A phosphor phthalocyanine@AIPH@siRNA composite material, a preparation method and application thereof

Abstract

The application discloses a kind of phosphor phthalocyanine AIPH siRNA composite material, its preparation method and application, comprising: the chloroform solution of phosphor phthalocyanine (PPc), SDS solution, CTAB solution, 2,2-azo [2- (2-imidazoline-2-yl) propane] dihydrochloride (AIPH) and siRNA solution;Preparation of PPc NPs AIPH nanoparticle of AIPH molecule loading, add appropriate amount of water dispersion, obtain the water dispersion of PPc NPs AIPH nanoparticle;SiRNA solution is added to SDS solution, then add the water dispersion of PPc NPs AIPH nanoparticle, and stirring at room temperature, the obtained precipitate is centrifuged and separated, and it is PPc NPs AIPH siRNA composite material.The method can effectively destroy HSP70 protein structure due to the introduction of AIPH;The introduction of siRNA can effectively inhibit the overexpression of HSP70, and the three efficient synergistic promotion near-infrared second zone photothermal treatment effect.The method is simple, efficient, and can be prepared in large quantities, and finally used in near-infrared second zone photothermal treatment and siRNA delivery and efficient treatment of tumor drugs, with good photothermal performance, less dosage and other obvious advantages.

Description

Technical Field

[0001] This invention belongs to the fields of materials chemistry, organic supramolecular chemistry, and biomedicine and nanomedicine, specifically relating to a phosphorophthalocyanine@AIPH@siRNA composite material, its preparation method, and its application. Background Technology

[0002] With the rapid pace of social development, the incidence and mortality rates of cancer continue to rise. Currently, clinical cancer treatment mainly relies on surgery, chemotherapy, and radiotherapy; however, these methods all have unavoidable side effects in practical applications. How to achieve efficient and precise treatment has become an urgent problem to be solved in cancer treatment. Near-infrared photothermal therapy produces good therapeutic effects through local overheating, and has advantages such as high efficiency, simplicity, deep tissue penetration, and non-invasiveness. However, tumor cells produce a large amount of heat shock protein (HSP70), which weakens the therapeutic effect, and increasing the dosage easily leads to drug resistance. Developing a method to inhibit HSP70 protein, thereby enhancing the effect of photothermal therapy, is key in this field. Traditional inhibition methods target microRNAs (miRNAs) and small interfering RNAs (siRNAs) directly. However, this method leads to the degradation of miRNAs and siRNAs by nucleases within cells. Therefore, developing a nanomaterial with good photothermal properties and high siRNA delivery efficiency is a research challenge in this field.

[0003] Phthalocyanines are compounds with a large π-conjugated system, possessing diverse substituents, large cyclic structures, strong rigidity, excellent optical and electronic properties, outstanding chemical stability, and good biocompatibility. However, they are highly hydrophobic and easily aggregate in physiological environments. 2,2-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (AIPH) decomposes upon heating to generate hydroxyl radicals (·OH), which can disrupt the structure of the HSP70 protein. siRNA can silence the corresponding gene and inhibit the expression of the corresponding protein by directly pairing with the corresponding mRNA in the cell, thus synergistically promoting the photothermal therapy effect. However, how to integrate these three compounds, maintain good performance and high siRNA delivery efficiency, and endow it with excellent tumor therapeutic properties remains a research challenge. Summary of the Invention

