TWI500426B - A use of platinum nanoparticles for manufacturing anti-cancer drugs - Google Patents

Description

Use of platinum nanoparticles for the preparation of a medicament for treating cancer

The present invention relates to the use of a platinum nanoparticle, in particular to the preparation of platinum nanoparticle for poisoning cancer cells, and to a method for producing the platinum nanoparticle.

Cancer is the leading cause of death among Chinese people. Current methods of treating cancer include conventional radiation therapy and conventional chemotherapy. The conventional radiotherapy method irradiates a cancer cell with X-rays or high-energy rays, but kills cancer cells of a cancer patient and kills normal cells in the vicinity of the tissue. Therefore, the conventional radiation Therapeutic methods are extremely burdensome for cancer patients, and can also cause side effects that cause cancer patients to be uncomfortable (for example, headache, fatigue, nausea, vomiting, hair loss, or red, dry, itching, or tenderness in the skin of the irradiated area). And reduce the quality of life of cancer patients.

The conventional chemotherapeutic method kills cancer cells in cancer patients with a conventional chemotherapy drug to alleviate the spread of cancer cells and prolong the survival time of cancer patients. The conventional chemotherapeutic drugs are directed against cells that rapidly replicate, and in particular to inhibit the DNA replication machinery of such cells. However, since human hematopoietic cells, intestinal mucosal cells, hair cells, and germ cells are also rapidly replicating cells, these cells are also affected by the conventional chemotherapy drugs, so that cancer patients undergo chemotherapy treatment. Side effects such as nausea, vomiting, abdominal pain, diarrhea, anemia, hair loss or damage to the reproductive system cause low quality of life for cancer patients and are unwilling to continue treatment. Therefore, regardless of the conventional radiotherapy method or the conventional chemotherapeutic method, serious side effects are caused by killing normal cells.

When the material size is in the "nano" range, the material in the nano-layer will have many special physicochemical properties, such as a large atomic surface area and a high degree of activity. When the nanoparticle size of some materials is less than 100 nm, it has the ability to kill cells. However, the killed cells are not classified into cancer cells or normal cells, so the conventional nanoparticle is a non-selective drug. In view of this, the search for a drug for treating cancer has the ability to kill cancer cells, but has little effect on normal cells, that is, nanoparticles having selective killing of cancer cells, and has become the focus of industrial development.

The main object of the present invention is to improve the above problems, and to provide a use of a platinum nanoparticle for preparing a medicament for treating cancer, which is a method for selectively killing cancer cells in a light energy environment to reduce side effects of drug administration.

A further object of the present invention is to provide a method for preparing a platinum nanoparticle, which is capable of preparing a platinum nanoparticle which selectively poisons cancer cells in a light energy environment, and tends to cause cancer cells to enter apoptosis without causing Inflammatory responders around the cancer cells.

In order to achieve the foregoing object, the technical means and the effects achievable by the technical means include: the use of a platinum nanoparticle for preparing a medicament for treating cancer, the platinum nanometer The particles are administered to a living body, and a light energy is provided for 4 to 10 minutes in the affected part of the living body, and the light wavelength of the light energy is 1064 nm; wherein the platinum nanoparticle is prepared by a method comprising the following steps: mixing 200 mg One chloroplatinic acid hexahydrate and 90 mM, 10 ml of a protective agent to obtain a mixed solution; the mixture is subjected to a nucleophilic reduction reaction with 1 mM, 50 μl of sodium hydroxide at 100-140 ° C, and the reaction 15 ~60 minutes to generate a platinum nanoparticle.

Wherein, the protective agent comprises a water-soluble polymer and ethylene glycol, and the water-soluble polymer is selected from the group consisting of polyvinyl alcohol, gelatin, polypropylene decylamine, polyethyleneimine or polyvinylpyrrolidone.

Among them, the nucleophilic reduction reaction was carried out at 120 ° C for 30 minutes.

The method for preparing the platinum nanoparticle further comprises mixing the mixture of sodium hydroxide and acetone at a ratio of 1:3 by volume, and centrifuging to remove the supernatant to obtain a purified platinum nanoparticle.

