CN113694026A - Berberine long-circulating nano-liposome with brain targeting function and preparation method thereof - Google Patents

Berberine long-circulating nano-liposome with brain targeting function and preparation method thereof Download PDF

Info

Publication number
CN113694026A
CN113694026A CN202110919767.4A CN202110919767A CN113694026A CN 113694026 A CN113694026 A CN 113694026A CN 202110919767 A CN202110919767 A CN 202110919767A CN 113694026 A CN113694026 A CN 113694026A
Authority
CN
China
Prior art keywords
liposome
solution
column
mass
drug
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN202110919767.4A
Other languages
Chinese (zh)
Inventor
吴正治
王林
姜倩倩
乐卫东
汪梦霞
张淼
刘洁人
李利民
李子雯
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Institute of Gerontology
Original Assignee
Shenzhen Institute of Gerontology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Institute of Gerontology filed Critical Shenzhen Institute of Gerontology
Priority to CN202110919767.4A priority Critical patent/CN113694026A/en
Publication of CN113694026A publication Critical patent/CN113694026A/en
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography

Abstract

The invention belongs to the technical field of medicine and pharmacology, and discloses a preparation method of a berberine long-circulating nanoliposome BR-Lf with a brain targeting function, which comprises the following steps: preparing PL-COOH liposome by using ammonium sulfate as a hydration medium and adopting an improved ethanol injection method; taking 2 parts by mass of PL-COOH, adding 1 part by mass of EDC solution and 1 part by mass of NHS solution in an ice-water bath, magnetically stirring and activating for 0.5h, recovering to room temperature, adding 6 parts by mass of Lf, fully dissolving, adding 1.4 parts by mass of TEA, reacting for 4h, and terminating the reaction in the ice-water bath to obtain the lactoferrin modified PEG blank liposome PL-Lf; taking PL-Lf suspension, removing external water phase by anion and cation fiber column, adding berberine hydrochloride BR solution according to the drug-lipid ratio of 1:15, incubating at 40 deg.C for at least 15min, and stopping drug loading in ice water bath to obtain BR-Lf. BR-Lf with uniform particle size distribution, higher encapsulation efficiency and good stability is successfully prepared by optimizing the preparation process.

Description

Berberine long-circulating nano-liposome with brain targeting function and preparation method thereof
Technical Field
The invention belongs to the technical field of medicine and pharmacology, and particularly relates to a berberine long-circulating nano liposome with a brain targeting function and a preparation method thereof.
Background
Alzheimer's Disease (AD) is the most common senile dementia, a degenerative disease of the central nervous system, characterized mainly by progressive memory impairment, cognitive impairment, personality changes, and most frequently occurs in the elderly or in the pre-senile stages. According to the ' 2019 report of world Alzheimer's disease ', more than 5000 million people worldwide suffer from dementia, and by 2050, the number will increase to 1.52 hundred million. One person suffers from dementia every 3 seconds, and the current annual dementia cost is estimated to be $ 1 trillion, which doubles by 2030. AD has become one of the largest global public health and social health challenges facing humans today and in the future. At present, the clinical treatment medicines for the Alzheimer disease are mainly cholinesterase inhibitors and glutamate receptor antagonists, can only temporarily relieve clinical symptoms, cannot cure the symptoms, have large toxic and side effects, and are difficult to tolerate by patients taking the medicines for a long time. The lack of effective drugs for the prevention and treatment of AD imposes a heavy social and economic burden on AD patients and families, and thus the enhancement of the reuse of conventional anti-AD drugs and the development of new drugs is imminent.
