Manufacturing method of modified gold nanodandelions and use of the same

Abstract

A method for manufacturing modified flower-shaped gold nanoparticles includes: adding a gold seed suspension and auric acid to a gelatin solution to form a mixture; adding a reducing agent to the mixture; and adding a ligand to the mixture to react and form flower-shaped gold nanoparticles. The ligand system comprises a thiol compound. The modified flower-shaped gold nanoparticles are intended for use as a pharmaceutical composition for treating cancer in individuals with a need for such treatment.

Description

Technical Field

[0001] This disclosure relates to a method for manufacturing modified gold nanodandelion (GND) and its uses, particularly to a method for manufacturing ligand-modified gold nanodandelion and its use in cancer treatment. Prior Technology

[0002] Radiotherapy is a common method for treating tumors. It uses high-energy radiation to irradiate tumor tissue, thereby destroying the DNA structure of tumor cells, inhibiting their division and proliferation, and causing tumor cells to apoptosis or die, thus treating patients with cancer.

[0003] However, when using radiotherapy to treat tumors, problems may still arise such as the immune system's inability to effectively eliminate tumor cells and the development of radioresistance in tumor cells, leading to insufficient efficacy, incomplete tumor eradication, or recurrence. Furthermore, tumor tissue often presents a hypoxic microenvironment. Existing research indicates a high correlation between hypoxia in tumor tissue and increased tumor malignancy. The hypoxic microenvironment not only fosters radiotherapy resistance in tumor cells but also further promotes tumor cell proliferation and metastasis, thereby reducing overall treatment effectiveness.

[0004] Therefore, there is an urgent need in the art for a method for manufacturing modified GNDs that can enhance the phagocytic activity of tumor cells, increase the sensitivity of the tumor cells to radiation, and / or alleviate the hypoxia of the tumor microenvironment, as well as their uses. Summary of the Invention

[0005] In view of the above problems, this disclosure provides a method for manufacturing modified flower-shaped gold nanoparticles, comprising: adding a gold seed suspension and auronic acid to a gelatin solution to form a mixture; adding a reducing agent to the mixture; and adding a ligand to the mixture to react and form flower-shaped gold nanoparticles. The ligand system comprises a thiol compound.

[0006] This disclosure also provides the use of modified flower-shaped gold nanoparticles as a pharmaceutical composition for treating cancer in individuals with a need for such treatment.

[0007] In at least one embodiment disclosed herein, the phagocytic activity of tumor cells can be enhanced, the production of reactive oxygen species can be significantly increased under radiation irradiation, the hypoxia of the tumor microenvironment can be alleviated, and the radiosensitization effect on tumor cells can be enhanced, which helps to improve the effect of radiotherapy. Simple Explanation of the Diagram

[0008] Figure 1A is a schematic diagram of the structure of GNDs according to at least one embodiment of the present disclosure. Figure 1B is an ultraviolet-visible-near-infrared (UV-vis-NIR) absorption spectrum of GNDs according to at least one embodiment of the present disclosure. Figure 1C is a bar chart of absorbance ratios at specific absorption wavelengths A1064nm / A555nm and A808nm / A555nm for GNDs according to at least one embodiment of the present disclosure (**p < 0.01). Figure 1D is a transmission electron microscope (TEM) photograph of GNDs according to at least one embodiment of the present disclosure. Figure 1E is an optical microscope photograph of the phagocytosis of GNDs by C6 cells according to at least one embodiment of the present disclosure. The top left of Figure 2 shows the DPBF fluorescence intensity ratio measured under 10 MV photon irradiation according to at least one embodiment of this disclosure; the bottom left of Figure 2 shows the DPBF fluorescence intensity ratio measured under 160 MeV proton irradiation according to at least one embodiment of this disclosure; the top middle of Figure 2 shows the DHE fluorescence intensity ratio measured under 10 MV photon irradiation according to at least one embodiment of this disclosure; the bottom middle of Figure 2 shows the DHE fluorescence intensity ratio measured under 160 MeV proton irradiation according to at least one embodiment of this disclosure; the top right of Figure 2 shows the TA fluorescence intensity ratio measured under 10 MV photon irradiation according to at least one embodiment of this disclosure; and the bottom right of Figure 2 shows the TA fluorescence intensity ratio measured under 160 MeV proton irradiation according to at least one embodiment of this disclosure (*p < 0.05, **p < 0.05). 0.01, DPBF fluorescence: amount of O2 produced in reaction 1, DHE fluorescence: amount of O2·- produced in reaction , TA fluorescence: amount of ·OH produced in reaction ). The left image of Figure 3A is a TEM photograph of the monomeric state according to at least one embodiment of the present disclosure; and the right image of Figure 3A is a TEM photograph of the aggregated state according to at least one embodiment of the present disclosure. The upper left of Figure 3B shows the DPBF fluorescence intensity ratio measured under 10 MV photon irradiation according to at least one embodiment of this disclosure; the lower left of Figure 3B shows the DPBF fluorescence intensity ratio measured under 160 MeV proton irradiation according to at least one embodiment of this disclosure; the upper middle of Figure 3B shows the DHE fluorescence intensity ratio measured under 10 MV photon irradiation according to at least one embodiment of this disclosure; the lower middle of Figure 3B shows the DHE fluorescence intensity ratio measured under 160 MeV proton irradiation according to at least one embodiment of this disclosure; the upper right of Figure 3B shows the TA fluorescence intensity ratio measured under 10 MV photon irradiation according to at least one embodiment of this disclosure; and the lower right of Figure 3B shows the TA fluorescence intensity ratio measured under 160 MeV proton irradiation according to at least one embodiment of this disclosure (*p < 0.05, **p < 0.05). 0.01, DPBF fluorescence: amount of O2 produced in reaction 1, DHE fluorescence: amount of O2·- produced in reaction , TA fluorescence: amount of ·OH produced in reaction ). Figure 4 is a quantitative bar chart of oxygen concentration produced by the decomposition of H₂O₂ according to at least one embodiment of the present disclosure. The upper left image of Figure 5A is an H2DCFDA fluorescence photograph under 10 MV photon irradiation according to at least one embodiment of the present disclosure; the lower left image of Figure 5A is the H2DCFDA fluorescence intensity measured under 10 MV photon irradiation according to at least one embodiment of the present disclosure; the upper right image of Figure 5A is an SOSG fluorescence photograph under 10 MV photon irradiation according to at least one embodiment of the present disclosure; and the lower right image of Figure 5A is the SOSG fluorescence intensity measured under 10 MV photon irradiation according to at least one embodiment of the present disclosure (**p < 0.005). The upper left image of Figure 5B is a fluorescence photograph of H2DCFDA under 160 MeV proton irradiation according to at least one embodiment of the present disclosure; the lower left image of Figure 5B is the fluorescence intensity of H2DCFDA measured under 160 MeV proton irradiation according to at least one embodiment of the present disclosure; the upper right image of Figure 5B is a fluorescence photograph of SOSG under 160 MeV proton irradiation according to at least one embodiment of the present disclosure; the lower right image of Figure 5B is the fluorescence intensity of SOSG measured under 160 MeV proton irradiation according to at least one embodiment of the present disclosure (**p < 0.005, H2DCFDA: ROS production, SOSG: 1O2 production, blue: cell nucleus, green: ROS / 1O2). The top image of Figure 6A is an immunofluorescence staining photograph showing the changes in cell nuclei over culture time under 10 MV photon irradiation according to at least one embodiment of the present disclosure (blue: cell nucleus, green: DNA damage); and the bottom image of Figure 6A is a quantitative bar graph of cell nuclei measured under 10 MV photon irradiation according to at least one embodiment of the present disclosure (*p < 0.05, **p < 0.005). The upper image of Figure 6B is an immunofluorescence staining photograph showing the changes in cell nuclei over culture time under 160 MeV proton irradiation according to at least one embodiment of the present disclosure (blue: cell nucleus, green: DNA damage); and the lower image of Figure 6B is a quantitative bar chart of cell nuclei measured under 160 MeV proton irradiation according to at least one embodiment of the present disclosure (*p < 0.05, **p < 0.005). The upper image of Figure 7A is an immunofluorescence staining photograph showing the changes in cell nuclei over culture time under 10 MV photon irradiation according to at least one embodiment of the present disclosure (blue: cell nucleus, red: aggregated mitochondria, green: monosodium mitochondria, yellow: overlap of aggregated and monosodium mitochondria); and the lower image of Figure 7A is a quantitative histogram of mitochondria measured under 10 MV photon irradiation according to at least one embodiment of the present disclosure (*p < 0.05, **p < 0.005). The upper image of Figure 7B is an immunofluorescence staining photograph showing the changes in mitochondria over culture time under 160 MeV proton irradiation according to at least one embodiment of the present disclosure (blue: cell nucleus, red: aggregated mitochondria, green: monosodium mitochondria, yellow: overlap of aggregated and monosodium mitochondria); and the lower image of Figure 7B is a quantitative histogram of mitochondria measured under 160 MeV proton irradiation according to at least one embodiment of the present disclosure (*p < 0.05, **p < 0.005). The upper image of Figure 8A is a photograph of CT-2A cell community analysis (clonogenic assay) under 10 MV photon irradiation as a function of irradiation dose, according to at least one embodiment of the present disclosure; and the lower image of Figure 8A is a line graph of CT-2A cell viability fraction measured under 10 MV photon irradiation, according to at least one embodiment of the present disclosure (*p < 0.05). The upper part of Figure 8B is a photograph of CT-2A cell community analysis under irradiation dose variation at 160 MeV proton irradiation according to at least one embodiment of the present disclosure; and the lower part of Figure 8B is a line graph of CT-2A cell survival fraction measured under 160 MeV proton irradiation according to at least one embodiment of the present disclosure (*p < 0.05). The upper image of Figure 9A is a photograph of the U87-MG cell community under 10 MV photon irradiation as a function of the irradiation dose, according to at least one embodiment of the present disclosure; and the lower image of Figure 9A is a bar chart of the quantitative relative survival rate of U87-MG cells measured under 10 MV photon irradiation, according to at least one embodiment of the present disclosure. The upper image of Figure 9B is a photograph of the U87-MG cell community under 160 MeV proton irradiation as a function of the irradiation dose, according to at least one embodiment of the present disclosure; and the lower image of Figure 9B is a bar chart of the quantitative relative survival rate of U87-MG cells measured under 160 MeV proton irradiation, according to at least one embodiment of the present disclosure. Implementation

