Living cell drug delivery system, preparation method therefor, and use thereof

Abstract

A living cell drug delivery system, a preparation method therefor, and use thereof. The living cell drug delivery system comprises carrier cells, polymer micelles, and a metal polyphenol network coating. The polymer micelles are loaded on the surface of the carrier cells, and the metal polyphenol network coating covers the surface of the polymer micelles. The raw materials of the metal polyphenol network coating comprise a first metal salt, a second metal salt and a polyphenol.

Description

A living cell drug delivery system and its preparation method and use Technical Field

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

[0002] Malignant tumors are a major disease that poses a serious threat to human health. Cell therapy, exemplified by chimeric antigen receptor T cells (CAR-T), has achieved breakthroughs. Intelligent drug delivery via cell-based carriers has also garnered significant interest and attention from researchers both domestically and internationally. Compared to synthetic drug carriers, living cell-based carriers offer improved biocompatibility and safety. Immune cells such as macrophages, neutrophils, T cells, and NK cells can also respond to signaling molecules released at the tumor site, enabling targeted drug delivery and treatment. To minimize the effects of drugs on the carrier cells and ensure high cell activity and targeted chemotaxis to the tumor site, the amount of drug loaded per cell carrier is often less than 0.5 pg. This makes it difficult to achieve satisfactory therapeutic effects (effectiveness) when administered at low cell doses in vivo, while high cell doses carry risks (safety) such as venous thromboembolism and cytokine storms. These effectiveness and safety issues are the primary challenges facing efficient drug delivery via cell-based carriers.

[0003] Currently, leveraging the phagocytic uptake of drug-loaded nanoparticles by immune cells is a common drug delivery method. However, premature intracellular drug release can compromise the activity and chemotactic ability of the carrier cells. Furthermore, carrier cells often struggle to release the drug from within, sometimes even inactivating the drug in organelles such as lysosomes, limiting its therapeutic efficacy. By contrast, attaching drug-loaded nanoparticles to the cell surface can reduce damage to the carrier cells and facilitate efficient drug release at the lesion site. However, the adhesion of drug-loaded nanoparticles to the cell surface is often unstable, making them susceptible to phagocytosis by the carrier cells. Furthermore, since the drug loading of conventional lipid and polymer nanoparticles is often less than 10%, excessive excipients occupy the limited intracellular and extracellular space of the carrier cells, resulting in extremely low drug loading per cell, making it difficult to meet therapeutic dose requirements in vivo. Based on these analyses, developing a live cell delivery system with stable extracellular attachment of high-drug-loaded nanoparticles is of great research value.

[0004] Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a living cell drug delivery system and a preparation method and use thereof.

[0006] To achieve the above-mentioned purpose and other related purposes, the present invention is achieved through the following technical solutions.

[0007] The first aspect of the present invention protects a living cell drug delivery system, which comprises carrier cells, polymer micelles and a metal polyphenol network coating, wherein the surface of the carrier cells is loaded with the polymer micelles, and the surface of the polymer micelles is wrapped with the metal polyphenol network coating, and the raw materials of the metal polyphenol network coating include a first metal salt, a second metal salt and polyphenol.

[0008] In certain embodiments, the carrier cells are selected from one or more of macrophages, neutrophils, T cells, and NK cells.

[0009] Preferably, the carrier cells are selected from macrophages.

[0010] In certain embodiments, the raw material of the polymer micelle is selected from the common block copolymer Pluronic, and the Pluronic is selected from one or more of Pluronic L35, Pluronic L43, Pluronic L44, Pluronic L61, Pluronic L62, Pluronic L64, Pluronic F68, Pluronic L81, Pluronic P84, Pluronic P85, Pluronic F87, Pluronic F88, Pluronic L92, Pluronic F98, Pluronic L101, Pluronic P103, Pluronic P104, Pluronic P105, Pluronic F108, Pluronic L121, Pluronic P123, and Pluronic F127. Preferably, it is Pluronic F127 (F127).

[0011] Preferably, the raw material of the polymer micelles further comprises a stabilizer.

[0012] More preferably, the stabilizer is selected from mifepristone (MIF) or clotrimazole (CLT). Preferably, it is clotrimazole (CLT). The present application has found that the addition of a stabilizer can reduce the particle size of the polymer micelles to below 200 nm, prevent the subsequent crystallization of the hydrophobic anti-tumor drug, avoid the aggregation of the hydrophobic anti-tumor drug and the micelles, and maintain dispersion for a long time. Without a stabilizer, the hydrophobic drug forms micelles that are prone to aggregation, and both dispersibility and stability are reduced.

[0013] More preferably, the polymer micelles are prepared by freeze-exfoliation of a conventional block copolymer, Pluronic, and a stabilizer. The polymer micelles obtained using this freeze-exfoliation method exhibit no degradation or increase in size when stored at 4°C for 15 days, demonstrating high stability. Furthermore, the polymer micelles exhibit a dispersion coefficient of less than 0.2 in both water and PBS solution, demonstrating high dispersibility.

[0014] Further preferably, the freeze exfoliation method comprises: mixing a common block copolymer Pluronic and water to obtain a Pluronic aqueous solution;

[0015] The stabilizer is dissolved in an organic solvent to obtain a premixed solution, and the premixed solution is added to the Pluronic aqueous solution and ultrafiltered to obtain the polymer micelles.

[0016] Preferably, based on the total mass of the Pluronic aqueous solution, the proportion of the common block copolymer Pluronic is 5-15 wt%, for example, 5-8 wt%, 7-12 wt%, or 11-15 wt%, and in a specific embodiment, 10 wt%.

[0017] Preferably, the organic solvent is dichloromethane. Based on the total mass of the premix, the stabilizer accounts for 5-15wt%, for example, 5-8wt%, 7-12wt%, or 11-15wt%, and in a specific embodiment, 10wt%.

[0018] Preferably, the volume ratio of the premix to the Pluronic aqueous solution is 1:(50-400), for example, 1:(50-150), 1:(100-280), or 1:(250-400), and in a specific embodiment, 1:100.

[0019] Preferably, the ultrafiltration conditions are: 1000-4000 rpm, 0.1-4° C., specifically, 3000 rpm and 4° C.; the number of ultrafiltrations is 5. Ultrafiltration is performed to remove free or loose common block copolymer Pluronic.

[0020] In certain embodiments, the raw materials of the metal polyphenol network include a first metal salt, a second metal salt, and a polyphenol.

[0021] Preferably, the first metal salt and the second metal salt are each independently selected from a divalent metal salt or a trivalent metal salt. The first metal and the second metal salt may be the same or different.

[0022] More preferably, the divalent metal salt is selected from one or more of manganese salts, iron salts, cobalt salts, copper salts, and zinc salts. For example, it is manganese chloride, ferric chloride, cobalt chloride, copper chloride, and zinc chloride. In a specific embodiment, it is manganese chloride.

[0023] More preferably, the trivalent metal salt is selected from one or both of chromium salts and iron salts, such as chromium chloride and ferric chloride.

[0024] Preferably, the polyphenol is selected from one or more of tannic acid (TA), ellagic acid (EA), epigallocatechin gallate (EGCG), chlorogenic acid (ChA), gallic acid (GA), caffeic acid (CA), quercetin (QR), myricetin (My), fisetin (Fi), luteolin (Lu), resveratrol (Re), gossypol (Go) and proanthocyanidin (OPC). In a specific embodiment, the polyphenol is proanthocyanidin (OPC).

[0025] Preferably, every 2×10 6 Each carrier cell is loaded with (0.01-2) mg of polymer micelles wrapped with a metal polyphenol network coating.

[0026] In certain embodiments, the preparation method of the metal polyphenol network coating encapsulated by polymer micelles is: the polymer micelles, the first metal salt and the polyphenol undergo a first coordination reaction.

[0027] Preferably, the mass molar ratio of the polymer micelle to the polyphenol is 1 mg: (0.02-0.22) μmol, and in a specific embodiment, 1 mg: 0.12 μmol.

[0028] Preferably, the molar ratio of the polyphenol to the first metal salt is (1-4):1. In a specific embodiment, it is 1:1.

