Pathological responsive nano-carrier as well as preparation method and application thereof

Abstract

The invention relates to a pathologically responsive nano-carrier as well as a preparation method and application thereof, belongs to the technical field of biological medicines, and aims to solve the problems that in the prior art, nano-particles are enriched in livers and kidneys in quantity, the risk of damaging organs and tissues exists, toxic and side effects on patients with spinal cord injury are large, the treatment effect is not obvious and the like. The carrier is a self-assembled nanoparticle which takes PLGA as a skeleton structure, is externally modified with hyaluronic acid and is connected with RVG29 through a diselenide bond. The carrier can load an active substance, such as curcumin, can be used for treating spinal cord injury, and can target spinal cord tissues through intravenous administration, penetrate a blood spinal cord barrier, target CD44 through HA at the early stage of injury, increase immune cell uptake and inhibit inflammatory infiltration and inflammatory cascade. In the late stage of injury, the nerve cell uptake is increased through RVG29 targeted acetylcholine receptor (nAchR), and nerve repair and regeneration are promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a pathology-responsive nanocarrier and a preparation method and application thereof. Background Art

[0002] Spinal cord injury (SCI) often causes severe functional impairment and seriously affects the patient's quality of life. According to statistics, the global incidence of SCI is 10.5 cases per 100,000 people, with nearly 800,000 new cases each year, which has brought a serious economic burden to society and families. Although modern medicine has made great progress, SCI still lacks effective treatment methods.

[0003] The pathological process of spinal cord injury can be divided into two stages: primary injury and secondary injury. Secondary injury is a dynamic and progressive process, including early inflammatory infiltration (inflammation, oxidative stress, demyelination) and late neural regeneration (glial scar formation, axonal growth, remyelination). Microglia are activated and proliferated immediately after spinal cord injury, inducing peripheral immune cells to cross the damaged blood-spinal cord barrier (BSCB) and infiltrate the spinal cord parenchyma, jointly producing high levels of inflammatory factors and reactive oxygen species (ROS), leading to the death of a large number of neurons in the spinal cord, and activating astrocytes to proliferate in large numbers. Such astrocytes have also been shown to continuously damage neurons and hinder neural repair. Regulating the microenvironment after spinal cord injury, inhibiting inflammation, and eliminating adverse factors for neural regeneration are important research directions for the treatment of spinal cord injury.

[0004] Many drugs have been studied for the treatment of spinal cord injury to eliminate factors that inhibit regeneration after injury, or to directly stimulate axonal regeneration and remyelination. However, since these drugs are difficult to cross the blood-spinal cord barrier and accumulate in non-target organs, the effect of the drugs is limited. For example, curcumin has been shown to promote functional recovery after nerve injury by inhibiting inflammation, reducing oxidative stress, and promoting nerve regeneration. However, due to its poor water solubility, curcumin is unstable in the body environment and cannot cross the blood-spinal cord barrier. In order to achieve the therapeutic effect, high-dose oral medication is required, which leads to problems such as drug waste and gastrointestinal side effects.

[0005] As a modular, customizable, and multimodal platform, the physicochemical properties of nanoparticles, especially size, surface chemical structure, and loading material composition, can be customized through non-invasive methods to achieve effective accumulation at the site of injury, thereby limiting off-target effects; controlling release characteristics; increasing interaction with preferred target cells and improving uptake to improve drug delivery to low-proliferation and non-proliferation cells, especially neurons. Although there is a large amount of data in preclinical studies that nanoparticles are effective in repairing spinal cord injuries, nanotherapy has not been used in the clinical treatment of patients with spinal cord injury. The reasons include obstruction of the blood-spinal cord barrier, clearance by the liver and kidneys, and low uptake rate of neural tissue. The most important of these is the blocking effect of BSCB on nanoparticles. Although BSCB function decreases within a few hours after SCI, it recovers its shielding effect on large molecules and some small molecules in the blood about 1 week after injury, which shortens the drug treatment window for SCI patients. At the same time, nanoparticles are almost always enriched in the liver and kidneys, which may pose a potential risk of organ and tissue damage.

[0006] Therefore, it is necessary to improve the tissue and cell targeting of nanoparticles, reduce off-target distribution, and have adaptive capabilities to pathological changes after spinal cord injury to improve drug delivery efficiency. Summary of the invention

[0007] In view of the above analysis, the present invention provides a pathology-responsive nanocarrier and a preparation method and application thereof, which are used to solve the problems in the prior art that nanoparticles are difficult to penetrate the blood-spinal cord barrier, are enriched in the liver and kidneys, are not adapted to pathological changes, have great toxic and side effects on patients with spinal cord injury, and have unclear therapeutic effects.

[0008] In the first aspect, the present invention provides a pathology-responsive nanocarrier, which is a self-assembled nanoparticle with PLGA (polylactic-co-glycolic acid) as the skeleton structure, externally modified with hyaluronic acid (HA), and connected to RVG29 (rabies virus glycoprotein) through a selenide bond.

[0009] Furthermore, the nanocarrier is spherical, with a particle size of 81.7 to 86.3 nm and a surface charge of -16.70 to -15.34 mV.

[0010] In a second aspect, the present invention provides a method for preparing a pathology-responsive nanocarrier, comprising the following steps:

[0011] (1) dissolving RVG29-PEG-Se-Se-PLGA and HA-PLGA in DMSO to obtain an oil phase;

[0012] (2) 1× PBS solution as the aqueous phase;

[0013] (3) mixing the oil phase and the water phase to obtain a mixed solution, dialyzing, and obtaining an oil-in-water pathological responsive nanocarrier.

[0014] Furthermore, in step (1), the mass ratio of RVG29-PEG-Se-Se-PLGA to HA-PLGA is 1:1 to 1:5, and the total mass of RVG29-PEG-Se-Se-PLGA and HA-PLGA to the volume ratio of DMSO is 1 to 20 mg:1 mL.

[0015] Furthermore, in step (3), the volume ratio of the oil phase to the water phase is 1:10 to 1:4.

[0016] Furthermore, in step (3), the dialysis is performed by placing the mixed solution in a dialysis bag and dialyzing it in a 1×PBS solution at 4° C. for 4 to 72 hours.

[0017] Furthermore, in step (3), the mixing is performed by dripping the oil phase into the water phase at a uniform rate of 300 to 500 μL / min under magnetic stirring;

[0018] Furthermore, the speed of the magnetic stirring is 800-1200 rpm.

[0019] In a third aspect, the present invention provides an application of the pathological responsive nanocarrier, wherein the nanocarrier is used in one or more of the preparation of biological products that promote nerve repair, preparation of biological preparations that antagonize neuroinflammation, preparation of drugs for treating diseases related to nerve damage, drug delivery systems, or preparations that target neural tissue.

[0020] Further, the pathological responsiveness is a redox response;

[0021] Furthermore, the nerve injury-related diseases include at least one of spinal cord injury, brain trauma, cerebral infarction, and infectious encephalomyelitis;

[0022] Furthermore, the drug delivery system includes at least one of natural compounds, protein drugs, nucleic acid drugs, polypeptide drugs, and small molecule compounds;

[0023] It should be noted that the nanocarriers described in the present invention can carry one or more drugs to enhance the therapeutic effect or synergistically treat the disease according to the needs of the disease or treatment, thereby achieving better application performance.

