Functionalized targeting nanoparticle as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicine, and particularly relates to a functional targeting nanoparticle as well as a preparation method and application thereof. The functionalized targeting nanoparticle is formed by self-assembly of a reduction response amphiphilic polymer, wherein the core of the functionalized targeting nanoparticle wraps a first chemotherapeutic drug and / or a macrophage regulator; the shell of the functionalized targeting nanoparticle adsorbs or chelates a second chemotherapeutic drug and / or a macrophage regulator; the surfaces of the functionalized targeting nanoparticles are modified with targeting molecules. The preparation method of the functionalized targeting nanoparticles is simple and efficient, and can effectively improve the accumulation of therapeutic drugs in tumor tissues. After treatment, the growth of tumors is significantly inhibited, and the infiltration abundance of anti-inflammatory tumor-related macrophages in tumor tissues is significantly reduced. Therefore, the functionalized targeting nanoparticles have a wide application prospect in the field of chemical immunotherapy of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a functionalized targeted nanoparticle, a preparation method thereof, and an application thereof. Background Art

[0002] Macrophages are the main components of the tumor immune microenvironment and are closely related to the occurrence, development, treatment resistance, and poor prognosis of tumors. After tumor formation, cancer cells highly express a series of cytokines and chemokines, recruiting macrophages to the tumor site and inducing them to polarize into tumor-associated macrophages (TAMs) with the M2 phenotype. TAMs then create an immunosuppressive microenvironment favorable for tumor cells by producing various anti-inflammatory factors and matrix metalloproteinases, and secrete a large number of growth factors to induce angiogenesis in tumor tissues, thereby promoting tumor growth and metastasis. In addition, TAMs are also the key components mediating chemotherapy resistance, and reducing the infiltration abundance of TAMs can achieve chemotherapy sensitization.

[0003] Currently, a large number of small molecule drugs for regulating TAMs have been developed, but these small molecule drugs still face problems of in vivo delivery barriers (such as low drug solubility, short half-life, non-specific biodistribution, and poor cellular uptake, etc.), which greatly hinders their clinical application. Macrophages have high phagocytic activity and will actively recognize and uptake nanoparticles entering the body, which makes nanodrugs show incomparable advantages in regulating the functional phenotypes of macrophages. In addition, the efficacy of single therapy mediated by TAMs is limited, and combining it with chemotherapy is a very promising synergistic strategy. As mentioned above, TAMs are the key components mediating chemotherapy resistance, and reducing TAMs infiltration can effectively enhance the sensitivity of chemotherapy; at the same time, chemotherapy drugs can also synergistically amplify the anti-tumor immune response by enhancing tumor antigen exposure.

[0004] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a functionalized targeted nanoparticle, which is self-assembled by a reduction-responsive amphiphilic polymer, wherein:

[0006] The core of the functionalized targeted nanoparticle encapsulates a first chemotherapy drug and / or a macrophage regulator;

[0007] The shell of the functionalized targeted nanoparticle adsorbs or chelates a second chemotherapy drug and / or a macrophage regulator;

[0008] The surface of the functionalized targeted nanoparticle is modified with a targeting molecule.

[0009] Preferably, the structure of the reduction-responsive amphiphilic polymer is shown in Formula I:

[0010]

[0011] wherein n = 10-100 and n is an integer.

[0012] Preferably, the method for preparing the reduction-responsive amphiphilic polymer comprises the following steps:

[0013] (1) Reacting cyclobutane tetracarboxylic dianhydride, cystamine, and dimethyl sulfoxide to obtain a mixture;

[0014] (2) Subjecting the mixture to sedimentation and then drying to obtain the reduction-responsive amphiphilic polymer.

[0015] Preferably, in step (1), the molar ratio of cyclobutane tetracarboxylic dianhydride, cystamine, and dimethyl sulfoxide is 1:1:10-100;

[0016] and / or, in step (1), the reaction temperature is 25-60 °C and the reaction time is 1-5 d;

[0017] and / or, in step (2), the sedimentation is carried out using ethanol or ether;

[0018] and / or, in step (2), the drying is carried out by vacuum drying.

[0019] Preferably, the first chemotherapeutic drug includes but is not limited to one or a combination of two or more of doxorubicin, paclitaxel, camptothecin, and platinum drugs;

[0020] and / or, the second chemotherapeutic drug includes but is not limited to one or a combination of two of doxorubicin and cisplatin;

[0021] and / or, the macrophage modulator includes but is not limited to one or a combination of two of CSF-1R inhibitors, CCL2-CCR2 antagonists, and TLR4 agonists.

