KR102156920B1 - Pharmaceutical composition for enhancing the radiotherapy and chemotherapy of cancer including porous metal nanoparticle - Google Patents

Abstract

The present invention relates to a porous metal nanoparticle that generates oxygen, and relates to a use of the porous metal nanoparticle to simultaneously enhance the radiation and anticancer therapeutic effects.
The porous metal nanoparticles according to the present invention can overcome hypoxia by generating oxygen by reaction with hydrogen peroxide present in the tumor, and can significantly improve the radiation treatment effect by effectively absorbing the irradiated radiation. In addition, if an imaging contrast agent or an anticancer agent is included in the porous metal nanoparticles, the location of the tumor can be confirmed from the image, thereby improving the accuracy of diagnosis/treatment, and a synergy effect between the anticancer agent and radiation therapy can be expected.

Description

Pharmaceutical composition for enhancing the radiotherapy and chemotherapy of cancer including porous metal nanoparticles

The present invention relates to a porous metal nanoparticle that generates oxygen, and relates to a use of the porous metal nanoparticle to simultaneously enhance the radiation and anticancer therapeutic effects.

Radiation therapy is one of the three standard treatments along with surgery and chemotherapy, and is one of the most common treatments for cancer patients.

Through irradiation, X-ray radiation penetrates deep into the body and can cause DNA damage directly, and it also kills cancer cells by generating reactive oxygen species. Therefore, it is known that the degree of cell damage caused by irradiation is highly dependent on the oxygen concentration in the tumor. However, in the case of rapidly growing tumors, due to the high consumption of oxygen, the concentration of oxygen in the tumor is often insufficient, resulting in hypoxia, and thus the tumor exhibits resistance to radiation therapy and the patient's prognosis is also poor. .

Various methods have been attempted to enhance the radiation treatment effect, and it is well known that the combination treatment of an anticancer drug and radiation produces a synergistic effect on cancer treatment even at a low dose. In addition, attempts have been made to increase the radiation treatment effect by using gold nanoparticles that can absorb radiation well with a high atomic number. If gold nanoparticles are treated with cancer cells and then radiation treatment is performed, higher radiation treatment It is known to have an effect. However, in the case of gold nanoparticles, since they do not have the ability to generate oxygen, there is still a problem in that they cannot overcome the radiation treatment resistance characteristics due to hypoxia.

Republic of Korea Patent Publication 10-2018-0009183

An object of the present invention is to provide a porous metal nanoparticle generating oxygen.

It is an object of the present invention to provide a composition for enhancing radiation therapy and anticancer treatment for cancer, comprising the porous metal nanoparticles as an active ingredient.

Another object of the present invention is to provide a method for treating cancer using the porous metal nanoparticles.

The present invention intends to use porous platinum nanoparticles or porous platinum nanoparticles containing (mounted) an anticancer agent in order to significantly improve the radiation treatment effect while being able to overcome hypoxia in tumors. Since platinum nanoparticles have a high atomic number, they can absorb radiation such as X-rays well to increase the radiation treatment effect. In addition, according to the present invention, it is possible to generate oxygen by reacting with hydrogen peroxide present in the tumor. I can. Since the platinum nanoparticles having a porous structure have a very large surface area, not only can the reaction with hydrogen peroxide occur effectively, but also an anticancer agent as well as an imaging contrast agent can be mounted into the empty space of the porous structure.

In order to achieve the above object, the present invention is a porous metal nanoparticle that generates oxygen by reacting with hydrogen peroxide, a method of preparing the porous metal nanoparticle, and a use for promoting radiation therapy and anticancer treatment for cancer, and cancer using the same. Provide treatment methods.

In the present invention, "treatment" is included without limitation as long as all actions that improve or benefit the symptoms of cancer.

Specifically, the porous metal nanoparticles may have a catalase function to generate oxygen by reacting with hydrogen peroxide in cancer tissues.

In the present invention, the porous metal nanoparticles are metals having a high atomic number, and preferably, may be porous platinum nanoparticles.

In the present invention, the porous metal nanoparticles may be characterized in that they have a diameter range of 1 to 1000 nm, preferably 10 to 500 nm.

