Platelet targeting peptide coupled iridium oxide nanoparticles and preparation method and application thereof

Abstract

The present invention discloses a platelet-targeting peptide conjugated iridium oxide nanoparticle and its preparation method and application. The structural formula of the nanoparticle is as follows: IrO x -P; wherein, P represents a P-selectin targeting peptide, and the amino acid sequence is as shown in SEQ ID No.1; IrO x -P, in which Ir 3+ and Ir 4+ coexist. The IrO x -P nanoparticles provided by the present invention utilize photothermal-induced vascular damage to achieve secondary enrichment of IrO x -P, realizing the combined and highly efficient treatment of PTT and ferroptosis, while reducing the side effects brought by PTT. In addition, IrO x -P can achieve oxidation-enhanced photoacoustic imaging, which will provide new ideas and methods for tumor-targeted diagnosis and treatment integration.

Description

Technical Field

[0001] The invention relates to the field of nanomedicine, and in particular to a platelet targeting peptide coupled iridium oxide nanoparticle, and a preparation method and application thereof. Background Art

[0002] Photothermal therapy (PTT) uses the specific light absorption of exogenous photothermal agents to cause tissues to heat up rapidly, thereby killing tumors. Due to its advantages such as low invasiveness, high temporal and spatial precision, and controllable killing area, PTT has received extensive attention in recent years and is considered to be a highly promising new anti-tumor therapy.

[0003] In order to increase the local enrichment of photothermal agents in tumor tissues, a series of photothermal agents based on nanocarriers have been developed, such as gold nanoparticles, carbon dots, and metal oxide nanoparticles. They usually use the enhanced permeability and retention effect of tumor tissues to achieve passive transport, or achieve active targeting by coupling tumor-targeting ligands.

[0004] However, despite the fact that PTT has been studied for many years, its clinical transformation has been limited, and no nanophotothermal agent has been approved for clinical PTT. Among them, the low light penetration depth, the limited selectivity and targeting of photothermal agents to tumor tissues, and the damage of photothermal agents to the surrounding tumor and skin tissues are considered to be the main factors restricting the further clinical application of PTT.

[0005] Passive transport strategies based on the EPR effect (high permeability and retention effect of solid tumors) are widely used in the development of photothermal nanocarriers. However, the vascular permeability and maturity of tumor blood vessels are highly heterogeneous, and the influence of extracellular matrix and interstitial fluid pressure leads to limited enrichment effects and difficulty for photosensitizers to penetrate tumor tissues. Due to the ubiquitous heterogeneity of tumor cell surface marker expression, active targeting methods also face problems such as failure and off-target. The above reasons lead to incomplete PTT treatment and increased risk of tumor recurrence, further hindering the clinical transformation of PTT. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a platelet targeting peptide coupled iridium oxide nanoparticles and a preparation method and application thereof. The present invention provides photothermal-induced recruitment of P-selectin targeting peptide (PSN) coupled iridium oxide (IrOx) nanoparticles (IrOx-P), which have good anti-tumor effects.

[0007] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a platelet targeting peptide coupled to iridium oxide nanoparticles, wherein the structural formula of the nanoparticles is as follows: IrO x-P; wherein P represents P-selectin targeting peptide PSN, the amino acid sequence is shown in SEQ ID No.1, and x represents 3-4, i.e., IrO x -P in Ir 3+ and Ir 4+ Coexistence, among which Ir 4+ The proportion is 30-50%.

[0009] As a further description of the above scheme: the IrO x -P's Z average particle size is 40-55nm, The potential is -14~-16mV. Appropriate particle size and appropriate potential help IrO x -P plays a role in living organisms.

[0010] In a second aspect, the present invention provides a method for preparing the platelet targeting peptide coupled iridium oxide nanoparticles, the method is performed as follows:

[0011] Step (1) Synthesis of IrO x : Dissolve iridium trichloride in water, stir at room temperature, filter the iridium trichloride aqueous solution with a filter membrane, add sodium citrate, adjust the pH with alkali solution, heat and stir, and you can get IrO x Nanoparticles;

[0012] Step (2) Synthesis of IrO x -PEG: First synthesize IrO x -NH2, take IrO x The powder was ultrasonically dispersed in DMF, and (3-aminopropyl)triethoxysilane was added dropwise, and the reaction was stirred under heating in an oil bath; then the powder was washed with DMF, and then with ethanol, and freeze-dried to obtain IrO x -NH2;

[0013] Weigh NH2-PEG2000-COOH, EDC and NHS in DMF and disperse them by ultrasonic. Then, stir in the dark and add IrO x -NH2 continued to react; after the reaction was completed, it was dialyzed and freeze-dried to obtain the product IrO x -PEG;

[0014] Step (3) Synthesis of IrO x -P: P-selectin targeting peptide PSN, NHS and EDC were weighed in sequence, dissolved in DMF, stirred continuously in the dark to obtain P-selectin targeting peptide PSN after activation of carboxyl group, and then added dropwise to IrO x -PEG aqueous solution, then kept stirring in the dark; dialyzed with a dialysis bag to remove unreacted reagents and by-products to obtain nanoparticles of IrO x -P.

