Curcumin-loaded brain-targeted drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses a curcumin-loaded brain-targeted drug delivery system as well as a preparation method and application thereof. The delivery system comprises a carrier macrophage and a curcumin drug, the curcumin nano-drug is prepared by encapsulating curcumin with a lipoic acid modified cyclodextrin conjugate, then macrophages and the curcumin nano-drug are mixed, and co-incubation is carried out, so that the curcumin nano-drug is constructed. The brain-targeted macrophage drug delivery system loaded with curcumin is a multifunctional composite drug with brain targeting property for treatment of cerebral arterial thrombosis. A brain-targeted macrophage drug delivery system loaded with the curcumin nano-drug gathers in cerebral apoplexy tissues, phenotypic transformation of inflammatory microglial cells is promoted, and a pro-inflammatory microenvironment is improved; the oxidative stress pressure of the stroke part is improved; the two effects promote each other, the stroke microenvironment is improved, the functions of neuronal cells are saved, the synergistic nerve protection effect can be achieved in the aspect of treating ischemic cerebral apoplexy, and potential safety hazards are reduced.

Description

Technical Field

[0001] The present invention relates to a drug delivery system, a preparation method and an application thereof, and in particular to a drug delivery system using macrophages as carriers, a preparation method and an application thereof, and belongs to the field of medical technology. Background Art

[0002] Stroke is the second leading cause of death worldwide. Vascular occlusion leads to neuronal apoptosis, blood-brain barrier destruction and brain tissue necrosis, resulting in severe functional impairment in patients (Prabhakaran S., et al, JAMA, 2015, 313, 1451). Current drug treatments for stroke include antithrombotic drugs, thrombolytic drugs and neuroprotective agents, but their efficacy is usually very limited. For example, thrombolytic agents (e.g., rt-PA) can dissolve blood clots and prevent their growth, but the treatment time window is very narrow (G. Stoll, C, et al, Blood 2008, 112, 3555–3562). Neuroprotective factors such as neurotrophic factors that contribute to functional reconstruction of the infarct area cannot be enriched in the ischemic area. Therefore, effective treatments for ischemic stroke are urgently needed.

[0003] Microglia are a type of glial cell distributed throughout the central nervous system (CNS). Microglia are responsible for host defense, inflammation regulation, neurodegeneration and tissue repair, and are the earliest cells activated during CNS injury (H. Kettenmann, et al, Physiol. Rev. 2011, 91, 461–553). Microglia have two polarized phenotypes during ischemia / reperfusion injury, the classic M1 (pro-inflammatory) and the alternative M2 (anti-inflammatory), and their effects are different (Hu X, et al, Stroke 2012, 43, 3063–3474). M1 microglia upregulate pro-inflammatory cytokines, aggravate neuronal damage, and hinder cell repair, while the M2 phenotype confers neuroprotection by producing anti-inflammatory cytokines and promotes brain recovery. Therefore, the selective reprogramming of inflammatory microglia to the M2 phenotype is considered a potential strategy for repair and regeneration of the CNS after stroke. Currently, the anti-inflammatory drug curcumin is used to activate M2 microglia (Lu Y, et al, Adv. Sci. 2019, 6, 1801586).

[0004] The blood-brain barrier (BBB) ​​prevents most small-molecule drugs and macromolecule drugs (such as gene drugs, peptides and protein drugs) from entering the brain. During ischemia and reperfusion in stroke, the integrity of the BBB is partially destroyed, which provides an opportunity for therapeutic drugs to reach the lesion (Liebner S, et al, Acta Neuropathol. 2018, 135, 311). However, since the degree and time of BBB opening vary greatly between individuals with different ischemic severity, the therapeutic effect of nanomedicines based on temporary opening of the BBB is limited. It is worth noting that during ischemia and reperfusion, intercellular adhesion molecule-1 (ICAM-1) and P-selectin are overexpressed on the luminal side of stressed vascular endothelial cells, and these molecules can interact with the corresponding ligands on leukocytes (CD44 and CD11b) recruited from peripheral blood to promote their infiltration into the lesion, and their recruitment is not affected by BBB openness (Yilmaz G, et al, Neuromol. Med. 2010, 12, 193). Literature reports that macrophages are recruited and infiltrated into the ischemic brain within a few hours (Li R, et al, Nano Lett. 2019, 19, 124; Zhang Y, et al, Nano Lett. 2018, 18, 1908). In addition, macrophages themselves have good phagocytic properties and can achieve efficient loading of curcumin drugs. Therefore, the drug delivery system constructed based on macrophages can accurately deliver therapeutic agents to the ischemic part of the brain, promote the phenotypic transformation of microglia, reverse the pro-inflammatory microenvironment, and thus treat ischemic stroke.

[0005] Curcumin (CUR) is a natural compound, a diketone compound extracted from the rhizomes of some plants in the Zingiberaceae and Araceae families. It is one of the best-selling natural edible pigments in the world. As a non-steroidal anti-inflammatory drug, curcumin has chemopreventive properties and has a wide range of pharmacological activities such as anti-inflammatory, antioxidant, lipid-regulating, antiviral, anti-infective, anti-tumor, anti-coagulation, anti-liver fibrosis, and anti-atherosclerosis. It also helps treat drug-resistant tuberculosis; it has a wide range of preventive properties for diseases, and has low toxicity and few adverse reactions. Summary of the invention

[0006] In order to solve the technical defects that existing drugs are difficult to cross the blood-brain barrier and difficult to achieve effective targeted drug delivery in ischemic brain areas, the present invention provides a brain-targeted macrophage drug delivery system loaded with curcumin, a preparation method and application thereof. The drug delivery system prepared by the method of the present invention achieves targeted delivery and aggregation of drugs in the stroke area, improves the stroke microenvironment, and rescues the function of neuronal cells, and can achieve a synergistic neuroprotective effect in the treatment of ischemic stroke.

[0007] The first object of the present invention is to provide a brain-targeted drug delivery system loaded with curcumin, wherein the brain-targeted drug delivery system comprises macrophages and drug curcumin.

[0008] Curcumin has multiple pharmacological activities such as anti-inflammatory and antioxidant activities; cyclodextrin-lipoic acid conjugates have antioxidant activity, and their water-transporting cavities can encapsulate curcumin through host-guest interactions to prepare curcumin nanomedicines. Curcumin nanomedicines can be loaded into cells through the phagocytic action of macrophages, and through the targeting action of the macrophages' own brain stroke microenvironment, they can cross the blood-brain barrier and deliver curcumin nanomedicines to the ischemic stroke site, where they accumulate and activate in the ischemic stroke area. The brain-targeted macrophage drug delivery carrier loaded with curcumin nanodrugs accumulated in the stroke tissue, on the one hand, promotes the phenotypic transformation of inflammatory microglia from the pro-inflammatory M1 type to the immunomodulatory M2 type through the pharmacological effects of curcumin and macrophages, thereby improving the pro-inflammatory microenvironment; on the other hand, the curcumin and lipoic acid-modified cyclodextrin conjugate has antioxidant activity, which can relieve oxidative stress at the stroke site; the two effects promote each other, improve the stroke microenvironment, and rescue the function of neuronal cells, so as to achieve a synergistic neuroprotective effect in the treatment of ischemic stroke and reduce safety risks.

[0009] Wherein, the drug curcumin is a curcumin nano drug, namely, curcumin nanoparticles.

[0010] Particularly, the curcumin nanomedicine is encapsulated in the macrophage cytoplasm.

[0011] In particular, the curcumin nanomedicine is formed by encapsulating the drug curcumin through host-guest interaction of cyclodextrin-lipoic acid conjugate.

[0012] Wherein, the macrophages are mouse-derived macrophages, peritoneal-derived macrophages or bone marrow-derived macrophages; preferably, the mouse-derived macrophages are RAW264.7.

[0013] Curcumin is a loaded drug, and has multiple functions such as active oxygen scavenging, anti-inflammatory, anti-oxidation, and reversal of microglial phenotype; cyclodextrin-lipoic acid conjugate is a skeleton material that encapsulates curcumin through host-guest interaction, and is used for loading the drug curcumin, and the lipoic acid conjugated thereto has antioxidant activity and certain pharmacological effects; macrophages are living cell carriers with cerebral stroke targeting performance in the drug delivery system of the present invention, and they can regulate the inflammatory microenvironment of stroke.