[0004] The purpose of this invention is to provide a phosphorophthalocyanine@AIPH@siRNA composite material, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a phosphorophthalocyanine@AIPH@siRNA composite material includes the following steps: 1) Prepare chloroform solution, SDS aqueous solution, CTAB aqueous solution, 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) aqueous solution and small interfering RNA (siRNA) aqueous solution respectively, wherein the concentrations of SDS aqueous solution and CTAB aqueous solution are the same; 2) Under stirring at room temperature, add the chloroform solution of phosphorophthalocyanine in step 1) to the SDS aqueous solution, sonicate with a probe for 1-3 min, and then stir the sonicated mixture in a 62-70℃ water bath until the chloroform is completely evaporated. After that, transfer it to an ice water bath to cool to room temperature, centrifuge to separate, and add an appropriate amount of water to the precipitate to disperse it, thus obtaining an aqueous dispersion of phosphorophthalocyanine nanoparticles (PPc NPs). 3) Under stirring at room temperature, add the AIPH aqueous solution from step 1) to the CTAB aqueous solution to obtain the AIPH CTAB solution, then add the aqueous dispersion of phosphorophthalocyanine nanoparticles from step 2), and continue stirring at room temperature for 5-10 h. After centrifugation, the precipitate obtained is phosphorophthalocyanine@AIPH nanoparticles loaded with AIPH molecules. Add an appropriate amount of water to the precipitate to disperse it and obtain the aqueous dispersion of phosphorophthalocyanine@AIPH nanoparticles. 4) Under stirring at room temperature, add the siRNA aqueous solution from step 1) to the SDS aqueous solution to obtain the siRNA SDS solution, then add the aqueous dispersion of phosphorophthalocyanine@AIPH nanoparticles from step 3), and stir at room temperature for 5-10 h. The precipitate obtained by centrifugation is the phosphorophthalocyanine@AIPH@siRNA composite material.

[0006] Further, in step 1), the phosphorophthalocyanine is specifically 1,4,8,11,15,18,22,25-octa(p-methoxyphenylthio)-29H,31H-phthalocyanine phosphorus, with a chloroform solution concentration of 10~20 mg / mL; the concentrations of the SDS aqueous solution and CTAB aqueous solution are 0.015 mol / L~0.025 mol / L, the concentration of the AIPH aqueous solution is 5 mg / mL~20 mg / mL, and the concentration of the siRNA aqueous solution is 4 OD / mL~8 OD / mL.

[0007] Further, in step 2), the volume ratio of the chloroform solution of phosphophthalocyanine to the aqueous solution of SDS is 1:(8~10).

[0008] Further, in step 3), the volume ratio of AIPH aqueous solution to CTAB aqueous solution is 1:(1~2), the concentration of the aqueous dispersion of phosphorophthalocyanine nanoparticles is 1~3 mg / mL, and the volume ratio of the aqueous dispersion of phosphorophthalocyanine nanoparticles to the AIPH CTAB solution is 1:(2~3).

[0009] Further, in step 4), the volume ratio of siRNA aqueous solution to SDS aqueous solution is 1:(1~2), the concentration of the aqueous dispersion of phosphorophthalocyanine@AIPH nanoparticles is 1~3 mg / mL, and the volume ratio of the aqueous dispersion of phosphorophthalocyanine@AIPH nanoparticles to the SDS solution of siRNA is 1:(2~3).

[0010] The phosphorophthalocyanine@AIPH@siRNA composite material was prepared by the above method.

[0011] The above-mentioned phosphorophthalocyanine@AIPH@siRNA composite material is used in the preparation of antitumor drugs, wherein the tumor refers to glioma.

[0012] The aforementioned phosphorophthalocyanine@AIPH@siRNA composite material exerts its effects through the following pathways: (1) Using 1064 nm, 1.0 W / cm 2 Laser irradiation at a specific wavelength kills tumor cells; (2) siRNA and mRNA coordinate with each other through base complementarity, silencing related genes; (3) AIPH decomposes under heat and produces ROS, which in turn destroys the structure of HSP70 protein and kills tumor cells.

[0013] The above-mentioned phosphorophthalocyanine@AIPH@siRNA composite material is used in the preparation of near-infrared photothermal therapy for tumors and siRNA therapy for tumors, wherein the tumor refers to glioma.

