An oxygen-enhanced pH-responsive magnetic targeting photosensitizer delivery system, a preparation method and application thereof

Abstract

The application discloses an oxygen-enhanced pH-responsive magnetic targeting photosensitizer delivery system, which is recorded as Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp, and the delivery system takes the superparamagnetic Fe3O4 core as a magnetic targeting matrix, is covered with mesoporous silica outside, and is crosslinked with polyethylene glycol-polyaspartic acid after being modified by amino on the surface of the mesoporous silica, so that the loading of the photosensitizer is realized. The oxygen-enhanced pH-responsive magnetic targeting photosensitizer delivery system provided by the application realizes the magnetic targeting property and the pH responsiveness, and realizes the effect of increasing the concentration of intracellular reactive oxygen species.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery technology, specifically, it relates to an oxygen-enhanced pH-responsive magnetic targeting photosensitizer delivery system, its preparation method, and its application. Background Technology

[0002] Photodynamic therapy (PDT) is a novel approach to cancer treatment that emerged in the 20th century. After more than 40 years of development, it has gradually become a new treatment for cancer following chemotherapy, surgery, and radiotherapy. However, some problems still exist in its application. The main issues are that photosensitizers are difficult to accumulate efficiently at the tumor site, making them prone to strong phototoxic side effects; at the same time, the tumor site is prone to forming a hypoxic environment, which limits the scope of application of PDT.

[0003] In recent years, magnetic nanomaterials have attracted attention due to their unique physical properties and applications in catalysts, magnetic resonance imaging, and controlled drug release. Magnetic iron tetroxide (Fe3O4) nanoparticles possess excellent biocompatibility and magnetic properties, and are widely used in magnetic resonance imaging and targeted drug delivery.

[0004] Mesoporous silica (mSiO2) nanoparticles not only possess excellent biostability and biocompatibility, but also exhibit high pore volume and specific surface area, unique pore structures, and easily modifiable internal and external surfaces. Due to their favorable structural characteristics, they can serve as highly efficient carriers for hydrophobic photosensitizers, and their pore structure also enhances the permeability of oxygen molecules to singlet oxygen. Currently, they are widely used in biomedical fields such as bioimaging, biosensing, drug delivery, and photodynamic therapy. Summary of the Invention

[0005] To address the technical problems of poor targeting of existing photosensitizers and the lack of oxygen in tumor tissue leading to poor photodynamic effects, the present invention aims to provide an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system to improve the therapeutic effect of photodynamic therapy on tumors.

[0006] A second objective of this invention is to provide a method for preparing the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system.

[0007] A third objective of this invention is to provide an application of the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system in the preparation of photosensitizer delivery systems.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system, denoted as Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp. This delivery system uses a superparamagnetic Fe3O4 core as a magnetically targeted matrix, which is covered with mesoporous silica (mSiO2). After the surface of the mesoporous silica is aminated, it is cross-linked with polyethylene glycol-polyaspartic acid (PEG-b-PAsp) to achieve loading of photosensitizer (PS).

[0010] The oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system has a particle size of 100–200 nm and exhibits good particle size distribution.

[0011] The oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system provided by this invention uses a superparamagnetic Fe3O4 core as the magnetic targeting matrix, enabling the material to perform magnetic targeting. The outer layer of mesoporous silica (mSiO2) protects the magnetic core and adsorbs the oxygen-generating catalyst MnO2, which increases the concentration of reactive oxygen species within tumor cells. Simultaneously, after amylation modification of the mSiO2 surface, a cross-linked pH-responsive polymer, polyethylene glycol-polyaspartic acid (PEG-b-PAsp), is used to load and release the poorly soluble photosensitizer PS in a pH-responsive manner, achieving targeted drug delivery. Compared to single photosensitizers, this oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system exhibits excellent magnetic targeting and pH responsiveness, significantly reduced toxicity, effectively increases cellular oxygen production, and enhances the killing effect on tumor cells.

[0012] The photosensitizer is selected from at least one of pyrophoric acid A, temoporphyrin, dihydroporphyrin e6, etc., all of which are commercially available drugs.