[0009] The following embodiments are provided to illustrate this disclosure in detail. Those skilled in the art will readily understand the advantages and effects of this disclosure upon reading it, and it can be implemented or applied in other different embodiments. Therefore, the following embodiments for carrying out this disclosure can be modified and / or changed without departing from its various aspects and scope of application, and any element or method within the scope of this disclosure can be combined with any other element or method disclosed in any embodiment of this disclosure.

[0010] As used herein, the articles “a,” “one,” and “the” refer to one or more (i.e., at least one) grammatical object of the article, unless otherwise clearly stated, and the terms “or” and “and / or” are used interchangeably, unless otherwise clearly stated.

[0011] As used herein, the phrase "at least one" refers to one or more requirements and should be understood to mean at least one requirement selected from any one or more of the listed requirements, but not necessarily including at least one of each of the listed requirements, and does not exclude any combination of the listed requirements. This definition also allows for the presence of requirements other than those identified in the list of requirements referred to by the phrase "at least one," whether related to or unrelated to those identified requirements. Therefore, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B", or, equivalently, "at least one of A and / or B") in one embodiment may refer to at least one A, including more than one A as needed, with no B (and including elements other than B as needed); in another embodiment, it is at least one B, including more than one B as needed, with no A (and including elements other than A as needed); in yet another embodiment, it is at least one A, including more than one A as needed, and at least one B, including more than one B as needed (and including other elements as needed).

[0012] As described herein, the terms "comprising," "including," "containing," "having," and any other variations thereof are intended to cover non-exclusive inclusions. For example, when describing something as "comprising" a limitation, it may additionally include other components, elements, parts, structures, regions, portions, devices, systems, steps, or connections, unless otherwise stated, and should not exclude other limitations.

[0013] The numerical ranges used in this document are inclusive and composable. Any value falling within the numerical ranges described herein can be considered a maximum or minimum value from which subranges can be derived. For example, the numerical range "4 MV to 25 MV" includes any subrange between the minimum value of 4 MV and the maximum value of 25 MV, such as subranges from 4 MV to 15 MV, from 9 MV to 18 MV, or from 6 MV to 25 MV. Furthermore, multiple numerical values ​​used in this document can be selected as maximum and minimum values ​​as needed to derive numerical ranges. For example, numerical ranges of 4 MV to 20 MV, 4 MV to 15 MV, and 6 MV to 15 MV can be derived from values ​​of 4 MV, 6 MV, 15 MV, and 20 MV, respectively.

[0014] As used herein, the term "about" generally refers to a value encompassing a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% in a numerical value or range. Such variation may be due to, for example, experimental errors, typical errors in the measurement or processing procedures of the manufacture of the compound, composition, concentrate, or formulation, differences in the source, manufacture, or purity of the starting material or the ingredients used in this disclosure, or similar considerations. Alternatively, the term "about" means within an acceptable average standard deviation as considered by someone of ordinary skill in the art. Unless otherwise expressly stated, all numerical ranges, quantities, values, and percentages disclosed herein, such as the amount of material, the duration of time periods, temperature, operating conditions, ratios of quantities, etc., should be understood to be modified by the term "about" in all cases.

