CN114642634A - Blood brain barrier penetrating drug-carrying micelle and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a blood brain barrier penetrating drug-loaded micelle and a preparation method and application thereof, wherein the blood brain barrier penetrating drug-loaded micelle comprises a micelle formed by PLGA covalently modified by a GM1 hydrolysate and drugs encapsulated in the micelle; the medicine is a medicine for treating central nervous system diseases. The blood-brain barrier penetrating drug-loaded micelle can realize the delivery of blood-brain barrier penetrating drugs, can realize the combined administration purpose of anti-tumor and nerve repair under the condition of not introducing other nerve injury repair drugs and materials, realizes the double effects of central nerve drug delivery and nerve function improvement, avoids excessive drug and material tissue accumulation and potential toxicity, and has great potential for preventing or treating glioma and other central nervous system diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a blood-brain barrier permeable medicine carrying micelle and a preparation method and application thereof.
Background
Gliomas are the most common primary malignancies of the central nervous system with a highly aggressive and extremely poor prognosis. Conventional surgical removal of tumors is greatly limited due to its infiltration and invasiveness. Meanwhile, the blood brain barrier system formed by the close connection of brain capillary endothelial cells, pericytes, astrocyte peripodes and the like prevents toxic substances from invading the brain and prevents most of medicines from entering the brain, so that the application of treatment medicines for glioma and a plurality of central system diseases (such as Parkinson's disease, Alzheimer's disease and the like) is limited. Therefore, designing a safe carrier that can assist the drug to efficiently penetrate the blood brain barrier and effectively reach the focus is an important research direction for the treatment of glioma and intracranial diseases.
Currently, strategies for the blood-brain barrier mainly involve invasiveness and non-invasiveness, but most invasive methods present the potential risk of nerve damage and intracranial infection. With the development of nanotechnology, intracranial administration of non-invasive blood brain barrier-penetrating nanocarriers has rapidly progressed. However, most of the nano materials have the problems of nondegradable accumulation in brain tissues, unclear degradation and metabolism pathways and the like, and the problems of safety and stability also exist in the complicated preparation process of the nano materials.
Although more and more blood-brain barrier-permeable drug delivery systems are being developed for the treatment of gliomas and brain diseases, such as: liposome, polymer/inorganic material nanoparticles, nano-carriers of dendritic macromolecules and other materials, self-assembled micelles and the like. However, the effectiveness, safety, feasibility of large-scale preparation and stability of the carrier materials constituting the drug delivery system are still required to be further improved and clarified, and most of the carrier materials are in the laboratory research stage and are difficult to be clinically transformed. Therefore, there is an urgent need to find a new method for improving the blood-brain barrier permeation efficiency of a drug carrier while having excellent biosafety and stability.
Monosialotetrahexosylganglioside (GM 1) contains hydrophilic sugar chains and lipophilic sphingosine and fatty acid structures and is the major class of mammalian gangliosides. GM1 plays an essential role in neurogenesis, neuronal growth and differentiation, and can promote the survival of damaged neurons, and most of the current studies focus on the functional assessment of GM1 for nerve repair.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a blood-brain barrier permeable drug-carrying micelle and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a blood-brain barrier penetrating drug-loaded micelle, which comprises a micelle formed by PLGA covalently modified by a GM1 hydrolysate, and a drug encapsulated in the micelle; the medicine is a medicine for treating central nervous system diseases.
The blood-brain barrier penetrating drug-loaded micelle can realize the delivery of blood-brain barrier penetrating drugs, can realize the combined administration purpose of anti-tumor and nerve repair under the condition of not introducing other nerve injury repair drugs and materials, realizes the double effects of central nerve drug delivery and nerve function improvement, avoids excessive drug and material tissue accumulation and potential toxicity, and has great potential for preventing or treating glioma and other central nervous system diseases. The invention takes adriamycin as an anti-tumor model drug, and the blood brain barrier penetrating drug-carrying micelle obtained by a glioma model in-vivo evaluation test has very excellent anti-glioma effect and nerve function improvement effect.
Preferably, the central nervous system disease therapeutic agent comprises any one or a combination of at least two of doxorubicin, rapamycin, paclitaxel, docetaxel, hydroxycamptothecin, vinblastine, amantadine, or rivastigmine.
Preferably, the central nervous system disease comprises any one of glioma, pituitary tumour, meningioma, stroke, parkinson or alzheimer.
The blood brain barrier penetrating drug-loaded micelle can be used for preventing or treating central nervous system diseases including glioma, pituitary tumor, meningioma, cerebral apoplexy, Parkinson, Alzheimer and the like.
