Nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a nano-drug as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The nano-drug provided by the invention comprises nano-particles and an active drug wrapped in the nano-particles, the nanoparticle comprises a carrier and a targeting peptide modified on the surface of the carrier, the active medicine is a hydrophobic BCL-2 protein inhibitor; the carrier comprises a methoxy polyethylene glycol polylactic acid-glycolic acid copolymer; the targeting peptide comprises a cRGD peptide. Based on the important effect of activated platelets in tumor metastasis, the nano-drug provided by the invention adopts a cRGD peptide modified carrier to deliver ABT-737 in a targeted manner, specifically induces apoptosis of the activated platelets, reduces the influence on resting platelets and normal tissues, reduces the toxic and side effects of the drug, significantly inhibits lung metastasis of melanoma, and enhances the anti-metastasis curative effect of a PD-1 antibody; the nano-drug disclosed by the invention can be further expanded to treatment of advanced metastasis of other cancer types except melanoma based on the supporting effect of platelets in various tumor metastasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a nano-drug, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer metastasis is the main cause leading to the death of cancer patients. Cancer metastasis is a complex process, involving multiple consecutive related steps, and is affected by various factors such as cancer types and the in-vivo microenvironment. This also largely results in very limited prevention and treatment means for cancer metastasis at the present stage. Research shows that platelets participate in multiple stages of tumor metastasis and promote tumor metastasis. In the pre-metastatic stage, platelets promote the epithelial-mesenchymal transition of tumor cells and activate the tumor invasion and metastasis cascade reaction by direct contact and secreting factors. In the mid-metastatic stage, for tumor cells entering the circulation, platelets bind to and cover their surfaces, protecting them from shear stress damage from the bloodstream and interfering with the attack and clearance of immune cells on them, thus helping the circulating tumor cells survive. In the late-metastatic stage, platelets assist the adhesion of tumor cells to the endothelium at the distal site, secrete active substances to open the endothelial barrier, and promote the exudation of tumor cells; at the same time, they secrete chemotactic factors to recruit granulocytes to promote the formation of an early metastatic niche. In addition to covering the surface of tumor cells and interfering with the recognition and killing of tumor cells by immune cells, platelets also inhibit the function of immune cells by secreting immunosuppressive factors, promoting the formation of an immunosuppressive microenvironment for tumors. Based on the important supportive and immunosuppressive effects of platelets on the survival and spread of tumor cells, many studies have taken platelets as new targets for cancer treatment, aiming to enhance the existing anti-tumor therapies by inhibiting the function of circulating platelets or inducing platelet clearance. However, systemic inhibition or clearance of platelets has a high bleeding risk and easily causes bleeding in normal tissues, thus limiting the application of anti-platelet drugs in tumor treatment.

[0003] Therapies that block programmed death receptor 1 / programmed death ligand 1 (PD-1 / PD-L1) play an important role in the treatment of various malignant tumors, especially inoperable advanced metastatic melanoma, non-small cell lung cancer, etc. However, their clinical response rate is often no higher than 30%. How to improve their clinical response rate and thus expand the scope of application is an urgent problem to be solved clinically.

[0004] Recent research shows that there is an apoptosis phenomenon similar to that of nucleated cells in platelets. BCL-2 protein inhibitors (BCL-2 inhibitor), such as ABT-263 and ABT-737, can efficiently induce platelet apoptosis. However, due to their strong hydrophobicity and severe toxic side effects when administered systemically, their application in tumor treatment is limited. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a nano-drug, its preparation method and application, which can effectively solve the application problem of antiplatelet drugs in tumor metastasis treatment and provide a new strategy for enhancing the anti-metastasis effect of immunosuppressants.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a nano-drug, comprising nanoparticles and an active drug encapsulated in the nanoparticles; the nanoparticles comprise a carrier and a targeting peptide modified on the surface of the carrier; the active drug is a hydrophobic BCL-2 protein inhibitor; the carrier comprises methoxypolyethylene glycol poly(lactic-co-glycolic acid) (mPEG-PLGA); the targeting peptide comprises cRGD peptide.

[0007] Through a large number of experimental designs, the inventors of the present application modified the surface of the carrier with cRGD cyclic peptide (CNPRGDY(OEt)RC) that can target and bind to activated platelets to obtain a kind of nanoparticles that can target activated platelets, and then used them to efficiently deliver a hydrophobic BCL-2 protein inhibitor, so as to target and induce the apoptosis of activated platelets and be cleared in the body, avoiding the participation of activated platelets in tumor cell diffusion and immune escape, and achieving the purpose of inhibiting tumor metastasis and enhancing the efficacy of PD-1 antibody drugs.

