CN108815134B

CN108815134B - Preparation and application of a bio-camouflaged targeted nano-drug delivery system for ischemic stroke treatment

Info

Publication number: CN108815134B
Application number: CN201810766716.0A
Authority: CN
Inventors: 王伟; 苏杨楠
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2018-07-10
Filing date: 2018-07-10
Publication date: 2021-07-13
Anticipated expiration: 2038-07-10
Also published as: CN108815134A

Abstract

The invention relates to the preparation and application of a biologically camouflaged targeted nano-drug delivery system for ischemic stroke treatment, wherein the nano-drug delivery system is composed of a drug, a core drug carrier, a biologically camouflaged shell and a target-seeking material, and the drug Ginkgolide B; the inner core drug carrier is recombinant high-density lipoprotein, which forms a drug-loaded inner core after the drug is encapsulated by physical embedding; the biological camouflage shell is a platelet membrane, which is encapsulated by co-extrusion. After the drug-loaded inner core is loaded, biomimetic drug-loaded nanoparticles are formed; the targeting material is cerebral ischemia targeting peptide CITP, and the CITP is modified on the surface of the biomimetic drug-loaded nanoparticles to form a biological camouflage targeted nano-drug delivery system. This patent uses recombinant high-density lipoprotein-encapsulated ginkgolide B as the drug-carrying core, platelet membrane and CITP modified on the platelet membrane surface as the biomimetic shell to construct a new type of nanoparticles for the treatment of ischemic stroke. Circulation time in vivo and good targeting.

Description

Preparation and application of biological camouflage targeted nano drug delivery system for treating ischemic stroke

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and relates to preparation and application of a biological camouflage targeted nano drug delivery system for treating ischemic stroke.

Background

Cerebrovascular disease is also called stroke, and is a disease with cerebral function defect caused by pathological changes of cerebral vessels due to various etiological factors. About 80% of clinical cerebral apoplexy belongs to ischemic stroke, mainly because of hardening of cerebral artery blood vessel, stenosis gradually develops into obstruction; the embolus in the heart can be blocked when flowing along blood vessels in the brain after falling off, so that the blood supply of the brain is interrupted, and the brain tissue is anoxic or necrotic. At present, there are many methods for clinically treating ischemic stroke, such as drug therapy for resisting platelet aggregation, promoting blood circulation to remove blood stasis, improving cerebral circulation, scavenging oxygen free radicals, improving cerebral metabolism, reducing intracranial pressure by using a dehydrating agent, and the like, however, these drugs have good effects on prevention and protection of ischemic stroke, but the treatment effect on ischemic stroke diseases is not satisfactory, so that the clinical significance is still provided for drug therapy research on ischemic stroke. Ginkgolide B (Ginkgolide B, GB) belongs to a first-line medicine for treating cerebral apoplexy, is a strong antagonist of Platelet Activating Factor (PAF), has obvious curative effect on cardiovascular and cerebrovascular diseases, is considered to be a natural PAF receptor antagonist with the most clinical application prospect at present, and has the effects of scavenging oxygen free radicals, inhibiting lipid peroxidation and the like.

GB is an antagonist of PAF (the strongest platelet aggregation inducer is found so far), however, GB is extremely small in water solubility and greatly limited in clinical use, in order to enable GB to have wider application in the aspect of treating cerebral apoplexy, GB is loaded in recombinant high-density lipoprotein, encapsulation of drugs with low solubility is achieved so as to be beneficial to the drugs to reach lesion sites to exert curative effects better, and the recombinant high-density lipoprotein is an endogenous substance and can be completely biodegraded without causing immune response and the like, GB can well pass through a blood brain barrier after being prepared into nanoparticles, and has the effects of reducing cholesterol of peripheral blood vessels of brain, reducing platelet aggregation and protecting neurons. Although the recombinant high-density lipoprotein can reduce the recognition and clearance of reticuloendothelial tissues and has a long half-life in vivo, the recombinant high-density lipoprotein is inevitably phagocytized by immune cells as a foreign body, so that the development of a long-acting carrier with good biocompatibility and targeting property is important for the research of the present day.