[0022] Preferably, the CSF-1R inhibitor includes but is not limited to one or a combination of two or more of PLX3397, BLZ-945, and JNJ-40346527;

[0023] and / or, the CCL2-CCR2 antagonist includes but is not limited to one or a combination of two or more of PF-4136309, BMS-813160, and cenicriviroc;

[0024] and / or, the TLR4 agonist includes but is not limited to GSK1795901.

[0025] Preferably, the targeting molecule is a sugar targeting molecule and / or a tumor targeting molecule; wherein:

[0026] The sugar targeting molecule includes, but is not limited to, one or a combination of two or more of glucose, galactose, and mannose;

[0027] The tumor targeting molecule includes, but is not limited to, one or a combination of two of lactobionic acid and folic acid.

[0028] Based on the same technical concept, the present invention further provides a method for preparing a functionalized targeted nanoparticle, and the preparation method includes the following steps:

[0029] (S1) Modify the reduction-responsive amphiphilic polymer to obtain a reduction-responsive amphiphilic polymer modified with a targeting molecule;

[0030] (S2) Mix and dissolve the reduction-responsive amphiphilic polymer modified with a targeting molecule, a first chemotherapeutic drug and / or a macrophage regulator, and N,N-dimethylformamide to obtain a first reaction solution;

[0031] (S3) Add deionized water to the first reaction solution, then add the second chemotherapeutic drug and / or macrophage regulator dissolved in N,N-dimethylformamide to the reaction system, stir and then concentrate using an ultrafiltration tube to obtain the functionalized targeted nanoparticle.

[0032] Preferably, in step (S2), the molar ratio of the reduction-responsive amphiphilic polymer modified with a targeting molecule, the first chemotherapeutic drug and / or macrophage regulator, and N,N-dimethylformamide for mixing and dissolving is 1:1 to 100:1 to 100;

[0033] And / or, in step (S3), the molar ratio of N,N-dimethylformamide, the second chemotherapeutic drug and / or macrophage regulator is 1 to 100:1 to 100;

[0034] And / or, in step (S3), the stirring time is 0.5 to 4 h;

[0035] And / or, in step (S3), the cut-off molecular weight of the ultrafiltration tube is 1 to 10 kDa.

[0036] Based on the same technical concept, the present invention further provides an application of the functionalized targeted nanoparticle in the preparation of a drug for treating malignant tumors, and it is characterized in that the malignant tumors include, but are not limited to, primary and secondary liver cancers, breast cancers, melanomas, lung cancers, and / or gastric cancers.

[0037] The beneficial effects of the present invention are:

[0038] 1. The preparation method of the functionalized targeted nanoparticles of the present invention is simple, and the chemotherapy drugs and macrophage regulators in the core and shell can be released in a tumor microenvironment-responsive manner to achieve precise treatment of tumors.

[0039] 2. The functionalized targeted nanoparticles of the present invention can synergistically enhance the chemicothermal immunotherapy of tumors by regulating macrophages, thereby effectively inhibiting the growth of malignant tumors.

[0040] 3. The functionalized targeted nanoparticles of the present invention can effectively increase the accumulation of chemotherapy drugs and macrophage regulators at the tumor site and improve their bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0042] Figure 1 It is the gel permeation chromatogram of the reduction-responsive amphiphilic polymer.

[0043] Figure 2 It is the reaction process diagram.

[0044] Figure 3 It is the transmission electron micrograph of targeted or non-targeted nanoparticles loaded with different drugs.

[0045] Figure 4 It is the hydrated particle size and potential diagram of targeted or non-targeted nanoparticles loaded with different drugs.

[0046] Figure 5 It is the release curve diagram of PLX3397 and cisplatin of the targeted nanoparticles.

[0047] Figure 6 It is the phagocytosis effect diagram of macrophages on nano-drugs.

[0048] Figure 7 It is the targeting effect diagram of nano-drugs on tumor tissues.

[0049] Figure 8 It is the growth curve diagram of subcutaneous LLC lung cancer tumors in mice.

