CN109758582B - Polysorbate 80 penetrating through blood brain barrier and drug-loaded micelle delivery system formed by components of polysorbate 80 - Google Patents
Polysorbate 80 penetrating through blood brain barrier and drug-loaded micelle delivery system formed by components of polysorbate 80 Download PDFInfo
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Abstract
The invention discloses a drug-loaded micelle delivery system formed by polysorbate 80 penetrating through a blood brain barrier and components thereof. The invention claims the use of polysorbate 80 for the preparation of a product capable of penetrating the blood brain barrier and a product containing polysorbate 80 capable of penetrating the blood brain barrier. The invention discovers that the micelle formed by polysorbate 80 and the components thereof can form drug-loaded micelle with certain drugs which need to permeate the blood brain barrier but are difficult to permeate the blood brain barrier, helps the drug-loaded micelle to permeate the blood brain barrier, and improves the concentration and bioavailability of the drugs in the brain, thereby achieving the aim of treating brain-related diseases, enabling certain diseases which are still not treated by drugs to have the possibility of treatment at present, providing a new research thought and solving method for the research and difficulty of the brain-related diseases, and having important significance for the exploration and development of the region.
Description
Technical Field
The invention belongs to the technical field of biomedical high molecular materials, and particularly relates to a drug-loaded micelle delivery system formed by polysorbate 80 penetrating through a blood brain barrier and components thereof.
Background
With the increasing aging of the world population, diseases of the nervous system are becoming a major problem for threatening human health, and at the same time, brain diseases also seriously endanger human health, and especially, the treatment of brain diseases such as brain tumor, brain ischemia and the like has been one of the great challenges in the progress of medical development. One of the reasons for this is that many drugs cannot pass the blood brain barrier or that the throughput is insufficient to control the disease, increasing dose toxicity. The blood brain barrier is an important tissue structure for maintaining stable environment in the central nervous system, can effectively prevent harmful substances from entering brain tissues, but can limit the transportation of medicines, so that about 95% of medicines cannot enter the brain from blood, and is unfavorable for the treatment of diseases. How to increase the bioavailability of drugs is thus an important issue facing current treatments for neurological diseases. Some current researches show that the medicine can be promoted to pass through the blood brain barrier by changing the structure of the medicine, changing the administration route, promoting the opening of the blood brain barrier, increasing the transportation of the medicine and the like, which suggests that the medicine can adopt corresponding methods according to different properties of the medicine, thereby achieving the aim of safely and effectively treating diseases.
Currently, commonly used drug carriers include inorganic material nanoparticles, dendrimers, liposomes, polymer nanohydrogels, polymer micelles, and the like. Wherein, the polymer micelle is formed by the spontaneous assembly of an amphiphilic copolymer with a hydrophilic chain segment and a hydrophobic chain segment in water, and has the following advantages: the polymer micelle has an obvious core-shell structure, the hydrophobic block forms the inner core of the micelle in the self-assembly process, the hydrophilic block forms the outer shell of the micelle around the inner core of the micelle, and the obvious core-shell structure can effectively improve the water solubility of the drug; the polymer micelle has high stability, small and uniform particle size, has the function of overcoming the blood brain barrier, can wrap hydrophobic or water insoluble medicines according to different properties of the polymer hydrophobic chain segments by chemical, physical, electrostatic action and other methods, thereby entering the blood brain barrier, improving the medicine concentration, stability and bioavailability and having small toxic and side effects. Therefore, the polymer micelle used as a drug carrier penetrates through the blood brain barrier has good application prospect.
Many drugs, because of their hydrophilicity or relatively large molecular weight, are unable to or rarely penetrate the blood brain barrier and are not clinically effective in treating central nervous system disorders. The medicinal auxiliary material polysorbate 80 is a nonionic surfactant, is often used as a solubilizer in pharmacy, can increase the solubility of insoluble or fat-soluble medicaments, can prevent oxidation and hydrolysis of the medicaments, and has a protective effect on the stability of the medicinal preparation. This property of polysorbate 80 and its components has potential adjunctive therapeutic effects in the field of biological therapy.
Disclosure of Invention
The invention aims to provide a drug-loaded micelle delivery system formed by polysorbate 80 and components thereof penetrating the blood brain barrier.
The invention claims the use of polysorbate 80 for the preparation of a product capable of penetrating the blood brain barrier;
in addition, products capable of penetrating the blood brain barrier comprising polysorbate 80 of formula I are also within the scope of the present invention.
In particular, the product may be a drug or a pharmaceutical formulation or a pharmaceutical carrier.
The medicine can be specifically a central nervous system inhibitor, a central nervous system stimulant or a brain tumor anticancer medicine;
the central nervous system depressant is more specifically selected from any one of sedative hypnotic, antipsychotic, antiepileptic, anticonvulsant, antidepressant, anti-neurodegenerative disease drug and analgesic;
the central nervous system stimulant is specifically selected from any one of a cerebral cortex stimulant, a medulla oblongata respiratory central stimulant and a brain function restoring promoting drug;
the brain tumor anticancer drug is selected from any one of chemotherapy drugs and biological agents.
More specifically, the drug may be donepezil or nimodipine.