[0015] As described in this article, the term "reactive oxygen species (ROS)" refers to oxygen-containing chemically reactive substances. ROS can damage lipids, DNA, RNA, and proteins, thereby inducing apoptosis in tumor cells.

[0016] As described herein, the term "tumor with high MMP-2 and / or MMP-9 expression" refers to tumor tissue with significantly higher MMP-2 and / or MMP-9 concentrations than its corresponding normal tissue, for example, but not limited to: the MMP-2 and / or MMP-9 concentration in tumor tissue being more than twice that in its corresponding normal tissue.

[0017] As described herein, the term "treatment" refers to achieving a desired pharmacological or physiological effect, such as, but not limited to, inhibiting tumor cell growth or shrinking a tumor. This effect may be preventative, i.e., completely or partially preventing the condition, appearance, disease, or symptoms, and / or therapeutic, i.e., partially or completely curing the condition and / or side effects attributable to the condition or disease.

[0018] As described herein, "individual" and "patient" are used interchangeably and refer to animals, such as mammals. The term "individual" is intended to refer to both males and females unless a particular sex is specifically specified. In at least one embodiment of this disclosure, an individual may be a rodent, mouse, monkey, dog, cat, cow, horse, emu, sheep, deer, wolf, fox, pig, rabbit, chicken, ostrich, or human, but this disclosure is not limited thereto. In some embodiments, an individual may be a human.

[0019] As stated herein, the terms “flower-like gold nanoparticles”, “modified flower-like gold nanoparticles”, “GNDs”, “modified GNDs” and “gold-containing compounds” are used interchangeably unless otherwise clearly stated.

[0020] As described herein, the term "effective amount" refers to an amount of a gold compound or pharmaceutical composition sufficient to treat a particular symptom, condition, or illness associated with a disease in an individual with the need. In some embodiments, the effective amount of the gold compound or pharmaceutical composition results in the suppression or reduction of appearance and / or symptoms associated with an undesirable condition. In some embodiments, the effective amount may be varied by those skilled in the art and may depend on the use of the gold compound or pharmaceutical composition, the route of administration, and / or the condition to be treated, but this disclosure is not limited thereto.

[0021] As described herein, the terms "application" or "administration" refer to the introduction of a gold-containing compound or pharmaceutical composition into an individual by a method or route, such that at least a portion of the gold-containing compound or pharmaceutical composition is located at a desired site to produce a desired effect. For example, the gold-containing compound or pharmaceutical composition disclosed herein may be administered to an individual by injection, but this disclosure is not limited thereto.

[0022] As used herein, the term "pharmaceutically acceptable carrier" refers to pharmaceutically acceptable materials, media, or compositions, such as, but not limited to, solid or liquid fillers, binders, diluents, preservatives, biocompatible solvents, disintegrants, lubricants, suspending agents, flavoring agents, encapsulating materials, thickeners, acids, surfactants, complexing agents, wetting agents, or any combination thereof. In some embodiments, each component is "pharmaceutically acceptable" in the sense of compatibility with other components of a pharmaceutical formulation and is suitable for contact with the organs or tissues of an individual (e.g., but not limited to mammals) without causing excessive toxicity, allergic reactions, irritation, immunogenicity, or other complications or problems. See, for example, Remington: Pharmaceutical Science and Practice, 22nd ed.; Allen, ed.: Philadelphia, PA, 2012; Handbook of Pharmaceutical Excipients, 7th ed.; Rowe et al., ed.; Pharmaceutical Publishers and American Pharmaceutical Association: 2012; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash, ed.; Gower Publishing Company: 2007; Pre- and Formulation Drugs, 2nd ed.; Gibson, ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0023] In at least one embodiment of this disclosure, a method for treating cancer is provided, which may include: administering an effective amount of modified flower-shaped gold nanoparticles to an individual in need.

[0024] In at least one embodiment of this disclosure, the effective amount may be a concentration of modified flower-shaped gold nanoparticles from about 5 μg / mL to 400 μg / mL, for example, but not limited to: about 6 μg / mL to 140 μg / mL, about 7 μg / mL to 130 μg / mL, or about 8 μg / mL to 120 μg / mL. In some embodiments, the effective amount may be about 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, or 400 μg / mL, but this disclosure is not limited thereto.

[0025] In at least one embodiment of this disclosure, a method for treating cancer is provided, which may include: administering an effective amount of modified flower-shaped gold nanoparticles to an individual in need.

[0026] In at least one embodiment of this disclosure, the effective amount may be a concentration of modified flower-shaped gold nanoparticles from about 5 μg / mL to 250 μg / mL, for example, but not limited to: about 6 μg / mL to 240 μg / mL, about 7 μg / mL to 230 μg / mL, or about 8 μg / mL to 220 μg / mL. In some embodiments, the effective amount may be about 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, or 250 μg / mL, but this disclosure is not limited thereto.

[0027] In at least one embodiment of this disclosure, the auric acid may be tetrachloroauric acid.

[0028] In at least one embodiment of this disclosure, the gelatin solution may comprise type A gelatin.

[0029] In at least one embodiment of this disclosure, the reducing agent may be L-ascorbic acid.

[0030] In at least one embodiment of this disclosure, the concentration ratio of auric acid to gelatin solution ([auric acid](μM) / [gelatin](mg.mL⁻¹)) can be 25 to 50, for example, but not limited to: about 26 to 50, about 27 to 50, or about 28 to 50. In some embodiments, the concentration ratio of auric acid to gelatin solution can be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0031] In at least one embodiment disclosed herein, the compound containing a thiol group may be 3-mercaptopropionic acid and 6-mercapto-1-hexanol.

[0032] In at least one embodiment of this disclosure, prior to the reaction, the molar ratio of 3-mercaptopropionic acid and 6-mercapto-1-hexanol may be more than about 1:20, for example, but not limited to: about 1:20 to 20:1, about 1:55 to 19:1 or about 1:50 to 18:1. In some embodiments, the molar ratio of 3-mercaptopropionic acid and 6-mercapto-1-hexanol may be about 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1, but this disclosure is not limited thereto. In some embodiments, when the molar ratio of 3-mercaptopropionic acid and 6-mercapto-1-hexanol is less than 1:20, it is easy to cause excessive reaction, leading to GNDs aggregation.

[0033] In at least one embodiment of this disclosure, the reaction time may be from 30 minutes to 90 minutes, for example, but not limited to: about 35 minutes to 85 minutes, about 40 minutes to 80 minutes, or about 45 minutes to 75 minutes. In some embodiments, the reaction time may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 minutes, but this disclosure is not limited thereto.

[0034] In at least one embodiment of this disclosure, the reaction temperature may be from 30°C to 45°C, for example, but not limited to: about 35°C to 85°C, about 40°C to 80°C, or about 45°C to 75°C. In some embodiments, the reaction temperature may be about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45°C, but this disclosure is not limited thereto.