[0014] In particular, the curcumin nanomedicine is prepared according to the following method:

[0015] A) dissolving lipoic acid (LA) in dimethylformamide (DMF) to prepare a DMF solution of LA; then adding N,N'-diisopropylcarbodiimide (DIC) to the DMF solution of LA, stirring evenly for the first time, adding a dimethylformamide (DMF) solution of β-cyclodextrin (β-CD) and 4-dimethylaminopyridine (DMAP), and performing an esterification reaction under stirring to prepare a lipoic acid-modified cyclodextrin conjugate CD-LA;

[0016] B) adding a DMF solution of curcumin to a DMF solution of lipoic acid-modified cyclodextrin conjugate CD-LA, stirring for a second time at 37±5° C., the second stirring time being at least 12 hours; then heating in a water bath, adding pure water to a mixed solution of CD-LA (lipoic acid-modified cyclodextrin conjugate) and curcumin at 70±5° C., continuing a third stirring treatment for at least 4 hours, to obtain a curcumin nano drug.

[0017] Particularly, the concentration of the DMF solution of lipoic acid in step A) is 0.2-1 M, preferably 0.5 M; the molar ratio of lipoic acid to N,N'-diisopropylcarbodiimide is 1:(1-2), preferably 1:1.2.

[0018] In the present invention, DIC is used to activate LA. During the reaction process of DIC activating LA, the theoretical molar ratio is LA:DIC=1:1. To make the reaction proceed more thoroughly, the molar ratio of LA:DIC is 1:1-1:2, preferably 1:1.2.

[0019] In particular, the concentration of the DMF solution of β-cyclodextrin (β-CD) in step A) is 0.1-1M, preferably 0.1M; the molar ratio of lipoic acid LA and β-CD is (4-6):1, preferably 5:1.

[0020] In particular, the reaction time of the esterification reaction in step A) is 24-72h, preferably 48-60h; the reaction temperature is 15-50°C, preferably 30-40°C; and the stirring rate of the esterification reaction is 100-2000rpm.

[0021] In particular, in step A), the molar ratio of lipoic acid to β-cyclodextrin is (3-10):1, preferably (4-6):1, and more preferably 5:1; the molar ratio of lipoic acid to N,N'-diisopropylcarbodiimide is 1:(1-2), preferably 1:1.2.

[0022] Wherein, the 4-dimethylaminopyridine (DMAP) is a catalyst, and its usage amount is a catalytic amount.

[0023] In particular, the first stirring time is at least 30 min, preferably 30-90 min; the first stirring rate is 100-2000 rpm.

[0024] In particular, step A) further comprises adding water to the reaction compound after the esterification reaction, stirring evenly and then centrifuging, collecting the centrifugal precipitate, and obtaining CD-LA.

[0025] In particular, the ratio of the volume of water added after the esterification reaction to the sum of the volumes of the DMF solution of CD and the DMF solution of LA is (20-40):1, preferably 30:1.

[0026] In particular, it also includes adding water to the system after the esterification reaction, mixing and centrifuging, and collecting the centrifugal precipitate; then adding dichloromethane (for removing unreacted lipoic acid) to the centrifugal precipitate, mixing and letting it stand; then centrifuging and collecting the centrifugal precipitate to obtain the lipoic acid-modified cyclodextrin conjugate CD-LA.

[0027] In particular, the method further comprises adding dichloromethane to the centrifugal precipitate, mixing and stratifying, collecting the upper liquid phase, and centrifuging at 10,000 rpm for 3 minutes to collect the precipitate, thereby obtaining the lipoic acid-modified cyclodextrin conjugate CD-LA. ;

[0028] In particular, the mass ratio of curcumin and thioctic acid-modified cyclodextrin conjugate (CD-LA) in step B) is (1-3):10, preferably (1.5-2):10, that is, the mass ratio of curcumin in the DMF solution of curcumin to the mass ratio of CD-LA in the DMF solution of thioctic acid-modified cyclodextrin conjugate CD-LA is (1-3):10, preferably (1.5-2):10.

[0029] In particular, in step B), the prepared lipoic acid-modified cyclodextrin conjugate CD-LA is added to dimethylformamide, heated in a water bath to dissolve, and prepared into the DMF solution of the lipoic acid-modified cyclodextrin conjugate CD-LA; wherein the mass volume concentration of the DMF solution of the lipoic acid-modified cyclodextrin conjugate CD-LA is 5-20 mg / ml, preferably 10 mg / ml; and the water bath heating temperature is 70±5°C.

[0030] In particular, the second stirring rate at 37±5°C in step B) is 1000-2000 rpm for 12-24 hours; the third stirring rate at 70±5°C is 1000-2000 rpm for 4-12 hours.

[0031] In particular, the ratio of the volume of pure water added in step B) to the total volume of the DMF solution of CD-LA and the DMF solution of curcumin is (5-10):1; preferably (8-10):1.

[0032] In particular, the method further comprises subjecting the mixed solution system after the third stirring treatment at 70±5° C. to a first centrifugation treatment to remove unencapsulated curcumin and collect the supernatant; then subjecting the supernatant to a second centrifugation treatment to collect the lower precipitate to obtain the curcumin nano drug, and re-dissolving the mixture with pure water to obtain the curcumin nano drug solution.

[0033] Particularly, the first centrifugal treatment is carried out at a rate of 1000-2000 rpm, preferably 1500 rpm; the second centrifugal treatment is carried out at a rate of 4000-10000 rpm, preferably 5000±100 rpm.

[0034] The DMF solution of curcumin is added to the DMF solution of CD-LA, and stirred at 37±5°C for at least 12 hours, and the lipoic acid-cyclodextrin conjugate encapsulates the drug curcumin through the host-guest interaction. Pure water is added to precipitate the curcumin nanoparticles generated after the lipoic acid-cyclodextrin conjugate encapsulates the drug curcumin.

[0035] In particular, the method further comprises drying the centrifugal precipitate to obtain dry lipoic acid-modified cyclodextrin conjugate CD-LA.

[0036] Another aspect of the present invention provides a method for preparing a brain-targeted drug delivery system loaded with curcumin, comprising the following steps:

[0037] (I) utilizing the host-guest effect of the hydrophobic cavity of cyclodextrin in lipoic acid-cyclodextrin conjugate to encapsulate curcumin to prepare curcumin nanoparticles;

[0038] (II) co-incubating the carrier macrophages with the prepared curcumin nanoparticles, and after the macrophages actively phagocytize the curcumin nanoparticles, washing and collecting the macrophages loaded with the curcumin nanoparticles, thereby obtaining the brain-targeted drug delivery system loaded with curcumin nanoparticles.

[0039] Wherein, the curcumin nanoparticles described in step (I) are prepared according to the following steps:

[0040] IA) dissolving lipoic acid-cyclodextrin compound (CD-LA) in DMF to prepare a DMF solution of CD-LA;

[0041] 1B) adding the DMF solution of curcumin to the DMF solution of CD-LA, stirring and mixing, to prepare a mixed solution of CD-LA and curcumin;

[0042] 1C) heating in a water bath, adding water to the mixed solution of CD-LA and curcumin under stirring;

[0043] ID) After stirring for at least 4 hours, the reaction mixture is centrifuged to collect the precipitate to obtain the curcumin nanoparticles.

[0044] In particular, the lipoic acid-cyclodextrin compound in step (IA) is prepared according to the following steps: N,N'-diisopropylcarbodiimide (DIC) is added to a dimethylformamide (DMF) solution of lipoic acid (LA), and after the first stirring is uniform, a dimethylformamide (DMF) solution of β-cyclodextrin (β-CD) and 4-dimethylaminopyridine are added, stirred, and an esterification reaction is carried out to obtain a lipoic acid-modified cyclodextrin conjugate CD-LA.

[0045] The concentration of the DMF solution of LA is 0.2-1M, preferably 0.5M; the molar ratio of LA to DIC is 1:(1-2), preferably 1:1.2; the concentration of the DMF solution of β-cyclodextrin is 0.1-1M, preferably 0.1M.

[0046] Particularly, the macrophages are mouse macrophages, peritoneal macrophages or bone marrow macrophages.

[0047] In particular, the murine macrophages are RAW 264.7;

[0048] Particularly, during the co-incubation process, the concentration of curcumin in the curcumin nanoparticle drug is 5-30 μg / mL, preferably 10-20 μg / mL.

[0049] Particularly, the co-incubation treatment time is 1-6 hours, preferably 2 hours; and the co-incubation temperature is 37±1°C.

[0050] In particular, macrophages are inoculated in DMEM medium and cultured until the cell density reaches more than 70% (usually 70-95%), the original DMEM medium is discarded, and then DMEM medium containing curcumin is added to perform the co-incubation treatment.