[0013] The molecular weight of the polyethylene glycol-polyaspartic acid (PEG-b-PAsp) is 15000 Da.

[0014] A second aspect of the present invention provides a method for preparing the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system, comprising the following steps:

[0015] Step 1: Preparation of Fe3O4@mSiO2 nanocomposites

[0016] H2O was added to a Fe3O4 ethanol dispersion with a concentration of 20-100 mg / mL and ultrasonically dispersed. The volume ratio of Fe3O4 ethanol dispersion to H2O was 1:(3-10) (preferably 1:4). CTAB solution was then added and stirred for 5-15 min to mix evenly. Ammonia water was then added and stirred. TEOS ethanol solution was added dropwise. The mass ratio of Fe3O4, CTAB, ammonia water, and TEOS was (0.1-1):1:(1-5):(2-10) (preferably 0.25:1:1.82:4.67). The reaction was carried out at a temperature of 75-85℃ for 1-3 h. The mixture was separated by magnetism and washed three times each with anhydrous ethanol and deionized water. The precipitate was redispersed in anhydrous ethanol and refluxed for 1-24 h. The mixture was separated by magnetism and washed three times each with anhydrous ethanol and deionized water. The mixture was then vacuum dried to obtain Fe3O4@mSiO2 nanocomposite.

[0017] In the first step, the TEOS ethanol solution is prepared by dissolving TEOS (tetraethyl orthosilicate) in anhydrous ethanol to obtain a TEOS ethanol solution with a concentration of 0.1-1 g / mL (preferably 0.23 g / mL).

[0018] In the first step, the CTAB solution is prepared by dissolving CTAB (hexadecyltrimethylammonium bromide) in water and anhydrous ethanol at a volume ratio of 1:1 to obtain a CTAB solution with a concentration of 0.01-0.1 g / mL (preferably 0.025 g / mL).

[0019] The second step is the preparation of Fe3O4@mSiO2@MnO2.

[0020] The Fe3O4@mSiO2 prepared in the first step was dispersed in deionized water to prepare a solution with a concentration of 5-15 mg / mL (preferably 10 mg / mL). The solution was stirred at room temperature for 5-150 min, and MnCl2-4H2O was added. The mass ratio of Fe3O4@mSiO2 to MnCl2-4H2O was (6-10):1 (preferably 8.3:1). NaOH solution was added dropwise to adjust the pH to 9. The reaction was stirred for 10-60 min, separated by a magnet, and the precipitate was washed with deionized water and dried under vacuum to obtain Fe3O4@mSiO2@MnO2.

[0021] The concentration of the NaOH solution is 0.5–2 mol / L.

[0022] The third step is the amylation modification of Fe3O4@mSiO2@MnO2.

[0023] The Fe3O4@mSiO2@MnO2 obtained in the second step was added to an APTES ((3-aminopropyl)triethoxysilane) ethanol solution, with a mass ratio of Fe3O4@mSiO2@MnO2 to APTES of 1:(0.1-2) (preferably 1:0.47). The mixture was stirred at room temperature for 1-14 hours, separated by magnetism, and the precipitate was washed with deionized water and dried under vacuum to obtain the aminated product Fe3O4@mSiO2@MnO2-NH2.

[0024] Step 4: Synthesis of the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp

[0025] The Fe3O4@mSiO2@MnO2-NH2 prepared in step 3 was dissolved in deionized water and ultrasonically dispersed. The mixture was stirred for 10–40 min, then EDC·HCl ((1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide (NHS) were added, and the mixture was stirred for 5–20 min. PEG-b-PAsp (polyethylene glycol-polyaspartic acid) was then added, and the mixture was stirred for 1–6 h. Finally, a THF solution containing a photosensitizer was added, and the mixture was stirred for 5–15 min. The mass ratio of @mSiO2@MnO2-NH2, EDC·HCl, NHS, PEG-b-PAsp, and photosensitizer is 1:(0.1~0.5):(0.05~0.2):(0.5~1.5):(0.05~0.2) (preferably 1:0.33:0.1:0.83:0.083). The precipitate is separated by magnetization, ultrasonically washed with deionized water, and freeze-dried to obtain Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp.

