Hardness-controllable polyethylene glycol nano-drug carrier as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medical carriers, and relates to a hardness-controllable polyethylene glycol nano-drug carrier as well as a preparation method and application thereof. Adsorbing a layer of polyethyleneimine on the surface of the template to obtain a carrier precursor; performing at least one layer assembly on the surface of the carrier precursor; and finally, removing the template. The primary layer assembly process comprises the following steps: firstly, adding multi-arm polyethylene glycol o-phthalaldehyde to carry out a cross-linking reaction, and then adding multi-arm polyethylene glycol amino to carry out a cross-linking reaction; the cross-linking reaction is that the o-phthalaldehyde group reacts with the primary amino group to generate the isoindolinone group. The PEG nano-drug carriers with different hardness can be prepared through a layer-by-layer assembly method, and targeting molecules and / or chemotherapeutic drugs can be introduced in the preparation process; by adjusting the PEG nano-drug carrier with proper hardness and loading the chemotherapeutic drug, the interaction between the nano-drug carrier and a biological interface can be adjusted, so that good tumor treatment is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical carriers, and relates to a poly(ethylene glycol) nanopharmaceutical carrier with controllable hardness, a preparation method thereof and an application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Nanopharmaceutical carriers can reduce systemic side effects, increase tumor accumulation and improve drug delivery efficiency. Due to the complex biological barriers in vivo (e.g., clearance by the mononuclear phagocyte system, limited penetration at the tumor site, etc.), it is extremely difficult to design nanopharmaceutical carriers with expected functions. The size, shape and hardness of nanopharmaceutical carriers are several physicochemical properties of nanopharmaceutical carriers that are relatively easy to regulate. Among them, the exploration of regulating the hardness of nanopharmaceutical carriers for improving drug delivery is easily overlooked. However, with the development of cell mechanics and cell mechanical signaling pathways in recent years, researchers have increasingly paid attention to the interaction between the hardness of nanopharmaceutical carriers and cells and biological interfaces. However, the inventors have found that existing methods cannot finely regulate the hardness of nanopharmaceutical carriers, and matching the hardness of nanopharmaceutical carriers with the modulus of mammalian cells (2 - 26 kPa) is an urgent problem to be solved in constructing nanopharmaceutical carriers.

[0004] Poly(ethylene glycol) (PEG) is a biofouling - resistant material with high hydration ability, which can reduce protein interaction and prevent clearance by the mononuclear phagocyte system. Surface modification of nanopharmaceutical carriers with PEG is a commonly used method to prolong their blood circulation time. Currently, the molecular weight, structure and dispersity of PEG have important effects on the blood circulation time and the biodistribution of PEG nanopharmaceutical carriers. However, according to the inventors' research and understanding, there are few related studies on the influence of the hardness of PEG nanopharmaceutical carriers on their biological behaviors. Summary of the Invention

[0005] To solve the deficiencies of the prior art, the purpose of the present invention is to provide a poly(ethylene glycol) nanopharmaceutical carrier with controllable hardness, a preparation method thereof and an application thereof. The present invention can prepare PEG nanopharmaceutical carriers with different hardnesses by a layer - by - layer assembly method, and targeting molecules (hyaluronic acid (HA)) and / or chemotherapeutic drugs (monocarboxylate cisplatin (Pt)) can be introduced during the preparation process; by adjusting the PEG nanopharmaceutical carriers with appropriate hardness and loading chemotherapeutic drugs, the present invention can regulate the interaction between the nanopharmaceutical carriers and biological interfaces, thereby achieving good tumor treatment.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows:

[0007] In a first aspect, a method for preparing a poly(ethylene glycol) nanomedicine carrier with controllable hardness includes the following steps:

[0008] Adsorb a layer of polyethyleneimine (PEI) on the surface of a template to obtain a precursor of the carrier; perform at least one layer-by-layer assembly on the surface of the precursor of the carrier; and finally remove the template to obtain the product.

[0009] The process of performing one layer-by-layer assembly is as follows: first add multi-arm poly(ethylene glycol) phthalaldehyde (X-arm-PEG-OPA) for crosslinking reaction, and then add multi-arm poly(ethylene glycol) amine (Y-arm-PEG-NH2) for crosslinking reaction.