In summary, the platinum nanoparticle of the present invention is used for preparing a drug capable of selectively killing cancer cells in a light energy environment, and is converted into heat energy by absorbing light energy to generate a photothermal effect, thereby inducing cells to enter apoptosis, thereby Can achieve the efficacy of treating cancer. Furthermore, if the platinum nanoparticle of the present invention has no irradiation light energy, it does not significantly affect the unirradiated cancer cells, and has the effect of selectively inducing cancer cell death in a light energy environment. Since the side effects of the conventional treatment method are caused by the poisoning of the normal cell line together, the platinum nanoparticle of the present invention can reduce the side effects caused by the conventional treatment method and provide a good life for the cancer patient as compared with the conventional treatment method. The effect of quality.

The invention further provides a method for preparing a platinum nano particle, which is capable of preparing a platinum nano particle which selectively poisons a cancer cell in a light energy environment, and the platinum nano particle causes the cancer cell to die through apoptosis, and It does not cause an inflammatory reaction around the cancer cells, and thus omits the medical procedure for treating the inflammatory response, thereby improving the clinical medical level and is the efficacy of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a transmission electron micrograph of a platinum nanoparticle of a preferred embodiment of the present invention.

Fig. 2 is a histogram of particle size distribution of platinum nanoparticles according to a preferred embodiment of the present invention.

Fig. 3A shows a case where the platinum nanoparticle of the preferred embodiment of the present invention is administered to a nerve cell strain (Neuro 2A) and the temperature of the cell rises with time after irradiation with laser light.

Fig. 3B is a diagram showing the case where the platinum nanoparticle of the preferred embodiment of the present invention is administered to a nerve cell strain (Neuro 2A) and the temperature of the cell rises with different doses after irradiation with laser light.

Figure 4 is a diagram showing the administration of a platinum nanoparticle to a nerve cell strain (Neuro 2A) according to a preferred embodiment of the present invention. And there is no cell survival rate of irradiated laser light.

Fig. 5 is a MALDI-MS diagram of the platinum cell particles of the preferred embodiment of the present invention administered to a neuronal cell line (Neuro 2A) with or without irradiation of laser light to induce apoptosis of the neural cell line.

The above and other objects, features, and advantages of the present invention will become more apparent from the <RTIgt; Preferably, the platinum nanoparticle having selective poisoning of cancer cells can be prepared by mixing a platinum ion with a protective agent, applying a sodium hydroxide at a reaction temperature, and performing a nucleophilic reduction reaction ( Nucleation-reduction reaction). Preferably, the platinum nanoparticles are more precipitated to obtain a purified platinum nanoparticle.

In detail, after the platinum ion is added with a protective agent, it is mixed with sodium hydroxide, which can provide monovalent electrons, undergo a nucleophilic reduction reaction with the platinum ion, reduce the platinum ion, and use metal platinum. The form produces a precipitate; and, once the metal platinum is formed, it can be immediately combined with the protective agent to form the platinum nanoparticle. Wherein, the protective agent can be selected from, but not limited to, polyvinyl alcohol (PVA), gelatin, polyacrylamide (PAM), polyethylenmine (PEI). Or polyvinylpyrrolidone (PVP); the protective agent prevents the platinum ions from excessively agglomerating on the one hand, and forms the platinum platinum particles which are too large, and prevents the generated metal platinum particles from colliding with each other. Agglomeration occurs.

For example, in a preferred embodiment of the invention, platinum ions are provided as dihydrogen hexachloroplatinate (IV) hexahydrate, and polyvinylpyrrolidone is the protective agent. Thus, in this example, 200 mg of chloroplatinic acid hexahydrate and 10 ml of polyvinylpyrrolidone (90 mM) were stirred until dissolved to form a mixed solution; the mixed solution was placed in a reaction tank, and the reaction vessel was heated. To 120 ° C, 50 μl of aqueous sodium hydroxide solution (1 mM) was added dropwise. Platinum nanoparticles. Preferably, after the aqueous sodium hydroxide solution is added to the mixed solution, the stirring can be continued for more than 30 minutes to ensure that the nucleophilic reduction reaction is complete.

Next, the precipitated platinum nanoparticles are continuously precipitated to obtain purified platinum nanoparticles, and preferably, the platinum nanoparticles are precipitated by acetone. In the preferred embodiment, the above-mentioned mixed sodium hydroxide solution and acetone are mixed with each other at a volume ratio of 1:3, and the supernatant is continuously removed, and the purified platinum nanoparticle can be obtained. .