[0026] In the fourth step, the concentration of Fe3O4@mSiO2@MnO2-NH2 prepared in the third step dissolved in deionized water is 1-10 mg / mL (preferably 2.4 mg / mL).

[0027] A third aspect of the present invention provides the application of the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system in the preparation of photosensitizer delivery systems.

[0028] The oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system of the present invention utilizes the porous properties of the mesoporous silica layer prepared in the composite material to achieve loading and delivery of photosensitizer.

[0029] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0030] The oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system provided by this invention was tested using dynamic light scattering and high-resolution transmission electron microscopy. The results showed that the delivery system possesses excellent particle size distribution and uniform pores, confirming its effective loading of photosensitizer. UV-Vis absorption experiments confirmed the system's good pH responsiveness to photosensitizer release. Magnetic experiments demonstrated its superior magnetic targeting. Laser confocal microscopy showed the composite material exhibits high targeting efficiency and potent killing effect on tumor cells. Cellular ROS detection experiments indicated that the material effectively generates reactive oxygen species, thus killing cells. In summary, the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system provided by this invention achieves both magnetic targeting and pH responsiveness, and increases intracellular reactive oxygen species concentration, significantly improving the photosensitizer's killing effect on human non-small cell lung cancer cells A549. This provides new insights for the improvement and innovation of future clinical photodynamic therapy. Attached Figure Description

[0031] Figure 1 High-resolution transmission electron microscopy image of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system.

[0032] Figure 2 This is a particle size distribution diagram of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system.

[0033] Figure 3 This is a schematic diagram of the magnetic targeting results of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system.

[0034] Figure 4 This is a schematic diagram of the pH responsiveness of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system.

[0035] Figure 5 This is a schematic diagram of the cellular uptake results of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system.

[0036] Figure 6 This is a schematic diagram of the cytotoxicity detection results for an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system.

[0037] Figure 7 This is a schematic diagram illustrating the apoptosis effect of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system on cells.

[0038] Figure 8 This is a schematic diagram of the reactive oxygen species detection results for an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system. Detailed Implementation

[0039] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] A method for preparing the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system includes the following steps:

[0042] Step 1: Preparation of Fe3O4@mSiO2 nanocomposites

[0043] Fe3O4@mSiO2 nanocomposites were prepared using the sol-gel method: 5 mL of a 50 mg / mL Fe3O4 ethanol dispersion was added to a three-necked flask, followed by 20 mL of H2O and ultrasonic dispersion. Then, 1 g of CTAB (hexadecyltrimethylammonium bromide) dissolved in 20 mL of water and 20 mL of anhydrous ethanol was added, and the mixture was stirred for 10 min to achieve homogeneity. 2 mL of ammonia was then added, and the mixture was stirred. TEOS (tetraethyl orthosilicate) ethanol solution (5 mL of TEOS dissolved in 15 mL of anhydrous ethanol) was added dropwise. The mixture was reacted in an oil bath at 80 °C for 2 h, separated by magnetic separation, and washed three times each with anhydrous ethanol and deionized water. The precipitate was then redispersed in 60 mL of anhydrous ethanol and refluxed for 24 h. After magnetic separation, the precipitate was washed three times each with anhydrous ethanol and deionized water, and then dried under vacuum to obtain the Fe3O4@mSiO2 nanocomposites.

[0044] The second step is the preparation of Fe3O4@mSiO2@MnO2.

[0045] The 500 mg Fe3O4@mSiO2 prepared in the first step was dispersed in 50 mL of deionized water and stirred at room temperature. After 10 min, 0.06 g MnCl2-4H2O was added, followed by dropwise addition of 20 mL of 1 mol / L NaOH. The pH was adjusted to 9, and the reaction was stirred for 50 min. The precipitate was separated by a magnet, washed three times with deionized water, and dried under vacuum to obtain Fe3O4@mSiO2@MnO2.