Preferably, the GM1 hydrolysate covalently modified PLGA is obtained from a hydrolysate of monosialotetrahexosylganglioside linked to a polylactic acid-glycolic acid copolymer via an amide bond; the hydrolysis product of the monosialotetrahexosylganglioside is shown as the formula (I):
preferably, the number average molecular weight of the polylactic acid-glycolic acid copolymer is 6-12kDa, such as 6kDa, 8kDa, 10kDa or 12kDa, and other specific values within the numerical range can be selected, and are not repeated herein.
Further preferably, the molar ratio of the lactic acid structural unit to the glycolic acid structural unit in the polylactic acid-glycolic acid is (50-80): (20-50), wherein the specific value in (50-80) can be selected from 50, 60, 70, 75 or 80, etc., wherein the specific value in (20-50) can be selected from 20, 30, 40 or 50, etc., and other specific values in the above numerical range can be selected, which is not repeated herein.
In a second aspect, the present invention provides a method for preparing the blood-brain barrier permeable drug-loaded micelle according to the first aspect, the method comprises the following steps:
(1) dropping PLGA solution covalently modified by GM1 hydrolysate into the stirring water solution to form blank micelle;
(2) and (3) dropwise adding the drug solution into the blank micelle solution which is being stirred, standing and dialyzing to obtain the blood-brain barrier permeable drug-loaded micelle.
The preparation method of the blood brain barrier permeable drug-loaded micelle is relatively simple to operate, is suitable for industrial production, and has a good transformation application prospect.
Preferably, the solvent of the GM1 hydrolysate covalently modified PLGA solution of step (1) comprises any one or a combination of at least two of dimethylformamide, dichloromethane, acetone, tetrahydrofuran, chloroform or ethyl acetate.
Preferably, the concentration of the solution in step (1) is 4-40mg/mL, such as 4mg/mL, 5mg/mL, 8mg/mL, 10mg/mL, 12mg/mL, 15mg/mL, 18mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, etc., and other specific values in the numerical range can be selected, which is not described herein any more, and preferably 10-14 mg/mL.
The concentration of the PLGA solution covalently modified by the hydrolysate of GM1 is specifically selected to be 4-40mg/mL because the drug-loading rate and the encapsulation rate of the finally prepared blood-brain barrier permeable drug-loaded micelle are higher under the condition, and 10-14mg/mL is a range with better effect.
Preferably, the dropping rate in step (1) is 10-100 μ L/min, such as 10 μ L/min, 20 μ L/min, 40 μ L/min, 50 μ L/min, 60 μ L/min or 100 μ L/min, and other specific values in the numerical range can be selected, and are not described in detail herein, and preferably 40-60 μ L/min.
Preferably, the volume of the aqueous solution in step (1) is 2-40mL, such as 2mL, 10mL, 15mL, 20mL, 25mL, 30mL, 35mL or 40mL, and other specific values in the numerical range can be selected, which is not described herein, and is preferably 2-10 mL.
The specific selection of the volume of the aqueous solution is 2-40mL because the drug-loading rate and the encapsulation rate of the finally prepared blood brain barrier permeable drug-loaded micelle are higher under the condition, and 2-10mL is a more significant range of effect.
Preferably, the solvent of the drug solution of step (2) comprises any one of or a combination of at least two of dimethylformamide, dichloromethane, acetone, tetrahydrofuran, chloroform or ethyl acetate.
Preferably, the concentration of the drug solution in step (2) is 1-30mg/mL, such as 1mg/mL, 5mg/mL, 8mg/mL, 10mg/mL, 12mg/mL, 15mg/mL, 18mg/mL, 20mg/mL or 30mg/mL, and the like, and other specific points in the numerical range can be selected, which is not described herein again, and preferably 2-8 mg/mL.
The concentration of the drug solution is specially selected to be 1-30mg/mL because the condition can enable the drug-loading rate and the encapsulation rate of the finally prepared blood brain barrier permeable drug-loaded micelle to be higher, and 2-8mg/mL is a more obvious range of effect.
Preferably, the dropping rate in step (2) is 10-100. mu.L/min, such as 10. mu.L/min, 20. mu.L/min, 40. mu.L/min, 50. mu.L/min, 60. mu.L/min or 100. mu.L/min, etc., preferably 40-60. mu.L/min.
Preferably, the temperature of the standing in the step (2) is 2-8 ℃, for example, 2 ℃, 4 ℃, 8 ℃ and the like, and the time is 18-30h, for example, 18h, 24h or 30h and the like, and other specific values in the above numerical range can be selected, and are not repeated herein.