[0051] Particularly, the concentration of curcumin in the DMEM medium containing curcumin is 5-30 μg / mL, that is, 5-30 μg of curcumin is contained in each 1 mL of DMEM medium; preferably 10-20 μg / mL.

[0052] In particular, after co-incubation treatment, the drug loading capacity of macrophages reached (31.7±1.3) mg / g.

[0053] In particular, the concentration of curcumin in the suspension of the brain-targeted drug delivery system loaded with curcumin nanoparticles is greater than 62 μg / mL, preferably 86-212 μg / mL, and further preferably 96.26±9.61 μg / mL.

[0054] In particular, per ml of macrophage suspension (4×10 7 The curcumin concentration in the culture medium (cells / ml) is greater than 62 μg / mL, preferably 86-212 μg / mL, and further preferably 96.26±9.61 μg / mL.

[0055] In particular, after the co-incubation treatment was completed, the cells were washed at least three times with PBS buffer to collect macrophages.

[0056] Another aspect of the present invention provides a brain-targeted drug delivery system loaded with curcumin prepared according to the above method.

[0057] In another aspect, the present invention provides an application of a brain-targeted drug delivery system loaded with curcumin in the preparation of a targeted therapeutic drug preparation for ischemic stroke.

[0058] Among them, the targeted therapeutic drug preparation directly targets the ischemic stroke site, promotes the phenotypic conversion of inflammatory microglia from M1 type to M2 type, reverses the pro-inflammatory microenvironment, improves the stroke microenvironment, rescues the function of neuronal cells, and achieves a synergistic neuroprotective effect in the treatment of ischemic stroke.

[0059] The present invention also provides an application of a brain-targeted macrophage drug delivery system in the preparation of a targeted therapeutic preparation for ischemic stroke. The curcumin nano-drug-loaded macrophage drug delivery system of the present invention can be targeted and accumulated at the site of cerebral ischemia and stroke, and can specifically deliver therapeutic drugs and macrophages to the site of cerebral ischemia.

[0060] The present invention further provides a drug for treating ischemic stroke, wherein the drug for treating ischemic stroke is a curcumin drug loaded in the macrophage drug delivery system, a curcumin nano drug skeleton material lipoic acid-cyclodextrin conjugate, and macrophages themselves. According to tests, the drug for treating ischemic stroke of the present invention can be accumulated in the stroke site, promote the phenotypic conversion of microglia, thereby reversing the pro-inflammatory microenvironment, improving the stroke microenvironment, and saving the function of neuronal cells, and can achieve a synergistic neuroprotective effect in treating ischemic stroke.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] 1. The present invention uses curcumin nano-drugs as primary drug carriers, and utilizes the phagocytic properties of macrophages to load nano-drugs to prepare a macrophage-based live cell drug delivery system. The system has good targeting and high drug loading, with the drug loading reaching (31.7±1.3) mg / g; and retains the characteristics of macrophages' own activity, phenotype and function. After intravenous injection, it can cross the blood-brain barrier and target the ischemic part of the brain;

[0063] 2. The ischemic stroke therapeutic drug of the present invention is a multifunctional complex. Macrophages serve as delivery carriers of nanomedicines and have certain therapeutic effects. The lipoic acid-cyclodextrin conjugate can encapsulate the drug curcumin through host-guest interaction and has good antioxidant activity. The therapeutic effects of the ischemic stroke therapeutic drug on improving the stroke microenvironment and repairing neuronal function come from three aspects: macrophages, curcumin, and lipoic acid-cyclodextrin conjugate.

[0064] 3. The brain-targeted macrophage drug delivery system loaded with curcumin nano-drugs of the present invention gathers in the stroke brain tissue, and realizes the accumulation and activation of macrophage drug delivery carriers in the stroke ischemic area, thereby delivering therapeutic curcumin nano-drugs to the brain ischemic site. The brain-targeted macrophage drug delivery carrier loaded with curcumin nano-drugs gathered in the stroke tissue, on the one hand, promotes the phenotypic conversion of inflammatory microglia through the pharmacological effects of curcumin and macrophages, from the pro-inflammatory M1 type to the immunomodulatory M2 type, and improves the pro-inflammatory microenvironment; on the other hand, the curcumin and lipoic acid modified cyclodextrin conjugate have antioxidant activity, which improves the oxidative stress pressure in the stroke site; the two effects promote each other, improve the stroke microenvironment, and save the function of neuronal cells, which can achieve a synergistic neuroprotective effect in the treatment of ischemic stroke and reduce safety hazards.

[0065] The brain-targeted drug delivery system of the present invention is administered by intravenous injection or tail vein injection. After the brain-targeted drug delivery system loaded with curcumin is prepared into a cell suspension, it is administered by intravenous injection or tail vein injection.

[0066] 4. The method for preparing the brain-targeted macrophage drug delivery system of the present invention has a simple operation process, does not need to rely on complex equipment, and is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0068] Figure 1Schematic diagram of the synthesis of lipoic acid-cyclodextrin conjugate;

[0069] Figure 2 Lipoic acid-cyclodextrin conjugate 1 Schematic diagram of H NMR characterization (solvent: deuterated dimethyl sulfoxide);

[0070] Figure 3 Schematic diagram of the synthesis of curcumin nanomedicine;

[0071] Figure 4 This is the hydrated particle size diagram of curcumin nanomedicine and blank nanoparticles;

[0072] Figure 5 This is the surface Zeta potential result diagram of curcumin nanomedicine and blank nanoparticles;

[0073] Figure 6 The graph shows the test results of ABTS free radical scavenging ability of curcumin, cyclodextrin, CD-LA and curcumin nanomedicine;

[0074] Figure 7 The effect of curcumin nanomedicine on the cell survival rate of macrophages;

[0075] Figure 8 Representative flow cytometry images of curcumin nanomedicine uptake by macrophages;

[0076] Fig. 9 Confocal image of the macrophage drug delivery system loaded with curcumin nanoparticles;

[0077] Fig.10 The flow cytometric scatter plots show the recognition and fusion of microglia with different polarized phenotypes by macrophages not loaded with curcumin nanoparticles, where BV-2:M0 is resting microglia; LPS-induced BV-2:M1 is inflammatory M1-type microglia constructed by lipopolysaccharide stimulation;

[0078] Fig.11 The flow cytometric scatter plots show the recognition and fusion of microglia with different polarized phenotypes by the curcumin nano-drug loaded macrophage drug delivery system, among which BV-2:M0 is a resting microglia; LPS-induced BV-2:M1 is an inflammatory M1 type microglia constructed by lipopolysaccharide stimulation;

[0079] Fig.12 Confocal microscopy images of the transfer of curcumin nanomedicine between the macrophage drug delivery system and mouse microglia BV2;

[0080] Fig.13The flow cytometric images and analysis of the polarization regulation of inflammatory mouse microglia BV2-M1 by the macrophage drug delivery system RN loaded with curcumin nanomedicine and macrophage R0 at the cellular level, wherein A is the expression of CD86 detected by flow cytometry; B is the quantitative analysis of the fluorescence intensity of CD86 in each group in Figure A; C is the expression of CD206 detected by flow cytometry; D is the quantitative analysis of the fluorescence intensity of CD206 in each group in Figure C;

[0081] Fig.14 The cell survival rate bar graph of PC12 cells treated with different concentrations of hydrogen peroxide is used to construct the oxidative stress model of neuronal cells PC12.

