I-type photosensitizer of dye-modified semiconductor nanoparticles as well as preparation method and application of I-type photosensitizer

Abstract

The invention discloses an I-type photosensitizer of a dye-modified semiconductor nanoparticle as well as a preparation method and application of the I-type photosensitizer, the I-type photosensitizer comprises a semiconductor nanoparticle, a dye modified on the surface of the semiconductor nanoparticle and an organic reactant coated outside the semiconductor nanoparticle, the dye is modified on the surfaces of the semiconductor nanoparticles through carboxyl, sulfonic acid groups, phosphonic acid groups or pyridine groups. The I-type photosensitizer of the dye-modified semiconductor nanoparticles can be used for photodynamic therapy, and has a wide potential application prospect in the field of biomedicine, especially tumor treatment application; the synthesis method has the advantages of simplicity and easiness in operation, low raw material cost, easiness in product separation and the like, and the I-type photosensitizer is a nano-composite, is small in size, can absorb near-infrared light with high penetrability, is good in biocompatibility, has a wide potential application prospect in the field of biomedicine, and is suitable for industrial production. And the material can be applied to photodynamic therapy of near-infrared illumination at hypoxic tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and particularly to a type I photosensitizer of dye-modified semiconductor nanoparticles, a preparation method thereof, and an application thereof. Background Art

[0002] Photodynamic therapy is an emerging treatment modality that has attracted great research interest in the past few decades. Briefly, photodynamic therapy refers to the generation of reactive oxygen species by a photosensitizer under light irradiation to kill cells. Photodynamic therapy has the advantages of spatial and temporal selectivity, low systemic toxicity, minimal invasiveness, negligible drug resistance, and reduced long-term morbidity, improving the quality of life of patients. In the past few decades, with the approval of various photosensitizers in clinical trials, the clinical application of photodynamic therapy has increased significantly.

[0003] Photodynamic therapy has two mechanisms: type I photodynamic mechanism and type II photodynamic mechanism. Among them, the type I photodynamic mechanism refers to the generation of superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals by a photosensitizer after being photoexcited to kill cells. It should be noted that all of the above reactions can occur under hypoxic conditions, so the type I photodynamic mechanism is generally considered to have good hypoxic tolerance. Different from the type I photodynamic mechanism, the type II photodynamic mechanism mainly generates singlet oxygen, which then oxidizes biomolecules inside cells, triggering apoptosis to achieve the purpose of killing cells.

[0004] Compared with type I photosensitizers, the research on type II photosensitizers is more mature, and their synthesis routes and molecular designs have been highly standardized. The process of generating singlet oxygen through excited-state energy transfer to oxygen is easier to achieve in molecular design. Therefore, currently, most reported photodynamic therapy systems operate through the type II photodynamic mechanism. However, the therapeutic effect of traditional type II photodynamic therapy highly depends on the oxygen content level, so it can only be initiated under conditions of good oxygen concentration. However, due to insufficient oxygen supply and rapid tumor growth, the natural microenvironment of some solid tumors is hypoxic. Therefore, the hypoxic environment inside tumors will severely limit the efficacy of photodynamic therapy mainly based on the highly oxygen-dependent type II mechanism. Therefore, it is of great significance to research and develop type I photosensitizers with excellent performance. Summary of the Invention

[0005] The present invention provides a type I photosensitizer of dye-modified semiconductor nanoparticles, a preparation method thereof, and an application thereof to solve the above problems.

[0006] To achieve the above object, the technical solution of the present invention is:

[0007] A type-I photosensitizer of dye-modified semiconductor nanoparticles, comprising semiconductor nanoparticles, a dye modified on the surface of the semiconductor nanoparticles, and an organic reactant coated outside the semiconductor nanoparticles, wherein the dye is modified on the surface of the semiconductor nanoparticles through a carboxyl group, a sulfonic acid group, a phosphonic acid group or a pyridine group.

[0008] Further, the semiconductor nanoparticles are selected from any one of nano-zinc oxide, nano-titanium oxide, nano-cerium oxide, nano-aluminum oxide, nano-iron oxide, nano-copper oxide, nano-cuprous oxide, nano-manganese oxide, nano-nickel oxide, nano-tin oxide, nano-zinc sulfide, nano-copper sulfide, nano-barium titanate, nano-zinc titanate, and nano-copper titanate.

[0009] Further, the dye is a cyanine dye, a semi-cyanine dye, Nile blue, methylene blue, rhodamine or a fluorescein-based dye.

[0010] Further, the dye is a Cy7-COOH cyanine dye; the semiconductor nanoparticles are nano-zinc oxide; and the organic reactant is dopamine hydrochloride.

