Intracellular self-assembled cell motor drug delivery system suitable for inflammation and preparation method thereof

Abstract

The present invention discloses an intracellular self-assembled cell motor drug delivery system suitable for inflammation and a preparation method thereof, and relates to the technical field of supramolecular chemistry. The drug delivery system includes immune cells as carriers and nano-drugs with therapeutic ingredients. The nano-drugs are loaded into immune cells through the autonomous endocytosis of immune cells. The nano-drugs include a first nano-material modified by a host molecule and a second nano-material modified by a guest molecule. The host molecule and the guest molecule autonomously form an assembly in the immune cell. The first nano-material and the second nano-material are both nano-materials that produce gas under the stimulation of the inflammatory microenvironment or move under the drive of external energy. The drug delivery system can inhibit the rapid excretion of nano-drugs by cells, reduce drug leakage before transport to the target site, and improve drug delivery efficiency. The drug delivery system can construct the immune cells themselves as "cell motors" to penetrate deep into the lesions, promote the effective use of drugs, and achieve good therapeutic effects.

Description

Technical Field

[0001] The present invention relates to the technical field of supramolecular chemistry, and in particular to an intracellular self-assembled cell motor drug-carrying system suitable for inflammation and a preparation method thereof. Background Art

[0002] Inflammatory response is an important signal for a series of diseases including cardiovascular and cerebrovascular diseases and cancer. Due to the strong immune clearance ability of the body in the blood circulation system, the targeting efficiency of traditional nanoformulations for inflammatory sites is extremely low, so the therapeutic effect is difficult to be satisfactory. Bionic technology provides good support for the delivery of nanoformulations. From simple cell membrane coating to live cell delivery, it can increase the biocompatibility of nanoformulations and reduce the clearance of nanoformulations by the reticuloendothelial system. However, how to overcome the premature loss of drugs caused by the vigorous endocytosis and exocytosis of living cells is the biggest challenge facing the research on live cell delivery. Therefore, for serious diseases related to inflammation, seeking new delivery strategies and developing nanodrug delivery platforms with high biocompatibility, low biotoxicity and strong targeting are important directions for preclinical research.

[0003] In view of the above considerations, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide an intracellular self-assembled cell motor drug delivery system suitable for inflammation and a preparation method thereof to solve the above technical problems.

[0005] The present invention is achieved in that:

[0006] In the first aspect, the present invention provides an intracellular self-assembled cell motor drug delivery system suitable for inflammation, which includes immune cells as carriers and nanomedicines with therapeutic ingredients, wherein the nanomedicine is loaded into the immune cells through the autonomous endocytosis of the immune cells, and the nanomedicine includes a first nanomaterial modified with a main molecule and a second nanomaterial modified with a guest molecule, wherein the main molecule and the guest molecule form an assembly in the immune cells, and the first nanomaterial and the second nanomaterial are both nanomaterials that produce gas under the stimulation of an inflammatory microenvironment or move under the drive of external energy.

[0007] In an optional embodiment, the nanomaterial that generates gas under the stimulation of the inflammatory microenvironment is manganese dioxide, cerium oxide, manganese iron oxide, calcium carbonate or platinum nanoparticles;

[0008] Preferably, the nanomaterial that moves under the drive of external energy is ferroferric oxide, titanium oxide or nickel-based nanoparticles, and the energy includes one or more of chemical energy, biological energy, magnetic energy, thermal energy and light energy;

[0009] Preferably, the particle size of the nano drug is 100-200 nm;

[0010] Preferably, the concentration of the nanodrug is 5-200 μg / mL.

[0011] In an optional embodiment, the nanomaterial is one or more of solid or hollow nanoparticles, nanogels and nanocapsules;

[0012] Preferably, the nanomaterial is hollow or loaded with free drugs;

[0013] Preferably, the free drug comprises an anti-inflammatory drug or an antibiotic;

[0014] Preferably, the anti-inflammatory drug comprises quercetin, curcumin or atorvastatin;

[0015] Preferably, the antibiotic comprises aspirin or penicillin.