[0082] Fig.15 Transwell experiment: Confocal microscopy images of targeted recruitment of curcumin nanomedicine-loaded macrophage drug delivery system RN and macrophage R0 by neuronal cells PC12 under oxidative stress (PC12 normal neuronal cells; PC12+H2O2 neuronal cells under oxidative stress);

[0083] Fig.16 Flow cytometry images of sorting microglia and exogenous macrophages from mouse brain cells (A is the Control group without any staining, B is the CD11b antibody single staining group, C is the control group (Sham), and D is the left brain of the MCAO group);

[0084] Fig.17 This is a flow cytometric image of brain exogenous macrophages isolated from the brain of MCAO model mice with ischemic stroke (CD45 screening of brain exogenous macrophages in the brain);

[0085] Fig.18 Flow cytometry of the macrophage drug delivery system injected into exogenous brain macrophages sorted from mouse brain cells (CD45 screened exogenous brain macrophages in the brain, MCAO was the stroke model group, and RAW-IR was the injected macrophage drug delivery system);

[0086] Fig.19 Representative TTC staining images of brain slices of MCAO model mice treated with different therapeutic agents (Sham negative control group; MCAO ischemic stroke model group; R0 macrophage treatment group; RN curcumin nanodrug-loaded macrophage drug delivery system treatment group);

[0087] Fig. 20The figure is a quantitative analysis of the expression of iNOS antibody (expressed by M1 microglia) and CD206 antibody (expressed by M2 microglia) in the brain of mice with ischemic stroke MCAO model, where: ML / R is the left / right brain of the model MCAO group, RN-L / R is the left / right brain of MCAO model mice after treatment with curcumin nanodrug-loaded macrophage drug delivery system; A is the flow cytometry detection of CD11b in microglia in the brain + CD45 int A is a quantitative analysis of the expression of M1 surface antibody iNOS in the brain; B is the flow cytometry detection of CD11b in microglia in the brain + CD45 int Quantitative analysis of the expression of M2 antibody CD206. DETAILED DESCRIPTION

[0088] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0089] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0090] Example 1 Synthesis and Characterization of Lipoic Acid-Cyclodextrin Conjugate (CD-LA)

[0091] like Figure 1 The lipoic acid-cyclodextrin conjugate CD-LA is mainly prepared by the condensation reaction between the carboxyl group on lipoic acid and the hydroxyl group on cyclodextrin. The specific experimental steps are as follows:

[0092] 1. Weigh 0.5mmol lipoic acid (LA) and dissolve it in 1mL DMF to prepare a DMF solution of LA; then add 3.6mmol N,N'-diisopropylcarbodiimide (DIC), stir and activate at 35°C (usually 15-50°C) for 1h (usually 0.5-1.5h) to prepare a lipoic acid solution (LA solution); the concentration of the DMF solution of LA is 0.5M (usually 0.5-1M); the concentration of LA in the LA solution is 0.5M (usually 0.5-1M); the molar ratio of LA to DIC is 1:1.2 (usually 1:(1-2)); DIC activates the carboxyl group in LA;

[0093] 2. Weigh 0.1 mmol of β-cyclodextrin (β-CD) and dissolve it in 1 mL of DMF. Stir in a water bath at 35°C (usually 15-50°C) for 30 min (usually 10-45 min) to dissolve it, and prepare a cyclodextrin DMF solution (CD DMF solution), wherein the concentration of the cyclodextrin DMF solution is 0.1 M (usually 0.1-1.0 M);

[0094] 3. Drop the prepared DMF solution of β-cyclodextrin (β-CD) into the lipoic acid (LA) solution, add a catalytic amount of 4-dimethylaminopyridine (DMAP), stir, and carry out esterification reaction, wherein the esterification reaction temperature is 35° C. (usually 15-50° C.); the reaction time is 48 h (usually 24-72 h); the molar ratio of β-cyclodextrin to lipoic acid is 1:5 (usually 1:(3-10), preferably 1:(4-6));

[0095] 4-Dimethylaminopyridine was used as a catalyst, and a catalytic amount of DMAP was added to carry out an esterification reaction to generate lipoic acid-modified cyclodextrin conjugate LA-CD.

[0096] 4. After 48 hours of esterification reaction, the reaction system was cooled to room temperature, and then 60 mL of water was added dropwise to the reaction system and stirred to mix evenly; then the mixed system was transferred to multiple 50 mL centrifuge tubes, centrifuged at 3500 rpm for 10 min, and the precipitate in the lower layer of the centrifuge was collected; wherein, the ratio of the volume of the added water to the sum of the volumes of the DMF solution of CD and the DMF solution of LA is 30:1 (usually (20-40):1); the volume of the added water is 20-40 times the volume of the reaction system (DMF phase), preferably 30 times.

[0097] Then, 10 ml of dichloromethane was added to the lower precipitate (to remove unreacted lipoic acid), and the mixture was washed by shaking and centrifuged at 10,000 rpm for 10 min. The lower precipitate was collected and dried in an open air at room temperature to obtain lipoic acid-modified cyclodextrin conjugate, i.e., lipoic acid-cyclodextrin conjugate (CD-LA).

[0098] H NMR spectrum of lipoic acid-cyclodextrin conjugate CD-LA ( 1 H-NMR, d 6 -DMSO) Figure 2 The hydroxyl group of cyclodextrin and the carboxyl group of lipoic acid were successfully connected to obtain the product lipoic acid-cyclodextrin conjugate CD-LA.

[0099] Example 2 Preparation and Characterization of Curcumin Nanomedicine

[0100] The synthesized lipoic acid-cyclodextrin conjugate is used as the skeleton material, and curcumin is loaded through the host-guest interaction to prepare curcumin nanomedicine. β-cyclodextrin is the main body, which has a truncated cone-shaped cavity with relatively low polarity, and can well include guest molecules (such as curcumin, etc.), thereby achieving the purpose of improving the stability, dispersibility and solubility of the guest molecules.

[0101] The preparation of curcumin nanoparticles is mainly through the host-guest interaction between lipoic acid-cyclodextrin conjugate (CD-LA) and curcumin drugs to achieve drug loading and encapsulation, thereby forming curcumin nanoparticles (such as Figure 3 ), the specific steps are as follows:

[0102] 1) Weigh 10 mg of CD-LA and dissolve it in 1 mL of DMF, and dissolve it in a water bath at 70° C. (usually 50-70° C.) to prepare a DMF solution of CD-LA, wherein the mass volume concentration of CD-LA in the DMF solution of CD-LA is 10 mg / ml, that is, 10 mg of CD-LA is dissolved in every 1 ml of DMF (usually 5-20 mg of CD-LA is dissolved in every 1 ml of DMF);

[0103] 2) Adding the DMF solution of curcumin to the DMF solution of CD-LA, stirring overnight at (37±2)°C to prepare a mixed solution of CD-LA and curcumin, wherein the DMF solution of curcumin contains 0.2 ml; the mass ratio of curcumin to CD-LA is 2:10 (usually 1-3:10); the mass volume concentration of the DMF solution of curcumin is 10 mg / mL (i.e., 10 mg of curcumin is dissolved in every 1 ml of DMF, and usually 5-20 mg of curcumin is dissolved in every 1 ml of DMF);

[0104] 3) adding 12 mL of pure water to the mixed solution of CD-LA and curcumin under a water bath condition of 70° C. (usually 50-70° C.), wherein the volume ratio of the added water to the volume of DMF in the mixed solution of CD-LA and curcumin is 10:1 (usually (5-10):1); stirring the mixture at a stirring speed of 1000 rpm (usually 1000-2000 rpm) for 4 h (usually 4-12 h) to allow CD-LA loaded with curcumin to slowly precipitate to form nanoparticles, i.e., formed curcumin nanoparticles, to obtain a mixed system of curcumin nanoparticles;

[0105] 4) After the stirring treatment is completed, the curcumin nanoparticle mixed system is subjected to a first centrifugation treatment, centrifuged at 1500rpm for 40min, the supernatant is the curcumin nano drug solution, and the lower precipitate is the curcumin that has not been successfully encapsulated; the upper liquid phase is collected, and then the second centrifugation is carried out at 5000rpm for 30min, and the precipitate is collected, and the collected precipitate is the curcumin nanoparticles, i.e., the curcumin nano drug; the collected curcumin nano drug is re-dispersed and washed with pure water to remove the residual organic phase, and centrifuged again at 5000rpm for 30min, and the collected lower precipitate is the washed curcumin nano drug.

[0106] Usually, the first centrifugal treatment rate is 1000-2000 rpm; the first centrifugal treatment time is 30-60 minutes; the second centrifugal treatment rate is 4000-10000 rpm; the second centrifugal treatment time is 20-40 minutes.

[0107] Example 2A Preparation of Blank Nanoparticles

[0108] Except that the DMF solution of curcumin was not added to the CD-LA solution in step 2), and pure water was directly added to carry out stirring in step 3), the rest was the same as in Example 2 to obtain blank nanoparticles.

[0109] Example 2B Characterization of curcumin nanoparticles and blank nanoparticles

[0110] 1) The hydrated particle size and surface potential of the curcumin nanoparticles prepared in Example 2 and the blank nanoparticles prepared in Example 2A were measured using a Malvern particle size analyzer. Figure 4 , 5 , among which: the particle size of blank nanoparticles is (139.9±3.5) nm, and the particle size of curcumin nanodrug is (168.2±4.48) nm; the Zeta potential of blank nanoparticles is (-15.9±4.39) mV, and the Zeta potential of curcumin nanodrug is (-35.1±1.55) mV.

