WO2007108618A1

WO2007108618A1 - Water-soluble organometallic nanoparticles and method for preparing the same

Info

Publication number: WO2007108618A1
Application number: PCT/KR2007/001313
Authority: WO
Inventors: Soon Hai Hong; Jin Hyuk Chung
Original assignee: Soon Hai Hong; Jin Hyuk Chung
Priority date: 2006-03-17
Filing date: 2007-03-16
Publication date: 2007-09-27
Also published as: KR20070094235A; KR100943923B1

Abstract

The present invention relates to a novel non-toxic and global anticancer drug employing nanoparticles that consist of a chelating organic polymer and biologically active metals, and enhanced permeation and retention (EPR) effect. More specifically, the present invention relates to water-soluble organometallic particles having anticancer effect via the EPR effect, comprising a multidentate metal chelating organic polymer and at least one metal moiety.

Description

WATER-SOLUBLE ORGANOMET ALLIC NANOPARTICLES AND METHOD FOR PREPARING THE SAME

Technical Field

[1] The present invention relates to a novel non-toxic and global anticancer drug employing nanoparticles comprising a chelating organic polymer and biologically active metals, and enhanced permeation and retention (EPR) effect. More specifically, the present invention relates to water-soluble organometallic particles having anticancer effect via the EPR effect and consists of multidentate metal chelating organic polymer(s) and at least one metal moiety. Background Art

[2] Cancer is one of three major causes of death in the present society and is a disease defined as abnormal cells growing infinitely. Cancer originates from various parts of a body including the brain, intestines, stomach, liver, lungs, skin, and the like in various patterns, and in most cases induces pain. In the end, cancer leads to death. However, at present early diagnosis and early treatment are the only ways to treat cancer, although with low likelihood of success.

[3] A variety of medicines have been developed for the purpose of treating cancer, and are still being developed. However, noninvasive treatment through medicinal treatment is impossible due to strong toxicity and side effects, limited drug targets or fast development of resistance to medicines. And even when the medicinal treatment is co- conducted with the radiation therapy and surgery, it is difficult to expect more than a short-term life extension after the early-stage of cancer has passed (Hayman et al., Cost-effectiveness of Routine Radiation Therapy Following Conservative Surgery for Early-stage Breast Cancer , / Clin Oncol 16, 1022-9 (1998)).

[4] Moreover, according to studies, cancer incidence increases by geometric progression and is proportional to the age and the degree of environmental pollution. This means that more people will be exposed to the danger of the disease in developing and advanced countries where pollution and population aging have long been known issues (Katanoda et al., International Comparisons of Cumulative Risk of All-Site Cancer from Cancer Incidence in Five Continents Vol. VIII. , Jpn J Clin Oncol 36, 66-8 (2006)).

[5] Conventional anticancer/treatment drugs can be classified into approximately three types such as cytotoxin, enzyme inhibitor, and signal transducer (substance that are involved in cellular signals such as hormones and some carbohydrates). However, the use of conventional approaches are restricted because of strong toxicity, side effects, fast development of resistance to medicines, and lastly limitation to selective action on cancer cells. A great deal of research has been conducted on effective delivery system to overcome such problems and recent developments in nanotechnology have suggested viable solutions by using nanoparticles as drug carriers against cancer.

[6] In general, body cells measure 10 to 20 micrometer units. Thus, anticancer drugs on a nano scale can easily provide noninvasive interaction with the cell surface and biomolecules in the cell. There are two different methods of targeting cancer using nanoparticles and they are referred to as active and passive methods in general.

[7] Active drug targeting is a method of selectively delivering anticancer elements to cancer cells by conjugating nanoparticles containing anticancer agents to recognition groups that bind or react with cancer cells. Nanoparticles-based drugs designed in this manner have an extreme advantage in selective-effectiveness as the design allows for controlled local release of drugs at specific drug targets defined by the recognition groups. Prime examples of this targeting method are lectin and carbohydrate, ligand and acceptor, or antibody and antigen (Farhan J. Ahmad, et al., Nanotechnology: A Revolution in the Making , The Pharma Review December 2005).