[0010] The crosslinking reaction is that the phthalaldehyde group reacts with the primary amino group (-NH2) to form an isoindolinone group

[0011] The multi-arm poly(ethylene glycol) phthalaldehyde described in the present invention refers to a branched polymer formed by connecting at least three branched-chain structure heads, and each branched-chain structure is a poly(ethylene glycol) with a phthalaldehyde group at the end, which can be tetra-arm poly(ethylene glycol) phthalaldehyde (4-arm-PEG-OPA), hexa-arm poly(ethylene glycol) phthalaldehyde (6-arm-PEG-OPA), octa-arm poly(ethylene glycol) phthalaldehyde (8-arm-PEG-OPA), etc.

[0012] The multi-arm poly(ethylene glycol) amine described in the present invention can be tetra-arm poly(ethylene glycol) amine (4-arm-PEG-NH2), hexa-arm poly(ethylene glycol) amine (6-arm-PEG-NH2), octa-arm poly(ethylene glycol) amine (8-arm-PEG-NH2), etc.

[0013] In the present invention, a template is used to construct the shape of the poly(ethylene glycol) nanomedicine carrier. Polyethyleneimine (PEI) is used to modify the template, which can modify amino groups on the surface of the template and is beneficial to the preparation of the poly(ethylene glycol) nanomedicine carrier through the amino groups; multi-arm poly(ethylene glycol) is used to increase the crosslinking degree of the nanocarrier; at the same time, the multi-arm poly(ethylene glycol) phthalaldehyde with a phthalaldehyde group at the end is used as a building unit in the present invention, which can be connected and crosslinked with the primary amino group by forming an isoindolinone group. Compared with other connection methods (such as forming a Schiff base (-C=N-)), the connection method in the present invention has a faster reaction rate and is more beneficial to the crosslinking of poly(ethylene glycol) (especially at a low concentration) on the surface of the template, so as to be beneficial to adjusting the crosslinking density of the poly(ethylene glycol) nanomedicine carrier through multiple layer-by-layer assemblies, and further beneficial to realizing the hardness adjustment of the poly(ethylene glycol) nanomedicine carrier.

[0014] Experiments of the present invention show that by using octa-arm polyethylene glycol phthalaldehyde and octa-arm polyethylene glycol amine as building units, the hardness can be adjusted from 2 kPa to 31 kPa through the method of layer-by-layer assembly.

[0015] In a second aspect, a polyethylene glycol nano-drug carrier with controllable hardness is obtained by the above preparation method.

[0016] In a third aspect, a targeted PEG nano-drug carrier includes the above polyethylene glycol nano-drug carrier with controllable hardness and a targeting molecule.

[0017] In a fourth aspect, an application of the above polyethylene glycol nano-drug carrier with controllable hardness or targeted PEG nano-drug carrier in the preparation of drugs is to load drug molecules onto the PEI layer.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1. The present invention uses multi-arm polyethylene glycol phthalaldehyde and multi-arm polyethylene glycol amine as building units to precisely control the hardness of the PEG nano-drug carrier through the method of layer-by-layer assembly. The controlled modulus range can match the cell level, which has good clinical transformation significance.

[0020] 2. The PEG nano-drug carrier prepared by the present invention has a spherical structure with good dispersibility. After being placed at room temperature for one year, it still maintains its complete morphological structure and still has good dispersibility.

[0021] 3. The PEG nano-drug carrier provided by the present invention has a long circulation time in mice, and its blood half-life is about 9 hours. Therefore, it has the property of long circulation.

[0022] 4. The targeted PEG nano-drug carrier provided by the present invention has obvious enrichment at the tumor site and has a high tumor-targeted delivery effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0024] Figure 1 It is a diagram showing the preparation of PEG nano-drug carriers with different hardnesses by the method of layer-by-layer assembly in the embodiments of the present invention.

[0025] Figure 2Fluorescence detection results of preparing PEG nano-drug carriers with different hardnesses by the layer-by-layer assembly method in the embodiments of the present invention. a is a curve showing the change in the fluorescence intensity of the PEG nano-drug carrier during the layer-by-layer assembly process with the increase in the number of assembly layers detected by flow cytometry, and b is a fluorescence distribution diagram showing the change in the fluorescence intensity of the PEG nano-drug carrier during the layer-by-layer assembly process with the increase in the number of assembly layers detected by flow cytometry.