[0011] On the other hand, the present invention provides a preparation method of the type-I photosensitizer of the dye-modified semiconductor nanoparticles as described above, comprising the following steps:

[0012] S1: Add a cyanine dye and semiconductor nanoparticles into an organic solvent, stir overnight, centrifuge and wash, and obtain dye-modified semiconductor nanoparticles after drying;

[0013] S2: In a Tris buffer solution, add the dye-modified semiconductor nanoparticles of nano-zinc oxide modified with Cy7-COOH cyanine dye and dopamine hydrochloride, stir and react, centrifuge and wash, and obtain the type-I photosensitizer of the dye-modified semiconductor nanoparticles after drying.

[0014] Further, in S1, the mass ratio of the dye to the semiconductor nanoparticles is 1:10, and the volume ratio of the organic solvent to the mass of the dye is 4:1 (mL:mg);

[0015] The dye is a Cy7-COOH cyanine dye, the semiconductor nanoparticles are nano-zinc oxide, the dye-modified semiconductor nanoparticles are nano-zinc oxide modified with Cy7-COOH cyanine dye; the type-I photosensitizer is a nano-complex, and the organic solvent is acetonitrile.

[0016] Further, the specific steps of S2 are: in a 10 mM Tris buffer solution, add the nano-zinc oxide modified with Cy7-COOH cyanine dye and dopamine hydrochloride, stir and react for 3 h, centrifuge and wash twice, and obtain the type-I photosensitizer of the dye-modified semiconductor nanoparticles after drying;

[0017] The volume ratio of the Tris buffer to the Cy7-COOH cyanine dye-modified zinc oxide nanoparticles is 2:1 (mL:mg); the mass ratio of the Cy7-COOH cyanine dye-modified zinc oxide nanoparticles to the dopamine hydrochloride is 1:2.

[0018] Furthermore, the volume of the Tris buffer is 40 mL, the mass of the Cy7-COOH cyanine dye-modified zinc oxide nanoparticles is 20 mg, and the mass of the dopamine hydrochloride is 20 - 40 mg.

[0019] Another aspect of the present invention provides an application of the type I photosensitizer of the dye-modified semiconductor nanoparticles in a photodynamic product in the field of tumor treatment.

[0020] The beneficial effects of the present invention are as follows:

[0021] A type I photosensitizer of dye-modified semiconductor nanoparticles disclosed in the present invention, after the dye is modified onto the semiconductor nanoparticles, can generate a high concentration of type I reactive oxygen species and can be used as a type I photosensitizer for photodynamic therapy, having broad potential application prospects in the field of biomedicine, especially in the field of tumor treatment; at the same time, the synthesis method has the advantages of being simple and easy to operate, low raw material cost, and easy product separation. The prepared type I photosensitizer is a nanocomposite with a small size, can absorb near-infrared light with strong penetrability, has good biocompatibility, has broad potential application prospects in the field of biomedicine, and can be applied to photodynamic therapy with near-infrared light irradiation at hypoxic tumors. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 It is a mechanism diagram of using the dye-modified semiconductor nanoparticles as a type I photosensitizer;

[0024] Figure 2 It is a transmission electron microscopy image of the Cy7-COOH cyanine dye-modified zinc oxide nanoparticles and the nanocomposite, where: A is the transmission electron microscopy image of the Cy7-COOH cyanine dye-modified zinc oxide nanoparticles, and B is the transmission electron microscopy image of the nanocomposite;

[0025] Figure 3Absorption and emission spectra of Cy7-COOH cyanine dye-modified zinc oxide nanoparticles and nanocomposites, where A is the absorption spectrum of Cy7-COOH cyanine dye-modified zinc oxide nanoparticles and nanocomposites, and B is the emission spectrum of Cy7-COOH cyanine dye-modified zinc oxide nanoparticles and nanocomposites;

[0026] Figure 4 Test for the effect of generating type I reactive oxygen species after the same dye is modified onto different semiconductor nanoparticles;

[0027] Figure 5 Test results for the generation of type I reactive oxygen species by Cy7-COOH cyanine dye, zinc oxide nanoparticles, and Cy7-COOH cyanine dye-modified zinc oxide nanoparticles, where A is the test result of Cy7-COOH cyanine dye-modified zinc oxide nanoparticles, B is the test result of Cy7-COOH cyanine dye, and C is the test result of zinc oxide nanoparticles;

[0028] Figure 6 Electron paramagnetic resonance signal of Cy7-COOH cyanine dye-modified zinc oxide nanoparticles after illumination;

[0029] Figure 7 Detection of the time of cell uptake of nanocomposites;

