aqueous solution of 5-ALA-HCl having a concentration of 1M. Prepare 990 μl of RPMI 1640 liquid medium containing 10% fetal calf serum and no phenol red in 5 tubes, add 5.5 μl of 1M 5-ALA-HCl aqueous solution to each, 110 μl of the above five kinds of gold citrate colloid solutions (Table 1) having different diameters were added to each liquid medium. A liquid medium to which these 5-ALA-HCl aqueous solution and gold citrate colloid solution are added is a cell growth inhibitor containing 5-ALA-HCl and gold nanoparticles. Also, prepare 990 μl of RPMI 1640 liquid medium containing 10% fetal calf serum and no phenol red in two tubes, add 5.5 μl of 1M 5-ALA-HCl aqueous solution to each, and add citrate buffer (PH 3.3) or 110 μl of water was added to each liquid medium. These liquid media contain 5 mM 5-ALA-HCl. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. .

To prepare a liquid medium having the same pH as the above liquid medium containing 5 mM 5-ALA-HCl, 990 μl of RPMI 1640 liquid medium containing 10% fetal calf serum and no phenol red was added to five tubes. Each was prepared, 5.3 μl of about 1 M HCl aqueous solution was added to each, and the above five kinds of gold citrate colloidal solutions having different gold nanoparticle diameters were added to each liquid medium by 110 μl. In addition, 990 μl each of RPMI 1640 liquid medium containing 10% fetal calf serum and not containing phenol red was prepared in two tubes, and 5.3 μl of about 1 M HCl aqueous solution was added to each, and citrate buffer (pH 3. 3) Or 110 μl of water was added to each liquid medium.
In order to prepare a liquid medium containing gold nanoparticles containing neither 5-ALA-HCl nor HCl, 990 μl each of RPMI 1640 liquid medium containing 10% fetal calf serum and no phenol red in five tubes Prepared, 110 μl of each of the above five gold citrate colloidal solutions having different gold nanoparticle diameters was added to each liquid medium. Also, prepare 990 μl of RPMI 1640 liquid medium containing 10% fetal bovine serum and no phenol red in two tubes, and add 110 μl of citrate buffer (pH 3.3) or water to each liquid medium. It was.

MCF-7 cells were cultured in RPMI 1640 liquid medium containing 10% fetal calf serum. The number of MCF-7 cells used here doubles every 18 to 20 hours in an atmosphere maintained at 37 ° C. and 5% CO ₂ concentration. Cells suspended by treatment with Trypsin-EDTA were resuspended in an RPMI 1640 liquid medium not containing phenol red to obtain a cell suspension having a cell concentration of about 4 × 10 ⁴ cells / ml. This cell concentration is a value measured with a cell number measuring device (Coulter multisizer, model Z2). A 96-well cell culture plate (Corning Incorporated Corning, model 3596) was prepared, and 100 μl of this cell suspension was dispensed into each well. The cell culture plate was placed in an incubator maintained at 37 ° C. and 5% CO ₂ concentration for 24 hours.

Only the medium (supernatant) is removed from the cell sample in each well, and each of the liquid medium containing 5-ALA-HCl, the liquid medium containing HCl, and 5-ALA-HCl and HCl are used. 100 μl of liquid medium not containing was added.
Here, the operation from the start of adding the liquid medium containing 5-ALA-HCl to the cell sample until the cell sample is placed in the dark place in the following incubator is within 10 minutes under extremely dim white light. went. In particular, since a cell sample containing 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm was prepared last, the time of exposure to dim white light was about 1 minute. Preparation of cell samples containing HCl and cell samples containing neither 5-ALA-HCl nor HCl was performed under the same conditions as those for preparing cell samples containing 5-ALA-HCl.

The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 19 hours. After the 19 hour incubation of the cell sample was thus completed, the cell culture plate was removed from the incubator. After removing only the medium (supernatant) from the cell samples in each well and adding 100 μl of DPBS, a CCK-F assay using a cytotoxicity detection reagent (Cell Counting Kit F, Dojindo) was performed according to the instruction manual. . In this CCK-F assay, the work from the start of replacing the cell sample medium to DPBS until the addition of Calcein-AM to the cell sample is extremely dim white light (9.5 × 10 ⁻³ μW / cm ² ). Under 20 minutes. After adding Calcein-AM to the cell sample and leaving it in a completely dark place at room temperature for 1 hour, the fluorescence intensity of 535 nm emitted from the cell sample by the excitation light with a wavelength of 485 nm is measured with a fluorescence microplate reader. (HTS 7000, Perkin Elmer). In addition, when this fluorescence measurement was performed simultaneously also about the cell sample which has not added Calcein-AM, the fluorescence was not measured at all.

As shown in FIG. 10, the fluorescence intensity obtained with the cell sample retained in the presence of 5-ALA-HCl and gold nanoparticles with a particle size of 5-103 nm is It can be seen that it is significantly lower than the fluorescence intensity obtained with the comparative cell sample not containing at least one of the particles. This result indicates that dark toxicity was remarkably induced in the cells by maintaining the cells in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 103 nm. . In addition, when the fluorescence intensity was compared between cell samples held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 103 nm, the smaller the gold nanoparticles, the more fluorescence It can also be seen that the strength decreases. Among these cell samples, a cell sample using gold nanoparticles having a particle diameter of 17 nm in particular showed a fluorescence intensity of about 80% less than that of the comparative cell sample. Moreover, the cell sample using the gold nanoparticle having a particle diameter of 5 nm hardly emitted fluorescence. This indicates that almost all cells are dead.
From the above results, it can be seen that maintaining MCF-7 cells in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 103 nm suppresses their cell proliferation for 23 hours. It was. It has also been found that cell proliferation is extremely effectively suppressed by using gold nanoparticles having a particle size of 5 nm or 17 nm.

{Example 4-2}
In this example, 5-ALA-HCl and five types having different particle diameters were used in the same manner as in Example 4-1, except that the holding time was selected from 0 hours, 6 hours, 12 hours, and 18 hours. MCF-7 cells were maintained in the presence of 5 nm, 17 nm, 41 nm, 82 nm, and 103 nm gold nanoparticles.
Prepare 990 μl of RPMI 1640 liquid medium containing 10% fetal calf serum and no phenol red in 5 tubes, add 5.5 μl of 1M 5-ALA-HCl aqueous solution to each, 110 μl of the above five kinds of gold citrate colloid solutions (Table 1) having different diameters were added to each liquid medium. Also, prepare 990 μl of RPMI 1640 liquid medium containing 10% fetal calf serum and no phenol red in four tubes, add 5.5 μl of 1M 5-ALA-HCl aqueous solution to each, 110 [mu] l of three different citrate buffers (pH 3.1, 3.3, and 6.5) or water were added to each liquid medium. These liquid media contain 5 mM 5-ALA-HCl. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. .

In addition, RPMI 1640 liquid medium containing 10% fetal calf serum and no phenol red was prepared to prepare a liquid medium containing neither 5ALA-HCl nor HCl, but containing 5 nm gold nanoparticles. 990 μl was prepared in one tube, and 110 μl of gold citrate colloidal solution containing gold nanoparticles having a particle size of 5 nm was added. Also, 990 μl of RPMI 1640 liquid medium containing 10% fetal calf serum and no phenol red was prepared in one tube, and 110 μl of water was added.
MCF-7 cells were cultured in RPMI 1640 liquid medium containing 10% fetal calf serum. Cells suspended by treatment with Trypsin-EDTA were resuspended in an RPMI 1640 liquid medium not containing phenol red to obtain a cell suspension having a cell concentration of about 4 × 10 ⁴ cells / ml. Four 96-well cell culture plates (Corning Incorporated Corning, model 3596) were prepared, and 100 μl of the cell suspension was dispensed into each well of each cell culture plate. The cell culture plate was placed in an incubator where the temperature was maintained at 37 ° C. and 5% CO ₂ for 24 hours.