[0111] 2) Weigh 4 mg of the freeze-dried curcumin nanodrug, redissolve it in 4 mL of pure water, and use a UV absorbance meter to measure the ultraviolet absorption peak of the curcumin nanodrug to determine the loading amount of curcumin, which is (31.7±1.3) mg / g.

[0112] Example 2C ABTS Experiment and Results

[0113] Reagent 1 and reagent 2 in the ABTS kit (Elabscience) were mixed in a ratio of 1:1 and placed at room temperature for 14 hours to obtain a mother solution of ABTS working solution. The mother solution of ABTS working solution was diluted with 80% ethanol to obtain a ABTS working solution for standby use.

[0114] Then, the test samples were prepared with 80% ethanol solution: curcumin free drug and curcumin nanodrug solution with a curcumin concentration of 200 μg / mL; CD-LA and cyclodextrin solution with a cyclodextrin concentration of 6.4 mg / mL. 10 μL of different test samples were added to the 96-well plate, and then 200 μL of ABTS working solution was added. The absorbance value was detected at 734 nm using an enzyme reader. The experimental results are shown in the figure. Figure 6 .

[0115] Depend on Figure 6 It can be seen that compared with cyclodextrin, the scavenging rate of ABTS in lipoic acid-cyclodextrin conjugates increased, indicating that the modification of lipoic acid increased the antioxidant activity of the nanoparticle carrier material. At the same time, the scavenging ability of ABTS in curcumin nanomedicine was significantly higher than that of curcumin free drug and lipoic acid-cyclodextrin conjugate, indicating that the curcumin free drug and lipoic acid-cyclodextrin conjugate in curcumin nanomedicine synergistically enhanced the free radical scavenging performance and had good antioxidant activity in vivo.

[0116] Example 3A Construction of a macrophage-loaded curcumin drug delivery system

[0117] 1. Effect of curcumin nanomedicine on macrophage activity

[0118] Macrophages (RAW 264.7 cells) were inoculated in 96-well plates and cultured for 16-24 hours until the cell density reached 70-85%. The original DMEM medium was discarded and a DMEM medium solution containing curcumin nanodrugs (i.e., drug solution) was added, wherein the concentration of curcumin was 0, 5, 10, and 20 μg / mL, respectively. After 1, 2, or 4 hours of drug administration and culture, the drug solution was replaced with DMEM medium and cultured for 24 hours. The cells were washed twice with PBS buffer, and then MTS reagent (cell proliferation detection reagent) was added and incubated at 37°C for 30 minutes. The wavelength was detected at 490nm using an enzyme reader, and the cell survival rate was calculated based on the absorbance value. The test results are as follows: Figure 7 ,The cell viability data in the figure show that within the measured concentration range and ,administration time, curcumin nanomedicine has no effect on the viability of macrophages, and the cell survival rate is above 90%.

[0119] 2. Construction of macrophage drug delivery system

[0120] 2-1) Macrophages (RAW 264.7 cells) were inoculated into a 12-well plate containing DMEM medium and cultured for 20 h (usually 16-24 h). When the cell density reached about 90% (usually 80-95%), the original DMEM medium was discarded.

[0121] 2-2) The curcumin nanoparticles (i.e., curcumin nanoparticles) prepared according to the method of Example 2 were dissolved in DMEM culture medium to prepare culture medium containing curcumin nanoparticles with curcumin concentrations of 10 and 20 μg / mL, respectively.

[0122] 2-3) Adding DMEM medium containing curcumin nanoparticles into a 12-well plate to replace the macrophage medium, adding 1.5-3 mL to each well, and co-incubating to construct a macrophage drug delivery system loaded with curcumin nanoparticles; wherein the co-incubation time is 1, 2, 4, or 6 h; the concentration of curcumin in the medium containing curcumin nanoparticles is 10 and 20 μg / mL, respectively;

[0123] The changes in drug loading of macrophages at different incubation times (1, 2, 4, or 6 h) and different incubation concentrations (10 and 20 μg / mL of curcumin concentration) were observed. During the co-incubation process, macrophages engulfed curcumin nanoparticles to form a brain-targeted drug delivery system loaded with curcumin.

[0124] 2-4) After the incubation, the cells were washed three times with PBS, and the cells were scraped off with a cell scraper to collect the cells to obtain the constructed macrophage drug delivery system (RN).

[0125] The macrophages without curcumin nanodrug encapsulation were marked as R0, and the macrophage drug delivery system loaded with nanodrug was marked as RN.

[0126] 3. Evaluation of curcumin-loaded macrophage drug delivery system

[0127] 3-1) The cells collected in 2 (i.e., the constructed macrophage drug delivery system RN) were centrifuged at 1000 rpm for 5 min, and then the centrifuged cells were resuspended in 300 μL of PBS buffer, and the fluorescence intensity of curcumin in the macrophages was analyzed by flow cytometry. Figure 8 . Figure 8 The values ​​on the abscissa show the fluorescence intensity of curcumin in cells.

[0128] Depend on Figure 8 It can be seen that the fluorescence intensity of curcumin in macrophages is time-dependent and dose-dependent. The fluorescence intensity of curcumin in cells gradually moves to the right with the extension of incubation time and the increase of curcumin concentration, indicating that the fluorescence intensity of curcumin in cells gradually increases, that is, the amount of curcumin nanomedicine loaded in cells increases.

[0129] 3-2) In order to determine the distribution of curcumin nanomedicine in macrophages, the fluorescence distribution of curcumin in the macrophage drug delivery system was observed using a confocal microscope. Fig. 9 .

[0130] Depend on Fig. 9 It can be seen that the blue DAPI marks the nuclear region of the macrophages, and the green represents the fluorescence of curcumin, which is mainly distributed in the cytoplasm of macrophages, which can prove that the macrophages have successfully taken up the curcumin nanomedicine.

[0131] Example 3B Construction of macrophage-loaded curcumin drug delivery system and determination of intracellular curcumin concentration

[0132] A macrophage-loaded curcumin drug delivery system was constructed according to the method in Example 3A. Macrophages (RAW264.7 cells) were inoculated in a T75 culture flask filled with DMEM medium and cultured for 20 h (usually 16-24 h). When the cell density reached about 90% (usually 80-95%), the original DMEM medium was discarded.

[0133] The curcumin nano drug (i.e., curcumin nanoparticles) prepared according to the method of Example 2 were dissolved in DMEM culture medium to prepare a culture medium containing curcumin nano drug with a curcumin concentration of 20 μg / mL (usually 5-30 μg / mL); the DMEM culture medium containing curcumin nano drug was added to a T75 culture flask to replace the culture medium of macrophages, and the volume added to each flask was 20 mL (usually 16-25 mL), and co-incubation treatment was performed to construct a macrophage drug delivery system loaded with curcumin nano drug; wherein the co-incubation treatment time was 1, 2, and 4 h, respectively; the concentration of curcumin in the culture medium containing curcumin nanoparticle drugs was 20 μg / mL; after the incubation, the cells were washed three times with PBS, the cells were scraped off with a cell scraper, and the cells were collected to obtain a constructed macrophage drug delivery system (RN);

[0134] The cells were resuspended in physiological saline at a concentration of 4 × 10 7 cells / mL of cell suspension (i.e., RN cell suspension), the cells were broken by a tissue disruptor, and the concentration of curcumin in each mL of the cell suspension was determined by ultraviolet spectrophotometry (the curcumin concentration of the curcumin nanomedicine in the culture medium was 20 μg / mL). The determination results are shown in Table 1 below:

[0135] Table 1 Curcumin loading in macrophage drug delivery system (μg / mL)

[0136]

[0137]

[0138] According to the data in the table, combined with the subsequent administration time and dosage, in the subsequent examples, the concentration of curcumin in the culture medium containing curcumin nanoparticles was 20 μg / mL, and the incubation time was 2 hours, to construct a macrophage-loaded curcumin drug delivery system.

[0139] Example 4 Flow cytometry analysis of recognition and fusion of macrophage drug delivery system to microglia

[0140] 1. Construction of M1-polarized microglial cell model

[0141] In the cerebral ischemia model, microglia are affected by the surrounding microenvironment and transform from a resting state to an M1 phenotype under pathological conditions. In order to evaluate the recognition of macrophages on microglia under pathological conditions, lipopolysaccharide (LPS) was used to stimulate the microglia in vitro to construct an M1 microglia model under pathological conditions.

[0142] Mouse microglial cells BV-2 were passaged and cultured in T25 cell culture flasks for 24 h, the original MEM medium was discarded, and fresh MEM medium containing 1 μg / mL LPS (lipopolysaccharide) was used to stimulate and culture for 24 h to obtain the M1 polarized BV-2 cell model (BV2-M1).