[0035] In at least one embodiment of this disclosure, cancer may comprise tumors with high levels of MMP-2 and / or MMP-9 expression. In some embodiments, tumors with high levels of MMP-2 and / or MMP-9 expression may be malignant tumors, such as, but not limited to, nervous system cancers, lung cancer, breast cancer, colorectal cancer, gastrointestinal cancer, head and neck cancer, oral cancer, pancreatic cancer, and / or ovarian cancer. In some embodiments, nervous system cancers include, but are not limited to, primary or metastatic nervous system cancers, but this disclosure is not limited thereto. In some embodiments, primary or metastatic nervous system cancers may comprise central nervous system cancers and peripheral nervous system cancers, such as, but not limited to, skull cancer, meningeal cancer, brain cancer, spinal cord cancer, neurofibroma, meningioma, or sarcoma, but this disclosure is not limited thereto. In some embodiments, brain cancer may comprise, glioma, embryonal tumor, meningioma, brain lymphoma, or schwannoma, but this disclosure is not limited thereto.

[0036] In at least one embodiment of this disclosure, the concentration of MMP-2 and / or MMP-9 in tumor tissue with high MMP-2 and / or MMP-9 expression levels is more than twice the concentration of MMP-2 and / or MMP-9 in the corresponding normal tissue. In some embodiments, the concentration of MMP-2 and / or MMP-9 in tumor tissue with high MMP-2 and / or MMP-9 expression levels is 2 to 30 times, 2 to 6 times, or 16 to 26 times the concentration of MMP-2 and / or MMP-9 in the corresponding normal tissue, for example, but not limited to: 2 times, 4 times, 6 times, 8 times, 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times, or 30 times. In some embodiments, the concentration of MMP-2 in tumor tissue with high MMP-2 expression levels is 2 to 6 times the concentration of MMP-2 in the corresponding normal tissue. In some embodiments, the MMP-9 concentration in tumor tissues with high MMP-9 expression is 16 to 26 times higher than the MMP-9 concentration in corresponding normal tissues. In some embodiments, the MMP-2 and / or MMP-9 concentration in nervous system tumor tissues with high MMP-2 and / or MMP-9 expression is 2 to 26 times higher than the MMP-2 and / or MMP-9 concentration in corresponding normal tissues. In some embodiments, the MMP-2 and / or MMP-9 concentration in breast cancer tumor tissues with high MMP-2 and / or MMP-9 expression is 5 to 18 times higher than the MMP-2 and / or MMP-9 concentration in corresponding normal tissues. In some embodiments, the MMP-2 and / or MMP-9 concentration in colorectal cancer tumor tissues with high MMP-2 and / or MMP-9 expression is 5 to 22 times higher than the MMP-2 and / or MMP-9 concentration in corresponding normal tissues. In some embodiments, the average MMP-2 and / or MMP-9 concentration in all tumor tissues with high MMP-2 and / or MMP-9 expression levels is 2 to 20 times higher than the average MMP-2 and / or MMP-9 concentration in the corresponding normal tissues. In at least one embodiment of this disclosure, the treatment may be at least one selected from the group consisting of photothermal therapy, photon therapy, proton therapy, and heavy ion therapy.

[0037] In at least one embodiment of this disclosure, the treatment may be photon therapy or proton therapy.

[0038] In at least one embodiment of this disclosure, modified flower-shaped gold nanoparticles are used to prepare pharmaceutical compositions for treating cancer in individuals in need, thereby enhancing tumor cell phagocytosis, increasing tumor cell sensitivity to radiation, and / or alleviating tumor microenvironment hypoxia.

[0039] In at least one embodiment of this disclosure, the energy range of the radiation may be from 4 MV to 25 MV, such as, but not limited to, about 4 MV to 24 MV, about 4 MV to 20 MV, or about 4 MV to 18 MV. In some embodiments, the energy range of the radiation may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 MV, but this disclosure is not limited thereto.

[0040] In at least one embodiment of this disclosure, the energy range of the radiation may be from 70 MeV to 250 MeV, such as, but not limited to, about 70 MeV to 240 MeV, about 80 MeV to 230 MeV, or about 90 MeV to 220 MeV. In some embodiments, the energy range of the radiation may be about 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 MeV, but this disclosure is not limited thereto.

[0041] In at least one embodiment of this disclosure, radiation can induce the production of reactive oxygen species.

[0042] In at least one embodiment of this disclosure, the tumor cells may be tumor cells with high levels of MMP-2 and / or MMP-9 expression. In some embodiments, the tumor cells with high levels of MMP-2 and / or MMP-9 expression may be malignant tumor cells, such as, but not limited to: nervous system cancer cells, lung cancer cells, breast cancer cells, colorectal cancer cells, gastrointestinal cancer cells, head and neck cancer cells, oral cancer cells, pancreatic cancer cells, and / or ovarian cancer cells. In some embodiments, nervous system cancer cells include, but are not limited to, glioma cells.

[0043] In at least one embodiment of this disclosure, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0044] The pharmaceutical compositions described herein can be formulated into any dosage form suitable for topical administration to produce local or systemic effects, such as, but not limited to, emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, foams, films, aerosols, rinses, sprays, suppositories, bandages, or skin patches. Topical formulations of the modified GNDs or pharmaceutical compositions described herein may also be, for example, liposomes, micelles, microspheres, nanosystems, and any mixtures thereof, but this disclosure is not limited thereto.

[0045] As used herein, local administration may be performed, for example but not limited to, via (endothelial), conjunctival, intracorneal, intraocular, ocular, ear, transdermal, nasal, vaginal, urethral, ​​respiratory, or rectal administration.

[0046] The modified GNDs or pharmaceutical compositions provided herein can also be formulated for injection, such as, but not limited to, intraperitoneal, intravenous, arterial, intramuscular, or subcutaneous injection.

[0047] In at least one embodiment of this disclosure, suitable pharmaceutically acceptable carriers may include, but are not limited to, aqueous carriers, water-miscible carriers, non-aqueous carriers, antimicrobial agents or preservatives that inhibit microbial growth, stabilizers, solubilizers, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting agents or emulsifiers, complexing agents, sequestering agents or chelating agents, penetration enhancers, cryoprotectants, lyophilization protectants, thickeners or inert gases.