[0014] This invention, combining the characteristics of photothermal therapy systems, uses phosphophthalocyanine with a large π-conjugated structure as the assembly unit. Due to the poor dispersibility of phosphophthalocyanine, an emulsifier is used to improve its dispersibility. Based on this, AIPH and siRNA are loaded stepwise through electrostatic adsorption, thus constructing a phosphophthalocyanine@AIPH@siRNA composite material for the first time. In this invention, by adjusting the concentrations of emulsifier, AIPH, and siRNA, a phosphophthalocyanine@AIPH@siRNA composite material with uniform size, regular morphology, high yield, and large-scale production capability is obtained. Simultaneously, the addition of the emulsifier greatly improves the water dispersibility and biocompatibility of the composite material, thereby increasing the phagocytic capacity of nanomedicines within tumor cells. In this invention, the composite material exhibits a high absorption spectrum in the near-infrared II region. After assembly, the strong intermolecular π-stacking inhibits radiative transitions and enhances non-radiative transitions, generating a large amount of heat and thus achieving high photothermal conversion efficiency. Due to the introduction of AIPH and siRNA, the ROS generated by the thermal decomposition of AIPH can effectively disrupt the structure of HSP70 protein, killing tumor cells. On the other hand, siRNA, protected by an emulsifier, is not easily degraded by nucleases within tumor cells, thereby improving siRNA delivery efficiency. siRNA can also directly coordinate with mRNA through base complementarity, silencing related genes. These three factors synergistically promote the therapeutic effect of the composite material. Furthermore, the composite material exhibits fluorescence emission in the near-infrared II region, demonstrating excellent tissue penetration depth. This achieves a combined therapeutic effect of near-infrared II photothermal therapy, siRNA gene therapy, and near-infrared II fluorescence imaging, opening a new avenue for exploring intelligent, precise, minimally invasive, and highly efficient combined therapies. Attached Figure Description

[0015] Figure 1 SEM image of phosphophthalocyanine self-assembled nanoparticles ( Figure 1 A) and TEM image ( Figure 1 B); Figure 2 SEM image of phosphophthalocyanine@AIPH@siRNA composite material ( Figure 2 A) and TEM image ( Figure 2 B); Figure 3 Agarose gel electrophoresis image of siRNA loaded on phosphophthalocyanine@AIPH nanoparticles; Figure 4 The UV-Vis-NIR absorption spectra of PPc / CHCl3 solution, phosphophthalocyanine particles, phosphophthalocyanine@AIPH and phosphophthalocyanine@AIPH@siRNA composites are shown. Figure 5 Standard curves for the relationship between different phosphorophthalocyanine mass concentrations and absorbance; Figure 6Near-infrared II fluorescence emission spectrum of phosphophthalocyanine self-assembled nanoparticles; Figure 7 The temperature rise curve of an aqueous dispersion of phosphophthalocyanine self-assembled nanoparticles under 1064 nm laser irradiation is shown. Figure 8 Electron paramagnetic resonance (ESR) spectra of reactive oxygen species before and after 1064 nm laser irradiation of the phosphophthalocyanine@AIPH@siRNA composite material; Figure 9 Dark toxicity test results after co-incubating U87 cells with different concentrations of phosphophthalocyanine@AIPH@siRNA composite materials for 24 h; Figure 10 The image shows the survival rate of U87 cells after irradiation with 1064 nm laser for different durations using phosphophthalocyanine@AIPH@siRNA composite materials of different concentrations. Figure 11 Fluorescence changes in gliomas in the brains of tumor-bearing mice labeled with luciferase in the treatment and control groups. Figure 12 Figure showing the change in body weight of tumor-bearing mice during treatment ( Figure 12 A) Fluorescence intensity change curves of gliomas ( Figure 12 B); Figure 13 The survival rate curves of tumor-bearing mice in the treatment and control groups are shown. Figure 14 Images of hematoxylin-eosin (H&E) staining of brain sections from tumor-bearing mice in the treatment and control groups; Figure 15 H&E staining images of heart, liver, spleen, lung, and kidney sections from tumor-bearing mice in the treatment and control groups. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0017] Sodium dodecyl sulfate (SDS) in the following examples was purchased from Sigma-Aldrich, CAS No. 151-21-3; hexadecyltrimethylammonium bromide (CTAB) was purchased from Sigma-Aldrich, CAS No. 57-09-0; 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) was purchased from Adamas, CAS No. 27776-21-2; the small interfering RNA (siRNA) sequence was 5'-rUrUrC rArCrA rGrGrU rCrUrUrUrArA rUrCrUrArCrC rUrCrC rUrCrA rArUrGrGdTdT-3' was purchased from Qiangyao Biotechnology Co., Ltd.; 1,4,8,11,15,18,22,25-octa(p-methoxyphenyl sulfide)-29H,31H-phthalocyanine phosphorus (PPc) was purchased from Suzhou Zhuoxinya Technology Co., Ltd.; 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) was purchased from Adamas, CAS No.: 3317-61-1. Example 1

