CN114288264A - Brain injury inflammation part tropism biomimetic nano system and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a brain injury inflammation part tropism biomimetic nano system and a preparation method and application thereof. The preparation method adopts an antioxidant ambucol and an amphiphilic polymer PEG-b-PDPA to form PP/SCB nanoparticles by a thin film hydration method, and 4T1 cell films are coated on the outer layer to form biomimetic MPP/SCB nanoparticles with brain injury inflammation part tropism; by utilizing the inflammation tropism and the blood brain barrier crossing capacity of the 4T1 tumor cells, the problem that the existing bionic nano system has limited brain injury inflammation part targeting capacity or blood brain barrier crossing capacity is solved. The biomimetic nano system has a better and obvious treatment effect on cerebral arterial thrombosis reperfusion injury.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to a biomimetic nano system, and particularly relates to a biomimetic nano system for tropism of a brain injury inflammation part and a preparation method and application thereof.
Background
It is well documented that stroke is an acute cerebrovascular disease that causes damage to brain tissue due to the obstruction or rupture of cerebral blood vessels, which leads to the disturbance of cerebral blood circulation. Research shows that ischemic stroke accounts for about 60-80% of total stroke, and the disease has five characteristics of high morbidity, high disability rate, high mortality, high recurrence rate and high economic burden, and seriously affects the life quality and life health of patients. The gold standard for clinical treatment of ischemic stroke is to restore blood supply to ischemic brain tissue in time within a treatment time window (within 4.5 hours) by thrombolysis or mechanical embolectomy, etc. Clinical practice has shown that most patients have missed optimal treatment times when admitted due to strict thrombolysis time window limitations, at which point reperfusion injury will result if thrombolytic therapy is resumed. The pathological mechanisms of reperfusion injury comprise excitotoxicity, calcium overload, oxidative stress, inflammatory reaction and the like, and the clinical treatment effect of reperfusion injury is far from expected at present, so that the exploration and development of effective treatment drugs for ischemic stroke reperfusion injury are important subjects of the basic and clinical research of the related field of ischemic stroke.
A large body of research evidence suggests that oxidative stress and inflammatory responses play a significant role in reperfusion injury. Reperfusion results in the production of large amounts of reactive oxygen species in a short period of time, inducing oxidative stress and exacerbating the inflammatory response. Probucol is a blood fat reducing drug, has antioxidant and anti-inflammatory effects, and has a protective effect on cerebral ischemia reperfusion injury of rats proved by researches; succinbocucol (SCB) is a mono-succinate derivative of a blood fat reducing drug, namely probucol, has stronger antioxidant and anti-inflammatory effects than probucol, has great potential in the aspect of treating reperfusion injury caused by ischemic stroke, but has extremely limited clinical application because SCB has extremely low water solubility and cannot cross a blood brain barrier to reach ischemic brain tissues.
Researches in recent years show that the slightly soluble drug is prepared into nanoparticles, and cell membranes are wrapped on the surfaces of the nanoparticles to prepare a bionic nano system, so that the solubility of the drug can be improved, and the drug can cross blood brain barriers; commonly used cell membranes include: although the biomimetic nano systems have certain targeting capacity across a blood brain barrier and a brain injury inflammation part, the brain injury inflammation part has limited targeting capacity or blood brain barrier crossing capacity, and extra targeting modification such as cerebral ischemia targeting peptide or T7 Tag polypeptide is often required on the surface of a carrier to promote more drugs to be distributed to the brain injury inflammation part; practice shows that the biochemical-simulating nanometer preparation has complex components and complicated preparation process. Therefore, the development of a nano preparation with simple components and good brain injury inflammation site tropism and blood brain barrier crossing capability has attracted the attention of the technicians in the industry.
The 4T1 cell is a breast cancer cell with high brain metastasis capacity, and the VCAM-1 protein and the CD138 protein are highly expressed on the surface of the cell membrane. Research shows that VCAM-1 protein can be combined with VLA-4 receptors on the surfaces of leukocytes which are gathered in large numbers on vascular endothelium at a brain injury inflammation part, so that 4T1 cells have good brain inflammation part tropism; the CD138 protein may help 4T1 cells cross the blood brain barrier to reach the brain.