[0024] The drug delivery system is an intravenous preparation, a transdermal preparation, an implant or a mucosal patch.

[0025] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0026] (1) The pathologically responsive nanocarriers of the present invention can continuously penetrate the blood-spinal cord barrier and maintain lasting therapeutic effects after spinal cord injury; they have pathological responsiveness and can change cell targeting according to pathological changes to increase drug efficacy; they improve the neural tissue targeting of drugs and reduce peripheral side effects;

[0027] (2) The nanocarriers of the present invention can be loaded with active substances as needed. The active ingredients are selected from one or more of natural compounds, protein drugs, nucleic acid drugs, polypeptide drugs, and small molecule compounds. For example, curcumin solves the problems of poor water solubility, low bioavailability, poor blood-spinal cord barrier permeability, and difficulty in using the nanocarriers for central nervous system damage (CNS) diseases. It can also cooperate with other drugs to enhance the therapeutic effect.

[0028] (3) The carrier described in the present invention is a nanoparticle, which is an excellent drug delivery system after spinal cord injury. It achieves continuous penetration of the blood-spinal cord barrier and neural tissue targeting through the external modification of RVG29 polypeptide, and the surface modification structure changes caused by the ROS-responsive breakage of the diselenide bond cause RVG29 to fall off after penetrating the blood-spinal cord barrier, exerting the immune cell uptake effect of hyaluronic acid, increasing immune cell uptake in the early stage of injury, and inhibiting inflammatory infiltration and inflammatory cascade. In the late stage of injury, the oxidative stress level is reduced, the shedding of RVG29 is reduced, and RVG29 targets the acetylcholine receptor (nAchR) to increase neural cell uptake, promote neural repair and regeneration, and achieve the cooperation of nanoparticles with pathological processes.

[0029] In the present invention, the above-mentioned technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be described in the subsequent description, and some advantages can become obvious from the description, or can be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained through the contents particularly pointed out in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings are only for the purpose of illustrating particular embodiments and are not to be considered limiting of the present invention. Like reference symbols denote like components throughout the drawings.

[0031] Figure 1-1 is the RVG29-PEG-Se-Se-PLGA in Example 1 of the present invention 1 H-NMR spectrum;

[0032] Figure 1-2 is the HA-PLGA in Example 1 of the present invention 1 H-NMR spectrum;

[0033] Figure 2is the infrared spectra of RVG29-PEG-Se-Se-PLGA, HA-PLGA and PLGA in Example 1 of the present invention;

[0034] Figure 3 TEM and DLS images of the carrier prepared in Example 1 of the present invention;

[0035] Figure 4 TEM and DLS images of the carrier prepared in Example 1 of the present invention after being dialyzed against a 1×PBS solution containing 100 μM H2O2, and the BCA protein concentration of the dialyzate;

[0036] Figure 5 The in vivo imaging fluorescence signal images of free Cy5.5, Cy5.5-modified hyaluronic acid nanocarriers, Cy5.5-modified pathological responsive nanocarriers, and Cy5.5-modified pathological responsive nanocarriers blocked by free RVG29 in the in vitro blood-spinal cord barrier model in Experimental Example 2 of the present invention;

[0037] Figure 6 The graph is a graph showing the percentage of Cy5.5-positive cells in resting BV2 cells and activated BV2 cells induced by 100 ng / mL LPS, or in HT22 cells, of the pathological responsive nanocarrier modified with Cy5.5 or the pathological responsive nanocarrier modified with Cy5.5 pretreated with 100 μM H2O2 solution in Experimental Example 3 of the present invention;

[0038] Figure 7 The in vivo fluorescence images of the spinal cord tissue in vitro were obtained after injection of Cy5.5-modified hyaluronic acid nanocarriers, Cy5.5-modified pathological responsive nanocarriers or Cy5.5-modified non-pathological responsive nanocarriers into the tail vein of the spinal cord injury model mice 7 and 28 days after injury in Experimental Example 4 of the present invention.

[0039] Figure 8 The in vivo fluorescence images of the heart, kidney, liver, spleen, and lungs of the mice with spinal cord injury model in Experimental Example 4 of the present invention were obtained after 7 days of tail vein injection of Cy5.5-modified hyaluronic acid nanocarriers, Cy5.5-modified pathological responsive nanocarriers, or Cy5.5-modified non-pathological responsive nanocarriers. 6 hours later;

[0040] Fig. 9 Seven days after the spinal cord injury mouse model was established in Experimental Example 4 of the present invention, Cy5.5-modified hyaluronic acid nanocarriers, Cy5.5-modified pathological responsive nanocarriers or Cy5.5-modified non-pathological responsive nanocarriers were injected into the tail vein, and 6 hours later, the spinal cord tissue sections were stained with anti-F4 / 80 antibody or anti-MAP2 antibody, and the number of Cy5.5-positive cells was observed under a confocal microscope;

[0041] Fig.10 After 28 days of modeling the spinal cord injury mice in Experimental Example 4 of the present invention, Cy5.5-modified hyaluronic acid nanocarriers, Cy5.5-modified pathological responsive nanocarriers or Cy5.5-modified non-pathological responsive nanocarriers were injected into the tail vein, and 6 hours later, the spinal cord tissue sections were stained with anti-GFAP antibody, anti-GAP43 antibody or anti-MAP2 antibody, and the number of Cy5.5-positive cells was observed under a confocal microscope;

[0042] Fig.11 TEM and DLS images of the pathological responsive nanocarrier loaded with curcumin in Experimental Example 5 of the present invention;

[0043] Fig.12 TEM and DLS images of the pathological responsive nanocarrier loaded with curcumin in Experimental Example 5 of the present invention after pretreatment with 100 μM H2O2 solution; and release curves under different conditions;

[0044] Fig.13 H&E staining images of the heart, spleen, lung, kidney, and liver of common BALB / c mice injected with 1×PBS solution or nanocarriers loaded with different curcumin by tail vein in Experimental Example 6 of the present invention after continuous treatment for 28 days;

[0045] Fig.14 This is a graph showing changes in the motor function score (BMS) of mice after 28 days of treatment of the spinal cord injury model using different nanocarriers in Experimental Example 7 of the present invention;

[0046] Fig.15 The graphs of the changes in the footprint experiment of mice and the changes in the Nissl staining of the spinal cord injury area after 28 days of treatment of the spinal cord injury model with different nanocarriers in Experimental Example 7 of the present invention;

[0047] Fig.16 The immunofluorescence staining of spinal cord tissue was used to observe the pathological changes after the spinal cord injury model mice were treated with different nanocarriers in Experimental Example 7 of the present invention. The tissue sections of the injured tissue were stained with anti-F4 / 80 and anti-iNOS antibodies, and the iNOS-positive M1 differentiation rate was observed by confocal microscopy; the number of C3-positive astrocytes was observed using anti-GFAP and anti-C3 antibodies in the injured tissue 28 days ago; and the axon and nerve regeneration were observed by staining with anti-NF200 and anti-GAP43 antibodies. DETAILED DESCRIPTION

[0048] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, wherein the accompanying drawings constitute a part of the present invention and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not used to limit the scope of the present invention.

