CN109939082A - K can be activatedATPThe preparation method and application of the metastatic encephaloma targeting drug delivery system in channel - Google Patents

Abstract

The invention discloses one kind can activate K_ATPThe preparation method and application of the metastatic encephaloma targeting drug delivery system in channel.It is basic carrier with biodegradable high molecular material, inside loads ATP sensitive potassium channel (K_ATP) agonist, surface modification metastatic encephaloma targeting ligand, the administration nano-drug administration system is based on K_ATPIncrease to the double strategy and suggestions of across cell and cell bypass that channel activation mediates the permeability of blood-brain tumor barrier, administration nano-drug administration system in metastatic encephaloma region and enters tumour cell across the barrier, accumulation in mediate blood circulation.The administration nano-drug administration system can load anti-tumor drug, realize the drug delivery for the treatment of concentration；Administration nano-drug administration system can be effectively reduced in the non-specific accumulation in normal brain activity region simultaneously.Its method is simple, operability and economic benefit with higher.

Description

K can be activatedATPThe preparation method and application of the metastatic encephaloma targeting drug delivery system in channel

Technical field

The invention belongs to nano biological pharmaceutical technology fields, and in particular to one kind can activate K on blood-brain tumor barrier_ATPIt is logical The preparation method and applications of the metastatic encephaloma targeted nanometer drug delivery system in road.

Background technique

Metastatic encephaloma is the most common intracranial tumors and the most common neurological complication of systemic tumor；In recent years Come the continuous innovation of the continuous development and diagnostic techniques treated with systemic tumor, the disease incidence and diagnosis of metastatic encephaloma Constantly rise；Due to grade of malignancy height and locating intracerebral position, metastatic encephaloma lethality is high.Clinical preferred operative treatment at present The characteristics of with radiotherapy due to the more lesions of metastatic encephaloma and wellability, cannot effectively tumor eradication and have great side effect, this But also traditional chemotherapy and novel gene biological treatment become research direction.However, blood-brain barrier and blood brain tumor screen The presence of barrier limits delivering of most drugs to intracerebral, therefore develops the drug delivery system of efficient metastatic encephaloma targeting System becomes research hotspot now.For brain tumor, fast-growth and the new vessels generation of tumour cell cause brain swollen The blood-brain barrier in tumor region is by partial destruction, to form blood-brain tumor barrier.Endothelial cell gap can on blood-brain tumor barrier Anti-tumor drug and nanoparticle (nanoparticles, NPs) is improved to be accumulated in brain by bypassing diffusion and passive target respectively and swell Tumor, but it is not sufficient to realize the drug delivery for the treatment of concentration.Have been reported that administration nano-drug administration system loads blood-brain barrier regulatory factor, Such as Rui Jiadesong, the openable close link enhancement passive target efficiency of blood-brain barrier, however the big spininess of used regulatory factor It to blood-brain barrier, while enhancing brain tumor region drug accumulation, can also enhance the accumulation of its normal brain activity region, lead to malicious secondary work With raising, limits these administration nano-drug administration systems and further convert and apply.

Therefore, in view of the above-mentioned problems, it is necessary to propose further solution.

Summary of the invention

Object of the present invention is to for presently, there are cancer target administration nano-drug administration system be difficult to overcome blood-brain tumor barrier Problem, K on blood-brain tumor barrier can be activated by providing one kind_ATPThe preparation method of the metastatic encephaloma targeted nanometer drug delivery system in channel, One kind obtained can activate K on blood-brain tumor barrier_ATPThe metastatic encephaloma targeted nanometer drug delivery system in channel, can be based on across thin Born of the same parents and the double approach specificity of cell bypass overcome blood-brain tumor barrier.

The technical scheme is that

K on blood-brain tumor barrier can be activated_ATPThe metastatic encephaloma targeted nanometer drug delivery system in channel, with biodegradable High molecular material is basic carrier, and inside loads K_ATPChannel agonist, surface modification metastatic encephaloma targeting ligand.

It is of the invention another solution is that

K on blood-brain tumor barrier can be activated_ATPThe preparation method of the metastatic encephaloma targeted nanometer drug delivery system in channel, the party Method includes the following steps:

(1) high molecular material is weighed, is dissolved in organic solvent, oily phase is formed；

(2) by anti-tumor drug and K_ATPChannel agonist is soluble in water, forms water phase；

(3) water phase is added dropwise in the oily phase of vortex, ultrasonic emulsification forms the emulsion of water-in-oil type；

(4) emulsion is added dropwise in the outer aqueous phase of vortex again, ultrasonic emulsification forms the emulsion of water-in-oil-in water；

(5) emulsion is poured into volatilization water phase rapidly, is stirred overnight volatilization and removes ethyl acetate, it is mixed forms nanoparticle Suspension；

(6) the nanoparticle suspension is purified by high speed centrifugation, obtains the unmodified nanoparticle of purification；

(7) by the unmodified nanoparticle ultrasonic disperse in the phosphate buffer that pH value is 7.3, surface modification function Connection molecule removes excessive function connects molecule by high speed centrifugation after reaction 1h at room temperature, and precipitating obtains first nanometer Grain；

(8) by the first nanoparticle ultrasonic disperse in the phosphate buffer that pH value is 7.3, pass through function connects point Son is in surface modification targeting ligand, and high speed centrifugation removes excessive targeting ligand after reacting 1h at room temperature, and precipitating obtains second and receives The grain of rice；

(9) by the second nanoparticle ultrasonic disperse Yu Shuizhong, high speed centrifugation, precipitating obtain final nanoparticle；

(10) the final nanoparticle is dispersed in water, freeze drying protectant freeze-drying is added, blood brain can be activated by obtaining one kind K on tumor barrier_ATPThe metastatic encephaloma targeted nanometer drug delivery system in channel.

Further, high molecular material described in step (1) is in polylactic acid, poly lactic-co-glycolic acid or polycaprolactone Any one；The organic solvent is any one in ethyl acetate or methylene chloride.

Further, anti-tumor drug described in step (2) is adriamycin, taxol or antitumor siRNA, DNA, egg Any one in white, polypeptide, antibody, the adriamycin are the product after triethylamine desalination, the high molecular material with it is described The mass ratio of anti-tumor drug is 100:1-100:50.

Further, K described in step (2)_ATPChannel agonist is in minoxidil, minoxidil sulfate or diazoxiide Any one；The minoxidil is product of the minoxidil sulfate after triethylamine desulfurization acid, wherein the sulfuric acid minot The molar ratio of you and the triethylamine is 1:0.5-1:10, the high molecular material and the K_ATPThe mass ratio of channel agonist For 100:2.5-100:30.

Further, the outer aqueous phase in step (4) is the poly-vinyl alcohol solution of 2.5%-5%.

Further, the volatilization water phase in step (5) is the poly-vinyl alcohol solution of 0.01%-0.3%.

Further, function connects molecule described in step (7) is succinyl Asia by-polyethylene glycol-maleimide, institute Stating the molar ratio that function connects molecule is reacted with the high molecular material is 0.05:1-1.6:1.

Further, targeting ligand described in step (8) be metastatic encephaloma targeting ligand, integrin receptor target ligand, It is Her2 targeting ligand, TfR targeting ligand, any one in 1 targeting ligand of LDH receptor related protein Kind；The targeting ligand and the molar ratio of polyethylene glycol are 5:100-100:100；TCEP is added during the reaction simultaneously to open The molar ratio of disulfide bond in targeting ligand, the TECP and targeting ligand is 50:1-1:1.

Further, freeze drying protectant described in step (10) is trehalose, the matter of the trehalose and high molecular material Amount is than being 10:1-1:1.

K on blood-brain tumor barrier is activated prepared by aforesaid way_ATPThe metastatic encephaloma targeted nanometer drug delivery system in channel In the tumor cell preparations that can be applied to targeting human body source or animal origin.

The present invention provides one kind can activate K on blood-brain tumor barrier_ATPThe metastatic encephaloma targeted nanometer drug delivery system in channel Preparation method, the advantage is that inside contains K using biodegradable high molecular material as basic carrier_ATPChannel agonist Agent, surface modification metastatic encephaloma targeting ligand, so that K on blood-brain tumor barrier can be activated by constructing one kind_ATPThe administration system in channel System, by up-regulation blood-brain tumor barrier endothelial cell on caveolin expression, lower tight junction protein expression, based on across The double approach of cell and cell bypass penetrate blood-brain tumor barrier, in mediate blood circulation administration nano-drug administration system across the barrier, store Product enters metastatic encephaloma cell using the targeting ligand of administration nano-drug administration system surface modification in metastatic encephaloma region；It will give simultaneously Medicine system loading anti-tumor drug, it can be achieved that treatment concentration drug delivery, can avoid the wind accumulated in normal brain activity region of drug Danger, it is highly-safe, while preparation method of the present invention is simply controllable, operability and economic benefit with higher.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, required use in being described below to embodiment Attached drawing be briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for this For the those of ordinary skill of field, without any creative labor, it can also be obtained according to these attached drawings other Attached drawing.Wherein,

Fig. 1 is that one kind of the present invention can activate K on blood-brain tumor barrier_ATPThe metastatic encephaloma targeted nano in channel is given The building of medicine system and its internal behavior schematic diagram；

Fig. 2 be hyaluronic acid (HA, nanoparticle name in indicated with H) hydrogen spectrum；