Researches in recent years show that the platelet membrane wrapped nanoparticles can reduce phagocytosis of macrophages, more importantly, the platelet membrane can be selectively combined with damaged blood vessels in a human body and enhance adhesion to pathogens, and platelets at the focal part of ischemic stroke are activated and adhered to subendothelial tissues of the damaged blood vessels, so that the platelet membrane wrapped nanoparticles can directionally reach the damaged part of brain tissues, improve biocompatibility of the nanoparticles, avoid phagocytosis by a reticuloendothelial system, and achieve the purpose of long circulation.

Although the platelet membrane coated nanoparticles have certain targeting property and can actively target the damaged part of the tissue, the targeting efficiency is limited. In order to achieve better therapeutic effect, the drug for treating ischemic stroke is delivered to a target site, and the surface of the carrier needs to be subjected to targeted modification. The research shows that the Cerebral Ischemia Targeting Peptide (CITP) is screened from a cerebral ischemia animal model with Middle Cerebral Artery (MCA) blockage, and can be detected to be positioned in apoptotic neuron cells at a cerebral ischemia part and generally combined with damaged neuron cells with recoverability. Therefore, after the polypeptide is combined with a medicament for treating ischemic stroke, the concentration of the medicament in a diseased tissue area can be improved, and the occurrence of side effects can be reduced.

In summary, the patent uses the recombinant high-density lipoprotein coated GB as a drug-loaded core, a platelet membrane and a CITP (amino acid sequence: CLEVSRKNC) modified on the surface of the platelet membrane as a bionic shell to construct a novel nanoparticle for treating ischemic stroke, and the biological camouflage targeted nano drug delivery system can solve the problems: (1) the defect of low bioavailability of a fat-soluble medicament GB after direct intravenous injection administration is overcome, and the GB is wrapped in the recombinant high-density lipoprotein to better play the therapeutic effect of the medicament; (2) the platelet membrane is used as a drug carrier, has incomparable biocompatibility, degradability and long circulation capability compared with other drug carriers, can improve targeting property and better repair focal parts of cerebral ischemia by using the platelet membrane as the drug carrier, avoids being cleared by an immune system, and improves the long circulation effect of the nano preparation in vivo.

Disclosure of Invention

The invention aims to provide a biological camouflage nano drug delivery system for treating ischemic stroke and a preparation method thereof. The invention constructs a novel nanoparticle for treating cerebral arterial thrombosis by using the recombinant high-density lipoprotein coated GB as a drug-loaded inner core, a platelet membrane and CITP (amino acid sequence: CLEVSRKNC) modified on the surface of the platelet membrane as a bionic shell, and the nano drug delivery system improves the circulation time in vivo and has good targeting property.

In order to solve the problems, the invention adopts the following technical scheme:

a biological camouflage nanometer drug delivery system for treating ischemic stroke comprises a drug, an inner core drug carrier, a biological camouflage outer shell and a targeting material, wherein the drug is GB, the inner core carrier is recombinant high-density lipoprotein, and a drug-loaded inner core is formed after the drug is coated in a physical embedding way; the biological camouflage shell is a platelet membrane, and the platelet membrane is used for coating a drug-loaded inner core in a passive combination mode to form drug-loaded nanoparticles; the targeting material is CITP, and the amino acid sequence of the targeting material is CLEVSRKNC; the CITP, polyethylene glycol (PEG) and Stearic Acid (SA) are connected through covalent bonds to form a compound CITP-PEG-SA, and the compound CITP-PEG-SA is modified on the surface of the nanoparticle of the platelet membrane entrapped drug-carrying core in a CITP-PEG-SA mode to form a biological camouflage targeted nano drug delivery system.

Further, the recombinant high-density lipoprotein is prepared from phospholipid, cholesterol ester and apolipoprotein.

Further, the preparation process of the platelet membrane comprises the following steps: whole blood is extracted from healthy SD rats, platelets are separated by adopting a differential centrifugation method, platelet fragments are obtained by a freeze-thaw method, and finally platelet membranes are obtained by ultrasonic crushing and centrifugation.