[0143] 2. Fluorescent labeling of microglia

[0144] After 24 hours of stimulation culture, the culture medium in the culture flask was discarded and replaced with a basal culture medium containing 10 μM carboxyfluorescein diacetate succinimidyl ester (CFSE), and culture was continued for 30 minutes to construct fluorescent CFSE-labeled M1 microglia (denoted as: CFSE-BV2-M1).

[0145] A similar method was used for the fluorescent labeling of normal microglia (BV2-M0), i.e., mouse microglia BV-2 were passaged and cultured in T25 cell culture flasks for 24 h, the original culture medium was discarded, and cultured with CFSE-containing culture medium for 30 min to construct fluorescently labeled normal microglia (denoted as: CFSE-BV2-M0).

[0146] 3. Construction of macrophage drug delivery system:

[0147] RAW 264.7 cells were subcultured in T75 cell culture flasks for 16-24 h. When the cell density reached 80-95%, the original DMEM medium was discarded and serum-free basal DMEM medium containing 20 μg / mL IR820-NHS (new indocyanine green active ester) was used for 2 h to construct fluorescently labeled macrophages (denoted as: RAW-IR).

[0148] After the fluorescent staining labeling is completed, centrifugation is performed, and the culture medium containing the staining solution, i.e., the serum-free basic culture of IR820-NHS, is discarded. DMEM complete culture medium containing 20 μg / mL curcumin nanodrug is added to the culture bottle and cultured for 2 hours to construct a fluorescently labeled macrophage drug delivery system (denoted as: RN-IR).

[0149] Normally cultured macrophages without curcumin nanodrugs were labeled as R0, and the macrophage drug delivery system loaded with nanodrugs was labeled as RN. Fluorescently labeled macrophages without curcumin nanodrugs were labeled as R0-IR, and the fluorescently labeled macrophage drug delivery system loaded with curcumin nanodrugs was labeled as RN-IR.

[0150] 4. Cell fusion experiment:

[0151] Collect the microglia in step 2 and the macrophage drug delivery system in step 3, mix the collected macrophages and microglia in the macrophage drug delivery system in a ratio of 1:1, 1:2, and 1:4, respectively, and inoculate them in a 12-well plate. Continue to culture for 30 minutes, 5 hours, and 10 hours, then collect the cells in the well plate, and then use flow cytometry to analyze the proportion of macrophage RAW-IR in microglia to determine the fusion of the two cells.

[0152] The results of flow cytometry analysis were as follows Fig.10 , 11 :

[0153] In the scatter plot, the higher the proportion of macrophage RAW-IR, the better the fusion degree of macrophages and microglia. It can be seen from the figure that as the amount of macrophages increases, the higher the proportion of macrophage RAW-IR in the system, the more macrophages are fused or adsorbed by microglia. Compared with normal microglia, the macrophage drug delivery system has a higher degree of recognition and fusion of pathological M1 microglia after LPS stimulation and activation.

[0154] from Fig.10 It can be seen that as the proportion of microglia increases, the degree of fusion between macrophages and microglia / M1 microglia decreases; as time goes by, the degree of fusion between macrophages and microglia / M1 microglia increases. At the same time and proportion, the degree of fusion of macrophages to M1 microglia is higher than that of unpolarized microglia, which indicates that macrophages have a certain ability to target inflammation.

[0155] and Fig.10 The results are consistent. Fig.11It can be seen that as the proportion of microglia increases, the degree of fusion between the macrophage drug delivery system and microglia / M1 microglia decreases; as time goes by, the degree of fusion between the macrophage drug delivery system and microglia / M1 microglia increases. At the same time and proportion, the degree of fusion of the macrophage drug delivery system with M1 microglia is higher than that with unpolarized microglia, which indicates that the macrophage drug delivery system retains the inflammatory targeting ability of macrophages.

[0156] Example 4A Confocal microscopy analysis of macrophage drug delivery system recognition and fusion of microglia

[0157] The experimental steps were the same as those in Example 4. LPS was used to stimulate microglia to differentiate into pathological M1 type. Macrophages were loaded with curcumin nanomedicine to construct a macrophage drug delivery system. Then, according to the macrophage drug delivery system, RN and microglia were mixed in a ratio of 1:1, and the two cells were plated in a confocal dish. After culturing for 10 hours, DAPI staining was used to determine the position of the cell nucleus, and the fusion of the two cells was observed using a confocal microscope. The results are shown in Figure 2. Fig.12 shown.

[0158] As can be seen from the figure, the red fluorescence of IR-NHS can be marked as macrophages (shown as RAW-IR in the figure), and the blue fluorescence of DAPI marking the cell nucleus can mark all cells. It can be observed from the figure that the green fluorescence representing the curcumin nanomedicine (shown as curcumin in the figure) is present in all cells, indicating that the curcumin nanomedicine can be transferred from macrophages (obvious red IR-NHS fluorescence) to microglia. The red fluorescence of IR-NHS still exists independently in macrophages, instead of being transferred to all cells like the red fluorescence of curcumin nanomedicine. This result shows that macrophages only adhere to the surface of microglia, instead of engulfing microglia to form fused cells.

[0159] The transfer of drugs between the macrophage delivery system and microglia, as well as the adhesion of macrophages and microglia, are beneficial for macrophages to regulate the polarization phenotype of microglia, converting them from the M1 type under pathological conditions to the M2 type with anti-inflammatory and neuroprotective effects, thereby regulating the inflammatory microenvironment of cerebral stroke.

[0160] Example 4B Regulation of the polarization phenotype of microglia by macrophage drug delivery system

[0161] (1) Construction of M1 polarized microglial cell model:

[0162] Same as step 1 of Example 4.

[0163] (2) Fluorescent labeling of microglia:

[0164] Same as step 2 of Example 4.

[0165] (3) Construction of macrophage drug delivery system:

[0166] RAW 264.7 cells were passaged in T75 cell culture flasks and cultured for 24 h. The original culture medium was discarded and complete culture medium containing 20 μg / mL curcumin nanodrug was added to the culture flask and cultured for 2 h to construct a macrophage drug delivery system. Macrophages without curcumin nanodrugs were marked R0, and macrophage drug delivery systems loaded with nanodrugs were marked RN.

[0167] (4) Regulation of microglial polarization:

[0168] The microglia in step (2) and the macrophages in the macrophage drug delivery system in step (3) were mixed evenly at a ratio of 1:1 and 3:1, respectively, and then inoculated into 12-well plates, respectively, and cultured for 24 hours.

[0169] After the co-culture, the cells in the well plate were collected and antibody staining was performed. The antibody used was FITC-labeled anti-CD86 antibody to label M1 microglia, and APC-labeled anti-CD206 antibody to label M2 microglia.

[0170] Flow cytometry was used to analyze the fluorescence intensity of CD86 and CD206 antibodies in CFSE-labeled microglia, and the regulation of the polarization phenotype of microglia by the inflammatory macrophage drug delivery system.

[0171] The results are as follows Fig.13 :After LPS treatment, microglia polarized from the resting state BV2-M0 to the M1 type (BV2-M1), the iNOS antibody content expressed on the cell surface increased significantly, and the cell fluorescence intensity increased significantly. With the addition of the macrophage drug delivery system, the M1-related iNOS antibody expressed on the surface of microglia (BV2-M1) was significantly reduced, and the expression of the M2-related antibody CD206 increased, indicating that the macrophage drug delivery system R0 and RN can reverse the polarization of microglia, transforming them from M1 to M2, thereby promoting the regulation of the stroke microenvironment. Compared with the simple macrophage system R0, the curcumin-loaded macrophage RN showed enhanced reversal of microglia in vitro. It shows that the regulation of microglia phenotype by the macrophage drug delivery system is the joint effect of macrophages and curcumin nanomedicines.

[0172] Example 5 Evaluation of the repair of neuronal cell function PC12 by macrophage drug delivery system at the cellular level

[0173] 1. Construction of PC12 oxidative stress cell model

[0174] Using hydrogen peroxide as a stimulant, a cell model of PC12 oxidative stress was constructed at the cellular level:

[0175] 1×10 rat adrenal pheochromocytoma PC12 cells (highly differentiated) in the logarithmic growth phase were taken 4 Inoculate in a 96-well plate and culture for 24 hours; discard the old 1640 culture medium, add fresh RPMI-1640 culture medium containing 50, 100, 150, 200, 250, 300 μM hydrogen peroxide to treat the cells for 1.5 hours; then replace the above RPMI-1640 culture medium containing hydrogen peroxide with complete culture medium, and culture for another 24 hours; then aspirate the complete culture medium and wash twice with PBS; then add MTS reagent to the well plate and incubate at 37±1℃ for 1-1.5 hours; then use an enzyme marker to detect cell viability. According to the measurement results, draw a cell survival rate bar graph, the results are as follows Fig.14 .