  Only the medium (supernatant liquid) was removed from the cell sample in each well, and 100 μl of the liquid medium containing 5-ALA-HCl and the liquid medium not containing 5-ALA-HCl were added to each. Here, the operation from the start of adding the liquid medium containing 5-ALA-HCl to the cell sample until the cell sample is placed in the dark place in the following incubator is within 10 minutes under extremely dim white light. went. In particular, since a cell sample containing 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm was prepared last, the time of exposure to dim white light was about 1 minute. The cell sample containing no 5-ALA-HCl was prepared under the same conditions as those for preparing the cell sample containing 5-ALA-HCl.

Three of the cell culture plates with the cell sample prepared as described above and the comparative cell sample were transferred into a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration, They were placed for 6 hours, 12 hours, and 18 hours, respectively. One cell culture plate was immediately transferred to the following CCK-F assay without being transferred into the incubator. This was a cell sample that had been incubated for 0 hours. After the incubation of the cell sample and the comparative cell sample was thus completed, the cell culture plate was removed from the incubator.
After removing only the medium (supernatant) from the cell samples in each well and adding 100 μl of DPBS, a CCK-F assay using a cytotoxicity detection reagent (Cell Counting Kit F, Dojindo) was performed according to the instruction manual. . In this CCK-F assay, the work from the start of replacing the cell sample medium with DPBS to the addition of Calcein-AM to the cell sample is extremely dim white light (9.5 × 10 ⁻³ μW / cm ² ). Under 20 minutes. After adding Calcein-AM to the cell sample and leaving it in a completely dark place at room temperature for 1 hour, the fluorescence intensity of 535 nm emitted from the cell sample by the excitation light with a wavelength of 485 nm is measured with a fluorescence microplate reader. (HTS 7000, Perkin Elmer). In addition, when this fluorescence measurement was performed simultaneously also about the cell sample which has not added Calcein-AM, fluorescence was not measured at all.

  As shown in FIG. 11A, all cell samples showed almost the same fluorescence intensity at 0 hour retention, but at 6 hours retention time, 5-ALA-HCl and 5 nm Cell samples kept in the presence of gold nanoparticles with particle size showed lower fluorescence intensity than other cell samples. In addition, as shown in FIG. 11C, in the retention time of 12 hours, the cell sample retained in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm is different from the other cell samples. In addition to showing lower fluorescence intensity, the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles with a particle size of 17 nm also showed lower fluorescence intensity than other cell samples. In addition, as shown in FIG. 11D, at the retention time of 18 hours, the cell sample retained in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm almost showed no fluorescence. , Cell samples kept in the presence of 5-ALA-HCl and gold nanoparticles with a particle size of 17 nm also showed very low fluorescence intensity relative to other cell samples.

  From these results, when MCF-7 cells are retained in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm, cell proliferation can be suppressed with a retention time of 4 hours or more. In the case where cells are held in the presence of 5-ALA-HCl and gold nanoparticles having a particle diameter of 17 nm, it has been found that cell growth can be suppressed with a holding time of 12 hours or longer.

{Example 4-3}
In this example, MCF-7 cells were maintained for 26 hours in the presence of 5-ALA-HCl and 5 types (5 nm, 17 nm, 41 nm, 82 nm, and 103 nm) of gold nanoparticles having different particle sizes.
MCF-7 cells were cultured in RPMI 1640 liquid medium containing 10% fetal calf serum. Cells suspended by treatment with Trypsin-EDTA were resuspended in RPMI 1640 liquid medium to obtain a cell suspension having a cell concentration of the order of 10 ⁴ cells / ml. A 12-well cell culture plate (Corning Incorporated Corning, model 3512) was prepared, and 2 ml of the cell suspension was dispensed into each well. The cell culture plate was placed in an incubator where the temperature was maintained at 37 ° C. and 5% CO ₂ for 25 hours.
Immediately after removing the medium alone (supernatant) from the cell samples in each well, 2 ml of fresh RPMI 1640 liquid medium containing 10% fetal calf serum was added. 11 μl of 1M 5-ALA-HCl aqueous solution was added to each cell sample, and 220 μl of the above five kinds of gold citrate colloid solutions (Table 1) having different gold nanoparticle diameters were added to each culture solution. These culture solutions contain 5 mM 5-ALA-HCl. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. .

Here, the operation from the start of adding the liquid medium containing 5-ALA-HCl to the cell sample until the cell sample is placed in the dark place in the following incubator is within 10 minutes under extremely dim white light. went. In particular, since a cell sample containing 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm was prepared last, the time of exposure to dim white light was about 1 minute. The cell sample containing no 5-ALA-HCl was also prepared under the same conditions as those for preparing the cell sample containing 5-ALA-HCl.
The cell culture plate having the cell sample prepared as described above and the comparative cell sample was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ and left for 26 hours. After the 26-hour incubation of the cell samples was thus completed, the cell culture plate was removed from the incubator, and the morphological observation of the cells of each cell sample was performed using a stereo microscope (Nicon Eclipse TS100) equipped with a digital camera (Nikon Coolpix 4500). It was performed using.

  As shown in FIG. 12, the cells of the comparative cell sample that did not contain at least one of 5-ALA-HCl and gold nanoparticles all had a normal shape. However, in the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm, all cells contracted spherically and had an abnormal shape. Moreover, even in the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles having a particle diameter of 17 nm, most of the cells had an abnormal shape that contracted into a spherical shape. Furthermore, the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 41 nm also had an abnormal shape in which many cells contracted spherically. This result also shows that dark toxicity was remarkably induced in the cells by holding the cells in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 41 nm. . In addition, in the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles having particle diameters of 82 nm and 103 nm, cells having an abnormal shape contracted spherically were not observed.

  From the above results, it is shown that the growth of MCF-7 cells is suppressed for 26 hours in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 41 nm to suppress their cell growth. It was also found from observation. It has also been found that cell proliferation is extremely effectively suppressed by using gold nanoparticles having a particle size of 5 nm or 17 nm.

{Example 4-4}
In this example, cultured cells (MCF-7) derived from breast cancer cells in the presence of 5-ALA-Me and four types of gold nanoparticles with different particle sizes (5 nm, 17 nm, 41 nm, and 82 nm) were used. Hold for 19 hours.
Powdered 5-ALA-Me (Sigma-Aldrich) was dissolved in water to prepare a 1M 5-ALA-Me aqueous solution. Prepare 990 μl of RPMI 1640 liquid medium containing 10% fetal calf serum and no phenol red in 4 tubes, add 5.5 μl of 1M 5-ALA-Me aqueous solution to each, Four types (5 nm, 17 nm, 41 nm, and 82 nm) of gold citrate colloid solutions (Table 1) having different diameters were added to each liquid medium in an amount of 110 μl. A liquid medium to which these 5-ALA-Me aqueous solution and gold citrate colloid solution are added is a cell growth inhibitor containing 5-ALA-Me and gold nanoparticles. In addition, 990 μl each of RPMI 1640 liquid medium containing 10% fetal calf serum and no phenol red was prepared in two tubes, to each of which 5.5 μl of 1M5-ALA-Me aqueous solution was added, and citrate buffer ( 110 μl of pH 3.3) or water was added to each liquid medium. These liquid media contain 5 mM 5-ALA-HCl. In addition, gold nanoparticles having a particle size of 5 nm are contained at a concentration of 22 μM, and gold nanoparticles having a particle size of 17 nm, 41 nm, and 82 nm are contained at a concentration of 36 μM, respectively.

