Preparation method of platelet membrane modified platinum nanoparticle loaded iron-tungsten-oxygen ternary nanoplatform, product and application thereof

Abstract

The application discloses a preparation method of a platelet membrane modified iron-tungsten-oxygen ternary compound nano-platform loaded with platinum nanoparticles, and a product and application thereof, and comprises the following steps: 1) synthesizing the iron-tungsten-oxygen ternary compound through an organic phase thermal decomposition method; 2) synthesizing platinum nanoparticles; 3) adsorbing a dasatinib drug and the platinum nanoparticles on the iron-tungsten-oxygen ternary compound to form FPD; 4) preparing nanoscale platelet membrane vesicles; 5) preparing E5 peptide modified platelet membrane vesicles; and 6) ultrasonically dispersing the FPD and the E5 peptide modified platelet membrane vesicles to obtain the nano-platform. The nano-platform can efficiently load the chemotherapeutic drug dasatinib, has good biological safety and no toxic side effects, can target in-situ and circulating tumor cells, and can combine photothermal action, photodynamic action and drug chemotherapy to play the roles of resisting in-situ breast cancer and preventing breast cancer lung metastasis. Meanwhile, the excellent CT and MRI imaging characteristics of the nano-platform can be used for monitoring in-vivo tumors and drug conditions.

Description

Technical Field

[0001] This invention belongs to the fields of nanomaterials technology and biomedicine, and specifically relates to a method for preparing a platelet membrane-modified iron-tungsten-oxygen ternary compound nanoplatform loaded with platinum nanoparticles, as well as its products and applications. Background Technology

[0002] Breast cancer has become the most common cancer worldwide, accounting for 12.5% ​​of all new cancer cases globally each year. In 2020, there were 2.3 million new cases of breast cancer worldwide, with 685,000 deaths. The high mortality rate of breast cancer is related to its highly aggressive nature; approximately 30% of patients develop metastases during treatment. Lung metastasis is the second most common site of metastasis in breast cancer, and the prognosis after lung metastasis is very poor, with a 5-year survival rate of approximately 16.8%. With the development of treatment methods, current treatment for breast cancer primarily involves surgery, combined with adjuvant and neoadjuvant therapies. However, because tumor cells remain in the circulatory system and are difficult to eradicate, postoperative recurrence and metastasis are still possible. Furthermore, the high mortality rate from lung metastasis is related to the limited treatment options currently available. Therefore, there is an urgent need for new treatment methods to combat circulating tumor cells in breast cancer, reduce lung metastasis, and lower the mortality rate of breast cancer.

[0003] Lung metastasis of breast cancer is a complex, multi-step process closely related to local cell invasion, hematogenous and lymphatic spread, and distant colonization. Breast tumor cells can secrete various substances that degrade the extracellular matrix, causing them to detach from their original location, enter the circulatory system, and metastasize to distant organs. During this hypercoagulable state, activated platelets and vWF polymers can promote tumor cell migration and metastasis. The unique metabolic characteristics of tumors result in a hypoxic tumor microenvironment (TME), which leads to DNA methylation, the Warburg effect, and other factors that further promote tumor development and progression. Furthermore, during invasion, various proteases released by tumor cells or stromal cells, such as MMP-9 and cathepsins (CTS), degrade the basement membrane and connective tissue, breaking down barriers that hinder tumor cell migration. Moreover, high expression of CXCR4 in breast cancer cells enables them to migrate to target tissues rich in CXCL12 expression, such as the lung, liver, and bone, thus participating in breast cancer metastasis. In addition, studies have shown that changes in the lung microenvironment before breast cancer metastasis result in pre-metastatic lung niches exhibiting high CXCR4 expression. In recent years, many studies have explored the mechanisms of proteases and confirmed that they can serve as potential targets for inhibiting breast cancer metastasis and improving breast cancer prognosis. Dasatinib (DAS) is an FDA-approved tyrosinase (Scr) inhibitor. Recent studies have shown that DAS can inhibit the expression of proteases, thereby inhibiting breast cancer metastasis. However, its hydrophobicity limits its bioavailability, and it is usually used orally. It is also easily affected by diet and exhibits large individual variability.

[0004] In recent years, the development and application of nanomaterials have increased the applicability of many drugs by improving their bioavailability and reducing their toxic side effects, providing more possibilities for the treatment of diseases. However, due to their heterogeneity, they are easily recognized and eliminated by the body's immune system.

[0005] Photothermal therapy (PTT) utilizes photothermal conversion materials absorbed by cells to generate heat upon light absorption, leading to thermal damage and cell death in cancer cells. To avoid non-specific temperature increases in healthy cells, the material must have strong absorption in the near-infrared (NIR) light region. Compared to conventional cancer treatments (chemotherapy, radiotherapy, and surgery, etc.), the use of NIR light to induce localized temperature increases is particularly attractive because most biological systems lack absorption in the 700–1100 nm NIR region.