Based on the foundation and the current situation of the prior art, the inventor of the application plans to adopt 4T1 cell membrane-loaded nanoparticles to prepare a biochemical nano system so as to achieve the effects of improving the aggregation of the drug at the brain injury inflammation part and crossing the blood brain barrier; in particular to a brain injury inflammation part tropism biomimetic nano system and a preparation method and application thereof.
Disclosure of Invention
The invention aims to provide a biomimetic nano system for the tropism of a brain injury inflammation part and a preparation method and application thereof based on the foundation and the current situation of the prior art, and particularly relates to a simple biomimetic nano particle prepared by utilizing the inflammation tropism and the blood brain barrier crossing capability of 4T1 tumor cells, which improves the aggregation of amber at the brain injury inflammation part and the blood brain barrier crossing effect so as to realize better treatment effect of ischemic stroke reperfusion injury.
The invention uses amphiphilic high molecular copolymer PEG-b-PDPA to load SCB to prepare nano-particles PP/SCB, and uses 4T1 cell membrane to pack PP/SCB to form bionic nano-particles MPP/SCB with encephalitis part tropism.
Specifically, the antioxidant ambucol and the amphiphilic polymer PEG-b-PDPA are adopted to form PP/SCB nanoparticles by a thin film hydration method, and 4T1 cell films are wrapped on the outer layer to form tropism bionic MPP/SCB nanoparticles at brain injury inflammation parts; by utilizing the inflammation tropism and the blood brain barrier crossing capacity of the 4T1 tumor cells, the problem that the existing bionic nano system has limited brain injury inflammation part targeting capacity or blood brain barrier crossing capacity is solved.
More specifically, the invention provides a biomimetic nano system MPP/SCB with brain injury inflammation site tropism.
The biomimetic nano system comprises a medicament, a medicament carrier and a biomimetic cell membrane, wherein the medicament is an antioxidant medicament for treating ischemic stroke; the drug carrier is an amphiphilic polymer, and the drug carrier is coated by a thin film hydration method to form inner core nanoparticles; the biomimetic cell membrane is a tumor cell membrane, and the 4T1 tumor cell membrane is wrapped with the inner core nanoparticles in an extrusion mode to form a biomimetic nano system with tropism for brain injury inflammation parts.
In the invention, the antioxidant medicine for treating ischemic stroke is preferably probucol, succinobucol, curcumin and baicalein, and more preferably succinobucol.
In the invention, the amphiphilic polymer drug carrier is polyethylene glycol-poly (diisopropylamino ethyl methacrylate) (PEG-b-PDPA).
In the present invention, the tumor cell membrane is: breast cancer cells (4T1 cells) with high brain metastasis capacity are lysed by using a cell lysate, and are crushed into fragments by repeated extrusion, and finally, a 4T1 cell membrane is obtained by separating by using a differential centrifugation method.
The brain injury inflammation part tropism biomimetic nano system is prepared by the following method, and comprises the following steps:
(1) preparing PP/SCB nanoparticles: dissolving SCB and PEG-b-PDPA in ethanol together, performing reduced pressure rotary evaporation in a water bath to form a film, adding pure water, and hydrating to form PP/SCB;
(2) preparing MPP/SCB nanoparticles: ultrasonically mixing 4T1 cell membranes with PP/SCB, and repeatedly extruding by an extruder to obtain MPP/SCB;
the ratio of the SCB to the PEG-b-PDPA is 2:1-1:10, and the hydration time is 30 min;
the extraction steps of the 4T1 cell membrane are as follows: scraping 4T1 cells from the culture dish with a cell scraper and collecting into a centrifuge tube, adding membrane protein extraction reagent A (1ml) containing 1mM protease inhibitor (PMSF), and resuspending the cells in an ice bath for 15 min; then repeatedly extruding the cell suspension through a polycarbonate membrane for 30 times to break the cells, then centrifuging the cell debris suspension at 700g for 10min at 4 ℃, collecting supernatant to remove cell nuclei and uncleaved cells, finally centrifuging the supernatant at 14,000g for 30min at 4 ℃ to obtain cell membrane precipitates, and measuring the quality of the purified 4T1 cell membrane protein by using a BCA protein quantitative kit for subsequent use;
in the step (2), the mass ratio of the 4T1 cell membrane (based on the mass of the contained membrane protein) to the PP/SCB is 1:1-1:10, preferably, the mass ratio of the 4T1 cell membrane to the PP/SCB is 1: 5;
in the step (2), a polycarbonate film extruder with a pore diameter of 200nm is adopted, and the repeated extrusion times are 7-15 times, preferably 11 times.