[0049] A specific embodiment of the present invention discloses a pathology-responsive nanocarrier, which is a self-assembled nanoparticle with PLGA (polylactic acid-glycolic acid block copolymer) as the skeleton structure, hyaluronic acid (HA) modified on the outside, and RVG29 (rabies virus glycoprotein) connected by a selenide bond.

[0050] Compared with the prior art, the carrier of the present invention is a nanoparticle, which can be used as an excellent drug delivery system after spinal cord injury. Through the external modification of RVG29 polypeptide, the continuous penetration of the blood-spinal cord barrier and the targeting of neural tissue are achieved, and the surface modification structure changes caused by the ROS-responsive breakage of the diselenide bond cause RVG29 to fall off after penetrating the blood-spinal cord barrier, play the role of hyaluronic acid in promoting immune cell uptake, and inhibit inflammatory infiltration and inflammatory cascades by increasing immune cell uptake in the early stage of injury. In the late stage of injury, the oxidative stress level is reduced, the shedding of RVG29 is reduced, and RVG29 targets acetylcholine receptors to increase nerve cell uptake, promote nerve repair and regeneration, and realize the cooperation of nanoparticles with pathological processes.

[0051] In the carrier of the present invention, hyaluronic acid is used to achieve the targeting and endocytosis of immune cells, preferably inflammation-related cells such as macrophages and microglia. RVG29 is combined with neurotransmitter receptors to achieve transcerebral microvascular endothelial cell transport, and to achieve the targeting and endocytosis of target cells, targeting tissues or cells expressing neurotransmitter receptors, preferably nerve cells.

[0052] Specifically, the carrier of the present invention can load active ingredients such as curcumin, and can achieve the functions of anti-oxidative stress, anti-inflammatory, anti-apoptosis and promoting nerve regeneration. It can treat diseases related to nerve injury, such as spinal cord injury, brain trauma, cerebral infarction, infectious encephalomyelitis, etc., preferably brain trauma and spinal cord injury, especially spinal cord injury, and can also cooperate with other drugs to enhance treatment.

[0053] In a preferred embodiment, the active substance is selected from one or more of natural compounds, protein drugs, nucleic acid drugs, polypeptide drugs, and small molecule compounds.

[0054] In a more preferred embodiment, the active ingredient in the active substance is one or more of an anti-inflammatory drug, an immunosuppressive drug, and a cell growth factor.

[0055] It should be noted that the nanocarriers described in the present invention can carry one or more drugs to enhance the therapeutic effect or synergistically treat the disease according to the needs of the disease or treatment, thereby achieving better application performance.

[0056] The pathology-responsive nanocarrier of the present invention can achieve responsive drug release or rupture, such as oxidation or reduction response, pH response, temperature-sensitive response, preferably, oxidation or reduction response.

[0057] In a specific embodiment, the nanocarrier is spherical, with a particle size of 81.7 to 86.3 nm and a surface charge of -16.70 to -15.34 mV.

[0058] Another specific embodiment of the present invention discloses a method for preparing a pathology-responsive nanocarrier, comprising the following steps:

[0059] (1) dissolving RVG29-PEG-Se-Se-PLGA and HA-PLGA in DMSO to obtain an oil phase;

[0060] (2) 1× PBS solution as the aqueous phase;

[0061] (3) mixing the oil phase and the water phase to obtain a mixed solution, dialyzing, and obtaining an oil-in-water pathological responsive nanocarrier.

[0062] Wherein, the structural formula of RVG29-PEG-Se-Se-PLGA is shown in formula (I), and the structural formula of HA-PLGA is shown in formula (II):

[0063]

[0064] In formula (I) and formula (II), x:y is 50:50, and n is the degree of polymerization of PEG.

[0065] Among them, RVG29-PEG-Se-Se-PLGA is prepared using the following raw materials:

[0066] PLGA-Se-Se-PEG-MAL was purchased from Xi’an Ruixi Biotechnology Co., Ltd. (R-ROS-0135), PLGA (50:50) with a molecular weight of 15KD and PEG with a molecular weight of 5KD;

[0067] RVG29-Cys was purchased from Shanghai Taopu Biotechnology Co., Ltd. (TP-WW-2626), sequence: YTIWMPENPRPGTPCDIFTNSRGKRASNGC.

[0068] The synthesis method of RVG29-PEG-Se-Se-PLGA is as follows: 20 mg of PLGA-Se-Se-PEG-MAL and 5 mg of RVG29-Cys are dissolved in 5 mL of DMSO and incubated at room temperature for 48 h in the dark on a 30 rpm shaker. The resulting solution is placed in a dialysis bag with a molecular weight cutoff of 20KD and dialyzed in distilled water for 48 h (distilled water is renewed every 12 h). The dialyzed solution is lyophilized to obtain PLGA-Se-Se-PEG-RVG29 powder and stored at -20°C in the dark.

[0069] HA-PLGA was prepared using the following raw materials:

[0070] PLGA-HNS was purchased from Xi'an Ruixi Biotechnology Co., Ltd. (R-PL1183-X), where PLGA (50:50) had a molecular weight of 15KD; hyaluronic acid (HA, 6KD) was purchased from Huaxi Freda Biopharmaceutical Co., Ltd. (Nano Enzymatically cleaved oligomeric hyaluronate).

[0071] HA-PLGA was synthesized by the following method:

[0072] (1) Dissolve 1 g of HA in acetate buffer (30 mL, pH 5.6, 2% w / w), add 1.0 mL of 1,4-diaminobutane (11.4 mmol) under magnetic stirring (1000 rpm), and stir at 50°C for 24 hours. Subsequently, add 0.2 g of sodium cyanoborohydride (3.2 mmol) to the mixture daily under magnetic stirring (1000 rpm) at 50°C for 3 consecutive days. The resulting product was placed in a dialysis bag with a molecular weight cutoff of 2.5 KD and dialyzed in distilled water for 72 h (distilled water was renewed every 24 h). The dialyzed solution was lyophilized to obtain pretreated HA;

[0073] (2) Dissolve 0.5 g PLGA-NHS (0.042 mmol) in 20 mL DMSO, add 0.35 g pretreated HA (0.063 mmol) in step (1), and then add 20 mL N, N-diisopropylethylamine (0.16 mmol). The mixture is reacted at 50 ° C with magnetic stirring (1000 rpm) for 48 h. The resulting solution is placed in a dialysis bag with a molecular weight cutoff of 20KD and dialyzed in distilled water for 48 h (distilled water is renewed every 12 h). The dialyzed solution is freeze-dried to obtain HA-PLGA powder and stored at -20 ° C in the dark.