Fig. 3 is the hydrogen spectrum of the hyaluronic acid (HA-S-SH) of sulfhydrylation；

Fig. 4 is that single load adriamycin (DOX) of the present invention and altogether packet adriamycin and rice are characterized by dynamic light scattering method Promise you (MS, nanoparticle name in indicated with M) two kinds of administration nano-drug administration systems (H-NPs/DOX and M@H-NPs/DOX) grain Diameter figure；

Fig. 5 is to characterize the administration of the nanometer of packet adriamycin and minoxidil altogether of the present invention by dynamic light scattering method to be The grain-size graph of system (M@H-NPs/DOX) whether there is or not two kinds of trehalose after freeze-drying resuspension；

Fig. 6 is that one kind of the present invention of dialysis characterization can activate K on blood-brain tumor barrier_ATPThe brain metastes in channel Release profiles of the minoxidil contained in tumor targeted nanometer drug delivery system under the conditions of 7.4 pH；

Fig. 7 is that one kind of the load adriamycin of the present invention of dialysis characterization can activate K on blood-brain tumor barrier_ATPIt is logical Release profiles of adriamycin under the conditions of 7.4 pH in the metastatic encephaloma targeted nanometer drug delivery system in road；

Fig. 8 is the free adriamycin of quantitatively characterizing and whether there is or not each administration nano-drug administration system (NPs/ of the load adriamycin of HA modification DOX and H-NPs/DOX) intake on metastatic encephaloma cell, the concentration of adriamycin is 3 μ g/mL, administration time 90min；

Fig. 9 is the free adriamycin of quantitatively characterizing and whether there is or not each administration nano-drug administration system (NPs/ of the load adriamycin of HA modification DOX and H-NPs/DOX) intake on normal brain microvessel endothelial cells in vitro, the concentration of adriamycin is 3 μ g/mL, and administration time is 90min；

Figure 10 is the metastatic encephaloma targeted nanometer drug delivery system of the different HA degree of modification of quantitatively characterizing in metastatic encephaloma cell On intake, the concentration of adriamycin is 3 μ g/mL, administration time 90min；

Figure 11 is that the free adriamycin of quantitatively characterizing exists with the nanoparticle (NPs/DOX and H-NPs/DOX) for containing adriamycin Whether there is or not the intake in the presence of minoxidil on metastatic encephaloma cell, the concentration of adriamycin is 3 μ g/mL, and administration time is 90min；

The free adriamycin of Figure 12 quantitatively characterizing and the nanoparticle (NPs/DOX and H-NPs/DOX) for containing adriamycin are having Without the intake in the presence of minoxidil on normal brain microvessel endothelial cells in vitro, the concentration of adriamycin is 3 μ g/mL, administration time For 90min；

Figure 13 is the free adriamycin of MTT experiment characterization and the nanoparticle (NPs/DOX and H-NPs/DOX) for containing adriamycin To the toxicity of metastatic encephaloma cell in the presence of various concentration minoxidil；

Figure 14 is that system is administered in single metastatic encephaloma targeted nano for carrying adriamycin of the present invention of fluorescence microscope characterization System (H-NPs/DOX) co-cultures in the presence of whether there is or not minoxidil in metastatic encephaloma cell and normal brain microvessel endothelial cells in vitro thin Intake figure on born of the same parents, the concentration of adriamycin are 3 μ g/mL, and administration time 90min, scale is 50 μm；

Figure 15 is that the preincubate of characterization carries metastatic encephaloma targeted nanometer drug delivery system (the M@H-NPs) administration of minoxidil not The metastatic encephaloma targeted nanometer drug delivery system (H-NPs/DOX) of adriamycin is carried on metastatic encephaloma cell with quantitative determination after the time Intake, administration time 90min, the concentration of adriamycin is 3 μ g/mL；

Figure 16 is the metastatic encephaloma targeted nanometer drug delivery system (M@H-NPs) of the load minoxidil of preincubate difference feed ratio 30h, quantitative determination carry metastatic encephaloma targeted nanometer drug delivery system (H-NPs/DOX) taking the photograph on metastatic encephaloma cell of adriamycin It takes, administration time 90min, the concentration of adriamycin is 3 μ g/mL；

Figure 17 is the blank administration nano-drug administration system (H-NPs) of Laser Scanning Confocal Microscope characterization, free minoxidil and contains The administration nano-drug administration system (M@H-NPs) of minoxidil is to in-vitro simulated blood-brain barrier and blood-brain tumor barrier claudin-5 table The effect reached, red fluorescence indicate that claudin-5, scale are 25 μm；

Figure 18 is in administration nano-drug administration system (M@H-NPs) animal level for containing minoxidil of Laser Scanning Confocal Microscope characterization To the effect that normal brain activity region and metastatic encephaloma region claudin-5 are expressed, red fluorescence indicates that blood vessel, white fluorescent indicate Claudin-5, scale are 25 μm；

Figure 19 is the blank administration nano-drug administration system (H-NPs) of fluorescence microscope characterization and the metastatic encephaloma for containing minoxidil The effect picture of blood-brain tumor barrier is overcome in targeted nanometer drug delivery system (M@H-NPs) animal level, blue-fluorescence expression contains Hoechst33342, indicate that nanoparticle enters brain parenchym area, white arrow through blood-brain tumor barrier at yellow arrows meaning Indicate that nanoparticle is absorbed by tumour cell at meaning, scale is 50 μm；

Figure 20 is the free adriamycin of fluorescence microscope characterization and contains each administration nano-drug administration system of adriamycin normal small The distribution map of mouse brain area domain macroscopic scale and microscopic scale, scale are 50 μm；

Figure 21 be fluorescence microscope characterization free adriamycin and contain adriamycin each administration nano-drug administration system it is small in lotus knurl The distribution map of mouse brain area domain macroscopic scale, scale 1.5mm；

Figure 22 is that the average light of adriamycin of the Figure 21 through Image J quantitative normal brain activity region and metastatic encephaloma region is close Degree；

Figure 23 be fluorescence microscope characterization free adriamycin and contain adriamycin each administration nano-drug administration system it is small in lotus knurl The distribution map of the submicroscopic level in mouse brain area domain, scale are 310 μm；

Figure 24 is that the average light of adriamycin of the Figure 23 through Image J quantitative normal brain activity region and metastatic encephaloma region is close Degree；

Figure 25 be fluorescence microscope characterization free adriamycin and contain adriamycin each administration nano-drug administration system it is small in lotus knurl The distribution map of mouse brain area domain microscopic scale, scale are 50 μm；

Figure 26 is that the average light of adriamycin of the Figure 25 through Image J quantitative normal brain activity region and metastatic encephaloma region is close Degree；

Figure 27 is the blank nanoparticle, free minoxidil and the metastatic encephaloma target for containing minoxidil of MTT experiment characterization To nanoparticle (M@H-NPs) to the toxicity of metastatic encephaloma cell, time 48h；

Figure 28 is the nanoparticle of the load minoxidil of MTT experiment characterization, free adriamycin and with carrying adriamycin and minot altogether Your metastatic encephaloma targeted nano granule (M@H-NPs/DOX) is to the toxicity of metastatic encephaloma cell, time 48h；

Figure 29 is that free adriamycin and one kind of the present invention can activate K on blood-brain tumor barrier_ATPThe brain metastes in channel Tumor targeted nanometer drug delivery system (M H-NPs/DOX) gives medicament to the treatment survival rate of metastatic encephaloma model mice, adriamycin Amount is every kilogram of mouse 4mg adriamycin；

Figure 30 is that free adriamycin and metastatic encephaloma targeted nanometer drug delivery system of the present invention (M@H-NPs/DOX) are given The weight figure of metastatic encephaloma model mice after medicine.

Specific embodiment

Minoxidil sulfate (Minoxidil sulfate, MS), as K_ATPChannel activator is a kind of clinically to commonly use Blood-pressure drug.Some researches show that K on MS specific activation blood-brain tumor barrier endothelial cell and tumour cell_ATPIt is logical Road raises the expression of endothelial cell caveolin, lowers the expression of tight junction protein, is based on across cell and two kinds of cell bypass Approach enhances the permeability of blood-brain tumor barrier, while avoiding its toxic side effect to normal brain activity.However, there is no at present report or Person's invention, utilizes K on MS specific activation blood-brain tumor barrier_ATPChannel come construct can efficiently penetrate blood-brain tumor barrier to Medicine system and the drug therapy for being applied to metastatic encephaloma.

Administration nano-drug administration system has drugloading rate height, medicament slow release, circulation time length and protection drug compared with free drug The advantages that avoiding degradation.Administration nano-drug administration system can pass through nanoscale effect, surface modification targeting ligand, internal load function point Son realizes the combination that passive target, active targeting and targeting are adjusted.CD44, LDH receptor related protein 1, TfR, Her2, integrin receptor etc. over-express on breast cancer patients with brain transfer oncocyte；The specificity of these receptors Ligand such as CD44 ligand water solubility, biodegradable micromolecule hyaluronic acid (hyaluronic acid, HA), can be used for Administration nano-drug administration system is modified, metastatic encephaloma targeting is realized by active targeting after penetrating blood-brain tumor barrier.