[0015] Preferably, step (1) synthesizes IrO x : Dissolve iridium trichloride in water, stir overnight at room temperature, and then put it in the refrigerator for 2-5 days. Filter the iridium trichloride aqueous solution with a filter membrane, add sodium citrate, adjust the pH to 9.5-10.5 with alkaline solution, stir vigorously at 90-110℃, check the pH every 20-40 minutes, and adjust the pH to 9.5-10.5 until the pH is stable, and continue stirring for 1-3 hours to obtain IrO x Nanoparticles; the mass ratio of iridium trichloride to sodium citrate is 1:2-5. Adding sodium citrate to the reaction system can ensure that a suitable particle size is obtained instead of agglomerating into blocks.

[0016] Preferably, in step (2), IrO x The mass volume ratio of powder, DMF and (3-aminopropyl)triethoxysilane is 50-90 mg: 10-50 mL: 200-400 μL; react in an oil bath at 60-90° C. with stirring for 6-12 hours;

[0017] The NH2-PEG 2000 The mass volume ratio of -COOH, EDC, NHS, DMF and IrO2-NH2 is 0.3-0.8 g: 0.05-0.2 g: 0.1-0.2 g: 3-10 mL: 5-15 mg.

[0018] Preferably, step (3) synthesizes IrO x -P: Weigh the P-selectin targeting peptide PSN, NHS and EDC in turn, dissolve the weighed reagents in DMF, and stir continuously for 2-6 hours in the dark; add the activated carboxyl group PSN dropwise to the IrO x -PEG aqueous solution, then kept stirring for 8-16 hours in the dark; dialyzed for 40-60 hours using a dialysis bag with a molecular weight cutoff of 8000-12000 Da to obtain IrO x -P;

[0019] P-selectin targeting peptides PSN, NHS, EDC, DMF and IrO x -PEG mass volume ratio is 8-12mg:120-180mg:80-120mg:3-8mL:8-15mg; IrO x The concentration of the PEG aqueous solution is 8-12 mg / mL.

[0020] The third aspect of the present invention provides the use of the platelet targeting peptide coupled to iridium oxide nanoparticles in the preparation of anti-tumor drugs.

[0021] Preferably, the tumor is breast cancer, which is highly enriched in IrO x-P under low power illumination (808 nm, 450mW / cm 2 ) under the condition of rapid temperature rise to above 50℃, killing breast cancer cells.

[0022] In a fourth aspect, the present invention further provides the use of the platelet targeting peptide coupled iridium oxide nanoparticles in the preparation of a photoacoustic imaging contrast agent; the reaction of the nanoparticles with H2O2 to generate O2 can enhance the photoacoustic signal.

[0023] The present invention develops a photothermal-induced recruitment of P-selectin targeting peptide (PSN) coupled to iridium oxide (IrO x ) nanoparticles (IrO x -P). IrO x After intravenous injection, -P is first enriched in the tumor tissue through the EPR effect. After the first round of light irradiation (808nm), with the help of IrO x The photothermal effect of the tumor tissue gently raises the local temperature to 43°C to 44°C, inducing acute vascular damage and recruiting activated platelets. Activated platelets highly express P-selectin, which further recruits PSN-coupled IrO x -P reaches the injury site and promotes IrO x -P secondary targeting. At the same time, vascular damage can promote IrO x -P penetrates into the tumor parenchyma. After the second illumination, the highly enriched IrO x -P under low power illumination (808 nm, 450 mW / cm 2 ) under the condition of rapid temperature rise to above 50℃, killing tumor cells.

[0024] Ir x -P can consume glutathione (GSH) and has peroxidase (POD) activity. x -P contains Ir 3+ and Ir 4+ , Ir 4+ In the acidic microenvironment, it undergoes redox reaction with GSH, consumes GSH, inhibits GPX4 activity, and Ir 4+ During the reaction, it is converted into Ir 3+ .Ir 3+ In the acidic microenvironment, it can react with H2O2 in tumor tissue to produce a Fenton-like reaction, exerting the function of POD and generating hydroxyl radicals ( OH). GSH consumption and The production of OH will induce ferroptosis in cells, and these reactions are x -P is enhanced by the photothermal effect. Ultimately, PTT and ferroptosis synergize to improve the efficacy.

