A magnetized platelet drug delivery system, its preparation method and application

Abstract

The application discloses a magnetized platelet drug delivery system, a preparation method and application, and the magnetized platelet drug delivery system comprises platelets, magnetic nanoparticles and drug nanoparticles; and the magnetic nanoparticles and the drug nanoparticles are encapsulated in the cytoplasm of the platelets. In the application, the magnetized platelet drug delivery system utilizes the platelets to load the magnetic nanoparticles, so that the platelets have good magnetic migration ability; under the guidance of the tumor tendency of the platelets and an external magnetic field, the tumor targeting delivery of the magnetized platelets is realized, and the concentration of the drug delivery system at a lesion site is improved; the magnetized platelets load microenvironment active oxygen and drug nanoparticles with a dual response of photothermal response, so that the premature release of the drug is avoided, the toxic and side effects of the drug are reduced, the drug safety is improved, the light-controlled release of the drug at a target site is realized, and the combined treatment of photothermal-chemotherapy of the tumor is realized.

Description

Technical Field

[0001] The present invention relates to a drug delivery system, a preparation method and an application thereof, and in particular to a platelet drug delivery system, a preparation method and an application thereof, belonging to the field of medical technology. Background Art

[0002] At present, malignant tumors have become a major disease that seriously threatens human health. The difficulty in completely eliminating them and the easy metastasis of tumor cells lead to a high recurrence rate, which is an important problem faced by tumor treatment. In recent years, personalized precision treatment of malignant tumors with multidisciplinary collaboration has become a trend in tumor treatment. With the help of targeted drug delivery systems, therapeutic agents are precisely delivered to the tumor site; at the same time, external magnetic field guidance can further enhance the concentration of therapeutic agents in the lesion site. At the same time, the spatiotemporal specific response and targeted release of therapeutic agents at the lesion site can achieve the goal of high-efficiency and low-toxicity treatment. In order to achieve effective tumor treatment, it is of great significance to research and develop precision targeted drug delivery systems with targeting functions and controllable drug release.

[0003] Inspired by natural biological systems, biological cell delivery systems have garnered widespread attention in recent years. Platelets, as multifunctional blood cells, possess properties such as high abundance in the blood, rapid replenishment, high drug loading efficiency, and specific binding to biological cells, making them an attractive platform for constructing targeted drug delivery systems. Platelets' inherent wound aggregation properties can lead to increased platelet count and aggregation at tumor sites. Platelets in the tumor microenvironment can participate in tumor progression, allowing them to penetrate deep into the tumor. Tumor cells in the blood are readily recognized by platelets, forming aggregates that activate adhered platelets and release internal granular material and therapeutic agents. Inspired by this, multifunctional platelet-based drug delivery systems can be constructed, achieving high loading efficiency, long-term circulation in the body, and autonomous tumor targeting, potentially developing a precision cancer treatment platform.

[0004] Targeted tumor accumulation in vivo by delivery systems is another key issue in achieving precision drug delivery. Non-invasive and highly targeted magnetic nanoparticles can be used. Under the influence of an external magnetic field, magnetic nanoparticles, such as iron oxide (Fe3O4, γ-Fe2O3) nanoparticles, are attracted by the external magnetic field and effectively accumulate at the lesion site, achieving accumulation in specific locations of the delivery system, increasing drug concentration at the target site and reducing the drug's toxic side effects on normal tissue cells throughout the body. Summary of the Invention

[0005] The present invention aims to provide a magnetized platelet drug delivery system, preparation method, and application thereof. The magnetized platelet drug delivery system utilizes platelets to simultaneously load magnetic nanoparticles and drug nanoparticles. The platelets are loaded with magnetic nanoparticles, which have excellent magnetic mobility, enabling targeted delivery under the guidance of an external magnetic field and increasing drug concentration at the lesion site. The platelets are also loaded with drug nanoparticles that are dually responsive to microenvironmental reactive oxygen species and photothermal therapy, thereby preventing premature drug release and reducing the toxic side effects of drugs, particularly tumor radiotherapy and chemotherapy drugs, thereby improving drug safety. This system enables light-controlled drug release at the target site and combined photothermal and chemotherapy treatment for tumors.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0007] The first aspect of the present invention provides a magnetized platelet drug delivery system, which comprises platelets, magnetic nanoparticles and drug nanoparticles;

[0008] Preferably, the magnetic nanoparticles and drug nanoparticles are loaded into platelets.

[0009] Preferably, the drug nanoparticles are loaded in the cytoplasm of the platelets; and the magnetic nanoparticles are encapsulated in the cytoplasm and on the cell membrane of the platelets.

[0010] Preferably, the magnetic nanoparticles are ferrosoferric oxide nanoparticles encapsulated by polyethyleneimine; and the drug nanoparticles are drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticles.

[0011] Preferably, the drug is a drug containing a benzene ring structure, preferably an anti-tumor or anti-cancer drug containing a benzene ring structure.

[0012] Preferably, the drug is doxorubicin, puerarin, quercetin, paclitaxel, or geniposide, preferably doxorubicin.

[0013] Preferably, the drug nanoparticles are doxorubicin-loaded polydopamine nanoparticles that are dual-responsive to light, heat, and reactive oxygen species.

[0014] Doxorubicin is the loaded drug for therapeutic effects; in addition to doxorubicin, other drugs containing a benzene ring structure are suitable for the present invention, such as puerarin, quercetin, paclitaxel, gardenia glycoside, etc.; dopamine and the reactive oxygen species-responsive dopamine monomers synthesized in the present invention are the skeleton materials for preparing polydopamine nanoparticles, which are used to load drugs (such as doxorubicin) to achieve their photothermal-reactive oxygen species-responsive release and photothermal therapy; magnetic nanoparticles are used to realize magnetic migration of the drug delivery system under the action of an external magnetic field to achieve magnetic targeting; platelets are living cell carriers with certain tumor tropism properties in the drug delivery system of the present invention.

[0015] Preferably, the polyethyleneimine encapsulated ferroferric oxide magnetic nanoparticles are prepared by the following method:

[0016] (1) Under a nitrogen atmosphere, ferroferric oxide magnetic nanoparticles are prepared by a basic solution coprecipitation method, and freeze-dried to obtain ferroferric oxide nanoparticles;

[0017] (2) The ferroferric oxide nanoparticles are dissolved in water, mixed with a polyethyleneimine aqueous solution, and then subjected to ultrasonic treatment. Then, stirring treatment is performed at 60±5°C. Finally, centrifugal treatment is performed to obtain polyethyleneimine encapsulated ferroferric oxide magnetic nanoparticles.

[0018] The polyethyleneimine encapsulated ferroferric oxide magnetic nanoparticles are dissolved in physiological saline to prepare a polyethyleneimine encapsulated ferroferric oxide magnetic nanoparticle solution, and the concentration of ferroferric oxide in the solution is 40-80 μg / mL.

[0019] Preferably, the basic solution coprecipitation method in step (1) comprises the following steps:

[0020] 1a) After mixing a FeCl2 hydrochloric acid solution and a FeCl3 aqueous solution, nitrogen is introduced, and an aqueous ammonia solution is added under a nitrogen atmosphere to initiate a basic coprecipitation reaction;

[0021] 1b) After the initiation reaction mixture is allowed to stand for at least 10 min, a magnet is placed at the bottom of the mixture to adsorb the precipitate for at least 10 min, and the supernatant is poured out. The magnet-adsorbed aggregated precipitate is washed with pure water;

[0022] 1c) The magnet-adsorbed aggregated precipitate is collected, and perchloric acid solution is added under a nitrogen atmosphere for perchloric acid stirring treatment.

[0023] Preferably, in step 1a), the molar ratio of FeCl2 to FeCl3 is 1:1.95-2.05, preferably 1:2; the reaction temperature is room temperature; the reaction time is 1-2 days, preferably 1 day; and the molar ratio of FeCl2 to ammonia NH3 in the aqueous ammonia solution is 1:20-30, preferably 1:24.

[0024] In step 1b), the magnet adsorption stirring treatment temperature is room temperature; the stirring treatment time is at least 10 min; and the number of times of washing the precipitate with pure water is at least 3.

[0025] In step 1c), the concentration of the perchloric acid solution is 0.5-1.5 M; the number of times of treatment with perchloric acid is at least 2; and the molar ratio of FeCl2 to perchloric acid in the perchloric acid solution is 1:8-15, preferably 1:10.

[0026] In particular, the method further includes centrifuging the reaction mixture after adding a perchloric acid solution and stirring, adding a perchloric acid solution to the centrifugal precipitate for a second time under a nitrogen atmosphere, stirring with perchloric acid for a second time, and then centrifuging again; resuspending the precipitate in water, transferring it to a dialysis bag with a molecular weight cutoff of 14,000 Da, dialyzing it until the dialyzed fluid is neutral, collecting the precipitate in the dialysis bag, and freeze-drying it to obtain Fe3O4 nanoparticles.

[0027] The mass ratio of ferrosoferric oxide to polyethyleneimine in step (2) is 1:(0.25-2), preferably 1:(0.5-1); PEI is branched polyethyleneimine having an average molecular weight of 600 or 1800 Da, preferably 1800 Da; and the concentration of the polyethyleneimine solution is 10 mg / mL.

[0028] The ultrasonic treatment is performed using a cell disruptor; the ultrasonic treatment time is at least 10 minutes; the ultrasonic treatment power is 150W, ultrasonic treatment is performed once every 10 seconds, and the duration of each ultrasonic treatment is 3 seconds.

[0029] The stirring treatment temperature is 60±5° C., and the stirring time is 3-5 h.

[0030] Adding Fe3O4 nanoparticles to water to prepare an Fe3O4 nanoparticle aqueous solution, wherein the concentration of the Fe3O4 nanoparticle solution is 2-10 mg / mL, preferably 5 mg / mL;

[0031] The Fe3O4 nanoparticle solution was added to the polyethyleneimine aqueous solution (PEI solution), mixed evenly, and then ultrasonicated to make the Fe3O4 nanoparticles evenly dispersed in the PEI solution;

[0032] The ultrasonically treated mixture was then stirred and encapsulated at a temperature of 60±5° C. to prepare polyethyleneimine (PEI)-encapsulated ferroferric oxide magnetic nanoparticles Fe 3 O 4 @ PEI NPs.

[0033] In particular, the method further includes centrifuging the encapsulated product, washing the precipitate with deionized water, resuspending the precipitate with deionized water, and drying the precipitate to obtain Fe3O4@PEI NPs.

[0034] The method also includes resuspending the Fe3O4@PEI NPs nanoparticles in physiological saline to prepare a magnetic nanoparticle Fe3O4@PEINPs solution.

[0035] Preferably, the drug nanoparticles are prepared according to the following method:

[0036] A. Preparation of activated thioketal compounds

[0037] Add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to a dimethyl sulfoxide solution of thioketal 2,2'-[propane-2,2-diylbis(sulfo)yl]diacetic acid (TK-COOH), and stir for at least 2 hours to obtain an activated thioketal compound (TK-NHS).