[0006] Photodynamic therapy (PDT) is a cytotoxic treatment method that induces apoptosis and necrosis by generating reactive oxygen species (ROS). Materials, upon receiving light energy of a specific wavelength, convert this light energy into intramolecular energy through photochemical reactions and energy transfer. Under aerobic conditions, this generates various reactive oxygen species, including singlet oxygen, oxygen free radicals, and hydroxyl radicals, which damage cellular components such as proteins and nucleic acids. This represents a promising strategy for anti-tumor therapy. Summary of the Invention

[0007] In view of the aforementioned problems, this invention provides a method for preparing a platelet membrane-modified iron-tungsten-oxygen ternary compound nanoplatform loaded with platinum nanoparticles, as well as its products and applications. The nanoplatform of this invention, through the embedding of platelet membrane vesicles, avoids recognition and clearance by the body's immune system, exhibiting good biocompatibility and no toxic side effects. Through surface-targeting molecule modification, combined with photothermal, photodynamic, and chemoradioactive effects, this nanoplatform can exert anti-tumor activity against breast cancer and prevent lung metastasis of breast cancer. Simultaneously, its excellent CT and MRI imaging characteristics can be used to monitor tumor and drug status in vivo.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] The method for preparing the iron-tungsten-oxygen ternary nanoplatform modified with platinum nanoparticles provided by this invention includes the following steps:

[0010] 1) Iron-tungsten oxide ternary compound (FeWOx) was prepared by organic phase thermal decomposition;

[0011] 2) Platinum nanoparticles (PtNPs) were prepared;

[0012] 3) Mix the FeWOx obtained in step 1) with the PtNPs obtained in step 2) to obtain iron tungsten oxide ternary compound (FP) loaded with platinum nanoparticles. Then mix the FP with the drug DAS to obtain iron tungsten oxide ternary compound (FPD) loaded with platinum nanoparticles and dasatinib drug.

[0013] 4) Collect anticoagulated fresh whole blood, separate platelets by centrifugation and washing, and extract platelet membranes by repeated freeze-thaw cycles to prepare nanoscale platelet membrane vesicles (PLTm);

[0014] 5) Mix E5 peptide with distearylphosphatidylethanolamine-amino polyethylene glycol (DSPE-PEG2000-NH2) to obtain distearylphosphatidylethanolamine modified E5 peptide (DSPE-E5). Mix DSPE-E5 with PLTm obtained in step 4) to obtain E5 peptide modified platelet membrane vesicles (E-PLTm).

[0015] 6) Mix the E-PLTm obtained in step 5) with the FPD obtained in step 3) to obtain the platelet membrane modified iron-tungsten-oxygen ternary nanoplatform E-PLTm@FPD loaded with platinum nanoparticles.

[0016] Step 1) specifically involves mixing sodium tungstate dihydrate, ferrous sulfate heptahydrate, and sodium dodecylbenzenesulfonate (SDBS) in a molar ratio of (0.8–1.2):(0.8–1.2):(1.8–2.2), adjusting the pH to 10, reacting at 170–190°C for 8–16 h, centrifuging and washing to obtain FeWOx.

[0017] Step 2) specifically involves dissolving polyvinylpyrrolidone (PVP) in a solution of chloroplatinic acid hexahydrate to form solution A, wherein the mass ratio of polyvinylpyrrolidone to chloroplatinic acid hexahydrate is (62-66).

[0018] (18-22); Sodium borohydride was dissolved in ice water to prepare solution B with a concentration of 0.5-0.7 mg / mL; solution B was added dropwise to solution A, and after reacting at room temperature for 3-5 h, the mixture was centrifuged and washed to obtain PtNPs.

[0019] Step 3) specifically involves: dispersing FeWOx and PtNPs separately in water, then mixing them together, wherein the mass ratio of FeWOx to PtNPs is 1:(5-10), stirring overnight, washing, and obtaining FP; dispersing FP in water, then mixing it with the drug DAS, wherein the mass ratio of FP to DAS is 1:(1-8), stirring for 20-28 hours, and then dialysis for 20-28 hours to remove free DAS, thereby obtaining FPD.

[0020] Before mixing FeWOx and PtNPs in step 3), FeWOx and PtNPs are stored away from light.

[0021] The specific process of extracting platelet membranes by repeated freeze-thaw cycles in step 4) is as follows: platelets are suspended in phosphate buffer, frozen at -80℃ for 30 min, then thawed at 37℃ for 30 min, repeated 3 times, centrifuged, and platelet membranes are extracted.

[0022] The specific process of preparing platelet membrane into PLTm in step 4) is as follows: the platelet membrane is suspended in phosphate buffered saline, treated with ultrasound, and then extracted back and forth using a 200-400nm polycarbonate porous membrane filter to obtain PLTm.

[0023] In step 5), the E5 peptide has the sequence shown in Sequence 1, specifically: Gly-Gly-Arg-Ser-Phe-Phe-Leu-Leu-Arg-Arg-Ile-Gln-Gly-Cys-Arg-Phe-Arg-Asn-Thr-Val-Asp-Asp; the specific process of step 5) is as follows: E5 peptide and DSPE-PEG2000-NH2 are dissolved in phosphate buffered saline, wherein the mass ratio of E5 peptide to DSPE-PEG2000-NH2 is 1:(4-6), and reacted at 35-39℃ for 4-8h to obtain DSPE-E5; then DSPE-E5 and PLTm are mixed at a mass ratio of (2-5):1, and stirred at 35-39℃ for 4-8h to obtain E-PLTm.

