Nanoparticles capable of simultaneously delivering photosensitizer and targeting antibody as well as preparation method and application of nanoparticles

Abstract

The invention discloses a nanoparticle capable of simultaneously delivering a photosensitizer and a targeting antibody as well as a preparation method and application of the nanoparticle. The nanoparticle provided by the invention contains a photosensitizer, a nitric oxide precursor, siRNA (small interfering Ribonucleic Acid) and a macrophage membrane, the photosensitizer is a pyromellitic chlorophyll a molecule, the siRNA is siPD-L1, and the nitric oxide precursor is arginine nonamer. The targeting nanoparticle provided by the invention can realize efficient delivery of the photosensitizer and the siRNA. According to the invention, arginine and pyropheophorbide a are covalently linked according to a ratio of 1: 1, after nanoparticles are prepared, negatively charged siPD-L1 is loaded through an electrostatic adsorption principle, and by wrapping M1 macrophage membrane vesicles, surface charges of the nanoparticles are reduced, and meanwhile, the tumor targeting ability of the nanoparticles is improved. And under the irradiation of 650nm laser, the photodynamic and anti-PD-L1 immune combined treatment of malignant tumors can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a targeted nanoparticle capable of simultaneously delivering a photosensitizer and siRNA deep inside a body, and a preparation method and application thereof. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a common digestive tract malignancy characterized by insidious onset, rapid progression, and low surgical resection rate. The tumor microenvironment of PDAC is characterized by rich stroma, hypoxia, insufficient blood supply, and strong immunosuppression, which poses great challenges to its treatment. For example, the dense extracellular matrix (ECM) in the tumor hinders drug penetration, and the accompanying hypoxia reduces the efficacy of treatment. In the treatment of hypoxic solid tumors, photodynamic therapy using type I photosensitizers has significant advantages. Type II photosensitizers produce singlet oxygen by energy transfer to molecular oxygen, which requires sufficient oxygen concentration. Tumor tissues often have hypoxic areas, which reduces the efficacy of type II photosensitizers. Type I photosensitizers transfer electrons or hydrogen protons generated by photoexcitation to surrounding substrates to produce free radicals (such as superoxide (O 2 -· ) and hydroxyl (·OH) free radicals). Type I photosensitizers can maintain their therapeutic effects under hypoxic or even anaerobic conditions through an electron / proton transfer mechanism. A wider range of reactive substances can cause different types of damage to cells and biomolecules, such as oxidative stress and damage to proteins, lipids, and nucleic acids. At the same time, compared with traditional chemotherapy, this photodynamic therapy avoids the drug resistance problem of PDAC. At the same time, as a tumor with a dense extracellular matrix, PDAC has a severely hypoxic tumor microenvironment, and type I photosensitizer photodynamic therapy has a low oxygen demand. Therefore, type I photosensitizers have significant advantages in the treatment of PDAC.

[0003] The dense extracellular matrix of PDAC is an important factor affecting the efficacy of photodynamic therapy. Immunosuppressive cells in the tumor microenvironment promote the activation of pancreatic stellate cells by secreting cytokines and growth factors. Activated pancreatic stellate cells synthesize a large amount of extracellular matrix proteins in the tumor matrix, resulting in matrix hardening and compaction. Excessive deposition of extracellular matrix components forms a physical barrier that limits the diffusion of drugs. Nitric oxide is a multifunctional endogenous gas messenger that plays a vital role in vasodilation, angiogenesis, tumor formation and progression. At the same time, nitric oxide and its derivatives can degrade extracellular matrix components such as collagen and fibronectin. In order to effectively deliver high concentrations of nitric oxide, a series of nitric oxide donors have been developed, mainly including nitroglycerin, tetranitrate, l-arginine, etc. In addition, nitric oxide can react with superoxide anions to generate peroxynitrite anions (ONOO -), which is a major reactive nitrogen with stronger peroxidative capacity than reactive oxygen species, leading to apoptosis of cancer cells. However, nitric oxide and ONOO - The half-life of nitric oxide and ONOO is very short. - It is crucial to release the drug or carrier system to maintain the appropriate concentration and duration of action in the body. Nona-arginine reacts with superoxide anions and has the potential to produce nitric oxide.