[0029] Preferably, the first coordination reaction can be carried out at room temperature and pressure.

[0030] In certain embodiments, the method for loading the polymer micelles with the carrier cells is as follows: performing a second coordination reaction on the polymer micelles coated with the metal polyphenol network coating, the carrier cells, and the second metal salt.

[0031] Preferably, the mass molar ratio of the polymer micelles coated with the metal polyphenol network coating and the second metal salt is 1 mg: (0.01-0.5) μmol.

[0032] Preferably, the temperature of the second coordination reaction is 0.1-4°C.

[0033] The second aspect of the present invention protects a method for preparing the living cell drug delivery system as described above, comprising the following steps:

[0034] obtaining polymer micelles;

[0035] The polymer micelles, the metal salt and the first polyphenol undergo a first coordination reaction to obtain polymer micelles wrapped with a metal polyphenol network coating;

[0036] The polymer micelles wrapped with the metal polyphenol network coating, the carrier cells and the second metal salt undergo a second coordination reaction in an isotonic solution to obtain the living cell drug delivery system.

[0037] Preferably, the first coordination reaction further includes the addition of a promoter. The promoter is selected from a weak base, and the weak base is selected from 3-(N-morpholino)propanesulfonic acid (MOPS) or sodium hydroxide. The pH value of the weak base is less than 8.0. The present application found that the addition of 3-(N-morpholino)propanesulfonic acid or sodium hydroxide can promote the wrapping of the metal polyphenol network coating on the surface of the polymer micelle. During the second coordination reaction, MOPS is preferably used to reduce damage to the carrier cells.

[0038] When the nanoparticles are micelles formed by F127 (F127-NP), the base is sodium hydroxide, and NaOH must be used to provide an alkaline environment before adding polyphenols to avoid aggregation of F127-NP and polyphenols.

[0039] In certain embodiments, the isotonic solution is selected from 5% glucose aqueous solution, physiological saline, and 1×PBS, preferably 5% glucose aqueous solution.

[0040] In certain embodiments, the mass molar ratio of the polymer micelles to the polyphenols is 1 mg: (0.02-0.22) μmol, for example, 1 mg: (0.02-0.15) μmol, 1 mg: (0.08-0.18) μmol, or 1 mg: (0.16-0.22) μmol. In a specific embodiment, the mass molar ratio is 1 mg: 0.12 μmol.

[0041] In certain embodiments, the molar ratio of the polyphenol to the first metal salt is (1-4):1, for example, (1-2.3):1, (1.8-3.2):1, or (2.8-4):1. In a specific embodiment, it is 1:1.

[0042] In certain embodiments, the first coordination reaction is carried out at room temperature and pressure.

[0043] In certain embodiments, in the second coordination reaction, every 2×10 6 The carrier cells reacted with (0.01-2) mg of polymer micelles encapsulating the metal polyphenol network.

[0044] In certain embodiments, the temperature of the second coordination reaction is 0.1 to 4°C, for example, 0.1 to 1.5°C, 1.1 to 2.8°C, or 2.4 to 4°C, and in a specific embodiment, 4°C. The second coordination reaction of the present invention reduces carrier cell metabolism under low temperature conditions and locks the polymer micelles coated with the metal polyphenol network coating on the carrier cell surface through the second metal salt. When the second coordination reaction is carried out at room temperature and pressure and in the absence of the second metal salt, the polymer micelles are internalized by the carrier cells to form an intracellular delivery system.

[0045] In certain embodiments, the mass molar ratio of the polymer micelles encapsulating the metal polyphenol network and the second metal salt can be 1 mg: (0.01-0.5) μmol, or 1 mg: (0.01-0.15) μmol, or 1 mg: (0.1-0.35) μmol, or 1 mg: (0.2-0.45) μmol, or 1 mg: (0.32-0.5) μmol, and in a specific embodiment, it is 1 mg: 0.24 μmol.

[0046] The third aspect of the present invention protects the use of the living cell drug delivery system as described above in the preparation of anti-tumor drugs.

[0047] The fourth aspect of the present invention protects an anti-tumor drug, comprising the living cell drug delivery system as described above and a hydrophobic anti-tumor drug, wherein the living cell drug delivery system is loaded with the hydrophobic anti-tumor drug.

[0048] In certain embodiments, the polymer micelles are loaded with the hydrophobic anti-tumor drug.

[0049] In certain embodiments, the medicament further comprises a pharmaceutically acceptable excipient.

[0050] In certain embodiments, the hydrophobic anti-tumor drug is selected from paclitaxel drugs.

[0051] Preferably, the taxanes are selected from one or more of paclitaxel (PTX), cabazitaxel (CTX) and docetaxel (DTX). The drug delivery system of the present invention has a drug loading of 21 μg of the hydrophobic anti-tumor drug per million macrophages.

[0052] The fifth aspect of the present invention protects the use of the living cell drug delivery system as described above or the anti-tumor drug as described above in preventing and / or treating tumors.

[0053] The innovation of this study lies in the construction of a live-cell drug delivery system with highly drug-loaded polymer micelles stably attached to the extracellular space. This system effectively reduces the phagocytosis of drug-loaded polymer micelles by carrier cells, better maintaining the activity and targeted chemotaxis of live cells. Furthermore, the drug loading capacity of a single cell exceeds 10 pg, enabling efficient drug delivery at low cell doses and enhanced tumor-targeted immunochemotherapy. Furthermore, this study provides a new platform technology for the development of efficient live-cell-based drug delivery systems, and can also be used to flexibly "customize" live-cell-based drug delivery systems to meet the treatment needs of other diseases.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] 1) The carrier cells of the live cell drug delivery system of the present application can attach to drug-loaded polymer micelles with high stability outside the cells, effectively reduce the phagocytosis of drug-loaded polymer micelles, maintain the activity and targeted chemotaxis of the carrier cells, and the drug loading capacity of a single cell is greater than 10 pg, which can achieve efficient drug delivery and tumor-targeted immunochemotherapy when used at a low cell dose in vivo.

[0056] 2) The live cell drug delivery system of this application utilizes the coordination effect of metal ions and polyphenols to rapidly adsorb drug-loaded polymer micelles onto the surface of living cells. Fluorescence signal analysis shows that over 90% of the polymer micelles are adsorbed extracellularly. Furthermore, the metal ion polyphenol network (MPN)-mediated polymer micelle surface adsorption performance is excellent, achieving stable extracellular attachment for up to 10 days.

[0057] 3) The method of the present application is simple and quick, and the construction of the extracellular drug-loaded cell delivery system can be achieved in 2 minutes under ice bath conditions.

[0058] 4) The live cell drug delivery system of this application uses paclitaxel-loaded F127 nanomicelles prepared by the freeze-stripping method, with a drug loading of 54.8%. In comparison, the drug loading of conventional paclitaxel-loaded PLGA nanoparticles is only 3.1%. When the F127 nanomicelles are attached to the surface of macrophages, the drug loading of a single carrier cell exceeds 10 pg (20 to 300 times that of extracellular drug delivery methods reported in the literature). With the same carrier cell mass and nanoparticle mass, more drug can be carried, significantly reducing the cell dose during in vivo use (by more than 10 times), thereby improving the safety of in vivo use.

[0059] 5) In vivo anti-tumor studies have shown that the live cell drug delivery system loaded with paclitaxel The tumor immunochemotherapy effect is stronger, and it can effectively inhibit the growth of orthotopic breast tumors (4T1) (tumor inhibition rate 67.2%) and significantly prolong the survival time of mice (prolonged by 58.8%). BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 shows the characterization of the F127-NP nanomicelles in Example 1 of the present application, wherein a is the particle size and zeta potential of the F127-NP nanomicelles, and b is the dispersion index (PDI) of the F127-NP nanomicelles in different dispersion media.

[0061] Figure 2 shows the stability and drug loading of the F127-NP nanomicelles in Example 1 of this application. (a) shows the particle size change of the F127-NP nanomicelles over 15 days; (b) shows the dispersion coefficient of the F127-NP nanomicelles over 15 days; (c) shows the retention rate of coumarin 6 labeled on the F127-NP nanomicelles over 7 days; (d) shows the HPLC chromatogram of paclitaxel; and (e) shows a standard curve constructed by comparing paclitaxel concentration with the corresponding HPLC peak area. Data are expressed as mean ± SD, n = 3.