Berberine is a natural active ingredient extracted from the rhizome of Chinese herbal medicine Coptis, the Shennong's herbal medicine has a clear record of Coptis ' long-term forgetfulness ', and the pharmacological action of BR on the nervous system is first reported in 1970. A plurality of studies at home and abroad find that BR has the pharmacodynamic effects of protecting nerves, obviously inhibiting acetylcholinesterase, antagonizing Tau protein phosphorylation of AD core pathological products and the like and resisting AD at multiple targets. A series of researches of Durairajan and the like find that BR can inhibit phosphorylation caused by GSK3 beta through a PI3K/Akt pathway, inhibit the expression of Tau protein in neuron cells in brains of AD model mice, reduce the level of phosphorylated Tau protein, reduce the formation of neuron fiber tangles in the cells and play a role in protecting the nerve cells in the brains. Chen and other researches find that BR can inhibit insulin resistance of mice and improve cognitive ability of the mice. The influence of BR on the cytokines and oxidative stress of rat astrocytes induced by Abeta 25-35 is studied by crystalloid et al, and as a result, the oxidative stress, IL-1 beta, IL-6, TNF-alpha protein secretion and mRNA expression of the drug group are reduced, and the curative effect of the BR high-dose group is best. Perimorphic studies and the like research the influence of different dosages of BR on the learning and cognitive functions of AD rats and the influence on the expression of GSK3 beta and Tau protein, and the result shows that the BR can improve the learning and memory abilities of the AD rats. Sadraie and other researches find that BR can inhibit acetylcholinesterase, malondialdehyde, protein carbonyl activity and DNA breakage in the hippocampus of an AD model mouse, improve the oxidation resistance, improve the space recognition memory of a Y maze and improve the recognition of a new object.
However, BR is difficult to be absorbed by oral administration and has extremely low bioavailability, the conventional oral preparation is difficult to achieve effective drug concentration in brain, and the BR is widely distributed in vivo and generates toxic effects such as myocardial toxicity and the like, and particularly intravenous injection or instillation can cause reactions such as vasodilatation, blood pressure reduction, cardiac inhibition and the like. These problems severely restrict the popularization and application of BR in the clinical treatment of AD. Therefore, it is important to use effective drug carriers to reduce these limitations and improve the therapeutic efficacy of BR.
The liposome is an ideal drug carrier, has the advantages of low toxicity, high encapsulation rate, biodegradation and the like, and can improve the in vivo circulation time of the drug by using the surface modification of the polyethylene glycol. Wang et al prepared ordinary and poly-PEG modified long-circulating BR liposomes, compared and evaluated their efficacy and safety as potential antitumor agents, and found that the residence time of BR liposomes in the circulation is significantly increased compared to BR solution, and BR liposomes selectively increased the BR concentration in liver, spleen, lung and tumor of tumor-bearing mice, and reduced the distribution in heart and kidney. Yung and the like prepare the BR liposome by different methods, and the results show that the PEG modified long-circulating BR liposome has stronger growth inhibition rate on HepG2 cells than the common BR liposome, and effectively reduces the clearance rate of BR in blood plasma and tissues and the size and weight of tumors in experimental mice. Allijn et al prepared berberine liposome ethanol injection by gel method, and experiments prove that the long-circulating BR liposome improves the solubility buffer of berberine and retains the ejection fraction after myocardial infarction. Alba et al prepared BR liposomes for reducing liver damage and improving leishmaniasis-infected organ dosing concentration, and the results showed that the entrapment of the liposomes enhanced drug accumulation in the liver and spleen and reduced plasma triglyceride levels. Zhang et al construct berberine and adriamycin co-carried liposome (LipoBeDo), and the result shows that LipoBeDo can obviously inhibit tumor growth of 4T1 mouse breast cancer model, can completely inhibit the rapture toxicity of myocardial infarction doxillin to mice, and provides a new direction for clinical treatment of triple negative breast cancer and safe application of DOX.