[8] Lectin and carbohydrate binding is one of the conventional methods for specific drug delivery system. Lectin is a nonimmune protein that can recognize and bind with glycoprotein on the surfaces of cells. Interaction between lectin and a specified carbohydrate is achieved very specifically. Therefore, a carbohydrate moiety is used to bind the drug delivery system to lectin (direct lectin targeting), and the lectin is used again as a targeting moiety to bind with a carbohydrate on the surface of a targeting cell (reverse lectin targeting).

[9] Passive drug targeting, on the other hand, employees enhanced permeation and retention (EPR) effect to specifically target cancer cells. The EPR effect is a phenomenon which appears widely only in cancer cells and in angiogenic vascular structures of cancer. The EPR effect is characterized by non-selective absorption, permeation, and retention of macromolecules by cancer cells, and the size of the macromolecules has been suggested to be between 10 to 100 nm for optimal and selective delivery of nanoparticles to cancer cells. This range of particle size was assigned by considering absorption and excretion through the intestine, selective retention in the cancer cells, and easy permeation through angiogenic vascular walls. In addition the nanoparticles must be hydrophilic to prevent the nanoparticles from coagulation/retention in blood and removal by the macrophage (Farhan J. Ahmad, et al., Nanotechnology: A Revolution in the Making , The Pharma Review December 2005).

[10] There are three distinct advantages of passive targeting strategy over active ones.

First advantage is that passive designs do not require recognition groups, making production procedures and drug administration relatively simple (many biological recognition groups are fragile and hence require delicate modes of production/ storage/introduction). The second advantage is that the lack of specificity allows for general application of the passively designed anti-cancer drugs to any cancer types displaying EPR effect. Lastly, the consequent ability of such drug to accumulate in cancer that come in contact with blood supply makes it easier to combat highly metastatic or mutating cancer.

[11] Recently, there have been pioneering works that attempted to employee the passive targeting strategy of nanoparticles against cancer with conventional anti-cancer toxins such as radioactive/toxic heavy metals (Gadolinium, Holmium-166, Copper) or cytotoxins (FU-5) (H. Tokumitsu, et al., Chitosan-gadopentetic acid complex nanoparticles for gadolinium neutron capture therapy of cancer: preparation by novel emulsion droplet coalescence technique and characterization , Pharm. Res. 16 (1999) 1830-1835, Kim JK, et al., Long-term clinical outcome of phase lib clinical trial of percutaneous injection with holmium-166/chitosan comples (Milicam) for the treatment of small hepatocellular carcinoma Clin Cancer Res. 2006 Jan, 15; 12(2): 543-8, Qi L, et al., Cytotoxic activities of chitosan nanoparticle and copper-loaded nanoparticles , Bioorg Med Chem Lett. 2005 Mar, 1; 15(5): 1397-9). These studies commonly found encouraging improvements in selective effectiveness of the conjugated toxins against cancers through enhanced necrosis, growth inhibition, and reduced metastasis when compared to those that were not fused with nanoparticles. They also observed signific ant reduction in side effects through fusion with nanoparticles. Despite all the advantages, there is still a major shortcoming about this design as well. Due to the fact that passively designed anti-cancer nano-particles have small tendency to accumulate in brain/spinal cord/bone marrow (K. Ringe, et al. Nanoparticle Drug Delivery to the Brain , Encyclopedia of Nanotechnology volume 7: pages 91-104), studies with long- term or frequent use have shown evidence of critical toxicity and side effects in the respective organs that result from slow release of the toxic conjugates. Considering the fact that observation period of 5 years is considered necessary to safely assume cure from cancer, this disadvantage is something that must be overcome.