[0004] During the photodynamic process, photosensitizers are activated to produce reactive oxygen species, leading to nucleic acid damage, protein oxidation, mitochondrial or plasma membrane destruction, and inducing immunogenic cell death. However, tumor cells with high expression of PD-L1 can inhibit the activity of T cells by binding to PD-1, leading to immune escape. In order to solve this problem, some regulators that inhibit self-protection are urgently needed to effectively enhance the efficiency of tumor killing. PD-L1 (programmed cell death ligand 1) is an important immune checkpoint protein. Tumor cells can inhibit the activity of T cells by highly expressing PD-L1, thereby escaping the surveillance of the immune system. PD-L1 inhibitors exert anti-cancer effects by blocking the PD-1 / PD-L1 signaling pathway and enhancing the activity of T cells. Summary of the invention

[0005] The purpose of the present invention is to provide a targeted nanoparticle for deep delivery of photosensitizer, nitric oxide precursor and siRNA, and a preparation method and application thereof. The targeted nanoparticle for delivering photosensitizer, nitric oxide precursor and siRNA provided by the present invention has the effect of increasing the enrichment of photosensitizer, nitric oxide precursor and siRNA in tumors, and can produce reactive oxygen and reactive nitrogen under hypoxic conditions under laser irradiation to achieve the effect of treating tumors.

[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0007] A nanoparticle for deep delivery of a photosensitizer, a nitric oxide precursor and siRNA, wherein the nanoparticle is prepared from pyropheophorbide a and nona-arginine to prepare positively charged photosensitizer nanoparticles, and then negatively charged siRNA is loaded using the principle of electrostatic adsorption, the mass ratio of the microbubbles to the siRNA is 1:3, and the microbubbles are wrapped in M1 macrophage membrane vesicles.

[0008] In the above-mentioned targeted nanoparticles, the average particle size of the nanoparticles is 126±12nn. The molar ratio of the components of the nanoparticles to the photosensitizer and the NO precursor is 1:1.

[0009] The above-mentioned targeted nanoparticles can actively target the tumor site, while increasing the enrichment of photosensitizers and siRNA in the tumor, and produce reactive oxygen and nitric oxide under laser irradiation, thereby treating the tumor. The laser is 650nm and the power is 8mW / cm 2 , the irradiation time is 2min.

[0010] The present invention also provides a method for preparing the above-mentioned targeted nanoparticles for simultaneously delivering photosensitizer and targeting antibody, comprising the following steps:

[0011] Step 1, dissolving pyropheophorbide a in N,N-dimethylformamide to obtain solution A;

[0012] Step 2, dissolving the NO precursor in N,N-dimethylformamide to obtain solution B;

[0013] Step 3, dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in N,N-dimethylformamide to obtain solution C;

[0014] Step 4, incubating the macrophages with lipopolysaccharide for 24 hours to obtain pro-inflammatory macrophages, and extracting pro-inflammatory macrophage cell membrane vesicles (M1 macrophage cell membrane vesicles) by ultrasonic disruption and differential centrifugation;

[0015] Step 5, dissolving N-hydroxysuccinimide (NHS) in N,N-dimethylformamide to obtain solution D;

[0016] Step 6, mixing solutions A, C and D, reacting them under magnetic stirring for 12 hours, and then purifying the product by column chromatography to remove unreacted compounds and other by-products;

[0017] Step 7, mixing the product obtained in step 6) with solution C, reacting under magnetic stirring for 24 hours, and then injecting it into a PBS solution or physiological saline under magnetic stirring to form nanoparticles, and then dialyzing it in a PBS solution or physiological saline to remove the organic solvent to form nanoparticles loaded with a photosensitizer and a nitric oxide precursor;

[0019] Step 9: Mix the nanoparticles obtained in step 7) with siRNA in a PBS solution or physiological saline, and stir for half an hour in a dark environment to obtain a photosensitizer particle dispersion loaded with siRNA; and physically extrude the obtained siRNA-loaded photosensitizer particle dispersion and M1 macrophage membrane vesicles through an Avanti microliposome extruder to obtain targeted nanoparticles that simultaneously deliver photosensitizer and siRNA.