The key problem of using BR liposome for preventing and treating AD lies in how to improve the drug concentration of focal sites in brain. Lf is a globular glycoprotein composed of a single polypeptide chain, a lactoferrin receptor (LfR) is connected to the surface of endothelial cells of a Blood Brain Barrier (BBB), and the combination of Lf and LfR can mediate an Lf modified drug carrier system to cross the BBB, so that the Lf modified drug carrier system is improvedThe concentration of drugs in the brain, Lf, has been widely used in the study of targeted nano-therapies for the treatment of neurological diseases. Liu et al found that Lf modified daunorubicin and magnolol liposome can be transported across BBB, and promote accumulation of drug in brain tumor tissue. Karami et al have shown that Lf modified Innovir nanoemulsion can significantly enhance the brain penetration of Innovir. Huang et al modified Lf in encapsulation99mCompared with the unmodified Lf liposome, the Tc-BMEDA liposome has the advantages that the in-vitro cell uptake of the Lf modified liposome is improved by more than 3 times, and the intracerebral distribution of the Lf modified liposome is improved by 2 times. The Lf modified drug carrier has obvious advantages in AD diagnosis and treatment, and compared with the single use of memantine hydrochloride (MEM), the Lf coupled polymethyl methacrylate loaded MEM obviously improves the drug brain uptake of AD model mice. The combination of the lipid carrier PLGA and Lf can obviously increase the blood brain barrier permeability of the curcumin serving as an anti-AD medicament.
For the research on preventing and treating AD by BR, the research focuses on the pharmacodynamics and mechanism of BR raw medicines, and no report on the research on the dosage form improvement of BR synergistic attenuation is provided. The prior art proves the effectiveness of BR in preventing and treating AD at multiple target points, but does not solve the pharmacological toxicity and the treatment efficiency of BR, and slows down the clinical application pace of BR in treating AD.
In summary, the problems of the prior art are as follows:
the disease course of AD can not be effectively blocked at present, and the existing treatment medicines can only temporarily relieve the symptoms of AD and have great side effects; in the prior art, the natural traditional Chinese medicine active ingredient berberine and the modern advanced drug-loading technology are not combined to prepare the brain-targeting invisible nano-liposome which is applied to the prevention and treatment of AD; moreover, BR is widely distributed in the body, generates toxicity to multiple organs, and has little medicine reaching the brain, thereby limiting the clinical application of BR.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an invisible brain-targeted berberine nano-liposome and a preparation method thereof. The invention fills up a gap of dosage form improvement of BR synergy and attenuation for AD prevention and treatment, and solves the technical problem of preparation of the invisible PEG-modified BR nano liposome with brain targeting.
The invention is realized in such a way that a preparation method of the berberine long-circulating nanoliposome BR-Lf with a brain targeting function comprises the following steps:
preparing a blank liposome PL-COOH containing an antelope base end by using ammonium sulfate as a hydration medium and adopting an improved ethanol injection method;
taking 2 parts by mass of PL-COOH liposome, adding 1 part by mass of EDC aqueous solution and 1 part by mass of NHS aqueous solution under the condition of an ice-water bath, magnetically stirring and activating for 0.5h, recovering to room temperature, adding 6 parts by mass of lactoferrin Lf, fully dissolving, adding 1.4 parts by mass of TEA, reacting for at least 4h, and terminating the reaction in the ice-water bath to obtain the lactoferrin modified PEG blank liposome PL-Lf;
and removing an outer water phase from the PL-Lf suspension through an anion-cation fiber column, adding berberine hydrochloride BR solution according to the medicine-lipid ratio of 1: 15-30, incubating at the constant temperature of 40-55 ℃ for at least 15min, and stopping carrying the medicine in an ice-water bath to obtain the lactoferrin modified PEG berberine long-circulating nanoliposome BR-Lf.
Further, removing an external water phase through an anion-cation fiber column, wherein the ratio of medicine to lipid is 1:20 adding small chai alkali BR solution.
Further, 6.0mg/mL of hydrochloric acid small firewood alkali solution is added according to the medicine-fat ratio of 1:20 (w/w).