In the present invention, the porous metal nanoparticles may contain (mount) an anticancer agent. These anticancer agents include gosipol, nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nitrotinib, semasanib, bosutinib, axitinib, Cediranib, Restaurtinib, Trastuzumab, Gefitinib, Bortezomib, Sunitinib, Carboplatin, Bevacizumab, Cisplatin, Cetuximab, Viscumalbum, Asparaginase, Tretinoin, Hydroxycarbamide , Dasatinib, Estramustine, Gemtuzumab ozogamycin, Ibritumomab tucetan, Heptaplatin, Methylaminolevulinic acid, Amsacrine, Alemtuzumab, Procarbazine, Alprostadil, Holmium nitrate chitosan , Gemcitabine, doxyfluridine, pemetrexed, tegapur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine, enocitabine , Flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmophor, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinbla Steen, teniposide, doxorubicin, darubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pyrarubicin, aclarubicin, pepromycin, temsiroli Mousse, temozolomide, busulfan, ifosfamide, cyclophosphamide, melpharan, altrethmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitoractol, leucovorin, tretonin, exme Consisting of stan, aminoglutesimide, anagrelide, navelbin, padazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borosol, bicalutamide, lomustine and carmustine One or more selected from the group may be used, but the present invention is not limited thereto.

In the present invention, the porous metal nanoparticles may include an imaging contrast agent, and the imaging contrast agent is not limited, but, for example, a fluorescent molecule, a radioactive isotope for positron emission tomography (PET) contrast It may be a containing contrast agent, a contrast agent containing radioactive isotopes for single photon emission computer tomography (SPECT) contrast, or a magnetic resonance imaging (MRI) contrast agent.

For example, the fluorescent molecule may include a Xanthene derivative selected from the group consisting of fluorescein, FITC (fluorescein isothiocyanate), Oregon green, and Texas red; Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, indocarbocyanine, rhodamine, oxacarbocyanine, thiacarbocyanine and merocyanine Cyanine derivatives selected from the group consisting of merocyanine; Oxadiazole derivatives selected from the group consisting of pyridiloxazole, nitrobenzoxadiazole, and benzoxadiazole; An acridine derivative selected from the group consisting of Nile red, Nile orange, and acridine yellow; Arylmethine derivatives selected from the group consisting of aumarine, crystal violet, and malachite green; A tetrapyrrole derivative selected from the group consisting of porphin, phthalocyanine and bilirubin; And

Consisting of X-SIGHT, Pz 247, DyLight 750, DyLight 800, Alexa Fluor 680, Alexa Fluor 750, IRDye 680, IRDye 800CW, indocyanine green and zwitterionic near-infrared fluorophores. It may be one or more selected from the group consisting of a NIR fluorophore selected from the group.

The radioactive isotope-containing contrast agent for PET contrast is C-11, N-13, O-15, F-18, Ru-82, Ga-68, Cu-60, Cu-61, Cu-62, or Cu- It may be a contrast medium containing 64, and the radioactive isotope-containing contrast agent for SPECT contrast may be a compound containing 99mTc, I-123, I-131 or In-111 as a radioactive isotope.

The MRI contrast agent may be a compound containing superparamagnetic iron platinum particles (SIPPs), gadolinium (Gd) or manganese (Mn).

In the present invention, the pharmaceutical composition may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that for humans.

The pharmaceutical composition of the present invention is not limited thereto, but is formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injectable solutions according to conventional methods. I can. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffers, preservatives, painlessness, etc. for injections. Agents, solubilizers, isotonic agents, stabilizers, and the like can be mixed and used, and in the case of topical administration, a base agent, excipient, lubricant, preservative, etc. can be used. The formulation of the pharmaceutical composition of the present invention can be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixir, suspension, syrup, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. have. Others, solutions, suspensions, tablets, capsules, can be formulated as sustained-release preparations.

On the other hand, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives, and the like may additionally be included.

The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local , Sublingual or rectal. Oral or parenteral administration is preferred.

In the present invention, “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention depends on a number of factors including the activity of the specific compound used, age, weight, general health, sex, formulation, time of administration, route of administration, excretion rate, drug formulation and the severity of the specific disease to be prevented or treated. It may vary in various ways, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day. It can be administered in kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention. The pharmaceutical composition according to the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.

According to another embodiment of the present invention, the step of administering a pharmaceutical composition for promoting radiation therapy and anticancer treatment according to the present invention to animals other than humans; And it relates to a method for treating cancer comprising the step of irradiating radiation.

In the case of irradiation with radiation after administering the pharmaceutical composition provided in the present invention, the radiation treatment effect and further cancer treatment effect can be remarkably improved according to the synergistic effect.

In addition, in the present invention, the animal subject to radiation treatment may be any mammal, including humans, livestock, and pets, but is not limited thereto.

In the present invention, the radiation irradiation may be any radiation irradiation method conventionally used for radiation treatment of cancer or a radiation radiation method to be developed later.

In the present invention, the type of cancer to be subjected to radiation treatment is not particularly limited, but for example, lung cancer, thyroid cancer, breast cancer, biliary tract cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, bladder cancer, It may be one or more cancers selected from the group consisting of kidney cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, blood cancer, and liver cancer.