[0025] In addition, IrO x-P itself can be used as a photoacoustic imaging contrast agent, and its reaction with H2O2 to generate O2 can enhance the photoacoustic signal.

[0026] Compared with the prior art, the present invention has the following beneficial effects: the IrO x -P nanoparticles use photothermal induced vascular damage to achieve IrO x -P secondary enrichment, to achieve the combined and efficient treatment of PTT and ferroptosis, while reducing the side effects of PTT. x -P enhanced photoacoustic imaging will provide new ideas and methods for the integrated targeted diagnosis and treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 IrO x Nanoparticle characterization: A is IrO x Transmission electron microscopy images; B and C are IrO x Elemental energy spectrum scanning of Ir and O in the medium; D is different concentrations of IrO x Absorption spectrum curve of IrO; E is the absorption spectrum of IrO detected by X-ray electron spectroscopy (XPS) x Medium 3+ and Ir 4 + F is the oxide or hydroxide of Ir detected by XPS; G is IrO x -P transmission electron microscope image; H is the Fourier transform infrared spectroscopy detection of IrO x 、IrO x -PEG and IrO x -P is the spectral transmittance; I is IrO x and IrO x -P hydrated particle size intensity distribution.

[0028] Figure 2 IrO x -P nanoparticles photothermal performance: A is the different powers of irradiation IrO x -P (100 g / mL), temperature change curve within 15 minutes; B is 1.0 W / cm 2 Under laser irradiation, different concentrations of IrO x -P solution temperature curve; C is 1.0 W / cm 2 Under laser irradiation, IrO x -P nanoparticles temperature variation curve during 5 switching cycles.

[0029] Figure 3 IrO x -P nanoparticles consume GSH: A is the effect of different pH on IrO x-P consumption of GSH, GSH content is shown by the absorption spectrum of DTNB (5,5'-dithiobis(2-nitrobenzoic acid), the higher the absorbance at 412 nm, the higher the GSH content; B is the detection and statistical results of DTNB absorbance at 412 nm for different pH groups; C is the detection of IrO by XPS x -P in Ir 3+ and Ir 4+ D is the ratio of IrO after reaction with GSH detected by XPS x -P in Ir 3+ and Ir 4+ The ratio of E is the ratio of IrO irradiated with light of different wavelengths. x -P influence on the consumption of GSH; F is the detection and statistical DTNB absorbance results at 412 nm for different power density groups.

[0030] Figure 4 IrO x -P nanoparticles produce hydroxyl radicals: AC is used for TMB color reaction to detect peroxidase activity, IrO x -P reacts with H2O2 to produce OH catalyzes TMB, and the enzyme activity is reflected by detecting the change in absorbance at 652 nm; AC is different IrO x -P concentration, different pH, and different H2O2 concentrations. x -P peroxidase activity detection. EG were detected by methylene blue to detect IrO x -P reacts with H2O2 to produce OH capacity; D is the capacity of IrO under different pH conditions x -P reacts with H2O2 to produce OH. E is the IrO x -P first reacts with GSH and then with H2O2; F is the IrO x -P reacts with H2O2 to produce Figure G shows the ESR detection of OH.

[0031] Figure 5 IrO x -P nanoparticles induce cell death and ROS production: A is IrO x -P-Cy5 and breast cancer cell 4T1 cells were incubated for different time periods, and the cell uptake was shown. The stronger the red fluorescence in the cell, the greater the uptake. B is CCK8 detection; C is the detection of IrO by DCFH-DA and HPF respectively. x -P produces ROS and OH situation.

[0032] Figure 6 IrO x -P photothermal effect enhances cell ferroptosis: A is IrO x -P caused changes in intracellular GSH content under non-illumination and illumination conditions; B is IrO x -P caused changes in the expression of GPX4 protein in cells under non-light and light conditions; C is IrO x -P shows the changes of intracellular LPO caused by non-illumination and illumination conditions; DH shows the rescue of cell death by treating cells with different concentrations of ferroptosis inhibitors such as GSH, Fer-1, DFOM, Lip-1 and VE, followed by photothermal reaction; IK shows the rescue of cell death by treating cells with different concentrations of apoptosis inhibitor Z-VAD-FMK, autophagy inhibitor 3-MA and necrosis inhibitor Necrostatin-1, followed by photothermal reaction.