[0008] As a preferred embodiment of the nano-drug of the present invention, the molecular weight of methoxypolyethylene glycol (mPEG) in the methoxypolyethylene glycol poly(lactic-co-glycolic acid) is 4000-6000, and the molecular weight of poly(lactic-co-glycolic acid) (PLGA) is 12000-18000. The methoxypolyethylene glycol poly(lactic-co-glycolic acid) adopted by the present invention is a preference, and the prepared nano-drug has a high drug encapsulation rate and drug loading amount, and at the same time has good biodegradability and biocompatibility.

[0009] As a preferred embodiment of the nano-drug of the present invention, the drug loading amount of the nanoparticles is 1-6%.

[0010] As a preferred embodiment of the nano-drug of the present invention, the hydrophobic BCL-2 protein inhibitor comprises at least one of ABT-263 and ABT-737.

[0011] More preferably, the hydrophobic BCL-2 protein inhibitor comprises ABT-737.

[0012] As a preferred embodiment of the nano-drug of the present invention, the particle size of the nano-drug is 60-300 nm, the average particle size is 103.9±1.0 nm, and the morphology is spherical.

[0013] The present invention also provides a preparation method of the above-mentioned nano-drug, comprising the following steps:

[0014] (1) Dissolve PLGA-PEG-NHS, cRGD peptide and a catalyst in an organic solvent. After the reaction, dialyze and freeze-dry to obtain PLGA-PEG-cRGD;

[0015] (2) Dissolve PLGA-PEG-cRGD, mPEG-PLGA and ABT-737 in an organic solvent, add ultrapure water and perform ultrasonic emulsification to obtain a primary emulsion;

[0016] (3) Add an emulsifier to the primary emulsion and perform a second ultrasonic emulsification to obtain a double emulsion;

[0017] (4) Dropwise add the double emulsion to an aqueous phase containing an emulsifier under stirring. After stirring and dispersing, rotary evaporate until the solution is clear and no bubbles are generated, and centrifuge to obtain a precipitate;

[0018] (5) Wash the precipitate with ultrapure water and centrifuge to obtain the nanodrug.

[0019] As a preferred embodiment of the preparation method of the nanodrug of the present invention, it includes at least one of (a)-(b):

[0020] (a) The molar ratio of PLGA-PEG-NHS to cRGD peptide in step (1) is 1:0.5 - 2;

[0021] (b) The mass ratio of PLGA, PEG, and NHS in PLGA-PEG-NHS in step (1) is PLGA:PEG:NHS = (12000 - 18000):(4000 - 6000):115.

[0022] As a preferred embodiment of the preparation method of the nanodrug of the present invention, the catalyst in step (1) includes triethylamine.

[0023] As a preferred embodiment of the preparation method of the nanodrug of the present invention, the weight ratio of mPEG-PLGA to PLGA-PEG-cRGD in step (2) is mPEG-PLGA:PLGA-PEG-cRGD = (0.67 - 4):1.

[0024] More preferably, the weight ratio of mPEG-PLGA to PLGA-PEG-cRGD in step (2) is mPEG-PLGA:PLGA-PEG-cRGD = 1.5:1.

[0025] The inventors of the present application have found through research that the weight ratio of PLGA-PEG-cRGD to mPEG-PLGA has a great influence on the targeting of activated platelets of the prepared nano-drugs. When mPEG-PLGA:PLGA-PEG-cRGD = (0.67 - 4):1, the targeting of activated platelets of the nano-drugs is better; preferably, the weight ratio of mPEG-PLGA:PLGA-PEG-cRGD can be one of 0.67:1, 1:1, 1.5:1, 2:1, 4:1 or the range value of any two of them; more preferably, when the weight ratio of PLGA-PEG-cRGD to mPEG-PLGA is 1:1.5, the effect of the nano-drugs being enriched in activated platelets is the best.

[0026] As a preferred embodiment of the preparation method of the nano-drugs of the present invention, in the step (2), the mass ratio of the sum of the masses of PLGA-PEG-cRGD and mPEG-PLGA to the mass of ABT-737 is 10:(0.25 - 1.5).