The polysorbate 80 may specifically be polysorbate 80 micelles, polysorbate 80 containing polyoxyethylene fatty acid ester micelles, or polysorbate 80 containing a complex or mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride, and polyoxyethylene alcohol.
The invention also claims a drug carrier or drug comprising at least one of polysorbate 80 micelles, polyoxyethylene fatty acid ester micelles, and a complex or mixture of polyethylene glycol, polyoxyethylene alcohol anhydride, and polyoxyethylene alcohol.
Specifically, the drug carrier or drug can be polysorbate 80 micelle, polyoxyethylene fatty acid ester micelle and a compound or mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol.
The drug carrier or drug is in particular a drug carrier or drug capable of penetrating the blood brain barrier.
The apparent forms of the polysorbate 80 micelle and the polyoxyethylene fatty acid ester micelle are of spheroid-like structures;
the polyoxyethylene fatty acid ester micelle is specifically polyoxyethylene oleic acid ester micelle.
The polyoxyethylene oleate micelle is specifically polyoxyethylene alcohol oleate micelle or polyoxyethylene alcohol anhydride oleate micelle.
The polyoxyethylene alcohol oleate micelle is specifically polyoxyethylene alcohol monooleate micelle;
the polyoxyethylene alcohol anhydride oleate micelle is specifically polyoxyethylene alcohol anhydride monooleate micelle or polyoxyethylene alcohol anhydride dioleate micelle.
The polyoxyethylene alcohol monooleate micelle is specifically a PIM micelle; the PIM represents polyoxyethylene isosorbide monooleate with a molecular formula of C 24 H 42 O 5 (OC 2 H 4 ) n N is 5-27; the structural formula is shown in formula III;
the polyoxyethylene alcohol anhydride monooleate micelle is specifically a PSM micelle; the PSM represents polyoxyethylene sorbitan monooleate with a molecular formula of C 24 H 44 O 6 (OC 2 H 4 ) n N is 15-33; the structural formula is shown in formula II;
the polyoxyethylene alcohol anhydride dioleate micelle is specifically a PSD micelle; the PSD represents polyoxyethylene sorbitan dioleate with a molecular formula of C 42 H 76 O 7 (OC 2 H 4 ) n N is 11-35; the structural formula is shown in formula IV.
The micelle can be prepared according to various conventional methods when used as a drug carrier or a drug, for example, can be prepared according to a method comprising the following steps:
1) Dissolving polysorbate 80, polyoxyethylene fatty acid ester or a mixture of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol with sodium chloride injection to obtain solution I with concentration of 10mg/ml;
2) Dissolving a proper amount of medicine to be loaded with ethanol to obtain solution II;
3) Mixing the solution I, the solution II, the ethanol and the sodium chloride injection, and evaporating to remove the ethanol to obtain the polysorbate 80 micelle, the polyoxyethylene fatty acid ester micelle or a compound consisting of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol.
Specifically, the concentration of the solution I can be 10mg/ml;
in the step 3), the volume ratio of the solution I to the ethanol to the sodium chloride injection is 0.5:0.2:0.5.
in the evaporation step, the temperature was 35 ℃.
The drug loading concentration of the polysorbate 80 micelle, the polyoxyethylene fatty acid ester micelle or the compound or the mixture consisting of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol is 0.3mg/ml.
In the compound or the mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol, the polyoxyethylene alcohol anhydride is PS; the polyoxyethylene alcohol is PI;
the PS represents polyoxyethylene sorbitan with a molecular formula of C 6 H 12 O 5 (OC 2 H 4 ) n N is 19-30;
the PI represents polyoxyethylene isosorbide, and the molecular formula is C 6 H 10 O 4 (OC 2 H 4 ) n N is 11-25;
the molecular formula of the polyethylene glycol is H (OCH) 2 CH 2 ) n OH, n is 5-18.
The PSM, PIM, PSD and the compound or the mixture consisting of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol can be prepared according to the following steps:
performing HPLC detection on the tetrahydrofuran solution of the polysorbate 80;
the conditions for the HPLC detection are as follows: the chromatographic column is a Gemini-NXC18 chromatographic column;
the eluent consists of a mobile phase A and a mobile phase B; wherein the mobile phase A is a mixed solution composed of methanol and water in a volume ratio of 95:5; mobile phase B is tetrahydrofuran;
the elution mode is gradient elution, and the elution speed is 25mL/min;
the gradient elution mode is as follows:
from 0 minutes to the end of 59 seconds at 14 minutes, the eluent is mobile phase A;
from 15 minutes to 15.09 minutes, the eluent consisted of mobile phase A and mobile phase B; the volume ratio of the mobile phase A to the mobile phase B is 50:50;
from 15.1 minutes to 16.99 minutes, the eluent is mobile phase B;
from 17 minutes to 17.09 minutes, the eluent is mobile phase B;
from 17.1 minutes to 18.99 minutes, the eluent is mobile phase A;
starting from 19 minutes to the end of 19 minutes, the eluent is mobile phase A;
collecting components with retention time of 1.10-1.40 min, and removing solvent to obtain the compound or mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol;
collecting components with retention time of 1.90-2.25min, and removing solvent to obtain PSM;
collecting components with retention time of 2.70-3.30min, and removing solvent to obtain PIM;
collecting components with retention time of 4.50-6.60min, and removing solvent to obtain PSD;
specifically, the concentration of the tetrahydrofuran solution of the polysorbate 80 is 500mg/ml;
the chromatographic column has a length of 150 mm and an inner diameter of21.2 mm, C18 particle size of 5 μm and pore diameter of
Carrier gas pressure is 60.0ps; the temperature of the drift tube is 60 ℃;
the method for removing the solvent is various conventional methods such as evaporation.