Further, the phospholipid is one or more of lecithin, cephalin, inositol phospholipid, phosphatidyl serine or phosphatidic acid; the cholesterol ester is one or more of cholesterol decanoate, cholesterol laurate, cholesterol myristate, cholesterol palmitate, cholesterol stearate, cholesterol oleate or cholesterol linoleate; the apolipoprotein is apoA-I.

The invention provides a preparation method of a biological camouflage nano drug delivery system for treating ischemic stroke, which comprises the following steps:

a, preparation of a drug-loaded core:

(1) dissolving phospholipid, cholesterol and cholesterol ester in chloroform to obtain a material A, dissolving GB in methanol to obtain a material B, dripping the material B into the material A at the speed of 3-5 ml/min, carrying out reduced pressure rotary evaporation in a water bath until a uniform honeycomb-shaped oil film is formed, and then placing the material in a vacuum drier for drying overnight to obtain a material C;

(2) adding pure water into the material C, carrying out reduced pressure rotary evaporation for 10-30 min, carrying out probe ultrasonic treatment to obtain a clear solution with opalescence, adding apolipoprotein apoA-I by using a post-insertion method, stirring overnight at 2-10 ℃, adding into a protein dialysis bag with the aperture of 50-100 kDa, and standing in PBS solution at 2-10 ℃ for dialysis overnight to obtain a drug-loaded core;

b, preparing a biological camouflage targeted nano drug delivery system:

(3) mixing the drug-loaded core and the extracted platelet membrane together according to the volume ratio of 1: 1-1: 5, repeatedly extruding for 3-9 times by an extruder to realize the fusion of the platelet membrane and the drug-loaded core, and then centrifuging at the speed of 3000r/min to remove the redundant platelet membrane to prepare a material D;

(4) and dissolving CITP-PEG-SA in a PBS solution to prepare a material E, dropwise adding the material E into the material D, stirring overnight, then adding into a protein dialysis bag with the aperture of 1000-3500 Da, and dialyzing overnight in the PBS solution to obtain the biological camouflage nano drug delivery system.

Further, in the step A, the mass ratio of the phospholipid to the cholesterol is 4: 1-8: 1, the mass ratio of the phospholipid to the cholesterol ester is 25: 1-15: 1, the mass ratio of the phospholipid to the ginkgolide B is 25: 1-15: 1, and the mass ratio of the phospholipid to the apolipoprotein apoA-I is 25: 1-15: 1.

Further, in the step A, the ultrasonic power of the probe is 200w, the work time is 2s, the pause time is 1s, and the total ultrasonic time is 10 min.

Further, the extruder is an extruder containing a polyester carbonate film with a pore diameter of 400 nm; the pH of the PBS solution was 7.4.

Further, the steps of extracting the platelet membrane by the freeze-thaw method are as follows: placing the obtained platelets in an anticoagulation centrifuge tube, standing for 30min, centrifuging for 20min by 100g, sucking supernatant, dropwise adding PBS buffer solution containing 1mM EDTA and 2mM prostaglandin E1, centrifuging for 20min by 800g, discarding supernatant, resuspending the platelets in hypotonic PBS solution, freezing the platelets in a refrigerator at-80 ℃ for 6-12 h, unfreezing at 25 ℃ for 1-6 h, repeating for 3 times, centrifuging for 30min by 3000r/min of freeze-thaw solution, respectively collecting platelet fragments and supernatant, suspending the platelet fragments in the PBS solution, washing and centrifuging, ultrasonically crushing for 5min by a 50/60Hz probe-type ultrasonic instrument, centrifuging for 3000r/min, and collecting supernatant.

The invention principle is as follows:

after the biological camouflage nano drug delivery system provided by the invention is injected into a human body, the nano particles can be brought into a cerebral ischemia part under the action of CITP; due to the phenomena of vascular injury and thrombosis at the focal part of cerebral ischemia, the platelet membrane in the nano drug delivery system plays a role in targeting the vascular injury part. Under the cooperative targeting action of CITP and platelet membrane, the nanoparticles enter the cerebral ischemia focus part accurately, and the nano carrier has biodegradability, so that the medicament GB can be released, and the aim of treating diseases is fulfilled.