The invention further provides application of the tropism bionic nano system MPP/SCB of the brain injury inflammation part in preparation of a medicine for treating cerebral arterial thrombosis.
The invention carries out the in-vitro ROS eliminating capability of MPP/SCB and the protection capability test on ROS damaged cells, and the results (shown in figures 2A and 2B) show that PP/SCB and MPP/SCB can obviously eliminate ROS in PC12 cells, and compared with PP/SCB, MPP/SCB has stronger ROS eliminating capability; the results shown in FIGS. 2C and 2D show that both PP/SCB and MPP/SCB significantly reduced apoptosis of PC12 by tBHP, and that MPP/SCB was more protective of PC12 cells damaged by ROS than PP/SCB.
The MPP/SCB is measured by the blood brain barrier crossing capability in vitro, and the results of quantitative detection and qualitative observation are shown in figures 3B and 3C, so that compared with free DiR, the blood brain barrier crossing capability of PP/DiR and MPP/DiR is obviously improved; with the prolonged incubation time, the fluorescence in PC12 cells incubated with PP/DiR and MPP/DiR is obviously enhanced, which shows that the amount of PP/DiR and MPP/DiR crossing the blood brain barrier is obviously increased; in addition, the fluorescence intensity of PC12 cells incubated with MPP/DiR is stronger than that of PP/DiR group at different incubation times, which indicates that MPP/DiR has stronger blood brain barrier crossing capability than that of PP/DiR.
The in vivo distribution experimental results of the MPP/DiR are shown in fig. 4A and 4C, the free DiR group brain basically has no fluorescence, which indicates that the free DiR is difficult to cross the blood brain barrier to reach the brain; fluorescence can be observed in the brain by the PP/DiR group and the MPP/DiR group, which shows that the PP/DiR and the MPP/DiR can cross the blood brain barrier and enter the brain; in addition, the brain fluorescence intensity of the MPP/DiR group is stronger than that of the PP/DiR group, and the MPP/DiR group is further proved to have stronger blood brain barrier crossing capability; the distribution results of DiR, PP/DiR and MPP/DiR in cerebral ischemia positions are shown in figure 4B, the fluorescence of free DiR group and PP/DiR group has no specificity in brain distribution, and the fluorescence specificity of MPP/DiR group is distributed in brain injury areas, so that the MPP/DiR has strong ability of crossing blood brain barrier and good inflammation tropism;
the results of the quantitative analysis of fluorescence intensity of each group of major organs in the present invention are shown in fig. 4D, the fluorescence intensity of the MPP/DiR group brain is higher than that of the DiR group and PP/DiR group, and the fluorescence intensity of the MPP/DiR group ischemic brain is about 7.2 times of that of the normal brain.
As shown in FIGS. 5A and 5B, the pharmacodynamic experiment results of the MPP/DiR of the invention show that PP/SCB and MPP/SCB can both significantly reduce the cerebral infarction area of tMCAO rats compared with the model group; and the MPP/SCB group had a smaller percentage of cerebral infarct size than the PP/SCB group.
The invention provides a biomimetic nano system for tropism of a brain injury inflammation part, which utilizes the inflammation tropism of 4T1 tumor cells and the ability of crossing a blood brain barrier to prepare a simple biomimetic nano particle, can improve the functions of succinogenes on the brain injury inflammation part and the blood brain barrier, and further can realize better treatment effect of ischemic stroke reperfusion injury.