[0046] The third step is the amylation modification of Fe3O4@mSiO2@MnO2.

[0047] 100 mg of Fe3O4@mSiO2@MnO2 obtained in the second step was added to an APTES ethanol solution (50 μl APTES ((3-aminopropyl)triethoxysilane) was added to 30 mL of anhydrous ethanol), stirred at room temperature for 12 h, separated by magnetism, the precipitate was washed with deionized water and dried under vacuum to obtain the aminated product Fe3O4@mSiO2@MnO2-NH2;

[0048] Step 4: Synthesis of the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp

[0049] 60 mg of Fe3O4@mSiO2@MnO2-NH2 prepared in step 3 was added to a round-bottom flask, followed by 25 mL of deionized water. The mixture was ultrasonically dispersed and stirred for 30 min. Then, 20 mg of EDC·HCl ((1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 6 mg of N-hydroxysuccinimide (NHS) were added, and the mixture was stirred for 10 min. Next, 50 mg of PEG-b-PAsp (polyethylene glycol-polyaspartic acid, molecular weight 15000 Da) was added, and the mixture was stirred for 4 h. Finally, 2.5 mL of THF solution containing 5 mg of photosensitizer (pyromellitic chlorophyll A) was added, and the mixture was stirred for 10 min. The mixture was then separated by magnetic separation, and the precipitate was ultrasonically washed with deionized water and freeze-dried to obtain Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp. Transmission electron microscopy and dynamic light scattering experiments confirmed the successful synthesis of this product.

[0050] The oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system prepared in this invention is denoted as Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp. This delivery system uses a superparamagnetic Fe3O4 core as a magnetic targeting matrix, which is covered with mesoporous silica (mSiO2). After the surface of the mesoporous silica is modified with amino group, it is cross-linked with polyethylene glycol-polyaspartic acid (PEG-b-PAsp) to achieve loading of photosensitizer (PS).

[0051] The oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp (referred to as the composite material) prepared in this embodiment was observed by high-resolution transmission electron microscopy as follows: Figure 1 As shown, Figure 1 High-resolution transmission electron microscopy image of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system. Figure 1 In the middle, 'a' is Fe3O4 with a particle size of about 100nm and is uniformly dispersed. Figure 1 In the middle, b represents Fe3O4@mSiO2 encapsulated with mesoporous silicon, with a particle size of approximately 130 nm. Figure 1 In the image, c represents manganese dioxide-coated nanoparticles, Fe3O4@mSiO2@MnO2. From... Figure 1 As can be clearly seen in image c, sheet-like black manganese dioxide nanoparticles are attached to the surface of the silicon layer.

[0052] Figure 2 This is a particle size distribution diagram of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system. Figure 2As can be seen from the data, the particle size of the delivery system measured by the Malvern laser particle size analyzer is 190 nm, indicating that the material has a good particle size distribution.

[0053] Figure 3 This is a schematic diagram of the magnetic targeting results of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system, demonstrating that the material has good magnetic targeting properties.

[0054] Figure 4 This is a schematic diagram of the pH responsiveness of the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system, showing that the material has good pH responsiveness to the release of photosensitizer.

[0055] The pH-responsive drug release rate of this delivery system was measured to be 89.2%, and it also exhibited good magnetic targeting properties.

[0056] The present invention conducted dynamic light scattering and high-resolution transmission electron microscopy experiments on the delivery system, which showed that the delivery system of the present invention has good particle size distribution and uniform pores, confirming the effective loading of photosensitizers; the ultraviolet-visible absorption experiment confirmed that the delivery system has good pH responsiveness to photosensitizer release; the magnetic test showed that the delivery system has better magnetic targeting.