Has the advantages that:

compared with the prior art, the invention has the following advantages:

(1) the medicament for treating cerebral ischemia diseases adopted by the invention is GB, is one of main active ingredients of traditional Chinese medicine ginkgo leaf extract, and is an effective medicament capable of inhibiting platelet aggregation, resisting inflammation, protecting heart and cerebral vessels, treating acute and chronic cerebral ischemia diseases and the like. GB is a platelet activating factor specific receptor antagonist, but GB has little water solubility and limited clinical use. In order to enable GB to be widely applied to the treatment of ischemic stroke diseases, GB is wrapped in recombinant high-density lipoprotein, so that the water solubility of GB can be increased, and the in-vivo bioavailability of GB is improved.

(2) The platelet membrane and the nanoparticles are combined to form a novel bionic nano targeting preparation, and the platelet membrane has the advantages of incomparable biocompatibility and degradability and the like compared with other drug carriers, and can tend to the damaged part of the blood vessel and effectively repair the damaged blood vessel. Therefore, in conclusion, the platelet membrane coated nanoparticles can enhance targeting property to better repair the focal part of ischemia, avoid immunological rejection reaction and improve the long circulation effect of the nano preparation in vivo.

Drawings

Fig. 1 is a transmission electron microscope image of the biological camouflage targeted nano drug delivery system of the invention. Wherein, the A picture is medicine-carrying inner core (rHDL/GB), the B picture is Platelet Membrane (PM), and the C picture is biological camouflage targeting nano medicine delivery system (CITP-PEG-SA/PM/rHDL/GB).

Fig. 2 is a graph of particle size and potential in the biosigneous targeted nano drug delivery system of the present invention. Wherein the left graph is a particle size graph of rHDL/GB and CITP-PEG-SA/PM/rHDL/GB, and the right graph is a potential graph of rHDL/GB, PM and CITP-PEG-SA/PM/rHDL/GB.

Fig. 3 is a cellular uptake map in a biomassaging targeted nano-drug delivery system of the present invention. Wherein the upper left graph is the uptake of CITP-PEG-SA/PM/rHDL cells without fluorescent markers, and the upper right graph is the uptake of CITP-PEG-SA/DiI-PM/Coumarin-6-rHDL cells with double fluorescent markers; the bottom left panel is the DiI fluorescence intensity in cellular uptake and the bottom right panel is the Coumarin-6 fluorescence intensity in cellular uptake.

Fig. 4 is a confocal view of laser in the biological camouflage targeted nano drug delivery system of the invention. Wherein the upper left graph is red fluorescence of DiI in the dual-fluorescence labeled CITP-PEG-SA/DiI-PM/Coumarin-6-rHDL, the lower left graph is green fluorescence of Coumarin-6 in the dual-fluorescence labeled CITP-PEG-SA/DiI-PM/Coumarin-6-rHDL, the right graph is the superposition of the green fluorescence and the red fluorescence of the dual-fluorescence labeled CITP-PEG-SA/DiI-PM/Coumarin-6-rHDL,

fig. 5 is a targeting distribution map of the biological camouflage targeting nano drug delivery system of the invention in the brain of a model rat. The upper row of the graph is the fluorescence distribution graph of each administration group in the rat brain when the administration is carried out for 6h, and the lower row of the graph is the fluorescence distribution graph of each administration group in the rat brain when the administration is carried out for 24 h.

Fig. 6 is a graph of the biomimic targeted nano drug delivery system of the present invention staining a brain slice of a molding rat. Wherein, from top to bottom, the group of pseudo surgery (Sham), the group of Model (MCAO), the group of vector (Vehicle), the group of GB, the group of rHDL/GB, the group of PM/rHDL/GB and the group of CITP-PEG-SA/PM/rHDL/GB are respectively.