[0176] Normal cultured cells were used as a control. According to the data in the figure, the IC50 value was calculated to be 100 μM. In subsequent experiments, PC12 oxidative stress cells were constructed by treating cells with 100 μM hydrogen peroxide for 1.5 h.

[0177] 2. Evaluation of the migration performance of macrophage drug delivery system on PC12 oxidative stress cells

[0178] The Transwell chamber experiment was used to evaluate the migration performance of the macrophage drug delivery system on PC12 oxidative stress cells.

[0179] 2-1) 12×10 rat adrenal pheochromocytoma PC12 cells (highly differentiated) in the logarithmic growth phase were taken. 4 The cells were inoculated in a 12-well plate and cultured for 24 hours; the old culture medium was discarded, and fresh culture medium containing 100 μM hydrogen peroxide was added to treat the cells for 1.5 hours to construct PC12 oxidative stress model cells.

[0180] 2-2) Take the macrophages in the logarithmic growth phase and inoculate them in a T75 culture flask for 24h, discard the original culture medium, and use a serum-free basal medium containing 5μg / mL CFSE for 0.5h to construct fluorescently labeled macrophages. After the staining is completed, the culture medium containing the staining solution is discarded, and 20mL of complete medium containing 20μg / mL curcumin curcumin nano drug is added to the T75 culture flask, cultured for 2h, and a macrophage drug delivery system is constructed. Macrophages without curcumin nano drug are marked as R0, and the macrophage drug delivery system loaded with nano drug is marked as RN.

[0181] 2-3) Collect the cells in step 2-2) using a cell scraper and centrifuge at a rate of 5×10 4 The cells were seeded at a density of 1 μm in 8 μm Transwell chambers, which were then transferred to 12-well plates as described in step 2-1).

[0182] 2-4) Place the PC12 system containing the macrophage drug delivery system chamber in the incubator, and after culturing for another 24 hours, take out the upper chamber, wash twice with PBS, add 500 μL tissue fixative, and fix at room temperature for 30 minutes. Wash twice with PBS, wipe off the upper cells of the chamber with a cotton swab, and wash twice with PBS. Finally, tear off the membrane of the Transwell chamber, place it on a slide, and observe the number of cells under an upright fluorescence microscope.

[0183] Observation results such as Fig.15 : The CFSE-labeled macrophage delivery system can be clearly seen on the lower membrane of the Transwell chamber, indicating that it has successfully migrated into the PC12 cells. Compared with the normal PC12 cell group, a stronger fluorescence signal can be observed in the PC12 oxidative stress model group, indicating that the oxidative stress microenvironment can promote the recruitment of more macrophage drug delivery systems to aggregate in the stroke microenvironment.

[0184] Example 6 Animal Level Evaluation of Macrophage Drug Delivery System for Treatment of Ischemic Stroke Model Mice

[0185] 1. Preparation of mouse middle cerebral artery occlusion (MCAO) model

[0186] Transient cerebral artery occlusion in mice: The animals were fixed on the operating table in a supine position. The neck was shaved and more hair was removed in the mid-right area. The mice were disinfected with cotton swabs dipped in iodine. The mice were placed on an electric blanket and the temperature was maintained at about (36.5±5)℃. The ventral midline was incised and the superficial fascia was removed. The adipose tissue was cut upward and downward along the digastric muscle to separate the common carotid artery and then the external carotid artery and the internal carotid artery. Then the two ECA branches were dissected, the first branch facing inward (occipital artery OA) and the second branch facing laterally (superior thyroid artery ST). The ST and OA branches of the ECA were cut off by electrocoagulation. The proximal end of the CCA was clamped with a micro-arteriolar clip. The beak (hui) of the distal ECA was dissected. The distal end of the ECA was ligated with 6-0 silk sutures, and the proximal end of the ECA (near the Y-shaped bifurcation) was gently tied with 5-0 silk sutures. First, a V-shaped incision was made in the middle segment of the ECA. Use elbow-tipped forceps to gently insert the suture plug from the incision along the CCA direction and cut off the distal end of the ECA. Clamp the proximal silk thread of the ECA to fix the suture plug in the lumen and prevent bleeding, then gently remove the ICA microvascular clamp. Use micro-tweezers to gently continue to insert the suture plug from the ECA to the ICA until the middle cerebral artery, with a length of about 8-10mm. Stop inserting the thread until you feel a slight resistance. Use 6-0 suture to ligate the blood vessel at the end of the ICA. Give each group drug intervention 5 minutes after infarction. Start timing the infarction time, which is 1 hour.

[0187] Reperfusion operation: Re-anesthetize the mouse, disinfect the incision once with iodine and 70% alcohol, remove the sutures and reopen the incision. Clamp the CCA again with an artery clamp. Gently pull the suture back along the original route to where the ICA is visible. Be careful not to pull it out completely. Clamp the distal end of the ICA with an artery clamp. Pull out the suture from the ECA and tie the ECA stump. Record the reperfusion time. Remove the ICA artery clamp and the CCA artery clamp. Suture the incision and scalp (dermis, fascia, subcutaneous tissue) with 5-0 silk sutures, and wipe the wound with a cotton swab dipped in iodine. Use a simple suture method. Intraperitoneally inject approximately 1 mL of preheated saline. 2. Targeted aggregation of macrophage drug delivery system at the site of cerebral ischemia in the mouse MCAO model

[0188] (1) Establishment of mouse MCAO model. According to the experimental method in (I), a mouse MCAO model was established.

[0189] (2) Construction of macrophage drug delivery system: The macrophage drug delivery system RN-IR (IR820-NHS) was constructed according to the method in Example 3, and the cells were collected by cell scraping, centrifuged, and resuspended in physiological saline to a concentration of 4×10 7 cells / mL of cell suspension (i.e., RN cell suspension), wherein the curcumin concentration is approximately 96.26±9.61 μg / mL.

[0190] (3) After 5 min of recovery and reperfusion in MCAO mice, 0.2 mL of 4 × 10 7 cells / ml RN-IR cell suspension.

[0191] (4) 24 h or 48 h after the tail vein injection, the mice were perfused by heart, the brains of the mice were removed, the left and right brains of the mice were cut and separated, and 2 mL of serum-free DMEM medium was added to each of them for grinding. The ground cell suspension was filtered through a cell sieve. The filtered cells were centrifuged at 1500 rpm for 5 min, and the lower layer of cells was collected.

[0192] (5) Add blocking solution to the lower layer of cells: 600 μL / tube, block at 4°C for 10 min.

[0193] (6) After blocking, antibody staining was performed with PE-F4 / 80, Percp-CD11b, and FITC-CD45 at 4°C for 30 min. 1 mL of PBS was added to each sample to terminate the staining, and the cell pellet was collected by centrifugation at 1500 rpm for 5 min.

[0194] (7) The precipitated cells were fixed with 600 μL of 4% tissue fixative, and the content of the drug delivery system in brain macrophages was analyzed using flow cytometry.

[0195] The analysis results are as follows Figure 16-18 The control group (Sham group) was normal mice; the blank group (Control group) was brain tissue cells that were not stained with antibodies, which were used for flow cytometry to determine the distribution of brain tissue cells; the CD11b single staining group was brain tissue cells stained with CD11b antibodies, which were used to separate all monocytes from brain tissue cells, including microglia in the brain and exogenous monocytes;

[0196] Depend on Fig.16 It can be seen that: in CD11b + Inside the door, CD45 int Microglia in the brain, CD45 hi As can be seen from the figure, there is no exogenous macrophage infiltration in the brain of mice in the control group, while there is CD45 infiltration in the left brain of the MCAO model group. hi The cell population showed that there was infiltration of exogenous macrophages, which proved that the MCAO model was successfully established.

[0197] Depend on Fig.17 It can be seen that as the culture time after administration increases, the infiltration of exogenous macrophages into the mouse brain increases, as shown by CD45 hiThe proportion of cells in the gate gradually increased. In addition, the infiltration of exogenous macrophages in the left brain was more obvious than that in the right brain, and the difference in the amount of exogenous macrophages in the left and right brains increased over time. This data proves that modeling leads to the destruction of the blood-brain barrier, thereby recruiting exogenous macrophages into the brain, and macrophages accumulate in the area of ​​cerebral ischemia.