[12] Therefore, development of an anticancer drug having wide and effective anticancer effect without short or long-term toxicity needs to be developed in order to effectively combat cancer. We aim to achieve this goal by proposing passively designed water- soluble organometallic nanoparticles with biological metals with little known toxicity. Disclosure of Invention Technical Problem

[13] The present invention has been made in view of the above problems, and it is an advantage of the present invention to provide water-soluble organometallic nanoparticles having anticancer effect via the enhanced permeation and retention (EPR) effect, comprising a multidentate metal chelating organic polymer and at least one metal moiety.

[14] It is another advantage of the invention to provide a brief mechanism of the respective anti-cancer nanoparticles, some of which are new and have not been known to academic community yet. Brifely, the nanoparticles are fused with biological metals that are capable of inducing indirect and/or direct oxidative stress in cells. Direct oxidative stress is carried out by oxidatively active metals such as iron, which is a strong oxidizing agent and stressor. The major advantage of our proposition, however, comes from biological metals that serve as signaling molecules or signal-mediators such as calcium and zinc (but not limited to), which can potentially jam cellular cycles and reactions using respective metals and consequently induce oxidative stress in the cancer cells upon sufficient accumulation.

[15] It is a further advantage of the invention is to provide a method for preparing water- soluble organometallic nanoparticles with required properties for the proposed anticancer activity. Briefly, organic acids or amino acids having multiple metal chelating groups are processed through esterification-polymerization to prepare a multidentate metal chelating organic polymer, and the resulting polymer is fused with metal source of various forms. Brief Description of the Drawings

[16] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[17] FIG. 1 is a graph illustrating the measurement of the distribution of nanoparticle size using DLS according to one embodiment of the present invention;

[18] FIG. 2 is a TEM image of nanoparticles according to one embodiment of the present invention;

[19] FIG. 3 is a high resolution TEM image of nanoparticles according to one embodiment of the present invention;

[20] FIG. 4 is a graph illustrating inorganic qualitative analysis of nanoparticles, which are not subjected to EDTA treatment, using XRDF according to one embodiment of the present invention;

[21] FIG. 5 is a graph illustrating inorganic qualitative analysis of nanoparticles, which are subjected to EDTA treatment, using XRDF according to one embodiment of the present invention;

[22] FIG. 6 is a graph illustrating relative survivability of B16/F10 melanoma cells treated with nanoparticles according to one embodiment of the present invention; [23] FIG. 7 is a graph illustrating relative survivability of B16/F10 melanoma cells treated with dialyzed nanoparticles according to one embodiment of the present invention;

[24] FIG. 8 is a photograph showing anticancer effect in C BL mice which orally took nanoparticles according to one embodiment of the present invention;

[25] FIG. 9 is a graph illustrating the weight and cancer cell increase in C BL mice which orally took nanoparticles according to one embodiment of the present invention;

[26] FIG. 10 is a photograph showing anticancer effect in C BL mice which continuously took nanoparticles passively according to one embodiment of the present invention;

[27] FIG. 11 is a photograph showing a cancer growth in C BL mice which did not inject OFeCa-I in the hypodermic transplantation model; and

[28] FIG. 12 is a photograph showing a cancer growth in C BL mice which directly injected OFeCa-I in the hypodermic transplantation model. Best Mode for Carrying Out the Invention

[29] Center subject of this invention is water-soluble organometallic nanoparticle having anticancer effect via enhanced permeation and retention (EPR) effect, and it comprises a multidentate metal chelating organic polymer and at least one metal moiety.

[30] Nanoparticles of the present invention are retained selectively in cancer cells via

EPR effect. And upon sufficient accumulation the nanoparticles give direct/indirect oxidative stress to the cancer cells, causing specific toxicity at site. The nanoparticles of the present invention having such anticancer mechanism consist of a multidentate metal chelating organic polymer and a plurality of minerals, some of which are capable of generating direct oxidative stress to the cancer cells, and others with biological functionality to trigger biological reactions/cycles/cascades.