[0026] Figure 3 Transmission electron microscope images of PEG1, PEG3, PEG5, and PEG8 prepared in the embodiments of the present invention.

[0027] Figure 4 Topography characterization diagrams and height statistical diagrams of PEG1, PEG3, PEG5, and PEG8 in the gas phase state in Application Experimental Example 1 of the present invention by atomic force microscopy.

[0028] Figure 5 Result diagrams of the extrusion deformation experiment in Application Experimental Example 1 of the present invention. a is a schematic diagram of the filter head extrusion transmittance of PEG1 and PEG8 nano-drug carriers, and b is the transmittance of PEG1 and PEG8 nano-drug carriers quantified by fluorescence signals.

[0029] Figure 6 Result diagrams of characterizing Young's modulus by AFM using a spherical probe in Application Experimental Example 1 of the present invention. a is a schematic diagram of the polystyrene microsphere probe used in the atomic force microscope, and b is the calculation of Young's modulus of PEG1, PEG3, PEG5, and PEG8 nano-drug carriers through the Hertzian model.

[0030] Figure 7 Result diagrams of the change in the concentration of PEG nano-drug carriers in the blood at different times after intravenous administration to mice in Application Experimental Example 2 of the present invention.

[0031] Figure 8 Statistical data diagrams of the interaction between PEG nano-drug carriers with different concentrations and macrophages (RAW264.7) (a) and monocytes (THP-1) (b) in Application Experimental Example 2 of the present invention.

[0032] Figure 9 Statistical data diagrams of the interaction between PEG nano-drug carriers modified with different concentrations of targeting molecules and tumor cells (4T1) in Application Experimental Example 3 of the present invention.

[0033] Figure 10This is the organ distribution map of HPEG1, HPEG3, HPEG5, and HPEG8 nano-drug carriers in mice in Application Experimental Example 3 of the present invention. Figure a is the fluorescence imaging photos of the heart, liver, spleen, lungs, kidneys, and tumor sites of mice 12 hours after intravenous administration of HPEG1, HPEG3, HPEG5, and HPEG8. Figure b is the fluorescence quantitative statistical analysis of the main organs.

[0034] Figure 11 This is the cytotoxicity experimental results of Pt@HPEG nano-drug modified with the targeting molecule HA and loaded with the chemotherapeutic drug Pt and free chemotherapeutic drug against 4T1 cells in Application Experimental Example 4 of the present invention.

[0035] Figure 12 This is the mouse tumor suppression experimental results of PBS, Pt, Pt@HPEG1, and Pt@HPEG8 in Application Experimental Example 4 of the present invention.

[0036] Figure 13 This is the 1 1H NMR spectrum of 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid-n-succinimide prepared in the embodiment of the present invention.

[0037] Figure 14 This is the 1 1H NMR spectrum of 8-arm-PEG-OPA prepared in the embodiment of the present invention.

[0038] Figure 15 This is the Fourier transform infrared spectra of 8-arm-PEG-OPA and PEG8 prepared in the embodiment of the present invention. Detailed implementation manners

[0039] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0040] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0041] The present invention discovers through research that by adjusting a PEG nano-drug carrier with an appropriate hardness and loading a chemotherapeutic drug, a good match with tumor cells can be achieved, thereby realizing good tumor treatment. In view of this research result, the present invention proposes a poly(ethylene glycol) nano-drug carrier with controllable hardness, its preparation method and application.

[0042] A typical embodiment of the present invention provides a preparation method of a poly(ethylene glycol) nano-drug carrier with controllable hardness, comprising the following steps:

[0043] Adsorb a layer of polyethylenimine (PEI) on the surface of the template to obtain a carrier precursor; perform at least one layer-by-layer assembly on the surface of the carrier precursor; and finally remove the template to obtain the product.

[0044] The process of performing one layer-by-layer assembly is as follows: first add multi-arm poly(ethylene glycol) phthalaldehyde (X-arm-PEG-OPA) for cross-linking reaction, and then add multi-arm poly(ethylene glycol) amine (Y-arm-PEG-NH2) for cross-linking reaction.