[0024] The specific process of step 6) is as follows: E-PLTm and FPD are mixed at a mass ratio of (1-4):1, dispersed and fused by ultrasound, squeezed back and forth with a 200-400nm needle filter, and then centrifuged to remove the supernatant to obtain E-PLTm@FPD.

[0025] A platelet membrane-modified iron-tungsten-oxygen ternary nanoplatform loaded with platinum nanoparticles was prepared according to the above preparation method.

[0026] Application of the iron-tungsten-oxygen ternary nanoplatform modified with platelet membrane and loaded with platinum nanoparticles as a targeted cancer drug.

[0027] The beneficial effects of this invention are:

[0028] The E-PLTm@FPD nanoplatform of this invention is a multifunctional nanoplatform that exhibits excellent photothermal (PTT) and photodynamic (PDT) effects under 808nm laser irradiation. Furthermore, it possesses peroxidase-like activity, capable of decomposing H2O2 into O2. This improves tumor hypoxia, creating a favorable metastatic microenvironment for breast cancer, and provides more raw materials for photodynamic therapy. Its Pt can also be converted into Pt2. 2+ It binds to DNA and induces DNA damage. Due to its Fe and W content, it also possesses excellent MRI and CT imaging capabilities, aiding in the diagnosis and drug tracking of breast cancer. To achieve high targeting of circulating tumors, this invention constructs a novel drug delivery system, E-PLTm@FPD, by camouflaging and modifying FPD with a novel platelet membrane. This system targets in situ and circulating tumor cells, as well as pre-metastatic niches in the lungs, via E5 peptide and PLTm, competitively inhibiting the binding of breast tumor cells to platelets, thereby mitigating the invasive process. It releases the small molecule drug dasatinib (DAS) to inhibit tumor cell growth. Furthermore, it combines with FP to enhance PDT and PTT effects, killing tumor cells. In addition to its killing effect on in situ tumors, E-PLTm@FPD can also capture tumor cells in the bloodstream, preventing lung metastasis of breast cancer. Simultaneously, this multifunctional nanoplatform has MRI and CT imaging capabilities, allowing for the tracking of disease progression and treatment efficacy.

[0029] This invention utilizes the platelet membrane to retain characteristic proteins on the platelet surface, thus preventing the nano-drug delivery system from being recognized and cleared by the body's mononuclear macrophage system. Furthermore, PLTm, through adhesion molecules, can specifically bind to tumor cells, thereby achieving auxiliary targeting of both in situ and circulating tumor cells. E5 peptide is a novel antagonistic peptide that specifically binds to invasive tumor-expressed (CXCR4) markers, targeting circulating tumor cells and even lung cells in a pre-metastatic niche state to prevent breast cancer metastasis. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the preparation method of the present invention;

[0031] Figure 2 TEM image of nanoparticles in Example 2;

[0032] Figure 3 UV-vis image of the nanoparticles in Example 2;

[0033] Figure 4Drug loading and release in Example 2: (A) Loading efficiency (EE) and encapsulation efficiency (LE) of DAS loaded on E-PLTm@FPD; (B) DAS release from E-PLTm@FPD under different conditions; (C) Intracellular distribution of DAS, FPD, PLTm@FPD, and E-PLTm@FPD after incubation with breast cancer tumor cells for 24 hours. Red fluorescence represents DAS, blue fluorescence represents cell nuclei, and green fluorescence represents lysosomes.

[0034] Figure 5 Example 2: In vivo targeting of the nanoplatform;

[0035] Figure 6 This is a photothermal effect diagram of the nanoplatform in mouse tumor treatment in Example 2;

[0036] Figure 7 The tumor volume changes of the nanoplatform in breast cancer-bearing mice in Example 2: (1) PBS, (2) FP, (3) DAS, (4) FPD, (5) PLTm@FPD, (6) E-PLTm@FPD, (7) E-PLTm@FPD+Laser;

[0037] Figure 8 The following is a diagram showing the effect of the nano-platform in preventing spontaneous lung metastasis of breast cancer in Example 2: (1) PBS, (2) FP, (3) DAS, (4) FPD, (5) PLTm@FPD, (6) E-PLTm@FPD, (7) E-PLTm@FPD+Laser;

[0038] Figure 9 Here are the CT imaging results of the nanometer platform in Example 2: (A) In vitro CT imaging results, (B) In vivo CT imaging results of tumor sites;

[0039] Figure 10 This is an image showing the tumor MRI imaging effect of the nanometer platform in Example 2.

[0040] Figure 11 The image shows the H&E staining of major organs in mice after the nanoplatform of Example 2 was injected into them. Detailed Implementation

[0041] As a preferred embodiment of the present invention, a method for preparing a platelet membrane-modified iron-tungsten-oxygen ternary compound nanoplatform loaded with platinum nanoparticles includes the following steps:

[0042] 1) Sodium tungstate dihydrate, ferrous sulfate heptahydrate, and sodium dodecylbenzenesulfonate (SDBS) were mixed in a molar ratio of (0.8–1.2):(0.8–1.2):(1.8–2.2). The pH was adjusted to 10 with sodium hydroxide aqueous solution. The mixture was then transferred to a reaction vessel and reacted at 170–190 °C for 8–16 h. After centrifugation and washing, the precipitate was dispersed in water to obtain a FeWOx dispersion. The dispersion was stored in the dark at 2–8 °C for later use.