[0020] In step 9, nanoparticles and siRNA were mixed at a mass ratio of 1:3;

[0021] In step 9, the concentration of the targeted photosensitizer particle dispersion is 2 mg / mL, and it is mixed with the M1 macrophage membrane vesicles in a 1:1 ratio.

[0022] The present invention also provides the use of the targeted nanoparticles prepared according to the above technical scheme in diagnostic reagents or treatments of cancer, such as for the treatment of pancreatic cancer.

[0023] In the present invention, positively charged photosensitizer nanoparticles are prepared using pyropheophorbide a and NO precursor, and then negatively charged siRNA is loaded using the principle of electrostatic adsorption, and the uptake of photosensitizer and PDL1 siRNA by tumor cells is improved through macrophage membrane wrapping. At the same time, active oxygen is generated under the action of laser irradiation, and the active oxygen promotes the NO precursor to release NO, further promoting the deep delivery of photosensitizer and siRNA in the tumor, achieving the effect of targeted photodynamic therapy for tumors, and PDL1 siRNA can block the PD-1 / PD-L1 signaling pathway, enhance the activity of T cells, and amplify the photodynamic killing effect of tumors.

[0024] The beneficial effects of the present invention are as follows: the present invention covalently links pyropheophorbide a with a NO precursor to prepare nanoparticles, thereby improving the efficiency of the photosensitizer in generating superoxide anions, possessing the characteristics of a type I photosensitizer, and being able to effectively kill tumor cells under hypoxic conditions. The reactive oxygen generated by the nitric oxide precursor in response to photodynamics can further generate nitric oxide and peroxynitrite. The generated nitric oxide can improve the infiltration of the nanoparticles, and the nitric oxide and peroxynitrite also have a tumor cell killing effect. By electrostatically adsorbing siRNA and wrapping the macrophage membrane, the high positive potential caused by nona-arginine is significantly reduced without affecting the function of the NO precursor, which is beneficial to improving biosafety. While using macrophage membranes to wrap the nanoparticles without affecting the functions of the photosensitizer and the NO precursor nona-arginine, the tumor targeting of the macrophage membrane is utilized to improve the delivery efficiency of the photosensitizer and siRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a functional schematic diagram of the multifunctional nanoparticles in specific embodiment 1; Figure 2 is a mass spectrum of a compound obtained by covalently linking a photosensitizer and a nitric oxide precursor in the multifunctional nanoparticles in specific embodiment 2; Figure 3 is the potential and hydrated particle size of the multifunctional nanoparticles prepared in specific Example 3; Figure 4 is an in vitro electron spin resonance image of superoxide anion generation by the multifunctional nanoparticles in specific embodiment 4; Figure 5 is a fluorescence spectrum of the multifunctional nanoparticles in specific embodiment 5 that produce peroxynitrite in response to laser irradiation in vitro; Figure 6 is a flow cytometry picture of the multifunctional nanoparticles in specific example 6 being taken up by cancer cells; Figure 7 is a fluorescence microscope image of nitric oxide produced after the multifunctional nanoparticles are taken up by cancer cells in specific embodiment 7; Figure 8 is a flow cytometry picture of cancer cell uptake of multifunctional nanoparticles in specific example 8 in response to laser irradiation to kill tumor cells; Fig. 9 This is a fluorescence imaging image of the enrichment of multifunctional nanoparticles in animal tumor tissues in specific embodiment 9; Fig.10 This is the tumor growth curve after the multifunctional nanoparticles in specific embodiment 10 were used for photodynamic therapy. DETAILED DESCRIPTION

[0027] The following specific embodiments will help to understand the present invention, but will not limit the contents of the present invention.