The present invention selects the biodegradable high molecular material of synthesis as basic carrier, ultrasonic emulsification-solvent volatilization Method preparation is internal to load K_ATPThe nanoparticle of channel agonist, nanoparticle surface amino abundant are sub- by double-functional group succinyl Amine-polyethylene glycol-maleimide (NHS-PEG-MAL) connection sulfhydrylation metastatic encephaloma targeting ligand, to construct one Kind can activate K on blood-brain tumor barrier_ATPThe drug delivery system in channel can overcome blood brain based on across cell and the double approach of cell bypass Tumor barrier, mediate blood circulation in nanoparticle across the barrier, accumulation in metastatic encephaloma region, utilize nanoparticle surface modification Targeting ligand enter metastatic encephaloma cell；Simultaneously by drug delivery system loading anti-tumor drug, it can be achieved that the drug for the treatment of concentration Delivering.The present invention is expected to improve the metastatic encephaloma targeting efficiency of the drug delivery system based on nanoparticle.

Lower mask body, which introduces one kind, can activate K on blood-brain tumor barrier_ATPThe metastatic encephaloma targeted nanometer drug delivery system in channel Structure, preparation method and application.

The present invention, which provides one kind, can activate K on blood-brain tumor barrier_ATPThe metastatic encephaloma targeted nanometer drug delivery system in channel, It is basic carrier with biodegradable high molecular material, inside loads K_ATPChannel agonist, the targeting of surface modification metastatic encephaloma Ligand, while the administration nano-drug administration system can load anti-tumor drug, realize the drug delivery for the treatment of concentration, which is characterized in that by High molecular material, polyethylene glycol, metastatic encephaloma targeting ligand, K_ATPChannel agonist and anti-tumor drug composition.

Wherein, the molar ratio of the high molecular material and polyethylene glycol is 1:0.05-1:1.6, the polyethylene glycol point Son amount is 2000,3500,5000, and the molar ratio of the metastatic encephaloma targeting ligand and polyethylene glycol is 5:100-100:100, The high molecular material and K_ATPThe mass ratio of channel agonist is 100:2.5-100:30, the high molecular material and anti- The mass ratio of tumour medicine is 100:1-100:50；

The high molecular material is polylactic acid (molecular weight be 0.3-1.5 ten thousand, 1.5-3 ten thousand, 3-5.5 ten thousand, 5.5-9 ten thousand), poly- (molecular weight is 0.1-1.5 ten thousand, 1.5-2.4 ten thousand, 2.4-3.8 ten thousand, 3.8-5.3 ten thousand, 5.3-7 ten thousand, 7-8.8 to lactic-co-glycolic acid 50/50) or polycaprolactone ten thousand, the ratio of lactide and glycolide is 75/50 or (molecular weight is 0.4-4 ten thousand, 4-10.6 ten thousand)；

The polyethylene glycol is succinimide-polyethylene glycol-maleimide；

The metastatic encephaloma targeting ligand, in the embodiment of the present invention by taking hyaluronic acid (HA) as an example, with poly- second two The molar ratio of alcohol is 5:100-100:100；

The K_ATPChannel agonist is minoxidil, minoxidil sulfate or diazoxiide；With sulphur in the embodiment of the present invention For sour minoxidil, contained in administration nano-drug administration system in the form of minoxidil after triethylamine desulfurization acid, wherein sulfuric acid The molar ratio of minoxidil and triethylamine is 1:0.5-1:10；

The anti-tumor drug is anti-tumor drug, including adriamycin, taxol or antitumor siRNA, DNA, egg White, polypeptide, antibody etc., in the embodiment of the present invention by taking adriamycin as an example.

Targeted nanometer drug delivery system of the invention be suitable for target human body source and animal origin metastatic encephaloma cell and Other cells.

One kind can activate K on blood-brain tumor barrier_ATPThe preparation method of the metastatic encephaloma targeted nanometer drug delivery system in channel, Include:

Step 1: weighing the high molecular material of synthesis, be dissolved in organic solvent, forms oily phase；

In one embodiment, which specific as follows can execute: weighing the high molecular material of synthesis, be dissolved in organic molten In agent, oily phase is formed.Wherein, the high molecular material be polylactic acid (molecular weight is 0.3-1.5 ten thousand, 1.5-3 ten thousand, 3-5.5 ten thousand, 5.5-9 ten thousand), poly lactic-co-glycolic acid (molecular weight be 0.1-1.5 ten thousand, 1.5-2.4 ten thousand, 2.4-3.8 ten thousand, 3.8-5.3 ten thousand, 5.3- 70000, the ratio of 7-8.8 ten thousand, lactide and glycolide be 75/50 or 50/50), (molecular weight is 0.4-4 ten thousand, 4- to polycaprolactone 10.6 ten thousand), and weighed amount is 50mg, and the organic solvent is ethyl acetate or methylene chloride.

Step 2: drug is soluble in water, form water phase；

In one embodiment, which specific as follows can execute: by K_ATPChannel agonist and anti-tumor drug are dissolved in In water, water phase is formed.Wherein, K_ATPChannel agonist includes minoxidil, minoxidil sulfate or diazoxiide；The high score Sub- material and K_ATPThe mass ratio of channel agonist is 100:2.5-100:30；Wherein, minoxidil sulfate is through triethylamine desulfurization acid It is contained in administration nano-drug administration system in the form of minoxidil afterwards, wherein the molar ratio of minoxidil sulfate and triethylamine is 1: 0.5-1:10.The anti-tumor drug includes adriamycin, taxol or antitumor siRNA, DNA, albumen, polypeptide, antibody etc.； The mass ratio of the high molecular material and anti-tumor drug is 100:1-100:50；Wherein, the adriamycin is hydrochloric acid Ah mould Product of the element after triethylamine desalination acid.

Step 3: the water phase is added dropwise in the oily phase of vortex, and ultrasonic emulsification forms water-in-oil emulsion；

In one embodiment, which specific as follows can execute: the water phase is added dropwise in the oily phase of vortex, Ultrasound condition is 300W, every ultrasound 10S, is spaced 3S, ultrasound 3 times, and emulsification forms water-in-oil emulsion.

Step 4: the emulsion being added dropwise in the outer aqueous phase of vortex again, and it is multiple that ultrasonic emulsification forms water-in-oil-in water Cream；

In one embodiment, which specific as follows can execute: vortex is added dropwise in the emulsion again In the poly-vinyl alcohol solution of 2.5%-5%, ultrasonic emulsification forms water-in-oil-in water emulsion.

Step 5: rapidly pouring into emulsion described in step 4 in volatilization water phase, is stirred overnight volatilization and removes ethyl acetate, shape At nanoparticle suspension；

In one embodiment, which specific as follows can execute: emulsion described in step 4 being poured into 0.01%- rapidly In 0.3% poly-vinyl alcohol solution, it is stirred overnight volatilization and removes ethyl acetate, form nanoparticle suspension.

Step 6: the nanoparticle suspension is purified by high speed centrifugation；

Step 7: by the unmodified nanoparticle ultrasonic disperse of the purification in appropriate 7.3 phosphate buffer of pH, Surface modification function connects molecule removes excessive function connects molecule by high speed centrifugation after reacting 1h at room temperature and precipitates, obtains Obtain the first nanoparticle；

In one embodiment, which specific as follows can execute: by the unmodified nanoparticle ultrasound of the purification It is scattered in appropriate 7.3 phosphate buffer of pH, surface modification function connects molecule, after reacting 1h at room temperature, is in speed It is centrifuged 20min under conditions of 13000rpm-35000rpm and removes excessive function connects molecule and precipitated nanocrystals grain.Wherein, described Function connects molecule be succinimide-polyethylene glycol-maleimide, the molar ratio reacted with the high molecular material For 0.4:1-1.6:1, the mechanism of reaction is the succinimide of amino and polyethylene glycol end that polymer surface is rich in Group combines.

Step 8: by the first nanoparticle ultrasonic disperse described in step 7 in appropriate 7.3 phosphate buffer of pH, lead to Function connects molecule is crossed in surface modification targeting ligand, high speed centrifugation removes excessive targeting ligand and sinks after reacting 1h at room temperature It forms sediment, obtains the second nanoparticle；

In one embodiment, which specific as follows can execute: by the first nanoparticle ultrasound described in step 7 point It dissipates in appropriate 7.3 phosphate buffer of pH, by function connects molecule in surface modification targeting ligand, reacts 1h at room temperature Afterwards, centrifugation 20min removes excessive targeting ligand and precipitated nanocrystals grain under conditions of speed is 13000rpm-35000rpm.Its In, the targeting ligand, in the embodiment of the present invention by taking hyaluronic acid as an example, the molar ratio with polyethylene glycol is 5:100- TCEP is added when reaction and disconnects disulfide bond in the hyaluronic acid of sulfhydrylation by 100:100, the hyaluronic acid of TCEP and sulfhydrylation Molar ratio is 50:1-1:1, and the mechanism of reaction is that the maleimide base group of polyethylene glycol end and the hyaluronic acid of sulfhydrylation break The sulfydryl exposed after disulfide bond is opened to combine.

Step 9: by the second nanoparticle ultrasonic disperse Yu Shuizhong described in step 8, high speed centrifugation is to precipitate final receive The grain of rice；

In one embodiment, which specific as follows can execute: by precipitated nanocrystals grain ultrasound described in step 8 point Yu Shuizhong is dissipated, 20min is centrifuged under conditions of speed is 13000rpm-35000rpm, to precipitate final nanoparticle.

Step 10: final precipitated nanocrystals grain described in step 9 is dispersed in water, and freeze drying protectant freeze-drying is added, for after It is continuous to use.