[0043] 2) Dissolve polyvinylpyrrolidone (PVP) in chloroplatinic acid hexahydrate at a mass ratio of (62-66):(18-22) to form solution A; dissolve sodium borohydride in ice water to prepare solution B with a concentration of 0.5-0.7 mg / mL; slowly add solution B dropwise to solution A, react at room temperature for 3-5 hours, centrifuge and wash, disperse the precipitate in water to obtain PtNPs dispersion, store in the dark at 2-8℃ for later use;

[0044] 3) Mix FeWOx dispersion with PtNPs dispersion at a mass ratio of 1:(5-10), stir overnight, wash, and disperse the precipitate in water to obtain FP dispersion. Store in the dark at 2-8℃ for later use. Mix FP with dasatinib (DAS) at a mass ratio of FP:(1-8), stir for 20-28 h, dialyze for 20-28 h to remove free DAS, and obtain FPD.

[0045] 4) Take fresh anticoagulated whole blood from the human body, separate platelets from the whole blood by centrifugation and washing, and extract platelet membranes by freezing at -80℃ for 30 min and then thawing at 37℃ for 30 min three times. Suspend the platelet membranes in phosphate-buffered saline (PBS) and sonicate (2-20 min, 42 kHz, 100 W). Then, use a 200-400 nm polycarbonate porous membrane filter to extract the nanoscale platelet membrane vesicles PLTm.

[0046] 5) E5 peptide and distearate phosphatidylethanolamine-amino polyethylene glycol (DSPE-PEG2000-NH2) were dissolved in PBS at a mass ratio of 1:(4-6) and stirred slowly at 35-39°C for 4-8 hours to obtain DSPE-E5; then DSPE-E5 was mixed with PLTm vesicles at 35-39°C for 4-8 hours at a mass ratio of (2-5):1 to obtain E-PLTm.

[0047] 6) The E-PLTm vesicle suspension and FPD were dispersed and fused by ultrasonic treatment (2-20 min, 42 kHz, 100 W). The mixture was then squeezed back and forth 20 times through a 200-400 nm needle filter. The mass ratio of E-PLTm to FPD was (1-4):1. Excess E-PLTm was removed by centrifugation and discarding the supernatant to obtain the platelet membrane modified iron-tungsten-oxygen ternary compound nanoplatform E-PLTm@FPD loaded with platinum nanoparticles.

[0048] The present invention will be further explained and described below with reference to specific embodiments.

[0049] Example 1

[0050] 1) Synthesis of the iron-tungsten-oxygen ternary compound FeWOx:

[0051] Add 8 ml of 0.01 M sodium tungstate dihydrate to a mixture of 8 ml of 0.01 M ferrous sulfate heptahydrate and 9 ml of SDBS (0.02 M). Adjust the pH to 10 with 1 M sodium hydroxide aqueous solution, then transfer the mixture to a reaction vessel and react at 170 °C for 8 h. After centrifugation and washing at 10,000 rpm, disperse the precipitate in 0.5 mL of water to obtain FeWOx dispersion. Store at 2 °C in the dark for later use.

[0052] 2) Synthesis of platinum nanoparticles (PtNPs):

[0053] 62 mg PVP was dissolved in 0.9 ml of chloroplatinic acid hexahydrate solution (20 mg / ml) to form solution A; 18 mg sodium borohydride was dissolved in 36 ml of ice water to form solution B; solution B was slowly added dropwise to solution A, and after reacting at room temperature for 3 h, the mixture was centrifuged at 8000 rpm for 20 min to remove excess PVP. The precipitate was dispersed in 0.5 mL of water to obtain a PtNPs dispersion, which was stored at 2 °C protected from light for later use.

[0054] 3) Dasatinib (DAS) and platinum nanoparticles (PtNPs) adsorb onto iron-tungsten oxide ternary compound (FeWOx) to form FPD:

[0055] FeWOx dispersion and PtNPs dispersion were mixed, with FeWOx at a mass of 1 mg and PtNPs at a mass of 5 mg. The mixture was stirred overnight, centrifuged at 13000 rpm for 5 min, washed, and the precipitate was dispersed in water to obtain FP dispersion. The FP dispersion was stored at 2°C in the dark for later use. 1 mg of FP was added to 1 mL of DAS (1 mg / mL, DMSO solvent), stirred for 20 h, and dialyzed for 20 h to remove free DAS, yielding FPD.

[0056] 4) Preparation of nanoscale platelet membrane vesicles:

[0057] Anticoagulated fresh whole blood was collected from the human body. Platelets were separated from the whole blood by centrifugation and washing. 1 mL of platelet suspension was prepared by adding phosphate-buffered saline (PBS). The platelet membrane was extracted by freezing at -80℃ for 30 min and then thawing at 37℃ for 30 min three times. The platelet membrane suspension was sonicated (2 min, 42 kHz, 100 W) and then extracted back and forth using a 200 nm polycarbonate porous membrane filter to obtain nanoscale platelet membrane vesicles (PLTm).