[31] The metal component of the present invention may include metals such as iron, magnesium, manganese, titanium, cesium, silver, gold, platinum, or nickel that are relatively low in toxicity and can be easily removed/detoxified from a body and capable of directly causing oxidation in cells. The metal component may also include minerals such as calcium, zinc, potassium or sodium that have no inherent toxicity but are capable of causing indirect oxidation stress by triggering biological reactions/ cycles/cascades in any cell upon significant accumulation and steady release.

[32] Specifically in the case of iron, there is an example where the iron has nontoxic anticancer function via superoxide generation when it is applied to a cell through liposome in a cationic porphyrin form (Yuasa M., et al., Liposomal surface-loading of water-soluble cationic iron (III) porphyrins as anticancer drugs , MoI Pharm. 2004 Sep- Oct; 1(5): 387-9). In the case of calcium, calcium cascade is among the most powerful and well known biological reactions that trigger numerous other biological activities, and hence the effect of over- accumulating calcium in cancer cells in our proposed manner is likely to be effective. In fact, similar mechanism of toxicity and cell death occur in neuronal cells of neurodegenerative diseases where increased level of intracellular calcium is the main cause of stress-induced cell death in the respective cells (Van Damme P. et al., Excitotoxicity and amyotrophic lateral sclerosis , Neurodegener Dis. 2005 Mar- Apr; 2 (3-4): 147-59).

[33] The multidentate metal chelating organic polymer of the present invention has a plurality of chelating sites by the inherent three-dimensional structure of the compound such that the organic polymer functions as a transporter for the metals. The organic polymer should have relatively strong affinity to the minerals to prevent excessive mineral retention in the body or cell without easily losing minerals in the body or cell. Thus, the organic polymer should be able to form a stable complex with ionic and/or small crystalline metals.

[34] Furthermore, the organic polymer should be stable in water and acid to increase retention time in the body, to prevent premature release of metals during circulation, for suitability of oral intake and such. The degraded component of the organic polymer should be harmless to human body and the elimination of the component should be easy.

[35] Therefore, the unit groups for the organic polymer are limited to substances that can be easily digested or eliminated in the body /cell, and hence carboxylic acid, alcohols, or amines with multiple metal chelating groups are good candidates. Unfortunately, these substances polymerize through esterization, and since ester bonds are fragile in water, acid, or base, careful selection is needed. Thus, it is important that the polymer forms through stable ester bonds and forms a stable complex with the metals/minerals.

[36] Meanwhile, the organic polymer may have a flexible size in the range of 2 to 300 nm, but in order to optimize selective retention in cancer cells according to the EPR effect, a size in the range of 10 to 100 nm is preferred.

[37] Using the organic polymer having the above properties and the metals, it is possible to achieve both the advantages from the passive EPR effect and the advantages of using biological metals. The resulting nanoparticles are hence expected to little or no side effects even on frequent or long-term use while retaining anti-cancer activity.

[38] Hereinbelow, a possible synthesis procedure of OFeCa-I and its physical/chemical properties will be described in detail as one embodiment of nanoparticles according to the present invention. Moreover, effects of OfeCa-1 on cell and animal models of cancer are examined. It must be noted, however, that the scope of the present invention is not limited to the stated embodiments, and those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the present specification.

[39]

[40] Example 1 : Preparation and synthesis of OFeCa-I

[41] A carboxylic acid represented by the following chemical formula 1 or a carbonyl amine represented by the chemical formula 2 was dissolved in ether that has the pH of 1.5 to 2 at 40 C until saturation. Rl, R2, Ll, and L2 groups in the carboxylic acid used herein will determine the amount and proportion of metal/mineral to be bound with a polymer, and the size and stability of nanoparticles depending on the selection as to a multidentate metal chelating group and linkage group, respectively.