[0198] Depend on Fig.18 It can be seen that in CD45 hi RAW-IR can be observed inside the door + The positive cell group indicates that the injected macrophage drug delivery system RN has entered the brain. As time goes by, the amount of RN entering the brain increases. Comparing the left and right brains, it was found that the RAW-IR on the left side of the MCAO model + The proportion of cells in the portal area was significantly higher than that in the right brain side. This result indicated that after cerebral ischemia modeling, the macrophage drug delivery system was more likely to accumulate in the ischemic stroke site and achieve targeted accumulation in the stroke lesion site.

[0199] 3. In vivo therapeutic effect of macrophage drug delivery system on ischemic stroke in mouse MCAO model

[0200] (1) Establishment of mouse MCAO model. According to the experimental method in (I), a mouse MCAO model was established.

[0201] (2) Construction of macrophage drug delivery system: Same as step 2 of experiment (II). The construction of macrophage R0 is the same as R0 in step 3 of Example 4, that is, normally cultured macrophages.

[0202] (3) After 5 min of recovery and reperfusion in MCAO mice, 0.2 mL of RN or R0 cell suspension was slowly injected through the tail vein; the cell concentration was 4×10 7 cells / mL, and the concentration of curcumin in RN was 96.26±9.61μg / mL.

[0203] (4) 72 hours after the tail vein injection, the mice were perfused by heart, the brains were removed and placed in the brain trough, and coronal sections were cut at a thickness of 2 mm before and after the visual chiasm. About 5 slices were cut, and the brain slices were removed and immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37°C in the dark for 10 min. Then the brain slices were removed and arranged in order, and photographed with a camera. Fig.19 .

[0204] Depend on Fig.19It can be seen that: after the mouse model was established, there was infarction in the brain area, which was shown as a white area in the figure; when the therapeutic cells (i.e., injection of RN cell suspension) were given for treatment, the area of ​​the white infarction area in the brain became smaller, indicating a good therapeutic effect on ischemic stroke. In different cell treatment groups, macrophages R0 (the same as R0 in 3 of Example 4, that is, normally cultured macrophages) can reduce the area of ​​the infarction area to a certain extent; and the macrophage delivery system RN loaded with curcumin nanodrugs showed the smallest infarction area, indicating that the synergistic therapeutic effect of curcumin and macrophages is significantly improved. 4. Regulation of microglia polarization in the stroke site by the macrophage drug delivery system in the mouse MCAO model

[0205] (1) Establishment of mouse MCAO model. According to the experimental method in (I), a mouse MCAO model was established.

[0206] (2) Construction of macrophage drug delivery system: Same as step 2 of experiment (II).

[0207] (3) After 5 min of recovery and reperfusion in MCAO mice, 0.2 mL of RN cell suspension was injected into the tail vein at a cell concentration of 4 × 10 7 cells / mL, and the concentration of curcumin in RN was 96.26±9.61μg / mL.

[0208] (4) 72 hours after the tail vein injection, the mice were perfused by heart, the brains of the mice were removed, the left and right brains of the mice were cut and separated, and 2 mL of serum-free medium was added to each of them for grinding. The cell suspension was filtered through a cell sieve. The filtered cells were centrifuged at 1500 rpm for 5 min, and the lower layer of cells was collected.

[0209] (5) Add blocking solution to the cells: 600 μL / tube, block at 4°C for 10 min.

[0210] (6) After blocking, antibody staining was performed with the following antibodies: Percp-anti-CD11b, FITC-anti-CD45, PE-anti-iNOS, and APC-anti-CD206. The staining was carried out at 4°C for 30 min. 1 mL of PBS was added to each sample to terminate the staining, and the cells were collected by centrifugation at 1500 rpm for 5 min.

[0211] (7) The precipitated cells were fixed with 600 μL of 4% tissue fixative and the polarization of microglia in the brain was analyzed by flow cytometry. Fig. 20 .

[0212] from Fig. 20It can be seen that compared with the control group (sham group, i.e. normal mouse group), the proportion of iNOS expressed by microglia in the model group (MCAO) was significantly increased, indicating that stroke induced the activation of microglia and turned them into inflammatory M1 cells under pathological conditions. After treatment with the therapeutic agent RN, it was found that the iNOS signal was significantly reduced and the CD206 signal was enhanced, indicating that the microglia in the stroke microenvironment were polarized from the inflammatory M1 type to the therapeutic M2 type.

[0213] This result indicates that the macrophage drug delivery system RN has an effective regulatory effect on inflammatory microglia in the stroke site, which can improve the inflammatory environment of the stroke microenvironment and promote the repair of brain function.

[0214] In summary, the macrophage drug delivery system loaded with curcumin nanoparticles of the present invention can achieve effective targeted aggregation and targeted treatment of ischemic stroke. The macrophage drug delivery system of the present invention is a multifunctional composite drug for the treatment of ischemic stroke. On the one hand, it can be used as a transport carrier for the therapeutic drug curcumin, and on the other hand, it itself has a certain therapeutic effect. The brain-targeted macrophage drug delivery system loaded with curcumin nanoparticles accumulates and activates in the ischemic area of ​​stroke, which can promote the phenotypic conversion of microglia from the pro-inflammatory M1 polarized phenotype to the anti-inflammatory M2 polarized phenotype, thereby reversing the pro-inflammatory microenvironment, improving the stroke microenvironment, and saving the function of neuronal cells, achieving a synergistic and neuroprotective effect in the treatment of ischemic stroke.

[0215] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A brain-targeted drug delivery system loaded with curcumin, characterized in that: Including carrier macrophages and drug curcumin.

2. The brain-targeted drug delivery system according to claim 1, characterized in that: The drug curcumin is a curcumin nano drug.

3. The brain-targeted drug delivery system according to claim 2, characterized in that: The curcumin nano drug is prepared according to the following method: A) adding N,N'-diisopropylcarbodiimide to a DMF (dimethylformamide) solution of lipoic acid LA, stirring evenly for the first time, adding a DMF (dimethylformamide) solution of β-cyclodextrin and 4-dimethylaminopyridine, stirring, and performing an esterification reaction to obtain a lipoic acid-modified cyclodextrin conjugate CD-LA; B) Adding the DMF solution of curcumin to the DMF solution of lipoic acid-modified cyclodextrin conjugate CD-LA, stirring at 37±5° C. for at least 12 h; then heating in a water bath, adding pure water to the mixed solution of CD-LA and curcumin at 70±5° C., and continuing stirring for at least 4 h to obtain a curcumin nanomedicine.

4. The brain-targeted drug delivery system according to claim 3, characterized in that: The concentration of the DMF solution of lipoic acid in step A) is 0.2-1M, preferably 0.5M; the molar ratio of lipoic acid LA and β-CD is (4-6):1, preferably 5:1; the molar ratio of lipoic acid to N,N'-diisopropylcarbodiimide is 1:(1-2), preferably 1:1.

2.

5. The brain-targeted drug delivery system according to claim 3, characterized in that: In the preparation process of the curcumin nanomedicine in step B), the mass ratio of curcumin to lipoic acid modified cyclodextrin conjugate CD-LA is (1-3):10, preferably (1.5-2):

10.

6. A method for preparing a brain-targeted drug delivery system loaded with curcumin, characterized in that: The steps include: (I) utilizing the host-guest effect of the hydrophobic cavity of cyclodextrin in lipoic acid-cyclodextrin conjugate to encapsulate curcumin to prepare curcumin nanoparticles; (II) co-incubating the carrier macrophages with the prepared curcumin nanoparticles, and after the macrophages actively phagocytize the curcumin nanoparticles, washing and collecting the macrophages loaded with the curcumin nanoparticles, thereby obtaining the brain-targeted drug delivery system loaded with curcumin nanoparticles.

7. The preparation method according to claim 6, characterized in that: The macrophages described in step (II) are mouse macrophages, peritoneal macrophages or bone marrow macrophages.

8. A brain-targeted drug delivery system loaded with curcumin, characterized in that: Prepared according to the method as claimed in claim 6 or 7.

9. Application of a brain-targeted drug delivery system loaded with curcumin in the preparation of a targeted therapeutic drug preparation for ischemic stroke.

10. The use according to claim 9, characterized in that: The targeted therapeutic drug preparation directly targets the ischemic stroke site, promotes the phenotypic conversion of inflammatory microglia from M1 to M2, reverses the pro-inflammatory microenvironment, improves the stroke microenvironment, rescues the function of neuronal cells, and achieves a synergistic neuroprotective effect in the treatment of ischemic stroke.

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