[0062] FIG3 shows a schematic diagram of the construction of the high-drug-loading macrophage delivery system in Example 1 of the present application.

[0063] Figure 4 shows the construction and characterization of the high-drug-loading macrophage delivery system in Example 1 of the present application. Wherein, a is the UV absorption of the proanthocyanidin manganese ion network dispersion system under different molar ratios; b is the particle size and Zeta potential of the F127-NP nanomicelles after being coated with the proanthocyanidin manganese ion network; c is Confocal microscopy 3D view; d is the constructed under different molar ratio conditions Confocal microscopy images (F127-NP nanomicelles labeled with coumarin 6, green). Data are expressed as Mean ± SD, n = 3.

[0064] FIG5 shows macrophages in Example 1 of the present application and high-drug-loaded macrophage delivery systems Scanning electron microscope image of .

[0065] FIG6 shows the drug loading of macrophage surface adhesion and intracellular loading in Example 3 of the present application.

[0066] FIG7 shows the high drug loading macrophage delivery system in Example 4 of the present application. and targeting of F127-NP nanomicelles. and fluorescence imaging of F127-NP nanomicelles at the tumor site; b is each time point, Fluorescence intensity of F127-NP nanomicelles at the tumor site. Data are expressed as Mean ± SD, n = 5.

[0067] FIG8 shows macrophages in Example 5 of the present application PLGA-NP nanoparticles, F127-NP nanomicelles, and high-drug-loaded macrophage delivery systems and low-drug-loading macrophage delivery systems In vitro killing effect on breast cancer cells. (a) Bioluminescence and viability of 4T1 cells at 24 hours; (b) Bioluminescence and viability of 4T1 cells at 36 hours; (c) Bioluminescence and viability of 4T1 cells at 48 hours. Data are expressed as Mean ± SD, n = 4.

[0068] FIG9 shows macrophages in Example 6 of the present application. Highly loaded drug delivery system for macrophages and low-drug-loaded macrophage delivery systems In vivo antitumor effect. (a) shows the change in mouse body weight over time; (b) shows the change in tumor volume over time; and (c) shows the mouse survival curve. Data are expressed as Mean ± SD, n = 5-7. DETAILED DESCRIPTION

[0069] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0070] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0071] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0072] Paclitaxel, also known as Paclitaxel in English, abbreviated as PTX;

[0073] Clotrimazole, also known as Clotrimazole or CLT in English;

[0074] 100 μg of paclitaxel was dissolved in a mixture of polyethylene castor oil and ethanol to form a paclitaxel solution, which was labeled as Taxol solution; wherein the volume ratio of polyethylene castor oil to ethanol was 50:50.

[0075] Macrophages are labeled Low drug loading macrophage delivery system labeled Group, high drug loading macrophage delivery system marked as In the 2 groups, the paclitaxel-loaded F127 nanomicelles were labeled as F127-NP nanomicelles, the paclitaxel-loaded PLGA nanoparticles were labeled as PLGA-NP nanoparticles, and the metal polyphenol network coating was labeled as MPN.

[0076] In the following examples of the present application, the isotonic solution is a 5 wt % glucose aqueous solution.

[0077] Example 1 Preparation and characterization of a high-drug-load macrophage delivery system

[0078] In this Example 1, paclitaxel-loaded F127 nanomicelles (F127-NP) were first prepared, and then a metal polyphenol network coating (NP@MPN) was formed on the outer surface of the paclitaxel-loaded F127-NP nanoparticles. The NP@MPN was then dispersed in an isotonic solution and allowed to incubate with macrophages. Incubate, and under the action of the second metal salt, NP@MPN is locked on the surface of macrophages to obtain a high-drug-load macrophage delivery system. The raw materials of the metal polyphenol network coating include a first metal salt and polyphenol, including the following:

[0079] 1.1 Preparation and characterization of F127 nanomicelles

[0080] 1.1.1 Preparation of F127 Nanomicelles without Paclitaxel

[0081] Preparation of 10 wt% F127 aqueous solution: Accurately weigh 10 g of F127 powder in a 100 mL volumetric flask and add triple-distilled water to make up to volume.

[0082] 1 mL of F127 aqueous solution was pipetted into a reaction bottle and a magnetic rotor was added. The reaction bottle was placed on a magnetic stirrer and the speed was adjusted to 350 rpm to allow the rotor to rotate smoothly to obtain micelles formed by F127 itself (F127 aqueous solution).

[0083] 100 μL of a 10 mg / mL clotrimazole (CLT) premix was added dropwise to the reaction flask with stirring, causing the F127 aqueous solution to become turbid. The hydrophobic stabilizer gradually intercalated into the micelles formed by the F127 during stirring. The clotrimazole premix was prepared by dissolving clotrimazole in dichloromethane.

[0084] When the solution becomes clear and transparent, stop stirring immediately, transfer the clear solution to a 4 mL ultrafiltration tube, and add 4°C pre-cooled PBS solution to the highest scale line.

[0085] The ultrafiltration tube was placed in a centrifuge and ultrafiltration was performed at 3000 rpm and 4°C. The loading of hydrophobic substances can stabilize the F127 micelles. When the temperature is lower than 4°C, the free and loosely bound F127 will dissociate and "strip off". After 5 ultrafiltrations, F127 nanomicelles embedded with stabilizers were obtained.

[0086] 1.1.2 Preparation of Paclitaxel-Loaded F127 Nanomicelles

[0087] 1) Prepare solution

[0088] Paclitaxel solution: Three taxane drugs, paclitaxel (PTX), cabazitaxel (CTX) and docetaxel (DTX), were dissolved in dichloromethane to prepare 40 mg / mL paclitaxel solution.

[0089] Stabilizer solvent: Dissolve mifepristone (MIF) and clotrimazole (CLT) in dichloromethane to prepare a 10 mg / mL stabilizer solution.

[0090] Induced luminescence dye HPS solution: Dissolve HPS in dichloromethane to prepare a 1 mg / mL HPS solution.

[0091] Prepare the premix according to the recipe in Table 1.

[0092] Table 1

[0093] 2) Pipette 1 mL of 10% F127 aqueous solution into a reaction flask and insert a magnetic rotor. Place the reaction flask on a magnetic stirrer and adjust the speed to 350 rpm to ensure smooth rotation of the rotor to obtain micelles formed by F127 itself (F127 aqueous solution).

[0094] 3) The premixed solution in Table 1 was added dropwise to the reaction flask in step 2) with stirring, so that the F127 aqueous solution became turbid. The hydrophobic paclitaxel drug, stabilizer, and HPS were also gradually incorporated into the micelles formed by the F127 itself in step 2) during the stirring process.

[0095] 4) When the solution in step 3) becomes clear and transparent, stop stirring immediately, transfer the clarified solution to a 4 mL ultrafiltration tube, and add 4°C pre-cooled PBS solution to the highest scale line.

[0096] 5) Place the ultrafiltration tube in a centrifuge and perform ultrafiltration at 3000 rpm and 4°C. The loading of the hydrophobic substance stabilizes the F127 micelles. When the temperature is below 4°C, the free and loosely bound F127 will dissociate and "strip off". After five ultrafiltration cycles, F127 nanomicelles embedded with the paclitaxel drug and stabilizer are obtained.

[0097] The results showed that when paclitaxel drugs were used alone to insert into F127 micelles, particles with a size exceeding 200 nm would be formed, which was not suitable for drug delivery; when mixed with mifepristone (MIF) or clotrimazole (CLT) as stabilizers, the particle size of F127 nanomicelles loaded with paclitaxel drugs decreased to below 200 nm, probably because the stabilizer prevented the paclitaxel drugs from forming crystals; PTX and CLT were finally selected.

[0098] PTX and CLT were mixed with HPS in a mass ratio of 4:1 (i.e., formula 8) and then inserted into the micelles formed by F127 itself to obtain paclitaxel-loaded F127 nanomicelles (the paclitaxel-loaded F127 nanomicelles were labeled F127-NP) for subsequent experiments.