[0027] More preferably, in the step (2), the mass ratio of the sum of the masses of PLGA-PEG-cRGD and mPEG-PLGA to the mass of ABT-737 is 10:1.

[0028] As a preferred embodiment of the preparation method of the nano-drugs of the present invention, the emulsifier includes an aqueous sodium cholate solution. More preferably, the mass concentration of the aqueous sodium cholate solution is 2%.

[0029] As a preferred embodiment of the preparation method of the nano-drugs of the present invention, when ultrasonic emulsification is used to form the primary emulsion, the ultrasonic power is 285W; when ultrasonic emulsification is used to form the double emulsion, the ultrasonic power is 332.5W. Specifically, when ultrasonic emulsification is used to form the primary emulsion, a ultrasonic crusher is used, the power is set to 285W, it is turned on for 1s and turned off for 2s, and this lasts for 3min. When ultrasonic emulsification is used to form the double emulsion, a ultrasonic crusher is used, the power is set to 332.5W, it is turned on for 1s and turned off for 2s, and this lasts for 5min. The inventors of the present application have found through research that in the preparation process of the nano-drugs, the parameter settings of ultrasonic emulsification have a great influence on the morphology and particle size of the finally prepared nano-drugs. The above ultrasonic emulsification conditions are a kind of preference, which can make the particle size distribution of the prepared nano-drugs uniform, and the average particle size is about 104nm, which is beneficial to delivering the drug to platelets.

[0030] As a preferred embodiment of the preparation method of the nano-drugs of the present invention, the centrifugation operation is centrifugation at 12000 rpm at room temperature for 15 min.

[0031] As a preferred embodiment of the preparation method of the nano-drug of the present invention, the organic solvent in the step (1) includes N,N-dimethylformamide. In the step (1), the reaction is carried out in the presence of a solvent. Those skilled in the art can select a suitable solvent and determine the amount of the solvent according to the reaction system (for example, according to the raw materials). The solvent can be an organic solvent, including but not limited to N,N-dimethylformamide, etc. The amount of the solvent can be determined according to the amount of the raw materials used.

[0032] As a preferred embodiment of the preparation method of the nano-drug of the present invention, the organic solvent in the step (2) includes dichloromethane. In the step (2), the reaction is carried out in the presence of a solvent. Those skilled in the art can select a suitable solvent and determine the amount of the solvent according to the reaction system (for example, according to the raw materials). The solvent can be an organic solvent, including but not limited to dichloromethane, etc. The amount of the solvent can be determined according to the amount of the raw materials used.

[0033] The present invention also provides the application of the nano-drug in the preparation of a tumor treatment drug and / or a drug for targeting and inducing platelet apoptosis.

[0034] Preferably, the tumor includes melanoma.

[0035] The nano-drug of the present invention targets and induces the apoptosis of activated platelets so as to clear them, exposing the tumor cells covered by platelets, promoting the recognition and killing of tumor cells by T cells, and at the same time avoiding the secretion of immunosuppressive factors by platelets to inhibit the functions of immune cells, thereby activating the immune system, reducing side effects while enhancing the efficacy of PD-1 antibody, and finally enhancing the inhibitory effect on melanoma lung metastasis.

[0036] Advantages of the present invention: The present invention prepares for the first time a nano-drug that can target activated platelets. The nano-drug of the present invention efficiently carries and delivers hydrophobic BCL-2 protein inhibitors (ABT-263, ABT-737) through a PLGA core, and at the same time utilizes the targeting function of the surface cRGD peptide to efficiently deliver the carried drug into activated platelets, improving its bioavailability. Compared with traditional drug delivery strategies, its toxic and side effects are reduced; based on the important role of activated platelets in tumor metastasis, the nano-drug of the present invention uses cRGD peptide to target and deliver hydrophobic BCL-2 protein inhibitors (ABT-263, ABT-737), specifically inducing the apoptosis and in vivo clearance of activated platelets, reducing the impact on resting platelets and normal tissues, reducing the toxic and side effects of the drug, significantly inhibiting melanoma lung metastasis, and enhancing the anti-metastasis efficacy of PD-1 antibody; based on the supporting role of platelets in the metastasis of various tumors, the nano-drug of the present invention can be further extended to the treatment of late metastasis of other cancer types beyond melanoma. Description of the Drawings

[0037] Figure 1 It is the NMR spectrum of PLGA-PEG-cRGD.