[0018] A method for preparing a phosphorophthalocyanine@AIPH@siRNA composite material includes the following steps: 1) Dissolve phosphophthalocyanine in chloroform to prepare 5 mg / mL phosphophthalocyanine (PPc) chloroform solution, 0.025 mol / L SDS aqueous solution, 0.025 mol / L CTAB aqueous solution, 10 mg / mL AIPH aqueous solution and 4 OD / mL small interfering RNA (siRNA) aqueous solution respectively; 2) Under stirring at room temperature, 1 mL of PPc / CHCl3 solution was added to 9 mL of SDS aqueous solution. The solution was sonicated with a probe for 1 min (power 80 W), then transferred to a 60℃ water bath and rapidly evaporated for 35 min with stirring to evaporate all the chloroform solution. The solution was then rapidly cooled to room temperature in an ice-water bath and centrifuged at 13000 r / min for 30 min. The resulting precipitate was the phosphophthalocyanine self-assembled nanoparticles (PPc NPs). 1 mL of water was added to disperse the precipitate to obtain an aqueous dispersion of phosphophthalocyanine nanoparticles with a concentration of 2 mg / mL. 3) While stirring at room temperature, add 1 mL of the AIPH aqueous solution from step 1) to 1.5 mL of the CTAB aqueous solution, then add 1 mL of the 2 mg / mL phosphophthalocyanine nanoparticle aqueous dispersion from step 2), and continue stirring at room temperature for 8 h. Centrifuge to separate the precipitate, and the resulting precipitate is the phosphophthalocyanine@AIPH nanoparticle loaded with AIPH molecules. Add 1 mL of water to the precipitate to disperse it, and obtain an aqueous dispersion of 2 mg / mL phosphophthalocyanine@AIPH nanoparticles (PPc NPs@AIPH).

[0019] 4) While stirring at room temperature, add 1 mL of the siRNA aqueous solution from step 1) to 1.5 mL of the SDS aqueous solution, then add 1 mL of the 2 mg / mL phosphorophthalocyanine@AIPH nanoparticle aqueous dispersion from step 3), stir at room temperature for 8 h, centrifuge to separate, and the resulting precipitate is the phosphorophthalocyanine@AIPH@siRNA composite material (PPc NPs@AIPH@siRNA).

[0020] The phosphorophthalocyanine@AIPH@siRNA composite material was dispersed in 1 mL of water to obtain an aqueous dispersion of the phosphorophthalocyanine@AIPH@siRNA composite material, which was then subjected to scanning electron microscopy and TEM scanning. SEM image of the phosphorophthalocyanine self-assembled nanoparticles (…). Figure 1 A) and TEM image ( Figure 1 B), SEM image of the phosphophthalocyanine@AIPH@siRNA composite material ( Figure 2 A) and TEM image ( Figure 2 B), agarose gel electrophoresis of siRNA loaded with different concentrations of PPcNPs@AIPH composite materials (B) Figure 3 (B) Morphological characterization confirmed that the diameter of the self-assembled phosphorophthalocyanine nanoparticles was approximately 30 nm, while the diameter of the PPc NPs@AIPH@siRNA nanoparticles was approximately 35 nm, with a slight increase in size after loading AIPH and siRNA. Agarose gel electrophoresis results showed that as the concentration of PPc NPs@AIPH nanomaterials gradually increased, the corresponding electrophoretic bands gradually disappeared, indicating that the amount of electrostatically adsorbed siRNA gradually increased, eventually achieving complete siRNA adsorption.