[0033] Figure 7 To promote the photothermal x -P enrichment in tumor sites: A is the detection of IrO by flow cytometry x P、IrO x -S or PSN polypeptide pretreatment with activated or unactivated platelets; B is the tail vein injection of IrO x -P or IrO x -S mice underwent the first round of illumination, and the temperature was maintained at 43-44 degrees; C is the vascular damage reflected by Dextran after the first round of illumination; D and E are the comparisons of tumor enrichment on the left and right sides before and after the first round of illumination (the right tumor of the mouse was illuminated).

[0034] Figure 8 In vivo efficacy results: A and B are the temperature changes of tumors in different groups of mice during illumination; C and D are the changes in tumor volume in different groups after treatment; E is the H&E and Ki67 staining results of tumor tissues in different groups after treatment; F is the change in body weight of mice in different groups after treatment.

[0035] Fig. 9 The safety of treatment was rated as follows: A and B for hemolysis assessment; C for major organ testing.

[0036] Fig.10 IrO x -P oxygen production and photoacoustic imaging: A is the detection of IrO in a test tube using a probe x -P is the comparison of oxygen production before and after adding NAC to remove ROS; B is the detection of IrO by probe x -P oxygen production in cells; C and D are the detection of IrO in test tubes x-P and H2O2 reaction on the intensity of photoacoustic signal; E and F are the effects of adding NAC to remove ROS on the intensity of photoacoustic signal in a test tube; G and H are the effects of injecting NAC to remove ROS on the intensity of photoacoustic signal in a 4T1 mouse subcutaneous tumor model. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution.

[0038] Example 1 Iridium oxide (IrO x ) Nanoparticle synthesis steps and characterization

[0039] Synthesis of IrO x :298.56 mg of iridium trichloride was dissolved in 20 mL of ultrapure water, stirred magnetically at room temperature overnight, and then placed in a 4°C refrigerator for 3 days. The iridium chloride aqueous solution was filtered with a 0.22 m filter membrane, 895.68 mg of sodium citrate was added, and the pH was adjusted to 10.0 with NaOH. The solution was stirred vigorously at 100°C, the pH was checked every 30 minutes, and the pH was adjusted to 10.0. After the pH stabilized, the stirring was continued for another 2 hours to obtain IrO x Nanoparticles.

[0040] Synthesis of IrO x -P: First synthesize IrO x -NH2 takes 70 mg IrO x The powder was ultrasonically dispersed in 30 mL DMF, and 280 μL (3-aminopropyl)triethoxysilane was added dropwise, and stirred in an 80 °C oil bath for 8 h. Then, it was washed three times by centrifugation with DMF at 13000 rpm for 10 min, and then washed three times with ethanol, and freeze-dried to obtain IrO x -NH2.

[0041] Weigh 0.5 g NH2-PEG 2000 -COOH (0.25 mmol) was added with 0.1 g (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) EDC (0.5 mmol) and 0.15 g N-hydroxysulfosuccinimide NHS (1.25 mmol), and ultrasonically dispersed in 5 mL DMF (dimethylformamide) for 30 min; then stirred in dark for 12 h, and 10 mg IrO x -NH2 continued to react for 3 days; after the reaction was completed, it was dialyzed for 48 hours and freeze-dried to obtain the product IrO x -PEG.

[0042] Weigh 10 mg of PSN (P-selectin targeting peptide PSN, amino acid sequence as shown in SEQ ID No. 1, DAEWVDVS), 150 mg of NHS, and 100 mg of EDC in turn, dissolve the weighed reagents in 5 mL of DMF, and stir continuously for 4 h in the dark; add the activated carboxyl peptide dropwise to 1 mL of 10 mg / mL IrO x -PEG aqueous solution, then kept stirring for 12 h in the dark; dialyzed for 48 h using a dialysis bag with a molecular weight cutoff of 10000 Da to remove unreacted reagents and by-products.

[0043] In step (1) of the above-mentioned synthesis method, sodium citrate is mainly used as a stabilizer. If the pH is not controlled during the reaction, agglomeration will occur, and nanoparticles of IrO with good dispersion cannot be prepared. x .

[0044] Prepared IrO x Nanoparticle Characterization: Figure 1 A is IrO x The transmission electron microscopy (TEM) image of the nanoparticles shows that they are spherical nanoparticles with good dispersion and a particle size of 31.7 4.5 nm. Figure 1 B and C are element mappings. It can be seen that IrO x The nanoparticles are composed of Ir and O elements. Figure 1 D shows that the synthesized IrO x The nanoparticles have the strongest absorption peak at ~580 nm. From the Ir 4f spectrum ( Figure 1 E) can be seen in IrO x Nanoparticles contain Ir 4+ (~63.73 eV, 66.84 eV) and Ir 3+ (~62.16 eV, 65.19 eV), O 1s spectrum ( Figure 1 F) can be seen in IrO x The nanoparticles contain Ir-O (~530.69 eV) and Ir-OH (~531.72 eV).