[0074] In a specific embodiment, the molecular weight of hyaluronic acid (HA) is 6KD. Those skilled in the art can select hyaluronic acid of different molecular weights according to the purpose of the experiment, preferably 1-6KD. The molecular weight of hyaluronic acid needs to be less than the sum of the molecular weight of PEG and the molecular weight of RVG29 (3.5KD); the molecular weight of PLGA (50:50) is 15KD. Those skilled in the art can select PLGA of different molecular weights and ratios according to actual needs, and the molecular weight is preferably greater than or equal to 10KD;

[0075] The molecular weight of PEG connecting RVG29 and the diselenide bond is 5KD; technicians in this field can choose PEG of different molecular weights according to actual needs. The sum of the molecular weight of PEG and the molecular weight of RVG29 (3.5KD) must be greater than the molecular weight of hyaluronic acid.

[0076] The synthesis of RVG29-PEG-Se-Se-PLGA and HA-PLGA in the present invention is explained by taking the above as an example. The synthesis method of RVG29-PEG-Se-Se-PLGA and HA-PLGA is not specifically limited in the present invention. As long as the compounds of the present application can be obtained, they are within the protection scope of the present invention. In the following specific embodiments, RVG29-PEG-Se-Se-PLGA and HA-PLGA are prepared by the above method as an example for explanation. It should be noted that RVG29-PEG-Se-Se-PLGA and HA-PLGA in the present invention can be purchased from commercially available products, such as Xi'an Ruixi Biotechnology Co., Ltd.

[0077] In a specific embodiment, in step (1), the mass ratio of RVG29-PEG-Se-Se-PLGA to HA-PLGA is 1:1 to 1:5, and the total mass of RVG29-PEG-Se-Se-PLGA and HA-PLGA to the volume ratio of DMSO is 1 to 20 mg: 1 mL.

[0078] In a specific embodiment, in step (3), the volume ratio of the oil phase to the water phase is 1:10 to 1:4.

[0079] By using the ratio of the oil phase to the water phase within the above range, the dispersion can be uniform, and the prepared nanocarrier solution has a moderate concentration and is not too dilute.

[0080] In a specific embodiment, in step (3), the dialysis is performed by placing the mixed solution in a dialysis bag and dialyzing it in a 1×PBS solution at 4° C. for 4 to 72 hours.

[0081] In a preferred embodiment, magnetic stirring is used during dialysis, and the stirring rate is 100-200 rpm.

[0082] In a specific embodiment, in step (3), the mixing is performed by dripping the oil phase into the water phase at a uniform rate of 300-500 μL / min under magnetic stirring.

[0083] In a specific embodiment, the speed of the magnetic stirring is 800-1200 rpm.

[0084] Another specific embodiment of the present invention discloses an application of the pathological responsive nanocarrier, wherein the nanocarrier is used in one or more of the following: preparing biological products that promote nerve repair, preparing biological products that antagonize neuroinflammation, preparing drugs for treating diseases related to nerve damage, drug delivery systems, or preparing preparations that target neural tissue.

[0085] In a specific embodiment, the nerve injury-related disease includes at least one of spinal cord injury, brain trauma, cerebral infarction, and infectious encephalomyelitis;

[0086] In a specific embodiment, the drug delivery system includes at least one of a natural compound, a protein drug, a nucleic acid drug, a polypeptide drug, and a small molecule compound;

[0087] The drug delivery system is an intravenous preparation, a transdermal preparation, an implant or a mucosal patch.

[0088] In a specific embodiment, the carrier is added with medically acceptable excipients to form one of intravenous preparations, transdermal preparations, implants, and mucosal patches. Those skilled in the art can implement different administration methods according to the lesion site of the disease, such as intravenous preparations for spinal cord injury, implants, patches, etc. for peripheral nerve injury.

[0089] In a specific embodiment, the use of the carrier in preparing a drug delivery system includes nanoparticles or nanomicroparticles.

[0090] It should be noted that all raw materials in the present invention are commercially available raw materials. The technical solution of the present invention is further explained below in conjunction with specific embodiments.

[0091] Example 1 Preparation of pathology-responsive nanocarriers

[0092] A pathology-responsive nanocarrier of this embodiment is prepared by the following method:

[0093] (1) 5 mg HA-PLGA and 5 mg RVG29-PEG-Se-Se-PLGA were dissolved in 1 mL DMSO and mixed evenly to obtain an oil phase;

[0094] (2) 4 mL of 1× PBS solution was used as the aqueous phase;

[0095] (3) The oil phase is uniformly dripped into the water phase at a rate of 300 μL / min under magnetic stirring at 1200 rpm to obtain a mixed solution, and the mixed solution is placed in a dialysis bag with a molecular weight cutoff of 20 KD, and dialyzed in a 1×PBS solution at 4°C for 48 hours under magnetic stirring at 200 rpm to obtain water-in-oil pathological responsive nanocarriers, and the nanocarriers are freeze-dried for storage, and can also be used directly or diluted with 1×PBS solution as required.

[0096] Take 5 mg RVG29-PEG-Se-Se-PLGA and 5 mg HA-PLGA from the raw materials of this example, dissolve them in DMSO-D6, and collect NMR images at 298K. 1H spectra; RVG29-PEG-Se-Se-PLGA and HA-PLGA 1 H-NMR spectrum, such as Figure 1-1 and Figure 1-2 As shown, specific peaks can be observed in PLGA (methylene, O=C-CH2-O, 4.8 ppm, 5.2 ppm and methyl, CH3, 1.5 ppm), -CH2-se- (3.1 ppm), PEG (methylene, O-CH2-CH2-O, 3.5 ppm) and HA (glycoside, 3.2-4.5 ppm).

[0097] Take 5 mg RVG29-PEG-Se-Se-PLGA, 5 mg HA-PLGA and 5 mg PLGA, mix them with 50 mg potassium bromide powder respectively, grind them thoroughly, put them in a dryer to remove water, press them into tablets, and measure them in an infrared spectrometer. The infrared spectrum is as follows: Figure 2 As shown. Figure 2 As can be seen in the figure, Se-Se (550 cm -1 ) and PLGA: C=O (1750 cm -1 ), CH2, CH3(2914cm -1 ), special peak of PEG: COC (1096cm -1 ).

[0098] The particle size of the carrier prepared in this example was measured by a laser particle size analyzer (DLS) to be 84 nm, the Zeta potential was -16.02±0.68 mV, the dispersion was 0.11, and the transmission electron microscope (TEM) observation showed a smooth spherical morphology (such as Figure 3 shown).

[0099] Example 2

[0100] Preparation of pathology-responsive nanocarriers modified with Cy5.5 fluorescent group

[0101] The preparation method of the pathology-responsive nanocarrier modified with Cy5.5 fluorescent group in this example is the same as that in Example 1, except that, in step (3), 250 μg Cy5.5-NHS is added to the mixed solution and mixed thoroughly.