[0021] The UV-Vis-NIR absorption spectra of phosphophthalocyanine monomers, phosphophthalocyanine self-assembled nanoparticles (PPc NPs), phosphophthalocyanine@AIPH composites (PPc NPs@AIPH), and phosphophthalocyanine@AIPH@siRNA composites (PPc NPs@AIPH@siRNA) are shown in the figure. Figure 4The results showed that the phosphophthalocyanine monomer exhibited a broad Q-band characteristic absorption peak in the near-infrared II region of 800–1100 nm, with the strongest absorption peak at 1036 nm. After self-assembly into nanoparticles, the near-infrared absorption peak broadened due to π-π stacking, confirming that the phosphophthalocyanine monomer was successfully assembled into nanoparticles. After loading AIPH to obtain the phosphophthalocyanine@AIPH composite material, the full-band near-infrared characteristic absorption peaks of the sample were consistent with those of the phosphophthalocyanine monomer, and no additional impurity peaks appeared, proving that AIPH was successfully loaded onto the phosphophthalocyanine nanoparticles, and that the phosphophthalocyanine conjugated framework structure was intact. Further electrostatic adsorption of siRNA yielded the phosphophthalocyanine@AIPH@siRNA composite material, whose characteristic peak positions remained unchanged, indicating that siRNA was modified on the particle surface by electrostatic interaction, and the phosphophthalocyanine@AIPH@siRNA composite material fully retained the excellent near-infrared II light absorption characteristics.

[0022] Using N,N-dimethylformamide (DMF) as solvent, standard solutions of phosphophthalocyanine with concentrations of 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, and 60 μg / mL were prepared, and the absorbance (A) of each solution was measured at a wavelength of 984 nm. A standard curve of absorbance versus mass concentration was plotted with phosphophthalocyanine mass concentration (c) on the x-axis and absorbance (A) on the y-axis. The results are as follows: Figure 5 As shown, where R 2 = 0.99999, which can be used for the quantitative determination of phosphorophthalocyanine sample concentration.

[0023] Phosphophthalocyanine self-assembled nanoparticles exhibited excellent near-infrared II fluorescence emission characteristics under 1064 nm light source excitation, such as... Figure 6 As shown, its maximum emission peak is located at 1128 nm.

[0024] Aqueous dispersions were prepared by dispersing self-assembled phosphophthalocyanine nanoparticles in ultrapure water, and analyzed using a 1064 nm laser at 1.0 W / cm². 2 Laser irradiation was performed for 10 minutes, and temperature changes were monitored, such as... Figure 7 As shown, the temperature of the solution increases with increasing concentration, and the heating rate accelerates, exhibiting a concentration-dependent temperature change. At a concentration of 100 μg / mL for the self-assembled phosphophthalocyanine nanoparticles, the temperature of the nanomaterial can rise to 73.4℃ in 10 min, indicating that the nanomaterial possesses excellent photothermal properties, with a photothermal conversion efficiency of 41.2%.

[0025] The phosphophthalocyanine@AIPH@siRNA composite material was dispersed in ultrapure water at a concentration of 100 μg / mL. 1 mL of this solution was then mixed with 10 μL of DMPO and subjected to a 1064 nm laser at 1.0 W / cm². 2After laser irradiation for 5 minutes, the ROS generated by the composite material was detected using a paramagnetic resonance spectrometer (made in Germany, manufactured by Brookbyspin GmbH). The results showed that after 5 minutes of irradiation, a characteristic ·OH peak of 1:2:2:1 was observed. Figure 8 As shown, under near-infrared II laser irradiation, the heat generated by the phosphophthalocyanine nanoparticles causes AIPH to decompose thermally, producing a large amount of ·OH.