[42] [Chemical Formula 1]

[43]

[44] [Chemical Formula 2]

[45]

[46] More specifically, Rl and R2 groups are metal chelating groups, which may be identical with or different from each other. Rl and R2 groups may be selected from the group including alkyl-carbonyl, alkyl-cyano, carbonyl, cyano, alkyl-amine, alkyl thiocyanide, amine, thiocyanide, alkyl-carbonyl-amide, carbonyl-amide, alkyl-thiol, alkyl-alcohol, thiol, and alcohol.

[47] Ll and L2 groups are linkage groups, and more specifically may include ether, methylene, or combinations thereof.

[48] The carboxylic acid represented by the chemical formula 1 is reacted at high temperature using a dehydration medium such as silica bead to induce esterification polymerization. This process allows the chemical reaction in the equilibrium to proceed to the right according to Le Chatellier s Principle.

[49] [Chemical Reaction 1]

[50]

[51] Similarly, the carbonyl amine represented by the chemical formula 2 is subjected to esterification polymerization as in the chemical reaction 2 by removing ammonia gas at high temperature in vacuum state. When ammonia gas is removed, the chemical reaction in the equilibrium proceeds to the right according to Le Chatellier s Principle.

[52] [Chemical Reaction 2]

[53]

[54] The prepared organic polymer is fused slowly over a long period of time with the metals/minerals in a highly concentrated liquid or solid state after carrying out the stabilization process since the organic polymer is sensitive to water, acid, and base. Herein, in the case of using dissolved metal, a dehydration element must be used as described above.

[55] The stable organic polymer fused with the metals/minerals has water resistance and acid resistance. This is possible because carbonyl oxygen in the backbone of the polymer forms coordination with metals/minerals such that the electron density of oxygen is transferred to the metals/minerals, thereby reducing nucleophilicity of carbonyl oxygen.

[56] The prepared organic polymer and the metals/minerals complex (OFeCa-I) can be separated by removing ether at the evaporation temperature to obtain a powder which is then dissolved in water, or the mixture is diluted with water and heated to remove ether thereby preparing an aqueous solution. The prepared aqueous solution of OFeCa- 1 is adjusted to pH of 3.5 to 4 using a buffer to prevent oxidation of the chelated metal/ mineral and degradation of the polymer. In this process, the raw material and the excessive amount of metals/minerals are removed using dialysis or other suitable methods.

[57] [58] Example 2: Physical/chemical properties of OFeCa- 1 [59] OFeCa-I is water-soluble nanoparticles containing mainly iron, calcium, magnesium, and the like as an example of anticancer drug synthesized according to the present invention. When inorganic components in OFeCa- 1 prepared by the above method are analyzed, about 10,000 ppm of iron, calcium, and magnesium are detected respectively in an 11% w/w aqueous solution of OFeCa-I as shown in Table 1. Additionally, potassium (K), Manganese (Mn), copper (Cu), silver (Ag), and the like are also detected, although relatively small, as shown in Table 1 (FIG. 1).

[60] Table 1

[61] The size of OFeCa-I nanoparticles were measured using a dynamic light scattering method, and the results showed binodal distribution of about 40 to 100 nm (FIG. 1). When the size was measured through transmission electron microscopy (TEM), the nanoparticles having about 10 nm in size were observed in addition to about 50 nm and 100 nm nanoparticles (FIG. 2).

[62] OFeCa- 1 particles shown in TEM exhibited a strong difference in contrast even though no staining agent was used. This means that the OFeCa-I nanoparticles contain many elements with large atomic weight such as metals. As a matter of fact, the comb pattern made by the metal- specific resonance shown in the high resolution TEM, also support the above fact (FIG. 3). The comb pattern with different spacings shown in FIG. 3 means that OFeCa-I contains a variety of metals.