In addition, the invention also claims the application of the drug carrier or the drug in preparing the drug carrier as well as the application of the polysorbate 80 micelle, the polyoxyethylene fatty acid ester micelle or the compound or the mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol in preparing the drug carrier or the drug.
The polyoxyethylene fatty acid ester is specifically polyoxyethylene oleate.
The polyoxyethylene oleate is specifically polyoxyethylene alcohol oleate or polyoxyethylene alcohol anhydride oleate.
The polyoxyethylene alcohol oleate is specifically polyoxyethylene alcohol monooleate;
the polyoxyethylene alcohol anhydride oleate is polyoxyethylene alcohol anhydride monooleate or polyoxyethylene alcohol anhydride dioleate.
The polyoxyethylene alcohol monooleate is specifically PIM; the PIM represents polyoxyethylene isosorbide monooleate with a molecular formula of C 24 H 42 O 5 (OC 2 H 4 ) n N is 5-27;
the polyoxyethylene alcohol anhydride monooleate is specifically PSM; the PSM represents polyoxyethylene sorbitan monooleate with a molecular formula of C 24 H 44 O 6 (OC 2 H 4 ) n N is 15-33;
the polyoxyethylene alcohol anhydride dioleate is specifically PSD; the PSD represents polyoxyethylene sorbitan dioleate with a molecular formula of C 42 H 76 O 7 (OC 2 H 4 ) n N is 11-35.
The product capable of penetrating the blood brain barrier is specifically a compound or a mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol.
In the compound or the mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol, the polyoxyethylene alcohol anhydride is PS; the polyoxyethylene alcohol is PI;
the PS represents polyoxyethylene sorbitan with a molecular formula of C 6 H 12 O 5 (OC 2 H 4 ) n N is 19-30;
the PI represents polyoxyethylene isosorbide, and the molecular formula is C 6 H 10 O 4 (OC 2 H 4 ) n N is 11-25;
the molecular formula of the polyethylene glycol is H (OCH) 2 CH 2 ) n OH, n is 5-18.
The polysorbate 80 may include the following components: PSM, PIM, PSD, PEG, PS and PI;
the PSM represents polyoxyethylene sorbitan monooleate with a molecular formula of C 24 H 44 O 6 (OC 2 H 4 ) n N is 15-33; the structural formula is shown as the formula II;
the PIM represents polyoxyethylene isosorbide monooleate with a molecular formula of C 24 H 42 O 5 (OC 2 H 4 ) n N is 5-27; the structural formula is shown in the formula III;
the PSD represents polyoxyethylene sorbitan dioleate with a molecular formula of C 42 H 76 O 7 (OC 2 H 4 ) n N is 11-35; the structural formula is shown as the formula IV.
The PEG represents polyethylene glycol, and has a molecular formula of H (OCH) 2 CH 2 ) n OH, n is 5-18;
the PS represents polyoxyethylene sorbitan with a molecular formula of C 6 H 12 O 5 (OC 2 H 4 ) n N is 19-30;
the PI represents polyoxyethylene isosorbide, and the molecular formula is C 6 H 10 O 4 (OC 2 H 4 ) n N is 11-25.
The polysorbate 80 can comprise the following components in addition to the components:
polyethylene glycol monooleate (Polyethylene glycol monooleate, PM) of formula V, PM formula C 18 H 33 O 2 (OC 2 H 4 ) n H, n is 3-21;
polyoxyethylene isosorbide dioleate of formula VI (Polyoxyethylene isosorbide dioleate, PID) having formula C 42 H 74 O 6 (OC 2 H 4 ) n N is 6-25; fully esterified PIDs are insoluble in water because of the lack of hydrophilic hydroxyl groups in the structure;
polyethylene glycol dioleate (Polyethylene glycol dioleate, PD) of formula VII, PD formula C 36 H 66 O 3 (OC 2 H 4 )O n H, n is 4-18; the esterification degree is high, and the solubility is poor;
polyoxyethylene sorbitan trioleate (Polyoxyethylene sorbitan trioleate, PSTri) of formula VIII, PSTri having formula C 60 H 108 O 8 (OC 2 H 4 ) n N is 17-35; the esterification degree is high, and the solvent is very slightly dissolved;
polyoxyethylene sorbitan tetraoleate (Polyoxyethylene sorbitan tetraoleate, PStetra) represented by formula IX, PStetra having formula C 78 H 140 O 9 (OC 2 H 4 ) n N is 28-35; fully esterified PStetra is insoluble in water due to the lack of hydrophilic hydroxyl groups in the structure.