[0048] In at least one embodiment disclosed herein, GNDs exhibit high accumulation in malignant tumor tissue and inhibit cancer cell growth, while showing low toxicity to fibroblasts and / or cell lines exhibiting low levels of matrix metalloproteinases (MMPs), demonstrating selective killing ability and good biocompatibility and safety. Modified GNDs can accumulate extensively within tumor cells and, under radiation irradiation, effectively enhance ROS production, thereby improving the destructive efficacy of radiotherapy against tumor cells. Furthermore, modified GNDs possess peroxidase-mimicking activity, helping to improve the hypoxic state in the tumor region and further overcoming the limitations imposed by hypoxia on the effectiveness of radiotherapy. Therefore, the modified GNDs disclosed herein, in addition to their therapeutic effect on tumor cells, can also serve as drug carriers to further enhance therapeutic efficacy. In some embodiments, the modified GNDs established in this disclosure also have matrix metalloproteinase (MMP) responsiveness, which can be used to enhance intracellular accumulation (the cells may be tumor cells overexpressing MMP-2 / 9, such as, but not limited to, C6, U87-MG), because gelatin (the receptor for MMP-2 / MMP-9) is used as a biological target to synthesize GNDs.

[0049] The following examples further demonstrate the effectiveness of this disclosure, but do not limit the scope of this disclosure.

[0050] The present disclosure is further described through the following embodiments. However, these embodiments are for illustrative purposes only and do not limit the scope and significance of the disclosure in any way. In fact, those skilled in the art will understand many modifications and variations of the present disclosure after reading it, and many modifications and variations of the present disclosure can be made without departing from its scope.

[0051] Material

[0052] The materials and / or pharmaceuticals used in this disclosure are shown in Table 1 below.

[0053] [Table 1] Materials and / or medicines Manufacturers serial number Tetrachloroauric acid (HAuCl4) Sigma-Aldrich 520918 Sodium citrate Sigma-Aldrich 1613859 Gelatin derived from pigskin (Type A gelatin) Sigma-Aldrich G2500 L-Ascorbic Acid Sigma-Aldrich 255564 6-Mercapto-1-hexanol (MCH) Sigma-Aldrich 451088 3-Mercaptopropionic acid (MPA) Sgma-Aldrich M5801 1,3-Diphenylisobenzofuran (DPBF) Sigma-Aldrich 105481 Dihydroethidium (DHE) Thermo Fisher D11347 Terephthalic acid (TA) Sigma-Aldrich 185361 Hydrogen peroxide (H2O2) 33% PanReac 141077 Singlet oxygen sensor (SOSG) Thermo Fisher S36002 H2DCFDA dye Thermo Fisher D399 Hoechst 33342 dye Thermo Fisher H3570 4% paraformaldehyde fixative (4% PFA in PBS buffer) (pH=7.4) Huizhong D460-PFA Phospho-Histone H2A.X (Ser139) Rabbit mAb Zenbio R381558 Anti-rabbit IgG Fab2 Alexa Fluor® 488 Cell Signaling 4412 Bovine serum albumin (BSA) Sigma-Aldrich A7030 JC-1 dye Selleckchem S9784 Crystal violet dye Sigma-Aldrich C6158

[0054] Preparation example: Unmodified GNDs

[0055] Dissolve 1 g of HAuCl4 in double-distilled water to prepare a 10% HAuCl4 solution. Add 200 μL of HAuCl4 solution to 49.8 mL of double-distilled water and mix thoroughly. Then add 3 mL of 38.8 mM sodium citrate solution and mix thoroughly to form a mixed solution. Place the mixed solution in a microwave oven and heat at 600 W for 80 seconds. Shake evenly and heat for another 10 seconds. Then cool at room temperature to obtain a gold nanoparticle (AuNPs) suspension.

[0056] In a 45°C water bath, 5 g of gelatin was dissolved in 500 mL of double-distilled water to prepare a 1% gelatin solution. At room temperature, the gelatin solution was placed on a hot plate and stirred at 600 rpm for the subsequent reaction: 1000 μL of 10% HAuCl4 and 37.5 mL of AuNPs were added to the gelatin solution, and the reaction was allowed to proceed for 15 minutes. Next, 12.5 mL of 10 mM L-ascorbic acid solution was added, mixed thoroughly for approximately 5 seconds, and then allowed to stand for at least 6 hours to form a reaction solution containing GNDs. During the reaction, the solution gradually turned deep purple. The reaction solution was centrifuged at 10000 g for 10 minutes to purify and concentrate the reaction solution, and unreacted matter was removed to obtain the prepared GNDs.

[0057] GNDs can be sterilized by storing them in a phosphate buffered solution (PBST) containing 0.1% polysorbate 20 (tween 20) and phosphate buffered saline (PBS) and reacting them in a water bath at 65°C for 30 minutes for subsequent use.

[0058] Example 1: Modified GNDs (MCH:MPA = 1:19)

[0059] Centrifuge 100 mL of the reaction solution as described in the preparation example at a relative centrifugal force of 10000 g for 10 minutes, and then remove unreacted matter to obtain GNDs. Next, reconstitute the GNDs with an equal volume of 100 mL PBST to form a mixed solution. Add 950 μL of 10 mM MPA and 50 μL of 10 mM MCH to the mixed solution. Place the mixed solution in a 37°C water bath and stir for 1 hour. Then centrifuge the mixed solution at a relative centrifugal force of 10000 g for 10 minutes to purify and concentrate the reaction solution, and then remove unreacted matter to obtain the GNDs of Example 1.

[0060] Example 2: Modified GNDs (MCH:MPA = 1:9)

[0061] The GNDs of Example 2 were manufactured using the method described in Example 1. However, the volumes of their MPA and MCH were varied to 900 μL and 100 μL, respectively.

[0062] Example 3: Modified GNDs (MCH:MPA = 1:3)

[0063] The GNDs of Example 3 were manufactured using the method described in Example 1. However, the volumes of their MPA and MCH were varied to 750 μL and 250 μL, respectively.

[0064] Example 4: Modified GNDs (MCH:MPA = 1:1)

[0065] The GNDs of Example 4 were manufactured using the method described in Example 1. However, the volumes of their MPA and MCH were varied to 500 μL and 500 μL, respectively.

[0066] Example 5: Modified GNDs (MCH:MPA = 3:1)

[0067] The GNDs of Example 5 were manufactured using the method described in Example 1. However, the volumes of their MPA and MCH were varied to 250 μL and 750 μL, respectively.

[0068] Example 6: Modified GNDs (MCH:MPA = 9:1)

[0069] The GNDs of Example 6 were manufactured using the method described in Example 1. However, the volumes of their MPA and MCH were varied to 100 μL and 900 μL, respectively.

[0070] Example 7: Monomer GNDs

[0071] The monomeric GNDs of Example 7 were manufactured using the method described in Example 1.

[0072] Example 8: Aggregated GNDs

[0073] GNDs from Example 1 were added to PBST to prepare a 10 mL solution of 1 mg / mL GNDs. The GNDs solution was placed in a 37°C water bath, and 0.025% trypsin was added. The reaction was allowed to proceed for 12 hours to degrade the gelatin on the surface of the GNDs, thus forming a reaction solution. After the reaction was complete, the reaction solution was centrifuged at a relative centrifugation force of 10,000 g for 10 minutes. The supernatant was then removed, and the GNDs were reconstituted using an equal volume of PBST at pH 5.5 to obtain the aggregated GNDs of Example 8.