Fig. 7 is a graph showing the effect of the biological camouflage targeting nano drug delivery system of the invention on the cerebral infarction of model-making rats, and the graph is a cerebral infarction area graph calculated by staining the brain sections of the groups in fig. 6.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

A preparation method of the biological camouflage nano drug delivery system comprises the following steps:

examples 1 to 7

1. Preparing a drug-loaded core-recombinant high-density lipoprotein/ginkgolide B (rHDL/GB) nanoparticle:

respectively dissolving phospholipid, cholesterol and cholesterol ester in chloroform, dissolving GB in methanol, slowly adding dissolved GB dropwise into chloroform solution, removing organic solvent by rotary evaporation under reduced pressure in 37 deg.C water bath to form uniform cellular oil film, and drying overnight in vacuum drier to remove residual organic solvent. And then adding pure water, carrying out reduced pressure rotary steaming for 10-30 min, carrying out ultrasonic treatment by using a probe, wherein the ultrasonic power of the probe is 200w, the work time is 2s, the intermission time is 1s, and the total ultrasonic time is 10min, adding apolipoprotein apoA-I by using a post-insertion method, stirring overnight at 4 ℃, then adding into a protein dialysis bag with the aperture of 50-100 kDa, and dialyzing overnight in PBS (pH 7.4) at 4 ℃ to obtain rHDL/GB.

Wherein, the process conditions adopted for preparing the drug-loaded core in the examples 1 to 7 are shown in the table 1:

table 1 shows the process conditions used to prepare drug loaded cores of examples 1-7

Examples 8 to 10

2. The extraction step of the platelet membrane:

extracting platelet membrane by a freeze-thaw method, placing collected blood in an anticoagulation centrifuge tube, standing for 30min, centrifuging for 20min at 100g, sucking supernatant, dropwise adding PBS buffer solution containing 1mM EDTA and 2mM prostaglandin E1 to prevent platelet activation, centrifuging for 20min at 800g, discarding supernatant, resuspending platelets in isotonic PBS solution (pH 7.4), freezing for 6-12 h in a refrigerator at-80 ℃, unfreezing for 1-6 h at 25 ℃, repeating for 3 times, centrifuging for 30min at 3000r/min for freeze-thaw solution, collecting platelet fragments and supernatant respectively, suspending the platelet fragments in PBS solution (pH 7.4), washing and centrifuging, ultrasonically crushing for 5min by a probe type ultrasonic instrument at 50/60Hz, centrifuging at 3000r/min, and collecting supernatant.

TABLE 2 Process conditions used for the preparation of the platelet membranes of examples 8-10

Process conditions	Example 8	Example 9	Example 10
				Freezing time/h	6	12	10
Thawing time/h	2	6	4

Examples 9 to 12

3. Preparation of biological camouflage nano drug delivery system-CITP-PEG-SA/platelet membrane/recombinant high density lipoprotein/ginkgolide B (CITP-PEG-SA/PM/rHDL/GB) nanoparticle:

(1) mixing the prepared drug-loaded core and the extracted platelet membrane together according to the volume ratio of 1: 1-1: 5, repeatedly extruding for 3-9 times by an extruder containing a polyester carbonate membrane with the aperture of 400nm to realize the fusion of the platelet membrane and the nanoparticles, and centrifuging at a high speed to remove the redundant platelet membrane to obtain the drug-loaded nanoparticles.

(2) Dissolving CITP-PEG-SA in a PBS solution (pH 7.4), dropwise adding the solution into the drug-loaded nanoparticles, stirring overnight, adding the solution into a protein dialysis bag with the aperture of 1000-3500 Da, and dialyzing overnight in the PBS solution to obtain CITP-PEG-SA/platelet membrane/recombinant high-density lipoprotein/ginkgolide B nanoparticles (CITP-PEG-SA/PM/rHDL/GB), namely the biological camouflage targeted nano drug delivery system.