  For comparison, in order to prepare a liquid medium containing 5 mM 5-ALA-HCl, RPMI 1640 liquid medium containing 10% fetal calf serum and not containing phenol red was prepared in four tubes, 990 μl each. 1 M 5-ALA-HCl aqueous solution was added in an amount of 5.5 μl, and the above-mentioned four types of gold citrate colloidal solutions having different gold nanoparticle diameters were added to each liquid medium in an amount of 110 μl. In addition, 990 μl each of RPMI 1640 liquid medium containing 10% fetal calf serum and not containing phenol red was prepared in two tubes, and 5.5 μl of about 1M 5-ALA-HCl aqueous solution was added to each tube. 110 μl of buffer (pH 3.3) or water was added to each liquid medium.

MCF-7 cells were cultured in RPMI 1640 liquid medium containing 10% fetal calf serum. The number of MCF-7 cells used here doubles every 18-20 hours in an atmosphere in which 37 ° C. and a CO ₂ concentration of 5% are maintained. Cells suspended by treatment with Trypsin-EDTA were resuspended in an RPMI 1640 liquid medium not containing phenol red to obtain a cell suspension having a cell concentration of about 4 × 10 ⁴ cells / ml. A 96-well cell culture plate (Corning Incorporated Corning, model 3596) was prepared, and 100 μl of this cell suspension was dispensed into each well. The cell culture plate was left for 24 hours in an incubator maintained at 37 ° C. and 5% CO ₂ concentration.
Only the medium (supernatant liquid) was removed from the cell sample in each well, and 100 μl each of the liquid medium containing 5-ALA-Me and the liquid medium containing 5-ALA-HCl was added to each.
Here, the operation from the start of adding the liquid medium containing 5-ALA-Me to the cell sample until the cell sample is placed in the dark place in the following incubator is within 10 minutes under extremely dim white light. went. The cell sample containing 5-ALA-HCl was also prepared under the same conditions as those for preparing the cell sample containing 5-ALA-Me.

The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 19 hours. After the 19 hour incubation of the cell sample was thus completed, the cell culture plate was removed from the incubator. After removing only the medium (supernatant) from the cell samples in each well and adding 100 μl of DPBS, a CCK-F assay using a cytotoxicity detection reagent (Cell Counting Kit F, Dojindo) was performed according to the instruction manual. . In this CCK-F assay, the work from the start of replacing the cell sample medium with DPBS to the addition of Calcein-AM to the cell sample is extremely dim white light (9.5 × 10 ⁻³ μW / cm ² ). Under 20 minutes. After adding Calcein-AM to the cell sample and leaving it in a completely dark place at room temperature for 1 hour, the fluorescence intensity of 535 nm emitted from the cell sample by the excitation light with a wavelength of 485 nm is measured with a fluorescence microplate reader. (HTS 7000, Perkin Elmer). In addition, when this fluorescence measurement was performed simultaneously also about the cell sample which has not added Calcein-AM, fluorescence was not measured at all.

  As shown in FIG. 13, the fluorescence intensity obtained for each cell sample retained in the presence of 5-ALA-Me and gold nanoparticles with a particle size of 5-82 nm is determined by the 5-ALA-Me and the quencher. It can be seen that the fluorescence intensity is remarkably lower than that obtained with a cell sample for comparison using a liquid medium to which an acid buffer (pH 3.3) is added or a liquid medium to which 5-ALA-Me and water are added. . This result indicates that dark toxicity was remarkably induced in the cells by holding the cells in the presence of 5-ALA-Me and gold nanoparticles having a particle size of 5 to 82 nm. . Moreover, when the fluorescence intensity was compared between the cell samples held in the presence of 5-ALA-Me and gold nanoparticles having a particle size of 5-82 nm, the smaller the particle size of the gold nanoparticles, the greater the fluorescence intensity. It can also be seen that the strength decreases. Among these cell samples, a cell sample using gold nanoparticles having a particle diameter of 17 nm in particular showed a fluorescence intensity of about 80% less than that of the comparative cell sample. Moreover, the cell sample using the gold nanoparticles having a particle size of 5 nm hardly emitted fluorescence. This indicates that almost all cells are dead.

  From the above results, it can be seen that by maintaining MCF-7 cells for 19 hours in the presence of 5-ALA-Me and gold nanoparticles having a particle size of 5-82 nm, their cell proliferation is suppressed. It was. At this time, it was also found that cell proliferation is extremely effectively suppressed by using gold nanoparticles having a particle size of 5 nm or 17 nm.

{Example 5-1}
In this example, 24 normal human fibroblasts (1522) were present in the presence of 5-ALA-HCl and 5 types of gold nanoparticles having different particle sizes (5 nm, 17 nm, 41 nm, 82 nm, and 103 nm). Held for hours.
Powdered 5-ALA-HCl (Sigma-Aldrich) was dissolved in water to prepare an aqueous solution of 5-ALA-HCl having a concentration of 1M. In addition, an F12 liquid medium containing 20% non-heated fetal calf serum was prepared. In the following, what is simply written as “F12 liquid medium” contains 20% of non-heated fetal calf serum. Take 480 μl of this F12 liquid medium in 5 tubes, add 3 μl of 1M 5-ALA-HCl aqueous solution to each, and add the above 5 kinds of gold citrate colloid solutions (Table 1) with different gold nanoparticle diameters. 120 μl was added to each F12 liquid medium. The F12 liquid medium to which these 5-ALA-HCl aqueous solution and gold citrate colloid solution are added is a cell growth inhibitor containing 5-ALA-HCl and gold nanoparticles. In addition, 480 μl of F12 liquid medium was taken in two tubes, 3 μl of 1M 5-ALA-HCl aqueous solution was added to each tube, and 120 μl of citrate buffer (pH 3.3) or water was added to each liquid medium. These liquid media contain 5 mM 5-ALA-HCl. In addition, gold nanoparticles having a particle size of 5 nm contain gold nanoparticles at a concentration of 44 μM, and gold nanoparticles having a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 72 μM. .

In order to prepare F12 liquid medium having the same pH as the above-mentioned F12 liquid medium containing 5 mM 5-ALA-HCl, 480 μl each of F12 liquid medium is taken into five tubes, and about 3 μl each of about 1 M HCl aqueous solution is taken. 120 μl each of the above five types of gold citrate colloidal solutions having different gold nanoparticle diameters were added to each F12 liquid medium. Further, 480 μl each of F12 liquid medium was taken in two tubes, about 3 μl of about 1 M HCl aqueous solution was added to each tube, and citrate buffer (pH 3.3) or water was added to each liquid medium 120 μl.
In order to prepare F12 liquid culture medium containing neither gold nor nanoparticles containing 5-ALA-HCl nor HCl, 480 μl each of F12 liquid culture medium is taken into five tubes, and the above five kinds of gold nanoparticles having different particle sizes are used. 120 μl of gold citrate colloid solution was added to each F12 liquid medium. Further, 480 μl each of F12 liquid medium was taken in two tubes, and 120 μl each of citrate buffer (pH 3.3) or water was added to each F12 liquid medium.

1522 cells cultured in F12 liquid medium were treated with Trypsin-EDTA, suspended, and resuspended in fresh F12 liquid medium to obtain a cell suspension having a cell concentration of about 5 × 10 ⁴ cells / ml. This cell density | concentration is the value measured with the cell number measuring device (Coulter multisizer, model Z2). A 96-well cell culture plate (Corning Incorporated Corning, model 3596) was prepared, and 100 μl of this cell suspension was dispensed into each well. The cell culture plate was placed in an incubator where the temperature was maintained at 37 ° C. and 5% CO ₂ for 24 hours.
Only the medium (supernatant) is removed from the cell sample in each hole, and the F12 liquid medium containing 5-ALA-HCl, the F12 liquid medium containing HCl, and 5-ALA prepared in advance as described above. -100 μl of F12 liquid medium without both HCl and HCl was added to each well.