[0038] Figure 2 It is the transmission electron microscope (a) and hydrodynamic diameter distribution (b) diagrams of the nano-drug of the present invention.

[0039] Figure 3 It is the schematic diagram of the activated platelet targeting of the nano-drug of the present invention.

[0040] Figure 4 It is the schematic diagram of the specific induction of apoptosis of activated platelets (manifested as mitochondrial depolarization) by the nano-drug of the present invention.

[0041] Figure 5 It is the diagram of the nano-drug of the present invention inhibiting melanoma lung metastasis and enhancing the anti-metastatic efficacy of PD-1 antibody: including the diagram of melanoma metastatic nodules on the lung surface (a), the statistical chart of the number of metastatic foci on the lung surface (b), and the statistical chart of the lung metastasis index (c). Detailed implementation manners

[0042] The above content of the present invention will be further described in detail below through specific implementation manners in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

[0043] Experimental reagents:

[0044] Poly (lactic-co-glycolic acid) - polyethylene glycol active ester (PLGA-PEG-NHS): PLGA15k-PEG5k-NHS, Xi'an Ruixi Biotechnology Co., Ltd.

[0045] Methoxypolyethylene glycol poly (lactic-co-glycolic acid) (mPEG-PLGA): mPEG5k-PLGA15k, Jinan Daigang Bioengineering Co., Ltd.

[0046] cRGD peptide: Guoping Pharmaceutical Co., Ltd.

[0047] Triethylamine: Shanghai Macklin Biochemical Co., Ltd.

[0048] N,N-Dimethylformamide (analytical pure): Shanghai Macklin Biochemical Co., Ltd.

[0049] Dichloromethane (analytical pure): Shanghai Macklin Biochemical Co., Ltd.

[0050] ABT-737: selleck;

[0051] Sodium cholate: Shanghai Macklin Biochemical Co., Ltd.

[0052] Example 1

[0053] This example provides a method for preparing a nano-drug, which includes the following steps:

[0054] (1) Weigh 100 mg of PLGA15k-PEG5K-NHS and dissolve it in 3 mL of N,N-dimethylformamide. Add 1.1 eq. of cRGD peptide and 3.0 eq. of triethylamine, dissolve completely, and react at room temperature for 12 h. Transfer the reaction solution to a dialysis bag with a molecular weight cut-off of 2000 Da and dialyze it in pure water for 24 h. Collect the dialysis solution and freeze-dry it to obtain PLGA-PEG-cRGD;

[0055] (2) Weigh 6 mg of mPEG-PLGA, 4 mg of PLGA-PEG-cRGD, and 1 mg of ABT-737 and dissolve them in 1 mL of dichloromethane. Add 200 μL of ultrapure water and perform primary emulsification with an ultrasonic crusher. Set the power to 285 W, turn it on for 1 s, turn it off for 2 s, and continue for 3 min to obtain the primary emulsion;

[0056] (3) Add 2 mL of a 2% sodium cholate aqueous solution to the primary emulsion and perform secondary emulsification with an ultrasonic crusher. Set the power to 332.5 W, turn it on for 1 s, turn it off for 2 s, and continue for 5 min to obtain the double emulsion;

[0057] (4) Drop the double emulsion drop by drop into 10 mL of a 0.5% sodium cholate aqueous solution under stirring and stir for 15 min to disperse. Rotate and evaporate to remove dichloromethane in the solution. Centrifuge the obtained transparent and clear solution at 12000 rpm at room temperature for 15 min, discard the supernatant to obtain the precipitate;

[0058] (5) Wash the precipitate twice with ultrapure water, centrifuge again, take the precipitate to obtain the nano-particle cRGD-PLGA-ABT, and resuspend it with 1 mL of ultrapure water for standby.

[0059] Example 2

[0060] (1) Weigh 100 mg of PLGA15k-PEG5K-NHS and dissolve it in 3 mL of N,N-dimethylformamide. Add 1.1 eq. of cRGD peptide and 3.0 eq. of triethylamine, dissolve completely, and react at room temperature for 12 h. Transfer the reaction solution to a dialysis bag with a molecular weight cut-off of 2000 Da and dialyze it in pure water for 24 h. Collect the dialysis solution and freeze-dry it to obtain PLGA-PEG-cRGD;

[0061] (2) Weigh 8 mg of mPEG-PLGA, 2 mg of PLGA-PEG-cRGD, and 1 mg of ABT-737 and dissolve them in 1 mL of dichloromethane. Add 200 μL of ultrapure water and perform primary emulsification using an ultrasonic disrupter. Set the power to 285 W, turn it on for 1 s, turn it off for 2 s, and continue for 3 min to obtain the primary emulsion.