[0058] 5) Preparation of E5 peptide-modified platelet membrane vesicles E-PLTm:

[0059] 5 mg of E5 peptide and 20 mg of DSPE-PEG2000-NH2 were weighed and dissolved in 5 ml of PBS. The mixture was stirred slowly at 35 °C for 4 h to obtain DSPE-E5. Then, 1 mg of DSPE-E5 and 0.5 mg of PLTm vesicles were mixed and stirred at 35 °C for 4 h to finally obtain E-PLTm.

[0060] 6) The iron-tungsten-oxygen ternary nanoplatform E-PLTm@FPD, which is modified with platelet membrane and loaded with platinum nanoparticles, was finally obtained by ultrasonic dispersion:

[0061] E-PLTm vesicle (1 mg) suspension and FPD (1 mg) were dispersed in 1 mL PBS by sonication (2 min, 42 kHz, 100 W). After fusion, the mixture was squeezed back and forth 20 times through a 200 nm needle filter. Excess E-PLTm was removed by centrifugation (2500 rpm for 10 min, 4 °C) and the supernatant was discarded to obtain PLTm-coated E-PLTm@FPD.

[0062] Example 2

[0063] 1) Synthesis of the iron-tungsten-oxygen ternary compound FeWOx:

[0064] Add 10 ml of sodium tungstate dihydrate (0.01 M) to a mixture of 10 ml of ferrous sulfate heptahydrate (0.01 M) and 10 ml of SDBS (0.02 M). Adjust the pH to 10 with sodium hydroxide aqueous solution (1 M). Transfer the mixture to a reaction vessel and react at 180 °C for 12 h. After centrifugation and washing at 10,000 rpm, disperse the precipitate in 0.5 mL of water to obtain FeWOx dispersion. Store at 4 °C in the dark for later use.

[0065] 2) Synthesis of platinum nanoparticles (PtNPs):

[0066] 64 mg PVP was dissolved in 1 ml of chloroplatinic acid hexahydrate solution (20 mg / ml) to form solution A; 22 mg sodium borohydride was dissolved in 36 ml of ice water to form solution B; solution B was slowly added dropwise to solution A, and after reacting at room temperature for 4 h, the mixture was centrifuged at 8000 rpm for 20 min to remove excess PVP. The precipitate was dispersed in 0.5 mL of water to obtain a PtNPs dispersion, which was stored at 4 °C protected from light for later use.

[0067] 3) Dasatinib (DAS) and platinum nanoparticles (PtNPs) adsorb onto iron-tungsten oxide ternary compound (FeWOx) to form FPD:

[0068] FeWOx (1 mg) and PtNPs (7.5 mg) were mixed and stirred overnight. After centrifugation at 13000 rpm for 5 min, the precipitate was washed and dispersed in water to obtain FP. The precipitate was stored at 4 °C in the dark for later use. 1 mg of FP was added to 1 mL of DAS (4 mg / mL, DMSO solvent), stirred for 24 h, and dialyzed for 24 h to remove free DAS to obtain FPD.

[0069] 4) Preparation of nanoscale platelet membrane vesicles:

[0070] Anticoagulated fresh whole blood was collected from the human body. Platelets were separated from the whole blood by centrifugation and washing. 1 mL of platelet suspension was prepared by adding phosphate-buffered saline (PBS). The platelet membrane was extracted by freezing at -80℃ for 30 min and then thawing at 37℃ for 30 min three times. The platelet membrane suspension was sonicated (5 min, 42 kHz, 100 W). The platelet membrane was then extracted back and forth through 400 nm and 200 nm polycarbonate porous membrane filters to obtain nanoscale platelet membrane vesicles (PLTm).

[0071] 5) Preparation of E5 peptide-modified platelet membrane vesicles E-PLTm:

[0072] 5 mg of E5 peptide and 25 mg of DSPE-PEG2000-NH2 were weighed and dissolved in 5 ml of PBS. The mixture was stirred slowly at 37 °C for 6 h to obtain DSPE-E5. Then, 1 mg of DSPE-E5 and 0.3 mg of PLTm were mixed and stirred at 37 °C for 6 h to obtain E-PLTm.

[0073] 6) A novel iron-tungsten-oxygen ternary nanoplatform, E-PLTm@FPD, was finally obtained by ultrasonic dispersion of platelet membrane-modified platinum nanoparticles:

[0074] E-PLTm vesicle (2 mg) suspension and FPD (1 mg) were dispersed in 1 mL PBS by ultrasonic treatment (5 min, 42 kHz, 100 W). After fusion, the mixture was squeezed back and forth 20 times through a 200 nm needle filter. Excess E-PLTm was removed by centrifugation (2500 rpm for 10 min, 4 °C) and the supernatant was discarded to obtain PLTm-coated E-PLTm@FPD.

[0075] Example 3

[0076] 1) Synthesis of the iron-tungsten-oxygen ternary compound FeWOx:

[0077] Add 12 ml of sodium tungstate dihydrate (0.01 M) to a mixture of 12 ml of ferrous sulfate heptahydrate (0.01 M) and 11 ml of SDBS (0.02 M). Adjust the pH to 10 with sodium hydroxide aqueous solution (1 M). Transfer the mixture to a reaction vessel and react at 190 °C for 16 h. After centrifugation and washing at 10,000 rpm, disperse the precipitate in 0.5 mL of water to obtain FeWOx dispersion. Store at 8 °C in the dark for later use.