The porous metal nanoparticles according to the present invention can overcome hypoxia by generating oxygen by reaction with hydrogen peroxide present in the tumor, and can dramatically improve the radiation treatment effect by effectively absorbing the irradiated radiation. In addition, if an imaging contrast agent or an anticancer agent is included in the porous metal nanoparticles, the location of the tumor can be confirmed from the image, thereby improving the accuracy of diagnosis/treatment, and a synergy effect between the anticancer agent and radiation therapy can be expected.

1 is a schematic diagram of the enhancement of the radiation treatment effect of porous platinum nanoparticles.
FIG. 2 is a photograph of an analysis of the shape of the prepared porous platinum nanoparticles with a transmission electron microscope.
3 is a graph showing the hydrodynamic size of the prepared porous platinum nanoparticles in an aqueous solution.
Figure 4 is to analyze the ability of the prepared porous platinum nanoparticles to generate oxygen, by adding and mixing porous platinum nanoparticles at different concentrations in an aqueous hydrogen peroxide solution, and measuring the concentration of oxygen generated in the solution over time. It is data.
5 is a result of analyzing the cytotoxicity of platinum nanoparticles in Example 3 of the present invention. The lung cancer cell line was treated with platinum nanoparticles at various concentrations, and cell viability was measured.
6 is a result of analyzing the cell safety and effects of the platinum nanoparticles as a radiosensitizer in Example 3 of the present invention. The survival rate for lung cancer cell lines was confirmed by increasing the concentration of platinum nanoparticles and the amount of irradiation.
7 is an analysis of the effect on DNA damage by treatment with platinum nanoparticles (50 μg/mL) and irradiation (5 Gy) alone or in combination of Example 4 of the present invention.
8 is an analysis of the effect on the cell cycle of cancer cells according to treatment with platinum nanoparticles (50 μg/mL) and irradiation (5 Gy) alone or in combination of Example 4 of the present invention.
9 is an analysis of the effect on the production of intracellular reactive oxygen species (ROS) according to the treatment of platinum nanoparticles (50 μg/mL) and irradiation (5 Gy) alone or in combination of Example 4 of the present invention. will be.
10 is an analysis of the effect on the tumor growth inhibitory effect according to the treatment with platinum nanoparticles (50 mg/kg) and irradiation (5 Gy) alone or in combination of Example 5 of the present invention.
11 is an analysis of the effect of the platinum nanoparticles (50 mg/kg) and irradiation (5 Gy) alone or in combination treatment on the effect of inducing apoptosis in tumor tissue of Example 5 of the present invention.
12 is an analysis of the effect of the treatment of platinum nanoparticles (50 mg/kg) in Example 5 of the present invention on the effect of improving the hypoxic environment in tumor tissues.
13 is an analysis of toxicity to each organ of mice according to treatment with platinum nanoparticles (50 mg/kg) and irradiation (5 Gy) alone or in combination of Example 6 of the present invention.
14 is a measurement of the weight change of a mouse according to treatment with platinum nanoparticles (50 mg/kg) and irradiation (5 Gy) alone or in combination of Example 6 of the present invention.
15A is a UV-vis absorption spectrum according to the concentration of rhodamine 6G in Example 7 of the present invention.
15B is a calibration curve between the concentration of rhodamine 6G in Example 7 of the present invention and the absorbance at the absorption peak (530 nm).
16A is a fluorescence spectrum according to the concentration of rhodamine 6G in Example 7 of the present invention (excitation wavelength: 480 nm).
16B is a curve for the concentration of rhodamine 6G and the fluorescence intensity value in fluorescence (558 nm) of Example 7 of the present invention.
17A is a UV-vis absorption spectrum according to a weight ratio of rhodamine 6G to platinum nanoparticles of Example 7 of the present invention (platinum nanoparticles: rhodamine 6G).
17B is a fluorescence spectrum according to the weight ratio of rhodamine 6G to the platinum nanoparticles of Example 7 of the present invention (platinum nanoparticles: rhodamine 6G) (excitation wavelength: 480 nm).
18A is a UV-vis absorption spectrum according to the concentration of gosifol in Example 7 of the present invention.
18B is a calibration curve between the concentration of gosifol in Example 7 of the present invention and the absorbance at the absorption peak (370 nm).
19 is a UV-vis absorption spectrum according to the weight ratio of platinum nanoparticles to gosifol (platinum nanoparticles: gosipol) of Example 7 of the present invention.