The invention has the following beneficial effects:
(1) the biomimetic nano system MPP/SCB with the tropism of the brain injury inflammation part is prepared, and the problem that the inflammation tropism or the blood brain barrier crossing capability of the biomimetic nano preparation is limited is solved.
(2) The prepared brain injury inflammation part tropism biomimetic nano system MPP/SCB can be used for treating cerebral arterial thrombosis reperfusion injury, and the biomimetic nano system is simple and rapid in preparation method and easy for batch production.
Drawings
FIG. 1 characterization of a biomimetic nanosystem MPP/SCB tropism for brain injury inflammation sites, wherein,
(A) TEM images of PP/SCB and MPP/SCB,
(B)4T1 cell membrane, PP/SCB and MPP/SCB,
(C) surface proteins of 4T1 cells, 4T1 cell membrane, PP/SCB and MPP/SCB.
FIG. 2 in vitro ROS scavenging results for PP/SCB and MPP/SCB, wherein,
(A) intracellular ROS inverted fluorescence plots after co-incubation of PP/SCB and MPP/SCB with tert-butyl hydroperoxide (tBHP) treated PC12 cells, respectively, Scale: the thickness of the film is 50 mu m,
(B) the flow-type quantitative determination result of intracellular ROS level after respectively incubating PP/SCB and MPP/SCB with the PC12 cells treated by tBHP,
(C) the result of the apoptosis flow measurement after respectively incubating PP/SCB and MPP/SCB with the PC12 cells treated by tBHP,
(D) cell viability assay results for PP/SCB and MPP/SCB after co-incubation with tBHP-treated PC12 cells, respectively.
FIG. 3 in vitro blood brain barrier crossing results for PP/DiR and MPP/DiR, wherein,
(A) schematic diagram of Transwell experiment for in vitro construction of blood brain barrier model,
(B) the average fluorescence intensity of the PC12 cell uptake under different treatment modes is measured,
(C) confocal images of laser uptake by PC12 cells under different treatment modalities, scale: 25 μm.
Fig. 4 in vivo distribution results of MPP/DiR, wherein,
(A) representative fluorescence images of major organs of a rat model of ischemia reperfusion (tMCAO) 24h after intravenous injection of various formulations,
(B) representative fluorescence images of brain sections from tMCAO rats 24h after intravenous injection of the different formulations,
(C) representative fluorescence images of the brain of the tMCAO rats 24h after intravenous injection of the different preparations,
(D) after intravenous injection of different preparations for 24h, the fluorescence of the main organs and brain of the tMCAO rats was quantified.
FIG. 5 in vivo pharmacodynamic graph of MPP/SCB, wherein,
(A) representative images of TTC staining of rat brain sections 24h after intravenous injection of the different formulations,
(B) the results of quantifying the percentage of cerebral ischemic area in rats 24 hours after intravenous injection of different preparations.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, elements, and/or combinations thereof, unless the context clearly indicates otherwise.
As described in the background art, the existing biomimetic nano system has the problem of limited brain injury inflammation part targeting capability or blood brain barrier crossing capability, and in order to solve the problem, the invention provides the biomimetic nano system MPP/SCB with brain injury inflammation part tropism, which consists of SCB, amphiphilic polymer PEG-b-PDPA and 4T1 cell membranes.
The invention aims at providing a preparation method of a biomimetic nano system MPP/SCB with brain injury inflammation part tropism, which comprises the following steps:
(1) preparation of PP/SCB nanoparticles: dissolving SCB and PEG-b-PDPA in ethanol, performing reduced pressure rotary evaporation in a water bath to form a film, adding pure water, and hydrating for 30min to form PP/SCB.
Preferably, the mass ratio of the SCB in the step (1) to the PEG-b-PDPA is 1: 3-10.
(2) Preparation of MPP/SCB nanoparticles: MPP/SCB was prepared by mixing 4T1 cell membranes with PP/SCB and repeatedly squeezing.