[0057] Example 2

[0058] Cellular uptake experiment of oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system

[0059] Human non-small cell lung cancer cells were selected, specifically A549 cells in the logarithmic growth phase, at a cell concentration of 1×10⁻⁶. 5 / mL was inoculated into 2mL culture dishes and cultured for 12h. The culture medium was discarded, and 5μM of the composite material and 5μM of free photosensitizer were added. The culture was continued for 2h. The culture medium was discarded, and the sample was washed three times with PBS. Lysosomal green fluorescent probe was added for staining for 45min. The culture medium was discarded, and the sample was washed three times with PBS. Then 1mL of PBS was added and the sample was observed using a laser confocal microscope.

[0060] The results are as follows Figure 5 As shown, Figure 5 This diagram illustrates the cellular uptake results of the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system. The results show that the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system can be effectively taken up by lysosomes and enter cells. The laser confocal microscopy experiments provided by this invention demonstrate that the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system has high targeting efficiency and high killing effect on tumor cells. Figure 5 In the diagram, a is a bright-field plot, b is the overlap of lysosomal staining and material, c is lysosomal staining, and d is the composite material itself (i.e., the oxygen-enhanced pH-responsive magnetic-targeted photosensitizer delivery system). This indicates that the oxygen-enhanced pH-responsive magnetic-targeted photosensitizer delivery system can be taken up by lysosomes and enter the cell.

[0061] Example 3

[0062] Cytotoxicity assays of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system

[0063] Human non-small cell lung cancer cells were selected, specifically A549 cells in the logarithmic growth phase, at a cell concentration of 5 × 10⁻⁶. 4 Inoculate with 1 / mL of the culture medium into two 96-well plates and incubate for 24 h. Discard the culture medium. For the light-controlled group, add different concentration gradients of free Pha (pyromellitic chlorophyll A) (1.00 μg / mL, 0.50 μg / mL, 0.25 μg / mL, 0.13 μg / mL, 0.06 μg / mL, 0.03 μg / mL) and blank material and composite material equivalent to the free photosensitizer concentration. For the dark-controlled group, add free Pha (5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.62 μg / mL, 0.31 μg / mL, 0.15 μg / mL) and blank material and composite material equivalent to the free photosensitizer concentration. Three subwells are set for each gradient. Incubate in the dark for 24 h. Discard the culture medium, wash three times with PBS, and add 100 μL of complete culture medium. For the light-controlled group, use a wavelength λ = 660 nm and a power density P = 200 mW / cm². 2 After irradiation with a laser for 400 s, the samples were cultured in the dark for 24 h. The group in the dark was washed three times with PBS and then cultured for another 24 h with 100 μL of complete culture medium. Both groups were then cultured in the dark with 100 μL of serum-free culture medium containing 10% CCK-8 for another 2 h, and the absorbance of each well at 450 nm was measured using a microplate reader.

[0064] The results are as follows Figure 6 As shown, Figure 6 This is a schematic diagram showing the cytotoxicity test results of the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system. The results indicate that the composite material's tumor cell-killing effect is eight times that of the free photosensitizer and twice that of Fe3O4@mSiO2@PS@PEG-b-PAsp, demonstrating a strong tumor cell-killing ability while also showcasing the safety of Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp. Figure 6 Through a comparison of experiments in the light-shielded group and the light-illuminated group, it was found that the light-illuminated group could effectively kill tumor cells, while the light-shielded group experiment showed that the material was safe under the same drug dosage conditions as the light-illuminated group. Figure 6 The data shows that the photosensitizer is pyromethiazolinone A, and the composite material is an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system.

[0065] Example 4

[0066] Apoptosis experiments using an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system

[0067] Human non-small cell lung cancer cells were selected, specifically A549 cells in the logarithmic growth phase, at a cell concentration of 2 × 10⁻⁶. 5 / mL was seeded into two 6-well plates and cultured for 24 h. The culture medium was discarded, and three concentrations of photosensitizer (pyromellitic chlorophyll A) and corresponding concentrations of composite material were added, respectively. A blank cell group was also set up and cultured in the dark for 24 h. The culture medium was discarded, and the cells were washed three times with PBS. 2 mL of complete culture medium was added, and the light group was controlled at a wavelength λ = 660 nm and a power density P = 200 mW / cm². 2 After 400 seconds of laser irradiation (total light intensity 10 J / cm²), 2 Cells were cultured in the dark for 24 hours. The dark-protected group was washed three times with PBS, and 2 mL of complete culture medium was added for further culture for 24 hours. The Annexin V-FITC / PI apoptosis detection kit was added to the cells, and the cells were treated in the dark for 10 minutes, vortexed for 30 seconds, and then detected by flow cytometry.