Wherein, the process conditions used to prepare the biosignal nano-drug delivery system in examples 9-12 are shown in table 2:

table 2 Process conditions used to prepare the Biocamouflage targeted nano-drug delivery systems of examples 9-12

Secondly, the performance characterization of the biological camouflage targeting nano drug delivery system:

A. characterization of recombinant high density lipoprotein/ginkgolide B (rHDL/GB) nanoparticles:

(1) particle size and potential measurement: the particle size and Zeta potential of the prepared rHDL/GB nanoparticles are measured by a laser particle size analyzer, the particle size of the rHDL/GB nanoparticles can be seen from an attached drawing 2 to be about 80nm, the potential size is about-4 mv, the morphology of the nanoparticles is observed by a Transmission Electron Microscope (TEM) and a picture A in the attached drawing 1 is photographed, and therefore the particle size of the nanoparticles can be proved to meet the requirement of being capable of entering a blood brain barrier.

(2) And (3) determining the encapsulation efficiency of the ginkgolide B: transferring 1ml of the rHDL/GB nanoparticles prepared into a 10ml volumetric flask with methanol, shaking up, measuring and recording peak areas of samples by an HPLC method according to certain chromatographic conditions, and measuring the GB content in the solution by substituting a GB standard curve to obtain the total drug dosage M1 in the preparation; then filtering the prepared nanoparticles by a 0.45-micron polycarbonate filter membrane to remove free drugs, then transferring 1mL of the filtrate, using methanol to fix the volume of the filtrate in a 10-mL volumetric flask, shaking up to obtain the drug quantity M2 wrapped in the carrier, and calculating the drug Encapsulation Efficiency (EE) by the following formula:

according to the data obtained by the operation, the encapsulation rate of the nanoparticles can be calculated to be about 88 percent, so that the encapsulation rate of the nanoparticles can meet the requirement of the administration dosage.

CITP-PEG-SA/platelet membrane/recombinant high density lipoprotein/ginkgolide B (CITP-PEG-SA/PM/rHDL/GB)

Characterization of the nanoparticles:

(1) particle size and potential measurement: the particle size and Zeta potential of the prepared CITP-PEG-SA/PM/rHDL/GB nanoparticles are measured by a laser particle size analyzer, and the particle size and the potential of the CITP-PEG-SA/PM/rHDL/GB nanoparticles are about 100nm and about-10 mv in figure 2, so that the CITP-PEG-SA/PM/rHDL/GB nanoparticles have obvious difference in comparison with the rHDL/GB nanoparticles in figure 2.

(2) Transmission electron microscopy characterization: to further prove that the nanoparticle-coated modified film was observed by Transmission Electron Microscopy (TEM) and photographed as A, B, C in fig. 1, the nanoparticle-coated modified film is clearly shown in the three figures.

(3) Characterization of the streaming dual uptake: taking a proper amount of CITP-PEG-SA/PM/rHDL and CITP-PEG-SA/DiI-PM/Coumarin-6-rHDL suspension with double fluorescence labels to detect in a flow cytometer, wherein the inner core of the nanoparticle marked by the Coumarin-6 is green fluorescence, the cell membrane marked by the DII is red fluorescence, the combination of the DiI-PM and the Coumarin-6-rHDL can be identified by the flow cytometer, and the fluorescent qualitative analysis is carried out on the nanoparticle according to the intensity of fluorescein carried by the nanoparticle.

Examining the degree of cell membrane adsorption on the nanoparticles, as shown in fig. 3, it can be seen that the nanoparticles labeled by double fluorescence are distinguished from the particles not labeled by carried double fluorescence, and are mainly distributed in the upper right region of the flow density diagram, indicating that a vast proportion of the complexes are labeled by double fluorescence, and further verifying that the nanoparticles labeled by double fluorescence have green fluorescence and red fluorescence at the same time, thereby showing that the nanoparticles are wrapped by the modified membrane.

(4) Characterization of laser confocal positioning: the CITP-PEG-SA/DiI-PM/Coumarin-6-rHDL nanoparticle with double fluorescence labels is placed in a confocal dish for shooting, as shown in figure 4, the nanoparticle with double fluorescence labels has green fluorescence and red fluorescence at the same time, and therefore, the modified film is further proved to be wrapped by the nanoparticle.