[0028] Example 1

[0029] Dissolve pyropheophorbide a in N,N-dimethylformamide to obtain solution A; NO precursor is dissolved in N,N-dimethylformamide to obtain solution B; 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) is dissolved in N,N-dimethylformamide to obtain solution C; macrophages are co-incubated with lipopolysaccharide for 24 hours to obtain pro-inflammatory macrophages, and pro-inflammatory macrophage cell membrane vesicles (M1 macrophage membrane vesicles) are extracted by ultrasonic disruption and differential centrifugation; N-hydroxysuccinimide (NHS) is dissolved in N,N-dimethylformamide to obtain solution D; solutions A:C:D are mixed in a ratio of 1:1.2:1, reacted under magnetic stirring for 12 hours, and then the product is purified by column chromatography to remove unreacted compounds and other by-products; then, mixed with solution B in a ratio of 1:1.2 , react under magnetic stirring conditions for 24 hours, then inject into PBS solution or physiological saline under magnetic stirring conditions to form nanoparticles, then dialyze in PBS solution or physiological saline to remove the organic solvent to form pyropheophorbide a covalently linked to nona-arginine nanoparticles; the obtained nanoparticles are mixed with siRNA in PBS solution or physiological saline, and stirred for half an hour under light-proof conditions to obtain pyropheophorbide a covalently linked to nona-arginine nanoparticles adsorbing PDL1 inhibitors; the obtained pyropheophorbide a covalently linked to nona-arginine nanoparticles dispersion adsorbing PDL1 inhibitors and M1 macrophage membrane vesicles are physically extruded by an Avanti microliposome extruder to obtain macrophage membrane vesicle-encapsulated pyropheophorbide a covalently linked to nona-arginine nanoparticles (attached Figure 1).

[0028] Example 2

[0028] The compound obtained by covalently linking the photosensitizer pyropheophorbide a obtained in Example 1 with the nitric oxide precursor nona-arginine was purified and freeze-dried. 1 mg of the compound was redissolved in 1 ml of methanol and the molecular weight of the compound was characterized by high-resolution mass spectrometry (see Appendix). Figure 2 ), it can be seen that the highest peak appears at a mass-to-charge ratio of 1940, which is the signal of the compound obtained by covalently linking the product pyropheophorbide a and nona-arginine through an amide bond. This indicates that the small molecule compound in which the photosensitizer and the nitric oxide precursor are covalently linked was successfully synthesized.

[0028] Example 3

[0028] The pyropheophorbide a covalently linked to nona-arginine nanoparticles obtained in Example 1 and the pyropheophorbide a covalently linked to nona-arginine nanoparticles encapsulated by macrophage membrane vesicles and adsorbed PDL1 inhibitor were measured by dynamic light scattering instrument to determine the hydrated particle size of the nanoparticles; the pyropheophorbide a covalently linked to nona-arginine nanoparticles obtained in Example 1, the pyropheophorbide a covalently linked to nona-arginine nanoparticles adsorbed PDL1 inhibitor and the pyropheophorbide a covalently linked to nona-arginine nanoparticles adsorbed PDL1 inhibitor and the macrophage membrane vesicles and adsorbed PDL1 inhibitor were measured by dynamic light scattering instrument to determine the potential (attached Figure 3 ). It shows that the average hydrated particle size of pyropheophorbide a covalently linked to nona-arginine nanoparticles is 126.7±12.1 nm, the average hydrated particle size of pyropheophorbide a covalently linked to nona-arginine nanoparticles encapsulated by macrophage membrane vesicles with adsorbed PDL1 inhibitor is 145.5±16.3 nm, the potential of pyropheophorbide a covalently linked to nona-arginine nanoparticles is about 24±1.8 mV, the potential of pyropheophorbide a covalently linked to nona-arginine nanoparticles with adsorbed PDL1 inhibitor is 18±1.2 mV, and the potential of pyropheophorbide a covalently linked to nona-arginine nanoparticles encapsulated by macrophage membrane vesicles with adsorbed PDL1 inhibitor is -13.6±1.2 mV, indicating the successful preparation of several nanoparticles, and the adsorbed PDL1 inhibitor and the encapsulated cell membrane reduce the positive charge of pyropheophorbide a covalently linked to nona-arginine nanoparticles.