[0050] Figure 9 It is the infiltration abundance diagram of different phenotypic macrophages in tumor tissues. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0051] To make the objectives, technical solutions and advantages of the present invention more clear, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other implementation manners obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope protected by the present invention.

[0052] Example 1

[0053] This example provides a preparation method of functionalized targeted nanoparticles, and the preparation method includes the following steps:

[0054] (I) Preparation of mannose-modified reduction-responsive amphiphilic polymer

[0055] (I-1) Cystamine (0.916 g, 4.67 mmol) and cyclobutanetetracarboxylic dianhydride (0.711 g, 4.67 mmol) were respectively dissolved in 1.5 mL of anhydrous DMSO and successively added to the reaction flask.

[0056] (I-2) Under nitrogen protection, the reaction was carried out at 45 °C for 48 h, and then it was precipitated and washed in absolute ethanol to obtain the reduction-responsive amphiphilic polymer.

[0057] (I-3) Its molecular weight was characterized by gel permeation chromatography and the like, and the results are as Figure 1 shown. Subsequently, the reduction-responsive amphiphilic polymer (0.324 g, 0.93 mmol structural units) obtained in the previous step was dissolved in 3 mL of anhydrous DMSO, then EDC / NHS was added, and the carboxyl group was activated by stirring at room temperature for 30 min. Then, amino mannose hydrochloride (60 mg, 0.28 mmol) and triethylamine (117 μL, 0.84 mmol) were added to the reaction system, and the reaction was continued for 24 h to obtain the mannose-modified reduction-responsive amphiphilic polymer. The reaction process is as Figure 2 shown.

[0058] (II) Preparation of functionalized targeted nanoparticles

[0059] (II-1) 20 mg of the mannose-modified reduction-responsive amphiphilic polymer and PLX3397 were dissolved in 0.5 mL of N,N-dimethylformamide.

[0060] (II-2) Then it was ultrasonically added dropwise to 20 mL of deionized water with an ultrasonic power of 30 W. Subsequently, cisplatin dissolved in 0.2 mL of N,N-dimethylformamide was added dropwise to the above reaction. After magnetic stirring for 2 h, targeted nanoparticles with PLX3397 encapsulated in the core and cisplatin adsorbed on the shell were obtained by ultrafiltration, and named Man-CDDP / PLX NPs.

[0061] Comparative Example 1

[0062] The control particle 1 in which PLX3397 is encapsulated by a non-targeted reduction-responsive amphiphilic polymer was named PLX NPs.

[0063] Comparative Example 2

[0064] The control particle 2 in which cisplatin is adsorbed on the surface of a non-targeted reduction-responsive amphiphilic polymer was named CDDP NPs.

[0065] Comparative Example 3

[0066] The control particle 3 in which PLX3397 is encapsulated by a non-targeted reduction-responsive amphiphilic polymer and cisplatin is adsorbed on the surface was named CDDP / PLX NPs.

[0067] Verification Example

[0068] (I) Transmission electron microscopy (TEM) detection of nanoparticles:

[0069] An appropriate amount of the above four nanoparticle dispersions was diluted to 1 mg / mL, 10 μL was aspirated and dropped onto the surface of a copper grid, air-dried naturally at room temperature, and then the sample was placed under a transmission electron microscope for photographing. The results are as Figure 3 shown.

[0070] It can be seen from Figure 3 that: all the nanoparticles exhibit a spherical morphology and have a uniform particle size distribution.

[0071] (II) Detection of the hydrodynamic diameter and potential of nanoparticles:

[0072] An appropriate amount of the above four nanoparticle dispersions was diluted to 1 mg / mL. The hydrodynamic diameter and potential of the four nanoparticles were detected by a Malvern nanoparticle size analyzer respectively, and the results are as Figure 4 shown.

[0073] It can be seen from Figure 4 that: the hydrodynamic diameter of all the nanoparticles is between 60 - 80 nm, and the potential is between -40 and -20 mV, with small differences in particle size and potential.

[0074] (III) Evaluation of the release of PLX3397 and cisplatin from targeted nanoparticles:

[0075] Targeted nanoparticles Man-CDDP / PLX NPs with known drug concentrations were separately taken and added into dialysis bags (with a molecular weight cut-off of 0.5 kDa). Subsequently, the dialysis bags were immersed in buffer solutions of different media (pH 7.4, pH 6.5, pH 5.5, pH 6.5 1 mM GSH, and pH 5.5 10 mM GSH). At different time points (1, 2, 4, 8, 24, 48 h, etc.), part of the buffer solution was taken out and the contents of PLX3397 and platinum were detected by ultraviolet absorption and ICP-MS, so as to calculate the release rates of PLX3397 and cisplatin. The results are as Figure 5 shown.