[0016] In an optional embodiment, the preparation method of the first nanomaterial and the second nanomaterial comprises: modifying the main molecule or the guest molecule on the surface of the nanomaterial directly or via a polymer; wherein the modification via a polymer comprises first modifying the surface of the nanomaterial via a polymer, then connecting the main molecule or the guest molecule to the active site of the polymer via a chemical bond and then grafting it to the surface of the nanomaterial; or, connecting the main molecule or the guest molecule to the active site of the polymer via a chemical bond, and then modifying the surface of the nanomaterial;

[0017] Preferably, the polymer comprises amino-terminally modified polyethylene glycol, carboxyl-terminally modified polyethylene glycol, polyallylamine phosphate or polyacrylic acid.

[0018] In an optional embodiment, the host molecule is cyclodextrin, cucurbituril, calixarene, pillararene or crown ether; the guest molecule is adamantane, ferrocene, spermine or azobenzene;

[0019] Preferably, the modification method of the host molecule and the guest molecule on the surface of the nanomaterial is covalent bond modification.

[0020] In an optional embodiment, the molar ratio of the host molecule on the surface of the first nanomaterial to the guest molecule on the surface of the second nanomaterial is 1-100:1-100; preferably 1-10:1-10;

[0021] In an alternative embodiment, the immune cells are cells with phagocytic ability and inflammatory tendency ability;

[0022] Preferably, the immune cells include macrophages, neutrophils, leukocytes, dendritic cells or lymphocytes.

[0023] In a second aspect, the present invention provides a method for preparing an intracellular self-assembled cell motor drug delivery system suitable for inflammation as described in any of the aforementioned embodiments, comprising: mixing immune cells with a first nanomaterial modified with a main molecule and a second nanomaterial modified with a guest molecule for incubation.

[0024] In an optional embodiment, the incubation includes: loading the immune cells with the first nanomaterial and the second nanomaterial by stepwise incubation;

[0025] Preferably, the immune cells are first incubated and loaded with the first nanomaterial, and then incubated and loaded with the second nanomaterial;

[0026] Preferably, the immune cells and the first nanomaterial are mixed in a culture medium for incubation, and after incubation, fresh culture medium is replaced to remove the first nanomaterial that has not been internalized, and then the second nanomaterial is added for incubation, and after incubation, the second nanomaterial that has not been internalized is removed;

[0027] Preferably, the immune cells are loaded with the first nanomaterial and the second nanomaterial for 1-8 hours, preferably 3-5 hours.

[0028] The present invention has the following beneficial effects:

[0029] The intracellular self-assembly cell motor drug delivery system suitable for inflammation provided by the present application utilizes the endocytosis of immune cells to encapsulate the first nanomaterial and the second nanomaterial in the immune cells, and the present application realizes the self-assembly of the first nanomaterial and the second nanomaterial in the immune cells through the main molecule and the guest molecule, thereby "locking" the nano drug in the immune cells, inhibiting its rapid excretion by the cells, reducing the drug leakage before being transported to the target site, avoiding the premature release of the nano drug in living cells, prolonging the circulation time, and improving the targeting of the nano drug to the inflammatory lesions through its homing effect, reducing the clearance of the nano drug by the reticuloendothelial system, and improving the delivery efficiency of the drug. Further, a nano material that generates gas under the stimulation of the inflammatory microenvironment or moves under the drive of external energy is selected as the first nano material and the second nano material. After the first nano material and the second nano material are loaded on the immune cells, they are transported to the corresponding disease location, and the immune cells themselves can be constructed as "cell motors" by generating gas or external energy drive. The immune cell drug delivery platform itself is driven by gas or energy to penetrate deep into the lesion, promoting the effective use of drugs and achieving good therapeutic effects. The intracellular self-assembled cell motor drug delivery system suitable for inflammation proposed in the present invention makes full use of the response characteristics of immune cells to inflammatory diseases, and is expected to provide innovative cell therapy for the targeted treatment of major acute diseases such as acute pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 This is a transmission electron microscope image of the intracellular self-assembled cell motor drug delivery system suitable for inflammation according to Example 1 of the present application;

[0032] Figure 2 A schematic diagram of the movement of the intracellular self-assembled cell motor drug-carrying system suitable for inflammation in a solution driven by gas provided in Experimental Example 1 of this application;