[0030] Example 4

[0031] The ability of the targeted nanoparticle macrophage membrane vesicles obtained in Example 1 to produce superoxide anions was tested by covalently linking pyropheophorbide a to nona-arginine nanoparticles encapsulated with PDL1 inhibitor under 650 nm laser irradiation, using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap at a laser power of 5 mW / cm 2, the laser action time was 1 min, and the electron spin resonance spectroscopy was used for detection. The electron spin resonance image showed that the prepared targeted nanoparticles had a superoxide anion-specific ultrastructure (attached Figure 4 ), which is shown as a four-line spectrum. In the group without laser irradiation, the electron spin resonance image has no characteristic signal, indicating that the prepared targeted nanoparticles produce superoxide anions in response to laser irradiation.

[0030] Example 5

[0031] The macrophage membrane vesicles obtained in Example 1 encapsulated the PDL1 inhibitor-adsorbed pyropheophorbide a and covalently linked the nona-arginine nanoparticles 4 μM and 5 μM peroxynitrite detection probes. O56 was mixed in 2 mL of ultrapure water and irradiated with a 650 nm laser (5 mW / cm2). The fluorescence spectra were measured at different time points with an excitation wavelength of 488 nm. The fluorescence spectra obtained showed that the fluorescence intensity of the probe increased with increasing laser irradiation time, and had a time-dependent property (see Appendix). Figure 5 ). This indicates that the obtained macrophage membrane vesicles encapsulated pyropheophorbide a covalently linked to nona-arginine nanoparticles adsorbing PDL1 inhibitor have the ability to produce peroxynitrite in response to 650nm laser irradiation.

[0032] Example 6

[0033] In order to track the uptake of targeted nanoparticles by tumor cells, macrophage membrane vesicles were encapsulated with PDL1 inhibitor-adsorbed pyropheophorbide a covalently linked to nona-arginine nanoparticles and cultured in a 37°C incubator. After the cells adhered to the wall, the nanoparticles were co-incubated with the cells for 0 h, 1 h, 2 h, and 4 h. The residual nanoparticles were then washed away, and the cells were digested with trypsin and the fluorescence of pyropheophorbide a was tracked by flow cytometry. The specific results are shown in the attached figure. Figure 6 As shown, the cells displayed a time-dependent uptake of the nanoparticles, with the fluorescence from the photosensitizer pyropheophorbide a in the nanoparticles increasing over time.

[0034] Example 7

[0035] To evaluate the ability of nanoparticles to produce NO from NO precursors after being taken up by tumor cells, macrophage membrane vesicles were encapsulated with PDL1 inhibitor-adsorbed pyropheophorbide a covalently linked to nona-arginine nanoparticles and incubated with tumor cells for 4 h, and then the un-ingested nanoparticles were washed away with serum-free medium. DAF-FM DA probe was used as a nitric oxide detection probe. After incubation with cells at a concentration of 5 μM for 40 min, the cells were irradiated with a 650 nm laser at a laser power of 5 mW / cm 2 The laser action time was 1 min. The fluorescence from the probe was observed using a fluorescence microscope with an excitation light of 488 nm (see attached Figure 7), demonstrating that targeted nanoparticles have a good ability to produce nitric oxide within cells.