[0074] Comparative example: AuNPs

[0075] AuNPs of the comparative example were manufactured by the method described in the preparation example.

[0076] Experimental Example 1: The Influence of Surface Modification on GNDs

[0077] As shown in Figure 1A, the flower-shaped gold nanoparticles 10 disclosed herein comprise a core 101, a ligand 102, and a coating layer 103. The core 101 may be gold nanodandelion, the ligand 102 may be MCH / MPA, and the coating layer 103 may be gelatin. As shown in Figure 1B, the GNDs of Examples 1 to 6 have different UV-vis-NIR absorption spectral characteristics. Among them, the GNDs of Example 2 (with a feed mole ratio of MCH to MPA of 1:9) have a lower absorbance in the near-infrared region (approximately 800 nm to 1100 nm). As the feed mole ratio increases, the absorbance of the GNDs in the near-infrared region (approximately 800 nm to 1100 nm) increases. As shown in Figure 1C, the GNDs of Examples 1 to 3 (with a feed mole ratio of MCH to MPA between 1:19 and 1:3) have relatively stable near-infrared absorption capabilities. As shown in Figure 1D, the GNDs of Examples 1 to 6 have the same particle size and morphology, differing only in their aggregation state, indicating that the ratio of MCH to MPA feed moles only affects the aggregation state of GNDs. As shown in Figure 1E, the GNDs of Examples 1 to 6 can all be taken up by C6 glioma cells, with fewer extracellular GNDs. The cellular uptake of GNDs increases with increasing feed mole ratio, with the GNDs of Example 6 (feed mole ratio of MCH to MPA 9:1) exhibiting the highest cellular uptake. Therefore, the GNDs disclosed herein can enter tumor cells via cellular uptake; and their tumor cell uptake can be further regulated by altering the composition of the GND surface ligands.

[0078] Experimental Example 2: ROS Detection

[0079] (1) Under irradiation with 10 MV photons and 160 MeV protons, the radiosensitizing effects of GNDs on singlet oxygen (1O2), superoxide anion (O2·-), and hydroxyl radicals (·OH) were detected using DPBF, DHE, and TA probes, respectively, to evaluate the potential of the singlet GNDs disclosed herein as radiosensitizers. 500 μL of 1 mg / mL GNDs solution dissolved in pH 5.5 PBST was added to a final concentration of 40 μM for 1O2 detection. 500 μL of 1 mg / mL GNDs solution dissolved in pH 5.5 PBST was added to a final concentration of 10 μM for O2·- detection. 500 μL of 1 mg / mL GNDs solution dissolved in double-distilled water (d2H2O) was added to a final concentration of 5 μM for TA detection. The samples were placed in 2.0 mL eppendorf tubes and kept on ice to protect them from light, awaiting detection. The radiation conditions were as follows: a linear accelerator with a photon energy of 10 MV and a cumulative dose of 10 Gy; and a proton energy of 160 MeV and a cumulative dose of 10 Gy. After irradiation, the samples were centrifuged at 12000 g for 10 minutes, and 200 μL of the supernatant was collected for fluorescence analysis using a 96-well fluorescence disk. The fluorescence detection conditions were as follows: DPBF: 414 nm / 458 nm; DHE: 490 nm / 585 nm; and TA: 315 nm / 420 nm.

[0080] As shown in Figure 2, under 10 MV photon irradiation and 160 MeV proton irradiation conditions, the monomeric GNDs of Example 7 can increase the production of 1O2, O2·- and ·OH, especially the production of 1O2 and O2·-.

[0081] (2) The effect of the aggregation of GNDs on its radiosensitization effect was detected by means of the method described in point (1).

[0082] The results are shown in the upper left, upper middle, and upper right images of Figures 3A and 3B. Under 10 MV photon irradiation, both the monomeric GNDs of Example 7 and the aggregated GNDs of Example 8 can increase the production of 1O2, O2·-, and ·OH, especially 1O2 and O2·-. As shown in the lower left, lower middle, and lower right images of Figures 3A and 3B, under 160 MeV proton irradiation, both the monomeric GNDs of Example 7 and the aggregated GNDs of Example 8 can increase the production of 1O2, O2·-, and ·OH, especially 1O2. Therefore, both monomeric and aggregated GNDs can effectively promote ROS generation under 10 MV or 160 MeV radiation irradiation. Thus, the aggregated nature of the disclosed GNDs has a radiosensitizing effect.

[0083] Experimental Example 3: Catalytic Activity of Catalase

[0084] The GNDs of Example 7, AuNPs of the Comparative Example, double-distilled water, 10 μM FeCl2, and 100 μM H2O2 were prepared as follows: Control group: double-distilled water (1.5 mL); H2O2 only: double-distilled water (1.4 mL) + 100 μM H2O2 (100 μL); Positive control group: double-distilled water (1.39 mL) + 100 μM H2O2 (100 μL) + 10 μM FeCl2 (10 μL); Example 7: double-distilled water (0.5 mL) + GNDs (1000 μL); Example 7 + H2O2: double-distilled water (0.4 mL) + GNDs (1000 μL) + 100 μM H2O2 (100 μL); Comparative example: double-distilled water (0.5 mL) + AuNPs (1000 μL); and Comparative example + H2O2: double-distilled water (0.4 mL) + 100 μM FeCl2 (10 μL). The reaction mixture was prepared by adding AuNPs (1000 μL) and 100 μM H2O2 (100 μL) to AuNPs (1000 μL) and allowing it to stand at 37°C for 1 hour. The concentration of O2 produced in each group was then calculated.

[0085] As shown in Figure 4, compared to AuNPs with a lower specific surface area, GNDs with a higher specific surface area can promote the decomposition of H2O2 and generate a higher concentration of O2. Therefore, the GNDs disclosed in this paper have superior catalase-like catalytic activity.

[0086] Experimental Example 4: In Vitro (In Vitro) 1O2 and Total ROS Measurement

[0087] CT-2A mouse glioma cells were cultured in 6 cm culture dishes. When the cell concentration reached approximately 70%, the medium was removed, and Dulbecco's modified Eagle's medium (DMEM) containing 20 µg / mL GNDs and 10% fetal bovine serum (FBS) was added. After 16 hours of incubation, the medium was removed, and the cells were washed twice with PBS to remove GNDs. Then, SOSG (final concentration 5 μM) or H2DCFDA (final concentration 5 μM) was added to the cell culture medium, and the cells were incubated at 37°C in the dark for 30 minutes. Immediately after culturing, the cells were irradiated with 10 Gy of 10 MV photons or 160 MeV protons. After irradiation, the medium was removed, and the cells were washed twice with PBS to remove SOSG or H2DCFDA. Subsequently, Hoechst 33342 (diluted 1:10000) was added, and the mixture was incubated at 37°C for 10 minutes. After staining, the sample was rinsed twice with PBS, replaced with fresh DMEM medium containing 10% FBS, and kept in the dark. Finally, the fluorescence was observed using a laser scanning confocal microscopy (LSCM). The observation conditions were set as follows: SOSG: 504 nm / 525 nm; H2DCFDA: 488 nm / 525 nm; and Hoechst 33342: 405 nm / 420 nm.