[0078] 2) Synthesis of platinum nanoparticles (PtNPs):

[0079] Dissolve 66 mg PVP in 1.1 ml of chloroplatinic acid hexahydrate solution (20 mg / ml) to form solution A; dissolve 25.2 mg sodium borohydride in 36 ml of ice water to form solution B; slowly add solution B dropwise to solution A, react at room temperature for 5 h, centrifuge at 8000 rpm for 20 min to remove excess PVP, disperse the precipitate in 0.5 mL of water to obtain PtNPs dispersion, protect from light, store at 8 °C for later use.

[0080] 3) Dasatinib (DAS) and platinum nanoparticles (PtNPs) adsorb onto iron-tungsten oxide ternary compound (FeWOx) to form FPD:

[0081] FeWOx (1 mg) and PtNPs (10 mg) dispersion were mixed and stirred overnight. After centrifugation at 13000 rpm for 5 min, the precipitate was washed and dispersed in water to obtain FP dispersion. The FP dispersion was stored at 8°C in the dark for later use. 1 mg of FP was added to 1 mL of DAS (8 mg / mL, DMSO solvent), stirred for 28 h, and dialyzed for 28 h to remove free DAS, thus obtaining FPD.

[0082] 4) Preparation of nanoscale platelet membrane vesicles:

[0083] Fresh, anticoagulated whole blood was collected from the human body. Platelets were separated from the whole blood by centrifugation and washing. 1 mL of platelet suspension was prepared by adding phosphate-buffered saline. Platelet membranes were extracted by freezing at -80℃ for 30 min and then thawing at 37℃ for 30 min three times. The platelet membrane suspension was sonicated (20 min, 42 kHz, 100 W) and then extracted back and forth using a 200 nm polycarbonate porous membrane filter to obtain nanoscale platelet membrane vesicles (PLTm).

[0084] 5) Preparation of E5 peptide-modified platelet membrane vesicles E-PLTm:

[0085] 5 mg of E5 peptide and 30 mg of DSPE-PEG2000-NH2 were weighed and dissolved in 5 ml of PBS. The mixture was stirred slowly at 39 °C for 8 h to obtain DSPE-E5. Then, 1 mg of DSPE-E5 and 0.2 mg of PLTm were mixed and stirred at 39 °C for 8 h to obtain E-PLTm.

[0086] 6) A novel iron-tungsten-oxygen ternary nanoplatform, E-PLTm@FPD, was finally obtained by ultrasonic dispersion of platelet membrane-modified platinum nanoparticles:

[0087] E-PLTm vesicle (4 mg) suspension and FPD (1 mg) were dispersed in 1 mL PBS by sonication (20 min, 42 kHz, 100 W). After fusion, the mixture was squeezed back and forth 20 times through a 400 nm needle filter. Excess E-PLTm was removed by centrifugation (2500 rpm for 10 min, 4 °C) and the supernatant was discarded to obtain PLTm-coated E-PLTm@FPD.

[0088] Example 4

[0089] The particles and nanoplatform prepared in Example 2 were characterized and tested, and the results are as follows:

[0090] 1. Microscopic testing of particles

[0091] The microstructure of the E-PLTm@FPD prepared in Example 2 is as follows: Figure 2 As shown, the FPD was successfully encapsulated by E-PLTm, resulting in an E-PLTm@FPD with a core-shell structure.

[0092] 2. Characteristics of particles

[0093] The UV-vis image of the E-PLTm@FPD prepared in Example 2 is shown below. Figure 3 As shown, E-PLTm@FPD exhibits characteristic absorption peaks at 215 nm, 260 nm, and 330 nm, which are consistent with the characteristic absorption peaks of FP, E5, and DAS, indicating the successful preparation of E-PLTm@FPD.

[0094] 3. Drug loading and release characteristics

[0095] E-PLTm@FPD load and release DAS characteristics such as Figure 4 As shown in A and 4B, the loading rate (EE) and encapsulation efficiency (LE) of DAS in E-PLTm@FPD were 122.6% ± 10.2% and 87.0% ± 4.1%, respectively. Under physiological conditions of pH 7.4, DAS release from E-PLTm@FPD was slow, but under simulated tumor acidic microenvironment (pH 5.4), DAS was released in large quantities from E-PLTm@FPD. After irradiation with 808 nm near-infrared light (2 W / cm²) at 48 h, DAS was significantly released. 2 The DAS release rate was further increased after 5 min, indicating that the DAS release from E-PLTm@FPD is pH-responsive and light-dependent.