In one embodiment, which specific as follows can execute: by final precipitated nanocrystals grain described in step 9 point Yu Shuizhong is dissipated, trehalose freeze-drying is added, for subsequent use.Wherein, the mass ratio of trehalose and the high molecular material is 10:1- 1:1。

Above-mentioned one kind prepared can activate K on blood-brain tumor barrier_ATPThe metastatic encephaloma targeted nanometer drug delivery system in channel It can be used for preparing treatment breast cancer patients with brain metastatic tumor preparation, applied to targeting human body source or the tumor cell preparations of animal origin.

Above-mentioned one kind prepared can activate K on blood-brain tumor barrier_ATPThe metastatic encephaloma targeted nanometer drug delivery system in channel Experimental data please refer to Fig. 1,4,5.Fig. 1 is that one kind of the present invention can activate K on blood-brain tumor barrier_ATPThe brain in channel The building of metastatic tumor targeted nanometer drug delivery system and its internal behavior schematic diagram；Fig. 4 is to characterize this hair by dynamic light scattering method Bright single two kinds of administration nano-drug administration systems (H-NPs/DOX and M@H- for carrying adriamycin and total packet adriamycin and minoxidil NPs/DOX grain-size graph)；Fig. 5 is that packet adriamycin altogether and minoxidil of the present invention are characterized by dynamic light scattering method The grain-size graph after being resuspended is lyophilized in administration nano-drug administration system (M@H-NPs/DOX) whether there is or not two kinds of trehalose.As shown in Figure 1, One kind of the present invention can activate K on blood-brain tumor barrier_ATPIt is loaded inside the metastatic encephaloma targeted nanometer drug delivery system in channel Anti-tumor drug and K_ATPChannel agonist, after reaching blood-brain tumor barrier region, the K of release_ATPChannel agonist is based on across thin Born of the same parents and the double approach of cell bypass penetrate blood-brain tumor barrier, and nanoparticle turns across the barrier, accumulation in brain in mediate blood circulation Tumor region is moved, the anti-tumor drug of release plays antitumor action；As shown in figure 4, the administration nano-drug administration system is not loaded than inside K_ATPThe partial size of the administration nano-drug administration system of channel agonist is more bigger, and the administration nano-drug administration system as shown in Figure 5 is added trehalose and freezes It is smaller that surveyed partial size is hanged after dry again.

Using biodegradable high molecular material as basic carrier, the ultrasonic emulsification-internal loading of solvent evaporation method preparation K_ATPThe administration nano-drug administration system of channel agonist, surface modification metastatic encephaloma targeting ligand, so that blood brain can be activated by constructing one kind K on tumor barrier_ATPThe drug delivery system in channel can penetrate blood-brain tumor barrier based on across cell and the double approach of cell bypass, mediate Nanoparticle utilizes the targeting of administration nano-drug administration system surface modification across the barrier, accumulation in metastatic encephaloma region in blood circulation Ligand enters metastatic encephaloma cell；Simultaneously by drug delivery system loading anti-tumor drug, it can be achieved that the drug delivery for the treatment of concentration.This Invention is expected to become the potential nano-carrier for the treatment of metastatic encephaloma.

In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to the accompanying drawings and examples Further illustrate technical solution of the present invention.But the present invention is not limited to listed embodiments, should also be included in institute of the present invention It is required that interest field in other any well known change.

Firstly, " one embodiment " or " embodiment " referred to herein, which refers to, may be included at least one realization side of the invention A particular feature, structure, or characteristic in formula." in one embodiment " that different places occur in the present specification not refers both to The same embodiment, nor the individual or selective embodiment mutually exclusive with other embodiments.

Secondly, the present invention is described in detail using structural schematic diagram etc., when describing the embodiments of the present invention, for convenient for saying Bright, schematic diagram can disobey general proportion and make partial enlargement, and the schematic diagram is example, should not limit the present invention herein The range of protection.In addition, the three-dimensional space of length, width and depth should be included in actual fabrication.

Embodiment 1

The implementation case shows that one kind can activate K on blood-brain tumor barrier as follows_ATPThe metastatic encephaloma in channel targets The preparation method of administration nano-drug administration system:

50mg poly lactic-co-glycolic acid-polylysine is weighed in 2mL ethyl acetate as oily phase, weigh 5mg hydrochloric acid Ah Mycin is dissolved in 100 μ L water, and 12 μ L triethylamine desalination acid weigh 1.25mg minoxidil sulfate and are dissolved in 200 μ L water, 0.32 μ L Triethylamine desulfurization acid, Doxorubicin solution and minoxidil solution are added dropwise in the oily phase of vortex, and ultrasonic emulsification forms Water-In-Oil Emulsion；The emulsion is added dropwise in 2.5% poly-vinyl alcohol solution of 4mL of vortex again, ultrasonic emulsification forms W/O/W Type emulsion；This emulsion is poured into 0.3% poly-vinyl alcohol solution of 100mL rapidly, volatilization is stirred overnight and removes ethyl acetate；By institute Nanoparticle suspension is obtained to purify by the high speed centrifugation 20min of 30000rpm；By the unmodified nanoparticle ultrasound of the purification It is scattered in appropriate 7.3 phosphate buffer of pH, the NHS-PEG5000-MAL of the 0.3125mg buffer solution, room temperature is added Excessive function connects molecule and precipitated nanocrystals grain are removed by high speed centrifugation after lower reaction 1h；Resulting precipitated nanocrystals grain is surpassed again Sound is scattered in appropriate 7.3 phosphate buffer of pH, is surface modified targeting ligand, be added 0.06mg HA-S-SH and High speed centrifugation removes excessive targeting ligand and precipitated nanocrystals grain after 0.0895mg TCEP reacts 1h at room temperature；It again will be resulting heavy Shallow lake nanoparticle ultrasonic disperse Yu Shuizhong, high speed centrifugation is to precipitate final nanoparticle；Again by the final precipitated nanocrystals grain point Yu Shuizhong is dissipated, the freeze-drying of freeze drying protectant 10mg trehalose is added.Freeze-drying nanoparticle is taken to be resuspended in water, using Malvern nanoparticle Degree-potentiometric analyzer carries out granularmetric analysis, and the embodiment conclusion is referring to Fig. 4, Fig. 4 is to characterize this hair by dynamic light scattering method Bright single two kinds of administration nano-drug administration systems (H-NPs/DOX and M@H- for carrying adriamycin and total packet adriamycin and minoxidil NPs/DOX grain-size graph)；As shown in figure 4, K on blood-brain tumor barrier can be activated made from the preparation method_ATPThe brain in channel turns The hydration partial size for moving tumor targeted nanometer drug delivery system is about 180nm.

Embodiment 2

50mg poly lactic-co-glycolic acid-polylysine is weighed in 2mL ethyl acetate as oily phase, weigh 5mg hydrochloric acid Ah Mycin is dissolved in 100 μ L water, and 12 μ L triethylamine desalination acid weigh 1.25mg minoxidil sulfate and are dissolved in 200 μ L water, 0.32 μ L Triethylamine desulfurization acid, Doxorubicin solution and minoxidil solution are added dropwise in the oily phase of vortex, and ultrasonic emulsification forms Water-In-Oil Emulsion；The emulsion is added dropwise in 2.5% poly-vinyl alcohol solution of 4mL of vortex again, ultrasonic emulsification forms W/O/W Type emulsion；This emulsion is poured into 0.3% poly-vinyl alcohol solution of 100mL rapidly, stirring volatilization removes ethyl acetate；Gained is received Grain of rice suspension is purified by the high speed centrifugation 20min of 30000rpm；By the unmodified nanoparticle ultrasonic disperse of the purification In appropriate 7.3 phosphate buffer of pH, the NHS-PEG5000-MAL of the 2.5mg buffer solution is added, reacts at room temperature Excessive function connects molecule and precipitated nanocrystals grain are removed by high speed centrifugation after 1h；Again by resulting precipitated nanocrystals grain ultrasonic disperse In appropriate 7.3 phosphate buffer of pH, it is surface modified targeting ligand, 1.2mg HA-S-SH and 1.7875mg is added High speed centrifugation removes excessive targeting ligand and precipitated nanocrystals grain after TCEP reacts 1h at room temperature；Resulting precipitated nanocrystals grain is surpassed again Sound is dispersed in water, and high speed centrifugation is to precipitate final nanoparticle；The final precipitated nanocrystals grain is dispersed in water again, is added Enter the freeze-drying of freeze drying protectant 10mg trehalose.Freeze-drying nanoparticle is taken to be resuspended in water, using Malvern nano particle size-potentiometric analysis Instrument carries out granularmetric analysis, and the embodiment conclusion is referring to Fig. 4, Fig. 4 is to characterize list of the present invention by dynamic light scattering method Carry adriamycin and the altogether grain of two kinds of administration nano-drug administration systems (H-NPs/DOX and M@H-NPs/DOX) of packet adriamycin and minoxidil Diameter figure；As shown in figure 4, K on blood-brain tumor barrier can be activated made from the preparation method_ATPThe metastatic encephaloma targeted nano in channel The hydration partial size of drug delivery system is about 180nm.