[0038] B. Preparation of reactive oxygen species-responsive dopamine monomers

[0039] Adding an activated thioketal compound TK-NHS solution to a dopamine DA solution, stirring and reacting for 10-14 hours (preferably 12 hours) in the dark; then placing the reaction mixture in a dialysis bag and dialyzing it with ultrapure water; collecting the liquid in the dialysis bag and freeze-drying it to obtain an active oxygen-responsive dopamine monomer DA-TK-DA;

[0040] C. dissolving dopamine, the drug to be loaded, and a dopamine monomer responsive to active oxygen in water and mixing them uniformly to prepare a dopamine-drug mixed system; then adding sodium hydroxide solution and performing a polymerization reaction under stirring to prepare drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticles;

[0041] Preferably, in step A), the concentration of the solution formed by dissolving TK in DMSO is 0.05-0.15 M, preferably 0.1 M;

[0042] In the reaction process of activating TK-COOH using NHS and EDC, the theoretical molar ratio is TK-COOH:EDC:NHS=1:2:2. In order to make the reaction complete and thorough, the molar ratio of ketal TK-COOH to N-hydroxysuccinimide NHS is 1:2-2.2, preferably 1:2.1; the molar ratio of ketal TK-COOH to N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDC is 1:2-2.2, preferably 1:2.1; the stirring time is 2-4h; and the stirring rate is 600-1000rpm, preferably 800rpm.

[0043] The dopamine DA solution in step B) is prepared as follows: dopamine is dissolved in a phosphate buffer solution (PBS buffer) at pH 5.5.

[0044] In particular, the molar concentration of the dopamine solution is 0.1-0.3M, preferably 0.2M.

[0045] The stirring reaction temperature is 40-50 DEG C, preferably 45 DEG C; the stirring reaction time is preferably 12h; the molar ratio of dopamine to the ketone thio-ketone in the activated ketone thio-ketone compound is greater than or equal to 2:1, preferably (2-2.2):1; the molecular weight cut-off of the dialysis bag is 200Da; and the dialysis time is 40-60h.

[0046] In step C), the mass ratio of dopamine to the active oxygen-responsive dopamine monomer DA-TK-DA is 1:1; the mass ratio of dopamine to the drug is 5:(0.5-3), preferably 5:1; and the ratio of the mass of the drug in the dopamine-drug mixed system to the sum of the mass of dopamine and the active oxygen-responsive dopamine monomer is greater than 0, preferably 5-30:100, further preferably 10-20:100, and further preferably 10:100.

[0047] The mass ratio of dopamine to the drug doxorubicin is 5:(0.5-3), preferably 5:1; and the ratio of the mass of doxorubicin in the dopamine-drug mixed system to the sum of the mass of dopamine and the active oxygen-responsive dopamine monomer is greater than 0, preferably 5-30:100, further preferably 10-20:100, and further preferably 10:100.

[0048] In step C), the molar ratio of dopamine to the added sodium hydroxide is 1:(1-4), preferably 1:(2-4).

[0049] In step C), the polymerization reaction temperature is 50-70 DEG C; and the reaction time is 1-4h.

[0050] In particular, the compound after the polymerization reaction is subjected to centrifugal treatment, and the precipitate is washed with water to obtain the drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticle.

[0051] The drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticle is resuspended in water or physiological saline to prepare a drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticle solution, wherein the concentration of the loaded drug (doxorubicin) is 10-20 μg / mL.

[0052] The second aspect of the present application provides a preparation method of a magnetized platelet drug delivery system, comprising the following steps:

[0053] (I) mixing and incubating platelets and a drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticle solution, and centrifuging to obtain a drug-loaded platelet drug delivery system PDN@PLT;

[0054] (II) The drug-loaded platelet drug delivery system and the polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticle solution are mixed and incubated to obtain the magnetized platelet drug delivery system PDN-FeN@PLT.

[0055] In the present invention, there is no strict restriction on the preparation of platelets and ferroferric oxide magnetic nanoparticles. For example, they can be prepared by conventional preparation methods in the art or purchased commercially. The preparation method of drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticles is prepared according to the technical means of this patent.

[0056] Preferably, the mixing ratio of the platelets and the reactive oxygen species / photothermal dual-responsive polydopamine nanoparticles in step (I) is 5×10 6 1 mL of drug-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticle solution was added to each platelet, wherein the drug (doxorubicin) concentration in the drug-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticle solution was 10-20 μg / mL; the incubation time was 1-4 h.

[0057] Preferably, the mixing ratio of the platelets and the polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticles in step (II) is 5×10 6 1 mL of polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticle solution is added to each platelet, wherein the concentration of ferroferric oxide in the polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticle solution is 40-80 μg / mL; the incubation time is 1-4 hours.

[0058] Preferably, the platelets in step I) are prepared from ex vivo blood according to the following method:

[0059] After adding sodium heparin to the ex vivo blood for anticoagulation, the blood is placed in a centrifuge tube and centrifuged at 100-200g for 20-30 minutes at room temperature; the platelet-rich plasma supernatant is collected and centrifuged again at 100-200g for 20-30 minutes to remove red blood cells; then, the platelet-rich plasma is centrifuged at 3200-3800 rpm for 20-30 minutes, the precipitate is collected and washed 2-3 times with PBS buffer to obtain the platelets.

[0060] Preferably, the platelets are extracted from ex vivo blood.

[0061] All magnetic nanoparticles are suitable for the present invention. In the present invention, magnetic ferroferric oxide nanoparticles encapsulated by polyethyleneimine are used as an example for illustration.

[0062] Preferably, the magnetic nanoparticles are prepared according to the following method:

[0063] Under a nitrogen atmosphere, ferroferric oxide magnetic nanoparticles were prepared by co-precipitation with an alkaline solution, and the ferroferric oxide nanoparticles were obtained by freeze-drying. The ferroferric oxide nanoparticles were dissolved in water, and polyethyleneimine was dissolved in water. The two solutions were mixed at a mass ratio of ferroferric oxide to polyethyleneimine of 1:(0.25-2). The solution was ultrasonically reacted for 10 minutes using a cell disruptor, and then transferred to a 60°C water bath for magnetic stirring for 4 hours. The product was collected by centrifugation to prepare polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticles.

[0064] In particular, the method further comprises resuspending the prepared polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticles in physiological saline to prepare a polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticle resuspension.

[0065] Preferably, the drug nanoparticles are drug-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticles, wherein the drug is a drug containing a benzene ring in its molecular structure.

[0066] Preferably, the drug is doxorubicin, paclitaxel, puerarin, quercetin, or geniposide, preferably doxorubicin.

[0067] Preferably, the drug nanoparticles are drug-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticles, and the drug nanoparticles are prepared according to the following method:

[0068] (1A) Synthesis of reactive oxygen species-responsive dopamine monomers:

[0069] Dissolving 2,2'-[propane-2,2-diylbis(sulfide)]diacetic acid as thioketal in a dimethyl sulfoxide solution to prepare a thioketal solution; wherein the concentration of the thioketal is 0.05-0.15 M (preferably 0.1 M); then adding N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and stirring at room temperature for at least 2 hours to prepare an activated thioketal compound;

[0070] (1B) Dopamine is dissolved in a pH 5.5 phosphate buffer solution (PBS buffer) to prepare a dopamine solution, to which an activated thioketal compound is added. The solution is stirred in the dark for 10-14 hours, and the reaction mixture is placed in a dialysis bag and dialyzed against ultrapure water for 40-60 hours. The liquid in the dialysis bag is collected and dried to obtain an active oxygen-responsive dopamine monomer (DA-TK-DA).

[0071] (1C) dissolving dopamine, the drug to be loaded (e.g., doxorubicin), and the prepared reactive oxygen species-responsive dopamine monomer in water, mixing them evenly, adding sodium hydroxide solution, and performing a polymerization reaction under stirring to prepare drug-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticles;

[0072] Wherein, the concentration of the ketal in step 1A) is 0.05-0.15 M (preferably 0.1 M); the molar ratio of the ketal to N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 1:2; the molar ratio of the ketal to N-hydroxysuccinimide is 1:2; and the stirring rate is 600-1000 rpm, preferably 800 rpm.

[0073] Wherein, the concentration of the dopamine solution in step 1B) is 0.1-0.3M, preferably 0.2M; the stirring temperature under light-proof conditions is 45±5°C; the dialysis molecular weight cut-off is 200Da; and the drying treatment is preferably freeze-drying.

[0074] In step 1C), the drug to be loaded is a drug containing a benzene ring structure, preferably doxorubicin, paclitaxel, puerarin, quercetin, or gardenia glycoside, preferably doxorubicin; the mass ratio of dopamine to reactive oxygen species-responsive dopamine monomer is (0.5-2):1, preferably 1:1; the mass ratio of the drug to be loaded to dopamine is (0.5-3):5, preferably 1:5; the polymerization reaction temperature is 50-70°C; and the reaction time is 1-4 hours.

[0075] Wherein, the molar ratio of dopamine to the added sodium hydroxide is 1:(1-4), preferably 1:(2-4).

[0076] In particular, the method further comprises centrifuging the compound after the polymerization reaction, washing the centrifugal precipitate with water for 2-4 times, and the precipitate is the drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticles.

[0077] In particular, the method further comprises resuspending the prepared drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticles in physiological saline to prepare a drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticle resuspension.

[0078] Wherein, the mixing of the platelets and the drug-loaded active oxygen / photothermal dual-responsive polydopamine nanoparticles in step (I) is as follows: 6 1 mL of drug-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticle solution is added to each platelet, wherein the concentration of doxorubicin in the drug-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticle solution is 10-20 μg / mL.

[0079] Particularly, the incubation time in step (I) is 1-4 h; the incubation temperature is room temperature.

[0080] Wherein, in step (II), the platelets and polyethyleneimine encapsulated ferroferric oxide magnetic nanoparticles are mixed as follows:6 1 mL of magnetic nanoparticle solution is added to each platelet, wherein the concentration of ferroferric oxide in the magnetic nanoparticle solution is 40-80 μg / mL.

[0081] In particular, the incubation time in step (II) is 1-4 hours; the incubation temperature is room temperature.

[0082] The third aspect of the present invention provides a method for preparing a magnetized platelet drug delivery system, comprising the following steps:

[0083] (i) mixing platelets and polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticles, incubating the mixture, and centrifuging the mixture to prepare the ferroferric oxide-loaded platelet drug delivery system FeN@PLT;

[0084] (ii) The platelet drug delivery system FeN@PLT loaded with ferroferric oxide was mixed and incubated with drug-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticles, and then centrifuged to prepare the magnetized platelet drug delivery system FeN-PDN@PLT.

[0085] A fourth aspect of the present invention provides the use of the magnetized platelet drug delivery system or the magnetized platelet drug delivery system prepared by the above preparation method in targeted therapy of malignant tumors.