[0030] Figure 8 Killing effect of nanocomposites at different concentrations on human breast cancer cells, where A is the experimental result under normoxic conditions and B is the experimental result under hypoxic conditions;

[0031] Figure 9 Experimental results of the imaging location and time guidance of the tumor location in mice by nanocomposites. Detailed implementation manners

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] The principle of this solution is as follows:

[0034] The Cy7-COOH cyanine dye is first chemically bonded to the surface of semiconductor nano-zinc oxide through an anchoring group (such as a carboxyl group, a phosphonic acid group, etc.) to enhance electron coupling and improve the electron injection efficiency. After the Cy7-COOH cyanine dye molecule absorbs photons, its electrons jump from the ground state (HOMO, the highest occupied molecular orbital) to the excited state (LUMO, the lowest unoccupied molecular orbital), forming an excited-state dye. At this time, the conduction band position of the semiconductor nano-zinc oxide needs to be lower than the LUMO energy level of the dye to form an energy gradient. The excited-state electrons are directly transferred to the conduction band of the semiconductor nano-zinc oxide through quantum tunneling or thermally assisted transition. The electron transfer between the Cy7-COOH cyanine dye and the semiconductor nano-zinc oxide increases the generation of type I ROS, such as Figure 1 as shown

[0035] Example:

[0036] A specific synthesis method for preparing Cy7-COOH cyanine dye-modified nano-zinc oxide (type I photosensitizer) for photodynamic therapy at hypoxic tumors includes the following steps:

[0037] S1: Add 5 mg of Cy7-COOH cyanine dye and 50 mg of nano-zinc oxide to 20 mL of acetonitrile, stir overnight, centrifuge and wash twice, and dry to obtain Cy7-COOH cyanine dye-modified nano-zinc oxide;

[0038] S2: Prepare a nano-complex: Take 40 mL of 10 mM Tris buffer, add 20 mg of Cy7-COOH cyanine dye-modified nano-zinc oxide and 40 mg of dopamine hydrochloride, stir and react for 3 h, centrifuge and wash twice, and dry to obtain a type I photosensitizer of cyanine dye-modified nano-zinc oxide.

[0039] Comparative example:

[0040] Use Cy7-COOH cyanine dye to modify different semiconductor nanoparticles (nano-copper oxide, nano-cerium oxide, nano-aluminum oxide, nano-iron oxide, nano-tin oxide, nano-zinc sulfide, nano-barium titanate, nano-nickel oxide, nano-manganese oxide) respectively. The parameters and steps are the same as those in the example to prepare type I photosensitizers of cyanine dye-modified nano-oxides. The ability of the obtained products to generate type I reactive oxygen species is detected using a dihydroethidium probe and compared with the type I photosensitizer prepared in the example;

[0041] Detection of the ability of type I reactive oxygen species:

[0042] Dihydroethidium is one of the most commonly used fluorescence detection probes for type I reactive oxygen species (superoxide anion). Dihydroethidium dehydrogenates under the action of superoxide anion to form ethidium bromide. Ethidium bromide can bind to RNA or DNA to produce red fluorescence at 610 nm. Therefore, the change in the fluorescence intensity at 610 nm can be used to calibrate the amount of superoxide anion generated in the system;

[0043] The specific experimental method is as follows: Add 3 mL of the solution of nanoparticles modified with the dye (100 μg / mL) into a cuvette, then add 6 μL of dihydroethidium solution (5 mM) and 600 μg of ctDNA, and irradiate with a laser at 808 nm and 50 mW / cm 2 . Therefore, an increase in the fluorescence intensity at 610 nm indicates that type I reactive oxygen species (superoxide anions) are generated in the system, and the ability to generate type I reactive oxygen species can be judged by comparing the amount of fluorescence increment at 610 nm;

[0044] The results are as Figure 4 shown. As Figure 4 can be seen, the type I reactive oxygen species of the nanocomposite (type I photosensitizer) prepared with zinc oxide nanoparticles are the highest, and it can efficiently generate superoxide anions, which means that in the field of photodynamic therapy, it can more effectively kill diseased cells and destroy the structure and function of tumor cells through the oxidation of biomolecules in cells by superoxide anions.