Embodiment 3

50mg poly lactic-co-glycolic acid-polylysine is weighed in 2mL ethyl acetate as oily phase, weigh 5mg hydrochloric acid Ah Mycin is dissolved in 100 μ L water, and 12 μ L triethylamine desalination acid weigh 15mg minoxidil sulfate and are dissolved in 200 μ L water, 3.8 μ L tri- Ethamine desulfurization acid, Doxorubicin solution and minoxidil solution are added dropwise in the oily phase of vortex, and ultrasonic emulsification forms water in oil emulsion Agent；The emulsion is added dropwise in 2.5% poly-vinyl alcohol solution of 4mL of vortex again, ultrasonic emulsification forms water-in-oil-in water Emulsion；This emulsion is poured into 0.3% poly-vinyl alcohol solution of 100mL rapidly, volatilization is stirred overnight and removes ethyl acetate；By gained Nanoparticle suspension is purified by the high speed centrifugation 20min of 30000rpm；By the unmodified nanoparticle ultrasound of the purification point It dissipates in appropriate 7.3 phosphate buffer of pH, the NHS-PEG5000-MAL of the 0.3125mg buffer solution is added, at room temperature Excessive function connects molecule and precipitated nanocrystals grain are removed by high speed centrifugation after reaction 1h；Again by resulting precipitated nanocrystals grain ultrasound Be scattered in appropriate 7.3 phosphate buffer of pH, be surface modified targeting ligand, be added 0.06mg HA-S-SH and High speed centrifugation removes excessive targeting ligand and precipitated nanocrystals grain after 0.0895mg TCEP reacts 1h at room temperature；It again will be resulting heavy Shallow lake nanoparticle ultrasonic disperse Yu Shuizhong, high speed centrifugation is to precipitate final nanoparticle；Again by the final precipitated nanocrystals grain point Yu Shuizhong is dissipated, the freeze-drying of freeze drying protectant 10mg trehalose is added.Freeze-drying nanoparticle is taken to be resuspended in water, using Malvern nanoparticle Degree-potentiometric analyzer carries out granularmetric analysis, and the embodiment conclusion is referring to Fig. 4, Fig. 4 is to characterize this hair by dynamic light scattering method Bright single two kinds of administration nano-drug administration systems (H-NPs/DOX and M@H- for carrying adriamycin and total packet adriamycin and minoxidil NPs/DOX grain-size graph)；As shown in figure 4, K on blood-brain tumor barrier can be activated made from the preparation method_ATPThe brain in channel turns The hydration partial size for moving tumor targeted nanometer drug delivery system is about 180nm.

Embodiment 4

50mg poly lactic-co-glycolic acid-polylysine is weighed in 2mL ethyl acetate as oily phase, weigh 5mg hydrochloric acid Ah Mycin is dissolved in 100 μ L water, and 12 μ L triethylamine desalination acid weigh 15mg minoxidil sulfate and are dissolved in 200 μ L water, 3.8 μ L tri- Ethamine desulfurization acid, Doxorubicin solution and minoxidil solution are added dropwise in the oily phase of vortex, and ultrasonic emulsification forms water in oil emulsion Agent；The emulsion is added dropwise in 2.5% poly-vinyl alcohol solution of 4mL of vortex again, ultrasonic emulsification forms water-in-oil-in water Emulsion；This emulsion is poured into 0.3% poly-vinyl alcohol solution of 100mL rapidly, volatilization is stirred overnight and removes ethyl acetate；By gained Nanoparticle suspension is purified by the high speed centrifugation 20min of 30000rpm；By the unmodified nanoparticle ultrasound of the purification point It dissipates in appropriate 7.3 phosphate buffer of pH, the NHS-PEG5000-MAL of the 0.3125mg buffer solution is added, at room temperature Excessive function connects molecule and precipitated nanocrystals grain are removed by high speed centrifugation after reaction 1h；Again by resulting precipitated nanocrystals grain ultrasound Be scattered in appropriate 7.3 phosphate buffer of pH, be surface modified targeting ligand, be added 1.2mg HA-S-SH and High speed centrifugation removes excessive targeting ligand and precipitated nanocrystals grain after 1.7875mg TCEP reacts 1h at room temperature；It again will be resulting heavy Shallow lake nanoparticle ultrasonic disperse Yu Shuizhong, high speed centrifugation is to precipitate final nanoparticle；Again by the final precipitated nanocrystals grain point Yu Shuizhong is dissipated, the freeze-drying of freeze drying protectant 10mg trehalose is added.Freeze-drying nanoparticle is taken to be resuspended in water, using Malvern nanoparticle Degree-potentiometric analyzer carries out granularmetric analysis, and the embodiment conclusion is referring to Fig. 4, Fig. 4 is to characterize this hair by dynamic light scattering method Bright single two kinds of administration nano-drug administration systems (H-NPs/DOX and M@H- for carrying adriamycin and total packet adriamycin and minoxidil NPs/DOX grain-size graph)；As shown in figure 4, K on blood-brain tumor barrier can be activated made from the preparation method_ATPThe brain in channel turns The hydration partial size for moving tumor targeted nanometer drug delivery system is about 180nm.

Embodiment 5

50mg poly lactic-co-glycolic acid-polylysine is weighed in 2mL ethyl acetate as oily phase, weigh 5mg hydrochloric acid Ah Mycin is dissolved in 100 μ L water, and 12 μ L triethylamine desalination acid weigh 7.5mg minoxidil sulfate and are dissolved in 200 μ L water, 3.8 μ L tri- Ethamine desulfurization acid, Doxorubicin solution and minoxidil solution are added dropwise in the oily phase of vortex, and ultrasonic emulsification forms water in oil emulsion Agent；The emulsion is added dropwise in 2.5% poly-vinyl alcohol solution of 4mL of vortex again, ultrasonic emulsification forms water-in-oil-in water Emulsion；This emulsion is poured into 0.3% poly-vinyl alcohol solution of 100mL rapidly, volatilization is stirred overnight and removes ethyl acetate；By gained Nanoparticle suspension is purified by the high speed centrifugation 20min of 30000rpm；By the unmodified nanoparticle ultrasound of the purification point It dissipates in appropriate 7.3 phosphate buffer of pH, the NHS-PEG5000-MAL of the 5mg buffer solution is added, reacts at room temperature Excessive function connects molecule and precipitated nanocrystals grain are removed by high speed centrifugation after 1h；Again by resulting precipitated nanocrystals grain ultrasonic disperse In appropriate 7.3 phosphate buffer of pH, it is surface modified targeting ligand, 0.96mg HA-S-SH and 1.433mg is added High speed centrifugation removes excessive targeting ligand and precipitated nanocrystals grain after TCEP reacts 1h at room temperature；Resulting precipitated nanocrystals grain is surpassed again Sound is dispersed in water, and high speed centrifugation is to precipitate final nanoparticle；The final precipitated nanocrystals grain is dispersed in water again, is added Or freeze drying protectant 10mg trehalose is not added and is lyophilized respectively.Two kinds of freeze-drying nanoparticles are taken to be resuspended in water, using Malvern nanometer Granularity-potentiometric analyzer carries out granularmetric analysis, and the embodiment conclusion is referring to Fig. 5, Fig. 5 is to characterize this by dynamic light scattering method The administration nano-drug administration system (M@H-NPs/DOX) of the invention total packet adriamycin and minoxidil is whether there is or not two kinds of situations of trehalose Grain-size graph after lower freeze-drying resuspension, as shown in figure 5, the presence of trehalose keeps the hydration partial size of the nanoparticle hanged again smaller.

Embodiment 6

It disperses 500mg HA (52.04 μm of ol) in the PBS solution of 10mL pH 7.4, is added 2- (two sulphur of 2- pyridine) Ethylamine hydrochloride (52.4mg, 235.23 μm of ol), EDC (75mg, 391.24 μm of ol) and sulfo group NHS (85mg, 391.45 μm of ol), Reaction mixture 4h is stirred at room temperature.Then it is dialysed with bag filter (MWCO 3500) to mixture.Then by bag filter Interior solution freeze-drying, obtained lyophilized products are dissolved in D2O, carry out hydrogen nuclear magnetic resonance spectrum analysis.The embodiment conclusion please join Fig. 2, Fig. 3 are read, the hydrogen that Fig. 2 is hyaluronic acid HA is composed；Fig. 3 is the hydrogen spectrum of the hyaluronic acid (HA-S-SH) of sulfhydrylation.Such as Fig. 2, figure Shown in 3, which is that this synthetic schemes successfully synthesizes the hyaluronic acid of sulfhydrylation, each molecular weight be 9608 it is transparent 3.75 disulfide bond have been grafted on matter acid.

Embodiment 7

In order to evaluate the metastatic encephaloma targeted nanometer drug delivery system physiological condition in vitro of total load adriamycin and minoxidil The ability of lower release drug, investigates drug release using dialysis then.It is 150 μ g/ by freshly prepared 1.9mL doxorubicin concentration ML nanoparticle solution is placed in the bag filter that molecular cut off is 3500Da, contains 0.5% lauryl sodium sulfate respectively with 100mL PH 7.4 PBS buffer solution be dissolution medium.It is discharged under the conditions of 37 DEG C, 100r/min, in different times point, with 1mL Fresh above-mentioned buffer replaces dissolution medium.The amount of the adriamycin of each time point release is measured using all-wave length microplate reader, and Using the amount of the minoxidil of each time point release of high-efficient liquid phase analysis.Wherein all-wave length microplate reader uses excitation wavelength 537nm, Launch wavelength 584nm；Efficient liquid phase chromatographic analysis uses 18 reverse chromatograms column of carbon, and mobile phase is 10mM Ammoniom-Acetate and methanol solution (60:40), flow velocity 1mL/min.The embodiment conclusion please refers to Fig. 6, Fig. 7, and Fig. 6 is the of the present invention of dialysis characterization Contain adriamycin activates K on blood-brain tumor barrier_ATPThe minot contained in the metastatic encephaloma targeted nanometer drug delivery system in channel Release profiles of ground that under the conditions of 7.4 pH；Fig. 7 be dialysis characterization the nanometer of the present invention for containing adriamycin to Release profiles of adriamycin under the conditions of 7.4 pH in medicine system.As shown in Figure 6, Figure 7, which is nanometer administration System has slow-release function.