  Here, the operation from the start of adding the above-mentioned F12 liquid medium containing 5-ALA-HCl to the cell sample until the cell sample is placed in the dark place in the following incubator is within 10 minutes under extremely dim white light. I went there. In particular, since a cell sample containing 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm was prepared last, the time of exposure to dim white light was about 1 minute. Preparation of cell samples containing HCl and cell samples containing neither 5-ALA-HCl nor HCl was performed under the same conditions as those for preparing cell samples containing 5-ALA-HCl.

The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 24 hours. Thus, after the 24-hour incubation of the cell sample was completed, the cell culture plate was removed from the incubator. After removing only the medium (supernatant) from the cell samples in each well and adding 100 μl of DPBS, a CCK-F assay using a cytotoxicity detection reagent (Cell Counting Kit F, Dojindo) was performed according to the instruction manual. . In this CCK-F assay, the work from the start of replacing the cell sample medium with DPBS to the addition of Calcein-AM to the cell sample is extremely dim white light (9.5 × 10 ⁻³ μW / cm ² ). Under 20 minutes. After adding Calcein-AM to a cell sample and leaving it in a completely dark place at room temperature for 2 hours, the fluorescence intensity of 535 nm emitted from the cell sample by excitation light with a wavelength of 485 nm is measured with a fluorescence microplate reader. (HTS 7000, Perkin Elmer). In addition, when this fluorescence measurement was performed simultaneously also about the cell sample which has not added Calcein-AM, fluorescence was not measured at all.

As shown in FIG. 14, the fluorescence intensity obtained with the cell sample retained in the presence of 5-ALA-HCl and gold nanoparticles with a particle size of 5 nm is the same as that of 5-ALA-HCl and gold nanoparticles. The fluorescence intensity was reduced by about 40% compared to the fluorescence intensity obtained with the comparative cell sample not containing at least one. The fluorescence intensity obtained with the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles with a particle size of 17 nm is a comparative cell that does not contain at least one of 5-ALA-HCl and gold nanoparticles. The fluorescence intensity was about 20% less than that obtained with the sample. On the other hand, the fluorescence intensity obtained in the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 41 to 103 nm was compared with the fluorescence intensity obtained in the comparative cell sample. It is not so low. This result shows that retention of 1522 cells in the presence of 5-ALA-HCl and gold nanoparticles with a particle size of 5-17 nm significantly induced dark toxicity in the cells. is there.
From the above results, it was found that maintaining 1522 cells in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 5 to 17 nm for 24 hours suppresses their cell growth.

{Example 5-2}
In this example, the concentration of gold nanoparticles having a particle size of 5 nm was set to 22 μM, 33 μM, or 44 μM, and the concentration of 5-ALA-HCl was set to 2.5 mM, 5 mM, or 10 mM. Similar to 5-1, 1522 cells were maintained in the presence of 5-ALA-HCl and gold nanoparticles with a particle size of 5 nm.
First, for gold citrate colloidal solution (Table 1) containing gold nanoparticles with a particle size of 5 nm, the concentration of gold nanoparticles was diluted to 3/4 and 1/2 respectively with citrate buffer (pH 3.3). I prepared what I did. Next, 480 μl each of the F12 liquid medium is taken into 16 tubes, and 1.5 μl of 1M5-ALA-HCl aqueous solution is added to the F12 liquid medium of 4 tubes, and gold citrate colloid solution and their diluted solutions ( The gold nanoparticle content concentration was diluted to 3/4 or 1/2), and citrate buffer (pH 3.3) was added to each F12 liquid medium in an amount of 120 μl.

In another 4 tubes of F12 liquid medium, add 3 μl of 1M 5-ALA-HCl aqueous solution, colloidal gold citrate solution, their dilutions (concentration of gold nanoparticles to 3/4 or 1/2) Dilution), and citrate buffer (pH 3.3) were added in an amount of 120 μl to each F12 liquid medium. In addition, 6 μl of 1M 5-ALA-HCl aqueous solution was added to another 12 tubes of F12 liquid medium, and gold citrate colloid solutions and their diluted solutions (the concentration of gold nanoparticles was 3/4 or 1/2). And citrate buffer (pH 3.3) were added to each liquid medium in an amount of 120 μl.
Without adding 5-ALA-HCl aqueous solution to the remaining four tubes of F12 liquid medium, colloidal gold citrate solutions and their dilutions (the concentration of gold nanoparticles was diluted to 3/4 or 1/2) ), And citrate buffer (pH 3.3) were added in an amount of 120 μl to each F12 liquid medium. In this way, 16 types of F12 liquid media were prepared in combinations of concentrations of 0 μM, 22 μM, 33 μM, and 44 μM for gold nanoparticles and concentrations of 0 mM, 2.5 mM, 5 mM, and 10 mM for 5-ALA-HCl. did.

1522 cells cultured in F12 liquid medium were suspended by treatment with Trypsin-EDTA and resuspended in fresh F12 liquid medium to obtain a cell suspension having a cell concentration of about 5 × 10 ⁴ cells / ml. . A 96-well cell culture plate (Corning Incorporated Corning, model 3596) was prepared, and 100 μl of this cell suspension was dispensed into each well. The cell culture plate was placed in an incubator maintained at 37 ° C. and 5% CO ₂ concentration for 24 hours.
Only the medium (supernatant) was removed from the cell sample in each well, and 100 μl of the above 16 types of liquid medium were added to each. Here, the operation from the start of adding the liquid medium to the cell sample until the cell sample was placed in the dark place in the following incubator was performed within 30 minutes under extremely dim white light.

The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 24 hours. Thus, after the 24-hour holding of the cell sample was completed, the cell culture plate was taken out of the incubator. After removing only the medium (supernatant) from the cell samples in each well and adding 100 μl of DPBS, a CCK-F assay using a cytotoxicity detection reagent (Cell Counting Kit F, Dojindo) was performed according to the instruction manual. . In this CCK-F assay, the work from the start of replacing the cell sample medium with DPBS to the addition of Calcein-AM to the cell sample is extremely dim white light (9.5 × 10 ⁻³ μW / cm ² ). Under 20 minutes. After adding Calcein-AM to the cell sample and leaving it in a completely dark place at room temperature for 2 hours, the fluorescence intensity of 535 nm emitted from the cell sample by the excitation light with a wavelength of 485 nm is measured with a fluorescence microplate reader. (HTS7000, Perkin Elmer). In addition, when this fluorescence measurement was performed simultaneously also about the cell sample which has not added Calcein-AM, fluorescence was not measured at all.

As shown in FIG. 15, when a liquid medium to which 5-ALA-HCl was not added was used, fluorescence was not observed between cell samples to which gold nanoparticles having a particle size of 5 nm were added at a concentration of 44 μM or less. There was no significant difference in strength. On the other hand, when the concentration of 5-ALA-HCl is 2.5 mM, a cell sample using a liquid medium to which 5-ALA-HCl and gold nanoparticles are not added in a cell sample to which gold nanoparticles are not added With respect to the fluorescence intensity, a decrease in fluorescence intensity of about 10% was observed, and slight cytotoxicity was observed with 5-ALA-HCl alone. There was no significant difference in fluorescence intensity between cell samples to which gold nanoparticles were further added at a concentration of 44 μM or less.
When the concentration of 5-ALA-HCl was 5 mM, a liquid medium to which 5-ALA-HCl and gold nanoparticles were not added was used in a cell sample to which gold nanoparticles having a particle size of 5 nm were not added. With respect to the fluorescence intensity of the cell sample, a decrease in fluorescence intensity of about 20% was observed, and some cytotoxicity was observed with 5-ALA-HCl alone. In the cell sample in which gold nanoparticles were further added at a concentration of 44 μM to this cell sample, a decrease in fluorescence intensity of about 50% was observed. From this result, it was found that when the concentration of 5-ALA-HCl is 5 mM, dark toxicity is remarkably synergized by adding gold nanoparticles at a concentration of 44 μM.