Hereinafter, preferred embodiments according to the present invention will be described in more detail with reference to the accompanying drawings in order to describe the present invention in more detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

[Example 1] Preparation of porous platinum nanoparticles

K ₂ PtCl ₄ aqueous solution (0.01 M, 6 mL) was mixed with hexadecyltrimethylammonium bromide (CTAB) aqueous solution (0.1 M, 15 mL), and the mixture was heated at 70° C. until it became clear. Then, ascorbic acid (0.02 M, 9 mL) was added, and the reaction was further performed at 70° C. for 3 hours and 30 minutes. Excess CTAB was removed through three stirring and washing, and nanoparticles were dispersed in the third distilled water. To modify the surface of the nanoparticles, 30 mL of an aqueous solution of porous platinum nanoparticles (540 μg Pt/mL) and 0.5 mL of an aqueous solution of monomethoxy (polyethylene glycol) thiol (10 mg/mL) were mixed and reacted for 20 hours by stirring. After that, the nanoparticles were centrifuged and dispersed in distilled water. The Pt concentration of the nanoparticles dispersed in the aqueous solution was analyzed using ICP-MS.

2 is a transmission electron micrograph of the prepared nanoparticles, and FIG. 3 is a size distribution in an aqueous solution phase. According to FIG. 2, it was confirmed that the platinum nanoparticles have a porous structure, so that a surface area capable of causing a catalytic reaction by contacting hydrogen peroxide is very large, as well as a space for carrying a drug inside the particles. In addition, the size of the aqueous solution of the nanoparticles was measured to be 115.6 nm.

[Example 2] Analysis of oxygen generation performance of porous platinum nanoparticles

For analysis, a sample obtained by diluting the porous nanoparticle concentrate to various concentrations in an aqueous solution from which oxygen was removed was prepared, and hydrogen peroxide was added to each solution, followed by stirring. The final concentration of hydrogen peroxide contained in the sample was 1 mM, and the final volume of the sample was 8 mL. To analyze the oxygen evolution in each sample, oxygen concentration was measured using a portable dissolved oxygen (DO) meter every 60 seconds.

3 is a graph showing an increase in oxygen concentration over time in an aqueous solution in which platinum nanoparticles and hydrogen peroxide are mixed. It can be seen that the oxygen concentration in the aqueous solution increases due to the porous platinum nanoparticles, and it can be seen that when the concentration of the platinum nanoparticles increases, the amount of oxygen generated increases.

[ Example 3] Porosity Of platinum nanoparticles Cell test safety and toxicity analysis

An experiment was conducted to confirm the cytotoxicity of the porous platinum nanoparticles against cancer cells. To determine whether platinum nanoparticles that can be used as radiotherapy sensitizers themselves cause toxicity to cells, platinum nanoparticles were treated by concentration in lung cancer cell lines, and after 24 hours, cell viability was checked to determine whether toxicity was induced. The group without any treatment was used as a control.

1) cell culture

The human lung cancer cell line NCI-H460 was obtained from the American Type Culture Collection (ATCC, USA). NCI-H460 cells were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies) at 37° C., 5% carbon dioxide and standard humidity conditions. .

2) Cytotoxicity and radiation treatment efficacy test

NCI-H460 cells were placed in each well of a 96-well plate by 1×10 ⁴ cells, and cultured for 24 hours so that the cells adhere well. Platinum nanoparticles were prepared at a concentration of 0-100 μg/mL, and the cells were treated for 4 hours. Thereafter, platinum nanoparticles that were not ingested into the cells were removed by washing, and after adding a new cell culture solution, they were further cultured for 24 hours. Cell viability was measured using the CCK-8 assay kit.

In order to evaluate the efficacy of platinum nanoparticles as a radiosensitizer, NCI-H460 cells were placed in a 96-well plate as much as 1×10 ⁴ cells/well, and cultured for 24 hours so that the cells adhere well. Platinum nanoparticles were prepared at a concentration of 0-100 μg/mL, treated on cells, and cultured for 4 hours. After that, platinum nanoparticles that were not ingested into the cells were removed through washing, and then irradiated with radiation at 1, 5, and 10 Gy, cultured in an incubator for 72 hours, and cytotoxicity was determined by CCK-8 assay.

5 is a graph showing the analysis of cell viability in lung cancer cells treated with porous platinum nanoparticles at various concentrations. According to the results of FIG. 5, it was confirmed that the nanoparticles did not exhibit cytotoxicity within 24 hours as such in the test concentration range.

6 is a graph showing the survival rate of lung cancer cells according to nanoparticles alone or combined treatment with irradiation. 6, it was confirmed that toxicity to cancer cells was increased according to the radiation dose in NCI-H460 cells, and when comparing the irradiation groups of the same dose, when the platinum nanoparticles were treated or the case was not treated. In comparison, it was confirmed that the cancer cell killing effect was increased. This shows that the platinum nanoparticles themselves do not cause cytotoxicity, but can act as a radiation sensitizer that can increase the effect of radiation. Subsequent experiments were carried out by selecting 50 μg/mL of platinum nanoparticles and 5 Gy of irradiation dose.