[0033] Figure 3Schematic diagram of frozen sections of lung tissue of mice with acute pneumonia provided in Experimental Example 2 of the present application, wherein A is a frozen section of lung tissue of mice with acute pneumonia treated with ordinary macrophages (non-motor) provided in Comparative Example 1, and B is a frozen section of lung tissue of mice with acute pneumonia treated with macrophages loaded with intracellular aggregates of manganese dioxide provided in Example 1. In the figure, the blue fluorescent part is the nucleus of lung cells labeled with Hochest33342, and the red fluorescence is the macrophage or motor macrophage labeled with Cy5. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0035] Cells are the smallest structural units that maintain physiological activities of living organisms. Due to their typical biological origin, cells themselves have natural advantages as carriers for delivering nanomedicines. There have been reports on the use of living cells as carriers in existing studies, and most of the construction methods have certain limitations. It is reported that drug delivery by living cells can be achieved by grafting covalent bonds with drug carriers on the surface of living cells through specific ligands-receptors or by chemical methods. However, specific ligand-receptor binding pairs are only expressed in specific cell lines and have a very limited range of applications; and the chemical grafting method involves bioorthogonal reactions and chemical synthesis on the cell surface, which may affect the physiological activity and function of cells. In addition, the method of connecting drug carriers outside cells is prone to partial internalization of the carriers due to cell endocytosis. In addition, cell loading of nanomedicines can be achieved through the cell's own endocytosis in living cells. However, the vigorous endocytosis and exocytosis of living cells can easily lead to premature leakage of internalized nanomedicines, and their drug utilization will be greatly reduced.

[0036] In view of this, the present invention provides an intracellular self-assembled cell motor drug delivery system suitable for inflammation, which includes immune cells as carriers and nanomedicines with therapeutic ingredients, the nanomedicines are loaded into the immune cells through the autonomous endocytosis of the immune cells, the nanomedicines include a first nanomaterial modified by a host molecule and a second nanomaterial modified by a guest molecule, the host molecule and the guest molecule autonomously form an assembly in the immune cells, and in the present application, the first nanomaterial and the second nanomaterial are both nanomaterials that produce gas under the stimulation of the inflammatory microenvironment or move under the drive of external energy. The particle size of the nanomedicine is 100-200nm; the concentration of the nanomedicine is 5-200μg / mL.

[0037] The present application realizes self-assembly of the first nanomaterial and the second nanomaterial in immune cells through the main molecule and the guest molecule, thereby "locking" the nano drug in the immune cell. The present invention avoids the premature release of the nano drug in living cells, and at the same time, due to the good biocompatibility of the cells themselves and the targeting of the immune cells themselves to the inflammatory site, the clearance of the nano drug by the reticuloendothelial system is reduced, and the delivery efficiency of the drug is improved. In addition, the present application is also different from directly loading the main molecule or the guest molecule on the surface of the immune cell. In the present application, the main molecule and the guest molecule are self-assembled in the immune cell, thereby preventing the nano drug from being released from the cell before reaching the inflammatory lesion.

[0038] Furthermore, in addition to the regulatory effect, the physical properties of the nanomaterials also affect the therapeutic efficiency of the living cell drug-carrying platform. In the present application, nanomaterials that produce gas under the stimulation of the inflammatory microenvironment or move under the drive of external energy are selected as the first nanomaterial and the second nanomaterial. The first nanomaterial and the second nanomaterial are loaded on the immune cells and transported to the corresponding disease location. The immune cells themselves can be constructed as "cell motors" by generating gas or external energy driving. The immune cell drug-carrying platform itself is driven by gas or energy to penetrate deep into the lesion, thereby promoting the effective use of drugs. Preferably, in the present application, the nanomaterials that produce gas under the stimulation of the inflammatory microenvironment are manganese dioxide, cerium oxide, manganese iron oxide, calcium carbonate or platinum nanoparticles; the nanomaterials that move under the drive of external energy are ferroferric oxide, titanium oxide or nickel-based nanoparticles, and the energy includes one or more of chemical energy, bioenergy, magnetic energy, thermal energy and light energy. The nanomaterials in the present application can be purchased commercially or prepared independently according to conventional methods.

[0039] In addition, the nanomaterial is one or more of solid or hollow nanoparticles, nanogels and nanocapsules; in the present application, the nanomaterial itself has a certain therapeutic effect, so solid nanomaterials can be selected, preferably, the nanomaterial is hollow and free drugs are loaded in the hollow; free drugs can be used as auxiliary treatment means, preferably, free drugs include anti-inflammatory drugs or antibiotics; anti-inflammatory drugs include quercetin, curcumin or atorvastatin; antibiotics include aspirin or penicillin; anticancer drugs include doxorubicin, paclitaxel or camptothecin.