[0045] The cross-linking reaction is that the phthalaldehyde group

[0046] reacts with the primary amino group (-NH2) to form an isoindolinone group.

[0047] In some embodiments, the template is mesoporous silica (MS). Research shows that using MS as the template has better effects. Specifically, the method for removing the template is: removing it in a buffer solution of hydrofluoric acid and ammonium fluoride (HF / NH4F).

[0048] In some embodiments, when adding multi-arm poly(ethylene glycol) phthalaldehyde for cross-linking reaction, the concentration of multi-arm poly(ethylene glycol) phthalaldehyde in the reaction system is 1.8 - 2.2 mg / mL. Research shows that under this condition, the hardness regulation effect of the obtained poly(ethylene glycol) nano-drug carrier is better.

[0049] In some embodiments, the multi-arm poly(ethylene glycol) phthalaldehyde is octa-arm poly(ethylene glycol) phthalaldehyde. Research shows that the poly(ethylene glycol) nano-drug carrier prepared by using octa-arm poly(ethylene glycol) phthalaldehyde has a better hardness regulation effect. Specifically, the molecular weight of octa-arm poly(ethylene glycol) phthalaldehyde is 9 - 11 kDa.

[0050] In some embodiments, the multi-arm poly(ethylene glycol) amine is octa-arm poly(ethylene glycol) amine. Research shows that the poly(ethylene glycol) nano-drug carrier prepared by using octa-arm poly(ethylene glycol) amine has a better hardness regulation effect.

[0051] In some embodiments, when adding multi-arm polyethylene glycol amine for cross-linking reaction, the concentration of multi-arm polyethylene glycol amine in the reaction system is 1.8 - 2.2 mg / mL. Research shows that under this condition, the hardness control effect of the obtained polyethylene glycol nano-drug carrier is better.

[0052] In some embodiments, the cross-linking reaction is carried out in a carbonate buffer solution as the solvent, and the pH of the carbonate buffer solution is 9.0 - 9.5.

[0053] Another embodiment of the present invention provides a polyethylene glycol nano-drug carrier with controllable hardness, obtained by the above preparation method.

[0054] In some embodiments, the Young's modulus is 2.0 - 3.0 kPa. Research shows that using this polyethylene glycol nano-drug carrier can better match with tumor cells, and the tumor treatment effect is better.

[0055] The third embodiment of the present invention provides a targeted PEG nano-drug carrier, including the above polyethylene glycol nano-drug carrier with controllable hardness and a targeting molecule.

[0056] In some embodiments, the targeting molecule is folic acid, monosaccharide, oligosaccharide, hyaluronic acid (HA), transferrin or RGD peptide, preferably HA. Specifically, the molecular weight of the HA is 60 - 120 kDa, preferably 60 - 65 kDa.

[0057] In some embodiments, during the preparation process, after the layer-by-layer assembly is completed, a targeting molecule and a coupling agent are added for coupling reaction, and then the template is removed. Specifically, the coupling agent is 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride.

[0058] The fourth embodiment of the present invention is an application of the above polyethylene glycol nano-drug carrier with controllable hardness or targeted PEG nano-drug carrier in the preparation of a drug, and a drug molecule is loaded onto the PEI layer.

[0059] In some embodiments, during the preparation process, a drug molecule is loaded onto the obtained carrier precursor, and then at least one layer-by-layer assembly is carried out on the surface of the carrier precursor loaded with the drug molecule.

[0060] In some embodiments, the drug is an anti-tumor drug. Specifically, the tumor is breast cancer.

[0061] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the following will specifically describe the technical solution of the present invention in detail with reference to specific examples and comparative examples.

[0062] Example 1

[0063] A method for preparing PEG nanomedicine carriers with different hardness using MS as a template, as Figure 1 shown, the steps are as follows:

[0064] (1) Disperse 300 mg of MS into 150 mL of carbonate buffer (0.1 M, pH 9.2) to prepare a suspension.

[0065] (2) Add 1 mL of PEI solution (concentration 3 mg / mL) to the above suspension and incubate at room temperature for 4 h to obtain MS-PEI nanoparticles.