[0026] To improve the uptake efficiency of PPc NPs@AIPH@siRNA composite material by tumor cells, the PPc NPs@AIPH@siRNA composite material was targeted modified. First, 1 mL of 6 mg / mL maleamide (Mal) and 1 mL of 6 mg / mL apolipoprotein E (ApoE) were mixed thoroughly for 3 h. Then, 1 mL of 2 mg / mL PPc NPs@AIPH@siRNA composite material was added and mixed for 6 h. After centrifugation at 13000 r / min for 30 min, the resulting precipitate was PPc NPs@AIPH@siRNA targeted to ApoE. The targeted PPc NPs@AIPH@siRNA was then co-incubated with U87 cells for dark toxicity (DTC) testing. Figure 9 ) and phototoxicity test ( Figure 10 U87 cells were cultured in a high-glucose medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% non-essential amino acids (this medium was used throughout the experiment; specifically, 50 mL of FBS, 5 mL of penicillin-streptomycin, and 5 mL of non-essential amino acids were added to 500 mL of DMEM and mixed thoroughly, resulting in a final medium volume of 560 mL). The medium was then cultured at a rate of 1 × 10⁻⁶ cells per cell line. 4Cells were seeded per well in 96-well plates and incubated for 12 hours in a 5% CO2 incubator at 37°C. Different concentrations of the PPc NPs@AIPH@siRNA composite material (0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL) were prepared in the aforementioned culture medium and dispersed in 100 μL of each concentration of the aforementioned sample solution. The cells were then co-incubated with the U87 cells in each well for 24 hours. Subsequently, the culture medium was removed, and the cells were slowly washed twice with PBS. Then, 100 μL of CCK-8 solution (diluted to 1 / 10 concentration using the aforementioned culture medium) was added to each well, and the cells were incubated for another 30 minutes. The absorbance was measured at 450 nm using a multi-sensor microplate reader, and the viability of live cells was calculated to determine the dark toxicity of the material to cells (e.g., % ... Figure 8 As shown in the figure). The cell viability of the PPc NPs@AIPH@siRNA composite material at concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL were 99.6%, 100%, 98.3%, 100%, 95.8%, 96.8%, 95.4%, and 98.6%, respectively, showing good safety in the dark reaction. Figure 9 ).

[0027] A 1064 nm wavelength laser (1.0 W / cm²) was used. 2 Cells were continuously irradiated for different durations of 3, 6, and 9 minutes, and compared with the unirradiated group. Subsequently, the cells were incubated for another 12 hours. After incubation, the culture medium was removed, and 100 μL of CCK-8 solution (diluted to 1 / 10 concentration using the aforementioned culture medium) was added to each well. Cells were incubated for another 30 minutes before removal. The absorbance was measured at 450 nm using a multi-sensor microplate reader, and the viability of live cells was calculated to determine the photothermal killing effect of the material on cells. Results are as follows: Figure 10As shown, after 3 min of light exposure, the cell viability rates at nanocomposite concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL were approximately 99.52%, 95.86%, 98.02%, 89.13%, and 93.92%, respectively; after 6 min of light exposure, the cell viability rates at nanocomposite concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL were approximately 98.31%, 90.32%, 89.05%, 63.75%, and 50.87%, respectively; and after 9 min of light exposure, the cell viability rates at nanocomposite concentrations of approximately 0 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL were approximately 100%, 98.63%, 77.59%, 61.84%, and 30.26%, respectively. With increasing concentration and prolonged illumination time, cell viability gradually decreased, exhibiting a significant concentration- and light-time-dependent cell-killing effect. This indicates that the PPc NPs@AIPH@siRNA composite material possesses good near-infrared photothermal therapeutic efficacy.