[63] Moreover, the OFeCa-I nanoparticles shown in the TEM image do not have any specific crystal structure. This means that the particles are not simple nanoparticles with metal crystal. The results in the organic element analysis of OFeCa- 1 in Table 2 show that about 25% of OFeCa-I weight is carbon. This means that the nanoparticles of OFeCa-I are fused of an organic components and inorganic components. [64] Table 2

[65] <Analysis of Organic Elements in 12% w/w Aqueous Solution of OFeCa- 1> [66] In the above analytic experiment, C, H, N, and S were analyzed using FIONS EA- 1108 Elemental Analyzer (detection limit of N and S is 0.1%), and O was analyzed using Thermo Finnigan FLASH EA-1112 Elemental Analyzer. Here, O and H data were detected when OFeCa-I was in aqueous state, and thus they do not represent the chemical properties of the OFeCa-I.

[67] Finally, 10 mM of EDTA, which is one of the strongest chelators of iron existing, was used to treat OFeCa-I at pH 7.0 for 3 hours followed by dialysis to carry out the experiment for removing iron and magnesium of OFeCa-I. When the metal signals for iron and other metals of OFeCa- 1 before and after the treatment with EDTA were compared as in FIGs. 4 and 5, it was known that the difference is about 20 to 30%. As from the result, it was known that OFeCa-I forms very stable structure with metal/ mineral components such as iron, calcium, magnesium, or zinc.

[68] [69] Example 3: Anticancer effect of OFeCa-I on B16/F10 melanoma cells [70] In order to observe the cancer toxicity of OFeCa-I, B16/F10 melanoma cells were artificially cultured using 3 10 cells per well in a 24-well plate. After inducing stability in the surface of the cells, 0, 10, 20, and 40 L of 13% w/w OFeCa-I were treated per 1 mL of the culture solution (RPMI 1640 with 10% FBS). Then, the survivability of B 16/F10 melanoma cancer cells based on the incubation time was examined. FIG. 6 shows the survivability of the B16/F10 melanoma cells treated with OFeCa-I based on the incubation time compared with the B16/F10 melanoma cells without any treatment.

[71] In the case where the B16/F10 melanoma cells were treated with the most amount of OFeCa-I (40 L/mL), complete necrosis of the cancer cells within 24 hours of the OFeCa-I treatment could be observed. In the case where 20 L/mL of OFeCa-I was treated on the cancer cells, it was observed that the number of surviving cancer cells decreased persistently based on the incubation time as with the case of 40 L/mL treatment such that most cells were extinct by the fourth day.

[72] Meanwhile, in the case where the B16/F10 melanoma cells were treated with 10 L/ mL of OFeCa-I, the number of cells decreased until 48 hours. However, the cancer cells proliferated dramatically after 72 hours, and showed over 20% growth compared with the control group. This rebound phenomenon is likely to have occurred since OFeCa-I employs the metal/mineral retention by the EPR effect and oxidation stress resulting from the retention. Thus, when the metal concentration of OFeCa-I retained in the cancer cells does not reach the proliferation speed of the cancer cells, the amount of OFeCa- 1 retained in the cells reduces by half, thereby giving insufficient amount of oxidation stress for the cancer cell death. The insufficient amount of oxidation stress for cell death leads acceleration to the growth/metabolism in mammalian cells (Burdon, et al., Superoxide and hydrogen peroxide in relation to mammalian cell proliferation , Free Radic Biol Med 18, 775-94 (1995)).

[73]

[74] Example 4: Experiment of cell toxicity using dialvzed OFeCa-I through 14.000

MWC membrane

[75] In order to confirm that the cell toxicity of Example 3 is resulted by a macromolecule, the experiment having dialyzed OFeCa- 1 with water using a dialysis membrane of 14,000 MWC was carried out in the same manner as in Example 2.

[76] The experiment result as shown in FIG. 7 using the dialyzed OFeCa-I exhibited similar effect as in Example 3. That is, the cell toxicity of OFeCa-I on the cancer cells was caused by macromolecules.