[0066] (3) Disperse 10 mg of MS-PEI nanoparticles in 200 μL of carbonate buffer, then add 400 μL of 8-arm-PEG-OPA for reaction to make the final concentration of 8-arm-PEG-OPA 2 mg / mL. Place the reaction solution of this step on a mixer, after reacting for 15 min, wash it 3 times with water.

[0067] Synthesis steps of 8-arm-PEG-OPA: The precursor small molecule 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid-N-succinimidyl ester was synthesized according to the method reported in the reference (Zhen Zhang et al. A fast and versatile cross-linking strategy via o-phthalaldehyde condensation for mechanically strengthened and functional hydrogels. Natl. Sci. Rev, 2021, 8, nwaa128), as Figure 13 shown. Dissolve 8-arm-PEG-NH2 (300 mg) in 5 mL of anhydrous dichloromethane and 150 μL of triethylamine. After stirring for 30 min, add 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid N-succinimidyl ester (114 mg), stir at room temperature for 48 h, precipitate with cold ether to form 8-arm-PEG-(1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxamide). Dissolve it with deionized water (5 mL) and trifluoroacetic acid (5 mL), stir for 1 h, dilute, and dialyze for 2 days. After freeze-drying, 8-arm-PEG-OPA is obtained, as Figures 14 - 15 shown.

[0068] (4) Redisperse the above-mentioned nano-drug carriers into 200 μL of carbonate buffer solution, add 400 μL of 8-arm-PEG-NH2 for reaction, so that the final concentration of 8-arm-PEG-NH2 is 2 mg / mL. Then place the reaction solution of this step on a mixer, after reacting for 15 min, wash it three times with water; obtain a layer of assembled MS@PEG nano-drug carriers, denoted as MS@PEG1.

[0069] By repeating step (3) and step (4), MS@PEG nano-drug carriers with different numbers of assembled layers are obtained. Repeating step (3) and step (4) once obtains a two-layer assembled MS@PEG nano-drug carrier, repeating step (3) and step (4) twice obtains a three-layer assembled MS@PEG nano-drug carrier, repeating step (3) and step (4) three times obtains a four-layer assembled MS@PEG nano-drug carrier... Repeating step (3) and step (4) eight times obtains a nine-layer assembled MS@PEG nano-drug carrier. Among them, the obtained two-layer to nine-layer assembled MS@PEG nano-drug carriers are respectively denoted as MS@PEG2, MS@PEG3, MS@PEG4, MS@PEG5, MS@PEG6, MS@PEG7, MS@PEG8, MS@PEG9 in sequence.

[0070] (5) Resuspend the MS@PEG nano-drug carriers with different numbers of assembled layers in a solution of 2M HF / 8M NH4F (pH = 5) to remove MS to obtain PEG nano-drug carriers. Among them, the obtained one-layer to nine-layer MS@PEG nano-drug carriers are denoted as PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9 in sequence. The infrared spectrum of PEG8 is as Figure 15 shown.

[0071] Application Experimental Example 1

[0072] First, use a flow cytometer to characterize the assembly process of PEG nano-drug carriers. Modify Cy5 dye molecules on 8-arm-PEG-NH2, and characterize the assembly process of PEG on MS nanoparticles by detecting the fluorescence intensity on the surface of MS@PEG nanoparticles. As Figure 2 shown in Figures 2a and 2b, as the number of assembled layers increases, the fluorescence intensity of the MS@PEG nano-drug carriers gradually increases. When the number of assembled layers reaches 9 layers, the fluorescence intensity of the MS@PEG nano-drug carriers no longer changes. Therefore, nano-drug carriers with 1, 3, 5, and 8 layers of assembled layers are selected for subsequent applications. Characterize the morphology of PEG nano-drug carriers by transmission electron microscopy. As Figure 3 shown, the PEG nano-drug carriers exhibit a spherical morphology, and the average diameter is about 110 - 150 nm.