[0062] (3) Add 2 mL of a 2% sodium cholate aqueous solution to the primary emulsion and perform secondary emulsification using an ultrasonic disrupter. Set the power to 332.5 W, turn it on for 1 s, turn it off for 2 s, and continue for 5 min to obtain the multiple emulsion.

[0063] (4) Dropwise add the multiple emulsion to 10 mL of a 0.5% sodium cholate aqueous solution under stirring and stir for 15 min to disperse. Rotate and evaporate to remove dichloromethane from the solution. Centrifuge the resulting transparent and clear solution at 12000 rpm for 15 min at room temperature and discard the supernatant to obtain the precipitate.

[0064] (5) Wash the precipitate twice with ultrapure water, centrifuge again, and collect the precipitate to obtain the nanometer particles cRGD-PLGA-ABT, which are resuspended in 1 mL of ultrapure water for standby.

[0065] Example 3

[0066] (1) Weigh 100 mg of PLGA15k-PEG5K-NHS and dissolve it in 3 mL of N,N-dimethylformamide. Add 1.1 eq. of cRGD peptide and 3.0 eq. of triethylamine, dissolve completely, and react at room temperature for 12 h. Transfer the reaction solution to a dialysis bag with a molecular weight cut-off of 2000 Da and dialyze it in pure water for 24 h. Collect the dialysis solution and freeze-dry it to obtain PLGA-PEG-cRGD.

[0067] (2) Weigh 4 mg of mPEG-PLGA, 6 mg of PLGA-PEG-cRGD, and 1 mg of ABT-737 and dissolve them in 1 mL of dichloromethane. Add 200 μL of ultrapure water and perform primary emulsification using an ultrasonic disrupter. Set the power to 285 W, turn it on for 1 s, turn it off for 2 s, and continue for 3 min to obtain the primary emulsion.

[0068] (3) Add 2 mL of a 2% sodium cholate aqueous solution to the primary emulsion and perform secondary emulsification using an ultrasonic disrupter. Set the power to 332.5 W, turn it on for 1 s, turn it off for 2 s, and continue for 5 min to obtain the multiple emulsion.

[0069] (4) Dropwise add the multiple emulsion to 10 mL of a 0.5% sodium cholate aqueous solution under stirring and stir for 15 min to disperse. Rotate and evaporate to remove dichloromethane from the solution. Centrifuge the resulting transparent and clear solution at 12000 rpm for 15 min at room temperature and discard the supernatant to obtain the precipitate.

[0070] (5) Wash the precipitate twice with ultrapure water, centrifuge again, and take the precipitate to obtain the nanometer particles cRGD-PLGA-ABT, which are resuspended in 1 mL of ultrapure water for standby.

[0071] Effect Example 1

[0072] (1) Perform nuclear magnetic resonance hydrogen spectrum analysis on the product prepared in step (1) of Example 1. The results are as Figure 1 shown. In the nuclear magnetic resonance hydrogen spectrum of PLGA-PEG-cRGD, polypeptide characteristic peaks similar to those of cRGD peptide appear, indicating that the cRGD peptide is successfully conjugated to PLGA-PEG.

[0073] (2) Use a transmission electron microscope and a dynamic light scattering instrument to characterize the size and morphology of cRGD-PLGA-ABT nanoparticles. The results are as Figure 2 shown. The cRGD-PLGA-ABT nanoparticles of the present invention have a spherical structure with a uniform size distribution. The particle size is about 80 nm under the transmission electron microscope, and the measured hydrated particle size is slightly larger, which is 104 nm.

[0074] (3) Use a liquid phase-high performance chromatograph with acetonitrile and water as the mobile phase to determine the content of ABT-737 in the cRGD-PLGA-ABT nano-drug by measuring the ultraviolet absorption at a wavelength of 254 nm, and repeat three times. Measure the maximum absorption peak area of different concentration gradients of ABT-737 at a wavelength of 254 nm, draw a standard curve, and determine the content of ABT-737 in the cRGD-PLGA-ABT nano-drug according to the drawn standard curve.