[0045] Performance test:

[0046] (1) Perform transmission electron microscopy analysis on the prepared nanocomposite:

[0047] Use a transmission electron microscope to analyze the morphology of the Cy7-COOH dye-modified zinc oxide nanoparticles and nanocomposites in the examples. Figure 2 These are the transmission electron micrographs of the Cy7-COOH cyanine dye-modified zinc oxide nanoparticles and nanocomposites. Figure 2 Figure A shows that the morphology of the Cy7-COOH dye-modified zinc oxide nanoparticles is irregular particles about 40 nm in size. Figure 2 Figure B shows that the thickness of the polydopamine layer of the nanocomposite is about 6 nm, and the morphology of the zinc oxide nanoparticles inside the polydopamine layer has not changed. Therefore, the nanocomposite prepared by this method will have an enhanced permeability and retention effect, be more likely to penetrate into tumor tissues and stay for a long time, thereby improving the effect of tumor treatment.

[0048] (2) Measure the absorption spectrum of the nanocomposite prepared in the example:

[0049] Measure the absorption spectrum of the nanocomposite prepared in the example with a UV-visible spectrophotometer. The results are as Figure 3 shown. Figure 3 These are the absorption and emission spectra of the Cy7-COOH cyanine dye-modified zinc oxide nanoparticles and nanocomposites, where Figure 3A is the absorption spectrum of Cy7-COOH cyanine dye-modified zinc oxide nanoparticles and nanocomposites. As can be seen from the figure, both Cy7-COOH cyanine dye-modified zinc oxide nanoparticles and nanocomposites have absorption at 808 nm, which can be used for subsequent treatment under 808 nm laser;

[0050] Figure 3 B is the emission spectrum of Cy7-COOH cyanine dye-modified zinc oxide nanoparticles and nanocomposites. As can be seen from the figure, the maximum emission wavelength of Cy7-COOH cyanine dye-modified zinc oxide nanoparticles and nanocomposites is 810 nm, indicating that the nanocomposite also has the function of fluorescence imaging.

[0051] (3) Test the effect of generating type I reactive oxygen species of the dye, nanoparticles and dye-modified nanoparticles respectively:

[0052] Use dihydroethidium probe to detect the effect of generating type I reactive oxygen species of the dye, nanoparticles and dye-modified nanoparticles prepared in the examples. The test method is the same as the detection method in Comparative Example 1. Figure 5 are the test results of Cy7-COOH cyanine dye, zinc oxide nanoparticles, and Cy7-COOH cyanine dye-modified zinc oxide nanoparticles. From Figure 5 it can be seen that zinc oxide nanoparticles do not generate type I reactive oxygen species after illumination, Cy7-COOH cyanine dye generates a small amount of type I reactive oxygen species after illumination, while Cy7-COOH cyanine dye-modified zinc oxide nanoparticles generate a large amount of type I reactive oxygen species after illumination.

[0053] (4) Electron paramagnetic resonance spectroscopy to verify the generated type I reactive oxygen species:

[0054] Use an electron paramagnetic resonance spectrometer to further verify the type I reactive oxygen species generated by the dye-modified nanoparticles prepared in the examples after illumination. DMPO is used as a scavenger in the experiment. From Figure 6 it can be seen that Cy7-COOH cyanine dye-modified zinc oxide nanoparticles will generate a strong superoxide anion signal (superoxide anion belongs to one of the type I reactive oxygen species) after illumination.

[0055] (5) Experiment on the uptake time of the nanocomposite prepared in the example by human breast cancer cells:

[0056] Add the nanocomposite prepared in the example to the culture medium of human breast cancer cells and co-incubate in a carbon dioxide incubator. Use an inverted fluorescence microscope to perform cell imaging at 0 min, 30 min, 60 min, 90 min, 120 min, and 180 min after co-incubation respectively. From Figure 7By showing the uptake of the nanocomposite by human breast cancer cells over time, it can be found that at 120 min, the uptake reaches the maximum value. Continuing the observation for another 60 min, the uptake hardly changes, indicating that the uptake time of the nanocomposite by human breast cancer cells can reach the highest amount in about 2 hours.

[0057] (6) Experiment on the killing effect of the nanocomposite prepared in the example on human breast cancer cells:

[0058] Mix the nanocomposite prepared in the example into the cell culture medium and use it for cell incubation in a carbon dioxide incubator. After 2 hours of incubation, remove the supernatant, wash twice with phosphate buffered saline solution, replace with fresh medium, and then perform light irradiation (808 nm, 600 mW / cm 2 , 10 min), and then place it back in the incubator for 24 hours. Then add the medium containing thiazolyl blue (0.5 mg / volume) and continue to incubate for 4 hours. After that, carefully take out the solution in the 96-well plate, dissolve the crystals with DMSO, measure the absorbance of each well with an enzyme-linked immunosorbent assay reader and calculate the cell survival rate. The results are as Figure 8 shown. It can be found that the survival rate of human breast cancer cells after treatment with the nanocomposite is about 20%, indicating that the nanocomposite has good phototoxic and cell killing effects on human breast cancer cells. The survival rate of cells without light irradiation is above 80%, indicating that in the absence of light, the nanocomposite has no significant adverse effects on the normal growth and metabolism of cells, and the nanocomposite has good biocompatibility.