[0088] The results are shown in Figures 5A (top) and 5B (top). Under no irradiation with 10 MV or 160 MeV radiation (0 Gy), the ROS signal (green fluorescence) of cells without GNDs treatment (control group) and cells treated with GNDs (Example 7) was weak. However, after irradiation with 10 MV or 160 MeV radiation (10 Gy), the ROS signal (green fluorescence) of cells treated with GNDs (Example 7) was significantly enhanced compared to cells without GNDs treatment (control group), indicating that GNDs can effectively enhance ROS production induced by 10 MV or 160 MeV radiation. Furthermore, as shown in the lower figures of Figures 5A and 5B, the mean fluorescence intensity (MFI) of total ROS in cells treated with combined radiation and GNDs (Example 7) was 1.8 times that of the radiation-only group; and under clinical megavolt (MV) radiation conditions, GNDs also significantly increased 1O2 production by approximately 6 times. Therefore, the GNDs disclosed herein can significantly increase the production of total ROS and 1O2 in cells under clinical radiation conditions, further validating their potential to enhance the efficacy of radiotherapy at the cellular level and supporting their application value as clinical radiosensitizers.

[0089] Experimental Example 5: In vitro DNA damage detection

[0090] In this study, we observed the double-strand DNA breakage damage in glioma cells of radioresistant CT-2A mice 1 hour and 24 hours after irradiation with 10 MV photons or 160 MeV protons, in order to investigate the effect of ROS induced by GNDs on tumor cell DNA damage.

[0091] (1) Experimental group that was irradiated and cultured for 1 hour.

[0092] CT-2A mouse glioma cells were cultured in 6 cm culture dishes. When the cell concentration reached approximately 70%, the culture medium was removed, and DMEM containing 200 µg / mL GNDs and 10% FBS was added. After 16 hours of continuous culture, the culture medium was removed, and the cells were washed twice with PBS to remove GNDs. Immediately afterwards, the cells were irradiated with 10 Gy of 10 MV photons or 160 MeV protons. After irradiation, the culture medium was removed, and the cells were fixed at 4°C with 4% PFA for one hour. After fixation, the cells were washed twice with PBS to remove PFA. Following cell fixation, the cells were blocked at 4°C with a 3% BSA solution prepared in PBS for at least 12 hours. After blocking, the cells were washed five times with PBS to remove BSA. Then, at room temperature, the cells were shaken and reacted with a PBS solution containing 0.05% Triton (i.e., PBS-Triton solution) for one hour to perform cell membrane perforation. Subsequently, the slide was washed five times with PBS to remove PBS-Triton. Phospho-Histone H2A.X (Ser139) Rabbit mAb (1:1000 dilution) was added at 4°C and reacted in the dark for at least 12 hours for staining. After staining, the slide was washed five times with PBS to remove excess antibody. Anti-rabbit IgG Fab2 Alexa Fluor® 488 (1:2000 dilution) was added at room temperature and reacted in the dark with shaking for 1 hour for staining. After staining, the slide was washed five times with PBS to remove excess antibody. Finally, the slide was mounted and fluorescence was observed using LSCM.

[0093] (2) The experimental group that was irradiated and cultured for 24 hours.

[0094] CT-2A mouse glioma cells were recultured at 37°C for 24 hours. After removing the culture medium, the cells were fixed by reacting with 4% PFA for one hour. Next, following the same method as the experimental group culturing for 1 hour after irradiation, cell fixation, blocking, perforation, and staining were performed. After staining, the cells were further stained with Hoechst 33342 (1:10000 dilution) for 10 minutes at room temperature. After staining, the cells were washed five times with PBS to remove excess Hoechst 33342. Finally, the slides were mounted, and fluorescence was observed using LSCM.

[0095] The observation conditions were set as follows: Anti-rabbit IgG Fab2 Alexa Fluor® 488: 488 nm / 519 nm; and Hoechst 33342: 405 nm / 420 nm.

[0096] The results are shown in Figures 6A and 6B. Under no radiation exposure of 10 MV or 160 MeV (0 Gy), the number of γ-H2AX fluorescent spots in cells without GNDs treatment (control group) and cells treated with GNDs (Example 7) was extremely low, indicating very low DNA damage in tumor cells under no radiation exposure conditions. One hour after irradiation with 10 MV or 160 MeV (10 Gy), compared to cells without GNDs treatment (control group), the number of γ-H2AX fluorescent spots in cells treated with GNDs (Example 7) was significantly increased, indicating a higher degree of tumor cell DNA damage. Furthermore, compared to one hour after the aforementioned radiation exposure, the number of γ-H2AX fluorescent spots in cells irradiated with 10 MV or 160 MeV (10 Gy) was significantly higher. After 24 hours, the γ-H2AX fluorescence spots in cells without GNDs treatment (control group) and cells treated with GNDs (Example 7) both decreased significantly. However, cells treated with GNDs (Example 7) still maintained a high level of residual DNA damage signals. Since the GNDs disclosed herein can promote the production of high concentrations of ROS in the near-nuclear region of tumor cells, when GNDs are used in combination with radiation at MV or MeV levels, it can lead to more complex and difficult-to-repair DNA damage.

[0097] Experimental Example 6: In vitro detection of mitochondrial damage

[0098] CT-2A mouse glioma cells were cultured in 6 cm culture dishes. When the cell concentration reached approximately 70%, the culture medium was removed, and DMEM containing 200 µg / mL GNDs and 10% FBS was added. After 16 hours of continuous culture, the culture medium was removed, and the cells were washed twice with PBS to remove GNDs. Immediately afterwards, the cells were irradiated with 10 Gy of 10 MV photons or 160 MeV protons. After irradiation, the culture medium was removed. JC-1 (final concentration 2.5 μM, which was premixed with the culture medium and ultrasonically vibrated to ensure homogeneity) was then co-cultured with the cells at 37°C for 30 minutes. After staining, the cells were washed five times with PBS to remove excess JC-1. Subsequently, the cells were stained with Hoechst 33342 (1:10000 dilution) at 37°C for 10 minutes. After staining, the cells were washed five times with PBS to remove excess Hoechst 33342, and the culture medium was replaced with fresh medium. Finally, fluorescence was observed using LSCM. The observation conditions were set as follows: JC-1 aggregates: 535 nm / 595 nm; JC-1 monomers: 485 nm / 535 nm; and Hoechst 33342: 405 nm / 420 nm.