[0086] Compared with the prior art, the beneficial effects of the present invention include at least:

[0087] The platelet drug delivery system of the present invention utilizes platelets to simultaneously load two different nanoparticles. This system can be encapsulated by carrier cells to avoid premature drug release, reduce drug toxicity and side effects, improve drug administration safety, and achieve photo-controlled release of therapeutic agents and combined photothermal-chemotherapy treatment of tumors.

[0088] In the present invention, the inherent tumor tropism of platelets is conducive to the targeted accumulation of drugs in tumor tissues. The magnetic nanoparticles loaded with platelets give the platelet delivery system magnetism. In an external magnetic field environment, magnetic migration occurs, which is conducive to the magnetic targeted aggregation of tumors by the delivery system under the magnetic guidance of the external magnetic field. The specific recognition of tumor cells by platelets and the photothermal-microenvironment responsive release of therapeutic agents can effectively prevent tumor proliferation.

[0089] The magnetized platelet drug delivery system prepared by the present invention can achieve effective magnetic targeted tumor aggregation through the guidance of an external magnetic field; the platelets' own tumor recognition and deep tumor penetration properties facilitate the delivery of therapeutic agents deep into the tumor; the photothermal and reactive oxygen species responsive release of the drug (doxorubicin) enables spatiotemporally controlled release of the drug at the tumor site, enabling precise targeted combined therapy of tumors;

[0090] At the same time, the preparation method of the platelet drug delivery system of the present invention has a simple operation process, does not need to rely on complex equipment, and is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.

[0092] Figure 1 Schematic diagram of the synthesis of the active oxygen-responsive dopamine monomer DA-TK-DA in Example 1 of the present invention;

[0093] Figure 2 is the infrared spectrum of DA-TK-DA in Example 1 of the present invention;

[0094] Figure 3 Schematic diagram of the hydrated particle size of PDA-TK NPs synthesized in Examples 2-4 of the present invention;

[0095] Figure 4 Schematic diagram of the hydrated particle size of PDA-TK NPs synthesized in Example 5 and Dox@PDA-TK NPs synthesized in Example 6 of the present invention;

[0096] Figure 5 Schematic diagram of the hydrated particle size of the Fe3O4 nanoparticles prepared in Example 10 of the present invention and the polyethyleneimine-encapsulated ferrosoferric oxide Fe3O4@PEI NPs prepared in Example 11;

[0097] Figure 6 This is a bar graph of the doxorubicin Dox content in the platelet drug delivery system (PDN@PLT) in Example 14 of the present invention;

[0098] Figure 7 This is a bar graph showing the Fe3O4 content loaded by the magnetized platelet delivery system (FeN@PLT) in Example 15 of the present invention;

[0099] Figure 8 The fluorescence spectra of doxorubicin in aqueous phase and H2O2 phase in Experimental Example 1 of the present invention are shown;

[0100] Figure 9 The fluorescence spectra of the lower precipitate of nanoparticles PDA-TK NPs and Dox@PDA-TK NPs after incubation in water and H2O2 phases in Experimental Example 1 of the present invention are shown;

[0101] Figure 10The fluorescence spectrum of the centrifugal supernatant collected after incubation of the nanoparticles PDA-TK NPs and Dox@PDA-TK NPs in the water phase and the H2O2 phase in the test example 1 of the present application;

[0102] Figure 11 The release behavior curve of Dox@PDA-TK NPs under different release conditions in vitro for loading doxorubicin in the test example 2 of the present application;

[0103] Figure 12 The thermal imaging image of the in vitro photothermal conversion of Dox@PDA-TK NPs, PDN@PLT, FeN@PLT and PDN-FeN@PLT in the test example 3 of the present application;

[0104] Figure 13 The in vitro magnetic migration observation result image of PLT, PDN@PLT, FeN@PLT, PDN-FeN@PLT and Fe3O4@PEI NPs in the test example 4 of the present application;

[0105] Figure 14 The confocal image of the drug-loaded magnetized platelet drug delivery system PDN-FeN@PLT under the confocal microscope in the test example 5 of the present application;

[0106] Figure 15 The transwell chamber experiment result schematic diagram of the drug-loaded magnetized platelet drug delivery system under the guidance of a magnetic field in the test example 6 of the present application;

[0107] Figure 16 The transport confocal image of doxorubicin Dox between the magnetized platelet drug delivery system and tumor cells under laser irradiation of the drug-loaded magnetized platelet drug delivery system in the test example 6 of the present application;

[0108] Figure 17 The killing ability of the drug-loaded magnetized platelet drug delivery system on rat glioma cells C6-Luc in the test example 6 of the present application;

[0109] Figure 18 The thermal imaging image of the drug-loaded magnetized platelet drug delivery system inside tumor tissue in the test example 7 of the present application;

[0110] Figure 19 The tumor volume growth curve of a subcutaneous C6-Luc tumor-bearing mouse in the treatment process of intravenous injection of the drug-loaded magnetized platelet drug delivery system of the present application in the test example 7 of the present application;

[0111] Figure 20 The optical image of the tumor tissue removed after 18 days of treatment of a subcutaneous C6-Luc tumor-bearing mouse by intravenous injection of the magnetized platelet drug delivery system of the present application in the test example 7 of the present application;

[0112] Figure 21 This is the weight growth curve of mice during the treatment of subcutaneous C6-Luc tumor-bearing mice by intravenous injection of the magnetized platelet drug delivery system of the present invention in Experimental Example 7 of the present invention;

[0113] Figure 22 These are bioluminescent images of mouse tumors during the treatment of C6-Luc tumor-bearing mice with orthotopic brain tumors by intravenous injection of the magnetized platelet drug delivery system of the present invention in Experimental Example 8 of the present invention. DETAILED DESCRIPTION

[0114] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only used as examples and cannot be used to limit the scope of protection of the present invention.

[0115] It should be noted that, unless otherwise specified, the technical or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0116] Currently, in the clinical treatment of tumors, the efficacy of a single drug or treatment method is limited. Therefore, the combination of two or more therapeutic drugs or treatment methods has become an effective treatment method. At the same time, targeted drug delivery is a prerequisite for achieving effective treatment.

[0117] The magnetized platelet multifunctional drug delivery system can achieve tumor-targeted aggregation of the delivery system under the drive of an external magnetic field, thereby improving its tumor targeting; at the same time, the platelets' own tumor tendency and deep penetration into the tumor improve the deep distribution of the therapeutic agent in the tumor.

[0118] Platelet encapsulation of photothermal-active oxygen dual-responsive nanoparticles loaded with drugs (e.g., drugs containing a benzene ring structure, other oncology drugs containing a benzene ring structure, such as doxorubicin, puerarin, quercetin, paclitaxel, geniposide, etc.) reduces the risk of drug resistance, avoids toxic side effects caused by premature drug release, and increases drug safety.

[0119] In particular, anti-cancer and anti-tumor drugs can be controlled by external lasers to trigger the spatiotemporal controlled release of drugs at the tumor site, thereby achieving combined photothermal-chemotherapy treatment of tumors. The combined use of treatment methods complements each other and exerts better therapeutic effects.

[0120] In view of this, an embodiment of the present invention provides a magnetized platelet drug delivery system, which includes platelets, magnetic nanoparticles and drug nanoparticles; the magnetic nanoparticles and drug nanoparticles are encapsulated in the cytoplasm of the platelets.

[0121] The technical solution of the present invention is further described in detail below through specific embodiments.

[0122] Example 1 Preparation of Synthetic Drug Skeleton - Dopamine Monomer Responsive to Active Oxygen Species

[0123] The preparation process of dopamine monomers responsive to active oxygen species is as follows Figure 1 As shown, the following steps are included:

[0124] 1. Weigh 2,2'-[propane-2,2-diylbis(sulfide)]diacetic acid (TK-COOH, 1 mmol) and dissolve it in 10 mL of dimethyl sulfoxide solution; add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 2.1 mmol) and N-hydroxysuccinimide (NHS, 2.1 mmol), and stir at room temperature for 2 h to obtain an activated thioketal compound TK-NHS solution;

[0125] The concentration of the solution formed by dissolving TK-COOH in DMSO is 0.1M (usually 0.05-0.15M); during the condensation reaction of TK, NHS, and EDC, the theoretical molar ratio is 1:2. In order to ensure complete and thorough reaction, the molar ratio of ketal TK-COOH to N-hydroxysuccinimide NHS is 1:2-2.2, preferably 1:2.1; the molar ratio of ketal TK-COOH to N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDC is 1:2-2.2, preferably 1:2.1.

[0126] 2. Dissolve dopamine (DA, 2 mmol) in 10 mL of pH 5.5 phosphate buffer solution (PBS buffer) to prepare a dopamine solution; wherein the molar concentration of the dopamine solution is 0.2 M (usually 0.1-0.3 M);

[0127] 3. Add the activated ketal compound TK-NHS solution prepared in step 1 to the dopamine solution, stir at 45° C. (usually 40-50° C.) in the dark, and react for 12 hours (usually 10-14 hours); wherein the molar ratio of dopamine to the ketal compound in the activated ketal compound is 2:1 (usually (2-2.2):1);

[0128] 4. Place the light-proof reaction system in a dialysis bag (molecular weight cutoff of 200 Da). After dialysis with ultrapure water for 50 hours (usually 40 to 60 hours), collect the liquid in the dialysis bag and freeze-dry it to obtain an active oxygen-responsive dopamine monomer (DA-TK-DA).

[0129] The reactive oxygen species-responsive dopamine monomer (DA-TK-DA) was used as the skeleton for the subsequent synthesis of drug-loaded photothermal-reactive oxygen dual-responsive polydopamine nanoparticles.

[0130] The prepared DA-TK-DA, raw material TK, DA, and simple mixture of TK and DA were characterized by Fourier transform infrared spectroscopy. Figure 2 As shown:

[0131] from Figure 2 It can be seen that dopamine (DA) is at 1502 cm -1 The NH stretching vibration characteristic peak appears at 1705 cm -1 The characteristic peak of -C=O in carboxylic acid appears at 1580 cm-1. In the mixture of the two (DA+TK), the characteristic peaks of dopamine and thioketal still exist. In DA-TK-DA, the characteristic peak of NH stretching vibration of dopamine moves to 1580 cm-1. -1 At the same time, the characteristic peak of -C=O in the carboxylic acid in the thioketal is weakened, while at 1640 cm -1 The characteristic peak of -C=O in amide appeared at , which indicated the formation of amide bond between dopamine and ketal thiol, indicating the successful synthesis of DA-TK-DA.

[0132] Example 2 Preparation of reactive oxygen species / photothermal dual-responsive polydopamine nanoparticles PDA-TK NPs

[0133] This embodiment is a method for preparing polydopamine nanoparticles PDA-TK NPs with dual responses to active oxygen and light and heat. During the preparation process, the mass ratio of dopamine DA and active oxygen-responsive dopamine monomer DA-TK-DA is 1:2. The specific preparation method is as follows:

[0134] 1. Accurately weigh dopamine (5 mg) and DA-TK-DA (10 mg) prepared according to the method of Example 1, place them together in a 50 mL eggplant-shaped flask, add 10 mL of water to dissolve the sample, place the reaction flask in a 50±5°C water bath, and stir magnetically for 30 min to mix evenly;

[0135] In the embodiment of the present invention, the mass ratio of dopamine to the reactive oxygen species-responsive dopamine monomer DA-TK-DA is described as 1:2. Other ratios, such as 1:(0.5-2), preferably 1:(0.5-1), are also applicable to the present invention.