[Example 4] Cell test for evaluation of the performance of platinum nanoparticles as a radiation sensitive agent

4.1. Analysis of the effect of platinum nanoparticles on DNA damage of cancer cells

In Example 3.1, an experiment was conducted to analyze the effect of platinum nanoparticles and irradiation alone or combined treatment on DNA damage. When radiation is irradiated to cancer cells, the DNA present in the cell nucleus is damaged and the expression of the protein γ-H2A.X increases. Therefore, by analyzing the expression level of the protein γ-H2A.X, the effect of platinum nanoparticles as a radiation sensitive agent can be analyzed.

1) cell culture

Human lung cancer cell line NCI-H460 was cultured in RPMI (Roswell Park Memorial Institute) 1640 medium containing 10% FBS and 1% penicillin/streptomycin (Life Technologies) at 37° C., 5% carbon dioxide and standard humidity conditions. NCI-H460 lung cancer cells were placed in a cell culture container as much as 5×10 ⁴ cells/well, and then cultured for 24 hours so that the cells adhere well. The group consisted of 4 groups: negative control group, platinum nanoparticle treatment group, irradiation group, and platinum nanoparticle + irradiation group. First, platinum nanoparticles were prepared at a concentration of 50 μg/mL, treated on cells, and cultured for 4 hours. Thereafter, platinum nanoparticles not ingested into the cells were removed through washing, irradiated with 5 Gy of radiation, and cultured in an incubator for 3 hours. Cells were fixed with a phosphate buffer solution containing 4% paraformaldehyde and immunocytochemical staining was performed.

2) Immunocytochemical analysis

The expression level of each group of the target protein γ-H2A.X was measured by immunocytochemical analysis. The cells were fixed at room temperature for 15 minutes with a phosphate buffer containing 4% paraformaldehyde and washed twice. The fixed cells were reacted with PBST containing 0.1% triton X-100 for 10 minutes and washed 3 times for 5 minutes with a phosphate buffer. After that, the cells were treated with PBST buffer containing 1% bovine serum albumin for 30 minutes, and then reacted with γ-H2A.X antibody (1:500 dilution) for 2 hours, followed by 5 minutes with phosphate buffer. Washed 3 times. After that, after reacting with the secondary antibody (1:500 dilution) in the dark for 1 hour, it was washed 3 times for 5 minutes again with a phosphate buffer. Cell nuclei were stained with DAPI for 1 minute, washed with phosphate buffer, and mounted. The reaction-completed sample was imaged with a confocal microscope (LSM780, Carl Zeiss MicroImaging, Thornwood, NY). The acquired image images were selected for each group, counting γ-H2A.X foci per cell, and quantitative analysis was performed.

7 is an analysis of the effect of platinum nanoparticles and irradiation alone or combined treatment on DNA damage. When irradiated with radiation, it could be observed that DNA damage was caused, and it was confirmed that the platinum nanoparticles themselves did not show DNA damage. In addition, it was observed that DNA damage increased by 1.7 times in cells that were pretreated with platinum nanoparticles and irradiated with radiation compared to cells that were only irradiated with radiation, and it was confirmed that platinum nanoparticles can be used as a radiation sensitive agent (*** P <0.001).

4.2. Analysis of the effect of platinum nanoparticles on the cell cycle

In Example 6.2, an experiment was conducted to analyze the effect of platinum nanoparticles and irradiation alone or combined treatment on the cell cycle. When the cancer cells are irradiated with radiation, G2/M stage arrest occurs, cell division is temporarily stopped, and the ratio of G2/M stage cells increases. After radiotherapy with platinum nanoparticles, the performance of platinum nanoparticles was confirmed through cell cycle analysis.

1) cell culture

Human lung cancer cell line NCI-H460 was cultured in RPMI (Roswell Park Memorial Institute) 1640 medium containing 10% FBS and 1% penicillin/streptomycin (Life Technologies) at 37° C., 5% carbon dioxide and standard humidity conditions. The NCI-H460 lung cancer cell line was placed in a 10 cm dish as much as 1×10 ⁶ cells/well and cultured for 24 hours so that the cells adhere well. The group consisted of 4 groups: negative control group, platinum nanoparticle treatment group, irradiation group, and platinum nanoparticle + irradiation group. First, platinum nanoparticles were prepared at a concentration of 50 μg/mL, treated on cells, and cultured for 4 hours. Thereafter, platinum nanoparticles that were not ingested into the cells were removed by washing, and then irradiated with 5 Gy of radiation, and cultured in an incubator for 24 hours.