[0099] 1.1.3 Characterization of F127-NP Nanomicelles

[0100] 1) Investigation of particle size, potential, and dispersion coefficient of F127-NP nanomicelles

[0101] The particle size and potential of F127-NP nanomicelles were measured by a laser particle size analyzer, and the dispersion coefficient was determined after the F127-NP nanomicelles were dissolved in PBS and water, respectively. The results are shown in Figure 1.

[0102] As shown in Figure 1a, the DLS data showed that the particle size of F127-NP nanomicelles was 43.8±3.3nm and the Zeta potential was -5.2±0.7mV.

[0103] As can be seen from Figure 1b, the F127-NP nanomicelles dispersed in PBS solution and water showed relatively uniform particle size, with PDI of 0.12 and 0.11, respectively, both less than 0.2.

[0104] 2) Stability investigation of F127-NP nanomicelles

[0105] The F127-NP nanomicelles were stored in a refrigerator at 4°C. A small amount of F127-NP nanomicelles was taken daily and diluted with PBS. The particle size and dispersion index of the F127 nanomicelles were then measured using a laser particle size analyzer for 15 days. The results are shown in Figure 2 (a) and (b). (a) represents the particle size change of the F127-NP nanomicelles over 15 days; (b) represents the dispersion index (PDI) of the F127-NP nanomicelles over 15 days.

[0106] As can be seen from Figure 2a, the particle size of F127-NP nanomicelles remained at around 40 nm within 15 days, and there was no obvious decrease or increase in size.

[0107] In addition, to investigate the stability of the fluorescent dye labeled on the F127-NP nanomicelles, eight 1.5 mL centrifuge tubes were labeled 0 to 7, and 1 mL of F127 nanomicelle solution labeled with coumarin 6 fluorescent dye was added to each tube. 200 μL of F127 nanomicelle solution was aspirated from centrifuge tube No. 0 into a 96-well plate, and the fluorescence intensity of coumarin 6 (Ex 488 nm, Em 520 nm) was measured as the total fluorescence intensity. Subsequently, one centrifuge tube was removed daily and the liquid was transferred to an ultrafiltration tube for ultrafiltration at 3000 rpm. 200 μL of the ultrafiltrate was aspirated into a 96-well plate to measure the fluorescence intensity of coumarin 6. The retention rate of coumarin 6 labeled on the F127 nanomicelles over 7 days is shown in Figure 2c.

[0108] Coumarin 6 retention rate (%) = (total fluorescence intensity – ultrafiltrate fluorescence intensity) / total fluorescence intensity × 100%

[0109] As can be seen from Figure 2c, the fluorescence intensity of F127-NP nanomicelles was maintained at about 95% within 7 days, indicating that the labeling of coumarin 6 was very stable.

[0110] 1.2 Construction and characterization of nanomicelles coated with metal polyphenol networks

[0111] 1.2.1. Construction of Nanomicelles Encapsulated with Metal Polyphenol Network Coating

[0112] OPC and Mn 2+ The ions were reacted at molar ratios of 1:1, 2:1, 3:1 and 4:1 to investigate the interaction of OPC with Mn 2+ The optimal ratio of ions is as follows:

[0113] 1) Adsorption: Take four 1.5 mL centrifuge tubes and add 1 mL of F127-NP nanomicelle solution (1 mg / mL) labeled with coumarin 6 to each tube. Then, add 100 μL of NaOH solution (0.1 M) to provide an alkaline environment. Then, add 10, 20, 30, and 40 μL of OPC solution (24 mM) to each tube under water bath sonication (25°C).

[0114] 2) Locking: Then, 10 μL of MnCl 2 solution (24 mM, first metal salt) was added under water bath ultrasonication to allow the metal ions to react with the phenolic hydroxyl groups of the polyphenols.

[0115] 3) After the reaction, the centrifuge tube was placed on a shaker to avoid light and gently shaken. After incubation for 30 minutes, ultrafiltration was performed at a speed of 2000 rpm and re-dissolved with pre-cooled PBS to obtain F127@MPN (Mn 2+ -OPC) solution.

[0116] At the same time, a control group (marked as Mn 2+ group), and the control group was: 1 mL of F127-NP nanomicelle solution labeled with coumarin 6 (1 mg / mL), followed by the addition of 100 μL of NaOH solution (0.1 M), and then the addition of 10 μL of MnCl2 solution (24 mM) under water bath sonication (25 ° C), without the addition of polyphenols.

[0117] 1.2.2 Characterization of Nanomicelles Coated with Metal Polyphenol Networks

[0118] 1) Investigation of absorption spectrum

[0119] 200 μL of the F127@MPN solution obtained in step 1.2.1 was added to a 96-well plate, and the absorption spectrum of the solution was measured using a microplate reader. The results are shown in Figure 4a.

[0120] As can be seen from Figure 4a, as the amount of OPC input increases, the absorption of the formed metal polyphenol network coating nanomicelle solution becomes stronger.

[0121] 2) Investigation of particle size and potential

[0122] The F127@MPN obtained in step 1.2.1 was dissolved in triple-distilled water, and the particle size and potential of the coatings with different metal polyphenol ratios were measured using a laser particle size analyzer. The results are shown in Figure 4b and Table 2.

[0123] At the same time, F127-NP nanomicelles not coated with metal polyphenol network coating (F127-NP nanomicelles not coated with metal polyphenol network coating are denoted as F127-NP) were used as the control group.

[0124] As shown in Figure 4b, the thickness of the metal polyphenol network coating increases with increasing OPC dosage, and the absolute value of the zeta potential also gradually increases, indicating that OPC improves the stability of the F127 nanomicelles. Table 2 shows that a 1:1 molar ratio of OPC to the first metal salt is optimal, and this ratio was used for subsequent experiments.

[0125] Table 2

[0126] 1.3 Construction and Characterization of a High-Drug-Loading Macrophage Delivery System

[0127] 1.3.1 Construction of a high-drug-load macrophage delivery system

[0128] 1 mL of macrophage suspension (2×10 6 / mL), 1mL of F127@MPN(Mn 2+ -OPC) solution (1 mg / mL) was added to a 7 mL centrifuge tube, and then 10 μL of MnCl2 solution (24 mM, second metal salt) was added, followed by 100 μL of MOPS buffer (100 mM, pH 8.0). After stirring for 2 min in an ice bath, the centrifuge tube was removed and centrifuged at 1000 rpm at 4°C for 3 min to remove the nanomicelles that were not attached to the macrophage surface (Figure 3), thereby preparing a high-drug-loading macrophage delivery system (abbreviated as ).

[0129] 1.3.2 Characterization of high-drug-load macrophage delivery systems

[0130] 1) Laser scanning confocal microscopy

[0131] Observation using laser scanning confocal microscopy The results are shown in Figure 4c and d. (Ex 405 nm, Em 460 nm, blue pseudocolor) and F127 nanomicelles attached to the cell surface (Ex 488 nm, Em 520 nm, green pseudocolor, see Figure 4)

[0132] As can be seen from Figure 4c, the green F127-NP nanomicelles can be well connected to the surface of macrophages.

[0133] From Figure 4d, we can see that OPC and Mn 2+ The metal polyphenol network coatings prepared by coordination reaction of ions with molar ratios of 1:1, 2:1, 3:1 and 4:1 can all construct macrophage delivery systems well, and there is no obvious difference in the macrophage delivery systems constructed with each ratio.

[0134] 2) Scanning electron microscopy observation

[0135] Will and Inoculate into a 24-well plate with a glass slide and wash twice after the macrophages adhere to the wall. Add electron microscopy fixative and fix for 2 hours at room temperature in the dark and then transfer to 4°C for storage. and The surface morphology is photographed.

[0136] As shown in Figure 5, macrophages not connected to F127 nanomicelles The cells adhered to the wall and had extended pseudopodia. The cell surface was relatively smooth. The surface becomes rough and pseudopodia are produced, suggesting that the motility of macrophages is not greatly affected.

[0137] Comprehensive description of high drug loading macrophage delivery system Build successful.