[0076] It can be Figure 5 seen that the targeted nanoparticles Man-CDDP / PLX NPs are stable in the physiological environment simulation solution (pH 7.4), and rarely release PLX3397 and cisplatin. However, in the tumor microenvironment and tumor cell simulation solutions (pH 6.5, pH 5.5, pH 6.5 1 mM GSH, and pH 5.5 10 mM GSH), PLX3397 and cisplatin are rapidly released.

[0077] (IV) Targeting of mannose-functionalized nano-drugs to macrophages:

[0078] Raw264.7 macrophages were co-incubated with Man-CDDP / PLX NPs and CDDP / PLX NPs for 2, 6, 12, and 24 hours respectively, and then the content of Pt element in the cells was detected by inductively coupled plasma spectrometer (ICP) to evaluate the phagocytosis effect of macrophages on the nano-drug. The results are as Figure 6 shown.

[0079] It can be Figure 6 seen that the phagocytosis effect of macrophages on Man-CDDP / PLX NPs is significantly better than that of CDDP / PLX NPs (non-targeted control), indicating that Man-CDDP / PLX NPs can enhance the uptake of the nanoparticles by macrophages by targeting the mannose receptor on the surface of macrophages.

[0080] (V) Targeting of mannose-functionalized nano-drugs to tumors:

[0081] 5×10⁵ LLC lung cancer cells were injected behind the right axilla of C57 mice to establish a mouse lung cancer model. After the tumors grew to an appropriate size, Man-CDDP / PLX NPs and CDDP / PLX NPs were injected via the tail vein, respectively, at a dose of 5 mg Pt / kg. At 2 h, 6 h, and 24 h after the injection of the nanodrugs, the tumor tissues of the mice were isolated, digested, and the Pt element content in the tissues was detected by ICP to evaluate the targeting effect of the mannose-modified nanodrugs on the tumor tissues. The results are as Figure 7 shown.

[0082] It can be Figure 7 seen that the Pt content in the tumor tissues of the Man-CDDP / PLX NPs injection group was significantly higher than that of the CDDP / PLX NPs injection group, indicating that Man-CDDP / PLX NPs can significantly increase the accumulation of CDDP and PLX3397 at the tumor site by targeting the mannose receptor in the tumor tissues.

[0083] (VI) The nanodrug inhibits tumor growth by regulating macrophages in synergy with chemotherapy:

[0084] 5×10 5 LLC lung cancer cells were injected behind the right axilla of C57 mice to establish a mouse lung cancer model. After the tumors grew to about 50 mm 3 , Man-CDDP / PLX NPs, CDDP / PLX NPs, CDDP NPs, and PLX NPs were intravenously injected for treatment, and then the changes in tumor volume were monitored. The drug was administered once every 4 days at a dose of 5 mg Pt / kg. The results are as Figure 8 shown.

[0085] It can be Figure 8 seen that compared with the control group and the single-drug (CDDP NPs, PLX NPs) treatment groups, CDDP / PLX NPs significantly inhibited tumor growth, indicating that the nanodrug can achieve the synergistic effect of CDDP and PLX3397 and thus more effectively inhibit tumor growth; while Man-CDDP / PLX NPs improved the delivery efficiency of CDDP and PLX3397 through the targeting effect on the tumor tissues. Therefore, its anti-tumor effect is significantly better than that of the non-targeted CDDP / PLX NPs.

[0086] (VII) After the nanodrug treatment was completed, the tumor tissues of the mice were isolated and prepared into single-cell suspensions, and then the proportion of CD206-positive tumor-associated macrophages in the tumor tissues was detected by flow cytometry. The results are as Figure 9 shown.

[0087] It can be Figure 9It can be seen that after treatment with Man-CDDP / PLX NPs, CD206 in tumor tissues + the infiltration abundance of TAMs was significantly reduced, while the proportion of pro-inflammatory CD86 + macrophages was significantly increased, indicating that Man-CDDP / PLX NPs can inhibit the infiltration of TAMs and reshape their phenotypes.

[0088] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claimed rights.