2) flow cytometry

Flow cytometry was used to observe the effect of platinum nanoparticles on the cell cycle. The prepared cells of each group were collected by centrifugation (2,000 × g, 10 min, 4 °C), washed twice with phosphate buffer, and fixed with cold 70% ethanol for 2 hours or more. Then, wash twice with phosphate buffer and react with PI (50 μg/mL) and RNase (200 μg/mL) at 37°C for 30 minutes, and then perform cytofluorimetric analysis of cell cycle distributions using a BD LSRFortessa flow cytometer. Analysis and results were derived using BD FACS DIVA software. For each sample, it was confirmed how the percentage of cells at various stages in the cell cycle changed through flow cytometry.

8 is data analyzing the effect of platinum nanoparticles and irradiation alone or combined treatment on the cell cycle of cancer cells. When compared with the cell ratio of each phase of the control group, it was confirmed that the ratio of the G2/M phase was increased in the case of cells that were irradiated with radiation, but the platinum nanoparticles themselves did not affect the ratio of each phase of the cell. there was. In addition, in the case of cells that were irradiated after treatment with platinum nanoparticles, it can be observed that the ratio of the G2/M phase increased from 47.97% to 54.93% compared to the group treated with irradiation alone (*** P <0.001). . It can be seen that this is not the effect of platinum nanoparticles, but the result of increasing the effect of radiotherapy. That is, it can be leaked that it is a result of the increase in the amount of radiation absorption by the platinum nanoparticles and thus the sensitivity of cells to radiation is increased.

4.3. Analysis of the effect of platinum nanoparticles on the generation of active oxygen

In Example 6.3, an experiment was conducted to analyze the effect of platinum nanoparticles and irradiation alone or combined treatment on intracellular reactive oxygen species (ROS) production. When cancer cells are stimulated such as anticancer drugs or irradiation, active oxygen is generated within the cells. After performing radiotherapy with platinum nanoparticles, the performance of platinum nanoparticles was confirmed through ROS analysis.

1) Cell culture and treatment

Human lung cancer cell line NCI-H460 was cultured in RPMI (Roswell Park Memorial Institute) 1640 medium containing 10% FBS and 1% penicillin/streptomycin (Life Technologies) at 37° C., 5% carbon dioxide and standard humidity conditions. NCI-H460 lung cancer cells were placed in a cell incubator as much as 5×10 ⁴ cells/well and cultured for 24 hours so that the cells adhere well. The group consisted of 4 groups: negative control group, platinum nanoparticle treatment group, irradiation group, and platinum nanoparticle + irradiation group. First, platinum nanoparticles were prepared at a concentration of 50 μg/mL, treated on cells, and cultured for 4 hours. Thereafter, platinum nanoparticles that were not ingested into the cells were removed through washing, and then irradiated with 5 Gy of radiation and cultured in an incubator for 24 hours.

2) Active oxygen analysis

In order to analyze the amount of active oxygen produced, CellROX® Oxidative Stress Reagents were used. The prepared cells were treated with CellROX® green reagent (5 μM) and hoechst33342 at the same time and reacted at 37° C. for 30 minutes, washed with phosphate buffer, and replaced with cell culture solution. Images were taken with a confocal microscope (LSM780), and 4 images per group were selected and quantified using MetaMorph® image analysis software. As can be seen in FIG. 9, when irradiated with radiation, it can be observed that the intracellular reactive oxygen increases, and the platinum nanoparticles themselves do not induce the production of intracellular reactive oxygen. However, in cells treated with platinum nanoparticles and irradiated with radiation, it was observed that the production of reactive oxygen increased by 3.3 times compared to cells irradiated with only radiation (** P <0.01). This is data confirming that the platinum nanoparticles are capable of being a radiation sensitive agent.

[Example 5] Animal test on the effect of enhancing radiotherapy of platinum nanoparticles

In Example 7, an experiment was conducted to analyze the effect of platinum nanoparticles and irradiation alone or combined treatment on the antitumor effect. In the lung cancer subcutaneous tumor model, platinum nanoparticles and radiation were injected intravenously and localized, respectively, to observe the tumor suppressing effect in vivo using a mouse model. To evaluate the anti-tumor effect, the tumor size was measured every day until the 16th day after treatment, and the difference between each group was analyzed.

5.1. Construction of tumor model and analysis of tumor growth inhibition effect

The human lung cancer cell line NCI-H460 was obtained from the American Type Culture Collection (ATCC, USA). NCI-H460 cells were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium containing 10% FBS and 1% penicillin/streptomycin (Life Technologies) at 37° C., 5% carbon dioxide and standard humidity conditions. NCI-H460 lung cancer cells 5×10 ⁶ cells/100 μL of a nude mouse (Balb/c nude) was injected subcutaneously, and it was confirmed that a subcutaneous tumor was generated 10-14 days later. When the tumor size reached about 70-80 mm ³ , each treatment was performed in 4 groups (negative control group, platinum nanoparticle treatment group, irradiation group, and platinum nanoparticle + irradiation group). On the first day, 50 mg/kg of platinum nanoparticles were systemically administered through the tail vein of the mouse, and on the second day, 5 Gy of radiation was locally irradiated to the tumor site. Tumor growth graph was prepared by measuring the size of the tumor until the 16th day, and the tumor size between each group was analyzed. As can be seen from the results of FIG. 10, it was confirmed that the antitumor effect in the irradiated group after treatment with platinum nanoparticles was the most excellent, compared with the negative control group, the platinum nanoparticle treatment group, and the radiation irradiation group, respectively, The tumor growth inhibitory effect of 83.6% and 64.6% was confirmed, and there was a statistically significant difference (*** P <0.001).