[0068] The results are as follows Figure 7 As shown, Figure 7 This diagram illustrates the effect of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system on cell apoptosis. The results show a significant difference in tumor cell killing ability between the light-exposed group and the dark-protected group, indicating that the composite material enhances the anti-tumor effect of the drug. Figure 7 The top-middle image shows the results of the light-exposed group, and the bottom image shows the results of the apoptosis experiment in the dark-protected group. The light-exposed group showed significantly stronger tumor cell killing ability than the dark-protected group, indicating that the composite material enhanced the anti-tumor effect of the drug.

[0069] Example 5

[0070] Intracellular reactive oxygen species (ROS) detection in an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system

[0071] Human non-small cell lung cancer cells were selected, specifically A549 cells in the logarithmic growth phase, at a cell concentration of 3 × 10⁻⁶. 5 / mL was seeded into a 6-well plate and cultured for 24 h. The culture medium was discarded, and three concentrations of manganese dioxide-free composite material (high, medium, and low) were added to each well, along with a blank cell group. Cells were cultured in the dark for another 24 h. The culture medium was discarded, and the cells were washed three times with PBS to remove unabsorbed drugs. 2 mL of DCFH-DA reactive oxygen species detection reagent (DCFH-DA: culture medium 1:2000) was added to each well. The assay was performed at a wavelength λ = 660 nm and a power density P = 200 mW / cm². 2 The cells were irradiated with laser for 400 seconds, then cultured in an incubator for 20 minutes before being detected by flow cytometry.

[0072] The results are as follows Figure 8 As shown, Figure 8This is a schematic diagram showing the reactive oxygen species (ROS) detection results of an oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system. The results indicate that the ROS content generated by the composite material is significantly higher than that of the composite material without manganese dioxide, demonstrating that the composite material possesses strong oxygen-enhancing capabilities, achieving the expected experimental results. Figure 8 The content of reactive oxygen species (ROS) within cells is reflected by cell viability. ROS can induce apoptosis, thereby killing tumor cells. The ROS production of the composite material is significantly higher than that of the composite material without manganese dioxide. The cell ROS detection experiment provided by this invention shows that the composite material can effectively generate ROS and play a role in killing cells.

[0073] The oxygen-enhanced pH-responsive magnetic-targeting photosensitizer delivery system of the present invention achieves both magnetic targeting and pH responsiveness, and increases the concentration of reactive oxygen species in cells, greatly improving the killing effect of photosensitizers on human non-small cell lung cancer cells A549, and providing new ideas for the improvement and innovation of future clinical photodynamic therapy.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A preparation method of an oxygen-augmented pH-responsive magnetic-targeting photosensitizer delivery system, characterized in that, Includes the following steps: Step 1: Preparation of Fe3O4@mSiO2 nanocomposites H2O was added to a Fe3O4 ethanol dispersion with a concentration of 20-100 mg / mL and ultrasonically dispersed. The volume ratio of Fe3O4 ethanol dispersion to H2O was 1:(3-10). CTAB solution was then added and stirred for 5-15 min to mix evenly. Ammonia water was then added and stirred. TEOS ethanol solution was added dropwise. The mass ratio of Fe3O4, CTAB, ammonia water, and TEOS was (0.1-1):1:(1-5):(2-10). The reaction was carried out at a temperature of 75-85℃ for 1-3 h. The precipitate was separated by magnetism and washed three times each with anhydrous ethanol and deionized water. The precipitate was then redispersed in anhydrous ethanol and refluxed for 1-24 h. The precipitate was separated by magnetism and washed three times each with anhydrous ethanol and deionized water. The precipitate was then vacuum dried to obtain Fe3O4@mSiO2 nanocomposite. The second step is the preparation of Fe3O4@mSiO2@MnO2. The Fe3O4@mSiO2 prepared in the first step was dispersed in deionized water to prepare a solution with a concentration of 5-15 mg / mL. The solution was stirred at room temperature for 5-150 min. MnCl2-4H2O was added, with the mass ratio of Fe3O4@mSiO2 to MnCl2-4H2O being (6-10):