  In addition, when the concentration of 5-ALA-HCl is 10 mM, in a cell sample to which gold nanoparticles having a particle diameter of 5 nm are not added, a liquid medium to which 5-ALA-HCl and gold nanoparticles are not added is used. With respect to the fluorescence intensity of the cell sample used, a decrease in fluorescence intensity of nearly 50% was observed, and considerable cytotoxicity was observed only with 5-ALA-HCl. In contrast to this cell sample, a decrease in fluorescence intensity of 50% was observed in the cell sample added with gold nanoparticles at a concentration of 33 μM, and nearly 75% was observed in the cell sample added with gold nanoparticles at a concentration of 44 μM. A decrease in fluorescence intensity was observed. From this result, it was found that when the concentration of 5-ALA-HCl is 10 mM, dark toxicity is remarkably synergized by adding gold nanoparticles at a concentration of 33 μM or 44 μM.

{Example 5-3}
In this example, human fibroblast normal cells (1522) were maintained for 24 hours in the presence of 5-ALA-Me and gold nanoparticles having a particle size of 5 nm or 17 nm.
Powdered 5-ALA-Me (Sigma-Aldrich) was dissolved in water to prepare an aqueous solution of 5-ALA-Me having a concentration of 1M. Take 480 μl each of F12 liquid medium in two tubes, add 3 μl of 1M 5-ALA-Me aqueous solution to each, and add gold citrate colloidal solution (Table 1) containing gold nanoparticles of 5 nm or 17 nm in diameter. 120 μl was added to each F12 liquid medium. The F12 liquid medium to which these 5-ALA-Me aqueous solution and gold citrate colloid solution are added is a cell growth inhibitor containing 5-ALA-Me and gold nanoparticles. Further, 480 μl of F12 liquid medium was taken in one tube, 3 μl of 1M 5-ALA-Me aqueous solution was added, and 120 μl of citrate buffer (pH 6.5) was added. These liquid media contain 5 mM 5-ALA-Me. In addition, gold nanoparticles having a particle size of 5 nm contain gold nanoparticles at a concentration of 44 μM, and those containing gold nanoparticles of a particle size of 17 nm contain gold nanoparticles at a concentration of 72 μM.

On the other hand, to prepare a comparative F12 liquid medium containing 5 mM 5-ALA-HCl, 480 μl of the F12 liquid medium was taken in two tubes, and 3 μl of 1M 5-ALA-HCl aqueous solution was added to each tube. 120 μl of gold citrate colloidal solution containing gold nanoparticles with a particle size of 17 nm was added to each F12 liquid medium. In addition, 480 μl of F12 liquid medium was taken in one tube, 3 μl of 1M 5-ALA-HCl aqueous solution was added, and 120 μl of citrate buffer (pH 6.5) was added. These liquid media contain 5 mM 5-ALA-HCl.
1522 cells cultured in F12 liquid medium were suspended by treatment with Trypsin-EDTA and resuspended in fresh F12 liquid medium to obtain a cell suspension having a cell concentration of about 5 × 10 ⁴ cells / ml. . A 96-well cell culture plate (Corning Incorporated Corning, model 3596) was prepared, and 100 μl of this cell suspension was dispensed into each well. The cell culture plate was placed in an incubator where the temperature was maintained at 37 ° C. and 5% CO ₂ for 24 hours.

Only the medium (supernatant liquid) was removed from the cell sample in each well, and 100 μl of the above-described liquid medium containing 5-ALA-Me or the above-mentioned liquid medium containing 5-ALA-HCl was added thereto.
Here, the operation from the start of adding the liquid medium containing 5-ALA-Me to the cell sample until the cell sample is placed in the dark place in the following incubator is within 10 minutes under extremely dim white light. went. In particular, since a cell sample containing 5-ALA-Me and gold nanoparticles having a particle size of 5 nm was prepared last, the time of exposure to dim white light was about 1 minute. The cell sample containing 5-ALA-HCl was also prepared under the same conditions as those for preparing the cell sample containing 5-ALA-Me.

The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 24 hours. Thus, after the 24-hour holding of the cell sample was completed, the cell culture plate was taken out of the incubator. After removing only the medium (supernatant) from the cell samples in each well and adding 100 μl of DPBS, a CCK-F assay using a cytotoxicity detection reagent (Cell Counting Kit F, Dojindo) was performed according to the instruction manual. . In this CCK-F assay, the work from the start of replacing the cell sample medium with DPBS to the addition of Calcein-AM to the cell sample is extremely dim white light (9.5 × 10 ⁻³ μW / cm ² ). Under 20 minutes. After adding Calcein-AM to a cell sample and leaving it in a completely dark place at room temperature for 2 hours, the fluorescence intensity of 535 nm emitted from the cell sample by excitation light with a wavelength of 485 nm is measured with a fluorescence microplate reader. (HTS 7000, Perkin Elmer). In addition, when this fluorescence measurement was performed simultaneously also about the cell sample which has not added Calcein-AM, fluorescence was not measured at all.

As shown in FIG. 16, the fluorescence intensity obtained with cell samples containing 5-ALA-Me and citrate buffer (pH 6.5) includes 5-ALA-HCl and citrate buffer (pH 6.5). It was not significantly different from the fluorescence intensity obtained in the cell sample.
The fluorescence intensity obtained with a cell sample retained in the presence of 5-ALA-Me and gold nanoparticles with a particle size of 5 nm is a cell sample containing 5-ALA-Me and citrate buffer (pH 6.5). Compared to the fluorescence intensity obtained in Example 1, the fluorescence intensity decreased by nearly 85%. Moreover, the fluorescence intensity was almost the same as the fluorescence intensity obtained with the cell sample containing 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm. This result indicates that retention of 1522 cells in the presence of 5-ALA-Me and gold nanoparticles having a particle size of 5 nm significantly induced dark toxicity in the cells.

On the other hand, the fluorescence intensity obtained with the cell sample held in the presence of 5-ALA-HCl and gold nanoparticles having a particle size of 17 nm includes 5-ALA-Me and citrate buffer (pH 6.5). The fluorescence intensity was almost the same as that obtained with the cell sample. The fluorescence intensity is twice that obtained with a cell sample containing 5-ALA-HCl and gold nanoparticles with a particle size of 17 nm.
From the above results, it was found that retaining 1522 cells in the presence of 5-ALA-Me and gold nanoparticles having a particle size of 5 nm for 24 hours suppresses their cell growth.

{Example 5-4}
In this example, 5-ALA-Me and 5-nmA or 17 nm gold nanoparticles were mixed and administered to cells in the same manner as in Example 5-2, except that 5-ALA-Me and 1522 cells were retained in the presence of gold nanoparticles. Moreover, 5-ALA-Me and gold nanoparticles were used in the same manner as in Example 5-2, except that 5-ALA-HCl and gold nanoparticles having a particle size of 5 nm or 17 nm were mixed and administered to cells. 1522 cells were maintained in the presence of.
A 1M 5-ALA-Me aqueous solution and a gold citrate colloid solution (Table 1) containing gold nanoparticles having a particle diameter of 5 nm or 17 nm were mixed at a ratio of 1:20, and the mixture was allowed to stand at room temperature for 4 hours. . Further, a 1M 5-ALA-Me aqueous solution and a citrate buffer (pH 6.5) were mixed at a ratio of 1:20, and the mixture was allowed to stand at room temperature for 4 hours.