C. The biological camouflage nano drug delivery system targets the cerebral ischemia area of a model rat:

the ginkgolide B marked with DIR is wrapped in a nanoparticle carrier to investigate the targeting property of the nanoparticles at the cerebral ischemia part, and the specific preparation method is the same as the above. The prepared GB, rHDL/GB, PM/rHDL/GB, CITP-PEG-SA/PM/rHDL/GB with fluorescence markers are injected into a modeled SD male rat through tail vein, the rat at 6h and 24h after administration is directly decapitated and killed, the brain of the rat is taken out and placed in a living body imaging instrument, the rat is subjected to shooting and investigation, the experimental result is shown in figure 5, the fluorescence distribution is stronger than 24h when the drug is administered for 6h, and the targeting of a final preparation group is better than that of other control groups, so that the novel biological camouflage nano drug delivery system can well pass through the blood brain barrier and can effectively improve the targeting of the drug at a focus part.

D. The biological camouflage nano drug delivery system has the improvement effect on cerebral infarction of model rats:

calculating cerebral infarction area by adopting a 2, 3, 5-triphenyltetrazolium chloride (TTC) staining method, randomly grouping rats subjected to surgery modeling, setting different administration control groups which are respectively a Sham surgery group (Sham), a model group (MCAO), a carrier group (Vehicle), GB, rHDL/GB, PM/rHDL/GB, CITP-PEG-SA/PM/rHDL/GB, performing coronary section after administration for 24 hours at visual crossover and about 2mm before and after visual crossover, rapidly placing brain slices into phosphate buffer solution containing 1% of TTC after each brain slice is cut into 5 slices, separating a white area (infarct area) and a non-white area (normal area) by using ophthalmological forceps in constant-temperature water bath at 37 ℃, wherein the normal brain tissue is red, the infarct tissue is not colored, and calculating the percentage of infarct area. The experimental results are shown in figures 6 and 7, and it can be seen from the figures that the cerebral infarction area of the treatment group is smaller than that of the model group, which shows that the medicine has better treatment effect and can obviously improve cerebral infarction symptoms.

Claims

1. a biological camouflage targeting nano-drug delivery system for the treatment of ischemic stroke, is characterized in that, comprises medicine, inner core drug carrier, biological camouflage shell and homing material, and described medicine is fat-soluble anti-ischemia Stroke drug; the inner core carrier is recombinant high-density lipoprotein, which forms a drug-loaded inner core after the drug is encapsulated by physical embedding; the biological camouflage shell is a platelet membrane, and the platelet membrane is co-extruded After the drug-loaded core is encapsulated in the method, biomimetic drug-loaded nanoparticles are formed; the targeting material is cerebral ischemia targeting peptide CITP, and its amino acid sequence is CLEVSRKNC; the cerebral ischemia targeting peptide CITP and polyethylene glycol ( PEG) and stearic acid (SA) are connected by covalent bonds to form the compound CITP-PEG-SA, which is modified in the form of CITP-PEG-SA on the surface of the nanoparticle containing the drug-loaded core in the platelet membrane to form a biologically camouflaged targeting nanodelivery. medicine system.

2. a kind of biological camouflage targeted nano drug delivery system for ischemic stroke treatment according to claim 1, is characterized in that, described platelet membrane preparation process is: extract whole blood from healthy SD rat , using differential centrifugation to separate platelets, then using freeze-thaw method to obtain platelet fragments, and finally to obtain platelet membranes by ultrasonication and centrifugation.

3. A bio-camouflaged targeted nano-drug delivery system for the treatment of ischemic stroke according to claim 1, wherein the recombinant high-density lipoprotein is composed of phospholipids, cholesterol, cholesterol esters and carrier lipids prepared from protein.

4 . The biologically camouflaged targeted nano-drug delivery system for the treatment of ischemic stroke according to claim 1 , wherein the lipid-soluble anti-ischemic stroke drug is ginkgolide B. 5 .