Please refer to Figure 1 for an electrographic display of platinum nanoparticles by a transmission electron microscope (TEM). As can be seen from the figure, the platinum nanoparticles have a cubic shape with a particle size of 4 to 7 nm, and the platinum nanoparticles are uniformly uniform in particle shape. Referring to Fig. 2, the histogram of the particle size distribution of the platinum nanoparticles is further analyzed, and it is shown that the particle size distribution of the platinum nanoparticles is about 75% concentrated in 5-6 nm. In summary, the present invention can produce platinum nanoparticles by the nucleophilic reduction reaction of the preferred embodiment of the present invention.

The platinum nanoparticle prepared by the above method has the effect of poisoning cancer cells, and the platinum nanoparticle preferably can be used to administer the platinum nanoparticle to the cancer cell, and then apply a light energy to make the platinum nanoparticle. The light energy can be absorbed and converted into heat energy to raise the temperature of the cancer cells, and the temperature of the cancer cells rises to 41 to 47 ° C, thereby promoting the death of cancer cells.

In order to confirm that the platinum nanoparticle of the preferred embodiment can absorb light energy and convert it into heat energy to increase the temperature of the cancer cell, thereby promoting the death of the cancer cell, the test is carried out. In particular, this experiment selected a neuronal cell line (Neuro 2A) purchased from the Hsinchu Food Industry Science Institute in Taiwan. The nerve cell strain was cultured in Minimum Essential Medium (MEM) and supplemented with 10%. (volume ratio) Deactivated fetal bovine serum (Fetal Bovine Serum, FBS for short) and 1% (by volume) penicillin/streptomycin. The culture environment was 37 ° C, 5% carbon dioxide humidification incubator. The nerve cell strain is a kind of cancer cell, and the platinum nanoparticle of the preferred embodiment is administered to the nerve cell strain, and the light energy is selected to be yttrium aluminum garnet laser light (Nd:YAG laser) having a light wavelength of 1064 nm. Irradiation, respectively, observed the temperature rise of the nerve cell strain, and the state of death of the nerve cell strain.

Fig. 3A shows a case where the platinum nanoparticle of the present invention is administered to a nerve cell strain (Neuro 2A) and the temperature of the neuronal cell line (Neuro 2A) is raised after irradiation with a light beam having a wavelength of 1064 nm. In the group A1, the nerve cell strain to which the platinum nanoparticle is not administered is used as a control group, and the nerve cell strain to which 50 μg of the platinum nanoparticle is administered is used as a test group (group A2). The A1 and A2 groups were irradiated with laser light for 480 seconds, respectively, and the temperature of each of the nerve cell strains of each group was measured at 30, 60, 120, 180, 240, 300, 360, 420, and 480 seconds. As can be seen from the figure, in the A1 group, the laser light having a wavelength of 1064 nm was irradiated for 480 seconds, the temperature of the nerve cell strain was only increased by about 1 ° C, and the irradiation of the A2 group was 480 seconds, and the temperature of the nerve cell strain was increased by about 7.5 ° C. Referring to Figure 3B, the effect is on the administration of different doses of the platinum nanoparticles of the present invention to the neuronal cell line (Neuro 2A). In the B1 group, the nerve cell strain in which the platinum nanoparticle was not administered was used as a control group, and the B2 to B5 group was administered with 5, 10, 25 or 50 μg of the nerve cell strain of the platinum nanoparticle, respectively. The results showed that the temperature of the nerve cell strain of the B1 group only increased to about 38 ° C, the temperature of the nerve cell strain of the B2 group increased to about 44 ° C, and the temperature of the nerve cell strain of the B3 to B5 group rose to about 47~48 °C. It can be seen that the platinum nanoparticle system of the preferred embodiment can effectively absorb the laser light having a wavelength of 1064 nm and convert the light energy into heat energy, thereby increasing the temperature of the nerve cell strain up to 48 ° C (hereinafter referred to as This phenomenon is the thermal effect of 〝 light).

This test confirms that the cancer cells are elevated in temperature and further promote cancer cell death. Therefore, in this test, 5oμg of the platinum nanoparticles of the present invention were administered, and 1×10 ⁶ nerve cell strains (Neuro 2A) cells were administered, and after 8 hours, laser light having a wavelength of 1064 nm was irradiated, and after 24 hours, MTT analysis was performed. Cell viability.

Referring to Fig. 4, the platinum nanoparticle of the present invention is administered to a nerve cell strain (Neuro 2A), and the cell survival rate of the laser light having a wavelength of 1064 nm is irradiated. The test conditions of each group are shown in Table 1.