[0102] Example 3 Preparation of pathology-responsive nanocarriers

[0103] In this example, pathology-responsive nanocarriers with different raw material ratios are prepared. The specific method is the same as that in Example 1, except that in step (1), the mass ratio of RVG29-PEG-Se-Se-PLGA and HA-PLGA is changed to 1:3 or 1:5 under the premise that the total mass of the raw materials remains unchanged at 10 mg. The particle size, dispersity and potential detected by DLS are shown in Table 1. As shown in Table 1, there is no significant difference in particle size and dispersity at the ratios of 1:1, 1:3, and 1:5. In order to increase the RVG29 content to enhance the ability to penetrate the blood-spinal cord barrier, unless otherwise specified, the subsequent examples and test examples all use a mass ratio of 1:1.

[0104] Table 1 Pathological responsive nanocarriers with different raw material ratios

[0105]

[0106] Example 4 Preparation of pathology-responsive nanocarriers

[0107] In this embodiment, pathological responsive nanocarriers with different raw material mass and DMSO solution volume ratios are prepared. The specific method is the same as that in Example 1, except that in step (1), the total raw material mass and DMSO volume ratio are changed under the premise that the mass ratio of HA-PLGA and RVG29-PEG-Se-Se-PLGA remains unchanged at 1:1, and the particle size and dispersion are detected by DLS, as shown in Table 2. As can be seen from Table 2, when the total raw material mass and DMSO volume ratio exceed 10 mg: 1 mL, the dispersion increases significantly, indicating that the uniformity of the nanocarrier deteriorates. In order to obtain a pathological responsive nanocarrier solution with good uniformity and high concentration, unless otherwise specified, the subsequent embodiments and test examples all use a total raw material mass and DMSO volume ratio of 10 mg: 1 mL.

[0108] Table 2 Pathological responsive nanocarriers with different total raw material mass: DMSO volume

[0109]

[0110] Example 5 Preparation of pathology-responsive nanocarriers

[0111] In this embodiment, pathological responsive nanocarriers with different volume ratios of oil phase to water phase were prepared. The specific method was the same as that of Example 1, except that in step (2), the volume of the water phase was changed under the premise that the oil phase was kept at 1 mL. The particle size and dispersion were detected by DLS and shown in Table 3. As can be seen from Table 3, when the volume ratio of oil phase to water phase was 1:4 to 1:10, the particle size of the nanocarrier remained stable and the dispersion maintained a stable low value. In order to obtain a nanocarrier solution with the highest possible concentration, the subsequent embodiments and test examples all adopted an oil phase to water phase volume ratio of 1:4 (mL:mL) unless otherwise specified.

[0112] Table 3 Pathological responsive nanocarriers prepared with different oil phase:water phase volume ratios

[0113]

[0114] Comparative Example 1 Preparation of Sodium Hyaluronate Nanocarrier

[0115] The preparation method of the sodium hyaluronate nanocarrier in this comparative example is the same as that in Example 1, except that an equal mass of HA-PLGA is used instead of RVG29-PEG-Se-Se-PLGA.

[0116] Comparative Example 2 Preparation of non-pathologically responsive nanocarriers

[0117] The preparation method of the non-pathological responsive nanocarrier in this comparative example is the same as that in Example 1, except that an equal mass of RVG29-PEG-PLGA is used instead of RVG29-PEG-Se-Se-PLGA.

[0118] Comparative Example 3: Cy5.5 modified hyaluronic acid nanocarrier

[0119] The preparation method of the Cy5.5-modified hyaluronic acid nanocarrier in this comparative example is the same as that in comparative example 1, except that in step (3), 250 μg Cy5.5-NHS is added to the mixed solution and mixed thoroughly.

[0120] Comparative Example 4 Cy5.5 modified non-pathological responsive nanocarrier

[0121] The preparation method of the Cy5.5-modified non-pathological responsive nanocarrier of this comparative example is the same as that of comparative example 2, except that in step (3), 250 μg Cy5.5-NHS is added to the mixed solution and mixed thoroughly.

[0122] Unless otherwise specified, the pathology-responsive nanocarriers in the following test examples were all prepared by the method of Example 1.

[0123] Experimental Example 1 Redox Response of Pathological Responsive Nanocarriers

[0124] The pathology-responsive nanocarrier prepared in Example 1 was placed in a dialysis bag with a molecular weight cutoff of 20 KD, and dialyzed in a 1×PBS solution containing 100 μM H2O2 at room temperature for 20 minutes under magnetic stirring at 200 rpm. The particle size, charge and morphology were analyzed by DLS and TEM. Figure 4 shown.

[0125] from Figure 4It can be seen that the particle size of the pathological responsive nanocarrier after H2O2 redox treatment measured by DLS is 99nm, the Zeta potential is -21.82±0.74mV, and the dispersion PDI is 0.14. Compared with the particle size before redox treatment, the negative value of Zeta potential is significantly increased, and the dispersion is increased. TEM observation shows a smooth spherical morphology, and some fusion occurs, indicating that the structure of the pathological responsive nanocarrier changes after H2O2 treatment, and the positively charged RVG29 structure falls off. The exposure of HA makes the Zeta potential negative, and the exposure of HA promotes the adhesion of nanocarriers, thereby increasing the measured particle size.

[0126] After dialysis against 100 μM H2O2 in 1× PBS solution, 274 ± 6.2 μg / L of protein components were detected in the dialysate, which was the shed RVG29 peptide, indicating that the pathologically responsive nanocarrier can respond to redox stimulation.

[0127] Based on the above analysis, it can be seen that the pathology-responsive nanocarrier prepared by the present invention has redox responsiveness.

[0128] Experimental Example 2 In vitro blood-spinal cord barrier penetration of pathology-responsive nanocarriers

[0129] 1. Construction of in vitro blood-spinal cord barrier model

[0130] bEnd.3 cells were seeded in transwell chambers (6.5 mm in diameter, 0.4 μm in pore size) at a density of 1×10 4 Cells / chamber, cultured for 72 hours, transepithelial electrical resistance (TEER) was measured using Millicell-ERS voltage-ohmmeter from Millipore, TEER ≥ 200 Ω·cm 2 It is thought to have formed a monolayer of bEnd.3 endothelial cells that mimics the morphology and activity of the blood-spinal cord barrier.

[0131] 2. In vitro blood-spinal cord barrier permeability assay

[0132] The culture medium in the transwell chamber of the in vitro blood-spinal cord barrier model obtained above was replaced with 500 μL of serum-free culture medium containing free Cy5.5, Cy5.5-modified hyaluronic acid nanocarriers (Comparative Example 3), Cy5.5-modified pathology-responsive nanocarriers (Example 2), and Cy5.5-modified pathology-responsive nanocarriers blocked by RVG29 (referring to the addition of free RVG29 to the nanocarriers prepared in Example 2, wherein the nanocarrier concentration is 250 μg / ml and the RVG29 concentration is 50 μg / ml) (the free Cy5.5, Cy5.5-modified hyaluronic acid nanocarriers, Cy5.5-modified pathology-responsive nanocarriers, and Cy5.5-modified pathology-responsive nanocarriers blocked by RVG29 described in the following test examples are the same as those in this test example), and incubated for 4 hours. The transwell chamber was taken out, the surface liquid was blotted with absorbent paper, and it was inverted on a flat surface. The fluorescence signal was detected at a wavelength of 675 / 695 nm using an in vivo imaging (IVIS) system. At the same time, the fluorescence signal of the culture medium in the lower 24-well plate was detected. The results were as follows: Figure 5 shown.