The polysorbate 80 is polysorbate 80 commonly used in the art. The polysorbate 80 is commercially available from a variety of sources. Such as those available from Nanjing Wilker chemical Co., ltd., product lot number 2015123-3. Polysorbate 80, which is publicly available from various commercial sources, contains PEG, PS, PI, PM, PID, PD, PSTri, but the specific amounts of each component may vary somewhat. For example, in polysorbate 80 available from Nanjing Weibull chemical Co., ltd, product lot number 20151203-3, the content of each of the above components is shown in Table 1, and the PM and PD contents are not listed because they are trace amounts per se, and are limited by the detection method.
TABLE 1 Single component content Table in Polysorbate 80 samples calculated by area normalization
The product may in particular be a medicament or a pharmaceutical formulation.
The medicine can be specifically a central nervous system inhibitor, a central nervous system stimulant or a brain tumor anticancer medicine;
the central nervous system depressant is more specifically selected from any one of sedative hypnotic, antipsychotic, antiepileptic, anticonvulsant, antidepressant, anti-neurodegenerative disease drug and analgesic;
the central nervous system stimulant is specifically selected from any one of a cerebral cortex stimulant, a medulla oblongata respiratory central stimulant and a brain function restoring promoting drug;
the brain tumor anticancer drug is selected from any one of chemotherapy drugs and biological agents.
More specifically, the drug may be donepezil or nimodipine.
The product also comprises auxiliary materials;
the auxiliary materials are specifically selected from any one of polysorbate 20, polysorbate 40 and polysorbate 60.
The drug-loaded micelle is prepared by a physical method or a chemical method; the physical method or the chemical method is a conventional method.
The administration mode of the product or the drug capable of penetrating the blood brain barrier may be specifically nasal administration, transdermal administration, ocular administration, oral administration or injection administration.
The drug-loaded micelle delivery system formed by the polysorbate 80 penetrating through the blood brain barrier and the components thereof can be applied to the following administration routes to promote the drug to pass through, such as: nasal administration, transdermal administration, ocular administration, oral administration and injection administration. The nasal administration can improve the concentration of certain medicines in brain, and has the advantages of high bioavailability, rapid absorption, convenient use and the like; the application of the polymer micelle in transdermal administration is mainly in the aspect of local skin treatment, and can also be used as a carrier of a plurality of biological macromolecules or genes, and the polymer micelle can enter the whole body to play a role in transdermal; the polymer micelle can improve the solubility of the therapeutic drug and the bioavailability of the therapeutic drug in eyes, reduce adverse reactions and realize active targeted drug delivery; the application of the polymer micelle to an oral administration system is beneficial to reducing the irritation of the drug to the gastrointestinal tract, increasing the concentration of the drug at the absorption part, improving the stability of the drug in the gastrointestinal tract and further improving the bioavailability of the drug; the polymer micelle for injection has good biocompatibility and higher safety when being applied to injection administration.
The invention discovers that the micelle formed by polysorbate 80 and the components thereof can form drug-loaded micelle with certain drugs which need to permeate the blood brain barrier but are difficult to permeate the blood brain barrier, helps the drug-loaded micelle to permeate the blood brain barrier, and improves the concentration and bioavailability of the drugs in the brain, thereby achieving the aim of treating brain related diseases, and ensuring that certain diseases which are still not treated by drugs are possible to treat at present.
Drawings
FIG. 1 is a MRM profile (380.30 >91.00, 380.30>243.10, 380.30> 362.20) of a 0.1ng/ml donepezil control solution.
FIG. 2 is a MRM profile (380.30 >91.00, 380.30>243.10, 380.30> 362.20) of a 1.0ng/ml donepezil control solution.
FIG. 3 is a MRM profile (380.30 >91.00, 380.30>243.10, 380.30> 362.20) of a 10.0ng/ml donepezil control solution.
FIG. 4 is an MRM pattern of polysorbate 80 micelles-donepezil (380.30 >91.00, 380.30>243.10, 380.30> 362.20).
FIG. 5 is an MRM profile of PEG/PS/PI micelle-donepezil (380.30 >91.00, 380.30>243.10, 380.30> 362.20).
FIG. 6 is an MRM pattern of PSM micelle-donepezil (380.30 >91.00, 380.30>243.10, 380.30> 362.20).
FIG. 7 is an MRM profile of PIM micelle-donepezil (380.30 >91.00, 380.30>243.10, 380.30> 362.20).
FIG. 8 is an MRM pattern of PSD micelle-donepezil (380.30 >91.00, 380.30>243.10, 380.30> 362.20).
Fig. 9 is an MRM profile of polysorbate 80 micelles-nimodipine (419.30 >343.10, 419.30> 301.05).
FIG. 10 is an MRM profile of PEG/PS/PI complex-nimodipine (419.30 >343.10, 419.30> 301.05).
Fig. 11 is an MRM profile of PSM micelles-nimodipine (419.30 >343.10, 419.30> 301.05).
Fig. 12 is an MRM profile of PIM micelle-nimodipine (419.30 >343.10, 419.30> 301.05).
FIG. 13 is an MRM profile of PSD micelles-nimodipine (419.30 >343.10, 419.30> 301.05).
FIG. 14 is a bar graph of the average particle size of different blank micelles and drug-loaded micelles measured by a laser particle size analyzer.
Fig. 15 is a graph showing morphological characterization of different blank micelles and drug-loaded micelles measured by transmission electron microscopy.
FIG. 16 is a photograph of a mouse living body with polysorbate 80 micelle-fluorescent DiR injected into the tail vein.