[0040] In the present invention, immune cells are cells with phagocytic ability and inflammatory tendency; preferably, immune cells include any one of macrophages, neutrophils, leukocytes, dendritic cells and lymphocytes; wherein macrophages include M1 type or M2 type; lymphocytes include one or more of T cells, B cells and NK cells.

[0041] There are five methods for preparing the first nanomaterial and the second nanomaterial. One is to modify the host and guest molecules directly on the surface of the nanomaterial. This method uses the host molecule or the guest molecule to combine with the nanomaterial itself through coordination bonds or electrostatic effects to achieve the surface modification of the nanomaterial. The second method is to use polymers such as amino-terminated polyethylene glycol or polyallylamine phosphate and other amino polymers to graft amino groups on the surface of the nanomaterial, and then use the carboxylated host molecule and the guest molecule to be connected separately on the surface of the nanoparticle through an amidation reaction. The third method is to use polymers such as carboxyl-terminated polyethylene glycol or polyacrylic acid to graft carboxyl groups on the surface of the nanomaterial, and then use the aminoated host molecule and the guest molecule to be connected separately on the surface of the nanoparticle through an amidation reaction. The fourth method is to modify the amino-terminal host and guest molecules on the carboxyl-terminated polymer molecules, and then the polymers containing the host and guest molecules are mixed with the nanomaterials for surface modification. The fifth method is to modify the carboxyl-terminal host and guest molecules on the amino-terminated polymer molecules, and then the polymers containing the host and guest molecules are mixed with the nanomaterials for surface modification. In the present application, the polymer includes but is not limited to amino-terminally modified polyethylene glycol, carboxyl-terminally modified polyethylene glycol, polyallylamine phosphate or polyacrylic acid.

[0042] The host molecule includes but is not limited to cyclodextrin, cucurbituril, calixarene, pillararene or crown ether; the guest molecule includes but is not limited to adamantane amine, ferrocene, spermine or azobenzene. The host molecule and the guest molecule in the present application can be purchased commercially or prepared independently according to conventional methods. The host molecule and the guest molecule can recognize each other to form a host-guest complex. The molar ratio of the host molecule on the surface of the first nanomaterial to the guest molecule on the surface of the second nanomaterial is 1-100:1-100; preferably 1-10:1-10, specifically 1:10, 2:10; 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, 1:1, 10:1, 10:2, 10:3, 10:4, 10:5, 10:6, 10:7, 10:8, 10:9, any one or the range between any two. Preferably 1:1.

[0043] In addition, the present invention also provides a method for preparing the above-mentioned intracellular self-assembled cell motor drug delivery system suitable for inflammation, which comprises: mixing immune cells with a first nanomaterial modified with a main molecule and a second nanomaterial modified with a guest molecule for incubation.

[0044] Specifically, the following steps are included:

[0045] (1) Cell collection.

[0046] Collect 10 6 The corresponding immune cells can be collected by methods including but not limited to in vitro expansion and direct extraction.

[0047] (2) Cell incubation.

[0048] The immune cells are incubated and loaded with the first nanomaterial and the second nanomaterial in steps; the step-by-step incubation may be to first incubate the first nanomaterial and then the second nanomaterial; or the second nanomaterial may be incubated first and then the first nanomaterial. In the present application, it is preferred that the immune cells are incubated and loaded with the first nanomaterial first and then the second nanomaterial.

[0049] Specifically, the immune cells and the first nanomaterial are mixed in a culture medium and incubated for 1-8 hours (preferably 3-5 hours). After incubation, the fresh culture medium is replaced to remove the first nanomaterial that has not been internalized, and then the second nanomaterial is added for incubation for 1-8 hours (preferably 3-5 hours). After incubation, the second nanomaterial that has not been internalized is removed. After the second nanomaterial enters the immune cells, the guest molecules on the second nanomaterial will self-assemble with the host molecules on the first nanomaterial, thereby "locking" the nanomedicine in the immune cells. Since different nanomaterials are internalized by immune cells in different situations, and complete internalization cannot be achieved, therefore, in this application, the concentration of the first nanomaterial and the second nanomaterial, the incubation time, and the order of step-by-step incubation and other related incubation parameters are defined, and the self-assembled product finally obtained is the intracellular self-assembled cell motor drug delivery system suitable for inflammation of this application.