[0133] from Figure 5 It can be seen that the fluorescence signal intensity in the transwell chamber of the Cy5.5-modified pathological responsive nanocarrier group is significantly increased compared with the Cy5.5-modified hyaluronic acid nanocarrier group, indicating that the endothelial cell monolayer composed of bEnd.3 cells has increased the uptake of nanocarriers. At the same time, the fluorescence signal in the lower well plate increased, indicating that the number of nanocarriers penetrating the endothelial cell monolayer increased. This signal increase was significantly weakened after the addition of free RVG29 peptide, because the addition of free RVG29 and RVG29 on the nanocarrier competed for receptors and inhibited their function.

[0134] In summary, pathology-responsive nanocarriers can penetrate the blood-spinal cord barrier in vitro via surface-modified RVG29.

[0135] Experimental Example 3 In vitro cellular uptake of pathology-responsive nanocarriers

[0136] 1. Cellular uptake of pathologically responsive nanocarriers in microglia

[0137] Mouse microglial cell line BV2 cells were cultured at 1×10 5Cells / well were seeded in a 6-well plate, cultured for 12 hours, and then treated with 1×PBS solution or 100 ng / mL LPS for 24 hours, and then replaced with 500 μg Cy5.5-modified pathological responsive nanocarriers in Example 2, or 500 μg Cy5.5-modified pathological responsive nanocarriers in Example 2 treated according to the method of Experimental Example 1. After incubation for 4 hours, BV2 cells were digested with 0.25% trypsin without EDTA into a single cell suspension, centrifuged at 2000 rpm, and resuspended with 1×PBS solution three times. Cy5.5 was detected by flow cytometry. + The percentage of BV2 cells, the results are as follows Figure 6 LPS induction significantly increased the uptake of nanocarriers by BV2, and pretreatment with H2O2 to allow RVG29 to fall off also promoted the uptake of nanocarriers by BV2, indicating that RVG29 interfered with the affinity of HA for BV2.

[0138] 2. Cellular uptake of pathology-responsive nanocarriers in neural cells

[0139] Mouse hippocampal neuronal cell line HT22 cells were cultured at 1×10 5 The cells / well were inoculated in a 6-well plate and cultured for 12 hours, and then replaced with 500 μg of Cy5.5-modified hyaluronic acid nanocarriers in Comparative Example 3, the pathological responsive nanocarriers modified with Cy5.5 in Example 2, or 500 μg of Cy5.5-modified pathological responsive nanocarriers in Example 2 treated according to the method of Experimental Example 1. The Cy5.5 was detected by flow cytometry. + The percentage of HT22 cells, such as Figure 6 As shown, RVG29 modification can make Cy5.5 + HT22 cells increased from 2.63% to 31.3%. Using H2O2 pretreatment to remove RVG29 would cause Cy5.5 + The HT22 cells decreased to 3.13%.

[0140] In summary, pathology-responsive nanocarrier-modified RVG29 can promote the uptake of HT22 cells but interfere with the uptake of BV2 cells. Therefore, the shedding of RVG29 through the selenide bond is necessary for the early treatment of spinal cord injury.

[0141] Experimental Example 4 In vivo distribution and tissue uptake of pathology-responsive nanocarriers

[0142] 1. Construction of spinal cord injury mouse model

[0143] Female BABL / c mice weighing 18-22g and aged 6-8 weeks were anesthetized by intraperitoneal injection of sodium pentobarbital (50mg / kg). T11-12 laminectomy was performed. The spinal cord was then compressed with a 1.3mm diameter percussion rod, with a pressure of 50kdynes for 10 seconds. The wound was then sutured layer by layer. Abdominal massage was performed every day for 1 week after surgery to help urination and defecation.

[0144] 2. In vivo distribution and tissue uptake of pathology-responsive nanocarriers

[0145] (1) On the 7th and 28th days after spinal cord injury modeling, Cy5.5-modified hyaluronic acid nanocarriers (Comparative Example 3), Cy5.5-modified pathological responsive nanocarriers (Example 2) or Cy5.5-modified non-pathological responsive nanocarriers (Comparative Example 4) (6.25 mg / kg) were injected via the tail vein. Mice were killed by cervical dislocation 6 hours after injection, and the whole body was perfused through the left ventricle with 40 mL of ice-cold physiological saline, and then perfused with 20 mL of pre-cooled paraformaldehyde (4%, w / v). The spinal cord and internal organs such as heart, liver, spleen, lung, and kidney were quickly separated and placed in 4 degrees paraformaldehyde (4% w / v) to protect from light. The fluorescence signal was detected at a wavelength of 675 / 695 nm using an in vivo imaging (IVIS) system.

[0146] (2) Spinal cord tissue was obtained and fixed with paraformaldehyde (4%, w / v) for 24 hours. Then, it was dehydrated in a gradient manner (20% and 30% w / v) using sucrose-1×PBS solution, embedded in OTC gel overnight, and sliced ​​into 10 μm thick spinal cord slices using a freezing microtome. The slices were placed at room temperature overnight, fixed with paraformaldehyde (4%, w / v) for 15 minutes, permeabilized with 1×PBS solution containing 0.3% Triton-X 100 for 20 minutes, blocked with 1×PBS solution containing 5% BSA for 2 hours, and then incubated with primary antibody containing 5% BSA in 1×PBS solution at 4 degrees overnight in a wet box. The next morning, the slices were washed 3 times with 1×PBS solution, incubated with fluorescent secondary antibody containing 5% BSA in 1×PBS solution at room temperature for 2 hours in a wet box, and then washed 3 times with 1×PBS solution. Finally, the sections were stained with DAPT-1×PBS solution for 10 minutes, and then anti-fluorescence quencher was added, coverslips were used to seal the sections, and laser confocal fluorescence microscopy was used to observe and photograph the sections. The DAPT laser channel was 405 nm, the AlexaFluor488 laser channel was 488 nm, and the Cy5.5 laser channel was 633 nm.

[0147] The results of the tests in (1) and (2) above are as follows Figure 7-10 shown.

[0148] from Figure 7It can be seen that pathological responsive nanocarriers can penetrate the blood-spinal cord barrier more than hyaluronic acid nanocarriers and non-pathological responsive nanocarriers in the early stage of spinal cord injury, enter the spinal cord tissue and accumulate in the injury site. In the late stage of spinal cord injury, hyaluronic acid nanocarriers can no longer penetrate the blood-spinal cord barrier to enter the spinal cord tissue, while pathological responsive nanocarriers can still penetrate the blood-spinal cord barrier to enter the spinal cord tissue and accumulate in the injury site.

[0149] from Figure 8 It can be seen that RVG29 modification can significantly reduce the enrichment of nanocarriers in the liver and kidneys.