[0045] Ir x -P nanoparticle characterization: First, TEM analysis of IrO x -P morphology is imaged, and it can be seen that in IrO x The nanoparticles are covered with a shell ( Figure 1 G, indicated by a red arrow). Measured by Fourier transform infrared spectroscopy (FTIR), 1636 cm -1 、1538 cm -1The absorption peaks of amide I and amide II are at 2928 cm -1 、2875 cm -1 -CH stretching vibration, which proves that iridium oxide is successfully PEGylated and the peptide is subsequently connected to the PEG molecule through amidation reaction, 3265 cm -1 The peak at is the NH stretching vibration peak, which further indicates that the peptide coupling is successful ( Figure 1 H). Figure 1 I is the dynamic light scattering detection of IrO x and IrO x -P hydrated particle size results, IrO x The hydrated particle size is 34.6 nm, the potential is -31.6 mV, and the average particle size of IrOx-P Z is 49.7 nm. The potential is -15.6 mV.

[0046] Example 2 IrO x -P nanoparticles performance determination

[0047] 1. IrO x Photothermal properties of -P nanoparticles

[0048] IrO x The photothermal properties of IrO-P nanoparticles were tested. First, under 808 nm laser irradiation with different powers, x -P (100 g / mL) temperature changes were detected. The higher the irradiation power, the higher the IrO x -P nanoparticles heat up faster ( Figure 2 A), and at a fixed power of 1.0 W / cm 2 Under 808 nm laser irradiation, the temperature of 100 g / mL IrOx-P nanoparticles can be raised from room temperature to about 60°C within 5 minutes, and the higher the concentration, the faster the temperature rise ( Figure 2 B). To test IrO x -P nanoparticles photothermal stability, at 1.00 W / cm 2 Under 808 nm laser irradiation, IrO x -P nanoparticles (100 μg / mL) were used for 5 cycles of laser switching experiments and it was found that IrO x -P nanoparticles showed no significant changes in the photothermal heating and cooling curves ( Figure 2 C) by Figure 2 It can be seen that IrO x -P nanoparticles have good photothermal conversion and photothermal stability.

[0049] 2. IrO x -P nanoparticles consume GSH

[0050] IrO was detected by DTNB x -P nanoparticles have the ability to consume GSH (glutathione). The results show that under acidic conditions, IrO x -P can consume GSH ( Figure 3 A and Figure 3 B). From the Ir 4f spectrum, it can be seen that after incubation with GSH for 30 min, Ir 3+ The proportion increased from 68.62% to 85.24% ( Figure 3 C and Figure 3 D), proved that IrO x -P undergoes a redox reaction with GSH. As the 808nm laser power increases, IrO x -P's GSH consumption capacity increased, indicating that light and heat promoted GSH consumption ( Figure 3 E and Figure 3 F).

[0051] 3. IrO x -P nanoparticles produce hydroxyl radicals ( OH)

[0052] First, TMB color reaction was used to detect IrO x The peroxidase activity of -P was detected. Figure 4 AC is different for different IrO x -P concentration, different pH, and different H2O2 concentrations. x -P peroxidase activity test, the results showed that IrO x -P has good peroxidase activity under acidic conditions. IrO x -P reacts with H2O2 to produce OH's ability, Figure 4 D shows that under acidic conditions, IrO x -P reacts with H2O2 to produce OH. IrO x -P first reacts with GSH and then with H2O2 to Figure 4 Figure E shows that IrO x After the redox reaction between -P and GSH, it reacts with H2O2 to produce The ability of OH to increase is shown by the Ir 3+ The ratio is increased, which is beneficial to the reaction with H2O22 to produce OH. From Figure 4 As can be seen from Figure F, the increase in laser irradiation power, i.e., photothermal effect, is conducive to the reaction with H2O2 to produce OH. ESR detection further determined that IrO x -P can react with H2O2 to produce OH( Figure 4 G).

[0053] 4. IrO x Photothermal effect of -P induces cell death and ROS production in vitro