[0138] Example 2 Preparation and characterization of low drug loading macrophage delivery system

[0139] In this Example 2, paclitaxel-loaded PLGA-NP nanoparticles (PLGA-NPs) were first prepared, and then a metal polyphenol network coating was coated on the outside of the PLGA-NPs to obtain nanoparticles coated with a metal polyphenol network coating (the PLGA nanoparticles coated with a metal polyphenol network coating were labeled as NP@MPN). Then, the NP@MPNs were dispersed in an isotonic solution and compared with macrophages. A second coordination reaction is carried out to lock NP@MPN on the surface of macrophages under the action of the second metal salt, thereby obtaining a low-drug-loading macrophage delivery system. These include:

[0140] 2.1 Preparation and Characterization of PLGA-NP Nanoparticles (PLGA-NP)

[0141] 2.1.1 Preparation of PLGA-NP nanoparticles (PLGA-NP)

[0142] The PLGA-NP nanoparticles prepared by single ultrasonic emulsification are as follows:

[0143] The preparation method of PLGA-NP nanoparticles (PLGA-NP) is as follows:

[0144] 1) Pour 40 mL of 0.5% (m / v) sodium cholate solution into a 50 mL beaker and place in a magnetic rotor.

[0145] 2) Nanoparticle Formulation: Weigh 20 mg of PLGA powder and add 1 mL of dichloromethane to dissolve the PLGA powder to form a dichloromethane solution. Then, add 3 mL of 1% (m / v) sodium cholate solution. Add a fluorescent dye to the dichloromethane solution to prepare fluorescent dye-labeled PLGA-NP nanoparticles, or add a lipid-soluble drug to prepare drug-loaded PLGA-NP nanoparticles.

[0146] 3) Nanoemulsion: Insert the probe of the probe sonicator 1 to 1.5 cm below the liquid surface in step 2) and sonicate at a power of 200 W for 25 seconds to obtain PLGA nanoemulsion.

[0147] 4) Volatilization and Condensation: Pour the PLGA nanoemulsions into the beaker from step 1) and stir for 40 minutes to evaporate the dichloromethane in the PLGA nanoemulsions and solidify the polymer into nanoparticles. Finally, pour the contents of the beaker into a 50 mL centrifuge tube and centrifuge at 11,000 g for 30 minutes. Discard the supernatant, add triple-distilled water, and re-dissolve the precipitate under water bath sonication to prepare a PLGA nanoparticle solution (PLGA-NP).

[0148] 2.1.2 Characterization of PLGA-NP Nanoparticles (PLGA-NP)

[0149] PLGA-NP nanoparticles were examined by cryo-electron microscopy and scanning electron microscopy.

[0150] The results showed that the morphology of PLGA-NP nanoparticles was round.

[0151] The dynamic light scattering data of Malvern laser particle size analyzer showed that the particle size of PLGA-NP nanoparticles was 104.8±2.1nm.

[0152] 2.2 Construction and Characterization of PLGA Nanoparticles Coated with Metal Polyphenol Networks

[0153] 2.2.1. Construction of PLGA Nanoparticles Coated with Metal Polyphenol Networks

[0154] The difference from step 1.2.1 of the embodiment is:

[0155] In step 1), the PLGA-NP nanoparticle solution obtained in step 2.1 was used to replace the 1 mg / L F127-NP nanomicelle solution, and tannic acid was used to replace proanthocyanidins. That is, the 1 mg / L PLGA-NP nanoparticle solution was mixed with 5 μL tannic acid (TA) solution (24 mM) and vortexed for 10 s to allow TA to adsorb on the surface of the PLGA-NP nanoparticles. In addition, there was no need to pre-add NaOH solution to create an alkaline environment before adding the TA solution;

[0156] In step 2), 5 μL of copper chloride solution (24 mM) was used instead of 10 μL of MnCl2 solution (24 mM).

[0157] Step 3) Add 50 μL of MOPS buffer (100 mM, pH 8.0) to raise the solution pH to a slightly alkaline environment, which facilitates the coordination reaction between the metal ions and the polyphenols. After shaking for 20 minutes, centrifuge at 8000 g at 25°C for 10 minutes. Repeat the centrifugation twice to wash away free polyphenols and metal chlorides, producing nanoparticles coated with a metal polyphenol network (MPN) (NP@MPN). Finally, the precipitate was reconstituted into 1 mL for later use. This yielded PLGA-NP@CuPN nanoparticles with a copper ion tannic acid network coating (abbreviated as PLGA-NP@CuPN nanoparticles).

[0158] 2.2.2 Characterization of PLGA Nanoparticles Coated with Metal Polyphenol Networks (NP@MPN)

[0159] The particle size of PLGA-NP@CuPN nanoparticles coated with copper ion tannic acid network (PLGA-NP@CuPN nanoparticles) was 121.7±3.1 nm, which was approximately 18 nm larger than the particle size of PLGA-NP nanoparticles; the PDI of PLGA-NP nanoparticles before and after coating with copper ion tannic acid network coating were 0.11 and 0.12, respectively, both less than 0.20, indicating that the uniformity of PLGA-NP nanoparticles and PLGA-NP@CuPN nanoparticles was good and there was no particle aggregation; the Zeta potential of PLGA nanoparticles before and after coating with copper ion tannic acid network coating was -32.1±0.5 mV and -43.1±3.1 mV.

[0160] 2.3 Construction and Characterization of Low-Drug-Loading Macrophage Delivery System

[0161] 2.3.1 Construction of a low-drug-load macrophage delivery system

[0162] 1) An isotonic solution was mixed with RAW 264.7 macrophages to obtain a macrophage suspension. The concentration of the macrophage suspension was 2×10 6 / mL.

[0163] 2) PLGA nanoparticles coated with a metal polyphenol network coating (NP@MPN) were dispersed in an isotonic solution to obtain an NP@MPN solution, wherein the concentration of the NP@MPN solution was 1 mg / mL.

[0164] 3) Pipette 1mL of macrophage suspension and 1mL of NP@MPN solution into a 7mL centrifuge tube, turn on the magnetic stirrer to gently stir the suspension, quickly add 10μL of CuCl2 solution (24mM, second metal salt) to lock NP@MPN on the surface of macrophages, and then add 100μL of MOPS buffer (100mM, pH 8.0) to increase the pH value of the reaction system. After stirring for 2 minutes, remove the centrifuge tube and centrifuge at 1000rpm and 4℃ for 3 minutes to remove NP@MPN that is not attached to the surface of macrophages, and prepare a low drug loading macrophage delivery system (abbreviated as ).

[0165] 2.3.2 Characterization of Low-Drug-Loading Macrophage Delivery System

[0166] Laser scanning confocal microscopy showed that PLGA nanoparticles coated with a metal network coating could be well connected to the surface of macrophages. SEM showed that macrophages connected to PLGA nanoparticles also produced pseudopodia, suggesting that the motility of macrophages would not be greatly affected. Both showed that a low-drug-load macrophage delivery system was successfully established.

[0167] Example 3 Investigation of drug loading

[0168] The paclitaxel-loaded F127-NP nanomicelles (F127-NP) obtained in step 1.1 of Example 1 and the paclitaxel-loaded PLGA-NP nanoparticles (PLGA-NP) obtained in step 1.1 of Example 2, as well as the high-drug-loaded macrophage delivery system obtained in step 1.3 of Example 1 were prepared. The drug loading capacity was investigated, including the following:

[0169] 3.1. Investigation of drug loading capacity of F127-NP nanomicelles and PLGA-NP nanoparticles

[0170] The drug loading of PTX in F127-NP nanomicelles and PLGA-NP nanoparticles was determined by high performance liquid chromatography (HPLC).

[0171] 1) First, prepare a standard curve of PTX concentration versus peak area. Prepare PTX solutions at concentrations of 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μg / mL in acetonitrile. Pipette 200 μL of each solution into a sample vial. Detection was performed using the following chromatographic conditions: a C18 reversed-phase column; a 10 μL sample load; a mobile phase consisting of acetonitrile and triple-distilled water in a 55:45 ratio of organic phase to aqueous phase; a flow rate of 1 mL / min; a detection wavelength of 227 nm; and a column temperature of 25°C.