FIG. 17 is a chromatogram of an HPLC preparation of polysorbate 80 secondary component.
Detailed Description
The invention will be further illustrated with reference to the following specific examples, but the invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The starting materials are available from published commercial sources unless otherwise specified. Polysorbate 80 used in the examples below was purchased from Nanjing Weir chemical Co., ltd, product lot number 2015103-3 and the chromatogram of the next component was shown in FIG. 17.
Example 1,
Fluorescent dye DiR iodide, english name DiR iodide [1,1'-dioctadecyl-3, 3' -tetramethylindotricarbocyanine iodide ], molecular weight 1013.39, excitation wavelength 740nm, emission wavelength 780nm, liposoluble fluorescent probe for labeling cell membrane and hydrophobic tissue, and has structure shown in formula X:
donepezil is a hexahydropyridine oxide and has the chemical name of 1-benzyl-4- [ (5, 6-dimethoxy indanone-2-yl) methyl]Piperidine of formula C 24 H 29 NO 3 Molecular weight of 379.49, pKa value (Strongget acid) of 17.02, pKa value (Strongget Basic) of 8.62, polarityDonepezil belongs to a fat-soluble drug, is a reversible acetylcholinesterase inhibitor, and has a structure shown in a formula XI:
the chemical structural formulas of nimodipine, taxol and chlorogenic acid are shown in formulas XII, XIII and XIV respectively:
the above-mentioned Tween 80-DiR micelle, tween 80-donepezil Ji Jiaoshu and Tween 80-nimodipine micelle can be prepared according to various conventional methods. Such as may be made by a process comprising the steps of:
a1 10mg of donepezil and nimodipine are respectively taken and dissolved by ethanol to prepare 1mg/ml of donepezil drug stock solution and 1mg/ml of nimodipine drug stock solution.
B1 10mg of DiR was dissolved in ethanol to prepare 1mg/ml of DiR stock solution.
C1 Taking appropriate amount of Tween 80 and its secondary components, and dissolving with sodium chloride injection to obtain solution with concentration of 10 mg/ml.
D1 0.3ml of DiR stock solution was taken into a flask at the bottom, 0.2ml of ethanol, 0.5ml of Tween 80 and a solution of a secondary component thereof and 0.5ml of sodium chloride injection were added, and the ethanol was removed by suspension evaporation at 35℃with a rotary evaporator to prepare a DiR micelle solution, wherein 1ml of the solution contained 0.3mg of DiR.
E1 0.3ml of donepezil drug stock solution and nimodipine drug stock solution are taken, 0.2ml of ethanol, 0.5ml of tween 80 and a solution of a secondary component thereof and 0.5ml of sodium chloride injection are respectively added into a round bottom flask, and the ethanol is removed by suspension evaporation at 35 ℃ by a rotary evaporator to prepare a donepezil drug micelle solution and a nimodipine drug micelle solution, wherein 1ml of solution respectively contains 0.3mg of donepezil and 0.3mg of nimodipine.
FIG. 1 is a MRM profile (380.30 >91.00, 380.30>243.10, 380.30> 362.20) of a 0.1ng/ml donepezil control solution.
FIG. 2 is a MRM profile (380.30 >91.00, 380.30>243.10, 380.30> 362.20) of a 1.0ng/ml donepezil control solution.
FIG. 3 is a MRM profile (380.30 >91.00, 380.30>243.10, 380.30> 362.20) of a 10.0ng/ml donepezil control solution.
FIG. 4 is an MRM pattern of polysorbate 80 micelles-donepezil (380.30 >91.00, 380.30>243.10, 380.30> 362.20).
FIG. 5 is an MRM profile of PEG/PS/PI micelle-donepezil (380.30 >91.00, 380.30>243.10, 380.30> 362.20).
FIG. 6 is an MRM pattern of PSM micelle-donepezil (380.30 >91.00, 380.30>243.10, 380.30> 362.20).
FIG. 7 is an MRM profile of PIM micelle-donepezil (380.30 >91.00, 380.30>243.10, 380.30> 362.20).
FIG. 8 is an MRM pattern of PSD micelle-donepezil (380.30 >91.00, 380.30>243.10, 380.30> 362.20).
Fig. 9 is an MRM profile of polysorbate 80 micelles-nimodipine (419.30 >343.10, 419.30> 301.05).
FIG. 10 is an MRM profile of PEG/PS/PI complex-nimodipine (419.30 >343.10, 419.30> 301.05).
Fig. 11 is an MRM profile of PSM micelles-nimodipine (419.30 >343.10, 419.30> 301.05).
Fig. 12 is an MRM profile of PIM micelle-nimodipine (419.30 >343.10, 419.30> 301.05).
FIG. 13 is an MRM profile of PSD micelles-nimodipine (419.30 >343.10, 419.30> 301.05).
In the invention, the morphological research specific method of blank micelle and drug-loaded micelle comprises the following steps:
a1 Taking the blank micelle and the drug-loaded micelle, adding a proper amount of sodium chloride injection for dilution, and measuring the particle size by using a laser particle size tester.
B1 Using a Transmission Electron Microscope (TEM) to perform appearance form characterization on the blank micelle and the drug-loaded micelle, taking micelle solution with proper concentration, dripping the micelle solution onto a special copper net, dyeing with 4% phosphotungstic acid, and observing the form by using the TEM after natural drying.