The results showed that the cell lines of the C1 and C2 groups which were not administered with the platinum nanoparticles of the present invention showed no significant difference in cell viability regardless of the presence or absence of illumination. On the contrary, the cell survival rate of the nerve cell strain of the C3 group was higher than 90%, and the cell survival rate of the nerve cell strain of the C4 group was only about 10%, that is, the cell death rate was 90%. Furthermore, the cell survival rate of the nerve cell line of the group C2 and the nerve cell line of the group C4 was compared, and the cell survival rate of the nerve cell line of the group C4 was statistically significantly lower than that of the group C2. Strain (P < 0.0001). Therefore, the laser light having a wavelength of 1064 nm does not significantly affect the cell survival rate of the nerve cell strain to which the platinum nanoparticle of the present invention is not administered; and the nerve cell strain of the platinum nanoparticle of the present invention is administered and irradiated with the light. Laser light with a wavelength of 1064 nm did not significantly affect cell viability. However, by implanting the platinum nanoparticle of the present invention and irradiating the laser light having a wavelength of 1064 nm, the mortality of the nerve cell strain is 90%, and the effect of killing the nerve cell strain is remarkable. From this, it is understood that the platinum nanoparticle of the present invention kills the nerve cell strain only at the portion of the laser light having a wavelength of 1064 nm, which has no effect on the nerve cell strain of the unirradiated portion.

In summary, the platinum nanoparticle of the present invention can cause the nerve cell strain to rise by using a photothermal effect at a laser irradiation site of a light wavelength of 1064 nm to cause death of the nerve cell strain; at a wavelength of 1064 nm without the light wavelength At the irradiation site, although the nerve cell strain was still administered with the platinum nanoparticle of the present invention, the nerve cell strain showed no significant death. Therefore, the platinum nanoparticle of the present invention can be confirmed as a platinum nanoparticle which initially selectively kills the nerve cell strain in a light energy environment.

Cell death can be divided into two categories: necrosis and apoptosis. Among them, necrosis is a cell undergoing drastic changes in the environment, cells swell, eventually rupture and die, and the cells are triggered. The surrounding tissue is inflammatory, and severe cell necrosis can cause gangrene, ulcers, and even death of the organism; on the contrary, apoptosis is the way in which the body naturally removes cells, and the cell line shrinks. It is phagocytized by macrophages and does not cause inflammatory reactions in the tissues surrounding the cells, so it does not cause side effects such as gangrene and ulcers.

In order to confirm that the platinum nanoparticle system of the present invention can promote the death of a neuronal cell line in an apoptotic manner, the present experiment is administered to a neuronal cell line as a positive control group by an HTI drug known to induce apoptosis, and is untreated. This neuronal cell line of any drug acts as a negative control group. The two groups of the neural cell strains were selected as a mass-to-charge ratio (MMS) by a matrix-assisted laser desorption/ionization mass spectrometer (MALDI-MS). /z) Analysis, at the mass-to-charge ratios 5697, 11389, and 13897, when the negative control group has no signal and the positive control group appears signal, it indicates that the nerve cell strain has entered apoptosis, and these signals can be regarded as standard apoptosis. Signal.

In detail, 2×10 ³ nerve cell strains are placed on a target plate of the matrix-assisted laser desorption ionization mass spectrometer, covering a matrix to make the matrix and the nerve The cell line formed a crystalline solid; in this test, the substrate was subjected to 0.5 μl of α-cyano-4-hydroxycinnamic acid (concentration: 0.05 M) at an absorption wavelength of 337 nm. Next, the crystalline solid is irradiated with a laser beam having a wavelength of 337 nm, so that the crystalline solid can be excited to form a gas phase ion, and the gas phase ion can be accelerated into an air tube for free flight after being accelerated by the electric field. (free flight), the time-of-flight mass analyzer can detect the flight time of the gas phase ion to obtain the mass-to-charge ratio of the nerve cell strain to be tested.

Referring to Fig. 5, a MALDI-MS analysis chart of a neuron cell line (Neuro 2A) was administered with the platinum nanoparticles of the present invention, with or without a laser light having a wavelength of 1064 nm. The test conditions of each group are shown in Table 2.