Further, a 1M 5-ALA-HCl aqueous solution and a gold citrate colloidal solution containing gold nanoparticles having a particle size of 5 nm or 17 nm were mixed at a ratio of 1:20, and the mixture was allowed to stand at room temperature for 4 hours. Further, a 1M 5-ALA-HCl aqueous solution and a citrate buffer (pH 6.5) were mixed at a ratio of 1:20, and the mixture was allowed to stand at room temperature for 4 hours.
1522 cells cultured in F12 liquid medium were suspended by treatment with Trypsin-EDTA and resuspended in fresh F12 liquid medium to obtain a cell suspension having a cell concentration of about 5 × 10 ⁴ cells / ml. . A 96-well cell culture plate (Corning Incorporated Corning, model 3596) was prepared, and 100 μl of this cell suspension was dispensed into each well. The cell culture plate was placed in an incubator where the temperature was maintained at 37 ° C. and 5% CO ₂ for 24 hours.

  100 μl each of the F12 liquid medium containing 5-ALA-Me and the F12 liquid medium containing 5-ALA-HCl prepared as described above was removed from the cell sample in each well, and only the medium (supernatant) was removed. added. Here, the operation from the start of adding the liquid medium containing 5-ALA-Me to the cell sample until the cell sample is placed in the dark place in the following incubator is within 10 minutes under extremely dim white light. went. In particular, since a cell sample containing 5-ALA-Me and gold nanoparticles having a particle size of 5 nm was prepared last, the time of exposure to dim white light was about 1 minute. The cell sample containing 5-ALA-HCl was also prepared under the same conditions as those for preparing the cell sample containing 5-ALA-Me.

The cell culture plate with the cell sample prepared as described above was transferred to a completely dark incubator maintained at 37 ° C. and 5% CO ₂ concentration and left for 24 hours. Thus, after the 24-hour incubation of the cell sample was completed, the cell culture plate was removed from the incubator. After removing only the medium (supernatant) from the cell sample in each well and adding 100 μl of DPBS, a CCK-F assay using a cytotoxicity detection reagent (Cell Counting Kit F, Dojindo) was performed according to the instruction manual. . In this CCK-F assay, the work from the start of replacing the cell sample medium with DPBS to the addition of Calcein-AM to the cell sample is extremely dim white light (9.5 × 10 ⁻³ μW / cm ² ). Under 20 minutes. After adding Calcein-AM to a cell sample and leaving it in a completely dark place at room temperature for 2 hours, the fluorescence intensity of 535 nm emitted from the cell sample by excitation light with a wavelength of 485 nm is measured with a fluorescence microplate reader. (HTS 7000, Perkin Elmer). In addition, when this fluorescence measurement was performed simultaneously also about the cell sample which has not added Calcein-AM, fluorescence was not measured at all.

  As shown in FIG. 16, when a mixed solution of a 5-ALA-Me aqueous solution and a gold citrate colloid solution containing gold nanoparticles having a particle size of 5 nm is added to the liquid medium, cells using this liquid medium are used. It was found that the fluorescence intensity of the samples was slightly higher than when they were added to the liquid medium without mixing. In addition, when a gold citrate colloid solution containing gold nanoparticles with a particle size of 5 nm and a 5-ALA-HCl aqueous solution are mixed and added to a liquid medium, the fluorescence intensity of cell samples using this liquid medium is There was no significant difference compared to the case of adding to the liquid medium without mixing.

However, when a mixed solution of a 5-ALA-Me aqueous solution and a gold citrate colloid solution containing gold nanoparticles having a particle size of 17 nm is added to the liquid medium, the fluorescence intensity of the cell sample using this liquid medium is It was found that they were significantly lower than when added to the liquid medium without mixing. On the other hand, when a mixed solution of a 5-ALA-HCl aqueous solution and a gold citrate colloidal solution containing gold nanoparticles having a particle size of 17 nm is added to the liquid medium, the fluorescence intensity of the cell sample using the liquid medium Were slightly larger than those of the cell samples added without mixing them. In addition, when a mixed solution of 5-ALA-Me aqueous solution and citrate buffer (pH 6.5) was added to the liquid medium, the fluorescence intensity of the cell sample using this liquid medium was added without mixing them. There was no big difference compared to the case.
From these results, a 5-ALA-Me aqueous solution and a gold citrate colloid solution containing gold nanoparticles having a particle size of 17 nm were mixed and placed for 4 hours, and a liquid medium to which this mixture was added was used for 5 hours. It was found that cell proliferation can also be suppressed by maintaining 1522 cells in the presence of ALA-Me and gold nanoparticles having a particle size of 17 nm for 24 hours.

It is a graph which shows the effect of suppression of cell growth when HL-60 cell is hold | maintained for 23 hours in presence of 5-ALA-HCl and a gold nanoparticle. Gold nanoparticles are spherical and have any particle size of 5 nm, 17 nm, 41 nm, 82 nm, and 103 nm with a narrow particle size distribution. The cell sample containing 5-ALA-HCl was prepared by adding 5-ALA-HCl to the cell suspension and then adding gold citrate colloid solution, citrate buffer (pH 3.3), or water. Yes, containing 5-ALA-HCl at a concentration of 5 mM. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. . For comparison, cell samples prepared by adding HCl to the cell suspension and then adding gold citrate colloid solution, citrate buffer (pH 3.3), or water are also shown. Furthermore, the cell suspension is also shown in which the gold citrate colloid solution, citrate buffer (pH 3.3), or water was added without adding 5-ALA-HCl or HCl to the cell suspension. It was. Cell retention was performed in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained. (A) shows the cell concentration of each cell sample obtained using the cell number measuring apparatus. (B) shows the average viable cell concentration of each cell sample (n = 2) obtained by the trypan blue staining method. (C) shows the average fluorescence intensity of each cell sample (n = 5) obtained by the CCK-F assay.

The difference in the increase in cell concentration when the concentration of 5-ALA-HCl is set to various values of 10 mM or less when HL-60 cells are maintained in the presence of 5-ALA-HCl and gold nanoparticles. It is a graph to show. Gold nanoparticles are spherical and have a narrow particle size distribution with any particle size of 5 nm, 17 nm, 41 nm, and 82 nm. Cell samples were prepared by adding 5-ALA-HCl at various concentrations to the cell suspension, and then adding gold citrate colloid solution, citrate buffer (pH 3.3), or water. Cells were maintained for 23 hours in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained.

Graph showing the difference in increase in cell concentration when the concentration of gold nanoparticles is set to various values lower than 40 μM when retaining HL-60 cells in the presence of 5-ALA-HCl and gold nanoparticles. It is. Gold nanoparticles are spherical and have a narrow particle size distribution with any particle size of 5 nm, 17 nm, 41 nm, and 82 nm. Cell samples were prepared by adding 5-ALA-HCl to the cell suspension, and then adding gold citrate colloid solution containing gold nanoparticles at various concentrations, or citrate buffer (pH 3.3). Cells were maintained for 23 hours in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration.

It is a graph which shows the effect of suppression of cell growth when holding time is set to various values within 23 hours, when holding HL-60 cells in the presence of 5-ALA-HCl and gold nanoparticles. . Gold nanoparticles are spherical and have a narrow particle size distribution with any particle size of 5 nm, 17 nm, 41 nm, and 82 nm. Cell samples were prepared by adding 5-ALA-HCl to the cell suspension and then adding gold citrate colloidal solution containing various concentrations of gold nanoparticles, citrate buffer (pH 3.3) or water. Cell retention was performed in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained. (A) shows the time change of the cell concentration when the retention time of the cells is 0 hour, 4 hours, 8 hours, 12 hours, 18 hours, or 23 hours. (B) shows the average fluorescence intensity of each cell sample (n = 3) obtained by the CCK-F assay when the cell retention time is 4 hours.