[0054] Evaluation of IrO by confocal microscopy x -P cellular uptake of IrO x -P-Cy5 (100 μg / mL) (Cy5 anthocyanin 5 is a fluorescent dye conjugated to IrO x -P, the uptake of IrO by cells can be observed by fluorescence microscopy x -P) were co-cultured with mouse breast cancer 4T1 cells for 0 h, 4 h, and 8 h. Figure 5 The results of A show that the cell uptake rate increases with the increase of incubation time. x -P in vitro therapeutic effect, divided into IrO x -P and IrO x -P+Light lighting group, first IrO x Co-culture with 4T1 cells for 8 h, aspirate the medium, replace with fresh serum-free medium and continue culturing for 12 h. x -P concentration as Figure 5 As shown in B, the results show that pure IrO x -P effect, can produce a certain killing effect, 200μg / mL IrO x -P can cause about 49.3% cell death. On this basis, 808 nm light irradiation (1.0 W / cm 2 ) can significantly enhance the IrO x -P killing effect, 200μg / mL IrO x -P can cause about 81.5% cell death ( Figure 5 B). IrO was evaluated by DCFH-DA (2,7-dichlorofluorescein diacetate) and HPF (hydroxyphenylfluorescein) probes, respectively. x -P has the ability to produce total ROS (reactive oxygen species) and OH, and the results show that IrO x -P can generate ROS and OH, and photothermal therapy can significantly increase IrO x -P produces ROS and The ability of OH ( Figure 5 C). The above results show that IrO x The photothermal effect of -P has a good cell killing effect and can promote the production of ROS.

[0055] 6. IrO x -P photothermal effect enhances cellular ferroptosis

[0056] In order to clarify IrO x We first evaluated whether IrO-P induced ferroptosis by consuming GSH (glutathione) in cells. x -P was co-incubated with 4T1 cells, and the GSH content was detected by cell lysate. The results showed that IrO x -P can consume intracellular GSH ( Figure 6 A), down-regulation of GPX4 (glutathione peroxidase 4) protein expression ( Figure 6 B), while photothermal therapy can further promote GSH consumption and thus downregulate GPX4 ( Figure 6 A and Figure 6 B). During cell culture, the addition of GSH can rescue cell death caused by photothermal therapy to a certain extent ( Figure 6 D), proving that GSH consumption plays an important role in the cell death induced by the photothermal effect of IrOx-P. x -P-mediated photothermal therapy enhances cell death by promoting GSH depletion.

[0057] Then through C11-BODIPY 581 / 591 The probe detects intracellular lipid peroxide (LPO) levels, which promote C11-BODIPY 581 / 591 The fluorescence emission peak of the probe shifted from ~590 nm to ~510 nm. The experimental results showed that IrO x -P incubation can increase LPO production to a certain extent, while photothermal therapy significantly increases LPO levels ( Figure 6 C).

[0058] Further experiments found that the addition of ferroptosis inhibitors Fer-1, deferoxamine mesylate DFOM, lipid peroxide inhibitor Lip-1 and antioxidant VE (vitamin E) could rescue IrO x -P Photothermal therapy-induced cell death further demonstrated the important role of ferroptosis ( Figure 6 E- Figure 6 H). The apoptosis inhibitor Z-VAD-FMK can also rescue IrO x -P cell death caused by photothermal therapy, indicating that photothermal therapy also effectively induced cell apoptosis ( Figure 6 I). The autophagy inhibitor 3-MA and necrosis inhibitor Necrostatin-1 could not rescue the cell death induced by IrOx-P photothermal therapy, indicating that IrO x -P Photothermal therapy does not induce autophagy and necrosis ( Figure 6 J and Figure 6 K).

[0059] In summary, the above cell experiments have proved that IrO x The photothermal effect of -P can enhance cell ferroptosis.

[0060] Example 3 IrO x -P nanoparticles for the treatment of tumors

[0061] 1. Photothermal promotion of IrO x -P enrichment in tumor sites

[0062] In order to verify that IrO x To target the ability of activated platelets, mouse blood was collected and platelets were activated with ADP (adenosine diphosphate, 20 μM) at 37°C for 30 min, and then with IrO x -P-Cy 5+ or IrO x -S-Cy 5+ (S is scramble, is a polypeptide sequence control, the amino acid sequence is shown in SEQ ID No. 2, AWVSDEVD) were incubated together at 37°C, and IrO was detected by flow cytometry. x -P-Cy 5+ and CD61-FITC + The results showed that when incubated with blood containing unactivated platelets, IrO x -P-Cy 5+ / CD61-FITC + The proportion of IrO was only 2.12%, while when incubated with blood containing activated platelets, IrO x -P-Cy 5+ / CD61-FITC + The proportion of IrO increased to 42.32%. x -P-Cy, then IrO x -P-FITC + / CD61-FITC + The proportion of IrO decreased to 7.13%. x- The binding of P to activated platelets depends on the PSN polypeptide. x When IrOx-S-Cy5 was co-incubated with activated platelets, 5+ / CD61-FITC + The proportion of IrO is only 3.05%, which further proves that x -P binding to activated platelets depends on PSN polypeptide ( Figure 7 A).