Further, comprising:
weighing oxidized soybean phospholipid HSPC and cholesterol CH according to the mass ratio of 2-4: 1, and weighing the oxidized soybean phospholipid HSPC and the cholesterol CH according to the mass ratio of 1: 1-3 weighing mPEG2000-DSPE/DSPE-PEG2000-COOH, and mixing with 5-15% ethanol by volume, heating in 65 deg.C water bath for at least 10min to volatilize ethanol, adding (NH) preheated to the same temperature4)2SO4Stirring the solution in water bath for at least 20min to obtain empty liposome primary product PL;
treating PL with ultrasonic cell pulverizer, and grading sequentially with 0.8um, 0.45 μm, and 0.22 μm microporous filter membrane to obtain blank liposome PL-COOH containing antelope base end.
Further, the ultrasonic cell crusher operates according to the following working parameters when working: 200 Wx 2min, 400 Wx 6min, 1s of work and 1s of pause.
Further, the air conditioner is provided with a fan,weighing oxidized soybean phospholipid HSPC and cholesterol CH according to the mass ratio of 3:1, and weighing mPEG according to the mass ratio of 1:22000-DSPE/DSPE-PEG2000-COOH, in admixture with 10% by volume of ethanol, heating in a water bath at 65 ℃ for at least 10min to evaporate the ethanol, adding 200mM (NH) preheated to the same temperature4)2SO43mL of the solution is stirred in a water bath for at least 20mm to obtain an empty liposome primary product PL.
Further, 1. mu. mol of-COOH, EDC was 8mg/ml, and aqueous NHS solution was 2.4 mg/ml.
Further, taking PL-Lf suspension, removing an external water phase through an anion and cation fiber column, and comprising the following steps:
taking 0.2 part by mass of PL-Lf suspension, loading the PL-Lf suspension onto the top end of a centrifugally pretreated anion-cation exchange fiber column, simultaneously adding 0.2 part by mass of redistilled water, staying for at least 10min, centrifuging for at least 4min at a rotating speed of 1000-2000 r/min, adding 0.1 part by mass of redistilled water at the top end of the column, centrifuging for at least 4min at a rotating speed of 1000-2000 r/min, eluting, merging eluents, and uniformly mixing to obtain the compound (NH) with the structure (NH)4)2SO4Liposomal suspensions of transmembrane ion gradients.
Further, still include:
adopting anion and cation fiber column filtration and high performance liquid chromatography to measure BR encapsulation efficiency in BR-PL, which comprises the following steps:
chromatographic conditions are as follows: a chromatographic column: a C18 column (4.6X 250mm, 5 μm, Thermo Co.); mobile phase: acetonitrile-0.05 mol/L KH2PO4(23:77) (v/v), pH 3.0 with phosphoric acid; flow rate: 1.0 ml/min; column temperature: 30 ℃; sample introduction amount: 20 mu l of the mixture; detector wavelength: 212 nm;
linear investigation: precisely measuring 0.5, 1.0, 2.0, 3.0 and 4.0mL of berberine hydrochloride reference solution 50.0 μ g/mL, respectively placing in a 10mL measuring flask, diluting to scale with double distilled water, shaking, and filtering with 0.45 μm microporous membrane to obtain standard solutions with concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 μ g/mL. 20 mu L of sample injection high performance liquid chromatograph is respectively taken for analysis, and the peak area A is recorded. Linear regression of the concentration C (. mu.g/mL) with peak area A;
and (3) drawing an elution curve: taking l ml of the prepared BR-PL liposome to be loaded on a column, eluting with PBS (phosphate buffer solution) with the pH value of 7.4 at the speed of 1 ml/min; the eluate was collected in 2ml per tube. Taking 1ml and 10ml measuring flasks of each tube, breaking methanol into a fixed volume, filtering with a 0.45 μm microporous filter membrane, determining with a liquid chromatograph, recording peak area, and drawing an elution curve;
analysis of fiber column for recovery of free drug and liposomes: preparing 0.20mg/ml hydrochloric acid berberine PBS solution, ultrasonically reducing drug particles, and vortex vibrating to promote dissolution; eluting 0.2ml according to the above conditions, collecting free drug, determining with liquid chromatograph, recording peak area, and calculating column recovery rate; accurately weighing the berberine hydrochloride PBS solution and an equivalent amount of blank liposome, mixing, eluting on a column, collecting free drug components, diluting with methanol to a constant volume, carrying out sample injection measurement, and calculating the recovery rate;