[0059] (7) Experiment on guiding the tumor imaging position and time of the nanocomposite prepared in the example in mice:

[0060] Establish tumor implants by subcutaneously injecting 5×10 6 ~1×10 7 cells suspended in 100 μL PBS into mice. After 7 days, when the volume of the tumor reaches about 90 - 100 mm 3 , perform subsequent experiments on the tumor-bearing mice. Conduct in vivo experiments by injecting the nanocomposite via the tail vein. As Figure 9 can be seen, after 12 hours, under the excitation of 740 nm light, it can be observed that the nanocomposite accumulates at the tumor site to reach the maximum value, which can be used to accurately guide the time and position of near-infrared light irradiation.

[0061] (The mice in this experiment were purchased from the SPF Experimental Animal Center of Dalian Medical University. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The animal experiment protocol was approved by the local research ethics review committee, namely the Animal Ethics Committee of Dalian University of Technology (ethical approval number is 20220726).)

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type I photosensitizer of a dye-modified semiconductor nanoparticle, characterized in that, The type I photosensitizer includes semiconductor nanoparticles, a dye modified on the surface of the semiconductor nanoparticles, and an organic reactant coated outside the semiconductor nanoparticles, and the dye is modified on the surface of the semiconductor nanoparticles through a carboxyl group, a sulfonic acid group, a phosphonic acid group or a pyridine group.

2. The type I photosensitizer of the dye-modified semiconductor nanoparticles according to claim 1, wherein The semiconductor nanoparticles are selected from any one of nano zinc oxide, nano titanium oxide, nano cerium oxide, nano aluminum oxide, nano iron oxide, nano copper oxide, nano cuprous oxide, nano manganese oxide, nano nickel oxide, nano tin oxide, nano zinc sulfide, nano copper sulfide, nano barium titanate, nano zinc titanate, and nano copper titanate.

3. The type I photosensitizer of the dye-modified semiconductor nanoparticles according to claim 2, characterized in that, The dye is a cyanine dye, a semi-cyanine dye, Nile blue, methylene blue, rhodamine or a fluorescein dye.

4. The type I photosensitizer of the dye-modified semiconductor nanoparticles according to claim 3, characterized in that, The dye is a Cy7-COOH cyanine dye; the semiconductor nanoparticles are nano zinc oxide; the organic reactant is dopamine hydrochloride.

5. A method for preparing a type-I photosensitizer of dye-modified semiconductor nanoparticles according to claim 1, characterized in that, It includes the following steps: S1: Add a cyanine dye and semiconductor nanoparticles into an organic solvent, stir overnight, centrifuge and wash, and obtain dye-modified semiconductor nanoparticles after drying. S2: In a Tris buffer solution, add the dye-modified semiconductor nanoparticles of nano zinc oxide modified with Cy7-COOH cyanine dye and dopamine hydrochloride, stir and react, centrifuge and wash, and obtain a type I photosensitizer of dye-modified semiconductor nanoparticles after drying.

6. The preparation method of the type I photosensitizer of the dye-modified semiconductor nanoparticles according to claim 5, characterized in that, In S1, the mass ratio of the dye to the semiconductor nanoparticles is 1:10, and the volume ratio of the organic solvent to the mass of the dye is 4:1 (mL:mg). The dye is a Cy7-COOH cyanine dye, the semiconductor nanoparticles are nano zinc oxide, the dye-modified semiconductor nanoparticles are nano zinc oxide modified with Cy7-COOH cyanine dye; the type I photosensitizer is a nanocomposite, and the organic solvent is acetonitrile.

7. The preparation method of the type I photosensitizer of the dye-modified semiconductor nanoparticles according to claim 6, characterized in that, The specific steps of S2 are: In a 10 mM Tris buffer solution, add the nano zinc oxide modified with Cy7-COOH cyanine dye and dopamine hydrochloride, stir and react for 3 h, centrifuge and wash twice, and obtain a type I photosensitizer of dye-modified semiconductor nanoparticles after drying. The volume ratio of the Tris buffer solution to the nano zinc oxide modified with Cy7-COOH cyanine dye is 2:1 (mL:mg); the mass ratio of the nano zinc oxide modified with Cy7-COOH cyanine dye to the dopamine hydrochloride is 1:

2.

8. Application of the type I photosensitizer of the dye-modified semiconductor nanoparticles according to claim 1 in a photodynamic product in the field of tumor treatment.

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