Preferably, the step (2) of MPP/SCB preparation comprises the following specific steps:
s1, scraping 4T1 cells from a culture dish by using a cell scraper and collecting the cells, cracking the cells by using a cell membrane extraction kit and a repeated extrusion method, and separating and collecting the cells by using a differential centrifugation method to obtain 4T1 cell membranes.
S2, ultrasonically mixing a 4T1 cell membrane with PP/SCB, repeatedly extruding the mixture to pass through a polycarbonate membrane of 200nm to fully fuse PP/SCB nanoparticles and a 4T1 cell membrane to obtain the bionic MPP/SCB nanoparticles with encephalitis part tropism.
Preferably, the mass ratio of the 4T1 cell membrane (based on the mass of the contained membrane protein) to the PP/SCB in the step S2 is 1: 5-10.
Preferably, the number of times of the repeated pressing in step S2 is 7 to 15.
The second purpose of the invention is to provide the application of the tropism biomimetic nano system MPP/SCB in treating ischemic stroke reperfusion injury at the brain injury inflammation part.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions specified in the examples are not indicated, they are generally according to the conventional conditions, or according to the conditions recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of MPP/SCB
(1) Accurately weighing 6mg of PEG-b-PDPA and 2mg of SCB into a solanaceous bottle by an analytical balance, adding absolute ethyl alcohol for ultrasonic dissolution, carrying out reduced pressure rotary evaporation at 45 ℃ to form a film, and then adding 5ml of deionized water for hydration for 30min to obtain the PP/SCB nanoparticles.
(2) The 4T1 cells were scraped from the petri dish using a cell scraper and collected into a centrifuge tube, membrane protein extraction reagent a (1ml) containing 1mM protease inhibitor (PMSF) was added, and the cells were resuspended for 15min in an ice bath. The cell suspension was then repeatedly squeezed through the polycarbonate membrane 30 times to disrupt the cells. The cell debris suspension was then centrifuged at 700g for 10min at 4 ℃ and the supernatant collected to remove nuclei and unlysed cells. Finally, the supernatant was centrifuged at 14,000g for 30min at 4 ℃ to obtain a cell membrane precipitate. The purified 4T1 cell membrane protein mass was determined using BCA protein quantification kit for subsequent use.
(3) 5mg of PP/SCB nanoparticles and 1mg of 4T1 cell membranes are taken, mixed uniformly by ultrasonic treatment, and then repeatedly extruded for 11 times through a 200nm polycarbonate membrane to obtain the PP/SCB nanoparticles MPP/SCB coated with 4T1 cell membranes.
Example 2 physical and chemical Properties characterization of MPP/SCB
(1) Dropping a drop of PP/SCB or MPP/SCB solution (1mg/ml) on a carbon film copper net, naturally drying, then dropping a drop of phosphotungstic acid solution for dyeing for 2-3min, and then observing the forms of the two nanoparticles by using a transmission electron microscope. As shown in the electron micrographs of FIGS. 1A and 1B, both PP/SCB and MPP/SCB are spherical with round particle size, and MPP/SCB has an obvious membrane shell structure with an outer membrane thickness of about 20 nm.
(2) The particle sizes of the PP/SCB and MPP/SCB solutions are tested by a laser particle size analyzer, the particle size results are shown in figure 1C, the particle size of the PP/SCB is 54.1 +/-1.6 nm, and the polydispersity index is 0.22 +/-0.01; the particle size of MPP/SCB is 67.8 +/-0.6 nm, and the polydispersity index is 0.29 +/-0.01. The particle size result is consistent with the electron microscope picture, and further illustrates the success of the preparation of the bionic MPP/SCB nanoparticles.
(3) The western blot technique was used to detect surface proteins of 4T1 cells, 4T1 cell membranes, PP/SCB and MPP/SCB. As shown in FIG. 1D, CD47, CD138 and VCAM-1 proteins were detected on the surface of 4T1 cells, 4T1 cell membrane and MPP/SCB, while no protein was detected on the surface of PP/SCB, indicating that the integrity of the protein on the surface of 4T1 cell membrane was not destroyed during cell membrane extraction and MPP/SCB preparation.