[0136] 2. Slowly add NaOH solution (1 M, 84 μL) dropwise and stir at 50 ± 5 ° C for 4 h (usually 1-5 h); then centrifuge the reaction mixture (10,000 rpm for 10 min), wash the centrifugal precipitate with water three times (usually at least once), and collect the lower precipitate to obtain active oxygen / photothermal dual-responsive polydopamine nanoparticles PDA-TK NPs;

[0137] Among them, the molar ratio of DA to NaOH is 1:3.2 (usually the molar ratio of DA to NaOH is 1:(2-4)), which is suitable for the present invention.

[0138] NaOH was added to maintain the alkaline environment for dopamine polymerization. The hydrated particle size of PDA-TK NPs was measured using a Malvern particle size analyzer. Figure 3 As shown, the particle size spectrum has two peaks, indicating that the particle size uniformity is too poor and it is not used in subsequent synthesis experiments.

[0139] The active oxygen / photothermal dual-responsive polydopamine nanoparticles PDA-TKNPs prepared according to the method of Example 2 were added to water and resuspended to prepare a PDA-TK NPs resuspension.

[0140] Example 3 Preparation of reactive oxygen species / photothermal dual-responsive polydopamine nanoparticles PDA-TK NPs

[0141] In this embodiment, the mass ratio of dopamine DA to the reactive oxygen species-responsive dopamine monomer DA-TK-DA is 1:1. The specific preparation method is as follows:

[0142] 1. Accurately weigh 5 mg of dopamine and 5 mg of DA-TK-DA and dissolve them in 10 mL of water. Place DA in a 50±5℃ water bath and stir magnetically for 30 minutes to mix evenly.

[0143] 2. Slowly add 84 μL of 1 M NaOH solution, stir and react at 50±5°C for 4 h, centrifuge at 10,000 rpm for 10 min, wash the precipitate three times with secondary water, and collect the lower precipitate to obtain nanoparticles PDA-TK NPs.

[0144] All the prepared PDA-TK NPs were resuspended in 5 mL of water to prepare a PDA-TK NPs resuspension, and the hydrated particle size was measured using a Malvern particle size analyzer. Figure 3 As shown, the particle size is 229 nm and the PDI is 0.269.

[0145] Example 4 Preparation of Reactive Oxygen Species / Photothermal Dual-Response Polydopamine Nanoparticles PDA-TK NPs

[0146] The mass ratio of dopamine DA and dopamine monomer DA-TK-DA responsive to dopamine and active oxygen in the embodiment is 2:1, and the specific preparation method is as follows:

[0147] 1. 10 mg of dopamine and 5 mg of DA-TK-DA were accurately weighed and dissolved in 10 mL of water, and the DA was placed in a 50±5°C water bath and magnetically stirred for 30 min to mix uniformly;

[0148] 2. 84 μL of 1M NaOH solution was slowly added, and the reaction was stirred at 50±5°C for 4 h, centrifuged at 10000 rpm for 10 min, and the precipitate was washed with water three times, and the lower precipitate was collected to obtain the nanoparticle PDA-TK NPs.

[0149] The hydrated particle size was measured by a Malvern particle size analyzer, as shown in Figure 3 The particle size was 645 nm, and the PDI was 0.287.

[0150] Example 5: Preparation of active oxygen / photothermal dual-responsive polydopamine nanoparticles PDA-TK NPs

[0151] In the embodiment, the mass ratio of dopamine DA and DA-TK-DA is 1:1, and except that the stirring reaction condition after adding NaOH solution in step 2) is that the reaction temperature is 70°C and the reaction time is 1 h, the rest is the same as that in example 3.

[0152] The hydrated particle size was detected by a Malvern particle size analyzer, as shown in Figure 4 The particle size was 238.2 nm, and the PDI was 0.20.

[0153] The particle sizes of the nanoparticles prepared by different reactants are different, but the photothermal activity and active oxygen response ability still exist, in order to better carry out the subsequent drug encapsulation, the reactant ratio of DA and DA-TK-DA with a mass ratio of 1:1 is selected in the subsequent drug-loaded nanoparticle preparation experiment.

[0154] Example 6: Preparation of drug (doxorubicin) loaded active oxygen / photothermal dual-responsive polydopamine nanoparticles Dox@PDA-TK NPs

[0155] In the specific embodiment of the application, the loaded drug is doxorubicin, and other drugs, antitumor drugs, anticancer drugs are suitable for the application.

[0156] In the process of preparing Dox@PDA-TK NPs, the drug added is doxorubicin, and the dosage of doxorubicin is marked according to the mass ratio of doxorubicin to the total mass of (DA and DA-TK-DA), and 1 mg of doxorubicin is put in, and the dosage is 10%, and the specific preparation method is as follows:

[0157] 1. Weigh 5 mg of dopamine and 5 mg of DA-TK-DA (prepared according to the method of Example 1) into a 50 mL eggplant-shaped flask and add 10 mL of water to dissolve; then add an aqueous solution of doxorubicin (200 μL) to the eggplant-shaped flask, wherein the concentration of the aqueous solution of doxorubicin is 5 mg / mL, stir evenly, place the reaction compound system in a water bath at 70±5° C., magnetically stir for 30 minutes, and mix evenly to prepare a dopamine-doxorubicin mixed system, wherein the mass ratio of dopamine to the active oxygen-responsive dopamine monomer DA-TK-DA is 1:1; the mass ratio of dopamine to doxorubicin is 5:1; the ratio of the mass of dopamine to the sum of the mass of dopamine and the active oxygen-responsive dopamine monomer in the dopamine-doxorubicin mixed system is 10:100 (usually greater than 0, preferably 5-30:100, more preferably 10-20:100);

[0158] 2. Slowly add NaOH solution (1 M, 84 μL) dropwise to the dopamine-doxorubicin mixture and stir at 70 ± 5°C for 1 h (usually 1-5 h); then centrifuge the reaction mixture (10,000 rpm for 10 min); then wash the centrifugal precipitate three times (usually at least once) with water and collect the lower precipitate to obtain doxorubicin-loaded nanoparticles Dox@PDA-TKNPs;

[0159] The upper layer solution was collected after centrifugation, and the content of unencapsulated doxorubicin in the upper layer solution was measured using a fluorescence spectrophotometer. The drug encapsulation efficiency of doxorubicin in the doxorubicin-loaded nanoparticles Dox@PDA-TK NPs was found to be 98.16%.

[0160] All the prepared Dox@PDA-TK NPs were resuspended in 5 mL of normal saline to form a Dox@PDA-TK NPs resuspension. The hydrated particle size of the nanoparticles Dox@PDA-TK NPs was measured using a Malvern particle size analyzer. Figure 4 As shown, its particle size is 284.9 nm and PDI is 0.29.

[0161] Example 7 Preparation of doxorubicin-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticles

[0162] In the preparation process of Dox@PDA-TK NPs in this example, the amount of doxorubicin added was 0.5 mg (5%). The specific preparation method is as follows:

[0163] The same procedures as in Example 6 were followed, except that 100 μL of the doxorubicin aqueous solution was added to the eggplant-shaped flask in step 1); the reaction compound system was placed in a water bath at 60±5°C; the mass ratio of dopamine to doxorubicin was 5:0.5; the mass percentage concentration of doxorubicin in the dopamine-doxorubicin mixed system was 5%; the stirring reaction temperature after adding the NaOH solution in step 2) was 60±5°C; and the reaction time was 2 h. The lower precipitate was collected to obtain doxorubicin-loaded nanoparticles Dox@PDA-TK NPs.

[0164] The upper layer solution was collected after centrifugation, and the content of unencapsulated doxorubicin in the upper layer solution was measured using a fluorescence spectrophotometer. The results showed that the drug encapsulation efficiency of doxorubicin in the doxorubicin-loaded nanoparticles Dox@PDA-TK NPs was 98.14%.

[0165] The hydrated particle size of Dox@PDA-TK NPs was measured using a Malvern particle size analyzer, and the particle size was 362.1 nm and the PDI was 0.469.

[0166] Example 8 Preparation of Doxorubicin-Loaded Active Oxygen Species / Photothermal Dual-Response Polydopamine Nanoparticles

[0167] In the preparation process of Dox@PDA-TK NPs in this example, the amount of doxorubicin added was 2 mg (20%). The specific preparation method is as follows:

[0168] The same procedures as in Example 6 were followed, except that 400 μL of the doxorubicin aqueous solution was added to the eggplant-shaped flask in step 1); the reaction compound system was placed in a water bath at 70±5° C.; the mass ratio of dopamine to doxorubicin was 5:2; and the mass percentage concentration of doxorubicin in the dopamine-doxorubicin mixed system was 20%. The lower precipitate was collected to obtain doxorubicin-loaded nanoparticles Dox@PDA-TK NPs.

[0169] The upper layer solution was collected after centrifugation, and the content of unencapsulated doxorubicin in the upper layer solution was measured using a fluorescence spectrophotometer. The results showed that the drug encapsulation efficiency of doxorubicin in the doxorubicin-loaded nanoparticles Dox@PDA-TK NPs was 97.7%.

[0170] The hydrated particle size was measured using a Malvern particle size analyzer, and the particle size was 332.1 nm and the PDI was 0.287.

[0171] Example 9 Preparation of doxorubicin-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticles

[0172] In the preparation process of Dox@PDA-TK NPs in this example, the amount of doxorubicin added was 3 mg (30%), and the specific preparation method was as follows:

[0173] The same procedures as in Example 6 were followed, except that 600 μL of the doxorubicin aqueous solution was added to the eggplant-shaped flask in step 1); the reaction compound system was placed in a water bath at 70±5° C.; the mass ratio of dopamine to doxorubicin was 5:3; and the mass percentage concentration of doxorubicin in the dopamine-doxorubicin mixed system was 30%. The lower precipitate was collected to obtain doxorubicin-loaded nanoparticles Dox@PDA-TK NPs.

[0174] The upper layer solution was collected after centrifugation, and the content of unencapsulated doxorubicin in the upper layer solution was measured using a fluorescence spectrophotometer. The results showed that the drug encapsulation efficiency of doxorubicin in the doxorubicin-loaded nanoparticles Dox@PDA-TK NPs was 94.2%.

[0175] The hydrated particle size was measured using a Malvern particle size analyzer, and the particle size was 380.2 nm and the PDI was 0.302.