[0172] 2) Compile a standard curve using the detected peak areas and corresponding PTX concentrations. Sample pretreatment: Ultrafilter the nanoparticle solution and replace the PBS with triple-distilled water. Pipette 100 μL of the nanoparticle solution into 900 μL of acetonitrile and mix thoroughly to dissolve the PTX in the nanoparticles. Using the same chromatographic conditions, measure the PTX loading in F127-NP nanomicelles and PLGA-NP nanoparticles. The results are shown in Figure 2(d).

[0173] As can be seen from Figure 2d, the peak time of PTX is 6.7 min, the peak shape of the chromatographic peak is a normally distributed narrow peak, there is no front peak and tailing peak, the baseline is stable, and there is no obvious impurity peak, indicating that the chromatographic conditions are good.

[0174] A series of PTX solution concentrations were prepared by serial dilution, and a standard curve was prepared using the corresponding peak areas measured by HPLC. The results are shown in Figure 2e.

[0175] As shown in Figure 2e, with the PTX concentration as the abscissa and the corresponding peak area as the ordinate, linear regression was performed to obtain the standard curve equation: y = 21697x + 23.291; R 2 The standard curve was 1, indicating that the preparation was excellent and had a good linear relationship in the range of 0.78125 to 400 μg / mL.

[0176] After sample pretreatment, the F127-NP nanomicelles and PLGA-NP nanoparticles were each analyzed using HPLC to determine the PTX chromatographic peaks. The peak areas were integrated to obtain the drug loading. The respective peak areas were then substituted into the standard curve.

[0177] The drug loading capacity of F127-NP nanomicelles was 54.8%; the drug loading capacity of PLGA-NP nanoparticles was 3.08%.

[0178] 3.2 High-drug-load macrophage delivery system Investigation of drug loading

[0179] 3.2.1 Construction of intracellular and extracellular delivery systems

[0180] 1) Coumarin 6 was used to label F127-NP nanomicelles. The coumarin 6-labeled F127 nanomicelles were then coated with the metal polyphenol network coating according to the method for constructing the metal polyphenol network coating in Example 1. The coumarin 6-labeled F127-NP nanomicelles coated with the metal polyphenol network coating (coumarin 6-labeled F127@MPN) were serially diluted to form a series of nanoparticle solutions. 200 μL of each nanoparticle solution was then added to a 96-well plate, and the fluorescence intensity of coumarin 6 (Ex 488 nm, Em 520 nm) was measured using a microplate reader. A standard curve was created based on the concentration of the nanoparticle solution and the corresponding coumarin 6 fluorescence intensity.

[0181] 2) Control group: also known as the intracellular delivery system, the operation is as follows: macrophages were inoculated into 6-well plates, with 2×10 6 After the cells adhered to the wall, coumarin 6-labeled F127@MPN nanoparticles containing 9.38, 18.75, 37.5, 75, 150, and 300 μg of PTX (calculated based on a PTX loading rate of 54.8%) were added to each well. After incubation for 3 h, DPBS was added along the well wall to wash the cells twice, and the cells were then blown down from the bottom of the well plate to obtain the intracellular delivery system.

[0182] Experimental group: that is, the extracellular delivery system, referring to the construction method of the high-drug-loading macrophage delivery system in Example 1, 1 mL of PTX concentrations of 9.38, 18.75, 37.5, 75, 150 and 300 μg / mL coumarin 6-labeled F127@MPN nanoparticles (calculated based on PTX drug loading of 54.8%) were taken and mixed with 1 mL of macrophage suspension (2×10 6 / mL) for incubation to obtain the extracellular delivery system.

[0183] 3) Pipette 200 μL of each intracellular and extracellular delivery system into a 96-well plate and measure the fluorescence intensity of coumarin 6 (Ex 488 nm, Em 520 nm) using a microplate reader. Calculate the nanoparticle concentration based on the standard curve, and then calculate the PTX binding efficiency of a single macrophage based on the PTX loading of the nanoparticles.

[0184] 3.2.2 Results Analysis

[0185] The amount of nanoparticles is indirectly indicated by the fluorescence intensity of coumarin 6, which is then converted by the drug loading amount of the nanoparticles. The fluorescence intensity of coumarin 6 can then indirectly indicate the amount of PTX in the drug-loaded macrophage delivery system.

[0186] With PTX concentration as the abscissa and the fluorescence intensity of coumarin 6 as the ordinate, linear regression was performed, and the equation of the high-concentration standard curve was obtained in the concentration range of 20-300 μg / mL: y=314.23x+3520.4; R 2 The linear relationship between the standard curve and the concentration range of 20 to 300 μg / mL was good.

[0187] The equation of the low-concentration standard curve in the concentration range of 0.3125 to 10 μg / mL was: y = 496.02x + 143.67; R 2 The linear relationship between the standard curve and the concentration of the standard was 0.9983, indicating that the standard curve was well prepared and had a good linear relationship in the range of 0.3125 to 10 μg / mL.

[0188] The fluorescence intensities of the prepared extracellular delivery system and the intracellular delivery system as a control were substituted into the curve equation. The loading efficiency was calculated by measuring the fluorescence intensity of coumarin 6 before and after loading of F127-NP nanomicelles. The results are shown in Figure 6. Macrophage surface adhesion and intracellular loading.

[0189] As shown in Figure 6, when 150 μg PTX and 1×10 6 When 150 μg PTX was added to 1×10 6 By constructing an intracellular delivery system with 10 macrophages, approximately 10 μg of PTX can be obtained, with a connection efficiency of approximately 6.6%. The intracellular drug loading efficiency is approximately equal to 47% of the extracellular drug loading efficiency.

[0190] Example 4 Targeting of High Drug Loading Macrophage Delivery System

[0191] In this Example 4, the F127 nanomicelles (F127-NP) labeled with the fluorescent dye DiD and the high-drug-loaded macrophage delivery system labeled with the fluorescent dye DiD were obtained. Investigate its tumor targeting, including the following:

[0192] 4.1. F127-NP labeled with fluorescent dye DiD and Acquisition

[0193] DiD was used to replace HPS to obtain DiD-labeled fluorescent dye F127-NP, and then the method of Example 1 was used to obtain DiD-labeled fluorescent dye F127-NP. The cell concentration was 1.67×10 7 / mL.

[0194] 4.2 Construction of Breast Cancer Mouse Model

[0195] 1) Adjust the concentration of 4T1-luc breast cancer cell suspension to 1.33×10 7 / mL.

[0196] 2) Five-week-old BLAB / c female mice were anesthetized with ketamine solution and injected with 75 μL of breast cancer cell suspension through the fourth mammary fat pad in the inguinal region.

[0197] 3) The tumor volume of the mice grew to about 100mm 3 300 μL of DiD-labeled and DiD-labeled F127-NP (the concentration of DiD fluorescent dye was 50 ng / mL), and then 100 μL of D-luciferin potassium salt solution (10 mg / mL) was injected through the tail vein. The fluorescence intensity of DiD in the mice was measured using a small animal in vivo imager at 0.5, 1, 2, 3, 5, and 6 h after injection of D-luciferin potassium salt solution (Ex 605 nm, Em 660 nm). The tumor site was circled and the fluorescence intensity was counted. The results are shown in Figure 7.

[0198] 4.3 Results Analysis

[0199] F127 nanomicelles loaded with fluorescent dye (F127-NP) or high-drug-loaded macrophage delivery system Injected into mice through the tail vein, if the dye fluorescence appears at the tumor site, it indicates that the F127 nanomicelles (F127-NP) or high drug loading macrophage delivery system It has a certain degree of tumor targeting. The stronger the fluorescence intensity at the tumor site, the better the tumor targeting.

[0200] As shown in Figure 7a, F127-NP and DiD fluorescence indicates that F127-NP In addition, DiD fluorescence can also be detected in the liver, indicating that F127-NP and It will also be distributed to the liver. As time goes by, F127-NP and The fluorescence of DiD distributed to the tumor site was enhanced, indicating that more F127-NPs were Accumulated in tumor sites.

[0201] F127-NP and The changes in fluorescence intensity of DiD at the tumor site over time are shown in Figure 7b.