Example 3MPP/SCB in vitro ROS Elimination and protection against ROS-damaged cells
(1) A model of PC12 cell reperfusion injury was constructed by incubating PC12 cells with t-butyl hydroperoxide solution (tBHP, 100. mu.M) for 1 h. Then, the cells were incubated with PC12 cells in DMEM medium, DMEM medium containing PP/SCB and DMEM medium containing MPP/SCB for 4 hours, respectively. Then adding a cell ROS probe DCFH-DA, and qualitatively and quantitatively detecting the ROS level in each group of cells by using a fluorescence inverted microscope and a flow cytometer respectively; experimental results As shown in FIGS. 2A and 2B, both PP/SCB and MPP/SCB can obviously eliminate ROS in PC12 cells, and compared with PP/SCB, MPP/SCB has stronger ability to eliminate ROS.
(2) A model of PC12 cell reperfusion injury was constructed by incubating PC12 cells with t-butyl hydroperoxide solution (tBHP, 100. mu.M) for 1 h. Then, the cells were incubated with PC12 cells in DMEM medium, DMEM medium containing PP/SCB and DMEM medium containing MPP/SCB for 4 hours, respectively. Then the Annexin V-FITC/PI apoptosis kit is used for detecting the apoptosis and necrosis of each group of cells. The experimental results are shown in FIGS. 2C and 2D, and both PP/SCB and MPP/SCB can significantly reduce the apoptosis of PC12 cells caused by tBHP, and compared with PP/SCB, MPP/SCB has stronger protective capability on PC12 cells damaged by ROS.
Example 4 in vitro determination of MPP/SCB Cross blood brain Barrier Capacity
Respectively preparing PP/DiR nanoparticles and MPP/DiR nanoparticles by replacing SCB with a fluorescent dye DiR. An in vitro blood brain barrier model was constructed using a transwell plate, and as shown in FIG. 3A, mouse brain microvascular endothelial cells (bEnd.3 cells) were seeded into the upper chamber of the transwell plate and cultured until the transmembrane resistance reached 330ohm cm2PC12 cells were then seeded under the transwell plate. After the cells were attached, the free fluorescent dyes DiR, PP/DiR or MPP/DiR (corresponding to 10. mu.g/ml DiR, respectively) were added to the upper chamber. After culturing for 12h and 24h respectively, detecting the fluorescence intensity in PC12 cells at the lower layer of the transwell plate by using a flow cytometer; after fixing the cells with 4% paraformaldehyde, the nuclei of PC12 cells were stained with 5ug/ml DAPI staining solution, and then the uptake of PC12 cells in each group was observed by confocal laser microscopy. Quantitative detectionAnd qualitative observation results are shown in fig. 3B and 3C, compared with free DiR, the blood brain barrier crossing ability of PP/DiR and MPP/DiR is significantly improved; with the prolonged incubation time, the fluorescence in PC12 cells incubated with PP/DiR and MPP/DiR is obviously enhanced, which shows that the amount of PP/DiR and MPP/DiR crossing the blood brain barrier is obviously increased; in addition, the fluorescence intensity of PC12 cells incubated with MPP/DiR is stronger than that of PP/DiR group at different incubation times, which indicates that MPP/DiR has stronger blood brain barrier crossing capability than that of PP/DiR.