[0176] Example 10 Preparation of ferroferric oxide magnetic nanoparticles

[0177] The reaction equation of Fe3O4 nanoparticles is as follows:

[0178] FeCl2+2FeCl3+8NH4OH→Fe3O4+8NH4Cl+4H2O

[0179] 1. Weigh 1.1 g of FeCl2·4H2O (5 mmol) and dissolve it in 25 mL of 1.0 M hydrochloric acid solution to prepare a FeCl2 hydrochloric acid solution, wherein the concentration of the FeCl2 hydrochloric acid solution is 0.2 M (usually 0.1-0.4 M);

[0180] 2. Weigh 2.7 g of FeCl3·6H2O (10 mmol) and dissolve it in 25 mL of freshly boiled deionized water. Wait until the temperature of the freshly boiled deionized water returns to room temperature to prepare an FeCl3 aqueous solution; the concentration of the FeCl3 aqueous solution is 0.4 M (usually 0.2-0.8 M);

[0181] 3. Add the two solutions to a 250 mL three-necked flask and mix, wherein the molar ratio of FeCl2 to FeCl3 is 1:2; then pour 80 mL of 1.5 M ammonia solution into a constant pressure dropping funnel and load it on the three-necked flask; seal the system, evacuate, and introduce nitrogen for 1 hour; under magnetic stirring, add the ammonia solution dropwise to initiate the reaction; after the addition is completed, continue stirring at room temperature under nitrogen atmosphere for 1 day; wherein, the molar ratio of FeCl2 to ammonia NH3 in the ammonia solution is 1:24 (usually 1:20-30);

[0182] 4、After stirring at room temperature for 1 day, stop the reaction, let the system stand for 10 min to allow the system to separate into layers, then place a magnet at the bottom of the bottle, stand for 10 min of adsorption, slowly pour out the upper liquid, then re-add freshly boiled deionized water to wash the solid product, and repeat the washing 4-5 times.

[0183] 5、In the manner of step 3, reassemble the reaction system in a three-necked flask, add 50 mL of 1M perchloric acid solution to the constant pressure dropping funnel, and load the constant pressure dropping funnel on the three-necked flask; seal the system, vacuumize and introduce nitrogen for 30 min, then add the perchloric acid solution in the constant pressure dropping funnel to the three-necked flask, stir at room temperature for 15 min. Let the system stand for 10 min to allow the system to separate into layers, then place a magnet at the bottom of the bottle, stand for 10 min of adsorption, slowly pour out the upper liquid, then re-add freshly boiled deionized water to wash the solid product, and repeat the washing 4-5 times;

[0184] 6、Reassemble the device, introduce nitrogen again for 30 min, and again add 50 mL of 1M perchloric acid solution from the constant pressure dropping funnel, stir at room temperature for 15 min. Stop the reaction, transfer the product to a 50 mL centrifuge tube, centrifuge at 10000 rpm for 10 min, discard the supernatant, and re-disperse the lower solid in the centrifuge tube in deionized water, transfer to a dialysis bag (molecular weight cut-off: 14000 Da) for dialysis treatment until the outside liquid is neutral, to obtain a Fe3O4 nanoparticle solution, then freeze-dry to obtain Fe3O4 nanoparticles, and store at 4°C.

[0185] Weigh 2 mg of Fe3O4 nanoparticles and dissolve them in 2 mL of water to prepare a 1 mg / mL Fe3O4 nanoparticle solution. Use a Malvern particle size analyzer to measure the hydration particle size of the Fe3O4 nanoparticle solution, as shown in Figure 5 The particle size is 81.9 nm and the PDI is 0.23.

[0186] Example 11 Preparation of polyethyleneimine (PEI) encapsulated ferroferric oxide magnetic nanoparticles Fe3O4@PEI NPs

[0187] Weigh 30 mg of Fe3O4 nanoparticles prepared according to the method of Example 10 and dissolve them in water to prepare a 5 mg / mL Fe3O4 nanoparticle solution.

[0188] Remove 0.75 mL of 10 mg / mL polyethyleneimine (PEI, molecular weight 1800 Da) solution and add it to the Fe3O4 nanoparticle solution, mix well, and the mass ratio of Fe3O4 to PEI is 1:0.25.

[0189] Then the mixture is subjected to ultrasonic treatment using a cell disruptor, so that the Fe3O4 nanoparticles are uniformly dispersed, wherein the power of the ultrasonic treatment is 150 W, the ultrasonic treatment is performed once every 10 s, the duration of each ultrasonic treatment is 3 s, and the ultrasonic treatment is performed for 10 min;

[0190] Then the mixture subjected to the ultrasonic treatment is placed in a water bath at 60 ± 5 °C, and subjected to encapsulation treatment under magnetic stirring, wherein the stirring rate is 1500 rpm (typically 1000-1500 rpm), the temperature of the encapsulation treatment is 60 ± 5 °C, and the time of the encapsulation treatment is 3-5 h, preferably 4 h.

[0191] After the encapsulation treatment for 4 h (typically 3-5 h), the product of the encapsulation treatment is collected, centrifuged at 9000 rpm for 10 min, and the supernatant is discarded. The precipitate is washed with deionized water for 3 times, resuspended with deionized water, to obtain a resuspension solution of Fe3O4@PEI NPs, which is then freeze-dried to obtain polyethyleneimine (PEI) encapsulated Fe3O4 magnetic nanoparticles Fe3O4@PEI NPs.

[0192] The prepared Fe3O4@PEI NPs nanoparticles are resuspended in 6 mL of normal saline to obtain a resuspension solution of Fe3O4@PEI NPs (aqueous solution of Fe3O4@PEI NPs), which is stored at 4 °C for standby use.

[0193] The hydrated particle size is measured using a Malvern particle size analyzer, as shown in FIG. 1, and the particle size is 176.9 nm and the PDI is 0.19. Figure 5

[0194] Example 12: Preparation of polyethyleneimine (PEI) encapsulated Fe3O4 magnetic nanoparticles Fe3O4@PEI NPs

[0195] Except that the polyethyleneimine (PEI) solution and the Fe3O4 nanoparticle solution prepared according to the method of Example 10 are mixed in a mass ratio of Fe3O4 to PEI of 1:0.5, 1:1 and 1:2, respectively, the rest is the same as Example 11. The hydrated particle size is measured using a Malvern particle size analyzer, wherein:

[0196] When the mass ratio of Fe3O4 to PEI is 1:0.5, the hydrated particle size of Fe3O4@PEI NPs is 261.6 nm and the PDI is 0.269; when the mass ratio of Fe3O4 to PEI is 1:1, the hydrated particle size of Fe3O4@PEI NPs is 322.3 nm and the PDI is 0.353; and when the mass ratio of Fe3O4 to PEI is 1:2, the hydrated particle size of Fe3O4@PEI NPs is 451.8 nm and the PDI is 0.221.

[0197] ​The experimental results show that as the amount of PEI added increases, the hydrated particle size increases. Nanoparticles with a hydrated particle size below 400 nm are usually selected.

[0198] Example 13

[0199] Blood from healthy experimental rats was anticoagulated with sodium heparin, placed in a centrifuge tube, and centrifuged at 100g for 20 minutes at room temperature. The supernatant containing platelet-rich plasma was collected and centrifuged again at 100g for 20 minutes to further remove red blood cells. Thereafter, the platelet-rich plasma was centrifuged at 3500 rpm for 20 minutes, the precipitate was collected and washed twice with 10mM PBS buffer at pH 7.4. The platelet precipitate at the bottom was collected and resuspended in PBS buffer at pH 7.4. The platelet concentration was then counted using a cell counter. The platelet concentration was 1.5×10 8 pieces / mL.

[0200] Example 14 Preparation of platelet drug delivery system encapsulated drug nanoparticles (PDN@PLT), i.e., platelet drug delivery system (PDN@PLT) of doxorubicin-encapsulated polydopamine nanoparticles Dox@PDA-TK NPs

[0201] The Dox@PDA-TK NPs prepared according to the method of Example 6 were resuspended in physiological saline to form a Dox@PDA-TK NPs resuspension, wherein the concentration of doxorubicin in the Dox@PDA-TK NPs resuspension was 10 μg / mL or 20 μg / mL, respectively.

[0202] Platelets were mixed with Dox@PDA-TK NPs nanoparticle suspension and incubated at room temperature for 1, 2, and 4 h. The mixing ratio was 5×10 6 1 mL of Dox@PDA-TK NPs suspension was added to each platelet, where the content of doxorubicin in the nanoparticle suspension was 10 or 20 μg / mL;

[0203] The incubation product system was centrifuged, the supernatant was removed, and the system was washed three times with PBS buffer at pH 7.4. The precipitate at the bottom was collected to obtain the platelet drug delivery system encapsulating drug nanoparticles (PDN@PLT), which was resuspended in 2 mL of normal saline.

[0204] Take 1 mL of the above-mentioned appropriate amount of PDN@PLT, centrifugal collection of the bottom precipitate, after the precipitate (i.e. the platelet drug delivery system (PDN@PLT) encapsulating drug nanoparticles) is resuspended in water, the PDN@PLT is broken by using a cell disruptor, the nanoparticles are released from the platelets, and then the absorbance value of the nanoparticles in the supernatant is measured by using a UV spectrophotometer, and the doxorubicin loading capacity of the nanoparticles is calculated.

[0205] The analysis results are shown in Table 1 as follows: Figure 6 As shown in Table 1, with the extension of incubation time and the increase of drug concentration, the content of Dox@PDA-TK NPs in the platelets gradually increases.

[0206] Example 15 Preparation of platelet delivery system loaded with ferroferric oxide (FeN@PLT) (i.e. preparation of platelet delivery system loaded with magnetic nanoparticles Fe3O4@PEI NPs (abbreviated as FeN@PLT))

[0207] The polyethyleneimine (PEI) encapsulated ferroferric oxide magnetic nanoparticles Fe3O4@PEI NPs prepared according to the method of Example 11 are dispersed in water to prepare a Fe3O4@PEI NPs resuspension, wherein the mass-volume ratio of Fe3O4@PEI NPs to water is 10:10 (usually 10:(5-20));

[0208] Fluorescein isothiocyanate solution (dissolved in dimethyl sulfoxide) is added to the Fe3O4@PEI NPs resuspension, wherein the concentration of the fluorescein isothiocyanate solution is 1 mg / mL, and the reaction is carried out at room temperature in the dark for 6 h (usually 5-7 h);

[0209] The product after the fluorescence reaction is subjected to centrifugal treatment (9000 rpm for 10 min); the supernatant is removed, the precipitate is washed twice with deionized water, and the precipitate is resuspended with 5 mL of normal saline to prepare a fluorescently labeled Fe3O4@PEI NPs resuspension; the resuspension is diluted with normal saline to prepare a nanoparticle solution of fluorescently labeled Fe3O4@PEI NPs with ferroferric oxide concentrations of 40, 60, and 80 μg / mL, respectively;

[0210] The platelets are mixed with the nanoparticle solution of fluorescently labeled Fe3O4@PEI NPs, and then incubated at room temperature for 1, 2, and 4 h, respectively; wherein the mixing ratio is 5×10 6 The platelets are mixed with the nanoparticle solution of fluorescently labeled Fe3O4@PEI NPs, and then incubated at room temperature for 1, 2, and 4 h, respectively; wherein the mixing ratio is 5×10

[0211] The incubation product system was centrifuged, the supernatant was removed, and the system was washed three times with PBS buffer at pH 7.4. The precipitate at the bottom was collected to obtain the platelet drug delivery system loaded with ferroferric oxide (FeN@PLT), which was resuspended in 2 mL of normal saline.