[0028] Female BALB / c nude mice (6-8 weeks old, weighing approximately 20 g) were purchased from Spiford Biotechnology Co., Ltd. and housed in our laboratory mouse house for 7 days to eliminate the influence of environmental stress on the experiments (all animal experiments in this study were approved by the Biomedical Research Ethics Committee of Henan University (HUSOM2025-081) and strictly implemented in accordance with relevant regulations and rules; the animal husbandry environment in the laboratory strictly followed the "Practical Guidelines for Animal Experiment Management and Operation"). Next, each mouse was anesthetized by intraperitoneal injection of 100 μL of 80 mg / mL trichloroacetaldehyde; after the anesthesia took effect, 5 μL of U87 cell suspension (total cell count approximately 2.5 × 10⁻⁶ cells) was slowly injected into the brain. 5 A mouse model of glioma was constructed by injecting 100 μL of 15 mg / mL potassium fluorescein into each mouse intraperitoneally to mark the size of the glioma tumor. The fluorescence intensity at the tumor site was measured until it reached 2.5 × 10⁻⁶. 5 ~8×10 5 After 7 days, the tumor-bearing mice were randomly divided into 4 groups (n=8): PBS group, PBS + light group (PBS+L), PPc NPs@AIPH@siRNA group, and PPc NPs@AIPH@siRNA + light group (PPc NPs@AIPH@siRNA+L).

[0029] Mice in each group were administered the drug via tail vein four times on days 7, 9, 11, and 13. The PBS group and the PBS + light irradiation group were each injected with 100 μL of PBS. The PPc NPs@AIPH@siRNA composite material group and the PPc NPs@AIPH@siRNA composite material + light irradiation group were each injected with 100 μL of the targeted PPc NPs@AIPH@siRNA composite material at a concentration of 2 mg / mL. Eight hours after drug administration, the light irradiation group was treated with a 1064 nm laser at a power density of 1.0 W / cm² for 5 min.

[0030] The growth and size of gliomas were indirectly observed by measuring tumor fluorescence intensity at 533 nm on days 7, 9, 11, 13, 15, and 17 using a small animal in vivo imaging system (Made in the USA, by PerkinElmer). Figure 11 , 12 As shown, the bioluminescence intensity in the treatment group was significantly reduced, indicating that the PPc NPs@AIPH@siRNA composite material has a significant inhibitory effect on glioma. The body weight and survival status of mice were continuously recorded during the treatment period, and the results are as follows. Figure 12 As shown, no significant decrease in body weight was observed in any group of mice, and the survival time of mice in the treatment group was extended to 55 days. Figure 13 After treatment, the mice were further observed to check their mental state, whether they could move, eat and drink normally, and whether they showed any abnormal signs such as arched back, limb tremors, or movement disorders. No obvious abnormalities were found.

[0031] After the treatment period, four groups of mice were collected, and heart, liver, spleen, lung, kidney, and brain tissues were harvested for hematoxylin-eosin (H&E) staining (n=3). The staining results are as follows: Figure 14 , 15 As shown, the stained area of ​​glioma was significantly reduced, indicating that the PPc NPs@AIPH@siRNA composite material had a significant therapeutic effect; no significant damage was found in the heart, liver, spleen, lungs, and kidneys of the mice, indicating that the composite material has good biocompatibility.