Embodiment 8

In order to investigate the targeting of targeting ligand hyaluronic acid in the administration nano-drug administration system, by metastatic encephaloma cell line 231Br cell inoculation in 24 orifice plates, be separately added into free DOX, NPs/DOX, H-NPs/DOX (while add or be not added excessive Free HA) it is incubated for 90min, the concentration of adriamycin is 3 μ g/mL；Cell pyrolysis liquid lytic cell is used again, is existed with all-wave length microplate reader Doxorubicin fluorescence, the adriamycin of quantitative detection cellular uptake are measured under 537nm excitation wavelength and 584nm launch wavelength.Use BCA Method detects the protein content in cell, thus the adriamycin of normalized cellular uptake, and deducts in the cell of non-administration Fluorescence/protein value.The embodiment conclusion referring to Fig. 8, Fig. 8 be quantitatively characterizing free adriamycin and whether there is or not HA modification load Ah Intake of each administration nano-drug administration system (NPs/DOX and H-NPs/DOX) of mycin on metastatic encephaloma cell.As shown in figure 8, HA is repaired The nanoparticle of decorations is absorbed more compared to unmodified nanoparticle by tumour cell, is administered simultaneously free HA since competitiveness presses down The obvious less intake of H-NPs/DOX of the effect of system.

Embodiment 9

In order to investigate the targeting of targeting ligand hyaluronic acid in the administration nano-drug administration system, by normal brain micro blood vessel endothelium Cell line bEND.3 is inoculated in 24 orifice plates, be separately added into free adriamycin, NPs/DOX, H-NPs/DOX (while add or be not added Excessive free HA) it is incubated for 90min, the concentration of adriamycin is 3 μ g/mL；With cell pyrolysis liquid lytic cell, with all-wave length enzyme mark Instrument measures doxorubicin fluorescence, the adriamycin of quantitative detection cellular uptake under 537nm excitation wavelength and 584nm launch wavelength. The protein content in cell is detected with BCA method, thus the adriamycin of normalized cellular uptake, and deduct the cell of non-administration In fluorescence/protein value.The embodiment conclusion is referring to Fig. 9, Fig. 9 is the free adriamycin of quantitatively characterizing and whether there is or not HA modifications Carry intake of each administration nano-drug administration system (NPs/DOX and H-NPs/DOX) of adriamycin on normal brain microvessel endothelial cells in vitro.Such as Shown in Fig. 9, intake indistinction of the nanoparticle of HA modification compared with unmodified nanoparticle, on bEND.3 cell.The implementation Example illustrates that the nanoparticle of HA modification has targeting to the metastatic encephaloma cell that CD44 is overexpressed, to normal endothelial cell without work With.

Embodiment 10

In order to investigate the degree of modification of targeting ligand in the administration nano-drug administration system, metastatic encephaloma cell line 231Br cell is connect For kind in 24 orifice plates, the H-NPs/DOX for being separately added into different HA degree of modification (relative to PEG grafting degree) is incubated for 90min, adriamycin Concentration be 3 μ g/mL；With cell pyrolysis liquid lytic cell, emitted with all-wave length microplate reader in 537nm excitation wavelength and 584nm Doxorubicin fluorescence, the adriamycin of quantitative detection cellular uptake are measured under wavelength.The protein content in cell is detected with BCA method, To the adriamycin of normalized cellular uptake, and deduct fluorescence/protein value in the cell of non-administration.The embodiment conclusion Referring to Fig. 10, Figure 10 is thin in metastatic encephaloma for the metastatic encephaloma targeted nanometer drug delivery system of the different HA degree of modification of quantitatively characterizing Intake on born of the same parents.As shown in Figure 10, which illustrates that, compared to unmodified nanoparticle, the nanoparticle of different HA degree of modification is equal Its intake by tumour cell is added somewhat to, 10% and 20% degree of modification increases 1.47~1.60 times, and modifies Degree further increases the decline for resulting in increasing degree, thus it is speculated that is since higher HA degree of modification increases the negative electricity of nanoparticle Property and sterically hindered.

Embodiment 11

In order to investigate effect of the minoxidil to tumour cell and normal brain microvessel endothelial cells in vitro intake nanoparticle, by brain Metastatic tumor cell line 231Br cell or normal brain microvessel endothelial cells in vitro bEND.3 are inoculated in 24 orifice plates, in free minot In the case that your concentration is 0 μ g/mL and 3 μ g/mL, it is separately added into free adriamycin, NPs/DOX, H-NPs/DOX incubation 90min, The concentration of adriamycin is 3 μ g/mL；It inhales and abandons medical fluid, dye 30min with 2 μ g/mL Hoechst 33342, pass through fluorescence microscope Qualitative observation cellular uptake situation.Use cell pyrolysis liquid lytic cell again, with all-wave length microplate reader in 537nm excitation wavelength and Doxorubicin fluorescence, the adriamycin of quantitative detection cellular uptake are measured under 584nm launch wavelength.With in BCA method detection cell Protein content thus the adriamycin of normalized cellular uptake, and deducts fluorescence/protein value in the cell of non-administration.It should Embodiment conclusion please refer to Figure 11, Figure 12, Figure 11 is the free adriamycin of quantitatively characterizing and contains the nanoparticle of adriamycin The intake of (NPs/DOX and H-NPs/DOX) in the presence of whether there is or not minoxidil on metastatic encephaloma cell, Figure 12 is quantitatively characterizing Free adriamycin and contain the nanoparticle (NPs/DOX and H-NPs/DOX) of adriamycin in the presence of whether there is or not minoxidil just Intake on normal brain microvessel endothelial cells in vitro.As shown in figure 12, nanometer of the bEND.3 cell to free adriamycin and load adriamycin The intake of grain is in the presence of minoxidil without humidification.The embodiment illustrates that minoxidil acts only on K_ATPChannel is overexpressed Tumour cell, open K_ATPChannel, raises the expression of caveolin, so that nanoparticle be promoted to pass through in caveolin mediation It gulps down and enters tumour cell.

Embodiment 12

In order to which whether the effect for checking that minoxidil absorbs nanoparticle in tumour cell is since minoxidil may Potential cytotoxicity under conditions of there are 1~10 μ g/mL minoxidil, given by 231Br cell inoculation in 96 orifice plates The free adriamycin of medicine and the nanoparticle for carrying adriamycin, the concentration of adriamycin is 3 μ g/mL；It is incubated for 90min, is detected with MTT experiment thin Cellular toxicity.The embodiment conclusion please refers to Figure 13, and Figure 13 is the free adriamycin of MTT experiment characterization and the nanometer for containing adriamycin Grain (NPs/DOX and H-NPs/DOX) is in the presence of various concentration minoxidil to the toxicity of metastatic encephaloma cell.It is dense such as Figure 15 Degree is that the cytotoxicity of the minoxidil of 1~10 μ g/mL is extremely low (> 95% vigor).Minoxidil and free adriamycin or carry Ah The nanoparticle of mycin, which is jointly processed by, also seldom induces cell apoptosis (> 85% vigor).The embodiment proves minoxidil to nanometer Toxicity of the effect of grain intake not due to minoxidil.

Embodiment 13

The effect of the endothelial cell intake nanoparticle stimulated to investigate minoxidil to tumour, by 231Br cell and BEND.3 cells in vitro co-incubation is to simulate blood-brain tumor barrier, by two kinds of cell inoculations in 24 orifice plates, respectively whether there is or not H-NPs/DOX is administered in the presence of 3 μ g/mL minoxidils, is incubated for 90min, the concentration of adriamycin is 3 μ g/mL；It inhales and abandons medical fluid, 30min is dyed with 2 μ g/mL Hoechst 33342, passes through the intake situation of fluorescence microscope nanoparticle.The embodiment knot By Figure 14 is please referred to, Figure 14 is that single metastatic encephaloma targeted nano for carrying adriamycin of the present invention of fluorescence microscope characterization is given Medicine system (H-NPs/DOX) is trained in the presence of whether there is or not minoxidil in metastatic encephaloma cell and normal brain microvessel endothelial cells in vitro altogether Support the intake figure on cell.Such as Figure 14, in the presence of no minoxidil, only surely turn the 231Br cellular uptake nanoparticle of GFP, BEND.3 cell is few to the intake of nanoparticle；When minoxidil coprocessing, 231Br and bEND.3 cell takes the photograph nanoparticle It takes and is remarkably reinforced, wherein the fluorescence intensity on bEND.3 cell is close with 231Br cell.The embodiment illustrates that minoxidil can To enhance intake of the endothelial cell of tumour stimulation to nanoparticle.