Preferably, the cut-off molecular weight of the dialysis bag used in the dialysis in the step (2) is 8000Da, and the dialysis time is 65-96h, such as 65h, 72h, 80h, 90h or 96h, and other specific values in the numerical range can be selected, which is not described in detail herein.
Preferably, the dialysis in step (2) is followed by filtration of the sample through a membrane having a pore size of 0.45. mu.m.
In the present invention, the method for preparing the GM1 hydrolysate covalently modified PLGA comprises the following steps:
(1') mixing a hydrolysate of monosialotetrahexosylganglioside shown in the formula (I), PLGA, tributylamine and 1-hydroxybenzotriazole, and reacting at 15-37 ℃ for 1-3 h;
(2 ') dropping the reaction solution obtained in step (1') into pre-cooled distilled water to form a colloidal precipitate, i.e. the GM1 hydrolysate covalently modified PLGA.
Specifically, the molar ratio of the hydrolysate to the PLGA is 1 (0.2-0.4), such as 1:0.2, 1:0.3, or 1:0.4, and other specific values within the value range can be selected, which is not described herein again.
The molar ratio of the hydrolysate to the 1-hydroxybenzotriazole is 1 (1.6-2.0), such as 1:1.6, 1:1.8 or 1:2.0, and other specific values in the numerical range can be selected, and are not described in detail herein.
The molar ratio of the hydrolysate to tributylamine is 1 (1.2-1.8), such as 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, or 1:1.8, and other specific values within the numerical range can be selected, and are not described in detail herein.
The temperature of the above reaction is 15-37 deg.C, such as 15 deg.C, 20 deg.C, 24 deg.C, 25 deg.C, 28 deg.C, 30 deg.C, 32 deg.C, 35 deg.C or 37 deg.C; the reaction time is 1-3h, such as 1h, 2h or 3 h; other specific point values within the above numerical range can be selected, and are not described in detail herein.
The temperature of the pre-cooled distilled water is 2-8 ℃, such as 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃ or 8 ℃, and other specific values in the numerical range can be selected, and are not described in detail herein.
In the present invention, the method for preparing the hydrolysate of monosialotetrahexosylganglioside represented by the formula (i) comprises the steps of:
(S1) mixing GM1 with a buffer solution containing 30 to 40mM sodium acetate, 0.3 to 0.5% taurodeoxycholic acid hydrate, and 50 to 150mM calcium chloride at pH 5.5 to 6.0, and dissolving the mixture with stirring;
(S2) mixing the hydrolase SCDase with the solution obtained in the step (S1), carrying out shaking reaction at 30-40 ℃ for 10-15h, centrifuging, taking the supernatant, and freeze-drying to obtain the monosialotetrahexosylganglioside hydrolysate shown in the formula (I).
The concentration of the sodium acetate can be 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM or 40mM and the like; too high or too low a concentration of sodium acetate reduces the hydrolysis efficiency of the monosialotetrahexosylganglioside, and the hydrolysis efficiency is optimal in the above-mentioned concentration range.
The mass percentage content of the taurodeoxycholic acid hydrate in the buffer solution can be 0.3%, 0.38%, 0.4%, 0.42%, 0.45%, 0.5% and the like.
The concentration of the calcium chloride may be 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 120mM, 150mM, or the like.
The pH value of the buffer solution can be 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 and the like, and the pH value can be adjusted by glacial acetic acid.
The reaction temperature may be 30 ℃, 32 ℃, 35 ℃, 38 ℃ or 40 ℃ and the like.
The reaction time can be 10h, 11h, 12h, 13h, 14h or 15h, and the like.
Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
More specifically, the rotation speed of the centrifugation is 10000-14000r/min, such as 10000r/min, 11000r/min, 12000r/min, 13000r/min or 14000 r/min; centrifuging for 3-6min, such as 3min, 4min, 5min or 6 min; other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
In a third aspect, the present invention provides an application of the blood brain barrier penetrating drug-loaded micelle in preparation of a drug for preventing or treating central nervous system diseases.
In a fourth aspect, the present invention provides an application of the blood brain barrier penetrating drug-loaded micelle in the first aspect in preparing a drug for preventing or treating intracranial tumors;
preferably, the intracranial tumour is a glioma.