[0075] The calculation formula for the encapsulation efficiency is: Encapsulation efficiency (%) = mass of the drug loaded in the nanoparticles / total mass of the input drug * 100%.

[0076] The calculation formula for the drug loading is: Drug loading (%) = mass of the drug loaded in the nanoparticles / (mass of the drug loaded in the nanoparticles + total mass of the input PLGA) * 100%.

[0077] The measurement results of the encapsulation efficiency and drug loading of the cRGD-PLGA-ABT nano-drug are shown in Table 1.

[0078] Table 1

[0079] Entrapment efficiency (%) Drug loading (%) 59.10±1.92 5.58±0.17

[0080] Effect Example 2

[0081] (1) In this effect example, the influence of the dosage of PLGA-PEG-cRGD in the nanoparticles prepared in Examples 1-3 on the activation of platelet targeting of the nanoparticles was detected through experiments. The specific experimental method is as follows:

[0082] 1) Cy5.5 was used as a fluorescent dye to label nanoparticles, and cRGD-PLGA-Cy5.5 nanoparticles containing different mass ratios of PLGA-PEG-cRGD (0, 20%, 40%, 60%, and the corresponding mass ratios of mPEG-PLGA to PLGA-PEG-cRGD were 10:0, 8:2, 6:4, 4:6, respectively) were synthesized;

[0083] 2) Blood was collected from the mouse heart, and ACD solution (39 mM citric acid, 75 mM sodium citrate, 135 mM glucose, 2 μM prostaglandin E1, pH = 7.4) was added at a ratio of blood: ACD = 9:1. After gentle mixing, the mixture was centrifuged at 100 g for 20 min at room temperature. The upper platelet-rich plasma was taken and centrifuged again at 800 g for 20 min. The obtained platelets were resuspended in PBS, and 10 μM ADP was added to stimulate the mixture at 37°C for 15 min. The supernatant was removed after centrifugation. The obtained precipitate was activated platelets, which was resuspended in PBS for later use.

[0084] 3) Add cRGD-PLGA-Cy5.5 nanoparticles with different mPEG-PLGA / PLGA-PEG-cRGD ratios containing equal amounts of fluorescent dye Cy5.5 to the activated platelets obtained by stimulation and the control resting platelets suspension, incubate at 37°C for 1 hour, centrifuge at 800g for 20 minutes, discard the supernatant, wash the precipitate once with PBS containing 2μM prostaglandin E1, repeat the centrifugation, discard the supernatant, resuspend the precipitate with 200μL benchtop buffer, and detect the Cy5.5 fluorescence intensity by flow cytometry. The results are shown in Figure 3 As shown in a. Figure 3 As shown in a, the cRGD peptide-modified nanoparticles in the three ratios of 20%, 40% and 60% can be better enriched in activated platelets, among which the enrichment effect is best when the PLGA-PEG-cRGD ratio is 40%, that is, the nanosystem obtained when the mass ratio of mPEG-PLGA to PLGA-PEG-cRGD is 6:4 has the best targeting to activated platelets. This ratio is used to synthesize cRGD-PLGA-ABT nanoparticles for subsequent experiments.

[0085] (2) Experimental verification of the activated platelet targeting of cRGD-PLGA polymer nanoparticles:

[0086] 1) Cy5.5 was selected as a fluorescent dye to label nanoparticles, and PLGA-Cy5.5 and cRGD-PLGA-Cy5.5 nanoparticles were synthesized according to the aforementioned method, wherein the mass ratios of mPEG-PLGA to PLGA-PEG-cRGD were 10:0 and 6:4, respectively, and PLGA-Cy5.5 was used as a control group of nanoparticles for subsequent experiments.

[0087] 2) Blood was drawn from the mouse heart and ACD solution (39 mM citric acid, 75 mM sodium citrate, 135 mM glucose, 2 μM prostaglandin E1, pH = 7.4) was added at a ratio of blood:ACD = 9:1. After gently mixing, it was centrifuged at 100 g for 20 min at room temperature; the upper layer of platelet-rich plasma was taken and centrifuged continuously. The centrifugation conditions were 800 g for 20 min. The obtained platelets were resuspended with PBS and divided into two groups. One group was not treated with anything, and the other group was added with 10 μM ADP and stimulated at 37 °C for 15 min, then centrifuged to remove the supernatant. The obtained precipitate was activated platelets, which were resuspended with PBS for standby.