Example 5 in vivo distribution experiment of MPP/DiR
The rat tMCAO model was established by the wire-plug method. Male SD rats (250-280g) were fasted for 12h before surgery and were anesthetized by intraperitoneal injection of 1% pentobarbital at a dose of 40 mg/kg. After rat skin was disinfected with iodophor, the skin of the neck was cut open, and the left Common Carotid Artery (CCA), External Carotid Artery (ECA) and Internal Carotid Artery (ICA) were sequentially isolated, and the three arteries were distributed in a "Y" shape. Care was taken not to disrupt the vagus nerve when isolating the common carotid artery due to the concomitant movement of the common carotid artery and the vagus nerve. The CCA and ICA were then occluded by clipping the ECA distal end, cutting a V-shaped small opening at the ECA proximal end with an ophthalmic scissors, inserting a nylon wire plug from the small opening, steering to the ICA at the Y-shaped bifurcation, opening the artery clip at the ICA, and slowly pushing the wire plug inward about 18mm to block the blood supply to the middle cerebral artery. To prevent the plug from slipping, the plug was secured with suture at the proximal end of the ECA, from which time cerebral ischemia time was recorded. The arterial clamp at CCA was opened and the rat skin tissue was sutured. The plug was slowly withdrawn 2h after embolization to expose the plug to the dark spot marks on the plug to allow reperfusion. Rats were also injected separately with free DiR, PP/DiR and MPP/DiR (corresponding to 0.1mg/kg of DiR, respectively) via the tail vein.
(1) After administration for 22h, the rats were sacrificed and immediately decapitated to remove the brain and the major organs were removed, and the distribution of DiR, PP/DiR and MPP/DiR in the brain and major organs was observed with a small animal biopsy machine. The experimental results are shown in fig. 4A and 4C, the free DiR group brains have substantially no fluorescence, which indicates that the free DiR hardly crosses the blood brain barrier to reach the brain; fluorescence can be observed in the brain by the PP/DiR group and the MPP/DiR group, which shows that the PP/DiR and the MPP/DiR can cross the blood brain barrier and enter the brain; in addition, the brain fluorescence intensity of the MPP/DiR group is stronger than that of the PP/DiR group, and the MPP/DiR has stronger blood brain barrier crossing capability.
(2) The brain was sliced along the coronal plane into 2mm thick serial sections, the distribution of DiR, PP/DiR and MPP/DiR in the brain was observed with a small animal Living body imager, and the brain sections were stained with TTC staining solution which stains normal brain tissue red and ischemia white. And observing the distribution of the DiR, the PP/DiR and the MPP/DiR in the cerebral ischemia position. The experimental result is shown in fig. 4B, the fluorescence distribution of free DiR group and PP/DiR group in brain has no specificity, while the fluorescence specificity of MPP/DiR group is distributed in brain injury area, which proves that MPP/DiR not only has stronger ability of crossing blood brain barrier, but also has good inflammation tropism.
(3) The fluorescence intensity of the major organs in each group was quantitatively analyzed, and the experimental results are shown in fig. 4D, in which the fluorescence intensity of the MPP/DiR group brain was higher than that of the DiR group and the PP/DiR group, and the fluorescence intensity of the MPP/DiR group ischemic brain was about 7.2 times higher than that of the normal brain.
EXAMPLE 6 pharmacodynamic experiment of MPP/DiR
The rat tMCAO model was constructed as in example 3, and the wiretap was withdrawn 2h after embolization to achieve reperfusion. At the same time, the rats were injected with PP/SCB and MPP/SCB (corresponding to SCB of 10mg/kg, respectively) through the tail vein, respectively. At 22h after administration, the rats were sacrificed and the brains were immediately decapitated and harvested, the brains were cut into 2mm thick serial sections along the coronal plane, the brain sections were stained with TTC staining solution, the improvement of the ischemic brain tissue by PP/SCB and MPP/SCB was observed, and the percentage of the ischemic area of each group was quantitatively calculated. As shown in FIGS. 5A and 5B, compared with the model group, both PP/SCB and MPP/SCB can significantly reduce the cerebral infarction area of tMCAO rats; and the MPP/SCB group had a smaller percentage of cerebral infarct size than the PP/SCB group.
In conclusion, the biomimetic nano system MPP/SCB for the inflammation part of the brain injury is prepared for the first time, and the problem that the inflammation tendency or the blood brain barrier crossing capacity of the biomimetic nano preparation is limited is solved. And the MPP/SCB has good curative effect on cerebral arterial thrombosis reperfusion injury.