Compared with the prior art, the invention has the following beneficial effects:
the blood-brain barrier penetrating drug-loaded micelle can realize the delivery of blood-brain barrier penetrating drugs, can realize the combined administration purpose of anti-tumor and nerve repair under the condition of not introducing other nerve injury repair drugs and materials, realizes the double effects of central nerve drug delivery and nerve function improvement, avoids excessive drug and material tissue accumulation and potential toxicity, and has great potential for preventing or treating glioma and other central nervous system diseases. The blood brain barrier-permeable drug-loaded micelle obtained by taking adriamycin as an anti-tumor model drug through a glioma model in-vivo evaluation test has very excellent anti-glioma effect and nerve function improvement effect, and provides a new strategy with a good transformation application prospect for non-invasive drug delivery of glioma and other central nervous system diseases.
Drawings
FIG. 1 is a graph of the particle size characterization of the blank micelle prepared in example 2;
FIG. 2 is a graph showing the particle size characterization of the blood-brain barrier permeable drug-loaded micelle prepared in example 2;
FIG. 3 is a Zeta potential characterization plot of blank micelles made in example 2;
FIG. 4 is a Zeta potential characterization chart of the blood-brain barrier permeable drug-loaded micelle prepared in example 2;
FIG. 5 is a standard curve for DOX in dimethylformamide;
FIG. 6 is a photograph of an image of a live small animal observing the DiR fluorescence distribution and fluorescence intensity of each organ tissue of a mouse in example 11;
FIG. 7 is a graph showing the fluorescence distribution in the brain of zebrafish observed by confocal laser microscopy in example 12;
FIG. 8 is a graph showing the survival rate of groups of C6 glioma-bearing mice in example 13;
FIG. 9 is a neurobehavioral score chart of groups of C6 glioma-bearing mice in example 13.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Kunming mice referred to in the examples below were purchased from Chongqing university of medicine; wistar rats were purchased from Beijing Wittingle laboratory animals GmbH; zebra fish (flk 1: GFP) was provided by the national local Union engineering laboratory of vascular implants of the institute of bioengineering, Chongqing university; other reagents and starting materials required for the assay are commercially available.
Example 1
This example prepares GM1 hydrolysate covalently modified PLGA as follows:
(1) weighing appropriate amount of sodium acetate, taurodeoxycholic acid hydrate and calcium chloride, adding a certain amount of triple distilled water, and dissolving completely to obtain a solution containing 35mM NaOAc, 0.4% TDC and 100mM CaCl2Buffering the solution, and adjusting the pH to 5.8 by using a glacial acetic acid solution;
(2) weighing 100mg of GM1 powder, placing the powder in a beaker, adding 20mL of the buffer solution system, and fully stirring and dissolving;
(3) after being fully dissolved, the mixture is transferred into a 50mL centrifuge tube, 15 mu L of hydrolase SCDase is added, and the mixture is placed in a constant temperature (37 ℃) shaking box for shaking reaction for 12 hours;
(4) centrifuging at 12000 rpm for 5min, and collecting supernatant;
(5) freeze drying the prepared hydrolysate to obtain flocculent solid, namely GM1 hydrolysate;
(6) transferring 50mg of GM1 hydrolysate to 10mL of anhydrous dimethylformamide for dissolving, weighing 1g of PLGA solid, and fully stirring after adding to completely dissolve the PLGA solid;
(7) weighing 10.6mg of HOBt, adding into the system, carefully adding 15.3 mu L of tributylamine solution, and reacting for 2h under vigorous stirring at 25 ℃, wherein the molar ratio of the hydrolysis product of GM1 to HOBt, tributylamine and PLGA is 1:1.8:1.5: 0.3;
(8) slowly dripping the reaction solution into distilled water at 4 ℃ to form colloidal precipitate, repeatedly washing with distilled water, and freeze-drying the solid to obtain the GM1 hydrolysate-covalently modified PLGA.
Example 2
The method for preparing the blood brain barrier penetrating drug-loaded micelle comprises the following steps:
(1) accurately weighing PLGA covalently modified by GM1 hydrolysate prepared in example 1 by a precision balance, and dissolving the PLGA in 2.5mL of dimethylformamide with the concentration of 12 mg/mL;
(2) slowly dripping the solution obtained in the step (1) into 5mL of ultrapure water which is stirring by using a syringe pump at the speed of 50 mu L/min to form a blank micelle, and storing the blank micelle for 24h at 4 ℃ in a refrigerator for later use;
(3) dissolving DOX & HCI powder into 3mL of dimethylformamide solution under the condition of keeping out of the sun, wherein the concentration is 5 mg/mL; adding triethylamine (the molar ratio of triethylamine to DOX & HCI is 3:1), stirring for 18h, and removing hydrochloric acid;
(4) slowly dripping the DOX solution after hydrochloric acid removal into the blank micelle solution prepared in the step (2) which is stirring at the speed of 50 mu L/min by using a syringe pump;
(5) standing the solution in a refrigerator at 4 deg.C for 24h, transferring to dialysis bag (MWCO: 8000) for dialysis for 72h, and removing excessive organic solvent such as DOX and DMF;
(6) and (3) filtering the solution obtained in the step (5) by using a membrane filter with the pore diameter of 0.45 mu m to obtain the blood brain barrier penetrating medicine-carrying micelle (marked as PLGA-lysoGM1/DOX micelle).