determination of BR-PL Liposome encapsulation efficiency: taking 0.1mL of BR liposome, adding methanol solution into one part of BR liposome to destroy liposome, diluting mobile phase to 10mL, shaking, passing through 0.45 μm microporous membrane, performing high performance liquid chromatograph at 212nm for sample injection determination, and recording peak area as AtotSubstituting into standard curve to calculate concentration, and recording as Ctot. Loading the other part onto the top of the cation exchange fiber column, and centrifuging at 2000r/min for 4 min; continuously adding 0.4mL redistilled water to the top of the cation exchange fiber column, centrifuging at 2000r/min for 4min, continuously operating for 3 times, mixing eluates, performing sample injection measurement at 212nm of high performance liquid chromatograph, and recording peak area as A1Substituting into standard curve to calculate concentration, and recording as C1(ii) a The encapsulation efficiency was calculated according to the following formula: EE ═ 1-C1)/Ctot×100％。
Further, still include:
the Lf connectivity was determined by Coomassie Brilliant blue: taking 2 parts of 100 mu l BR-Lf reaction solution, passing one part through a Sephadex G-100 micro-column to remove unreacted Lf, and passing the other part through the column, and specifically operating as follows: 0.1ml of Lf-TP-PL is added with 5ml of acid Coomassie brilliant blue G250 developing solution, mixed evenly and then the absorbance A is measured at 595nm of an ultraviolet-visible spectrophotometerafterAnd AbeforeSubstituting into standard curve to calculate concentration CafterAnd CbeforeThe Lf grafting rate was calculated according to the formula: l isfgrafed＝Cafter/Cbefore×100％。
The invention has the advantages and positive effects that:
according to several pathomechanistic hypotheses for AD: the invention discloses a Tau protein hypothesis, a cholinergic hypothesis and an apoptosis hypothesis, and finds that berberine has the neuroprotective effects of resisting Tau protein phosphorylation, inhibiting acetylcholinesterase activity and neuronal apoptosis in traditional Chinese medicine treatment of AD, and is expected to become a potential medicine for treating AD. In order to perform synergistic attenuation on the AD resistance of BR, the invention prepares an AD-resistant BR brain-targeted nano-carrier medicament: nano liposome is used as a drug carrier; polyethylene glycol (PEG) is used for stealth modification, so that the in vivo circulation time is prolonged, and the drug distribution of tissues such as liver, spleen, kidney and the like is reduced; the lactoferrin is selected to carry out brain targeting modification on the PEG liposome, so that the brain targeting property of the stealth BR nano liposome is improved.
The preparation process is optimized, so that the stealth brain-targeting berberine nano-liposome with uniform particle size distribution, high encapsulation efficiency and good stability is successfully prepared, and related results are not reported in documents.
The anion and cation fiber column filtration-high performance liquid chromatography detection method disclosed by the invention is simple in operation, high in precision and good in repeatability, and is suitable for determining the BR entrapment rate in the BR-Lf liposome.
The invention determines a method for measuring the encapsulation efficiency of brain-targeted long-circulating berberine liposome. Establishing a high performance liquid phase determination method of berberine and drawing a standard curve; and (3) separating BR liposome and free triptolide by adopting an anion-cation fiber column filtration method, inspecting the recovery rate, detecting the content of BR by using a high performance liquid chromatography method, and calculating the entrapment rate. The linear equation obtained by linear relation investigation is as follows: 94459x-22315, R20.9996. The berberine hydrochloride shows a good linear relation in the concentration range of 2.5-20.0 mug/mL. The anion and cation fiber column has a recovery rate of 99.87% for free drug and a better recovery rate for liposome.