[0073] Next, an atomic force microscope (AFM) was used to characterize the hardness and deformability of PEG nanomedicine carriers with different assembly layers. Figure 4 Figs. 3a and 3b are AFM images of PEG nanomedicine carriers with 1, 3, 5, and 8 assembly layers in the gas phase. As the number of assembly layers increases, the height of the PEG nanomedicine carriers in the gas phase gradually increases. This is because as the number of assembly layers increases, the hardness of the PEG nanomedicine carriers gradually increases, and they can maintain a better morphology during the gas-phase drying process and are not easily collapsed. A squeezing deformation experiment was carried out using a 100-nm tip, and the deformability of the PEG nanomedicine carriers was reflected by the transmittance of the PEG nanomedicine carriers. As Figure 5 shown in Figs. 5a and 5b, as the number of assembly layers increases, the transmittance of the PEG nanomedicine carriers decreases, indicating a decrease in their deformability. Next, the Young's modulus of PEG1, PEG3, PEG5, and PEG8 nanomedicine carriers in the liquid phase was characterized by AFM. Using a spherical probe, the Young's modulus of PEG1, PEG3, PEG5, and PEG8 nanomedicine carriers was detected to increase from 2 kPa to 31 kPa, as Figure 6 shown. This indicates that as the number of assembly layers increases, the hardness of the PEG nanomedicine carriers gradually increases.

[0074] Application Experimental Example 2

[0075] The pharmacokinetics of PEG1, PEG3, PEG5, and PEG8 nanomedicine carriers in mice were detected.

[0076] The fluorescently labeled PEG drug carriers (concentration: 500 μg / mL) were injected into Kunming mice via the tail vein. Blood samples (100 μL) were collected by tail clipping at 2, 4, 8, 12, 24, and 48 h. Then, the blood was centrifuged (3000 g, 10 minutes) to collect the serum. The fluorescence intensity of the particles in the serum was detected using a microplate reader. As Figure 7 shown, the soft PEG1 nanomedicine carriers had the longest circulation half-life (t 1 / 2 = 9 h) and the largest area under the curve (AUC 0→∞ = 2085), while the hard PEG8 NPs had a t 1 / 2 of 7 h and an AUC 0→∞ of 1706.

[0077] The interactions of PEG1, PEG3, PEG5, and PEG8 nanomedicine carriers with macrophages (RAW 264.7) and monocytes (THP-1) were detected.

[0078] Flow cytometry was used to analyze the endocytosis of RAW 264.7 and THP-1 cells by PEG1, PEG3, PEG5, and PEG8 nano-drug carriers. RAW264.7 and THP-1 cells were cultured in a 24-well plate for 24 h and incubated with 0.5 mL of medium containing two concentrations (2.5 and 5 μg / mL) of cy5-labeled PEG nano-drug carriers. Then, the cells were washed twice with PBS and analyzed by flow cytometry. As Figure 8 shown in a, compared with the PEG1 nano-drug carrier, in the medium containing 10% FBS, the intracellular endocytosis of the PEG8 nano-drug carrier by RAW264.7 cells increased significantly, reaching 84.37%. Similarly, in the culture of PEG nano-drug carriers with THP-1 cells, although the endocytosis of the PEG nano-drug carrier decreased, the degree of endocytosis increased with the increase in the hardness of the PEG nano-drug carrier.

[0079] Example 2

[0080] A method for preparing targeted PEG nano-drug carriers with different hardness using MS as a template is as follows:

[0081] (1) Disperse 300 mg of MS into 150 mL of carbonate buffer (0.1 M, pH 9.2) to prepare a suspension.

[0082] (2) Add 1 mL of PEI solution (concentration 3 mg / mL) to the above suspension and incubate at room temperature for 4 h to obtain MS-PEI nanoparticles.

[0083] (3) Disperse 10 mg of MS-PEI nanoparticles in 200 μL of carbonate buffer, then add 400 μL of 8-arm-PEG-OPA for reaction to make the final concentration of 8-arm-PEG-OPA 2 mg / mL. Place the reaction solution of this step on a mixer, wash 3 times with water after reacting for 15 min.

[0084] (4) Redisperse the above nano-drug carrier in 200 μL of carbonate buffer, add 400 μL of 8-arm-PEG-NH2 for reaction to make the final concentration of 8-arm-PEG-NH2 2 mg / mL. Place the reaction solution of this step on a mixer again, wash 3 times with water after reacting for 15 min; obtain a monolayer-assembled MS@PEG nano-drug carrier, denoted as MS@PEG1.