Example 3
This example prepared a drug-loaded micelle that permeated blood brain barrier, and the method differed from example 2 only in that the concentration of PLGA covalently modified by GM1 hydrolysate in step (1) was 8mg/mL, and all other conditions remained the same.
Example 4
This example prepared a drug-loaded micelle that permeated blood brain barrier, and the method differed from example 2 only in that the concentration of PLGA covalently modified by GM1 hydrolysate in step (1) was 16mg/mL, and all other conditions remained the same.
Example 5
This example prepares a drug-loaded micelle which permeates blood brain barrier, and the method only differs from example 2 in that the volume of ultrapure water in step (2) is 20mL, and other conditions are kept unchanged.
Example 6
This example prepares a drug-loaded micelle which permeates blood brain barrier, and the method only differs from example 2 in that the volume of ultrapure water in step (2) is 30mL, and other conditions are kept unchanged.
Example 7
This example prepares a drug-loaded micelle that permeates the blood brain barrier, and the method only differs from example 2 in that the concentration of the DOX & HCI solution in step (3) is 1mg/mL, and other conditions are kept unchanged.
Example 8
This example prepares a drug-loaded micelle that permeates the blood brain barrier, and the method only differs from example 2 in that the concentration of the DOX & HCI solution in step (3) is 12mg/mL, and other conditions are kept unchanged.
Example 9
The blank micelles and blood-brain barrier permeable drug-loaded micelles obtained in example 2 were subjected to particle size analysis and surface charge analysis: the particle size and surface charge of the micelles were characterized using a dynamic light scattering instrument (DLS) and the micelle size and Zeta potential were measured by DLS using a Zetasizer (Nano ZS90, Malvern), setting the instrument parameters: the transmission was 1.57 and the temperature was 25 ℃. The results are shown in FIGS. 1 to 4, and it can be seen that: the average sizes before and after drug loading were 110nm (fig. 1) and 246.8nm (fig. 2), respectively; the potential measurements showed negative surface charges of-34.9 mV (FIG. 3), -33.2mV (FIG. 4), respectively.
Example 10
The blood-brain barrier penetrating drug-loaded micelles prepared in examples 2 to 8 were subjected to determination of drug loading and encapsulation efficiency:
(1) dissolving DOX powder in a dimethylformamide solution, diluting the solution into a plurality of concentration gradients, and drawing a standard curve of DOX in the dimethylformamide solution by using an ultraviolet spectrophotometry, wherein the standard curve is shown in figure 5, and a regression equation is as follows: y is 0.021x-0.0089, R2When the concentration was 0.9999, the linear relationship between the concentration of DOX and the absorbance value was good in the range of 10 μ g/mL to 45 μ g/mL.
(2) And (3) determining the drug loading and the encapsulation efficiency:
measuring a certain volume of PLGA-lysoGM1/DOX micelle solution, freeze-drying, recording the weight of the dried micelle, adding a proper amount of dimethylformamide solution, stirring to release DOX into the dimethylformamide solution, detecting the absorbance of the solution by ultraviolet, and calculating the content of DOX in the micelle by referring to a standard curve, thereby obtaining the drug loading rate and the encapsulation efficiency of the micelle.
The results are shown in table 1:
TABLE 1
As can be seen from the data in Table 1: the research of the invention finds that the drug concentration, the copolymer concentration and the water phase volume can obviously influence the drug loading capacity and the encapsulation efficiency of the drug-loaded micelle, wherein the indexes of the drug loading capacity and the encapsulation efficiency of the drug-loaded micelle are relatively optimal under the matching of the process conditions shown in the embodiment 2, the drug loading capacity is 3.8%, and the encapsulation efficiency is 61.6%.
Example 11
In the embodiment, a small animal imaging system is adopted to observe the distribution condition of the blood brain barrier permeable drug-loaded micelle after entering the body: PLGA-lysoGM1/DiR micelles were prepared by loading fluorescein DiR in the same loading manner, replacing the drug with fluorescein DiR as in example 2.