[0099] The results are shown in Figures 7A and 7B. Without 10 MV or 160 MeV radiation (0 Gy), both untreated cells (control group) and cells treated with GNDs (Example 7) exhibited red fluorescence with very little green fluorescence, indicating that GNDs do not affect mitochondria without radiation exposure. After 1 hour of 10 MV or 160 MeV radiation (10 Gy), radiation induced excessive ROS (e.g., 1O2 and / or O2·-), decreasing the mitochondrial membrane potential. This resulted in significantly enhanced green fluorescence in cells treated with GNDs (Example 7), while the fluorescence signal of cells that only received radiation but were not treated with GNDs (control group) showed no significant change. After 24 hours of treatment with GNDs (Example 7), the mitochondrial depolarization / membrane potential decrease in cells was more pronounced, demonstrating that GNDs can induce mitochondrial damage and promote tumor cell death under clinical radiation conditions. Furthermore, tumor cells, due to their rapid proliferation and vigorous mitochondrial metabolism, readily produce large amounts of H2O2, which has a long diffusion distance and half-life within the tumor cell. In response, the GNDs disclosed herein possess catalase-like catalytic activity, catalyzing the decomposition of H2O2 into H2O and O2, and further converting O2 into cytotoxic IO2 under MV or MeV level radiation irradiation, thereby causing selective damage to the mitochondrial sites of tumor cells. On the other hand, since mitochondria play a crucial role in cellular energy production and the regulation of apoptosis pathways, they need to maintain redox homeostasis and are highly sensitive to oxidative stress, thus becoming a primary target for ROS attack. Therefore, the GNDs disclosed herein can improve the hypoxic conditions in the tumor microenvironment.

[0100] Experiment Example 7: Cell Colony Formation Assay

[0101] (1) Treatment of CT-2A mouse glioma cells: CT-2A cells were co-cultured overnight at 37°C in DMEM containing 10% FBS and 200 µg / mL GNDs. After culture, the culture medium was removed, and the cells were washed twice with PBS to remove GNDs, and then the culture medium was replaced with fresh medium. Subsequently, the cells were irradiated with 10 MV photons or 160 MeV protons at doses of 0, 2, 4, 6, 8, and 10 Gy, respectively. After irradiation, cells were washed twice with PBS, treated with 0.025% trypsin, and then the cell count for each dose was calculated using a cell counter. Cells were seeded into six-well plates according to radiation dose groups, for example: 0 Gy: 200 cells / well; 2 Gy: 300 cells / well; 4 Gy: 2000 cells / well; 6 Gy: 5000 cells / well; 8 Gy: 7000 cells / well; and 10 Gy: 10000 cells / well. 3 mL of DMEM containing 10% FBS was added to each well at 37°C and the cells were cultured for 14 days. After culture, the culture medium was removed. Cells were then fixed by reacting with 4% PFA at room temperature for 1 hour. After fixation, the PFA was removed. Next, cells were stained with 2% crystal violet for 30 minutes. After staining, cells were rinsed with water, and cell colony counts were performed to assess cell viability relative to the control group.

[0102] The results are shown in Figures 8A and 8B. The cell viability fraction of the cells treated with GNDs was significantly lower than that of the control group, indicating that GNDs enhanced the radiation killing effect of 10 MV photons and 160 MeV protons and increased the radiosensitivity of cells.

[0103] (2) Treatment of U87-MG human glioma cells: U87-MG human glioma cells were treated using the method described above for treating CT-2A mouse glioma cells. However, the irradiation doses were varied at 4, 6, 8, and 10 Gy, with 0 Gy equaling 2000 cells / well and 2 Gy equaling 2000 cells / well. After staining with crystal violet and washing with water, the crystal violet was dissolved using dimethyl sulfoxide (DMSO). The absorbance of each well was measured using a spectrophotometer (the absorbance was compared using the control group as 100%).

[0104] The results are shown in Figures 9A and 9B. At a radiation dose of 10 Gy, no significant inhibition of U87-MG cell growth was observed, indicating that this cell has high radioresistance. When combined with GNDs and 10 MV photons or 160 MeV protons, the proliferation of U87-MG cells was effectively inhibited, and GNDs showed a superior radiosensitizing effect compared to AuNPs. The above descriptions are merely some embodiments of this disclosure. Those skilled in the art will readily understand that various modifications and alterations can be made to each embodiment without departing from the teachings of this disclosure. Therefore, all equivalent changes and modifications made within the scope of the claims of this disclosure should fall within the scope of this disclosure.

[0105] none.

Claims

1. A method for manufacturing modified flower-shaped gold nanoparticles, comprising: A gold seed suspension and auric acid are added to a gelatin solution to form a mixture; a reducing agent is then added to the mixture. The mixture is further modified by adding ligands to form the flower-shaped gold nanoparticles. The ligand system includes thiol compounds, such as 3-mercaptopropionic acid and 6-mercapto-1-hexanol.

2. The method as described in claim 1, wherein, The auric acid is tetrachloroauric acid.

3. The method as described in claim 1, wherein, This gelatin solution includes type A gelatin.

4. The method as described in claim 1, wherein, The reducing agent is L-ascorbic acid.

5. The method as described in claim 1, wherein, The concentration ratio of the auric acid to the gelatin solution is 25 to 50.

6. The method as described in claim 1, wherein, Prior to the reaction, the molar ratio of the 3-mercaptopropionic acid to the 6-mercapto-1-hexanol was 1:20 to 20:

1.

7. The method as described in claim 6, wherein, The reaction takes between 30 and 90 minutes.

8. The method as described in claim 6, wherein, The reaction temperature is between 30°C and 45°C.

9. Use of the modified flower-shaped gold nanoparticles as described in claim 1 for the preparation of pharmaceutical compositions for treating cancer in individuals in need.

10. The use as described in claim 9, wherein, This cancer includes tumors with high levels of MMP-2 and / or MMP-9 expression.

11. The use as described in claim 10, wherein, Tumors with high levels of MMP-2 and / or MMP-9 expression include gliomas.

12. The use as described in claim 9, wherein, The treatment is selected from at least one of the group consisting of photothermal therapy, photon therapy, proton therapy, and heavy ion therapy.

13. The use as described in claim 9, wherein, The treatment is either photon therapy or proton therapy.

14. The use as described in claim 9, thereby enhancing tumor cell phagocytosis, increasing the tumor cell's sensitivity to radiation, and / or alleviating tumor microenvironment hypoxia.

15. The use as described in claim 14, wherein, The energy range of this radiation is 4 MV to 25 MV or 70 MeV to 250 MeV.

16. The use as described in claim 14, wherein, This radiation induces the production of reactive oxygen species.

17. The use as described in claim 14, wherein, The tumor cells were tumor cells with high levels of MMP-2 and / or MMP-9 expression.

18. The use as described in claim 17, wherein, Tumor cells with high levels of MMP-2 and / or MMP-9 are glioma cells.

Images