[0050] The intracellular self-assembled cell motor drug delivery system suitable for inflammation involved in the present invention has a simple self-assembly preparation process, high biocompatibility, high targeting efficiency, and a broad spectrum of effects on a variety of nanomaterials and immune cells. Therefore, it can be regarded as a potential inflammation treatment strategy.

[0051] Furthermore, acute diseases such as acute pneumonia often require timely drug penetration into the deep tissue of the disease for rapid release to achieve efficient treatment. This application selects the first nanomaterial and the second nanomaterial, and utilizes the characteristics of nanoparticles generating gas driving force under internal or external stimulation, so that the system forms a cell motor after reaching the inflammatory tissue, pushing the carrier cells to infiltrate deeper, and the drug molecules can also be selectively released at the lesion site to effectively treat the disease.

[0052] In addition, the drug release method involved in the present application can be that the nanodrug assembly is disassembled in the carrier cell and then released through active exocytosis; the nanoparticles can be degraded in the carrier cell and directly release the free drugs loaded therein; or the release of drug molecules loaded by the nanoparticles can be stimulated by external stimulation, such as near-infrared laser, ultrasound, etc.

[0053] The drug treatment methods involved in the present invention include but are not limited to one or more of the following methods: the nanoassemblies in the immune cells themselves responsively degrade at the inflammatory site, weaken the inflammatory signal, and regulate the inflammatory microenvironment; the degradation of nanoparticles in the immune cells leads to the release of free drugs loaded by the nanoparticles, which act on the corresponding cells at the inflammatory site; through external stimulation, such as near-infrared laser, ultrasound, etc., the therapeutic function of the nanoparticles themselves is stimulated or the drug molecules loaded by the nanoparticles are stimulated to release and act on inflammation-related cells.

[0054] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0055] Example 1

[0056] This embodiment provides an intracellular self-assembled cell motor drug delivery system suitable for inflammation and a preparation method thereof. In this embodiment, mono(6-ethylenediamine-6-deoxy)β-cyclodextrin was purchased from Shandong Zhiyuan Biology, DMEM cell culture medium was purchased from HyClone, and PAA was purchased from Sigma-Aldrich. In this example, macrophages were selected as carrier cells, macrophages were purchased from ATCC cell bank, and manganese dioxide nanoparticles were obtained by the laboratory according to conventional methods, and the particle size was about 100 nanometers.

[0057] Macrophages were cultured and expanded overnight in DMEM medium until the total number of cells was greater than 10 6 . Cyclodextrin-modified manganese dioxide nanoparticles (125 μg / mL) were incubated at 37°C for 4 hours. After incubation, fresh culture medium was replaced to remove the cyclodextrin-modified manganese dioxide nanoparticles that were not internalized, and then adamantane-modified manganese dioxide nanoparticles (125 μg / mL) were added and incubated for another 4 hours. After washing away the adamantane-modified manganese dioxide nanoparticles that were not internalized, macrophages loaded with intracellular aggregates of manganese dioxide nanoparticles were obtained. The activity and physiological function of the cells were not affected, and the biological transmission electron microscopy imaging results are shown in Figure 2. Figure 1 shown.

[0058] Example 2

[0059] This embodiment provides an intracellular self-assembled cell motor drug delivery system suitable for inflammation and a preparation method thereof. In this embodiment, DMEM cell culture medium was purchased from HyClone. The carrier cells were selected from macrophages, which were purchased from the ATCC cell bank, and the ferroferric oxide nanoparticles were obtained by the laboratory according to conventional methods.

[0060] Macrophages were cultured and expanded overnight in DMEM medium to make their cell fusion greater than 80%. Cyclodextrin-modified ferroferric oxide nanoparticles (150 μg / mL) were incubated at 37°C for 4 hours. After incubation, fresh culture medium was replaced to remove the cyclodextrin-modified ferroferric oxide nanoparticles that were not internalized, and then adamantane-modified ferroferric oxide nanoparticles (150 μg / mL) were added and incubated for another 4 hours. After washing away the adamantane-modified ferroferric oxide nanoparticles that were not internalized, macrophages carrying intracellular aggregates of ferroferric oxide were obtained.