When HL-60 cells are retained in the presence of 5-ALA-HCl and gold nanoparticles, the cells are mixed with 5-ALA-HCl and gold nanoparticles for 1 hour and then administered to the cells. It is a graph which shows the effect of suppression of proliferation. Gold nanoparticles are spherical and have a narrow particle size distribution with any particle size of 5 nm, 17 nm, and 41 nm. The cell sample is prepared by mixing a mixed solution of 5-ALA-HCl aqueous solution and gold citrate colloid solution, a mixed solution of 5-ALA-HCl aqueous solution and citrate buffer (pH 3.3), or 5-ALA-HCl aqueous solution with water. Each of the diluted solutions prepared by adding to the cell suspension was prepared. For comparison, cell samples prepared by adding 5-ALA-HCl to the cell suspension and then adding gold citrate colloid solution, citrate buffer (pH 3.3), or water are also shown. . All the cell samples shown in this graph contain 5-ALA-HCl at a concentration of 5 mM, and those containing gold nanoparticles with a particle size of 5 nm have a particle size of 17 nm, 41 nm, and 82 nm at a concentration of 22 μM. Those containing gold nanoparticles each contain gold nanoparticles at a concentration of 36 μM. Cells were maintained for 23 hours in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration. The bar graph shows the cell concentration of each cell sample obtained using the cell number counting device. The line graph shows the viable cell concentration of each cell sample obtained by the trypan blue staining method.

It is a graph which shows the difference in the increase in a cell density | concentration when HL-60 cell is hold | maintained in presence of 5-ALA-Me and a gold nanoparticle. Gold nanoparticles are spherical and have any particle size of 5 nm, 17 nm, 41 nm, 82 nm, and 103 nm with a narrow particle size distribution. The cell sample containing 5-ALA-Me was prepared by adding 5-ALA-Me to the cell suspension and then adding gold citrate colloid solution, citrate buffer (pH 3.3), or water. Yes, containing 5-ALA-Me at a concentration of 5 mM. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. . For comparison, cell samples prepared by adding gold citrate colloid solution, citrate buffer (pH 3.3), or water after adding 5-ALA-HCl to the cell suspension are also shown. Indicated. Cell retention was performed in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained.

When retaining HL-60 cells in the presence of 5-ALA-Me and gold nanoparticles, the difference in increase in cell concentration when the concentration of 5-ALA-Me is set to various values of 5 mM or less. It is a graph to show. Gold nanoparticles are spherical and have a narrow particle size distribution with any particle size of 5 nm, 17 nm, 41 nm, and 82 nm. Cell samples were prepared by adding 5-ALA-Me at various concentrations to the cell suspension, and then adding gold citrate colloid solution, citrate buffer (pH 3.3), or water. Cells were maintained for 24 hours in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained.

When holding HL-60 cells in the presence of 5-ALA-Me and gold nanoparticles, the cells are mixed with 5-ALA-Me and gold nanoparticles after various times within 3 hours. It is a graph which shows the difference in the increase in cell concentration when administered. Gold nanoparticles are spherical and have a narrow particle size distribution with any particle size of 5 nm, 17 nm, and 41 nm. A cell sample (n = 2) was prepared by mixing a 5-ALA-Me aqueous solution and a gold citrate colloid solution and leaving for 0 to 3 hours, a 5-ALA-Me aqueous solution and a citrate buffer (pH 6.5) And a mixed solution that was mixed for 0 to 3 hours, or a 5-ALA-Me aqueous solution diluted with water and diluted for 0 to 3 hours was added to the cell suspension, respectively. . For comparison, cell samples prepared by adding 5-ALA-Me to the cell suspension and then adding gold citrate colloid solution, citrate buffer (pH 6.5), or water are also shown. . Furthermore, a cell sample prepared by adding a mixed solution of an aqueous 5-ALA-HCl solution and a gold citrate colloidal solution containing gold nanoparticles having a particle diameter of 17 nm and placing the mixture for 0 to 3 hours to the cell suspension; A cell sample prepared by adding 5-ALA-HCl to the cell suspension and then adding a gold citrate colloid solution containing gold nanoparticles having a particle diameter of 17 nm is also shown. Any cell sample shown in this graph contains 5-ALA-Me or 5-ALA-HCl at a concentration of 5 mM, and contains gold nanoparticles with a particle size of 5 nm at a concentration of 22 μM, 17 nm and 41 nm. In the case of containing gold nanoparticles having a particle diameter of 36 μM, each contains gold nanoparticles at a concentration of 36 μM. Cells were maintained for 23 hours in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration. In this graph, the cell concentration measured in a cell sample prepared by adding citrate buffer (pH 6.5) without adding 5-ALA-Me and 5-ALA-HCl to the cell suspension is shown. The cell concentration obtained in all other cell samples was set to 1 (not shown), and the ratio was taken as an average value.

It is a graph which shows the difference in the increase in a cell concentration when HL-525 cell is hold | maintained for 22 hours in presence of 5-ALA-HCl and a gold nanoparticle. Gold nanoparticles are spherical and have any particle size of 5 nm, 17 nm, 41 nm, 82 nm, and 103 nm with a narrow particle size distribution. The cell sample containing 5-ALA-HCl was prepared by adding 5-ALA-HCl to the cell suspension and then adding gold citrate colloid solution, citrate buffer (pH 3.3), or water. Yes, containing 5-ALA-HCl at a concentration of 5 mM. In addition, gold nanoparticles having a particle size of 5 nm contain gold nanoparticles at a concentration of 44 μM, and gold nanoparticles having a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 72 μM. . For comparison, a cell suspension prepared by adding gold citrate colloid solution, citrate buffer (pH 3.3), or water without adding 5-ALA-HCl is also shown. It was. Cell retention was performed in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained.

It is a graph which shows the effect of suppression of cell growth when MCF-7 cell is hold | maintained for 24 hours in presence of 5-ALA-HCl and a gold nanoparticle. Here, the average fluorescence intensity of each cell sample (n = 2) obtained by the CCK-F assay is shown. Gold nanoparticles are spherical and have any particle size of 5 nm, 17 nm, 41 nm, 82 nm, and 103 nm with a narrow particle size distribution. Cell samples containing 5-ALA-HCl are liquid medium containing 5-ALA-HCl and gold citrate colloid solution, liquid medium containing 5-ALA-HCl and citrate buffer (pH 3.3), 5- A liquid medium to which ALA-HCl and water are added is prepared, and these liquid mediums are respectively administered to cells, and contain 5-ALA-HCl at a concentration of 5 mM. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. . For comparison, a liquid medium to which HCl and gold citrate colloid solution are added, a liquid medium to which HCl and citrate buffer (pH 3.3) are added, and a liquid medium to which HCl and water are added are prepared. It also showed together about the cell sample which administered each liquid medium to the cell. Furthermore, the liquid culture medium which added only the gold citrate colloid liquid, the citrate buffer (pH 3.3), or water was prepared, and the cell sample which administered each of these liquid culture media to the cell was also shown collectively. Cell retention was performed in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained. In this graph, the fluorescence intensity measured in a cell sample in which a liquid medium containing only water is administered to the cell is 1, and the fluorescence intensity obtained in all other cell samples is the ratio value relative thereto. Indicated.