Removing hair on the surface of a mouse, injecting 0.5mL of free DiR solution, PLGA/DiR nanoparticle solution and PLGA-LysoGM1/DiR micelle solution with the concentration of 50 mu g/mL into a tail vein of a healthy Kunming mouse according to the measurement of 25 mu g/mouse, putting the mouse into a dark box platform of a living body imaging instrument for 1, 2, 4, 6, 8 and 24 hours, respectively shooting a background picture under a bright field and a fluorescence picture of spontaneous specific photons DiR in the mouse under a dark field, carrying out exposure time of 400sm, collecting an incineration light signal at 780-900nm, and superposing the background pictures of the dark field and the bright field to visually display the distribution part and the intensity of the DiR in the animal body. The distribution and intensity of the DiR fluorescence of each organ tissue of the mouse can be observed and recorded under a small animal living body imaging instrument, and the condition that PLGA-lysoGM1/DiR micelle penetrates through BBB can be evaluated. The preparation method of the PLGA/DiR nano solution comprises the following steps: 40mg PLGA and 4.3mg DiR are dissolved in 2.5mL dimethylformamide, and the dissolved solution is slowly dripped into 5mL water solution which is stirring by a syringe pump at the speed of 50 mu L/min to obtain PLGA/DiR nanoparticle solution. Then, it was left standing in a refrigerator at 4 ℃ for 24 hours and transferred to a dialysis bag (MWCO: 8000) for dialysis for 24 hours to remove residual DiR and dimethylformamide. Finally, the solution was filtered through a 0.45 μm pore size filter and the PLGA/DiR solution was stored in a refrigerator at 4 ℃ until use.
The results are shown in FIG. 6: accumulation of DiR fluorescence signal was observed in the liver after DiR injection; after PLGA/DiR nanoparticles were injected, the same trend as DiR was observed, but a small amount of DiR fluorescence signal was also observed in the brain at 8h and 24 h; after PLGA-LysoGM1/DiR micelle injection, DiR fluorescence signals can be observed in mouse brains in the whole monitoring period, and the fluorescence signals of the brains are continuously increased along with the time. The results of this experiment demonstrate that PLGA-LysoGM1/DiR micelles are able to cross the blood brain barrier and deliver DiR to the brain.
Example 12
In the embodiment, zebra fish is adopted to evaluate the blood-brain barrier penetrating effect of the blood-brain barrier penetrating drug-loaded micelle disclosed by the invention: fish (flk 1: GFP) were allocated 5 days before injection and embryos were collected the next day and individually fed. After the zebra fish is fertilized for 96 hours, a proper amount of 0.4% fish stabilizing anesthetic is dripped to anesthetize the zebra fish, and then the heart of the zebra fish is arranged in an agarose groove in a direction towards a microinjector for later use. The glass capillary needle for injection is prepared by pulling the needle by a needle pulling instrument, the pulled capillary needle is opened by an operating blade under a microscope and is placed into an injection instrument, and free DOX solution to be injected, PLGA/DOX nano solution and the blood brain barrier permeable drug-loaded micelle solution (wherein the preparation method of the PLGA/DiR nano solution is the same as that in example 11) related to the invention are sucked into the glass capillary needle to start injection. The needle tip was inserted into the heart of zebrafish, and the injection amount per fish was 10 nL. Transferring the zebra fish after injection onto a glass slide, with the scalp facing upwards, and the brain being straightened and symmetrical, fixing the zebra fish with melted 1% agarose, dripping a proper amount of small fish water containing 0.4% diazepam, and finally putting a clean cover glass. And respectively observing the fluorescence distribution condition of the zebra fish brain by using a laser confocal microscope 20min after injection.
The results are shown in FIG. 7: the free DOX solution was consistently localized in the cerebral vessels and did not exude, and in the PLGA/DOX nanoparticle control group, there was very little DOX penetration into the vessels. In contrast, the blood brain barrier penetrating drug-loaded micelle group obviously has a large amount of DOX fluorescence exuded blood vessels to gather in the brain parenchyma, which shows that the blood brain barrier penetrating drug-loaded micelle can penetrate the blood brain barrier and is consistent with the imaging experiment result of small animals.