1. NaOH solution was added dropwise to adjust the pH to 9. The reaction was stirred for 10-60 min. The precipitate was separated by a magnet, washed with deionized water, and dried under vacuum to obtain Fe3O4@mSiO2@MnO2. The third step is the amylation modification of Fe3O4@mSiO2@MnO2. The Fe3O4@mSiO2@MnO2 obtained in the second step was added to the APTES ethanol solution, with the mass ratio of Fe3O4@mSiO2@MnO2 to APTES being 1:(0.1~2). The mixture was stirred at room temperature for 1~14h, separated by magnetism, and the precipitate was washed with deionized water and dried under vacuum to obtain the aminated product Fe3O4@mSiO2@MnO2-NH2. Step 4: Synthesis of the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp The Fe3O4@mSiO2@MnO2-NH2 prepared in step 3 was dissolved in deionized water and ultrasonically dispersed. After stirring for 10–40 min, EDC·HCl and N-hydroxysuccinimide NHS were added and stirred for 5–20 min. PEG-b-PAsp was then added and stirred for 1–6 h. Finally, a THF solution containing photosensitizer was added and stirred for 5–15 min. The mass ratio of Fe3O4@mSiO2@MnO2-NH2, EDC·HCl, NHS, PEG-b-PAsp, and photosensitizer was 1:(0.1–0.5):(0.05–0.2):(0.5–1.5):(0.05–0.2). The mixture was separated by magnetization, and the precipitate was ultrasonically washed with deionized water and freeze-dried to obtain Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp. The oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system is denoted as Fe3O4@mSiO2@MnO2@PS@PEG-b-PAsp. This delivery system uses a superparamagnetic Fe3O4 core as a magnetically targeted matrix, which is covered with mesoporous silica. After the surface of the mesoporous silica is modified by amylation, it is cross-linked with polyethylene glycol-polyaspartic acid to achieve the loading of photosensitizer. The photosensitizer is selected from at least one of pyrophoric acid A, temoporphyrin, and dihydroporphyrin e6.

2. The method for preparing the oxygen-augmented pH-responsive magnetic-targeting photosensitizer delivery system according to claim 1, wherein, In the first step, the preparation of TEOS ethanol solution involves dissolving TEOS in anhydrous ethanol to obtain a TEOS ethanol solution with a concentration of 0.1–1 g / mL.

3. The method for preparing the oxygen-augmented pH-responsive magnetic-targeting photosensitizer delivery system according to claim 1, characterized in that, In the first step, the CTAB solution is prepared by dissolving CTAB in water and anhydrous ethanol at a volume ratio of 1:1 to obtain a CTAB solution with a concentration of 0.01 to 0.1 g / mL.

4. The method for preparing the oxygen-augmented pH-responsive magnetic-targeting photosensitizer delivery system according to claim 1, characterized in that, In the fourth step, the concentration of Fe3O4@mSiO2@MnO2-NH2 prepared in the third step dissolved in deionized water is 1-10 mg / mL.

5. The method for preparing the oxygen-augmented pH-responsive magnetic-targeting photosensitizer delivery system according to claim 1, wherein, The particle size of the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system is 100–200 nm.

6. The method for preparing the oxygen-augmented pH-responsive magnetic-targeting photosensitizer delivery system according to claim 1, wherein, The molecular weight of the polyethylene glycol-polyaspartic acid is 15000 Da.

7. The application of the oxygen-enhanced pH-responsive magnetically targeted photosensitizer delivery system prepared by the method of any one of claims 1 to 6 in the preparation of photosensitizer delivery systems.

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