[0061] This intracellular self-assembled cell motor drug delivery system suitable for inflammation can produce a motor effect under the drive of external magnetic force, thereby achieving rapid infiltration at the disease site.

[0062] Example 3

[0063] This embodiment provides an intracellular self-assembled cell motor drug delivery system suitable for inflammation and a preparation method thereof. In this embodiment, DMEM cell culture medium was purchased from HyClone. The carrier cells were selected from macrophages, which were purchased from the ATCC cell bank, and the platinum nanoparticles were obtained by the laboratory according to conventional methods.

[0064] Macrophages were cultured and expanded in DMEM medium overnight to make their cell fusion greater than 80%. Cyclodextrin-modified platinum nanoparticles (25 μg / mL) were incubated at 37°C for 4 hours. After incubation, fresh culture medium was replaced to remove the cyclodextrin-modified platinum nanoparticles that were not internalized, and then adamantane-modified platinum nanoparticles (25 μg / mL) were added and incubated for another 4 hours. After washing away the adamantane-modified platinum nanoparticles that were not internalized, macrophages carrying intracellular aggregates of platinum nanoparticles were obtained.

[0065] In the treatment of acute pneumonia, the inflammatory tropism of macrophages leads to a large accumulation of the drug delivery system in the lungs. At the same time, due to the characteristic of platinum nanoparticles to produce gas in response to the inflammatory microenvironment, the motor effect produced by the intracellular self-assembled cell motor drug delivery system suitable for inflammation can achieve rapid penetration into the disease site in the treatment of acute pneumonia.

[0066] Example 4

[0067] This embodiment provides an intracellular self-assembled cell motor drug delivery system suitable for inflammation and a preparation method thereof. In this embodiment, DMEM cell culture medium was purchased from HyClone. Neutrophils were selected as carrier cells, and HL60 cells were used as a model of neutrophils purchased from ATCC cell bank. Platinum nanoparticles were obtained by the laboratory according to conventional methods.

[0068] Neutrophils were incubated with cyclodextrin-modified platinum nanoparticles (25 μg / mL) at 37°C for 4 hours. After incubation, fresh culture medium was replaced to remove the cyclodextrin-modified platinum nanoparticles that were not internalized, and then adamantane-modified platinum nanoparticles (25 μg / mL) were added and incubated for another 4 hours. After washing away the adamantane-modified platinum nanoparticles that were not internalized, neutrophils loaded with intracellular aggregates of platinum nanoparticles were obtained.

[0069] In the treatment of acute cerebral infarction, the drug delivery system accumulates at the disease site due to the tropism of neutrophils. At the same time, due to the characteristic of platinum nanoparticles to produce gas in response to the inflammatory microenvironment, the intracellular self-assembled cell motor drug delivery system suitable for inflammation produces a motor effect, resulting in rapid release and deep penetration of the drug, thereby achieving rapid thrombolysis.

[0070] Example 5

[0071] This embodiment provides an intracellular self-assembled cell motor drug delivery system suitable for inflammation and a preparation method thereof. In this embodiment, DMEM cell culture medium was purchased from HyClone, carboxyl-terminally modified polyethylene glycol and ferrocene were purchased from Sigma-Aldrich, nickel-based nanoparticles were homemade in the laboratory, and cucurbituril was a synthetic product of this laboratory. In this example, neutrophils were selected as carrier cells, and neutrophils used HL60 cells as a model, which were purchased from the ATCC cell bank.

[0072] The nickel-based nanoparticles are modified with polyethylene glycol modified with the carboxyl end, and then cucurbituril and ferrocene are grafted onto the nickel-based nanoparticles modified with polyethylene glycol with the carboxyl end, respectively, to form cucurbituril-modified nickel-based nanoparticles and ferrocene-modified nickel-based nanoparticles.

[0073] Neutrophils were cultured in DMEM medium and incubated at 37°C for 8 hours with cucurbituril-modified nickel-based nanoparticles (5 μg / mL). After incubation, fresh culture medium was replaced to remove the cucurbituril-modified nickel-based nanoparticles that were not internalized, and then ferrocene-modified nickel-based nanoparticles (5 μg / mL) were added and incubated for another 8 hours. After washing away the ferrocene-modified nickel-based nanoparticles that were not internalized, neutrophils loaded with nickel-based nanoparticle aggregates were obtained. The activity and physiological function of the cells were not affected.