Example 13
In this embodiment, the method for exploring the inhibitory effect of the blood brain barrier permeable drug-loaded micelle on glioma is as follows:
establishing a rat C6 glioma model: culturing C6 glioma cell to 80% fusion degree, digesting with 0.25% trypsin, collecting digestive juice, centrifuging, removing supernatant, adding 1mL cell culture solution, blowing to obtain cell suspension, sucking 10 μ L cell suspension, adding into cell counter, counting to ensure cell suspension concentration to be 1 × 10510 mul, placed in a constant temperature cell culture box at 37 ℃ to be inoculated. Before operation, rats are fasted for 12 hours without water prohibition, 10% chloral hydrate is injected into abdominal cavities (3mL/kg) of the rats, heads are fixed by a stereotaxic instrument after anesthesia, two ear needles penetrate into external auditory canals to be symmetrically fixed, hairs on tops of the heads are shaved, after alcohol disinfection, scalers are longitudinally cut along the median sagittal line of the heads by an operating knife, and the parietal bones are exposed by a separation film layer. Determining bregma point according to the stereotactic anatomical atlas of rat head, marking the coordinate site of tumor with bregma as origin, bregma 1.0cm in front, midline 3.0cm on right side. The skull drill is used for vertically drilling holes, the depth reaches the membrane, but the dura mater is not punctured, and the diameter of the bone hole is enlarged to form a small hole with the diameter of about 1.0 mm. Sucking 20 μ L C6 cell suspension with a micro syringe, vertically fixing on a stereotaxic apparatus, inserting needle through the center of bone hole to reach subdural 6.0mm, withdrawing needle by 1mm, and injecting at 1 μ L/min for cell to diffuse into brain completely, and reserving needle for 5 min. Slowly pulling out the needle at the speed of 1 μ L/min, sealing the bone hole with sterile bone wax, suturing scalp with sterile suture, sterilizing the incision, and keeping warm.
The survival curve of the rat is shown in FIG. 8, which shows that: the normal saline group, the DOX solution group, the GM1 solution group, the PLGA/DOX nanoparticle group and the PLGA-lysoGM1/DOX micelle group enable the median survival time of tumor-bearing rats to be 15.6 days, 21.4 days, 27.7 days, 23.7 days and 138.4 days respectively, wherein the survival time of the rats of the PLGA-lysoGM1/DOX micelle group is the longest, and the fact that the PLGA-lysoGM1 micelle can effectively deliver the antitumor drugs to the intracranial to play the drug effect is proved, and the survival time of the tumor-bearing rats is prolonged remarkably.
The neurobehavioral results are shown in fig. 9, from which it can be seen that: the physiological saline group, DOX solution group, GM1 solution group, PLGA/DOX nanoparticle group, and PLGA-lysoGM1/DOX micelle group maintained the nerve score of the tumor-bearing rats for the first 13 days after injection for at least 10 points, with no significant difference between the groups. After 15 days, significant differences in neurological scores occurred for each group: saline-treated tumor-bearing rats showed significant nerve damage before death, with the lowest nerve score of only 3 points. In contrast, rats with the DOX solution group and PLGA/DOX nanoparticle group had a higher neurological score than the saline group, with a neurological score of 5 points before death. The GM1 solution group had a slightly higher neurological score than the experimental group due to the effect of GM1 ganglioside in promoting the recovery of the function of the injured nerve. In contrast, the nerve score of the PLGA-lysoGM1/DOX micelle group was highest, and in particular, the nerve score of rats could be maintained around 12 points during the 15 to 35 days; although there was a decrease in the neurological score after 35 days, the neurological score was still significantly improved to around 7 points compared to the other groups.
The experimental results show that the PLGA-lysoGM1/DOX drug-loaded micelle can not only realize the delivery of the drug penetrating through the blood brain barrier, effectively inhibit the growth of tumors and prolong the survival time of tumor-bearing rats, but also realize the combined administration purpose of anti-tumor and nerve repair under the condition of not introducing other nerve injury repair drugs and materials, and obviously improve the survival state of the glioma rats. It was further demonstrated that PLGA-lysoGM1 micelles have great potential for the treatment of gliomas and other central nervous system diseases.
The applicant states that the present invention is illustrated by the above examples to provide a blood brain barrier permeable drug-loaded micelle, a preparation method and applications thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are all within the protection scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. The blood-brain barrier penetrating drug-loaded micelle is characterized by comprising a micelle formed by PLGA covalently modified by a GM1 hydrolysate and a drug encapsulated in the micelle; the medicine is a medicine for treating central nervous system diseases.