Embodiment 14

It, will in order to investigate whether the minoxidil contained remains to play the effect for promoting tumour cell to absorb nanoparticle M@H-NPs is administered in 96 orifice plates in 231Br cell inoculation, is incubated for 0,2,4,6,18,30,42,54h respectively, inhales and abandons medical fluid, then gives Medicine H-NPs/DOX, is incubated for 90min, and the concentration of adriamycin is 3 μ g/mL；With cell pyrolysis liquid lytic cell, with all-wave length enzyme mark Instrument measures doxorubicin fluorescence, the adriamycin of quantitative detection cellular uptake under 537nm excitation wavelength and 584nm launch wavelength. The protein content in cell is detected with BCA method, thus the adriamycin of normalized cellular uptake, and deduct the cell of non-administration In fluorescence/protein value.The conclusion of the embodiment please refers to Figure 15, and Figure 15 is the metastatic encephaloma targeting that preincubate carries minoxidil The metastatic encephaloma targeted nanometer drug delivery system of quantitative determination load adriamycin after different time is administered in administration nano-drug administration system (M@H-NPs) (H-NPs/DOX) intake on metastatic encephaloma cell.As shown in figure 15, with the extension of the administration time, minoxidil is gradually Release plays its effect for promoting tumour cell endocytosis nanoparticle.

Embodiment 15

In order to optimize the feed ratio of minoxidil, by 231Br cell inoculation in 96 orifice plates, different macromolecule materials is administered Material and M@H-NPs obtained by minoxidil mass ratio, are incubated for 30h, inhale and abandon medical fluid, then H-NPs/DOX is administered, be incubated for 90min, The concentration of adriamycin is 3 μ g/mL；With cell pyrolysis liquid lytic cell, with all-wave length microplate reader in 537nm excitation wavelength and Doxorubicin fluorescence, the adriamycin of quantitative detection cellular uptake are measured under 584nm launch wavelength.With in BCA method detection cell Protein content thus the adriamycin of normalized cellular uptake, and deducts fluorescence/protein value in the cell of non-administration.It should The conclusion of embodiment please refers to Figure 16, and Figure 16 is that the metastatic encephaloma targeted nano of the load minoxidil of preincubate difference feed ratio is given Medicine system (M@H-NPs) 30h, quantitative determination carry the metastatic encephaloma targeted nanometer drug delivery system (H-NPs/DOX) of adriamycin in brain Shift the intake on oncocyte.As shown in figure 16, the amount of the adriamycin of cellular uptake is positively correlated with feed ratio.

Embodiment 16

In order to investigate minoxidil levels in vitro lower tight junction protein expression effect, by bEND.3 cell inoculation in In 48 orifice plate creep plates, in vitro blood-brain barrier model is simulated with fresh culture culture, the culture medium crossed with tumor cell culture Culture carrys out the outer blood-brain tumor barrier model of analogue body, and when cell density reaches 100%, free minoxidil is administered respectively 1.5h, H-NPs and M@H-NPs 30h inhale and abandon medical fluid, fixes 10min with 4% paraformaldehyde room temperature, penetrating with -20 DEG C of ice methanol 10min is handled, closes 1h with confining liquid, is incubated for 4 DEG C of primary antibody of 2.5 μ g/mL claudin-5 overnight, then be incubated for the two of fluorescent marker Anti- 2h, contaminates DAPI 5min, and the effect expressed with confocal microscopy drug tight junction protein is fixed with Image J Amount.The conclusion of the embodiment please refers to Figure 17, Figure 17 be Laser Scanning Confocal Microscope characterization blank administration nano-drug administration system (H-NPs), Free minoxidil and the administration nano-drug administration system (M@H-NPs) of minoxidil is contained to in-vitro simulated blood-brain barrier and blood brain The effect of tumor barrier claudin-5 expression.As shown in figure 17, free minoxidil is administered and carries the metastatic encephaloma of minoxidil The expression of claudin-5 on targeted nanometer drug delivery system (M@H-NPs) energy specific downregulation blood-brain tumor barrier, to normal blood brain Barrier is without effect.

Embodiment 17

In order to investigate the effect that horizontal down-regulation tight junction protein is expressed in minoxidil body, by physiological saline and minot is carried Tail vein injection enters in metastatic encephaloma model mice body your metastatic encephaloma targeted nanometer drug delivery system (M H-NPs) of ground respectively, CD31 label vascular is injected, perfusion fixation after 1h takes mouse brain, sucrose dehydration, frozen section, the confining liquid room temperature closing of brain piece 1h is incubated for 4 DEG C of primary antibody of 2.5 μ g/mL claudin-5 overnight, then is incubated for the secondary antibody 2h of fluorescent marker, contaminates DAPI 5min, with altogether The effect that focusing microscope observation drug expresses tight junction protein.The conclusion of the embodiment please refers to Figure 18, and Figure 18 is total To normal brain activity region and brain in administration nano-drug administration system (M@H-NPs) animal level for containing minoxidil of focusing microscope characterization The effect of metastatic tumor region claudin-5 expression.As shown in figure 18, administration carries the metastatic encephaloma targeted nano administration of minoxidil The expression of claudin-5 on system (M@H-NPs) energy specific downregulation blood-brain tumor barrier, to normal blood-brain barrier without effect.

Embodiment 18

It carries the metastatic encephaloma targeted nanometer drug delivery system (M@H-NPs) of minoxidil in order to investigate and overcomes blood brain tumor screen The effect of barrier, will carry Hoechst33342 metastatic encephaloma targeted nano granule (H-NPs/Hoechst33342) individually or with M@ Tail vein injection enters in metastatic encephaloma model mice body after H-NPs mixing, injects CD31 label vascular, and perfusion fixation after 1h takes Mouse brain, sucrose dehydration, frozen section overcome blood-brain tumor barrier with fluorescence microscope H-NPs/Hoechst33342, It accumulates in the efficiency in metastatic encephaloma region.The conclusion of the embodiment please refers to Figure 19, and Figure 19 is the blank of fluorescence microscope characterization Administration nano-drug administration system (H-NPs) and metastatic encephaloma targeted nanometer drug delivery system (the M@H-NPs) animal for containing minoxidil are horizontal On overcome the effect picture of blood-brain tumor barrier, blue-fluorescence indicates the Hoechst33342 contained, indicates at yellow arrows meaning Nanoparticle enters brain parenchym area through blood-brain tumor barrier, indicates that nanoparticle is absorbed by tumour cell at white arrow meaning.Such as Shown in Figure 19, one kind of the present invention can activate K on blood-brain tumor barrier_ATPThe administration nano-drug administration system in channel can overcome blood Brain tumor barrier reaches metastatic encephaloma region, and is absorbed by tumour cell.

Embodiment 19

K on blood-brain tumor barrier can be activated in order to probe into_ATPWhether the administration nano-drug administration system in channel can exist to promotion nanoparticle Accumulation in normal brain activity, normal mouse tail vein injection dissociate in adriamycin and each administration nano-drug administration system for carrying adriamycin, 12h Perfusion fixation takes mouse brain, sucrose dehydration, frozen section, with accumulation of the fluorescence microscope nanoparticle in normal brain activity.It should The conclusion of embodiment please refers to Figure 20, Figure 20 be the free adriamycin of fluorescence microscope characterization and contain each nanometer of adriamycin to Distribution map of the medicine system in normal mouse brain area domain macroscopic scale and microscopic scale.As shown in figure 20, this to activate blood brain swollen K on tumor barrier_ATPThe administration nano-drug administration system in channel will not promote nanoparticle in the accumulation in normal brain activity region.

Embodiment 20

K on blood-brain tumor barrier can be activated in order to probe into_ATPThe internal blood-brain tumor barrier target of the administration nano-drug administration system in channel To efficiency, metastatic encephaloma model mice tail vein injection dissociate adriamycin, without HA modification nanoparticle (NPs/DOX), HA modification Targeted nano granule (H-NPs/DOX) and K on blood-brain tumor barrier can be activated_ATPMetastatic encephaloma targeted nano granule (the M@in channel H-NPs/DOX), perfusion fixation after 12h, takes mouse brain, sucrose dehydration, frozen section, with macroscopical fluorescence microscope and microcosmic glimmering Light microscope observes nanoparticle in the accumulation in metastatic encephaloma region.The average optical density of adriamycin is quantified with Image J.The implementation The conclusion of example please refers to Figure 21-26, Figure 21 be the free adriamycin of fluorescence microscope characterization and contain each nanometer of adriamycin to Distribution map of the medicine system in tumor-bearing mice brain area domain macroscopic scale；Figure 22 be Figure 21 through Image J quantitative normal brain activity region and The average optical density of the adriamycin in metastatic encephaloma region；Figure 23 is the free adriamycin of fluorescence microscope characterization and contains adriamycin Each administration nano-drug administration system the submicroscopic level in tumor-bearing mice brain area domain distribution map；Figure 24 is that Figure 23 is quantitative through Image J The average optical density of the adriamycin in normal brain activity region and metastatic encephaloma region；Figure 25 is the free adriamycin of fluorescence microscope characterization With distribution map of each administration nano-drug administration system in tumor-bearing mice brain area domain microscopic scale for containing adriamycin；Figure 26 is Figure 25 warp The average optical density of the adriamycin in Image J quantitative normal brain activity region and metastatic encephaloma region.Such as Figure 21-26, blood can be activated K on brain tumor barrier_ATPAccumulation of the metastatic encephaloma targeted nano granule in channel in metastatic encephaloma region is significantly stronger than other administrations Group.