Claims

1. A functionalized targeted nanoparticle, characterized in that, The functionalized targeted nanoparticles are self-assembled from reduction-responsive amphiphilic polymers, where: The core of the functionalized targeted nanoparticles encapsulates a first chemotherapeutic drug and / or macrophage regulator; The outer shell of the functionalized targeted nanoparticles adsorbs or chelates a second chemotherapeutic drug and / or macrophage regulator; The surface of the functionalized targeted nanoparticles is modified with a targeting molecule.

2. The functionalized targeted nanoparticle according to claim 1, wherein, The structure of the reduction-responsive amphiphilic polymer is shown in Formula I: Where n = 10 - 100 and n is an integer.

3. The functionalized targeted nanoparticle according to claim 2, wherein The preparation method of the reduction-responsive amphiphilic polymer includes the following steps: (1) Mix cyclobutane tetracarboxylic dianhydride, cystamine, and dimethyl sulfoxide and react to obtain a mixture; (2) After the mixture is sedimented and dried, the reduction-responsive amphiphilic polymer is obtained.

4. The functionalized targeted nanoparticle according to claim 3, characterized in that, In step (1), the molar ratio of cyclobutane tetracarboxylic dianhydride, cystamine, and dimethyl sulfoxide is 1:1:10 - 100; And / or, in step (1), the reaction temperature is 25 - 60 °C and the reaction time is 1 - 5 d; And / or, in step (2), the sedimentation method is to sediment with ethanol or ether; And / or, in step (2), the drying method is vacuum drying.

5. The functionalized targeted nanoparticle according to claim 1, wherein The first chemotherapeutic drug is one or a combination of two or more of doxorubicin, paclitaxel, camptothecin, and platinum drugs; And / or, the second chemotherapeutic drug is one or a combination of two of doxorubicin and cisplatin; And / or, the macrophage regulator is one or a combination of two of CSF-1R inhibitor, CCL2-CCR2 antagonist, and TLR4 agonist.

6. The functionalized targeting nanoparticle according to claim 5, wherein The CSF-1R inhibitor is one or a combination of two or more of PLX3397, BLZ-945, and JNJ-40346527; And / or, the CCL2-CCR2 antagonist is one or a combination of two or more of PF-4136309, BMS-813160, and Cenicriviroc; And / or, the TLR4 agonist is GSK1795901.

7. The functionalized targeted nanoparticle according to claim 1, characterized in that, The targeting molecule is a sugar targeting molecule and / or a tumor targeting molecule; where: The sugar targeting molecule is one or a combination of two or more of glucose, galactose, and mannose; The tumor targeting molecule is one or a combination of two of lactobionic acid and folic acid.

8. The preparation method of the functionalized targeted nanoparticles according to any one of claims 1 to 7, characterized in that, The preparation method includes the following steps: (S1) Modify the reduction-responsive amphiphilic polymer to obtain a reduction-responsive amphiphilic polymer modified with a targeting molecule; (S2) Mix and dissolve the reduction-responsive amphiphilic polymer modified with a targeting molecule, the first chemotherapeutic drug and / or macrophage regulator, and N,N-dimethylformamide to obtain a first reaction solution; (S3) Add deionized water to the first reaction solution, then add the second chemotherapeutic drug and / or macrophage regulator dissolved in N,N-dimethylformamide to the reaction system, stir, and concentrate with an ultrafiltration tube to obtain the functionalized targeted nanoparticles.

9. The preparation method of the functionalized targeted nanoparticles according to claim 8, characterized in that, In step (S2), the molar ratio of the reduction-responsive amphiphilic polymer modified with a targeting molecule, the first chemotherapeutic drug and / or macrophage regulator, and N,N-dimethylformamide for mixed dissolution is 1:1 to 100:1 to 100; And / or, in step (S3), the molar ratio of N,N-dimethylformamide, the second chemotherapeutic drug and / or macrophage regulator is 1 to 100:1 to 100; And / or, in step (S3), the stirring time is 0.5 to 4 h; And / or, in step (S3), the cut-off molecular weight of the ultrafiltration tube is 1 to 10 kDa.

10. Use of the functionalized targeted nanoparticles according to any one of claims 1 to 7 in the preparation of a medicament for treating malignant tumors, characterized in that, The malignant tumors include primary and secondary liver cancers, breast cancers, melanomas, lung cancers and / or gastric cancers.

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