It is a graph which shows the effect of suppression of cell growth when holding time is set to various values within 18 hours when holding MCF-7 cells in the presence of 5-ALA-HCl and gold nanoparticles. . Here, the average fluorescence intensity of each cell sample (n = 2) obtained by the CCK-F assay is shown. Gold nanoparticles are spherical and have any particle size of 5 nm, 17 nm, 41 nm, 82 nm, and 103 nm with a narrow particle size distribution. Cell samples containing 5-ALA-HCl are liquid medium supplemented with 5-ALA-HCl and gold citrate colloid solution, liquid medium supplemented with 5-ALA-HCl and citrate buffer (pH 3.3), 5- A liquid medium to which ALA-HCl and water are added is prepared, and these liquid mediums are respectively administered to cells, and contain 5-ALA-HCl at a concentration of 51 mM. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. . In addition, a liquid medium containing only gold citrate colloidal solution containing gold nanoparticles having a particle diameter of 5 nm or water was prepared, and cell samples in which these liquid mediums were respectively administered to cells were also shown. Cell retention was performed in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained. (A) shows the fluorescence intensity when the retention time is 0 hours. (B) shows the fluorescence intensity when the retention time is 6 hours. (C) shows the fluorescence intensity when the retention time is 12 hours. (D) shows the fluorescence intensity when the retention time is 18 hours.

It is a photograph which shows the morphological change of a cell when MCF-7 cell is hold | maintained in presence of 5-ALA-HCl and a gold nanoparticle. Gold nanoparticles are spherical and have any particle size of 5 nm, 17 nm, 41 nm, 82 nm, and 103 nm with a narrow particle size distribution. A cell sample containing 5-ALA-HCl is prepared by adding 5-ALA-HCl to a cell culture medium and then adding gold citrate colloid solution, citrate buffer (pH 3.3), or water. It contains 5-ALA-HCl at a concentration of 5 mM. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. . For comparison, a cell sample prepared by adding a gold citrate colloid solution containing gold nanoparticles having a particle size of 5 nm to a cell culture solution and a cell sample in which nothing was added to the cell culture solution A morphological observation was made. Cell retention was performed in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained.

It is a graph which shows the effect of suppression of cell growth when MCF-7 cell is hold | maintained for 24 hours in presence of 5-ALA-Me and a gold nanoparticle. Here, the average fluorescence intensity of each cell sample (n = 2) obtained by the CCK-F assay is shown. Gold nanoparticles are spherical and have a narrow particle size distribution with any particle size of 5 nm, 17 nm, 41 nm, and 82 nm. Cell samples containing 5-ALA-Me are liquid media containing 5-ALA-Me and gold citrate colloid solution, and liquid containing 5-ALA-Me and citrate buffer (pH 3.1 or 6.5). A liquid medium to which a medium, 5-ALA-Me and water are added is prepared, and these liquid mediums are respectively administered to cells, and contain 5-ALA-Me at a concentration of 5 mM. In addition, gold nanoparticles having a particle size of 5 nm are contained at a concentration of 22 μM, and gold nanoparticles having a particle size of 17 nm, 41 nm, and 82 nm are contained at a concentration of 36 μM, respectively. For comparison, a liquid medium containing 5-ALA-HCl and gold citrate colloid solution, a liquid medium containing 5-ALA-HCl and citrate buffer (pH 3.3 or 6.5), 5- A liquid medium to which ALA-HCl and water were added was prepared, and cell samples obtained by administering these liquid medium to cells were also shown. Cell retention was performed in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained.

It is a graph which shows the effect of suppression of cell growth when 1522 cells are hold | maintained for 24 hours in presence of 5-ALA-HCl and a gold nanoparticle. Here, the average fluorescence intensity of each cell sample (n = 6) obtained by the CCK-F assay is shown. Gold nanoparticles are spherical and have any particle size of 5 nm, 17 nm, 41 nm, 82 nm, and 103 nm with a narrow particle size distribution. Cell samples containing 5-ALA-HCl are liquid medium containing 5-ALA-HCl and gold citrate colloid solution, liquid medium containing 5-ALA-HCl and citrate buffer (pH 3.3), 5- A liquid medium to which ALA-HCl and water are added is prepared, and these liquid mediums are respectively administered to cells, and contain 5-ALA-HCl at a concentration of 5 mM. Also, those containing gold nanoparticles with a particle size of 5 nm contain gold nanoparticles at a concentration of 22 μM, and those containing gold nanoparticles with a particle size of 17 nm, 41 nm, 82 nm, and 103 nm contain gold nanoparticles at a concentration of 36 μM. . For comparison, a liquid medium to which HCl and gold citrate colloid solution are added, a liquid medium to which HCl and citrate buffer (pH 3.3) are added, and a liquid medium to which HCl and water are added are prepared. It also showed together about the cell sample which administered each liquid medium to the cell. Furthermore, the liquid culture medium which added only the gold citrate colloid liquid, the citrate buffer (pH 3.3), or water was prepared, and the cell sample which administered each of these liquid culture media to the cell was also shown collectively. Cell retention was performed in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained.

When retaining 1522 cells in the presence of 5-ALA-HCl and gold nanoparticles with a particle size of 5 nm, the gold nanoparticles are selected from concentrations of 0 μM, 22 μM, 33 μM, and 44 μM, and 5-ALA− About HCl, it is a graph which shows the effect of suppression of cell growth when selecting from the density | concentration of 0 mM, 2.5 mM, 5 mM, and 10 mM and hold | maintaining 1522 cells. Here, the average fluorescence intensity of each cell sample (n = 2) obtained by the CCK-F assay is shown. Gold nanoparticles are spherical and have a 5 nm particle size with a narrow particle size distribution. Cells were maintained for 24 hours in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained.

It is a graph which shows the effect of suppression of cell growth when 1522 cell is hold | maintained in presence of 5-ALA-Me and a gold nanoparticle. Here, the average fluorescence intensity of each cell sample (n = 2) obtained by the CCK-F assay is shown. Gold nanoparticles are spherical and have a particle size distribution of either 5 nm or 17 nm with a narrow particle size distribution. A cell sample containing 5-ALA-Me was prepared by adding a liquid medium containing 5-ALA-Me followed by a gold citrate colloid solution, and adding 5-ALA-Me followed by citrate buffer (pH 6.5). Liquid media added were prepared, and each of these liquid media administered to cells, 5-ALA-Me and gold citrate colloidal solution were mixed and added for 4 hours, and then added, 5-ALA- A liquid medium added with Me and citrate buffer (pH 6.5) added after 4 hours was prepared, and these liquid mediums were respectively administered to cells. On the other hand, a cell sample containing 5-ALA-HCl for comparison includes a liquid medium in which 5-ALA-HCl is added and then a gold citrate colloid solution is added, and 5-ALA-HCl is added and then a citrate buffer is added. A liquid medium added with (pH 6.5) was prepared, and each of these liquid mediums administered to the cells was mixed with 5-ALA-HCl and gold citrate colloid liquid, and the liquid added after 4 hours. A medium, 5-ALA-HCl, and citrate buffer (pH 6.5) were mixed, and a liquid medium added after 4 hours was prepared, and these liquid mediums were respectively administered to cells. Cell retention was performed in a completely dark incubator with 37 ° C. and 5% CO ₂ concentration maintained.

Claims (6)

A method for inhibiting cell growth, comprising retaining cells in the presence of at least one substance selected from 5-aminolevulinic acids and salts thereof and nanoparticles containing at least one substance selected from metals and metal compounds . The method for inhibiting cell growth according to claim 1, wherein the metal is a noble metal. The method for inhibiting cell growth according to claim 2, wherein the noble metal is gold. The method for inhibiting cell proliferation according to claim 1, wherein the nanoparticles have a particle size of 100 nanometers or less. The method for inhibiting cell growth according to claims 1 to 4, wherein at least one of the substances selected from the 5-aminolevulinic acids and salts thereof is 5-aminolevulinic acid hydrochloride and / or 5-aminolevulinic acid methyl ester hydrochloride. A cell growth inhibitor comprising: at least one substance selected from 5-aminolevulinic acids and salts thereof; and nanoparticles containing at least one substance selected from metals and metal compounds.