Embodiment 21

In order to evaluate the toxicity for the targeted nanometer drug delivery system for carrying minoxidil, by 231Br cell inoculation on 96 orifice plates, The free minoxidil, empty vectors (H-NPs) and the targeted nano granule for carrying minoxidil of various concentration is administered after adherent respectively (M@H-NPs) is incubated for 48h, measures cell survival rate with mtt assay.The conclusion of the embodiment please refers to Figure 27, and Figure 27 is MTT real Test the blank nanoparticle of characterization, free minoxidil and contain minoxidil metastatic encephaloma targeted nano granule (M@H-NPs) it is right The toxicity of metastatic encephaloma cell, time 48h.Such as Figure 27, carry the cytotoxicity of the targeted nanometer drug delivery system of minoxidil compared with Low, cell survival rate is still 80% or more when minoxidil concentration is up to 20 μ g/mL.

Embodiment 22

It is in order to evaluate the toxicity for the metastatic encephaloma targeted nanometer drug delivery system for carrying minoxidil and adriamycin, 231Br is thin Born of the same parents are inoculated on 96 orifice plates, and the free adriamycin of various concentration is administered after adherent respectively, carries targeted nano granule (the M@of minoxidil H-NPs the targeted nano granule (M@H-NPs/DOX) for) and altogether carrying minoxidil and adriamycin, is incubated for 48h, measures cell with mtt assay Survival rate.The conclusion of the embodiment please refers to Figure 28, Figure 28 be the load minoxidil of MTT experiment characterization nanoparticle, free Ah Mycin and altogether the metastatic encephaloma targeted nano granule (M@H-NPs/DOX) of load adriamycin and minoxidil are to the poison of metastatic encephaloma cell Property, time 48h.Cytotoxicity such as Figure 28, M@H-NPs/DOX is close with free adriamycin, and IC50 is respectively 0.348 μ g/ ML and 0.184 μ g/mL.

Embodiment 23

K on blood-brain tumor barrier can be activated in order to evaluate one kind of the present invention_ATPThe metastatic encephaloma targeted nano in channel Drug delivery system (M@H-NPs/DOX) is to the therapeutic effect of metastatic encephaloma, after nude mice injection of heart metastatic encephaloma cell 5 days respectively Tail vein is administered physiological saline, free adriamycin, single targeted nano granule for carrying minoxidil and carries adriamycin and minoxidil altogether Targeted nano granule, by adriamycin 5mg/kg dosage be administered, biweekly, treat seven weeks, record metastatic encephaloma model The life cycle of mouse and changes of weight situation.It is free adriamycin and Ben Fa that the conclusion of the embodiment, which please refers to Figure 29,30, Figure 29, K on blood-brain tumor barrier is activated described in bright_ATPThe metastatic encephaloma targeted nanometer drug delivery system (M@H-NPs/DOX) in channel is right The treatment survival rate of metastatic encephaloma model mice, the dosage of adriamycin are every kilogram of mouse 4mg adriamycin；Figure 30 is free Metastatic encephaloma model is small after adriamycin and metastatic encephaloma targeted nanometer drug delivery system of the present invention (M H-NPs/DOX) administration The weight figure of mouse.Such as Figure 29,30, K on blood-brain tumor barrier can be activated_ATPThe metastatic encephaloma targeted nano granule in channel can be significant Extend its life cycle, the potentiality with good treatment metastatic encephaloma.

Compared with prior art, the beneficial effects of the present invention are: the invention discloses one kind can activate blood-brain tumor barrier Upper K_ATPThe preparation method and applications of the metastatic encephaloma targeted nanometer drug delivery system in channel, by containing minoxidil in targeting Administration nano-drug administration system can efficiently overcome blood-brain tumor barrier based on across cell and the double approach of cell bypass, increase blood brain tumor The permeability of barrier, administration nano-drug administration system realizes treatment across the barrier, accumulation in metastatic encephaloma region in mediate blood circulation The drug delivery of concentration, while accumulation of the administration nano-drug administration system in normal brain activity can be reduced, and this method side easy to operate Just, so administration nano-drug administration system operability with higher and economic benefit that the present invention obtains.

It should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to preferable Embodiment describes the invention in detail, those skilled in the art should understand that, it can be to technology of the invention Scheme is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered in this hair In bright scope of the claims.

Claims (10)

1. K can be activated_ATPThe metastatic encephaloma targeting drug delivery system in channel, it is characterised in that: with biodegradable high molecular material For basic carrier, inside loads K_ATPChannel agonist, surface modification metastatic encephaloma targeting ligand.

2. K can be activated_ATPThe preparation method of the metastatic encephaloma targeting drug delivery system in channel, which is characterized in that this method includes as follows Step:

(1) high molecular material is weighed, is dissolved in organic solvent, oily phase is formed；

(2) by anti-tumor drug and K_ATPChannel agonist is soluble in water, forms water phase；

(3) water phase is added dropwise in the oily phase of vortex, ultrasonic emulsification forms the emulsion of water-in-oil type；

(4) emulsion is added dropwise in the outer aqueous phase of vortex again, ultrasonic emulsification forms the emulsion of water-in-oil-in water；

(5) emulsion is poured into volatilization water phase rapidly, is stirred overnight volatilization and removes ethyl acetate, formed nanoparticle and be suspended Liquid；

(6) the nanoparticle suspension is purified by high speed centrifugation, obtains the unmodified nanoparticle of purification；

(7) by the unmodified nanoparticle ultrasonic disperse in the phosphate buffer that pH value is 7.3, surface modification function connects Molecule removes excessive function connects molecule by high speed centrifugation after reaction 1h at room temperature, and precipitating obtains the first nanoparticle；

(8) the first nanoparticle ultrasonic disperse is existed in the phosphate buffer that pH value is 7.3 by function connects molecule Surface modification targeting ligand, high speed centrifugation removes excessive targeting ligand after reacting 1h at room temperature, and precipitating obtains second nanometer Grain；

(9) by the second nanoparticle ultrasonic disperse Yu Shuizhong, high speed centrifugation, precipitating obtain final nanoparticle；

(10) the final nanoparticle is dispersed in water, freeze drying protectant freeze-drying is added, blood brain tumor can be activated by obtaining one kind K on barrier_ATPThe metastatic encephaloma targeted nanometer drug delivery system in channel.

3. according to claim 1 activate K_ATPThe preparation method of the metastatic encephaloma targeting drug delivery system in channel, feature Be: high molecular material described in step (1) is any one in polylactic acid, poly lactic-co-glycolic acid or polycaprolactone；Institute Organic solvent is stated as any one in ethyl acetate or methylene chloride.

4. according to claim 1 activate K_ATPThe preparation method of the metastatic encephaloma targeting drug delivery system in channel, feature Be: anti-tumor drug described in step (2) is adriamycin, taxol or antitumor siRNA, DNA, albumen, polypeptide, antibody In any one, the adriamycin is the product after triethylamine desalination, the high molecular material and the anti-tumor drug Mass ratio is 100:1-100:50；The K_ATPChannel agonist is any one in minoxidil, minoxidil sulfate or diazoxiide Kind；The minoxidil is product of the minoxidil sulfate after triethylamine desulfurization acid, wherein the minoxidil sulfate and institute The molar ratio for stating triethylamine is 1:0.5-1:10, the high molecular material and the K_ATPThe mass ratio of channel agonist is 100: 2.5-100:30。

5. according to claim 1 activate K_ATPThe preparation method of the metastatic encephaloma targeting drug delivery system in channel, feature Be: outer aqueous phase described in step (4) is the poly-vinyl alcohol solution of 2.5%-5%.

6. according to claim 1 activate K_ATPThe preparation method of the metastatic encephaloma targeting drug delivery system in channel, feature It is: the poly-vinyl alcohol solution that volatilization water phase described in step (5) is 0.01%-0.3%.

7. according to claim 1 activate K_ATPThe preparation method of the metastatic encephaloma targeting drug delivery system in channel, feature Be: function connects molecule described in step (7) is succinyl Asia by-polyethylene glycol-maleimide, the function connects point The sub molar ratio reacted with the high molecular material is 0.05:1-1.6:1.

8. according to claim 1 activate K_ATPThe preparation method of the metastatic encephaloma targeting drug delivery system in channel, feature Be: targeting ligand described in step (8) be metastatic encephaloma targeting ligand, integrin receptor target ligand, Her2 targeting ligand, Any one in TfR targeting ligand, 1 targeting ligand of LDH receptor related protein；The targeting is matched The molar ratio of body and polyethylene glycol is 5:100-100:100；TCEP is added during the reaction simultaneously to open in targeting ligand The molar ratio of disulfide bond, the TECP and targeting ligand is 50:1-1:1.

9. according to claim 1 activate K_ATPThe preparation method of the metastatic encephaloma targeting drug delivery system in channel, feature Be: freeze drying protectant described in step (10) is trehalose, and the mass ratio of the trehalose and high molecular material is 10:1-1: 1。

10. activating K described according to claim 1-9_ATPThe preparation method institute of the metastatic encephaloma targeting drug delivery system in channel Preparation activates K on blood-brain tumor barrier_ATPThe metastatic encephaloma targeting drug delivery system in channel comes in targeting human body source or animal Application in the tumor cell preparations in source.