In the invention, the intravenous injection mode of the tail of the mouse is adopted, and the preparation method of the biological sample specifically comprises the following steps:
a1 Intravenous injection of mice at a dose of 3mg/kg, and 10min after administration except for the positive control mice, dissecting, collecting brain tissue of mice, washing with physiological saline, drying, and storing at-20deg.C. The mice in the positive control group were dissected 2 hours after administration, and the brain tissues of the mice were washed with physiological saline, wiped dry, and placed in a living body imager to measure the fluorescence intensity.
B1 Before the experiment, brain tissue was weighed, mashed, and an appropriate amount of physiological saline (1 g:2 ml), vortex and mix well. After centrifuging the homogenate at 10000rpm for 15min at 4deg.C, 200 μl of the supernatant was removed and placed in a 1.5ml vial, 600 μl of methanol was added, vortexed, and mixed well. Centrifuging at 10000rpm,4℃for 15min. The supernatant was removed in 1.5ml vials, N 2 And (5) blow-drying. 300. Mu.L of methanol was added to dissolve the precipitate, and the mixture was vortexed and mixed well. Centrifuging at 10000rpm,4℃for 15min. Supernatant was taken for UPLC-MS/MS analysis.
The UPLC-MS/MS method is preferably specifically as follows:
instrument: SHIMADZU LC-MS 8050
CE: the ionic reaction for donepezil characterization was m/z 380.3> m/z 91.00, m/z 380.3> m/z243.10, m/z 380.3> m/z 362.20; the ionic reaction for nimodipine characterization was m/z 419.00> m/z 343.10, m/z 419.00> m/z 301.05.
Ion source: ESI, positive ion detection, multiple Reaction Monitoring (MRM) scan
Ion source temperature: 300 DEG C
Chromatographic column: kimetex 2.6. Mu.C 18(50×2.1mm)
Flow rate: 0.3ml/min
Column temperature: 30 DEG C
Mobile phase: a:5mM ammonium acetate in water solution B: acetonitrile;
TABLE 2 gradient of mobile phases
In the present invention, the in vivo imager method is specifically:
a1 1 mouse is randomly taken, and anesthetic is injected into the abdominal cavity, wherein the injection dosage is 0.1-0.15ml/20g. After anesthesia, the tail vein was injected with a blank DiR solution at a dose of 3mg/kg. After administration, 0min,5min, 10min, 20min, 30min, 50min, and 60min were used for observing fluorescence distribution and fluorescence intensity, and after administration for 60min, the mice were dissected and brain tissues of the mice were taken out as blank groups.
B1 7 mice were given intraperitoneal injections of anesthetic. After anesthesia, the mice were randomly drawn from the polysorbate 80DiR drug-loaded micelle solution and single-component DiR drug-loaded micelle solution by intravenous injection at a dose of 3mg/kg. After administration, 0min,5min, 10min, 20min, 30min, 50min,60min,90min,120min were observed in a living imager (excitation wavelength: 740nm, emission wavelength: 790 nm) and the DiR fluorescence intensities of the brain tissues of each group of mice were calculated as experimental groups.
In the invention, the particle sizes of the blank micelle prepared by the film dispersion method, the polysorbate 80 loaded with donepezil, nimodipine, chlorogenic acid and taxol and the single-component PEG/PS/PI, PSM, PIM and PSD micelle thereof (shown in figure 14) show that the particle sizes of the blank micelle of the polysorbate 80 and the single-component thereof are below 200nm except for the PEG/PS/PI. The PEG/PS/PI particle size is about 1.1 mu m, and the PEG/PS/PI particle size is not amphiphilic and cannot form micelle, so that the PEG/PS/PI and drug molecules can possibly form a composite structure in a solution system, and the structure can improve the solubility of insoluble drugs and promote the drugs to enter brain tissues through blood brain barriers. In the drug-loaded micelle, the particle size of the polysorbate 80 and the single-component paclitaxel-loaded drug micelle thereof is distributed between 5 μm and 30 μm, and the particle size is overlarge, presumably because the paclitaxel-loaded micelle is unstable and the agglomeration phenomenon is serious. The appearance form of the polysorbate 80 and the single-component blank micelle and drug-loaded micelle thereof are both in a spheroid-like structure as shown in fig. 15 when the appearance form is detected by an electron microscope.
In the invention, diR micelles prepared from polysorbate 80 and single-component PEG/PS/PI, PSM, PIM and PSD thereof can be distributed in vivo and can reach the brain, and it is presumed that polysorbate 80 and single-component PEG/PS/PI, PSM, PIM and PSD thereof have brain targeting effect.
In the invention, through MRM maps of polysorbate 80 micelle-donepezil and polysorbate 80 micelle-nimodipine and in-vivo imaging images of mice with polysorbate 80 micelle-fluorescent DiR (shown in figure 16), it can be obtained that all of polysorbate 80 micelle-donepezil, polysorbate 80 micelle-nimodipine and polysorbate 80 micelle-fluorescent DiR enter the blood brain barrier of the mice, presumably because donepezil, nimodipine and DiR contain ring nitrogen atoms in chemical structural formulas, have molecular weights between 300 and 400 and are easy to be wrapped by the micelles.