Claims (10)
1. A biomimetic nano system with tropism for brain injury inflammation parts is characterized by comprising a medicine, a medicine carrier and a biomimetic cell membrane, wherein the medicine is an antioxidant medicine for treating ischemic stroke; the drug carrier is an amphiphilic polymer, and the drug carrier is coated by a thin film hydration method to form inner core nanoparticles; the biomimetic cell membrane is a tumor cell membrane, and the tumor cell membrane is wrapped with the inner core nanoparticles in an extrusion mode to form a biomimetic nano system with tropism at the brain injury inflammation part.
2. The brain injury inflammation site tropism biomimetic nano system according to claim 1, wherein the antioxidant drug for treating ischemic stroke is selected from probucol, amber bucco, curcumin, baicalein, and preferably amber bucco.
3. The biomimetic nanosystem according to claim 1, wherein the amphiphilic polymer drug carrier is polyethylene glycol-poly (diisopropylaminoethyl methacrylate) (PEG-b-PDPA).
4. The chemotaxis biomimetic nanosystem for sites of inflammation of brain injury according to claim 1, wherein the tumor cell membrane is: the breast cancer cell 4T1 cell with high brain metastasis ability is prepared by cracking the cell with cell cracking liquid, breaking the cell into fragments through repeated extrusion, and finally separating the fragments by using a differential centrifugation method to obtain a 4T1 cell membrane.
5. The method for preparing the biomimetic nanosystem for tropism of the inflammatory site of brain injury according to claims 1-4, comprising the steps of:
(1) preparing PP/SCB nanoparticles: dissolving SCB and PEG-b-PDPA in ethanol together, performing reduced pressure rotary evaporation in a water bath to form a film, adding pure water, and hydrating to form PP/SCB;
(2) preparing MPP/SCB nanoparticles: the MPP/SCB is prepared by ultrasonically mixing 4T1 cell membranes with PP/SCB and repeatedly extruding the mixture by an extruder.
6. The preparation method according to claim 5, wherein in the step (1), the ratio of SCB to PEG-b-PDPA is 2:1-1:10, and the hydration time is 30 min.
7. The method according to claim 5, wherein in the step (2), the extraction of the 4T1 cell membrane comprises the following steps: scraping 4T1 cells from a culture dish by using a cell scraper and collecting the cells into a centrifuge tube, adding a membrane protein extraction reagent A1ml containing 1mM protease inhibitor (PMSF), and resuspending the cells for 15min in an ice bath; then repeatedly extruding the cell suspension through a polycarbonate membrane for 30 times to break the cells; the cell debris suspension was then centrifuged at 700g for 10min at 4 ℃ and the supernatant collected to remove nuclei and unlysed cells; finally, the supernatant was centrifuged at 14,000g for 30min at 4 ℃ to obtain a cell membrane precipitate; and (4) determining the quality of the purified 4T1 cell membrane protein by using a BCA protein quantitative kit for later use.
8. The preparation method according to claim 5, wherein in the step (2), the mass ratio of the 4T1 cell membrane to PP/SCB is 1:1-1:10 based on the mass of the contained membrane protein.
9. The method according to claim 8, wherein the mass ratio of the 4T1 cell membrane to PP/SCB is 1: 5.
10. The production method according to claim 5, wherein in the step (2), the extruder is a polycarbonate film extruder having a pore diameter of 200nm, and the number of repeated extrusion is 7 to 15.
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| US20190290592A1 (en) * | 2018-03-23 | 2019-09-26 | University Of South Carolina | Nanoparticles for Brain Targeted Drug Delivery |
| CN111603454A (en) * | 2020-06-08 | 2020-09-01 | 上海交通大学医学院附属第九人民医院 | Multi-targeting fusion cell membrane modified bionic nano delivery system and preparation method and application thereof |
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| US20190290592A1 (en) * | 2018-03-23 | 2019-09-26 | University Of South Carolina | Nanoparticles for Brain Targeted Drug Delivery |
| CN111603454A (en) * | 2020-06-08 | 2020-09-01 | 上海交通大学医学院附属第九人民医院 | Multi-targeting fusion cell membrane modified bionic nano delivery system and preparation method and application thereof |
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