[0088] PLGA-Cy5.5 and cRGD-PLGA-Cy5.5 nanoparticles encapsulating equal amounts of the fluorescent dye Cy5.5 were respectively added to activated platelets and control resting platelets, incubated at 37 °C for 1 h, centrifuged at 800 g for 20 min, the supernatant was discarded, the precipitate was washed once with PBS containing 2 μM prostaglandin E1, centrifugation was repeated, the supernatant was discarded, the precipitate was resuspended with 200 μL of Tyrode's solution, and the fluorescence intensity of Cy5.5 was detected by flow cytometry. As Figure 3 shown in Fig. b, there was no difference in the enrichment of PLGA-Cy5.5 in the control group between resting platelets and activated platelets, while the cRGD-PLGA-Cy5.5 nanoparticles modified with cRGD peptide were significantly more enriched in activated platelets than in resting platelets, indicating that the cRGD-PLGA polymer nanodelivery platform of the present invention has good activated platelet targeting.

[0089] Effect Example 3

[0090] This effect example detected the specific induction of apoptosis of activated platelets (manifested as mitochondrial depolarization) of the cRGD-PLGA-ABT nanoparticles prepared in Example 1 through experiments. The specific experimental steps are as follows:

[0091] The cRGD-PLGA-ABT nano-drug was prepared according to the method of Example 1; the platelets were treated according to the method of Effect Example 2. The nano-drug containing different concentrations of ABT-737 (0.1, 0.5, 1, 2, 5 μM) was added to the platelet 1640 medium suspension and treated at 37 °C for 40 min; the staining solution of tetramethylrhodamine ethyl ester (TMRE, mitochondrial membrane potential detection dye) was added and incubated for another 20 min; centrifuged at 800 g for 20 min, the supernatant was discarded, the precipitate was washed twice with 1640 medium, centrifuged again, the precipitate was resuspended with 200 μL of Tyrode's solution, and the fluorescence intensity of TMRE was detected by flow cytometry.

[0092] The results are as Figure 4As shown in the figure, the mitochondrial depolarization ratios of resting platelets in the nano-drugs containing different concentrations (0.1 - 5 μM) of ABT-737 were 1.00 ± 0.59%, 3.90 ± 0.23%, 12.14 ± 3.08%, 14.17 ± 6.10%, and 19.90 ± 1.99% respectively; while the mitochondrial depolarization ratios of activated platelets in the nano-drugs containing different concentrations (0.1 - 5 μM) of ABT-737 were 2.74 ± 0.21%, 7.51 ± 1.20%, 17.50 ± 1.24%, 44.46 ± 4.88%, and 74.98 ± 2.57% respectively. From the above, it can be seen that compared with resting platelets, activated platelets showed significantly stronger mitochondrial depolarization after treatment with the cRGD-PLGA-ABT nano-drug, indicating that the cRGD-PLGA-ABT nano-drug of the present invention can specifically induce the apoptosis of activated platelets.

[0093] Effect Example 4

[0094] This effect example detected the inhibition of melanoma lung metastasis and the synergistic effect of PD-1 antibody by the cRGD-PLGA-ABT nano-drug prepared in Example 1 through experiments. The specific experimental methods are as follows:

[0095] 1) Digest and collect B16-F10 cells, make them into a PBS suspension, and slowly inject 2×10 5 cells into the tail vein of each mouse to construct a melanoma lung metastasis model.

[0096] 2) Randomly divide the mice into 7 groups (n = 5). On the second day after model construction, the mice were respectively given normal saline (Saline), cRGD-PLGA, free ABT-737, cRGD-PLGA-ABT, PD-1 antibody (aPD1), free ABT-737 + aPD1, cRGD-PLGA-ABT + aPD1 treatment (ABT-737: 10 mg / kg, PD-1 antibody: 200 μg / mouse), and administered once every 3 days for a total of 5 times. During this period, the body weight and status of the mice were observed and recorded. At the end of the treatment, the lung tissues of the mice were taken out, the number of surface metastasis foci was counted, and fixed with 4% paraformaldehyde for subsequent section analysis. The calculation formula of the metastasis index is as follows: Metastasis index (%) = Metastasis area / Lung area * 100%.