[0096] 4. Suborgan localization within tumor cells

[0097] The localization of E-PLTm@FPD in breast cancer tumor cells is as follows: Figure 4 As shown in Figure C, blue fluorescence represents the cell nucleus, green fluorescence represents lysosomes, and red fluorescence represents DAS. After 24 hours of incubation, a small amount of free DAS entered breast cancer cells, PLTm@FPD partially entered the cells and was mainly distributed in lysosomes in the cytoplasm, while E-PLTm@FPD entered the cells in large quantities and was distributed in lysosomes and the cell nucleus. After irradiation with 808nm near-infrared light (2W / cm²), 2 (5 min), E-PLTm@FPD almost completely entered the cell nucleus (E-PLTm@FPD+Laser group). This indicates that loading DAS into FPD, encapsulating it on the platelet membrane, and modifying it with E5 peptide facilitates the uptake of chemotherapeutic drugs by tumor cells, and that near-infrared light irradiation facilitates drug entry into the cell nucleus, which provides a basis for chemotherapeutic drugs and Pt to destroy tumor cell DNA.

[0098] 5. Targeted research

[0099] To verify the ability of E-PLTm@FPD to target breast cancer sites in vivo, 4T1 tumor-bearing mice were constructed, and FPD, PLTm@FPD, and E-PLTm@FPD loaded with fluorescently labeled DAS were injected into the mice via the tail vein. Figure 5 As shown, in vivo imaging at 6h, 24h, and 48h, along with ex vivo imaging of major organs and tumor tissues, revealed that E-PLTm@FPD mainly accumulated in the breast cancer tumor site at 48h, with very low concentrations in other sites, demonstrating that E-PLTm@FPD has good tumor targeting properties.

[0100] 6. Photothermal Effect Research

[0101] To verify the in vivo photothermal effect of E-PLTm@FPD, FPD, PLTm@FPD, and E-PLTm@FPD were injected into 4T1 tumor-bearing mice via the tail vein. 24 hours later, near-infrared light (808nm, 2W / cm²) was used to evaluate the effect. 2 After irradiating the tumor site for 0, 60, 180, and 300 seconds, the temperature rise at the tumor site was captured by an infrared camera. The results are as follows: Figure 6 As shown, in the PBS, FPD, PLTm@FPD, and E-PLTm@FPD groups, the temperatures rose to 46.8±0.5℃, 57.7±1.1℃, 57.8±0.8℃, and 59.7±0.2℃ respectively after 300s of near-infrared light irradiation. After 300s of irradiation following PBS injection, the temperature only increased by about 6℃, while the other groups all showed a temperature increase of more than 15℃ after 300s of irradiation. In particular, it was observed that the E-PLTm@FPD group showed a larger temperature increase at the tumor site compared to the PLTm@FPD and FPD groups. This is because E-PLTm@FPD targets breast cancer, resulting in a higher concentration at the breast cancer tumor site and thus exerting a photothermal effect.

[0102] 7. Antigen site effects on breast cancer

[0103] 1×10 5 Four T1 cells were orally injected into the left abdominal fat pad of nude mice. The mice were then treated every other day with PBS, FP, DAS, FPD, PLTm@FPD, E-PLTm@FPD, and E-PLTm@FPD+Laser groups, respectively, for three treatments. Tumor volume changes were recorded continuously for 14 days. Tumor tissue was harvested from the mice after euthanasia on day 14 for further experiments. E-PLTm@FPD+Laser refers to E-PLTm@FPD irradiation with 808nm near-infrared light (2W / cm²). 2 (5 min). Results are as follows Figure 7 As shown, compared with the PBS group, the tumor size in the FP group did not change significantly, while DAS, FPD, and PLTm@FPD delayed the growth of breast cancer tumor volume to a certain extent. The E-PLTm@FPD and E-PLTm@FPD+Laser groups could even reduce the size of in situ breast cancer to 0.623±0.10 and 0.41±0.04 times that of D0, respectively.

[0104] 8. Anti-spontaneous lung metastasis effect in breast cancer

[0105] To verify its anti-breast cancer metastasis effect, 1×10 54T1-LUC cells were orally injected into the left ventral fat pad of nude mice. On days 2, 4, and 6, mice were treated with PBS, FP, DAS, FPD, PLTm@FPD, E-PLTm@FPD, and E-PLTm@FPD+Laser. Bioimaging was performed every 7 days starting from day 14. E-PLTm@FPD+Laser refers to E-PLTm@FPD irradiation with 808nm near-infrared light (2W / cm²). 2 (5 min). Results are as follows Figure 8 As shown, distant metastases were detected in the PBS group starting on day 21, and by day 42, metastases had appeared in multiple sites including the lungs, spleen, and bones in the PBS group. However, after treatment with E-PLTm@FPD+Laser, no distant organ metastases occurred by day 42. E-PLTm@FPD+Laser can significantly inhibit spontaneous metastasis of breast cancer.

[0106] 9. The role of CT imaging

[0107] The CT imaging effect of E-PLTm@FPD is as follows Figure 9 As shown, E-PLTm@FPD exhibits concentration-dependent behavior under CT scans, with HU (Hounsfield Unit) increasing with increasing W concentration, and the slope being 1856.0 HU (mg / ml). -1 This indicates that it can be used as a good CT contrast agent. In mice, enhanced CT images observed after injecting E-PLTm@FPD into the tumor site showed that the tumor site was brighter after injection, and the CT intensity was 6.70 times that of the uninjected image.