[0150] from Fig. 9 It can be seen that the pathological responsive nanocarriers have the most obvious microglial uptake in the local area of ​​spinal cord injury, which is stronger than the non-pathological responsive nanocarriers, and further stronger than the hyaluronic acid nanocarriers. This shows that although RVG29 can promote the nanocarriers to penetrate the blood-spinal cord barrier and enter the spinal cord tissue in the early stage of spinal cord injury, it will interfere with the enrichment of nanocarriers in the local injury area infiltrated by inflammatory cells; in the early stage of spinal cord injury, no obvious neuronal uptake was observed in each group of nanocarriers, which shows that the early injury environment of spinal cord injury will significantly weaken the uptake ability of neurons. At this time, RVG29 cannot promote neuronal uptake, and retaining RVG29 cannot improve the treatment effect by affecting neurons.

[0151] from Fig.10 It can be seen that pathological responsive nanocarriers and non-pathological responsive nanocarriers were largely taken up by GAP43-positive newborn neurons at the site of spinal cord injury, and were taken up in small amounts by MAP2-positive mature neurons at the remote site of spinal cord injury, while no cell uptake was observed for hyaluronic acid nanocarriers; in the late stage of spinal cord injury, astrocytes did not significantly take up nanocarriers. This indicates that RVG29 is necessary for crossing the blood-spinal cord barrier in the late stage of spinal cord injury, and also has the function of targeting neurons.

[0152] From the above analysis, it can be seen that pathological responsive nanocarriers can penetrate the blood-spinal cord barrier, enter the spinal cord tissue and accumulate in the injury site in both the early and late stages of spinal cord injury, targeting microglia in the early stage of spinal cord injury and targeting neurons in the late stage of spinal cord injury.

[0153] Experimental Example 5 Preparation and Detection of Pathological Responsive Nanocarriers Loaded with Curcumin

[0154] 1. Preparation of curcumin-loaded pathological responsive nanocarriers and determination of encapsulation efficiency and drug loading rate

[0155] (1) The method for preparing curcumin-loaded pathology-responsive nanocarriers is similar to that of Example 1, except that in step (3) of preparing the pathology-responsive nanocarriers, 5-15% (w:w) curcumin is additionally added to the oil phase mixed solution and mixed thoroughly.

[0156] (2) The amount of free curcumin in the supernatant was detected by high performance liquid chromatography. The amount of drug loaded was obtained by subtracting the amount not loaded from the total amount of drug added. The calculation formulas for drug loading rate and encapsulation rate are as follows:

[0157] Encapsulation efficiency (%) = amount of drug in pathology-responsive nanocarrier / total mass of drug input × 100%;

[0158] Drug loading rate (%) = the amount of drug in the pathology-responsive nanocarrier / the total mass of the carrier (the mass of the carrier + the amount of drug loaded) × 100%.

[0159] The HPLC results show that the pathological responsive nanocarrier has a good encapsulation rate for hydrophobic small molecule drugs. As shown in Table 1, among the different curcumin input amounts, it can be seen that the encapsulation rate and drug loading rate of curcumin with a feed ratio of 10% (w:w) are close to the maximum. Unless otherwise specified, the subsequent test examples all use a feed ratio of 10% (w:w).

[0160] Table 4 Encapsulation efficiency and drug loading rate of pathological responsive nanocarriers at different curcumin contents

[0161] Curcumin feed ratio (%, w:w) Encapsulation rate (%) Drug loading rate (%) 5 88.85±3.02 5.45±0.19 10 85.49±2.55 9.02±0.06 15 56.34±2.24 8.96±0.33

[0162] 2. Nano-characterization of curcumin-loaded pathological responsive nanocarriers

[0163] The particle size, charge and morphology of the curcumin-loaded pathological responsive nanocarrier prepared in 1 were analyzed by DLS and TEM. Fig.11 As shown, the average particle size of the pathological responsive nanocarrier loaded with curcumin is 90.2 nm, the zeta potential value is -16±1.21 mV, and the morphology is a smooth regular sphere. Compared with the non-drug-loaded pathological responsive nanocarrier, the particle size is slightly larger, and the negative value of the Zeta potential does not change significantly.

[0164] 3. Pathological response and drug sustained release of curcumin-loaded pathological responsive nanocarriers

[0165] (1) The curcumin-loaded pathological responsive nanocarriers prepared in 1 were placed in a dialysis bag with a molecular weight cutoff of 20 KD, incubated in a 1×PBS solution containing 100 μM H2O2 at room temperature for 20 min under magnetic stirring at 200 rpm, and the particle size, charge and morphology were analyzed by DLS and TEM.

[0166] The results are as follows Fig.12As shown, DLS measured that the particle size of the pathological responsive nanocarrier loaded with curcumin after H2O2 redox treatment was 103.2nm, the Zeta potential was -22±1.74mV, and the dispersion was 0.22. Compared with before redox treatment, its particle size increased significantly, the negative value of Zeta potential increased significantly, and the dispersion increased. Transmission electron microscopy showed a smooth spherical morphology, and some fusion occurred. This is similar to the changes of the pathological responsive nanocarrier without drug loading.

[0167] (2) The pathological responsive nanocarrier loaded with curcumin prepared in 1 was placed in a dialysis bag with a molecular weight cutoff of 20KD at a concentration of 500 μg / mL, and then placed in a 15 mL centrifuge tube containing 1×PBS solution (pH 7.4) containing 1% (v / v) Tween 80, 1×PBS solution (pH 5.8) containing 1% (v / v) Tween 80, and 1×PBS solution (pH 7.4) containing 1% (v / v) Tween 80 and 100 μM H2O2, so that the liquid outside completely submerged the dialysis bag. The dialysis bag was placed in a constant temperature shaker with the shaker parameters set to 37°C and 200 rpm for in vitro simulation of drug release in vivo. Subsequently, 1 mL of the solution outside the dialysis bag was taken at 0, 6, 12, 18, 24, 48, 72, and 96 hours, and 1 mL of the corresponding solution was added. The solution taken out at all time points was measured by HPLC for the concentration of curcumin, and the drug release at each time point was calculated.

[0168] The results are as follows Fig.12 As shown in the figure, 100μM H2O2 and acidic environment can accelerate the release rate of curcumin and increase the maximum release rate of curcumin. Within 24 hours, the release rate of curcumin was 90% under 100μM H2O2 stimulation, 75% under pH5.8, and 55% under pH7.4. In terms of the maximum release rate, the release of curcumin under different conditions was saturated at 72 hours, and the maximum release rate was 92% under 100μM H2O2 stimulation, 91% under pH5.8, and 79% under pH7.4.

[0169] 4. Stability of curcumin-loaded pathological responsive nanocarriers

[0170] The pathological responsive nanocarrier loaded with curcumin prepared in 1 was placed in a dialysis bag with a molecular weight cutoff of 20KD at a concentration of 500 μg / mL and incubated in 1×PBS solution (pH 7.4) at room temperature for 30 days. The size and surface charge of the pathological responsive nanocarrier loaded with curcumin were measured at 0, 1, 3, 10, 20, and 30 days.