The invention optimizes the preparation process of the stealth berberine nano liposome BR-PL: a preparation method for screening BR-PL nano liposome by taking the entrapment rate as an assessment index; the preparation process of the BR-PL nano liposome is optimized by changing the drug-to-lipid ratio, the drug loading temperature and the drug loading time. In the screening of the preparation method, an active drug loading-ammonium sulfate gradient method is selected for BR-PL nano liposome; the best preparation conditions for preparing the liposome are as follows: the drug loading temperature is 55 ℃, the drug loading time is 15min, and the drug-to-lipid ratio is l: 20.
the invention optimizes the preparation process of the stealth berberine nano liposome BR-Lf with the brain targeting function, which comprises the following steps: the process of firstly connecting lactoferrin, removing external water phase and carrying medicine is determined by observing the influence of removing and not removing external water on the encapsulation efficiency. Investigating the influence of different carboxyl end contents and different Lf dosages on the Lf grafting rate, and determining to select mPEG2000-DSPE/DSPE-PEG2000-COOH ratio of 1:2, Lf is used in an amount of 6 mg. . According to optimized preparation conditions, the particle sizes of BR-CL, BR-PL and BR-Lf which are prepared by gradient optimization and are established by taking ammonium sulfate as a hydration medium are 129.5 +/-4.6, 100.4 +/-3.2 and 118.6 +/-4.5 nm respectively; PI is respectively 0.235 plus or minus 0.012, 0.216 plus or minus 0.015 and 0.246 plus or minus 0.012, and the particle size distribution is narrow; the BR encapsulation rates are respectively 92.1 +/-3.0%, 94.7 +/-2.4% and 90.4 +/-1.2%; the grafting ratio of Lf in BR-Lf is 65.9 +/-2.2%, and the modification of lactoferrin has little influence on the particle size and the entrapment rate of BR liposome. The key technical problem of preparing BR brain targeting nano-carrier medicaments is solved preliminarily.
The invention adopts a mouse fluorescence imaging experiment to evaluate the BR-Lf brain targeting; the influence of BR-Lf liposome on the behavioral ability of the AD mouse is observed through a Y maze experiment; the influence of BR-Lf on AChE activity, Tau protein phosphorylation, apoptosis protein Bcl-2 and Bax expression of hippocampal tissues of AD model mice is evaluated to discuss the anti-AD mechanism of BR-Lf. The Lf modification is proved to improve the brain targeting property of the BR liposome; BR-Lf can improve the behavioral ability of AD model mice by inhibiting AChE activity, antagonizing Tau protein hyperphosphorylation and inhibiting apoptosis, and is expected to become an ideal anti-AD drug. Provides a new method for the treatment of AD, opens up a new idea, and has important research value and wide application prospect.
Drawings
FIG. 1 is a flow chart of a preparation method of brain targeting PEG berberine liposome provided by the embodiment of the invention.
FIG. 2 is a standard graph of berberine provided by an embodiment of the present invention.
FIG. 3 is a graph showing the elution curve of the fiber column filtration method provided by the embodiment of the present invention.
FIG. 4 is a graph showing the effect of different drug lipid ratios on the encapsulation efficiency of BR-PL liposomes provided by the examples of the present invention.
FIG. 5 is a graph of the drug loading time versus the encapsulation efficiency of BR-PL liposomes as provided by the examples of the present invention.
FIG. 6 is a graph showing the effect of different drug loading temperatures on the encapsulation efficiency of BR-PL liposomes provided by the present invention.
Fig. 7 is a standard graph of Lf provided by an embodiment of the present invention.
FIG. 8 is a transmission electron micrograph of BR liposomes provided by embodiments of the present invention.