[0202] From Figure 7b, we can see that The fluorescence intensity peak of the F127-NP group was at 3 hours, indicating that the tumor was most concentrated at this time. The fluorescence intensity peak of the F127-NP group was at 2 hours, indicating that the tumor was most concentrated at this time. The fluorescence intensity of the group was higher than that of the F127-NP group.

[0203] Comprehensive description, It is enriched in the tumor site, has better targeting, and can carry more drugs to the tumor site, thereby improving the therapeutic effect of the drug.

[0204] Example 5 In vitro tumor killing study of macrophage delivery system

[0205] In this Example 5, the F127-NP nanomicelles (F127-NP) loaded with paclitaxel and the high drug loading macrophage delivery system were obtained by using Example 1. Example 2 Obtaining Paclitaxel-Loaded PLGA-NP Nanoparticles (PLGA-NP) and Low Drug Loading Macrophage Delivery System 4T1-luc breast cancer tumor cells were treated in vitro, including the following:

[0206] 5.1. In vitro tumor treatment

[0207] 1) 4T1-luc breast cancer cells with luciferase reporter gene, adjust the concentration of 4T1-luc breast cancer cell suspension to 2×10 5 / mL; pipette 1mL of breast cancer tumor cell suspension into a 6-well plate for culture. After the tumor cells adhere to the wall, discard the old culture medium and wash once with DPBS.

[0208] 2) Add 2 mL of serum-free DMEM medium (as the control group) and macrophage Suspension (2.5×10 5 / mL), Cell suspension (2.5×10 5 / mL) and its corresponding mass of PLGA-NP nanoparticles (10 μg), Cell suspension (2.5×10 5 / mL) and its corresponding mass of F127-NP nanomicelles (10 μg).

[0209] 3) After incubation for 24, 36, and 48 h, discard the old culture medium and wash once with DPBS. Add 500 μL of D-luciferin potassium salt solution (10 mg / mL) to each well.

[0210] 4) After incubation at 37°C in the dark for 20 min, the bioluminescence of 4T1-luc breast cancer tumor cells was detected using a small animal live imaging system.

[0211] 5.2 Detection and Analysis

[0212] Assays include bioluminescence images and viability statistics.

[0213] Assay Principle: 4T1-luc breast cancer cells harboring a luciferase reporter gene produce luciferase. Adding D-luciferin potassium salt as a substrate results in bioluminescence. The intensity of the bioluminescence is positively correlated with the number of luciferase-producing cells; higher bioluminescence intensity indicates a higher number of cells.

[0214] Measuring bioluminescence intensity can indirectly infer cell number. A series of 4T1-luc breast cancer cell suspensions were prepared by serial dilution, and the bioluminescence intensity of each concentration was measured using a small animal live imaging system.

[0215] Prepare 10 mg / mL D-luciferin potassium salt solution. Digest 4T1-luc tumor cells with luciferase reporter gene and adjust the concentration of cell suspension to 8×10 5 / mL. Then dilute to 4×10 5 / mL, 2×10 5 / mL, 1×10 5 / mL, 5×10 4 / mL, 2.5×10 4 mL and 1.25×10 4 Prepare a series of cell suspensions at different concentrations (100 μL / mL). Pipette 100 μL of each suspension into a 96-well black plate, then add 100 μL of D-luciferin potassium salt solution. Incubate at 37°C in the dark for 20 minutes. Detect cell bioluminescence using a small animal imaging system. Construct a standard curve based on bioluminescence intensity versus corresponding cell concentration.

[0216] Small animal in vivo imaging group, PLGA-NP group, F127-NP group, Group and The well plates were incubated for 24 h, 36 h, and 48 h for bioluminescence imaging and activity investigation. The results are shown in FIG8 .

[0217] 5.3 Results Analysis

[0218] The number of 4T1-luc breast cancer cells was used as the horizontal axis and the bioluminescence intensity of the corresponding number was used as the vertical axis. Linear regression was performed and the standard curve equation was obtained in the range of 1250 to 80000 4T1-luc breast cancer cells: y = 4.167x + 10484; R 2 The linear relationship between the standard curve and the number of 4T1-luc breast cancer cells was 0.989, indicating that the standard curve was acceptable and had a relatively good linear relationship in the range of 1250 to 80000 4T1-luc breast cancer cells.

[0219] The PTX carried by the macrophage delivery system kills tumor cells, and the higher the PTX content, the stronger the killing effect. The number of 4T1-luc breast cancer cells in the well plate decreases under the action of PTX, and the bioluminescence intensity after the addition of D-luciferin potassium salt also decreases.

[0220] As shown in Figure 8a, at 24h, compared with the Control group, The bioluminescence intensity was slightly reduced in the group, which may be due to Competition with 4T1-luc breast cancer cells for the growth environment may also be due to macrophages phagocytosing a small number of 4T1-luc breast cancer cells. Group and The bioluminescence intensity of the F127-NP group was weaker than that of the PLGA-NP group, indicating that both the low-drug loading macrophage delivery system and the high-drug loading macrophage delivery system can kill 4T1-luc breast cancer cells. The bioluminescence intensity of the F127-NP group was weaker than that of the PLGA-NP group.

[0221] As can be seen from Figure 8b and Figure 8c, the bioluminescence intensity of each group is similar to that of 24 h, but is slightly lower than that of 24 h. This may be due to the prolonged incubation time and the longer time for PTX to act on 4T1-luc breast cancer cells.

[0222] The bioluminescence intensity of the F127-NP group was weaker than that of the PLGA-NP group. This was because: under the same mass of nanoparticles, the drug loading capacity of the F127-NP group was more than 10 times higher than that of the PLGA-NP group, so it could carry more PTX to kill 4T1-luc breast cancer cells. Bioluminescence intensity ratio of the groups The bioluminescence intensity of the group was weak. This is because: under the same number of macrophages and the same mass of nanoparticles loaded, The drug loading of the group was also higher than The drug loading capacity of the group is more than 10 times higher, so it can carry more PTX to kill 4T1-luc breast cancer cells.

[0223] Example 6 Investigation of in vivo tumor killing by macrophage delivery system

[0224] In this Example 6, the high drug loading macrophage delivery system obtained in Example 1 was used. Low drug loading macrophage delivery system of Example 2 Conduct in vivo tumor killing studies, including the following:

[0225] 6.1. Construction and grouping of 4T1-luc breast cancer mouse model

[0226] 6.1.1. Mouse Model Construction

[0227] 1) Inject 75 μL of 4T1-luc breast cancer cell suspension (1.33×10 7 / mL), that is, each mouse was inoculated with about 1 million breast cancer cells.

[0228] 2) Return the mice to the cages for continued feeding, and write the vaccination date on the label outside the cages, with the vaccination date marked as -7 days.

[0229] 6.1.2 Grouping

[0230] Day 5 after inoculation of 4T1-luc breast cancer cells (recorded as day -2, when the breast tumor volume grew to 50-75 mm 3 ), tumor-bearing mice were randomly divided into the following 6 groups (n=10) and marked on the labels outside the mouse cages. The groups were as follows:

[0231] 1) Control group: 1× PBS solution;

[0232] 2) Group: 1×10 7 macrophages;

[0233] 3) Group: Low drug loading macrophage delivery system with 1×10 7 macrophages (10 μg PTX);

[0234] 4) Free PTX group: Taxol solution (100 μg PTX);

[0235] 5) Group: High drug loading macrophage delivery system with 1×10 7 macrophages (100 μg PTX);

[0236] 6) Group: High drug loading macrophage delivery system combined with PD-1 antibody (100 μg αPD 1×3).

[0237] After the grouping is completed, The group began to be injected with αPD1, with 100 μL of αPD1 solution (1 mg / mL) injected intraperitoneally every day for 3 consecutive days.

[0238] 7 days after inoculation of 4T1-luc breast cancer cells (marked as day 0, when the breast tumor volume is about 100 mm 3 ), refer to the construction method of macrophage delivery system mentioned above and use 5×10 6 Macrophages were connected with 1 mg of PLGA nanoparticles or F127 nanomicelles to produce cells with an extracellular load of about 100 μg of PLGA nanoparticles. Cell suspension (5 million macrophages-100 μg PLGA nanoparticles-5 μg PTX) and extracellular loading of about 100 μg F127 nanomicelles Cell suspension (5 million macrophages-100 μg F127 nanomicelles-50 μg PTX).