[0085] By repeating step (3) and step (4), MS@PEG nano-drug carriers with different numbers of assembled layers are obtained. Repeating step (3) and step (4) once gives a bilayer-assembled MS@PEG nano-drug carrier, repeating step (3) and step (4) twice gives a trilayer-assembled MS@PEG nano-drug carrier, repeating step (3) and step (4) three times gives a quadrilayer-assembled MS@PEG nano-drug carrier... Repeating step (3) and step (4) eight times gives a nonalayer-assembled MS@PEG nano-drug carrier. Among them, the bilayer to nonalayer-assembled MS@PEG nano-drug carriers obtained are denoted as MS@PEG2, MS@PEG3, MS@PEG4, MS@PEG5, MS@PEG6, MS@PEG7, MS@PEG8, and MS@PEG9 in sequence.

[0086] (5) Dissolve 10 mg of HA and 15 mg of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride in 1 mL of triple-distilled water, and mix for 30 min to obtain solution A. Take 10 mg of MS@PEG with different numbers of assembled layers and disperse it in 1 mL of carbonate buffer, then add 100 μL of A to obtain MS@HPEG nano-drug carriers with different numbers of assembled layers.

[0087] (6) Resuspend the MS@HPEG nano-drug carriers with different numbers of assembled layers in a solution of 2 M HF / 8 M NH4F (pH = 5) to remove MS and obtain HPEG nano-drug carriers with different numbers of assembled layers, denoted as HPEG1, HPEG2, HPEG3, HPEG4, HPEG5, HPEG6, HPEG7, HPEG8, and HPEG9 respectively.

[0088] Application Experimental Example 3

[0089] Detect the interaction between HPEG1, HPEG3, HPEG5, and HPEG8 nano-drug carriers and 4T1.

[0090] Culture 4T1 cells in a 24-well plate for 24 h, and incubate them with 0.5 mL of medium containing two concentrations (2.5 and 5 μg / mL respectively) of cy5-labeled HPEG nano-drug carriers. Then, wash the cells twice with PBS and analyze them using a flow cytometer. As Figure 9 shown, at a concentration of 25 μg / mL, the cell interaction of the HPEG1 nano-drug carrier reaches 60%, which is significantly higher than that of the other three HPEG nano-drug carrier groups.

[0091] Detect the organ distribution of HPEG1, HPEG3, HPEG5, and HPEG8 nano-drug carriers in mice.

[0092] Establish 4T1 tumor-bearing mice. When the tumor volume reaches 100 mm3 At about [time], Cy5.5-labeled HPEG nano-drug carriers (500 μg / mL) were intravenously injected into 4T1 tumor-bearing mice. After 12 h, the mice were euthanized, and the main organs (heart, liver, spleen, lung, kidney) and tumors were excised for in vitro imaging. As Figure 10 The fluorescence imaging in Fig. a shows that the HPEG1 nano-drug carriers effectively target tumors and exhibit high enrichment at the tumor site. Quantitative analysis shows that the accumulation amount of HPEG1 nano-drug carriers at the tumor site is 1.8 times that of HPEG8 nano-drug carriers and 1.4 times that of HPEG5 nano-drug carriers, as Figure 10 shown in Fig. b.

[0093] Example 3

[0094] A drug-loaded targeted PEG nano-drug carrier with different hardnesses was prepared in the same manner as in Example 2, except that: the MS-PEI nanoparticles obtained in step (2) were added with a chemotherapeutic drug, and then step (3) was carried out.

[0095] The specific process of adding the chemotherapeutic drug is as follows: 100 mg of monocarboxylate cisplatin was pre-dissolved with 120 mg of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride in 10 mL of triple-distilled water, and reacted for 30 min to obtain solution A. 10 mg of PEG-PEI was dispersed in 500 μL of carbonate buffer solution, 0.6 mL of solution A was added, and after reacting for 6 h, it was washed.

[0096] Application Experimental Example 4

[0097] Detect the cytotoxicity of Pt, Pt@HPEG1, and Pt@HPEG8 nano-drugs against 4T1.