[0063] Subsequently, whether photothermal energy can effectively promote the IrO x The enrichment of -P in the tumor site was verified. A mouse breast cancer subcutaneous tumor model was established using 4T1 cells. Ten days after tumor formation, IrO x -P or IrO x -S, 4 h later, the mice were irradiated with 808 nm light, and the temperature of the tumor tissue was monitored in real time by a thermal imager, and its temperature was controlled at 43-44°C ( Figure 7 B) Irradiation for 5 minutes. Immunofluorescence showed that IrO x -P or IrO x -S-induced photothermal effect can cause vascular endothelial damage and increase vascular permeability, which is manifested as increased Dextran signal penetration in tumor tissue ( Figure 7 C). Then, small animal in vivo imaging was used to measure the IrO in the tumor site of mice before and after illumination. x -P-Cy5 and IrO x -S-Cy5 fluorescence signal intensity was detected, and the analysis results showed that light can significantly increase the x -P-Cy5 accumulates in tumor sites, but does not promote IrO x -S-Cy5 enrichment ( Figure 7 D and Figure 7 E). The above results show that IrO x -P induced mild photothermal (43~44℃) can promote the IrO x -P targeting at tumor sites.

[0064] 2. IrO x -P In vivo evaluation of photothermal ferroptosis

[0065] Based on the above experimental results, the following treatment plan was designed: 4T1 cells were used to construct a mouse breast cancer subcutaneous tumor model. Ten days after tumor formation, IrO x -P, and 4 hours later, the mice were exposed to light for the first time, the temperature was controlled at 43-44°C, and 24 hours later, the mice were exposed to light for the second time. Figure 8 As shown in the figure, multiple control groups were designed to evaluate the efficacy. First, the photothermal effect of the living body after two illuminations was evaluated, such as Figure 8 A and Figure 8 As shown in B, the first light exposure promotes the IrO x -P accumulation at the tumor site, during the second illumination, using only 450 mW / cm 2 The power density can raise the temperature of tumor tissue to above 50°C within 2 minutes. This power density is significantly lower than the currently commonly used 1000 mW / cm 2, which can effectively reduce damage to normal tissues.

[0066] from Figure 8 C and Figure 8 As can be seen from D, IrO x -P combined with secondary light irradiation can significantly inhibit tumor growth, while Fer-1 inhibitors can rescue IrO x- P-induced tumor suppression, indicating that IrO x -P photothermal effect indeed promoted tumor ferroptosis, and Ki67 staining also verified this result ( Figure 8 E). IrO x During the treatment, there was no significant change in the body weight of mice ( Figure 8 F).

[0067] 3. IrO x -P Safety of photothermal therapy

[0068] IrO x The safety of IrO-P photothermal therapy was evaluated. x -P does not cause hemolysis ( Fig. 9 A and Fig. 9 B) had no significant effect on the liver and kidney functions of mice ( Fig. 9 B) will not cause damage to major organs ( Fig. 9 C) had no significant effect on the liver and kidney functions of mice.

[0069] Example 4 IrO x Enhanced photoacoustic imaging effect of -P nanoparticles

[0070] In vitro detection of IrO using Ru(ddp)3Cl2 (tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride) probe x- The ability of P to produce O2, from Fig.10 A can be seen that as IrO x -P reacts with H2O2, and the fluorescence of Ru(ddp)3Cl2 gradually quenches, proving the generation of O2. When NAC (N-acetylcysteine) is added to remove H2O2, the fluorescence of Ru(ddp)3Cl2 remains basically stable, proving that the generation of O2 depends on H2O2. The 4T1 cell experiment also proved that IrO x -P can produce O2 in cells ( Fig.10 B).

[0071] IrO x -P in vitro photoacoustic imaging showed that the photoacoustic signal intensity increased with the increase of IrO x -P concentration increased, and the addition of H2O2 (1 mM) significantly increased the photoacoustic signal intensity ( Fig.10 C and Fig.10 D). Will IrO x -P (50 μg / ml) reacted with different concentrations of H2O2. Fig.10 E and Fig.10 It can be seen from F that as the H2O2 concentration increases, the photoacoustic signal intensity increases, and after adding NAC, the photoacoustic signal intensity decreases. The above experiments show that IrO x -P reacts with H2O2 to produce O2, which can enhance the photoacoustic signal.

[0072] Subsequently, a breast cancer cell 4T1 subcutaneous tumor model was constructed. 14 days after inoculation, the control group was injected with IrO x -P, experimental group injected with IrO x NAC (500 mg / kg) was injected intraperitoneally 2 h before -P, and photoacoustic imaging was performed before, 1 h, 4 h, and 8 h after injection. The results showed that after injection, the photoacoustic signal increased with time, while the early injection of NAC could significantly reduce the photoacoustic signal, further proving that IrO x -P reacts with H2O2 to produce O2, which can enhance the photoacoustic signal ( Fig.10 G and Fig.10 H).