In the invention, four medicines for intravenous injection are respectively: donepezil, nimodipine, paclitaxel and chlorogenic acid, from the obtained in vivo imaging of mice, it was found that donepezil and nimodipine could on average enter the blood brain barrier of mice.
The other two drugs, paclitaxel and chlorogenic acid, which are unable to cross the blood brain barrier, presumably related to their chemical structures and relative molecular weights, are each free of ring nitrogen atoms, which are 418.44, 853.9 and 354.31, respectively. Chlorogenic acid has a pKa of 3.91, and its acidity may disrupt the complete structure of the micelles formed by polysorbate 80 and its single components, resulting in leakage of chlorogenic acid before it enters brain tissue. The pKa value of the taxol is 11.9, the alkaline medicine has larger molecular weight and complex structure, and on one hand, the micelle formed by the polysorbate 80 and a single component thereof is presumably unstable in the alkaline environment formed by the taxol itself, so that the complete structure of the micelle is destroyed, the micelle agglomeration is seriously developed, the particle size is overlarge, and the micelle is difficult to pass through the blood brain barrier of a mouse; on the other hand, the taxol has larger molecular size and is difficult to be wrapped by polysorbate 80 and single components thereof, so that the medicine is easy to leak; it is also possible because the accumulation of paclitaxel in the brain tissue of the mice is below the detection limit of the instrument.
Claims (12)
1. A medicine capable of penetrating blood brain barrier comprises at least one of polyoxyethylene fatty acid ester micelle and compound or mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol, and donepezil;
the polyoxyethylene fatty acid ester micelle is a polyoxyethylene alcohol monooleate micelle, a polyoxyethylene alcohol anhydride monooleate micelle or a polyoxyethylene alcohol anhydride dioleate micelle;
the polyoxyethylene alcohol monooleate micelle is a PIM micelle; the PIM represents polyoxyethylene isosorbide monooleate with a molecular formula of C 24 H 42 O 5 (OC 2 H 4 ) n N is 5-27;
the polyoxyethylene alcohol anhydride monooleate micelle is a PSM micelle; the PSM represents polyoxyethylene sorbitan monooleate with a molecular formula of C 24 H 44 O 6 (OC 2 H 4 ) n N is 15-33;
the polyoxyethylene alcohol anhydride dioleate micelle is a PSD micelle; the PSD represents polyoxyethylene sorbitan dioleate with a molecular formula of C 42 H 76 O 7 (OC 2 H 4 ) n N is 11-35;
in the compound or the mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol, the polyoxyethylene alcohol anhydride is PS; the polyoxyethylene alcohol is PI;
the PS represents polyoxyethylene sorbitan with a molecular formula of C 6 H 12 O 5 (OC 2 H 4 ) n N is 19-30;
the PI represents polyoxyethylene isosorbide, and the molecular formula is C 6 H 10 O 4 (OC 2 H 4 ) n N is 11-25;
the molecular formula of the polyethylene glycol is H (OCH) 2 CH 2 ) n OH, n is 5-18.
2. A medicine capable of penetrating blood brain barrier comprises polyoxyethylene fatty acid ester micelle and nimodipine;
the polyoxyethylene fatty acid ester micelle is a polyoxyethylene alcohol monooleate micelle or a polyoxyethylene alcohol anhydride monooleate micelle;
the polyoxyethylene alcohol monooleate micelle is a PIM micelle; the PIM represents polyoxyethylene isosorbide monooleate with a molecular formula ofC 24 H 42 O 5 (OC 2 H 4 ) n N is 5-27;
the polyoxyethylene alcohol anhydride monooleate micelle is a PSM micelle; the PSM represents polyoxyethylene sorbitan monooleate with a molecular formula of C 24 H 44 O 6 (OC 2 H 4 ) n N is 15-33.
3. A medicament according to claim 1 or 2, characterized in that: the apparent morphology of the polyoxyethylene fatty acid ester micelle is of a spheroid-like structure.
4. A medicament according to claim 1, characterized in that: the compound or the mixture consisting of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol is prepared according to the method comprising the following steps:
performing HPLC detection on the tetrahydrofuran solution of polysorbate 80;
the conditions for the HPLC detection are as follows: the chromatographic column is a Gemini-NXC18 chromatographic column;
the eluent consists of a mobile phase A and a mobile phase B; wherein the mobile phase A is a mixed solution composed of methanol and water in a volume ratio of 95:5; mobile phase B is tetrahydrofuran;
the elution mode is gradient elution, and the elution speed is 25mL/min;
the gradient elution mode is as follows:
from 0 minutes to the end of 59 seconds at 14 minutes, the eluent is mobile phase A;
from 15 minutes to 15.09 minutes, the eluent consisted of mobile phase A and mobile phase B; the volume ratio of the mobile phase A to the mobile phase B is 50:50;
from 15.1 minutes to 16.99 minutes, the eluent is mobile phase B;
from 17 minutes to 17.09 minutes, the eluent is mobile phase B;
from 17.1 minutes to 18.99 minutes, the eluent is mobile phase A;
starting from 19 minutes to the end of 19 minutes, the eluent is mobile phase A;
collecting the components with retention time of 1.10-1.40 min, and removing solvent.