[0036] Example 8

[0037] To evaluate the cell killing ability of nanoparticles in response to laser irradiation under normoxic and hypoxic conditions, tumor cells were cultured in a 21% normoxic incubator and a 1% hypoxic incubator, respectively. After 12 h of culture, macrophage membrane vesicles encapsulated with PDL1 inhibitor-adsorbed pyropheophorbide a covalently linked to nona-arginine nanoparticles were incubated with tumor cells for 12 h. The unabsorbed nanoparticles were washed with serum-free medium. The culture dish was sealed and irradiated with a 650 nm laser at a laser power of 5 mW / cm 2 After laser irradiation for 12 h, the cells were incubated with the Annexin V-FITC apoptosis detection kit for 20 min, and the apoptosis was detected by flow cytometry (see Appendix Figure 8 ).

[0038] Example 9

[0039] In order to evaluate the distribution of nanoparticles in vivo, in vivo near-infrared fluorescence imaging was performed. First, a mouse subcutaneous Panc02 tumor model was established, and the PDL1 inhibitor-adsorbed pyropheophorbide a covalently linked to nona-arginine nanoparticles encapsulated in macrophage membrane vesicles and the PDL1 inhibitor-adsorbed pyropheophorbide a covalently linked to nona-arginine nanoparticles not encapsulated in macrophage membrane vesicles were injected into the mice through the tail vein, and near-infrared fluorescence imaging of the mice was performed at 6h, 9h, 12h, and 36h, respectively (see Figure 2). Fig. 9 ), the nanoparticles wrapped in macrophage membranes had stronger fluorescence in the tumor site, indicating that the wrapping of macrophage membranes improved the tumor targeting of nanoparticles. The group of macrophage membrane vesicles wrapped with PDL1 inhibitor-adsorbed pyropheophorbide a covalently linked to nona-arginine nanoparticles plus laser irradiation was laser irradiated at 12h, with a laser intensity of 8mW / cm 2 The irradiation time was 2 min, and near-infrared fluorescence imaging of mice was performed at 6 h, 9 h, 12 h, and 36 h (see Appendix Figure 6 ). In vivo fluorescence imaging images are shown in the attached Figure 6 As shown, it can be seen that the enrichment of tumor tissue in the laser irradiation group was significantly increased under the mediation of nitric oxide produced in response to laser irradiation, which strongly proves that the nitric oxide produced by the prepared nanoparticles can effectively increase the enrichment in the tumor site.

[0038] Example 10

[0039] In vivo treatment combined experiments investigated whether the prepared M1@PAP nanoparticles can effectively inhibit tumor growth. Nude mice bearing SW1990 subcutaneous tumors were randomly divided into 7 groups: G1-G7, and given different treatment regimens, including G1: normal saline; G2: M1 macrophage membrane vesicles encapsulated pyropheophorbide a covalently linked to nona-arginine nanoparticles without laser irradiation; G3: M1 macrophage membrane vesicles encapsulated pyropheophorbide a covalently linked to nona-arginine nanoparticles adsorbed with PDL1 inhibitor without laser irradiation; G4: PDL1 inhibitor adsorbed pyropheophorbide a covalently linked to nona-arginine nanoparticles G5: M0 macrophage membrane vesicles encapsulated with pyropheophorbide a covalently linked to nona-arginine nanoparticles + laser irradiation; G6: M1 macrophage membrane vesicles encapsulated with pyropheophorbide a covalently linked to nona-lysine nanoparticles + laser irradiation; G7: M1 macrophage membrane vesicles encapsulated with pyropheophorbide a covalently linked to nona-arginine nanoparticles + laser irradiation. The administration method was 200ul injection through the tail vein. After treatment, the tumor volume and body weight changes of each group of mice were recorded every day (tumor volume = 1 / 2 × length × width 2 The M1@PAP+US group had a significant inhibitory effect on tumor growth, with an inhibition rate of ~93.7% after treatment. This therapeutic effect may be due to the fact that the combined use of PPA and laser irradiation can produce a certain amount of ROS at a lower dose of PPA (1 mg / kg), caused by nitric oxide and PDL1 small molecule inhibitor (siPDL1). The results are shown in the attached Figure 7 shown.