[0098] The MTT method was used to determine the cytotoxicity of Pt@HPEG nano-drugs and Pt. 4T1 cells were seeded in 96-well plates at a density of 1×10 4 cells per well and cultured for 12 h to allow the cells to adhere. The cells were incubated with different concentrations of Pt@HPEG nano-drugs and Pt (equivalent to Pt at 5, 10, 20, and 40 μg / mL) for 24 h, then 10 μL of MTT (5 mg / mL) solution was added to each well and incubated for 4 h. The cell culture medium was removed, and 100 μL of DMSO was added to dissolve the formazan crystals. Then the absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader to obtain the results of cytotoxicity. As Figure 11 shown, compared with the Pt group, Pt@HPEG8 and Pt@HPEG1 more significantly increased the killing effect on 4T1 cells.

[0099] Detect the tumor inhibition of Pt, Pt@HPEG1, and Pt@HPEG8 nano-drugs against 4T1 tumor-bearing mice.

[0100] On days 0, 2, and 6, PBS, Pt, Pt@HPEG1, and Pt@HPEG8 nanomedicines were intravenously injected into Balb / c mice bearing 4T1 tumors. The tumor volume of the mice was measured every other day. After sacrificing the mice on day 16, the intact tumors of each mouse were dissected and photographed. As Figure 12 shown, compared with the PBS group and the Pt group, the Pt@HPEG1 nanomedicine group had the most significant inhibitory effect on the growth of mouse tumors.

[0101] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a polyethylene glycol nano-drug carrier with controllable hardness, characterized in that It includes the following steps: Adsorb a layer of polyethyleneimine on the template surface to obtain a carrier precursor; perform at least one layer-by-layer assembly on the surface of the carrier precursor; finally, remove the template to obtain the product; The process of performing one layer-by-layer assembly is as follows: first add multi-arm polyethylene glycol phthalaldehyde for crosslinking reaction, and then add multi-arm polyethylene glycol amine for crosslinking reaction; The crosslinking reaction is that phthalaldehyde groups react with primary amino groups to form isoindolinone groups.

2. The preparation method according to claim 1, characterized in that, When adding multi-arm polyethylene glycol phthalaldehyde for crosslinking reaction, the concentration of multi-arm polyethylene glycol phthalaldehyde in the reaction system is 1.8 - 2.2 mg / mL; Or, the multi-arm polyethylene glycol phthalaldehyde is octa-arm polyethylene glycol phthalaldehyde.

3. The preparation method according to claim 1, characterized in that, The multi-arm polyethylene glycol amine is octa-arm polyethylene glycol amine; Or, when adding multi-arm polyethylene glycol amine for crosslinking reaction, the concentration of multi-arm polyethylene glycol amine in the reaction system is 1.8 - 2.2 mg / mL.

4. The preparation method according to claim 1, characterized in that, The template is mesoporous silica; preferably, the way to remove the template is: remove it in a buffer solution of hydrofluoric acid and ammonium fluoride; Or, the number of layer-by-layer assembly times is 1 - 2 times; Or, the crosslinking reaction is carried out in a solvent of carbonate buffer solution, and the pH of the carbonate buffer solution is 9.0 - 9.

5.

5. A polyethylene glycol nanomedicine carrier with controllable hardness, characterized in that, Obtained by the preparation method according to any one of claims 1 - 4.

6. The hardness-controllable polyethylene glycol nano-drug carrier according to claim 5, characterized in that, The Young's modulus is 2.0 - 3.0 kPa.

7. A targeted PEG nanomedicine carrier, characterized in that, It includes the hardness-controllable polyethylene glycol nanodrug carrier and targeting molecule according to claim 5 or 6.

8. The targeted PEG nano-drug carrier according to claim 7, wherein The targeting molecule is folic acid, monosaccharide, oligosaccharide, HA, transferrin or RGD peptide, preferably HA; Or, during the preparation process, after the layer-by-layer assembly is completed, add a targeting molecule and a coupling agent for coupling reaction, and then remove the template.

9. Use of the hardness-controllable polyethylene glycol nanodrug carrier according to claim 5 or 6 or the targeting PEG nanodrug carrier according to claim 7 or 8 in the preparation of a drug, by loading drug molecules onto the PEI layer.

10. The application according to claim 9, characterized in that, in During the preparation process, load drug molecules onto the obtained carrier precursor, and then perform at least one layer-by-layer assembly on the surface of the carrier precursor loaded with drug molecules; Or, the drug is an anti-tumor drug; preferably, the tumor is breast cancer.

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