FIG. 9 is a DiR-Lf liposome mouse fluorescence imaging diagram provided by the embodiment of the invention.
FIG. 10 is a graph showing the effect of BR-Lf liposomes provided in the examples of the present invention on the behavioral capacity of AD model mice.
FIG. 11 is a graph showing the effect of BR-Lf liposomes on Tau protein phosphorylation in AD model mice.
FIG. 12 is a graph showing the effect of BR-Lf liposomes on acetylcholinesterase activity in AD model mice according to the present invention.
FIG. 13 is a graph showing the effect of BR-Lf liposomes provided in the examples of the present invention on apoptotic proteins in neuronal cells of AD model mice.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
As shown in fig. 1, a preparation method of berberine long-circulating nanoliposome BR-Lf with brain targeting function comprises:
s110, preparing a blank liposome PL-COOH containing an antelope base end by using ammonium sulfate as a hydration medium and adopting an improved ethanol injection method;
s120, taking 2 parts by mass of PL-COOH liposome, adding 1 part by mass of EDC aqueous solution and 1 part by mass of NHS aqueous solution under the condition of an ice-water bath, magnetically stirring and activating for 0.5h, recovering to room temperature, adding 6 parts by mass of lactoferrin Lf, fully dissolving, adding 1.4 parts by mass of TEA, reacting for at least 4h, and terminating the reaction in the ice-water bath to obtain the lactoferrin-modified PEG blank liposome PL-Lf;
s130, removing an external water phase from the PL-Lf suspension through an anion-cation fiber column, adding a berberine hydrochloride BR solution according to a drug-lipid ratio of 1: 15-30, incubating at the constant temperature of 40-55 ℃ for at least 15min, and stopping drug loading in an ice-water bath to obtain the lactoferrin modified PEG (poly (ethylene glycol) berberine) long-circulating nanoliposome BR-Lf.
The first embodiment,
The berberine long-circulating nano-liposome BR-PL takes nano-liposome as a drug carrier; is prepared by stealth modification with polyethylene glycol PEG.
Preparing PEG blank liposome PL and carboxyl terminal PL-COOH by adopting an improved ethanol injection method with ammonium sulfate as a hydration medium, and preparing berberine PEG liposome BR-PL and BR-PL-COOH by adopting an ammonium sulfate gradient method, wherein the modified ethanol injection method specifically comprises the following steps:
the blank liposome is prepared by using ammonium sulfate as a hydration medium and adopting an improved ethanol injection method. Accurately weighing the film material according to the prescription in a penicillin bottle, wherein the HSPC/CH ratio is 3:1, mPEG2000-DSPE/DSPE-PEG2000-COOH ratio of 1:2, mixing with 10 percent (v/v) of ethanol, heating in a water bath at 65 ℃ (NH)4)2SO4The concentration of the solution is 200mM, and the solution is stirred in a water bath for 20min to obtain a blank liposome primary product.
After being treated by an ultrasonic cell crusher (200W multiplied by 2min, 400W multiplied by 6min, work for 1s and pause for 1s), the blank PEG blank liposome PL and the carboxyl terminal PL-COOH are respectively prepared by finishing the particles through 0.8 mu m, 0.45 mu m and 0.22 mu m microporous filter membranes in sequence.
Taking 0.2mL blank liposome suspension, loading onto the top of 3mL anion-cation exchange fiber column subjected to centrifugal pretreatment, adding 0.2mL redistilled water, standing for 10min, centrifuging at 2000r/min for 4min, and loading into columnAdding 0.1mL redistilled water 2000r/min at the top end, centrifuging for 4min, mixing the eluates, and mixing to obtain the final product with phospholipid concentration of about 20mg/mL (NH)4)2SO4Liposomal suspensions of transmembrane ion gradients. Taking a proper amount of gradient liposome PL, and mixing the gradient liposome PL with the medicine-lipid ratio of 1: adding 6.0mg/mL berberine hydrochloride solution into 20(w/w), incubating at constant temperature of 55 deg.C for 15min, and stopping drug loading in ice water bath to obtain BR-PL and BR-PL-COOH liposome.