[0212] Take 1 mL of the above-mentioned appropriate amount of FeN@PLT, centrifuge and collect the bottom precipitate, which is the platelet drug delivery system loaded with ferroferric oxide (FeN@PLT). After resuspending it in water, use a cell disruptor to break up the FeN@PLT to release the fluorescently labeled Fe3O4@PEI NPs nanoparticles from the platelets. Then use a fluorescence spectrophotometer to measure the absorbance value of the nanoparticles in the supernatant and calculate the loading amount of the nanoparticles.

[0213] The analysis results are as follows Figure 7 As shown in the figure, with the extension of incubation time and the increase of drug concentration, the content of Fe3O4@PEI NPs inside platelets gradually increased.

[0214] Example 16 Preparation of drug-loaded magnetized platelet drug delivery system (FeN-PDN@PLT)

[0215] Platelets were mixed with the fluorescently labeled Fe3O4@PEI NPs nanoparticle solution prepared in Example 15, and then incubated at room temperature for 1 hour (usually 0.5-2 hours). The mixing ratio of platelets to Fe3O4@PEI NPs nanoparticle solution was: 1×10 7 2 mL of a fluorescently labeled Fe3O4@PEI NPs nanoparticle solution was added to each platelet, wherein the concentration of the Fe3O4@PEI NPs nanoparticles in the fluorescently labeled Fe3O4@PEI NPs nanoparticle solution was 60 μg / mL (usually 40-80 μg / mL);

[0216] The incubation product system was centrifuged, the supernatant was removed, and the precipitate was washed three times with PBS buffer (pH 7.4). The precipitate at the bottom was collected to prepare a platelet drug delivery system loaded with ferroferric oxide (FeN@PLT). The prepared FeN@PLT was resuspended in physiological saline (1 mL).

[0217] Dox@PDA-TK NPs were prepared according to the method in Example 6 and resuspended in normal saline. The volume of normal saline added was determined based on the doxorubicin loading to achieve a final doxorubicin concentration of 20 μg / mL. 1 mL of the Dox@PDA-TK NPs suspension was added to the resuspended FeN@PLT solution and incubated at room temperature for 1 hour. The incubated product system was centrifuged to remove the supernatant, and the lower precipitate was the drug-loaded magnetized platelet drug delivery system (FeN-PDN@PLT) of the present invention. The system was resuspended in 2 mL of normal saline.

[0218] Example 17 Preparation of drug-loaded magnetized platelet drug delivery system (PDN-FeN@PLT)

[0219] The Dox@PDA-TK NPs prepared according to the method in Example 6 were resuspended in normal saline. The volume of the solution was determined according to the doxorubicin loading amount so that the concentration of doxorubicin in the prepared Dox@PDA-TK NPs resuspension was 20 μg / mL. Platelets (1×10 7 ) was mixed with 2 mL of Dox@PDA-TK NPs resuspension and incubated at room temperature for 1 h. The system was centrifuged to remove the supernatant to prepare PDN@PLT, which was then washed three times with pH 7.4 PBS buffer; finally, the prepared PDN@PLT was resuspended in 1 mL of normal saline;

[0220] 1 mL of the fluorescently labeled Fe3O4@PEI NPs solution prepared according to the method of Example 15 was added to the resuspended PDN@PLT solution. The Fe3O4 concentration in the fluorescently labeled Fe3O4@PEI NPs solution was 60 μg / mL. After incubation at room temperature for 1 hour, the incubation product system was centrifuged to remove the supernatant. The lower precipitate was the magnetized platelet drug delivery system (PDN-FeN@PLT). This was resuspended in 2 mL of normal saline.

[0221] Experimental Example 1 In vitro hydrogen peroxide response performance test of doxorubicin-loaded reactive oxygen species / photothermal dual-responsive polydopamine nanoparticles Dox@PDA-TK NPs

[0222] 1 mL each of the PDA-TK NPs resuspension in Example 5 and the Dox@PDA-TK NPs resuspension in Example 6 were transferred to 2 mL EP tubes, centrifuged at 12000 rpm for 10 min, and the supernatant was removed.

[0223] The lower precipitate was resuspended by adding 1 mL of 3% hydrogen peroxide solution and 1 mL of deionized water respectively, and placed at room temperature in the dark overnight. The EP tube was then centrifuged (12000 rpm, 10 min), the supernatant was collected, and the precipitate was resuspended with 1 mL of deionized water.

[0224] An appropriate amount of free Dox was weighed and divided into two groups. One group was dissolved in deionized water, and the other group was dissolved in 3% hydrogen peroxide solution. Both groups were left overnight to detect the effect of 3% hydrogen peroxide solution on Dox fluorescence.

[0225] Then, the fluorescence curves of the supernatant and precipitate of the nanoparticle sample and the doxorubicin sample were measured by a fluorescence spectrophotometer at an excitation wavelength of 488 nm. The hydrogen peroxide responsiveness of the nanoparticles PDA-TK NPs and Dox@PDA-TK NPs was verified based on the fluorescence curves. The experimental results are shown in Figure 2. Figure 5-7 shown.

[0226] from Figure 8 It can be seen that the fluorescence spectrum of the Dox sample incubated overnight in 3% hydrogen peroxide solution is almost the same as the fluorescence spectrum of Dox in water, indicating that hydrogen peroxide H2O2 does not interfere with the fluorescence properties of Dox.

[0227] from Figure 9 It can be seen that in PDA-TK NPs, no characteristic peak of doxorubicin was observed regardless of the presence of hydrogen peroxide, indicating that the skeleton of the nanoparticles did not interfere with the determination of doxorubicin under the test conditions; after incubation in the aqueous phase overnight, doxorubicin Dox in Dox@PDA-TK NPs was loaded in PDA-TK NPs through π-π bonds. Due to the shielding effect of the polymer skeleton, the characteristic peak of doxorubicin in the sample (Dox@PDA-TK NPs, aqueous phase in the figure) almost disappeared; but after incubation in the H2O2 phase overnight, the active oxygen response bond ketal thiol TK in the polymer skeleton of Dox@PDA-TK NPs broke, causing the polymer system structure to become loose and doxorubicin to be released, so that the characteristic peak of doxorubicin in the sample could be detected ( Figure 9 Dox@PDA-TK NPs, H2O2 phase).

[0228] from Figure 10 It can be seen from the fluorescence spectrum of the supernatant that Dox@PDA-TK NPs release the loaded doxorubicin after being treated with hydrogen peroxide, and the signal peak of doxorubicin can be detected in the supernatant.

[0229] The above results can prove that Dox@PDA-TK NPs have good hydrogen peroxide responsiveness and can achieve the active oxygen responsive release of drugs. Experimental Example 2 In vitro photothermal and hydrogen peroxide responsive drug release test of doxorubicin loaded active oxygen / photothermal dual responsive polydopamine nanoparticles Dox@PDA-TK NPs

[0230] The release medium conditions were divided into: phosphate buffered saline (PBS) group, PBS+Laser (808nm laser) group, PBS+H2O2 group, and PBS+H2O2+Laser (808nm laser) group.

[0231] The Dox@PDA-TK NPs prepared according to the method in Example 6 were resuspended in water to prepare a Dox@PDA-TK NPs resuspension, wherein the concentration of doxorubicin in the resuspension was 100 μg / mL.

[0232] Pipette 1 mL of the Dox@PDA-TK NPs resuspension into a 2 mL EP tube, centrifuge to remove the supernatant, and then add 1.5 mL of release medium to disperse the nanoparticles and shake on a 37°C shaker. Set sampling time points at 0, 2, 4, 6, 8, 11, 30, 60, 84, 108, 132, and 168 h. At each sampling time point, remove the EP tube and centrifuge. Collect 1.2 mL of the supernatant, then add 1.2 mL of fresh release medium to the EP tube and resuspend the system.

[0233] In the laser irradiation group, the samples were irradiated with an 808 nm semiconductor laser for 5 min at a radiation intensity of 1 W / cm 2 Continue shaking the shaker until the next sampling point. The supernatant of each release group collected at different time points was measured with a fluorescence spectrophotometer to determine the Dox fluorescence content in the sample and calculate the cumulative release of doxorubicin. The measurement results are shown in Figure 2. Figure 11 As shown:

[0234] Dox@PDA-TK NPs release Dox in a dual-responsive manner, responsive to both photothermal and hydrogen peroxide. External laser and hydrogen peroxide can accelerate the release rate and amount of doxorubicin from the nanoparticles.

[0235] Experimental Example 3: Evaluation of the in vitro photothermal performance of the magnetized platelet drug delivery system:

[0236] Platelets (PLT), doxorubicin-loaded nanoparticles Dox@PDA-TK NPs prepared according to the method of Example 6, and a platelet drug delivery system (PDN@PLT) encapsulating drug nanoparticles prepared according to the method of Example 14 were irradiated with 808 nm laser, wherein the content of doxorubicin in the Dox@PDA-TK NPs solution was 20 μg / mL; a platelet delivery system (FeN@PLT) loaded with ferroferric oxide prepared according to the method of Example 15, wherein the concentration of Fe3O4@PEI NPs nanoparticles in the Fe3O4@PEI NPs nanoparticle solution was 60 μg / mL, and a drug-loaded magnetized platelet drug delivery system (PDN-FeN@PLT) prepared according to the method of Example 17, wherein the laser power density was 1.0 W / cm 2 ; Irradiation for 5 minutes, temperature changes were recorded every minute using an infrared thermal imager (E6, FLIR System Inc, USA); in vitro thermal imaging results are as follows Figure 12 shown.

[0237] Depend on Figure 12 It can be seen that

[0238] The temperature of platelet PLT hardly changes with the extension of laser irradiation time; the temperature of platelet delivery system FeN@PLT loaded with ferroferric oxide increases slightly with the extension of irradiation time, and the temperature rises to about 35.2℃ after 5 minutes; while the temperature of platelet drug delivery system PDN@PLT encapsulating drug nanoparticles and drug-loaded magnetized platelet drug delivery system PDN-FeN@PLT gradually increases with the extension of irradiation time, and rises to about 45.2℃ after 5 minutes, which reveals the good photothermal conversion performance of PDN-FeN@PLT system.

[0239] Experimental Example 4 Evaluation of in vitro magnetic migration performance of magnetized platelet drug delivery system:

[0240] Platelets (PLT), the platelet drug delivery system encapsulating drug nanoparticles prepared by the method of Example 14 (PDN@PLT), and the platelet delivery system loaded with ferroferric oxide (FeN@PLT) prepared in Example 15, wherein the concentration of Fe3O4@PEI NPs nanoparticles in the Fe3O4@PEI NPs nanoparticle solution is 60 μg / mL, and 0.1 mL of each of the drug-loaded magnetized platelet drug delivery system (PDN-FeN@PLT) prepared in Example 17 were transferred to a 2 mL transparent glass vial, and 0.9 mL of normal saline was added;

[0241] 20 μL of Fe3O4@PEI NPs prepared according to the method of Example 11 was transferred to a 2 mL transparent glass vial and resuspended. 0.98 mL of normal saline was added. A magnet was placed on one side of the vial to apply an external magnetic field. The movement of the sample in the vial toward the side where the magnet was applied was observed to evaluate the magnetic migration performance of the sample.