5. A medicament according to claim 4, characterized in that: the concentration of the tetrahydrofuran solution of the polysorbate 80 is 500mg/ml;
the chromatographic column has a length of 150 mm, an inner diameter of 21.2 mm, a C18 particle diameter of 5 μm and a pore diameter
Carrier gas pressure is 60.0ps; the drift tube temperature was 60 ℃.
6. A medicament according to claim 1 or 2, characterized in that: the drug capable of penetrating the blood brain barrier is administered by injection.
7. Application of polyoxyethylene fatty acid ester micelle or a compound or mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol in preparing a medicament capable of penetrating the blood brain barrier;
the drug is donepezil;
the polyoxyethylene fatty acid ester micelle is a polyoxyethylene alcohol monooleate micelle, a polyoxyethylene alcohol anhydride monooleate micelle or a polyoxyethylene alcohol anhydride dioleate micelle;
the polyoxyethylene alcohol monooleate micelle is a PIM micelle; the PIM represents polyoxyethylene isosorbide monooleate with a molecular formula of C 24 H 42 O 5 (OC 2 H 4 ) n N is 5-27;
the polyoxyethylene alcohol anhydride monooleate micelle is a PSM micelle; the PSM represents polyoxyethylene sorbitan monooleate with a molecular formula of C 24 H 44 O 6 (OC 2 H 4 ) n N is 15-33;
the polyoxyethylene alcohol anhydride dioleate micelle is a PSD micelle; the PSD represents polyoxyethylene sorbitan dioleate with a molecular formula of C 42 H 76 O 7 (OC 2 H 4 ) n N is 11-35;
in the compound or the mixture composed of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol, the polyoxyethylene alcohol anhydride is PS; the polyoxyethylene alcohol is PI;
the PS represents polyoxyethylene sorbitan with a molecular formula of C 6 H 12 O 5 (OC 2 H 4 ) n N is 19-30;
the PI represents polyoxyethylene isosorbide, and the molecular formula is C 6 H 10 O 4 (OC 2 H 4 ) n N is 11-25;
the molecular formula of the polyethylene glycol is H (OCH) 2 CH 2 ) n OH, n is 5-18.
8. Application of polyoxyethylene fatty acid ester micelle in preparing medicine capable of penetrating blood brain barrier;
the medicine is nimodipine;
the polyoxyethylene fatty acid ester micelle is a polyoxyethylene alcohol monooleate micelle or a polyoxyethylene alcohol anhydride monooleate micelle;
the polyoxyethylene alcohol monooleate micelle is a PIM micelle; the PIM represents polyoxyethylene isosorbide monooleate with a molecular formula of C 24 H 42 O 5 (OC 2 H 4 ) n N is 5-27;
the polyoxyethylene alcohol anhydride monooleate micelle is a PSM micelle; the PSM represents polyoxyethylene sorbitan monooleate with a molecular formula of C 24 H 44 O 6 (OC 2 H 4 ) n N is 15-33.
9. Use according to claim 7 or 8, characterized in that: the apparent morphology of the polyoxyethylene fatty acid ester micelle is of a spheroid-like structure.
10. The use according to claim 7, characterized in that: the compound or the mixture consisting of polyethylene glycol, polyoxyethylene alcohol anhydride and polyoxyethylene alcohol is prepared according to the method comprising the following steps:
performing HPLC detection on the tetrahydrofuran solution of polysorbate 80;
the conditions for the HPLC detection are as follows: the chromatographic column is a Gemini-NXC18 chromatographic column;
the eluent consists of a mobile phase A and a mobile phase B; wherein the mobile phase A is a mixed solution composed of methanol and water in a volume ratio of 95:5; mobile phase B is tetrahydrofuran;
the elution mode is gradient elution, and the elution speed is 25mL/min;
the gradient elution mode is as follows:
from 0 minutes to the end of 59 seconds at 14 minutes, the eluent is mobile phase A;
from 15 minutes to 15.09 minutes, the eluent consisted of mobile phase A and mobile phase B; the volume ratio of the mobile phase A to the mobile phase B is 50:50;
from 15.1 minutes to 16.99 minutes, the eluent is mobile phase B;
from 17 minutes to 17.09 minutes, the eluent is mobile phase B;
from 17.1 minutes to 18.99 minutes, the eluent is mobile phase A;
starting from 19 minutes to the end of 19 minutes, the eluent is mobile phase A;
collecting the components with retention time of 1.10-1.40 min, and removing solvent.
11. The use according to claim 10, characterized in that: the concentration of the tetrahydrofuran solution of the polysorbate 80 is 500mg/ml;
the chromatographic column has a length of 150 mm, an inner diameter of 21.2 mm, a C18 particle diameter of 5 μm and a pore diameter
Carrier gas pressure is 60.0ps; the drift tube temperature was 60 ℃.
12. Use according to claim 7 or 8, characterized in that: the drug capable of penetrating the blood brain barrier is administered by injection.
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| "Stabilising cubosomes with Tween 80 as a step towards targeting lipid nanocarriers to the blood–brain barrier;Hanisah Azhar 等;《European Journal of Pharmaceutics and Biopharmaceutics》;20160507;第104卷;第149页左栏第3段 * |
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