[0239] Will and Adjusted to 3.33×10 7 Adjust the concentration of Taxol solution to 333.3 μg / mL and inject 300 μL of the solution into the tail vein of mice in each experimental group.

[0240] 6.2 Detection and Analysis

[0241] Detection indicators include mouse body weight, tumor volume and survival monitoring.

[0242] After the administration was completed, the body weight of mice in each group was monitored and recorded.

[0243] The length and width of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula:

[0244] Tumor volume = tumor length × tumor short diameter 2 ×0.5, and the body weight and tumor volume of mice were measured every other day.

[0245] When the tumor volume of the mouse is ≥2000mm 3 At 4 hr, the mice were euthanized and the survival curves were recorded. The median survival time of the mice in each group was calculated using the Kaplan-Meier method, and the survival time and tumor volume of the mice in each group were compared pairwise using the Gehan-Breslow-Wilcoxon test. The results are shown in Figure 9.

[0246] 6.3 Results Analysis

[0247] On day 22 after administration, the first tumor grew to 2000 mm 3 The mice were killed.

[0248] As shown in Figure 9a, compared with the Control group, Group, group, Free PTX group, Group and There was no significant difference in body weight between the two groups, indicating that no obvious damage was caused to the mice.

[0249] The change in tumor volume in tumor-bearing mice is an important indicator for pharmacodynamic investigation, and good drug efficacy can inhibit tumor growth.

[0250] As can be seen from Figure 9b, the Control group, Group, group, Free PTX group, Group and The tumor volumes of the two groups on day 22 were 1742.0±307.1mm 3 、1493.0±454.4mm 3 、1097.8±103.4mm 3 、1328.3±293.6mm 3 、571.6±220.7mm 3 and 410.8±191.7mm 3 The tumors in the mice in the control group grew fastest; The tumor inhibition effect of the group was the strongest; the group with the same cell number and nanomicelle quality Group comparison The tumor inhibition effect of the group was better, which may be due to the higher content of PTX delivered to the tumor site. Group comparison The tumor suppression effect of the 2-group was better, which may be because the immune checkpoint inhibitor αPD1 relieved the immunosuppressive state, thereby enhancing the immune response and better achieving tumor immunochemotherapy.

[0251] The survival time of mice is also an important indicator for evaluating the efficacy of drugs or preparations.

[0252] As can be seen from Figure 9c, the Control group, Group, group, Free PTX group, Group and The survival periods of the two groups were 25.5 days, 29 days, 32 days, 31 days, 40.5 days and 45 days, respectively. Group comparison The survival of the group was prolonged by 29.0%, which was a statistically significant difference.

[0253] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods and compositions in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A living cell drug delivery system, characterized in that: The living cell drug delivery system comprises carrier cells, polymer micelles and a metal polyphenol network coating, wherein the surface of the carrier cells is loaded with the polymer micelles, the surface of the polymer micelles is coated with the metal polyphenol network coating, and the raw materials of the metal polyphenol network coating comprise a first metal salt, a second metal salt and polyphenols.

2. The living cell drug delivery system according to claim 1, characterized in that The carrier cells are selected from one or more of macrophages, neutrophils, T cells and NK cells; and / or, the raw material of the polymer micelle is selected from the common block copolymer Pluronic, and the Pluronic is selected from one or more of Pluronic L35, Pluronic L43, Pluronic L44, Pluronic L61, Pluronic L62, Pluronic L64, Pluronic F68, Pluronic L81, Pluronic P84, Pluronic P85, Pluronic F87, Pluronic F88, Pluronic L92, Pluronic F98, Pluronic L101, Pluronic P103, Pluronic P104, Pluronic P105, Pluronic F108, Pluronic L121, Pluronic P123 and Pluronic F127; and / or, every 2×10 6 Each carrier cell is loaded with (0.01-2) mg of polymer micelles coated with metal polyphenol network coating; and / or, the first metal salt and the second metal salt are each independently selected from a divalent metal salt or a trivalent metal salt; and / or, the polyphenols are selected from one or more of tannic acid, ellagic acid, epigallocatechin gallate, chlorogenic acid, gallic acid, caffeic acid, quercetin, myricetin, fisetin, luteolin, resveratrol, gossypol and proanthocyanidins; And / or, the preparation method of the metal polyphenol network coating encapsulated by polymer micelles is: the polymer micelles, the first metal salt and the polyphenols undergo a first coordination reaction; And / or, the method for loading the polymer micelles with the carrier cells is: subjecting the polymer micelles coated with the metal polyphenol network coating, the second metal salt and the carrier cells to a second coordination reaction.

3. The living cell drug delivery system according to claim 2, characterized in that The raw materials of the polymer micelles also include a stabilizer; and / or, the divalent metal salt is selected from one or more of manganese salts, iron salts, cobalt salts, copper salts and zinc salts; And / or, the trivalent metal salt is selected from one or both of chromium salts and iron salts; and / or, the mass molar ratio of the polymer micelle to the polyphenol is 1 mg: (0.02-0.22) μmol; and / or, the molar ratio of the polyphenol to the first metal salt is (1-4):1; And / or, the mass molar ratio of the polymer micelles coated with the metal polyphenol network coating and the second metal salt is 1 mg: (0.01-0.5) μmol.

4. The living cell drug delivery system according to claim 3, characterized in that The preparation method of the polymer micelle is as follows: the polymer and the stabilizer are prepared by a cryo-stripping method; And / or, the stabilizer is selected from mifepristone or clotrimazole.

5. The living cell drug delivery system according to claim 4, characterized in that The cryo-stripping method comprises: mixing the polymer Pluronic with water to obtain a Pluronic aqueous solution; dissolving a stabilizer in an organic solvent to obtain a premixed solution; The premixed solution is added to the Pluronic aqueous solution and ultrafiltered to obtain the polymer micelles.

6. The method for preparing the living cell drug delivery system according to any one of claims 1 to 5, characterized in that: The steps include: obtaining polymer micelles; The polymer micelles, the first metal salt and the polyphenol undergo a first coordination reaction to obtain polymer micelles encapsulating a metal polyphenol network; The polymer micelles encapsulating the metal polyphenol network, the carrier cells and the second metal salt undergo a second coordination reaction in an isotonic solution to obtain the living cell drug delivery system.

7. The preparation method according to claim 6, characterized in that: The mass molar ratio of the polymer micelle to the polyphenol is 1 mg: (0.02-0.22) μmol; and / or, the molar ratio of the polyphenol to the first metal salt is (1-4):1; And / or, the temperature of the second coordination reaction is 0.1-4°C; And / or, the first or second coordination reaction further comprises adding a promoter, wherein the promoter is selected from 3-(N-morpholinyl)propanesulfonic acid or sodium hydroxide; and / or, the first metal salt and the second metal salt are each independently selected from a divalent metal salt or a trivalent metal salt; and / or, the mass molar ratio of the polymer micelles encapsulating the metal polyphenol network and the second metal salt is 1 mg: (0.01-0.5) μmol; And / or, in the second coordination reaction, every 2×10 6 Each carrier cell reacts with (0.01-2) mg of polymer micelles encapsulating the metal polyphenol network.

8. Use of the living cell drug delivery system according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

9. An anti-tumor drug, characterized in that: Comprising the living cell drug delivery system according to any one of claims 1 to 5 and a hydrophobic anti-tumor drug, wherein the living cell drug delivery system is loaded with the hydrophobic anti-tumor drug; Preferably, it further comprises pharmaceutically acceptable excipients.

10. The anti-tumor drug according to claim 9, characterized in that: The hydrophobic anti-tumor drug is selected from paclitaxel drugs; preferably, the paclitaxel drug is selected from one or more of paclitaxel (PTX), cabazitaxel (CTX) and docetaxel (DTX).

11. Use of the living cell drug delivery system according to any one of claims 1 to 5 or the anti-tumor drug according to claim 9 or 10 in preventing and / or treating tumors.

Images