[0108] 10. MRI imaging function

[0109] To verify its in vivo imaging function, E-PLTm@FPD was injected orally into tumor-bearing mice. One hour later, MRI imaging was performed using a 7T MRI scanner (PharmaScan70 / 16US, Burker). Figure 10 As shown, the MRI images showed that the tumor site was darker at both T1 and T2 compared to the uninjected group.

[0110] 11. Biosafety

[0111] To verify the biosafety of E-PLTm@FPD, it was injected into mice via the tail vein. After 42 days, the heart, liver, spleen, lungs, and kidneys were collected for H&E staining. Figure 11 As shown, no pathological changes were observed in the major organs, indicating that it has good biocompatibility.

Claims

1. A method for preparing a platelet membrane-modified iron-tungsten-oxygen ternary compound nanoplatform loaded with platinum nanoparticles, comprising the following steps: 1) Iron-tungsten oxide ternary compound FeWOx was prepared by organic phase thermal decomposition; 2) Platinum nanoparticles (Pt NPs) were prepared; 3) Mix the FeWOx obtained in step 1) with the Pt NPs obtained in step 2) to obtain the iron-tungsten-oxygen ternary compound FP loaded with platinum nanoparticles. Then mix the FP with the drug DAS to obtain the iron-tungsten-oxygen ternary compound FPD loaded with platinum nanoparticles and dasatinib drug. 4) Collect anticoagulated fresh whole blood, separate platelets by centrifugation and washing, and extract platelet membranes by repeated freeze-thaw cycles to prepare nanoscale platelet membrane vesicles (PLTm). 5) Mix E5 peptide and distearate phosphatidylethanolamine-amino polyethylene glycol DSPE-PEG2000-NH2 to obtain distearate phosphatidylethanolamine modified E5 peptide DSPE-E5. Mix DSPE-E5 with PLTm obtained in step 4) to obtain E5 peptide modified platelet membrane vesicle E-PLTm. 6) Mix the E-PLTm obtained in step 5) with the FPD obtained in step 3) to obtain the platelet membrane modified iron-tungsten-oxygen ternary nanoplatform E-PLTm@FPD loaded with platinum nanoparticles; Step 3) specifically involves: dispersing FeWOx and PtNPs separately in water, then mixing them together, wherein the mass ratio of FeWOx to PtNPs is 1:(5~10), stirring overnight, washing, and obtaining FP; dispersing FP in water, then mixing it with the drug DAS, wherein the mass ratio of FP to DAS is 1:(1~8), stirring for 20~28 hours, and then dialysis for 20~28 hours to remove free DAS, thereby obtaining FPD; The specific process of preparing platelet membrane into PLTm in step 4) is as follows: the platelet membrane is suspended in phosphate buffered saline, treated with ultrasound, and then extracted back and forth using a 200~400nm polycarbonate porous membrane filter to obtain PLTm. The sequence of the E5 peptide is GGRSFFLLRRIQGCRFRNTVDD, as shown in SEQ ID NO.1; the specific process of step 5) is as follows: E5 peptide and DSPE-PEG2000-NH2 are dissolved in phosphate buffered saline, wherein the mass ratio of E5 peptide to DSPE-PEG2000-NH2 is 1:(4~6), and reacted at 35~39℃ for 4~8h to obtain DSPE-E5; then DSPE-E5 and PLTm are mixed at a mass ratio of (2~5):1, and stirred at 35~39℃ for 4~8h to obtain E-PLTm; The specific process of step 6) is as follows: E-PLTm and FPD are mixed at a mass ratio of (1~4):1, dispersed and fused by ultrasound, squeezed back and forth with a 200~400nm needle filter, and then centrifuged to remove the supernatant to obtain E-PLTm@FPD.

2. The preparation method according to claim 1, characterized in that, Step 1) specifically involves mixing sodium tungstate dihydrate, ferrous sulfate heptahydrate, and sodium dodecylbenzenesulfonate in a molar ratio of (0.8~1.2):(0.8~1.2):(1.8~2.2), adjusting the pH to 10, reacting at 170~190℃ for 8~16 h, centrifuging and washing to obtain FeWOx.

3. The preparation method according to claim 1, characterized in that, Step 2) specifically refers to: Polyvinylpyrrolidone was dissolved in chloroplatinic acid hexahydrate to form solution A, wherein the mass ratio of polyvinylpyrrolidone to chloroplatinic acid hexahydrate was (62~66):(18~22); sodium borohydride was dissolved in ice water to prepare solution B with a concentration of 0.5~0.7 mg / mL; solution B was added dropwise to solution A, and after reacting at room temperature for 3~5 h, the mixture was centrifuged and washed to obtain Pt NPs.

4. The preparation method according to claim 1, characterized in that, The specific process of extracting platelet membranes by repeated freeze-thaw cycles in step 4) is as follows: platelets are suspended in phosphate buffer, frozen at -80℃ for 30 min, then thawed at 37℃ for 30 min, repeated 3 times, centrifuged, and platelet membranes are extracted.

5. A platelet membrane-modified iron-tungsten-oxygen ternary compound nanoplatform prepared by the preparation method according to any one of claims 1 to 4.

6. The application of the iron-tungsten-oxygen ternary nanoplatform modified with platelet membrane and loaded with platinum nanoparticles as described in claim 5 in the preparation of cancer-targeted drugs; wherein the cancer is breast cancer.

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