At the mass-to-charge ratio (m/z) 5697, 11389 and 13897, there is no obvious signal in the D1 group (negative control group), whereas the D2 group (positive control group) has obvious signals. Please continue to the D3 and D4 groups, the platinum nanoparticles of the present invention are administered to the nerve cell strain, but the irradiation light has a wavelength of 1064 nm laser light (Group D3) at mass-to-charge ratio (m/z) 5697, 11389 and 13897. There is no obvious signal. These signal patterns are the same as group D1, indicating that the nerve cell strain of this group survives. The nerve cell strain of the D4 group is irradiated with the platinum nanoparticle of the present invention and irradiated with a laser light having a wavelength of 1064 nm, and signals are generated at mass-to-charge ratios (m/z) 5697, 11389 and 13897, and the signal patterns are The group D2 is the same, indicating that the group promotes the death of the nerve cell line via the apoptotic pathway. Accordingly, the platinum nanoparticle of the preferred embodiment can induce cancer cells to enter apoptosis in an organism, and thus does not cause an inflammatory reaction. Furthermore, this test reaffirmed that the administration of the platinum nanoparticle of the present invention to the laser light having a wavelength of 1064 nm without irradiation of light does not induce the death of the cancer cell, and the cancer cell is induced only when the laser light having a wavelength of 1064 nm is irradiated. death. Therefore, the platinum nanoparticle of the present invention can be reconfirmed as a platinum nanoparticle which initially selectively kills the cancer cell in a light energy environment, and the cancer cell death is induced by a cell apoptosis mode.

The platinum nanoparticle system of the invention can effectively kill cancer cells, and thus can be used as an active ingredient for treating cancer, preferably the platinum nanoparticle can be used for preparing a medicament for treating or assisting tumor treatment, the platinum The nanoparticle can be combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition, wherein the platinum nanoparticle can be prepared into any convenient form such as a tablet, a capsule, a powder, or a granule. Liquid agent or the like, or the platinum nanoparticle In combination with other foods or beverages, the organism is administered orally in a form suitable for consumption; the platinum nanoparticles can be prepared into any preparation for injection for topical treatment.

In summary, the platinum nanoparticle of the present invention is used for preparing a drug capable of selectively killing cancer cells in a light energy environment, and is converted into heat energy by absorbing light energy to generate a photothermal effect, thereby inducing cells to enter apoptosis, thereby Can achieve the efficacy of treating cancer. Furthermore, if the platinum nanoparticle of the present invention has no irradiation light energy, it does not significantly affect the unirradiated cancer cells, and has the effect of selectively inducing cancer cell death in a light energy environment. Since the side effects of the conventional treatment method are caused by the poisoning of the normal cell line together, the platinum nanoparticle of the present invention can reduce the side effects caused by the conventional treatment method and provide a good life for the cancer patient as compared with the conventional treatment method. The effect of quality.

The invention further provides a method for preparing a platinum nano particle, which is capable of preparing a platinum nano particle which selectively poisons a cancer cell in a light energy environment, and the platinum nano particle causes the cancer cell to die through apoptosis, and It does not cause an inflammatory reaction around the cancer cells, and thus omits the medical procedure for treating the inflammatory response, thereby improving the clinical medical level and is the efficacy of the present invention.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

Claims (4)

The use of a platinum nanoparticle for preparing a medicament for treating cancer, wherein the platinum nanoparticle is administered to an organism, and a light energy is provided for 4 to 10 minutes in the affected part of the living body, the light energy The wavelength of the light is 1064 nm; wherein the platinum nanoparticle is obtained by a method comprising the steps of: mixing 200 mg of one chloroplatinic acid hexahydrate with 90 mM, 10 ml of a protective agent to obtain a mixed solution; The solution is subjected to a nucleophilic reduction reaction with 1 mM and 50 μl of sodium hydroxide at 100 to 140 ° C for 15 to 60 minutes to form a platinum nanoparticle. The use of the platinum nanoparticle according to claim 1, wherein the protective agent comprises a water-soluble polymer and ethylene glycol, and the water-soluble polymer is selected from the group consisting of polyvinyl alcohol, gelatin, and poly Acrylamide, polyethyleneimine or polyvinylpyrrolidone. The use of the platinum nanoparticle according to claim 1, wherein the nucleophilic reduction reaction is carried out at 120 ° C for 30 minutes. The method for preparing the platinum nanoparticle according to the first aspect of the invention, wherein the method for preparing the platinum nanoparticle further comprises mixing the mixture with sodium hydroxide in a ratio of 1:3 by volume, and acetone. The supernatant was removed by centrifugation to obtain a purified platinum nanoparticle.