Claims

1. A targeted nanoparticle for simultaneously delivering a photosensitizer and siRNA, wherein: The nanoparticles are prepared from pyropheophorbide a and nona-arginine to form positively charged photosensitizer nanoparticles, which can generate superoxide anions and hydroxyl radicals under hypoxic conditions and have the properties of a type I photosensitizer. Then, negatively charged siRNA is loaded using the principle of electrostatic adsorption, and the mass ratio of the nanoparticles to siRNA is 1:3, encapsulating M1 macrophage membrane vesicles.

2. A targeted nanoparticle for simultaneously delivering a photosensitizer and siRNA as claimed in claim 1, characterized in that: The photosensitizer in the lipidated photosensitizer is selected from pyropheophorbide a; the siRNA is selected from PDL1 siRNA, and the sequence is GGCGUUUACUGCUGCAUAATT; and the NO precursor is nona-arginine.

3. A targeted nanoparticle for simultaneously delivering a photosensitizer and siRNA as claimed in claim 2, characterized in that: The photosensitizer pyropheophorbide a is covalently linked to the nitric oxide precursor nona-arginine, and its structural formula is as follows:

4. A targeted nanoparticle for simultaneously delivering a photosensitizer and siRNA as claimed in claim 1, characterized in that: The average particle size of the nanoparticles is 126±12nn.

5. The targeted nanoparticle for simultaneously delivering a photosensitizer and siRNA according to claim 1, characterized in that: In the components constituting the targeted nanoparticles, based on a total amount of 100 micrograms, the content of pyropheophorbide a is about 11.2, and the content of the nitric oxide precursor nona-arginine is about 29.

6.

6. The method for preparing photodynamic nanoparticles for simultaneously delivering photosensitizer and siRNA according to any one of claims 1 to 5, comprising the following steps: 1) dissolving pyropheophorbide a in N,N-dimethylformamide to obtain solution A; 2) dissolving the NO precursor in N,N-dimethylformamide to obtain solution B; 3) dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in N,N-dimethylformamide to obtain solution C; 4) macrophages were co-incubated with lipopolysaccharide for 24 h to obtain pro-inflammatory macrophages, and pro-inflammatory macrophage cell membrane vesicles (M1 macrophage cell membrane vesicles) were extracted by ultrasonic disruption and differential centrifugation; 5) dissolving N-hydroxysuccinimide (NHS) in N,N-dimethylformamide to obtain solution D; 6) Solutions A, C, and D were mixed and reacted under magnetic stirring for 12 h, and then the product was purified by column chromatography to remove unreacted compounds and other by-products; 7) mixing the product obtained in step 6) with solution B, reacting under magnetic stirring for 24 hours, and then injecting into a PBS solution or a physiological saline under magnetic stirring to form nanoparticles, and then dialyzing in a PBS solution or a physiological saline to remove the organic solvent to form nanoparticles loaded with a photosensitizer and a nitric oxide precursor; 8) mixing the nanoparticles obtained in step 7) with siRNA in a PBS solution or physiological saline, and stirring for half an hour in a dark environment to obtain a photosensitizer particle dispersion loaded with siRNA; 9) The siRNA-loaded photosensitizer particle dispersion obtained in step 8) and M1 macrophage membrane vesicles are physically extruded through an Avanti microliposome extruder to obtain targeted nanoparticles that simultaneously deliver photosensitizer and siRNA.

7. The preparation method according to claim 6, characterized in that: The nanoparticles loaded with photosensitizer and nitric oxide precursor in step 7) are mixed in step 8) at a mass ratio of 1:3 between nanoparticles and siRNA; in step 9), the photosensitizer particle dispersion loaded with siRNA obtained in step 8) is mixed with M1 macrophage membrane vesicles at a mass ratio of 1:

1.

8. Use of the targeted nanoparticles for simultaneously delivering photosensitizer and siRNA as claimed in any one of claims 1 to 7 as a drug for diagnosing and / or treating cancer.

9. The use according to claim 8, characterized in that The loaded siRNA is PDL1 siRNA, and the cancer is pancreatic cancer.

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