[77]

[78] Example 5: Growth inhibition effect on B16/F10 melanoma hypodermically xenog "rafted in C 57. BL mice by oral intake of OFeCa- 1

[79] After confirming that OFeCa-I has direct anticancer effect on cancer cells in

Examples 3 and 4, the growth inhibition effect on B16/F10 melanoma transplanted hypodermically was observed using female C BL mice to find out whether or not the similar outcome resulted in vivo.

[80] First, 100,000 melanoma cells were injected hypodermically to the neck using a syringe. Further, 13% w/w aqueous solution of OFeCa-I was administered orally according to three schedules, namely 0.1 ml administration once per day (Tl), 0.2 ml administration once per day (T2), and 0.3 mL administration twice per day (T3: i.e., total of 0.6 mL per day), starting on the same day. After 33 days of observing the cancer cell growth, autopsy was performed on the mice, and the degree of the cancer cell growth was determined by measuring the weight of the mouse and cancer cells (FIG. 8).

[81] When the size of the cancer cells grown under the skin of the C BL mice was compared, it clearly showed the size in the order of Tl > control group > T2 > T3. Moreover, a yellow transparent solution surrounding the cancer cells could be observed during the autopsy. The mice did not show any special side effect until the day of autopsy which means the hypodermically transplanted cancer cells did not cause any other special effect on the mice. [82] The size and distribution of the cancer cells observed with bare eyes in the above was reconfirmed by the weight of the cancer cells and mouse measured through autopsy. Additionally, two important facts were found in addition to the cancer growth inhibition effect depending simply on the concentration (FIGs. 9 A and 9B).

[83] First, the acceleration of cancer growth was induced in Tl in spite of the cancer growth inhibition effect depending on the concentration was observed. The same phenomenon was observed in Example 3. The rebound phenomenon is caused, as readily described, since OFeCa- 1 employs the metal/mineral retention by the EPR effect. Thus, when the metal concentration retained in the cancer cells of OFeCa-I does not reach the proliferation speed of the cancer cells, the cancer cells rather recognize this oxidation stress as a stimulus to accelerate the growth in the cancer cells.

[84] Second, the total weight of the mouse including the weight of the cancer cells described the concentration dependence of the growth inhibition effect in a parabola as the administration dose increased. Meanwhile, the weight of the mouse excluding the weight of the cancer cells exhibited direct relation to the administration dose of OFeCa-I (FIG. 9C). That is, separate from the size of the cancer cells, the other normal tissues of the mice were protected according to the administration dose of OFeCa-I. At the same time, OFeCa-I had no negative effects on normal tissue cells, irrespective of its cancer growth inhibition effect on the cancer cells.

[85]

[86] Example 6: Growth inhibition and treatment effect on B16/F10 melanoma cells through passive and continuous administration of OFeCa-I in hypodermic trans - plantation model

[87] In order to confirm OFeCa-I causing the cancer cell toxicity through the metal retention by the EPR effect, an experiment to find out the effect by continuously injecting OFeCa-I to the mice with very low concentration was carried out. Metastatic cancer was used in lung post intravenous injection (LV.) model to apply the EPR model more effectively.

[88] Two days before carrying out the LV. injection, water containing 10% v/v of 13% w/w OFeCa-I solution was fed passively to the C BL mice. During the LV. injection, 100,000 B 16/F10 melanoma cells were injected to the vein from the tail of the C BL mice. 14 days after the LV. injection, autopsy was performed on the mice to observe metastatic cancer colony in the lung (FIG. 10). The amount of water containing 13% w/w of OFeCa-I, which the mice drank passively, was about 2 to 3 ml per day.