[0171] Table 5 Stability of pathological responsive nanocarriers loaded with curcumin

[0172] Time (day) Z-Average(nm) PDI ZetaPotential(mV) 0 92.93 0.09 -16.75±1.35 1 90.54 0.09 -16.58±2.87 3 92.07 0.12 -14.52±1.34 10 91.41 0.14 -17.64±1.44 20 93.82 0.15 -16.25±0.29 30 93.71 0.13 -16.53±1.31

[0173] The results are shown in Table 5. There was no significant change in the size and surface charge of the drug-loaded nanocarriers, indicating good stability. During the one-month observation process, the size was uniform and the morphology was complete.

[0174] Experimental Example 6 Biological Toxicity of Pathological Responsive Nanocarriers Loaded with Curcumin

[0175] 25 healthy BALB / c mice were randomly divided into 5 groups, and the tail vein was injected with 1×PBS solution, hyaluronic acid nanocarrier loaded with curcumin, pathological responsive nanocarrier loaded with curcumin, non-pathological responsive nanocarrier loaded with curcumin, or pathological responsive nanocarrier solution (9 mg / kg) without curcumin loading, once every 3 days, for a total of 10 times. Because curcumin is extremely insoluble in water and cannot be administered intravenously, no curcumin treatment group was set. The mice at the end of treatment were killed by cervical dislocation, and the heart, liver, spleen, lung, and kidney were quickly separated. The tissues were fixed with paraformaldehyde (4%, w / v) for 24 hours, paraffin embedded and sliced, and after dewaxing, repairing, sealing, etc., H&E staining, dehydration, sealing, and microscopic observation and photography were performed.

[0176] The results showed that no obvious visceral organ tissue damage occurred in each group, indicating that the pathological responsive nanocarriers loaded with curcumin had no obvious in vivo toxicity, such as Fig.13 shown.

[0177] Experimental Example 7 Treatment of spinal cord injury by curcumin-loaded pathological responsive nanocarriers

[0178] 25 BALB / c mice were subjected to spinal cord injury modeling, and the grouping and dosing regimen were the same as in Experimental Example 6. Motor function recovery and pathological changes were detected, including:

[0179] (1) The motor function of the spinal cord injury model was evaluated using the Basso mouse scale (BMS) score before surgery and 1, 3, 7, 14, 21, and 28 days after surgery;

[0180] (2) Footprint analysis was performed 28 days after surgery to assess the gait and motor coordination of mice. The mice were painted with blue dye on their forelimbs and red dye on their hindlimbs and placed on a running pad covered with blotting paper to encourage them to walk in a straight line. The spacing between the footprints of the mice's lower limbs and the distance they dragged were observed.

[0181] (3) Mice were killed 28 days after surgery, and the spinal cord tissue was removed and frozen sections were made. Nissel staining was performed to observe the changes in the area of ​​spinal cord injury.

[0182] (4) Mice were killed on the 7th and 28th days after surgery, and spinal cord tissues were obtained and made into frozen sections. Immunofluorescence staining was performed using anti-F4 / 80 antibodies, anti-iNOS antibodies, anti-GFAP antibodies, anti-C3 antibodies, anti-GAP43 antibodies, and anti-NF200 antibodies. Tissue repair was observed using a confocal microscope. The results are as follows: Figure 14-16 shown.

[0183] from Fig.14 It can be seen that the pathological responsive nanocarriers loaded with curcumin showed better motor function recovery than other groups starting from the 7th day and maintained this advantage until the end of the experiment.

[0184] from Fig.15 It can be seen that the pathological responsive nanocarriers loaded with curcumin significantly increased the distance between the lower limb footprints of mice, significantly shortened the dragging distance of the footprints, and had a smaller spinal cord injury area than those in other groups.

[0185] from Fig.16 It can be seen that spinal cord tissue immunostaining showed that the pathological responsive nanocarrier treatment group loaded with curcumin could reduce the M1 polarization of microglia after injury (characterized by iNOS and F4 / 80 double positivity) compared with other groups; it could significantly inhibit GFAP + The formation of glial scar was inhibited and the differentiation of neurotoxic A1 astrocytes (characterized by C3) was significantly inhibited. The GAP43 signal intensity of the regenerated tissue was significantly increased and more dense NF200-positive axon structures appeared.

[0186] In summary, the pathologically responsive nanocarriers loaded with curcumin promoted the recovery of motor function after spinal cord injury, inhibited the inflammatory polarization of microglia in the spinal cord injury area, inhibited the differentiation of toxic A1 astrocytes, and promoted nerve and axon regeneration.

[0187] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A pathology-responsive nanocarrier, characterized in that: The carrier is a self-assembled nanoparticle with PLGA as the skeleton structure, hyaluronic acid modified on the outside, and connected to RVG29 through a selenide bond.

2. A pathology-responsive nanocarrier according to claim 1, characterized in that: The nano-carrier is spherical, with a particle size of 81.7 to 86.3 nm and a surface charge of -16.70 to -15.34 mV.

3. A method for preparing a pathology-responsive nanocarrier, characterized in that: The steps include: (1) dissolving RVG29-PEG-Se-Se-PLGA and HA-PLGA in DMSO to obtain an oil phase; (2) 1× PBS solution as the aqueous phase; (3) mixing the oil phase and the water phase to obtain a mixed solution, dialyzing, and obtaining an oil-in-water pathological responsive nanocarrier.

4. The method according to claim 3, characterized in that In step (1), the mass ratio of RVG29-PEG-Se-Se-PLGA to HA-PLGA is 1:1 to 1:5, and the total mass of RVG29-PEG-Se-Se-PLGA and HA-PLGA to the volume ratio of DMSO is 1 to 20 mg: 1 mL.

5. The method according to claim 3 or 4, characterized in that: In step (3), the volume ratio of the oil phase to the water phase is 1:10 to 1:

4.

6. The method according to claim 3 or 4, characterized in that: In step (3), the dialysis comprises placing the mixed solution in a dialysis bag and dialyzing it in a 1×PBS solution at 4° C. for 4 to 72 hours.

7. The method according to claim 3 or 4, characterized in that: In step (3), the mixing is performed by dripping the oil phase into the water phase at a uniform rate of 300 to 500 μL / min under magnetic stirring.

8. The method according to claim 7, characterized in that The speed of magnetic stirring is 800-1200 rpm.

9. Use of the pathology-responsive nanocarrier according to claim 1 or 2 or the pathology-responsive nanocarrier prepared by any one of the methods 1 to 8, characterized in that: The nanocarrier is used in one or more of the following applications: preparing biological products that promote nerve repair, preparing biological products that antagonize nerve inflammation, preparing drugs for treating diseases related to nerve damage, drug delivery systems, or preparing preparations that target nerve tissue.

10. The use according to claim 9, characterized in that: The nerve injury-related diseases include at least one of spinal cord injury, brain trauma, cerebral infarction, and infectious encephalomyelitis; Preferably, the drug delivery system comprises at least one of natural compounds, protein drugs, nucleic acid drugs, polypeptide drugs, and small molecule compounds; The drug delivery system is an intravenous preparation, a transdermal preparation, an implant or a mucosal patch.

Images