[0097] The results are as Figure 5As shown, the numbers of metastatic foci in the mice of the normal saline, cRGD-PLGA, free ABT-737, cRGD-PLGA-ABT, PD-1 antibody (aPD1), free ABT-737 + aPD1, and cRGD-PLGA-ABT + aPD1 treatment groups were 122 ± 9, 115 ± 12, 101 ± 12, 96 ± 5, 110 ± 8, 67 ± 12, and 51 ± 20, respectively; the lung metastasis indexes were 28.9 ± 5.5%, 30.6 ± 4.4%, 18.3 ± 5.8%, 17.1 ± 2.8%, 16.9 ± 2.1%, 10.3 ± 2.8%, and 9.6 ± 1.2%, respectively. The cRGD-PLGA-ABT nanomedicine treatment group significantly inhibited the formation of melanoma lung metastasis, and compared with the free drug ABT-737, the metastasis inhibition effect was enhanced; the PD-1 antibody had a certain anti-metastasis effect, and when it was used in combination with the free drug ABT-737 or the cRGD-PLGA-ABT nanomedicine, its inhibitory effect on metastasis was significantly enhanced. The combined treatment group of the cRGD-PLGA-ABT nanomedicine and the PD-1 antibody of the present invention produced the strongest anti-metastasis effect, which was reflected in the least number of lung surface metastatic foci and the smallest metastasis index.

[0098] 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 the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A nano-drug, characterized in that, it comprises nanoparticles and an active drug encapsulated in the nanoparticles; the nanoparticles comprise a carrier and a targeting peptide modified on the surface of the carrier; the active drug is a hydrophobic BCL-2 protein inhibitor; the carrier comprises methoxypolyethylene glycol poly(lactic-co-glycolic acid); the targeting peptide comprises cRGD peptide.

2. The nano-drug according to claim 1, characterized in that, in the methoxypolyethylene glycol poly(lactic-co-glycolic acid), the molecular weight of methoxypolyethylene glycol is 4000 - 6000, and the molecular weight of poly(lactic-co-glycolic acid) is 12000 - 18000.

3. The nano-drug according to claim 1, characterized in that, the drug loading of the nanoparticles is 1 - 6%.

4. The nano-drug according to claim 1, characterized in that, the hydrophobic BCL-2 protein inhibitor comprises at least one of ABT-263 and ABT-737.

5. The nano-drug according to claim 1, characterized in that, the particle size of the nano-drug is 60 - 300 nm, the average particle size is 103.9 ± 1.0 nm, and the morphology is spherical.

6. The preparation method of the nano-drug according to any one of claims 1 - 5, characterized in that, it comprises the following steps: (1) Dissolve PLGA-PEG-NHS, cRGD peptide and a catalyst in an organic solvent, and after reaction, dialyze and freeze-dry to obtain PLGA-PEG-cRGD; (2) Dissolve PLGA-PEG-cRGD, mPEG-PLGA and ABT-737 in an organic solvent, add ultrapure water and perform ultrasonic emulsification to obtain a primary emulsion; (3) Add an emulsifier to the primary emulsion and perform secondary ultrasonic emulsification to obtain a double emulsion; (4) Dropwise add the double emulsion to an aqueous phase containing an emulsifier drop by drop under stirring, stir and disperse, then rotary evaporate until the solution is clear and there are no bubbles, and centrifuge to obtain a precipitate; (5) Wash the precipitate with ultrapure water and centrifuge to obtain the nano-drug.

7. The preparation method of the nano-drug according to claim 6, characterized in that, it comprises at least one of (a) - (b): (a) The molar ratio of PLGA-PEG-NHS to cRGD peptide in step (1) is 1:0.5 - 2; (b) In PLGA-PEG-NHS of step (1), the mass ratio of PLGA, PEG, and NHS is PLGA:PEG:NHS = (12000 - 18000):(4000 - 6000):

115.

8. The preparation method of the nano-drug according to claim 6, characterized in that, the weight ratio of mPEG-PLGA to PLGA-PEG-cRGD in step (2) is mPEG-PLGA:PLGA-PEG-cRGD = (0.67 - 4):

1.

9. The preparation method of the nano-drug according to claim 6, characterized in that, in step (2), the mass ratio of the sum of the masses of PLGA-PEG-cRGD and mPEG-PLGA to the mass of ABT-737 is 10:(0.25 - 1.5).

10. Use of the nano-drug according to any one of claims 1 to 5 in the preparation of a tumor therapeutic drug and / or a drug for targeting and inducing platelet apoptosis.

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