5. a kind of biological camouflage targeted nano-drug delivery system for ischemic stroke treatment according to claim 3, is characterized in that, described phospholipid is lecithin, cephalin, inositol phospholipid, phosphatidyl One or more of serine or phosphatidic acid; the cholesterol ester is cholesteryl caprate, cholesteryl laurate, cholesteryl myristate, cholesteryl palmitate, cholesteryl stearate, cholesteryl oleate or One or more of cholesteryl linoleate; the apolipoprotein is apoA-I.

6. the preparation method of a kind of biological camouflage targeted nano-drug delivery system for ischemic stroke treatment according to any one of claims 1-5, is characterized in that, comprises the following steps:

A. Preparation of drug-loaded cores:

(1) dissolving phospholipid, cholesterol and cholesterol ester in chloroform to obtain material A, dissolving ginkgolide B in methanol to obtain material B, then adding material B dropwise to material A at a speed of 3～5ml/min, Then under reduced pressure and rotary evaporation in a water bath to form a uniform honeycomb oil film, then placed in a vacuum desiccator and dried overnight to obtain material C;

(2) After adding pure water to feed C, vacuum rotary steaming for 10-30min, then carrying out probe ultrasonic treatment to obtain a clear solution with opalescence, then using post-insertion method to add apolipoprotein apoA-I, 2-10 Stir overnight at ℃, then add it to a protein dialysis bag with a pore size of 50 to 100 kDa, and place it in a PBS solution at 2 to 10 ℃ for overnight dialysis to obtain a drug-loaded core;

B. Preparation of bio-camouflage targeted nano-drug delivery system:

(1) Mix the drug-loaded inner core and the extracted platelet membrane in a volume ratio of 1:1 to 1:5, and repeatedly extrude the platelet membrane and the drug-loaded core for 3 to 9 times through an extruder to fuse the platelet membrane and the drug-loaded core. The excess platelet membrane was removed by centrifugation at a speed of 3000 r/min to prepare feed D;

(2) Dissolve CITP-PEG-SA in PBS solution to prepare feed E, then add feed E dropwise to feed D and stir overnight, and then add it to a protein dialysis bag with a pore size of 1000-3500Da, in PBS solution The bio-camouflage targeted nano-drug delivery system was obtained after overnight dialysis.

7. the preparation method of a kind of biological camouflage targeted nano drug delivery system for ischemic stroke treatment according to claim 6, is characterized in that, in step A, the mass ratio of described phospholipid and cholesterol is 4 : 1～8:1, the mass ratio of phospholipid to cholesterol ester is 25:1 to 15:1, the mass ratio of phospholipid to ginkgolide B is 25:1 to 15:1, and the mass ratio of phospholipid to apolipoprotein apoA-I is 25:1 to 15:1. The mass ratio is 25:1 to 15:1.

8. The preparation method of a biologically camouflaged targeted nano-drug delivery system for the treatment of ischemic stroke according to claim 6, wherein in step A, the ultrasonic power of the probe is 200w, the work is 2s, and the intermittent 1s, and the total ultrasonic time is 10min.

9. The preparation method of a biologically camouflaged targeted nano-drug delivery system for ischemic stroke treatment according to claim 6, wherein the extruder is a polyester carbonate containing a pore diameter of 400 nm Ester film extruder, the pH of the PBS solution is 7.4.

10. A bio-camouflage targeted nano-drug delivery system for the treatment of ischemic stroke according to claim 2, characterized in that, the step of extracting platelet membranes by the freeze-thaw method is as follows: the obtained platelets are placed in Place in an anticoagulated centrifuge tube for 30min, centrifuge at 100g for 20min to absorb the supernatant, add dropwise PBS buffer containing 1mM EDTA and 2mM prostaglandin E1, then centrifuge at 800g for 20min to discard the supernatant, and resuspend the platelets in a low Infiltrate the PBS solution, put it in a refrigerator at -80 °C for 6-12 hours, then thaw it at 25 °C for 1-6 hours, repeat 3 times, centrifuge the freeze-thaw solution at 3000 r/min for 30 minutes, and collect platelet fragments and supernatant respectively. , the platelet fragments were suspended in PBS solution, washed and centrifuged, sonicated for 5 min with a 50/60 Hz probe-type sonicator, centrifuged at 3000 r/min, and collected.