5.2. Observation of changes in induced apoptosis in tumor tissue through tissue staining

In order to confirm the change in apoptosis and apoptosis in the tumor tissue, the mouse was euthanized on the third day of the anti-tumor effect experiment to obtain tumor tissue, and paraffin blocks and tissue slides were prepared. Hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed using tissue slides. TUNEL assay-completed tissue slide samples were imaged with a tissue microscope, and 5 sheets per group were selected and quantified using MetaMorph® image analysis software. As a result, as shown in FIG. 11, compared to the other groups, it was confirmed that apoptosis occurred most in the platinum nanoparticles and the radiation treatment group, and it was increased 3.1 times compared to the radiation treatment group, and it was confirmed that a statistically significant difference was shown. (*** P <0.001).

5.3. Observation of hypoxia in tumor tissue through tissue staining

In order to confirm the change of apoptosis and apoptosis in the tumor tissue, the systemic administration of platinum nanoparticles at 50 mg/kg through the tail vein of the mouse was performed, and after 24 hours, the mouse was euthanized to obtain the tumor tissue. Block and tissue slides were prepared. Using tissue slides, CD31 staining to confirm vascular distribution and HIF-1α staining to confirm hypoxic environment were performed. CD31 staining and HIF-1α staining were performed overnight at 4°C using anti-CD31 antibody (abcam) and anti-HIF-1α antibody (abcam), respectively, and a secondary antibody (anti-rabbit IgG-HRP) was used at room temperature. After reacting for 1 hour, color was developed with diaminobenzidine (DAB) chromogen substrate (DAKO, Carpinteria, CA), counterstaining with Meyer's hematoxylin, dehydration with ethanol, and mounting. The stained tissue slide samples were imaged with a tissue microscope, and 5 sheets per group were selected and quantified using MetaMorph® image analysis software. As a result, as shown in FIG. 12, it was confirmed that the expression level of CD31 in the control group and the platinum nanoparticle-treated group was similar, but the expression level of HIF-1α decreased by 82.3% in the platinum nanoparticle-treated group compared to the control group. . This result could be predicted that platinum nanoparticles improved the hypoxic conditions in the tumor tissue by converting hydrogen peroxide, which is abundantly present in the tumor tissue, into oxygen (*** P <0.001).

[Example 6] In vivo safety evaluation of platinum nanoparticles

In Example 6, in order to evaluate the in vivo safety of platinum nanoparticles, an experiment was conducted to confirm the histological changes for each organ. At the same time as the experiment to confirm the tumor growth inhibitory effect of Example 5 was conducted, not only the size of the tumor but also the weight was measured until the 16th day, and the heart, lung, liver, spleen and kidney of the mouse were collected after euthanizing the mouse on the 16th day. Thus, paraffin blocks and tissue slides were prepared and H&E staining was performed.

13, it can be seen that the histological change does not occur in the platinum nanoparticles or the radiation-treated group compared to the control group. In addition, according to FIG. 14, it can be seen that the platinum nanoparticles or the radiation-treated group did not lose weight for 16 days when compared to the control group. Through these results, it was proved that the platinum nanoparticles are biocompatible and safe.

[ Example 7] Drug loading experiment on porous platinum nanoparticles

Porous platinum nanoparticles have enough space to mount drugs or various imaging contrast agents in the nanoparticles, and also mount various kinds of drugs and contrast agents by additional methods such as applying various polymer coatings known in the past. can do. In this example, rhodamine 6G and gosifol were mounted on porous platinum nanoparticles as representative contrast agents and anticancer agents.

7.1. Rhodamine 6G loading experiment and analysis

0.02 mg of porous platinum nanoparticles whose surface was modified with mPET and various masses of rhodamine 6G (0.02, 0.04, 0.10, 0.20 mg) were dissolved in 1 mL of distilled water. The solution was stirred at room temperature for 24 hours using a mixer to induce a fluorescent dye to be mounted in the porous platinum nanoparticles. Thereafter, centrifugation (16,000 rpm, 10 minutes) was repeated three times to remove the fluorescent dye that did not enter the nanoparticles, and only the platinum nanoparticles loaded with the fluorescent dye were separated.