[0242] The observation results are as follows Figure 13 As shown:

[0243] Platelet PLT and platelet drug delivery system PDN@PLT showed no signs of migration to the external magnetic field within 5 minutes. Platelets were dispersed in water and appeared as a milky white turbid system.

[0244] The migration of magnetic nanoparticles Fe3O4@PEI NPs (yellow-brown) can be observed at 30s, and almost all of the yellow-brown material can be seen gathering near the external magnetic field at 2min, indicating that the nanoparticles themselves have good magnetic migration properties.

[0245] The magnetic migration phenomenon of the magnetized platelet delivery system FeN@PLT and the magnetized platelet drug delivery system PDN-FeN@PLT group is also very outstanding. Obvious migration occurs after 10 seconds, and after 1 minute, it is completely enriched on the side of the external magnetic field.

[0246] The research results show that loading magnetic nanoparticles inside platelets can give the platelet drug delivery system good magnetic migration properties, which is conducive to achieving good aggregation of the platelet drug delivery system PDN-FeN@PLT near the external magnetic field under the magnetic guidance of the external magnetic field.

[0247] Test Example 5: Distribution of drug loading in the magnetized platelet drug delivery system:

[0248] The drug-loaded magnetized platelet drug delivery system PDN-FeN@PLT prepared in Example 17 was resuspended in physiological saline and dropped onto a coverslip. The drug distribution in the platelets was observed using a confocal microscope. The observation results are as follows: Figure 14 shown.

[0249] Depend on Figure 14 It can be seen that the red fluorescence of drug nanoparticles Dox@PDA-TKNPs is distributed inside the platelets, and magnetic nanoparticles Fe3O4@PEI NPs (black spheres, arrows indicate the position) are also dispersed in the platelet cytoplasm or adsorbed on the platelet membrane.

[0250] Figure 14 The first picture represents the fluorescence of drug nanoparticles, which are dispersed in the entire platelet system; the second picture is a bright field picture, and the black dots on it are ferroferric oxide nanoparticles, some of which are indicated by arrows. The third picture is the superposition of the two.

[0251] Test Example 6

[0252] This test example is the evaluation of the drug-loaded magnetized platelet drug delivery system (PDN-FeN@PLT) at the cellular level in Example 17:

[0253] Cell Culture: Luciferase-expressing rat glioma cells (C6-Luc) (purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China)) were cultured in Ham's F-12K medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 U / mL streptomycin. Mouse brain microvascular endothelial cells (bEnd.3) (purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China)) were cultured in DMEM high-glucose medium supplemented with 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin for 2-3 days. When the cell density reached 90% or higher, cells were passaged or plated for experiments. Culture conditions were: 37°C, 5% CO2, and 95% humidity in a CO2 incubator.

[0254] (1) Evaluation of the ability of the drug-loaded magnetized platelet drug delivery system PDN-FeN@PLT to migrate to tumor cells under magnetic field guidance:

[0255] C6-Luc cells were stained with new indocyanine green active ester for 30 min (labeled as IR-C6) and the cells were cultured at 3×10 5 The cells were seeded at a density of 10 cells / well on a 12-well plate and cultured in an incubator for 24 hours. bEnd.3 cells were added to the Transwell chamber and cultured for 24 hours. The Transwell chamber was transferred to a 12-well plate, and a platelet drug delivery system labeled with 5(6)-carboxydiacetate fluorescein-N-succinimidyl ester (CFSE) was added to the upper chamber. A magnet was placed at the bottom of the well plate and an external magnetic field (MF) was applied. After the system was cultured in a cell culture incubator for 24 hours, the Transwell chamber was removed, the original culture medium was aspirated, and after washing twice with PBS, 50 μL of serum-free Ham's F-12K culture medium was added. The well plate was placed under a laser confocal microscope for observation and photography. The results are shown in FIG. Figure 15 As shown:

[0256] We can observe the fluorescence of CFSE-labeled platelets in the lower well plate (labeled as CFSE-PLT), indicating that both platelets and platelet drug delivery systems can freely pass through the Transwell chamber simulating the blood-brain barrier and achieve migration to tumor cells. After applying an external magnetic field, in the drug-loaded magnetized platelet drug delivery system PDN-FeN@PLT treatment group, the fluorescence intensity of platelet CFSE-PLT in the lower cell layer is stronger than that of the group without a magnetic field. The magnetic guidance of the external magnetic field on the surface can enhance the ability of PDN-FeN@PLT to penetrate the Transwell chamber and enter the lower tumor cells. This shows that the drug-loaded magnetized platelet drug delivery system PDN-FeN@PLT of the present invention has good tumor magnetic targeting and tumor tropism.

[0257] (2) Transport of the therapeutic agent doxorubicin Dox between the magnetized platelet drug delivery system PDN-FeN@PLT and tumor cells:

[0258] C6-Luc cells (3×10 5 Cells / well) and the CFSE-stained platelet drug delivery system PDN@PLT of the present invention (prepared by the method of Example 14, the concentration of doxorubicin is 10 μg / mL, and the co-incubation time is 1 h) or PDN-FeN@PLT (the drug-loaded platelet drug delivery system in Example 17) were mixed in a ratio of 1:3 and then transferred to a confocal dish (Nest). In order to evaluate the effect of external laser on the drug transport process, the platelet drug delivery system PDN@PLT and PDN-FeN@PLT were irradiated with 808 nm laser for 3 min. After culturing for 24 h, the cells were washed three times with PBS and immediately observed by confocal microscopy (CLSM, ZEISS LSM 710). The observation results are shown in the following table. Figure 16 As shown;

[0259] Without laser treatment, red fluorescence of the chemotherapy drug doxorubicin (Dox) was present in the platelet delivery system (labeled as PLTCFSE), while the red fluorescence of the chemotherapy drug doxorubicin (Dox) was very weak in the tumor cells (labeled as C6-luc IR-NHS). After laser treatment, strong red fluorescence of the chemotherapy drug doxorubicin (Dox) was observed in the tumor cells (labeled as C6-luc IR-NHS), indicating that the therapeutic agent Dox was released from the platelet drug delivery systems PDN@PLT and PDN-FeN@PLT and transported into the tumor cells. This phenomenon demonstrates that external laser control can accelerate the release of the loaded therapeutic agent Dox from the magnetized platelet drug delivery system of the present invention, thereby promoting drug transport between the delivery system and tumor cells.

[0260] (3) The killing effect of magnetized platelet drug delivery system PDN-FeN@PLT on tumor cells:

[0261] C6-Luc cells (3×10 5 ) After being labeled with the fluorescent dye CFSE, the cells were mixed with the platelet drug delivery system PDN@PLT (prepared by the method of Example 14, with a doxorubicin concentration of 10 μg / mL and a co-incubation time of 1 h) or PDN-FeN@PLT (the drug-loaded platelet drug delivery system in Example 17) at a mixing ratio of 1:3; and then inoculated into a 6-well plate and cultured for 24 h. In order to regulate the killing effect of external laser on cells, the cells were irradiated with 808 nm laser for 0, 30 or 60 s, respectively, and then co-incubated for 24 h. The cells were then washed twice with PBS, dissociated and digested with trypsin, and the cells were collected. 1.5 mL of propidium iodide (PI) staining solution (5 μg / mL) was then added to the cells and incubated at 4°C in the dark for 30 min. Wash twice with PBS, collect the cells, resuspend in PBS, and record with flow cytometer FACS, analyze the proportion of PI-positive cells, evaluate cell apoptosis, and test the proportion of apoptotic cells. The higher the proportion of PI-positive cells, the greater the number of apoptotic cells. The analysis results are as follows Figure 17 .

[0262] like Figure 17As shown: Compared with the control group (blank C6-Luc cells), the number of PI-positive cells in tumor cells co-cultured with the platelet drug delivery system PDN@PLT or PDN-FeN@PLT increased significantly; at the same time, laser irradiation further increased the proportion of PI-positive tumor cells. The longer the laser irradiation time, the higher the tumor cell mortality rate. Moreover, the therapeutic effect of the PDN-FeN@PLT group was better than that of the PDN@PLT group. The main reason is that Fe3O4 nanoparticles also have certain photothermal conversion properties. The temperature is higher when laser irradiated, resulting in an increase in the apoptosis rate of tumor cells. The results show that the magnetized platelet drug delivery system PDN-FeN@PLT of the present invention can achieve combined photothermal-chemotherapy treatment of tumor cells and has good therapeutic effects.

[0263] Test Example 7

[0264] This test example is a study on the tumor inhibitory effect of the drug-loaded magnetized platelet drug delivery system (PDN-FeN@PLT) in Example 17 on ectopic brain glioma tumors (subcutaneous model)

[0265] Animal Husbandry: Male Balb / c nude mice (18-20 g) were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, China). Throughout the study, the nude mice were housed under SPF conditions with free access to standard chow and water. The mice were acclimated to the feeding for 3 days before the relevant experimental studies were performed. All studies adhered to the guidelines for the care and use of laboratory animals established by the Institutional Animal Care and Use Committee of Peking Union Medical College.

[0266] To establish an ectopic glioma model, 1×10 6 C6-Luc cells.

[0267] (1) In vivo photothermal imaging of the magnetized platelet drug delivery system PDN-FeN@PLT

[0268] When the tumor volume reaches 200 mm 3 At the same time, mice were randomly divided into 4 groups and injected with Dox@PDA-TK NPs (prepared according to the method of Example 6), PDN@PLT (prepared according to the method of Example 14), PN-FeN@PLT (prepared according to the method of Example 17, replacing Dox@PDA-TK NPs with PDA-TK NPs), and PDN-FeN@PLT (Example 17) through the tail vein. 24 hours after injection, the tumor site was irradiated with an 808 nm semiconductor laser for 5 minutes at a radiation intensity of 1 W / cm 2 , and used an infrared imager to monitor the temperature changes of each group of tumor sites in real time and take pictures.

[0269] like Figure 18As shown, 5 minutes after laser irradiation, the tumor temperature rose to 41.3°C in the Dox@PDA-TK NPs nanoparticle group and to 43.1°C in the platelet drug delivery system PDN@PLT group. In the magnetized platelet drug delivery systems PN-FeN@PLT and PDN-FeN@PLT, the local tumor temperature increased significantly, reaching over 45°C. This result suggests that, compared to nanoparticles, platelets can deliver nanoparticles to the tumor site in a targeted manner, increasing the concentration of nanoparticles in tumor tissue and thus improving the photothermal conversion performance of drugs at the tumor site. Furthermore, the guidance of an external magnetic field can further enhance the tumor aggregation effect of the magnetized platelet drug delivery system, further improving the photothermal conversion performance at the tumor site and ensuring the feasibility of in vivo photothermal therapy and photothermally controlled drug release.