[0032] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a phosphorophthalocyanine@AIPH@siRNA composite material, characterized in that, Includes the following steps: 1) Prepare chloroform solution, SDS aqueous solution, CTAB aqueous solution, AIPH aqueous solution and siRNA aqueous solution respectively. The concentrations of SDS aqueous solution and CTAB aqueous solution are the same. AIPH stands for 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride. 2) Under stirring at room temperature, add the chloroform solution of phosphorophthalocyanine in step 1) to the SDS aqueous solution, sonicate with a probe for 1-3 min, and then stir the sonicated mixture in a 62-70℃ water bath until the chloroform is completely evaporated. Then, transfer it to an ice water bath to cool to room temperature, centrifuge, and add an appropriate amount of water to the precipitate to disperse it, thus obtaining an aqueous dispersion of phosphorophthalocyanine nanoparticles. 3) Under stirring at room temperature, add the AIPH aqueous solution from step 1) to the CTAB aqueous solution to obtain the AIPH CTAB solution, then add the aqueous dispersion of phosphorophthalocyanine nanoparticles from step 2), and continue stirring at room temperature for 5-10 h. Centrifuge to separate the precipitate, and the obtained precipitate is phosphorophthalocyanine@AIPH nanoparticles. Add an appropriate amount of water to the precipitate to disperse it, and obtain the aqueous dispersion of phosphorophthalocyanine@AIPH nanoparticles. 4) Under stirring at room temperature, add the siRNA aqueous solution from step 1) to the SDS aqueous solution to obtain the siRNA SDS solution, then add the aqueous dispersion of phosphorophthalocyanine@AIPH nanoparticles from step 3), and stir at room temperature for 5-10 h. The precipitate obtained by centrifugation is the phosphorophthalocyanine@AIPH@siRNA composite material.

2. The method for preparing the phosphophthalocyanine@AIPH@siRNA composite material according to claim 1, characterized in that, In step 1), the phosphorophthalocyanine is specifically 1,4,8,11,15,18,22,25-octa(p-methoxyphenylthio)-29H,31H-phthalocyanine phosphorus, with a chloroform solution concentration of 10~20 mg / mL; the concentrations of the SDS aqueous solution and CTAB aqueous solution are 0.015 mol / L~0.025 mol / L, the concentration of the AIPH aqueous solution is 5 mg / mL~20 mg / mL, and the concentration of the siRNA aqueous solution is 4 OD / mL~8 OD / mL.

3. The method for preparing the phosphophthalocyanine@AIPH@siRNA composite material according to claim 1, characterized in that, In step 2), the volume ratio of the chloroform solution of phosphorophthalocyanine to the aqueous solution of SDS is 1:(8~10).

4. The method for preparing the phosphophthalocyanine@AIPH@siRNA composite material according to claim 1, characterized in that, In step 3), the volume ratio of AIPH aqueous solution to CTAB aqueous solution is 1:(1~2), the concentration of the aqueous dispersion of phosphorophthalocyanine nanoparticles is 1~3 mg / mL, and the volume ratio of the aqueous dispersion of phosphorophthalocyanine nanoparticles to the AIPH CTAB solution is 1:(2~3).

5. The method for preparing the phosphophthalocyanine@AIPH@siRNA composite material according to claim 1, characterized in that, In step 4), the volume ratio of siRNA aqueous solution to SDS aqueous solution is 1:(1~2), the concentration of the aqueous dispersion of phosphorophthalocyanine@AIPH nanoparticles is 1~3 mg / mL, and the volume ratio of the aqueous dispersion of phosphorophthalocyanine@AIPH nanoparticles to the SDS solution of siRNA is 1:(2~3).

6. The phosphorophthalocyanine@AIPH@siRNA composite material prepared by any one of the preparation methods according to claims 1 to 5.

7. The application of the phosphorophthalocyanine@AIPH@siRNA composite material according to claim 6 in the preparation of antitumor drugs, characterized in that, The tumor in question refers to a glioma.

8. The application according to claim 7, characterized in that, Phosphocyanine@AIPH@siRNA composites function through the following pathways: (1) Using 1064 nm, 1.0 W / cm 2 Laser irradiation at a specific wavelength kills tumor cells; (2) siRNA and mRNA coordinate with each other through base complementarity, silencing related genes; (3) AIPH decomposes under heat and produces ROS, which in turn destroys the structure of HSP70 protein and kills tumor cells.

9. The application of the phosphorophthalocyanine@AIPH@siRNA composite material according to claim 6 in the preparation of near-infrared photothermal therapy for tumors and siRNA therapy for tumors.

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