In order to analyze the amount of the loaded fluorescent contrast agent, rhodamine 6G, platinum nanoparticles loaded with rhodamine were redispersed in distilled water and stirred at room temperature for 24 hours to induce rhodamine 6G to escape from the nanoparticles. Thereafter, the nanoparticles were removed through centrifugation (16,000 rpm, 10 min), absorption and fluorescence measurements were performed on the supernatant in which rhodamine 6G was present, and compared with the calibration graph, the rhodamine 6G mounted on the nanoparticles. The amount of was calculated. According to FIG. 15A, the highest absorption peak of rhodamine 6G is 530 nm, and it can be seen that the concentration of rhodamine 6G charged in platinum nanoparticles can be calculated through a calibration curve using the absorption value at this time (FIG. 15B). In addition, it can be seen that intrinsic fluorescence can be confirmed using the fluorescence spectrum of Rhodamine 6G (FIG. 16) (λ _Ex . 480 nm, λ _Em .558 nm). For each sample loaded with 0.0162 (1:1), 0.0295 (1:2), 0.0982 (1:5), 0.212 (1:10), Rhodamine 6G of 0.04 μM, 0.4 μM, 2.0 μM and 4.8 μM was added. The result was obtained by filling platinum nanoparticles. FIG. 17 is a result of analyzing the absorption and fluorescence spectrum of the supernatant after extracting rhodamine 6G again from the porous platinum nanoparticles loaded with rhodamine at different ratios. Referring to FIG. 17, as the loading amount of rhodamine 6G increased, the absorbance and fluorescence spectra increased, and this is a result showing that rhodamine 6G is well loaded in a concentration-dependent manner on the porous nanoparticles.

7.2. Gosifol mounting experiment and analysis

In this example, the anticancer agent Gosipol was used as a model drug, and an experiment was conducted in which it was mounted in porous platinum nanoparticles. 0.02 mg of porous platinum nanoparticles and 0.02, 0.04, 0.10, and 0.20 mg of gosifol were dissolved in 1.0 mL of ethanol. The solution was stirred at room temperature for 24 hours using a mixer to mount gossypol on the porous nanoparticles. After stirring for 24 hours, each solution was centrifuged (16,000 rpm, 10 minutes) and washed three times to separate and obtain the drug-loaded nanoparticles. In order to analyze the amount of mounted gosifol, the platinum nanoparticles loaded with gosifol were redispersed in ethanol and stirred at room temperature for 24 hours again using a mixer to induce gosipol to escape from the nanoparticles. The nanoparticles were removed through centrifugation (16,000 rpm, 10 min), and the absorption spectrum of the supernatant with gosifol was analyzed, and the amount of gosifol loaded on the nanoparticles was calculated by comparing with the calibration graph. According to FIG. 18A, the highest absorption peak of posifol is 370 nm, and it can be seen that the concentration of gosipol charged in the platinum nanoparticles can be calculated through a calibration curve using the absorption value at this time (FIG. 18B). FIG. 19 is a result of analyzing the absorption spectrum of the supernatant after extracting the gosipole again from the porous platinum nanoparticles loaded with gossifol at different ratios. 0.0122 (1:1), 0.0188 (1:2), 0.0261 (1:5), 0.0522 (1:10) For each sample, 1.4 μM, 2.1 μM, 3.0 μM, 6.0 μM of Gosifol is platinum nano The result of filling in the particles was obtained. Referring to FIG. 19, the absorption spectrum increased with the increase in the loading amount of Gosifol, which is a result indicating that Gosifol is well loaded in a concentration-dependent manner on the porous nanoparticles.

Claims (8)

A pharmaceutical composition for enhancing radiotherapy for hypoxia solid cancer, comprising porous platinum nanoparticles that generate oxygen by reacting with hydrogen peroxide in cancer tissues as an active ingredient. delete The method of claim 1,
The porous platinum nanoparticles contain an anticancer agent, pharmaceutical composition. The method of claim 1,
The porous platinum nanoparticles include an imaging contrast agent, pharmaceutical composition. The method of claim 1,
The solid cancer is lung cancer, thyroid cancer, breast cancer, biliary tract cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, gastric cancer, brain cancer, rectal cancer, bladder cancer, kidney cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, and liver cancer. At least one selected from the group consisting of, a pharmaceutical composition. Administering a pharmaceutical composition for enhancing radiotherapy for hypoxia solid cancer comprising porous platinum nanoparticles as an active ingredient to animals other than humans; And
A treatment method for hypoxia solid cancer comprising the step of irradiating radiation,
The porous platinum nanoparticles react with hydrogen peroxide in the cancer tissue to generate oxygen. delete delete

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