[0270] (2) Determination of the in vivo tumor inhibition efficacy of the magnetized platelet drug delivery system PDN-FeN@PLT

[0271] When the tumor volume reaches 100 mm 3 At the same time, the tumor-bearing mice were weighed and randomly divided into 6 groups (n=4). Starting from day 0, each group of mice was intravenously injected with PBS, Dox, D Dox@PDA-TK NPs (prepared according to the method of Example 6), PDN@PLT (prepared according to the method of Example 14), PN-FeN@PLT (prepared according to the method of Example 17, replacing Dox@PDA-TK NPs with PDA-TK NPs) and PDN-FeN@PLT (Example 17) every 7 days. , wherein the dose of doxorubicin was 2 mg / kg. After injection, a magnet was fixed to the tumor site of the mice in the PN-FeN@PLT or PDN-FeN@PLT group with medical tape, and an external magnetic field was applied for magnetic guidance for 2 hours. And on the 1st and 4th day after drug injection, each group of mice was subjected to laser irradiation hyperthermia therapy, and the irradiation conditions were: 808 nm, 1.0 W / cm 2 , 5min. Tumor size and body weight were measured every two days, and the growth curve of subcutaneous glioma C6-Luc tumor was recorded. Figure 19 On the 18th day, the mice were killed, and the tumors were collected and weighed; the optical images of the tumors removed after the treatment on the 18th day are shown in Figure 20 The weight change curve of tumor-bearing mice during treatment is shown in Figure 21 shown.

[0272] Depend on Figure 19 and 20As shown, compared with the control group (PBS group), the nanoparticles Dox@PDA-TKNPs have a certain inhibitory effect on tumor proliferation; the PN-FeN@PLT (single photothermal therapy) group is enriched at the tumor site under the action of the platelet's own tumor tendency and the guidance of the external magnetic field, and laser irradiation has a certain photothermal therapeutic effect, which is equivalent to the tumor inhibition effect of the free drug Dox group (single chemotherapy); PDN@PLT realizes the photothermal-chemotherapy combination treatment of tumors under the control of external laser, and the inhibition of tumor proliferation is enhanced; and PDN-FeN@PLT achieves good aggregation at the tumor site under the action of dual targeting, and realizes the photothermal-chemotherapy combination treatment of tumors under the control of external laser, and the inhibition of tumor proliferation is most obvious. Figure 21 It can be seen that the overall weight of animals showed a significant upward trend, but the weight of mice in the Dox group was the lowest, indicating that doxorubicin treatment had certain toxicity to mice.

[0273] Test Example 8

[0274] This test example is a study on the tumor inhibition effect of the drug-loaded magnetized platelet drug delivery system (PDN-FeN@PLT) in Example 17 on in situ brain glioma tumors (in situ modeling)

[0275] Animal husbandry: The same as that of Experimental Example 7.

[0276] Establishment of in situ brain glioma model: Anesthetize the nude mouse and place it on a brain stereotaxic instrument to stereotactically locate the lateral ventricle of the nude mouse. After the nude mouse head is fixed stably, the scalp is disinfected with alcohol. A scalpel is used to cut along the midline of the scalp to expose the skull. The periosteum is peeled off with diluted H2O2 solution to accurately determine the position of the anterior fontanelle. The position of the nude mouse lateral ventricle is located at the following coordinates: 0.34 mm behind the anterior fontanelle, 0.8 mm lateral, and 3.1 mm below the dura mater. A microsyringe is fixed on the brain stereotaxic instrument and 5 μL (5×10 5 ) A C6-Luc glioma cell suspension was slowly injected at a rate of 0.5 μL / min. After the injection was complete, the microinjector was left in place for 8 minutes to allow for complete diffusion of the glioma cells and to prevent backflow of the cell suspension when the microinjector was withdrawn. The microinjector was then slowly removed. The burr hole was sealed with bone wax, and the nude mouse was removed from the stereotaxic apparatus. The scalp incision was sutured with absorbable surgical sutures, and the wound was disinfected with iodine. The mouse was then placed in a warm environment until it regained consciousness.

[0277] (1) Tumor growth monitoring in orthotopic glioma models

[0278] The luciferase substrate D-luciferin potassium salt was prepared with PBS to 15 mg / mL and 200 μL was injected into the abdominal cavity of each mouse. Anesthesia was induced using a small animal anesthesia machine, with an isoflurane concentration of 2% and an anesthesia flow rate of 0.8 L / min. After 5 minutes, the nude mice were placed in a living imaging device, with an anesthesia halothane concentration of 1% and an anesthesia flow rate of 0.25 L / min. The tumor growth size was monitored by bioluminescence fluorescence imaging. Confirm whether the model was successful. (2) Determination of the in vivo tumor inhibition efficacy of the magnetized platelet drug delivery system PDN-FeN@PLT

[0279] Nine days after the in situ injection of brain glioma cells, the tumor-bearing mice were weighed and randomly divided into four groups, namely PBS group, Dox group, PDN@PLT (Example 14) group and PDN-FeN@PLT (Example 17) group after bioluminescence fluorescence imaging confirmed the successful modeling. The dosage of Dox was 2 mg / kg, administered once through the tail vein every 5 days, for three times. After injection, the brains of mice in the PDN-FeN@PLT group were fixed with medical tape to the magnets, and an external magnetic field was applied for magnetic guidance for 2 hours. Laser irradiation hyperthermia was performed on the first day after drug injection, and the irradiation conditions were: 808 nm, 1.0 W / cm 2 , 5 min. Tumor size and body weight were measured every two days. On day 13, mice were sacrificed, brain tissue was collected, and brain tumor fluorescence intensity was measured using bioluminescence fluorescence imaging.

[0280] Bioluminescence images of C6-Luc tumors in situ brain gliomas Figure 22 As shown in the figure, the in situ tumor model in the PBS group progressed rapidly, and tumor cells metastasized to the spine along with the cerebrospinal fluid; this phenomenon also occurred in the doxorubicin (Dox) group. Tumor metastasis in the PDN@PLT group was reduced, while the PDN-FeN@PLT group showed almost no metastasis and significantly slowed tumor progression, demonstrating a strong inhibitory effect on the growth and metastasis of gliomas.

[0281] In summary, the magnetized platelet drug delivery system of the present invention can achieve tumor-targeted drug delivery and combined photothermal and chemotherapy treatment of tumors. Due to the adhesion of platelets to the tumor vascular endothelium, the platelet delivery system can reach the tumor site and accumulate at the vascular site damaged by the external laser. Under the guidance of the external magnetic field, the tumor targeting of the system can be further enhanced. Thereafter, the tumor microenvironment reactive oxygen species and photothermal-controlled doxorubicin are released at the target site of the tumor, which can achieve photothermal-chemotherapy combined treatment of the tumor. In the ectopic and in situ Balb / c mouse model of brain glioma, good effective therapeutic effects of combined chemical and photothermal therapy were demonstrated. This magnetized platelet drug delivery system can achieve controllable external magnetic field targeted recruitment and effective anti-cancer treatment, and it has broad application prospects in the development of anti-tumor drug preparations.

[0282] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A magnetized platelet drug delivery system, characterized in that: The invention comprises platelets, magnetic nanoparticles and drug nanoparticles, wherein the magnetic nanoparticles are ferroferric oxide magnetic nanoparticles encapsulated by polyethyleneimine; the drug nanoparticles are drug-loaded reactive oxygen species and photothermal dual-responsive polydopamine nanoparticles, and the drug nanoparticles are prepared according to the following method: A. Add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to a dimethyl sulfoxide solution of thioketal 2,2'-[propane-2,2-diylbis(sulfo)yl]diacetic acid (TK-COOH), and stir for at least 2 h to obtain an activated thioketal compound (TK-NHS). B. Add the activated thioketal compound TK-NHS to the dopamine DA solution and stir to react for 10-14 hours in the dark. Then, place the reaction mixture in a dialysis bag and dialyze it with ultrapure water. Collect the liquid in the dialysis bag and freeze-dry it to obtain the active oxygen-responsive dopamine monomer DA-TK-DA. C. Dopamine, the drug to be loaded, and the reactive oxygen species-responsive dopamine monomer DA-TK-DA are dissolved in water and mixed evenly to prepare a dopamine-drug mixed system. Then, sodium hydroxide solution is added and a polymerization reaction is carried out under stirring to prepare drug-loaded reactive oxygen species and photothermal dual-responsive polydopamine nanoparticles.

2. The magnetized platelet drug delivery system according to claim 1, characterized in that: The polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticles are prepared according to the following method: (1) Under nitrogen atmosphere, ferroferric oxide magnetic nanoparticles were prepared by alkaline solution co-precipitation method, and ferroferric oxide nanoparticles were obtained by freeze drying; (2) The ferroferric oxide nanoparticles were resuspended in water and mixed with a polyethyleneimine aqueous solution, and the mixture was then ultrasonically treated; then stirred at 60±5°C; and finally centrifuged to obtain polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticles.

3. The magnetized platelet drug delivery system according to claim 1, wherein The stirring time described in step B is 12 h.

4. A method for preparing the magnetized platelet drug delivery system according to claim 1, characterized in that: The steps include: (I) platelets, drug-loaded reactive oxygen species, and photothermal dual-responsive polydopamine nanoparticles are mixed and incubated, followed by centrifugation to obtain a drug-loaded platelet drug delivery system; (II) The drug-loaded platelet drug delivery system and the polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticles are mixed and incubated to obtain the magnetized platelet drug delivery system.

5. The preparation method according to claim 4, characterized in that The mixing ratio of the platelets, drug-loaded reactive oxygen species and photothermal dual-responsive polydopamine nanoparticles in step (I) is 5×10 6 1 mL of drug-loaded reactive oxygen species and photothermal dual-responsive polydopamine nanoparticle solution was added to each platelet, where the drug concentration in the drug-loaded reactive oxygen species and photothermal dual-responsive polydopamine nanoparticle solution was 10-20 μg / mL; the incubation time was 1-4 h.

6. The preparation method according to claim 4, characterized in that The mixing ratio of platelets and polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticles in step (II) is 5×10 6 1 mL of polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticle solution was added to each platelet, wherein the concentration of ferroferric oxide in the polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticle solution was 40-80 μg / mL; the incubation time was 1-4 h.

7. A method for preparing the magnetized platelet drug delivery system according to claim 1, characterized in that: The steps include: (i) Platelets and polyethyleneimine-encapsulated ferroferric oxide magnetic nanoparticles were mixed and incubated, and the mixture was centrifuged to prepare the ferroferric oxide-loaded platelet drug delivery system FeN@PLT; (ii) The platelet drug delivery system FeN@PLT loaded with ferroferric oxide was mixed and incubated with drug-loaded reactive oxygen species and photothermal dual-responsive polydopamine nanoparticles, and the mixture was centrifuged to prepare a magnetized platelet drug delivery system.

8. Use of the platelet drug delivery system according to any one of claims 1 to 3 or the magnetized platelet drug delivery system prepared by the preparation method according to any one of claims 4 to 7 in the preparation of drugs for the targeted treatment of malignant tumors.

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