[0074] Example 6

[0075] This embodiment provides an intracellular self-assembled cell motor drug delivery system suitable for inflammation and a preparation method thereof. In this embodiment, RPMI 1640 cell culture medium was purchased from HyClone, spermine was purchased from Sigma-Aldrich, pillar aromatics and cerium oxide nanoparticles were homemade in this laboratory, and in this example, dendritic cells were selected as carrier cells, and the dendritic cells were purchased from the ATCC cell bank.

[0076] Cerium oxide was modified with pillararene and spermine respectively to obtain pillararene-modified cerium oxide and spermine-modified cerium oxide.

[0077] Dendritic cells were cultured and expanded overnight in RPMI 1640 medium to a cell confluence greater than 80%. Cerium oxide modified with spermine (100 μg / mL) was incubated at 37°C for 2 hours. After incubation, fresh culture medium was replaced to remove the cerium oxide modified with calixarene that was not internalized, and then spermine-modified cerium oxide (100 μg / mL) was added and incubated for another 2 hours. After washing away the cerium oxide modified with columnararene that was not internalized, dendritic cells carrying intracellular aggregates of cerium oxide were obtained. The activity and physiological function of the cells were not affected.

[0078] Comparative Example 1

[0079] This comparative example only provides a common macrophage, which does not contain intracellular aggregates.

[0080] Experimental Example 1: Detecting the movement of the intracellular self-assembled cell motor drug delivery system under gas propulsion

[0081] The motility of macrophages loaded with intracellular aggregates of manganese dioxide provided in Example 1 was evaluated in a PBS solution containing 1% hydrogen peroxide.

[0082] 2-3 drops of the PBS solution were dripped onto a glass slide, and then the macrophages carrying intracellular aggregates of manganese dioxide provided in Example 1 were dispersed in the solution. Under the bright field of a laser confocal microscope, the cell movement was captured using a 4× objective lens magnification. The cell movement and gas production in the cells were obtained by capturing one field of view image every 0.1 seconds, and the total acquisition time was at least 25 seconds. The movement of the motor macrophage driven by gas in the solution is shown in FIG. Figure 2 As shown, from Figure 2 It can be seen that after hydrogen peroxide stimulation, macrophages produce uniform bubbles, and the positions of the bubbles change over time, showing a gas-driven movement trend.

[0083] Experimental Example 2: Targeting Effect in Mice with Acute Pneumonia

[0084] The targeting effects of the drug delivery systems provided in Example 1 and Comparative Example 1 were tested in mice with acute pneumonia.

[0085] The acute pneumonia mouse model was obtained by inhaling LPS in balb / c mice. The motor macrophages in Example 1 labeled with Cy5 and the common macrophages in Comparative Example 1 labeled with Cy5 were respectively injected into the model mice (10 6cells per mouse), 15 minutes after the injection, the model mice were killed, the lung tissues were fixed, and frozen sections of the lung tissues were obtained, with a thickness of 4 microns. The sections were incubated with Hochest33342 for 30 minutes and then imaged under the fluorescence mode of a laser confocal microscope. The infiltration of macrophages provided in Example 1 and Comparative Example 1 in the inflammatory lung tissue was determined by judging the co-localization of the red fluorescence of the Cy5 molecule and the fluorescence of Hoechst33342.

[0086] Test results can be found in Figure 3 ,from Figure 3 It can be seen that in the mouse tissue treated with Comparative Example 1, the red fluorescence was weak, while in the lung tissue treated with motor macrophages, the Cy5-labeled motor macrophages showed obvious red fluorescence, indicating that motor macrophages can enhance their deep infiltration into inflammatory tissues.