[89] In FIG. 10, A represents the control group which did not drink water containing the

OFeCa-I solution, and B represents the experiment group which drank water containing the 10% v/v of 13% w/w OFeCa-I solution. By far more B16/F10 melanoma colonies were observed in the lung of the control group when the experiment group and the control group were compared. Therefore, it was known that continuous and passive intake of low concentrated OFeCa- 1 prevented the metastatic cancer colonies from growing in the C BL mice' lungs effectively.

[90]

[91] Example 7: Experiment on growth inhibition and treatment effect through direct injection of OFeCa-I in hypodermic xenograft model

[92] The OFeCa-I, which killed B16/F10 melanoma cancer cell in the experiment of

Example 3, showing only the cancer growth inhibition effect in the in vivo experiment of Example 5 was thought to have main cause in the absorption problem to the gastrointestinal tract. Thus, an experiment was carried out using another hypodermic transplantation model to find out the effect of OFeCa-I when directly injected to the hypodermic cancer cells.

[93] B16/F10 melanoma cells were transplanted hypodermically to 8 female C BL mice per group in the neck, and the cells were grown to a size of about a grain of rice. Then, 50 L of 13% w/w aqueous solution of OFeCa-I was administered directly or close to the hypodermic cancer cells. Such injection was performed once per day for two continuous days. After four days since the first injection, the sizes of the cancer cells were compared by the photographs.

[94] First, the control group, which did not have drug administration after the cancer transplant, had the size of the cancer cells grown to 1 to 2 cm (FIG. 11). On the contrary, the experiment group, which had OFeCa-I injected, exhibited the following phenomenon. The cancer cells were not found in three mice among 8 mice. Instead, a wound or a portion with cell necrosis was found where the cancer had been placed (FIG. 12). The cancer cells were not found in three mice among the remaining 5 mice where drug had been injected. Instead, a portion far from the originally transplanted portion had cancer cells growing (FIG. 12). The remaining two mice died after three days of drug administration. The reason for the death seems to be caused by direct flow of an excessive amount of the solution into the blood by the EPR effect so as to cause pH shock, osmotic shock or the like.

[95] Accordingly, the nanoparticles of the present invention are specifically retained in the cancer cells by the enhanced permeation and retention (EPR) effect after being administered in the body. Thereby, the metals chelated to the nanoparticles generate oxidation stress and osmotic stress directly/indirectly to the cancer cells whereby exhibiting cancer cell-specific toxicity. In addition to having wide range of toxicity to cancer cells, the nanoparticles of the present invention function specifically to the cancer cells only, thus the side effects on the normal cells other than the cancer cells are rarely found. Therefore, the practical use of nanoparticles is high since various ap- plications are possible such as direct administration to the cancer cell portion or continuous intake with drinks. [96]

Claims

[1] Water-soluble organometallic nanoparticles having anticancer effect via the enhanced permeation and retention (EPR) effect, consisting of a multidentate metal chelating organic polymer and at least one metal moiety between hy- drodynamic size of 2 and 500 nm.

[2] The nanoparticles according to claim 1, wherein the metal is at least one selected from the group consisting of iron (Fe), calcium (Ca), magnesium (Mg), Manganese (Mn), potassium (K), sodium (Na), zinc (Zn), titanium (Ti), silicon (Si), cesium (Cs), copper (Cu), silver (Ag), gold (Au) platinum (Pt), nickel (Ni) and isotopes thereof.

[3] A method for preparing water-soluble organometallic nanoparticles comprising: subjecting organic acids or amino acids having multiple metal chelating groups through esterification-polymerization to prepare a multidentate metal chelating organic polymer; and fusing the organic polymer with metal components.

[4] The method according to claim 3, wherein the multidentate metal chelating group is at least one selected from the group consisting of alkyl-carbonyl, alkyl- cyano, carbonyl, cyano, alkyl-amine, alkyl thiocyanide, amine, thiocyanide, alkyl-carbonyl-amide, carbonyl- amide, alkyl-thiol, alkyl-alcohol, thiol, and alcohol.