2. The blood brain barrier permeable drug-loaded micelle of claim 1, wherein the central nervous system disease comprises any one of glioma, pituitary tumor, meningioma, stroke, parkinson, or alzheimer;
preferably, the central nervous system disease therapeutic agent comprises any one or a combination of at least two of doxorubicin, rapamycin, paclitaxel, docetaxel, hydroxycamptothecin, vinblastine, amantadine, or rivastigmine.
3. The blood brain barrier permeable drug-loaded micelle of claim 1 or 2, wherein the GM1 hydrolysate covalently modified PLGA is obtained by amide linkage of a hydrolysate of monosialotetrahexosylganglioside and a polylactic acid-glycolic acid copolymer; the hydrolysis product of the monosialotetrahexosylganglioside is shown as the formula (I):
preferably, the polylactic acid-glycolic acid copolymer has a number average molecular weight of 6-12 kDa;
preferably, the molar ratio of the lactic acid structural unit to the glycolic acid structural unit in the polylactic acid-glycolic acid is (50-80): (20-50).
4. The method of preparing the blood brain barrier permeable drug-loaded micelle of any one of claims 1-3, wherein the method of preparation comprises the steps of:
(1) dropping PLGA solution covalently modified by GM1 hydrolysate into the stirring water solution to form blank micelle;
(2) and (3) dropwise adding the drug solution into the blank micelle solution which is being stirred, standing and dialyzing to obtain the blood-brain barrier permeable drug-loaded micelle.
5. The method for preparing the blood brain barrier penetrating drug-loaded micelle of claim 4, wherein the solvent of the GM1 hydrolysate covalently modified PLGA solution in the step (1) comprises any one or a combination of at least two of dimethylformamide, dichloromethane, acetone, tetrahydrofuran, chloroform or ethyl acetate;
preferably, the concentration of the solution in the step (1) is 4-40mg/mL, preferably 10-14 mg/mL;
preferably, the dropping rate of the step (1) is 10-100 μ L/min, preferably 40-60 μ L/min;
preferably, the volume of the aqueous solution of step (1) is 2-40mL, preferably 2-10 mL.
6. The method for preparing the blood brain barrier penetrating drug-loaded micelle according to claim 4 or 5, wherein the solvent of the drug solution in the step (2) comprises any one or a combination of at least two of dimethylformamide, dichloromethane, acetone, tetrahydrofuran, chloroform or ethyl acetate;
preferably, the concentration of the drug solution in the step (2) is 1-30mg/mL, preferably 2-8 mg/mL;
preferably, the dropping rate of the step (2) is 10-100 μ L/min, preferably 40-60 μ L/min;
preferably, the standing temperature in the step (2) is 2-8 ℃, and the time is 18-30 h;
preferably, the cut-off molecular weight of the dialysis bag used in the dialysis in the step (2) is 8000Da, and the dialysis time is 65-96 h;
preferably, the dialysis in step (2) is followed by filtration of the sample through a membrane having a pore size of 0.45. mu.m.
7. The method for preparing the blood brain barrier penetrating drug-loaded micelle of claim 4, wherein the method for preparing the GM1 hydrolysate covalently modified PLGA comprises the following steps:
(1') mixing a hydrolysate of monosialotetrahexosylganglioside shown in the formula (I), PLGA, tributylamine and 1-hydroxybenzotriazole, and reacting at 15-37 ℃ for 1-3 h;
(2 ') dropping the reaction solution obtained in step (1') into pre-cooled distilled water to form a colloidal precipitate, i.e. the GM1 hydrolysate covalently modified PLGA.
8. The method for preparing the blood brain barrier penetrating drug-loaded micelle according to claim 7, wherein the method for preparing the hydrolysate of the monosialotetrahexosylganglioside shown in formula (I) comprises the following steps:
(S1) mixing GM1 with a buffer solution containing 30 to 40mM sodium acetate, 0.3 to 0.5% taurodeoxycholic acid hydrate, and 50 to 150mM calcium chloride at pH 5.5 to 6.0, and dissolving the mixture with stirring;
(S2) mixing the hydrolase SCDase with the solution obtained in the step (S1), carrying out shaking reaction at 30-40 ℃ for 10-15h, centrifuging, taking the supernatant, and freeze-drying to obtain the monosialotetrahexosylganglioside hydrolysate shown in the formula (I).
9. Use of the blood brain barrier permeable drug-loaded micelle of any one of claims 1-3 for the preparation of a medicament for the prevention or treatment of a central nervous system disorder.
10. Use of the blood brain barrier permeable drug-loaded micelle of any one of claims 1-3 for the preparation of a medicament for the prevention or treatment of an intracranial tumor;
preferably, the intracranial tumour is a glioma.
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