[0073] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modification, equivalent replacement or improvement without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. A platelet targeting peptide coupled to iridium oxide nanoparticles, characterized in that: The structural formula of the nanoparticles is as follows: IrO x -P; wherein P represents P-selectin targeting peptide PSN, the amino acid sequence is as shown in SEQ ID No.1, and x represents 3-4.

2. The platelet targeting peptide coupled iridium oxide nanoparticles according to claim 1, characterized in that: The IrO x -P's Z average particle size is 40-55nm, The potential is -14 to -16 mV.

3. A method for preparing platelet targeting peptide coupled iridium oxide nanoparticles according to claim 1 or 2, characterized in that: The method operates as follows: Step (1) Synthesis of IrO x : Dissolve iridium trichloride in water, stir at room temperature, filter the iridium trichloride aqueous solution with a filter membrane, add sodium citrate, adjust the pH with alkali solution, heat and stir, and you can get IrO x Nanoparticles; Step (2) Synthesis of IrO x -PEG: First synthesize IrO x -NH2, take IrO x The powder was ultrasonically dispersed in DMF, and (3-aminopropyl)triethoxysilane was added dropwise, and the mixture was heated and stirred in an oil bath for reaction; the mixture was then washed with DMF, then with ethanol, and freeze-dried to obtain IrO x -NH2; Weigh NH2-PEG 2000 -COOH, EDC and NHS were ultrasonically dispersed in DMF; then stirred in the dark, and IrO2-NH2 was added to continue the reaction; after the reaction was completed, the product IrO2 was dialyzed and freeze-dried to obtain the product IrO x -PEG; Step (3) Synthesis of IrO x -P: P-selectin targeting peptide PSN, NHS and EDC were weighed in sequence, dissolved in DMF, stirred continuously in the dark to obtain P-selectin targeting peptide PSN after activation of carboxyl group, and then added dropwise to IrO x -PEG aqueous solution, then kept stirring in the dark; dialyzed with a dialysis bag to remove unreacted reagents and by-products to obtain nanoparticles of IrO x -P.

4. The method for preparing platelet targeting peptide coupled iridium oxide nanoparticles according to claim 3, characterized in that: Step (1) Synthesis of IrO x : Dissolve iridium trichloride in water, stir overnight at room temperature, and then put it in the refrigerator for 2-5 days. Filter the iridium trichloride aqueous solution with a membrane, add sodium citrate, adjust the pH to 9.5-10.5 with alkaline solution, stir vigorously at 90-110℃, check the pH every 20-40 minutes, and adjust the pH to 9.5-10.5 until the pH is stable, and continue stirring for 1-3 hours to obtain IrO x Nanoparticles; the mass ratio of iridium trichloride to sodium citrate is 1:2-5.

5. The method for preparing platelet targeting peptide coupled iridium oxide nanoparticles according to claim 3, characterized in that: In step (2), IrO x The mass volume ratio of powder, DMF and (3-aminopropyl)triethoxysilane is 50-90 mg: 10-50 mL: 200-400 μL; react in an oil bath at 60-90° C. with stirring for 6-12 hours; The NH2-PEG 2000 The mass volume ratio of -COOH, EDC, NHS, DMF and IrO2-NH2 is 0.3-0.8g: 0.05-0.2g: 0.1-0.2g: 3-10mL: 5-15mg.

6. The method for preparing platelet targeting peptide coupled iridium oxide nanoparticles according to claim 3, characterized in that: Step (3) Synthesis of IrO x -P: Weigh the P-selectin targeting peptide PSN, NHS and EDC in turn, dissolve the weighed reagents in DMF, and stir continuously for 2-6 hours in the dark; add the activated carboxyl group PSN dropwise to the IrO x -PEG aqueous solution, then kept stirred for 8-16 hours in the dark; dialyzed for 40-60 hours using a dialysis bag with a molecular weight cutoff of 8000-12000Da to obtain IrO x -P; P-selectin targeting peptides PSN, NHS, EDC, DMF and IrO x -PEG mass volume ratio is 8-12mg:120-180mg:80-120mg:3-8mL:8-15mg; IrO x The concentration of the PEG aqueous solution is 8-12 mg / mL.

7. Use of the platelet targeting peptide coupled iridium oxide nanoparticles according to claim 1 or 2 in the preparation of anti-breast cancer drugs.

8. Use of the platelet targeting peptide coupled iridium oxide nanoparticles according to claim 1 or 2 in the preparation of a photoacoustic imaging contrast agent.

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