[0087] In summary, the intracellular self-assembled cell motor drug delivery system suitable for inflammation provided by the present application utilizes the endocytosis of immune cells to encapsulate the first nanomaterial and the second nanomaterial in immune cells, and the present application realizes the self-assembly of the first nanomaterial and the second nanomaterial in immune cells through the main molecule and the guest molecule, thereby "locking" the nano drug in the immune cell, inhibiting its rapid excretion by the cell, reducing the drug leakage before transport to the target site, avoiding the premature release of the nano drug in living cells, prolonging the circulation time, and improving the targeting of the nano drug to the inflammatory lesion through its homing effect, reducing the clearance of the nano drug by the reticuloendothelial system, and improving the drug delivery efficiency. Further, nano materials that produce gas under the stimulation of the inflammatory microenvironment or move under the drive of external energy are selected as the first nano material and the second nano material. After being loaded on the immune cell, the first nano material and the second nano material are transported to the corresponding disease location, and the immune cell itself can be constructed as a "cell motor" by generating gas or external energy drive. The immune cell drug delivery platform itself is driven by gas or energy to penetrate deep into the lesion, promoting the effective use of drugs and achieving good therapeutic effects. The intracellular self-assembled cell motor drug delivery system suitable for inflammation proposed in the present invention makes full use of the response characteristics of immune cells to inflammatory diseases, and is expected to provide innovative cell therapy for the targeted treatment of major acute diseases such as acute pneumonia.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An intracellular self-assembled cell motor drug delivery system suitable for inflammation, characterized in that: The invention comprises immune cells as carriers and nanometer medicines with therapeutic components, wherein the immune cells comprise macrophages; the nanometer medicines are loaded into the immune cells through the autonomous endocytosis of the immune cells; the nanometer medicines comprise a first nanometer material modified by a main molecule and a second nanometer material modified by a guest molecule, wherein the main molecule and the guest molecule form an assembly in the immune cells, and both the first nanometer material and the second nanometer material are manganese dioxide; the nanometer material is one or more of hollow nanometer particles, nanometer gels and nanometer capsules; the main molecule is cyclodextrin; the guest molecule is adamantane; the modification mode of the main molecule and the guest molecule on the surface of the nanometer material is covalent bond modification; the molar ratio of the main molecule on the surface of the first nanometer material to the guest molecule on the surface of the second nanometer material is 1-10:1-10.

2. The intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 1, characterized in that: The particle size of the nano drug is 100-200nm.

3. The intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 1, characterized in that: The concentration of the nano drug is 5-200 μg / mL.

4. The intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 1, characterized in that: The nano material is hollow or has free drugs loaded in the hollow part.

5. The intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 4, characterized in that: The free drugs include anti-inflammatory drugs or antibiotics.

6. The intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 5, characterized in that: The anti-inflammatory drugs include quercetin, curcumin or atorvastatin.

7. The intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 5, characterized in that: The antibiotics include aspirin or penicillin.

8. The intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 1, characterized in that: The preparation method of the first nanomaterial and the second nanomaterial includes: modifying the main molecule or the guest molecule on the surface of the nanomaterial directly or via a polymer; wherein the polymer modification includes first modifying the surface of the nanomaterial with a polymer, and then connecting the main molecule or the guest molecule to the active site of the polymer via a chemical bond and then grafting it to the surface of the nanomaterial; or, connecting the main molecule or the guest molecule to the active site of the polymer via a chemical bond, and then modifying the surface of the nanomaterial.

9. The intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 8, characterized in that: The polymer includes amino-terminally modified polyethylene glycol, carboxyl-terminally modified polyethylene glycol, polyallylamine phosphate or polyacrylic acid.

10. A method for preparing an intracellular self-assembled cell motor drug delivery system suitable for inflammation as claimed in any one of claims 1 to 9, characterized in that: It includes: The immune cells are mixed with the first nanomaterial modified with the host molecule and the second nanomaterial modified with the guest molecule for incubation.

11. The method for preparing the intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 10, characterized in that: The incubation includes: loading the immune cells with the first nanomaterial and the second nanomaterial by incubation in steps.

12. The method for preparing the intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 10, characterized in that: The immune cells are first incubated and loaded with the first nanomaterial, and then incubated and loaded with the second nanomaterial.

13. The method for preparing the intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 11, characterized in that: The immune cells and the first nanomaterial are mixed in a culture medium for incubation. After incubation, the culture medium is replaced with a fresh one to remove the first nanomaterial that has not been internalized. The second nanomaterial is then added for incubation. After incubation, the second nanomaterial that has not been internalized is removed.

14. The method for preparing the intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 11, characterized in that: The immune cells are loaded with the first nanomaterial and the second nanomaterial for 1-8 hours.

15. The method for preparing the intracellular self-assembled cell motor drug delivery system suitable for inflammation according to claim 11, characterized in that: The immune cells are loaded with the first nanomaterial and the second nanomaterial for 3-5 hours.

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