Example II,
The brain-targeted berberine long-circulating nanoliposome BR-Lf is prepared by connecting a brain-targeted ligand Lf to the surface of a carboxyl-terminal PEGylated BR liposome by adopting a covalent coupling method, and specifically comprises the following steps:
ammonium sulfate is used as a hydration medium, and an improved ethanol injection method is adopted to prepare the blank liposome PL-COOH containing carboxyl terminals. Accurately weighing the film material according to the prescription in a penicillin bottle, wherein the HSPC/CH ratio is 3:1, the ratio of mPEG2000-DSPE/DSPE-PEG2000-COOH is 1:2, mixing with 10% (v/v) ethanol, heating in 65 deg.C water bath, volatilizing most ethanol, adding 200mM (NH) preheated to the same temperature4)2SO43mL of the solution is stirred in a water bath for 20min to obtain a blank liposome primary product.
After treatment with an ultrasonic cell disruptor (200 Wx 2min, 400 Wx 6min, 1s of work, 1s of pause), the carboxyl terminal PL-COOH was prepared by sizing through 0.8 μm, 0.45 μm, 0.22 μm microporous membranes in that order.
Precisely transferring 2ml of the carboxyl terminal PL-COOH liposome (containing 3mg, 1 mu mol-COOH) prepared in the previous step into a 10ml penicillin bottle, adding 1ml of EDC aqueous solution (8mg/ml, 40 mu mol) and 1ml of NHS aqueous solution (2.4mg/ml, 20 mu mol) under the condition of an ice-water bath, activating by magnetic stirring for 0.5h, and removing the ice-water bath to restore the reaction system to the room temperature. 6mgLf was further added thereto, and after Lf was sufficiently dissolved, 1.4. mu.l (0.4. mu. mol) of TEA was added thereto. After reacting for 4h, terminating the reaction in an ice-water bath to obtain PL-Lf.
Taking a proper amount of PL-Lf liposome suspension, removing an external water phase by an anion-cation fiber column, adding 6.0mg/mL BR solution according to a drug-lipid ratio of 1:20(w/w), incubating at the constant temperature of 55 ℃ for 15min, and stopping drug loading in an ice-water bath to obtain BR-Lf.
In one embodiment of the invention, a method for detecting the BR-PL entrapment rate of a berberine long-circulating nanoliposome adopts anion and cation fiber column filtration and high performance liquid chromatography to measure the BR entrapment rate in BR-PL, and specifically comprises the following steps:
chromatographic conditions. A chromatographic column: a C18 column (4.6X 250mm, 5 μm, Thermo Co.); mobile phase: acetonitrile-0.05 mol/L KH2PO4(23:77) (v/v), pH 3.0 with phosphoric acid; flow rate: 1.0 ml/min; column temperature: 30 ℃; sample introduction amount: 20 mu l of the mixture; detector wavelength: 212 nm.
And (5) drawing an elution curve. Taking l ml of the prepared BR-PL liposome to be loaded on a column, eluting with PBS (phosphate buffer solution) with the pH value of 7.4 at the speed of 1 ml/min; the eluate was collected in 2ml per tube. Taking 1ml and 10ml measuring bottles of each tube, breaking methanol into a fixed volume, filtering with a 0.45 μm microporous filter membrane, measuring with a liquid chromatograph, recording peak area, and drawing an elution curve, as shown in FIG. 3.
Analysis of the recovery of free drug and liposomes with fiber column. Preparing 0.20mg/ml hydrochloric acid berberine PBS solution, ultrasonically reducing drug particles, and vortex vibrating to promote dissolution; eluting 0.2ml according to the above conditions, collecting free drug, determining with liquid chromatograph, recording peak area, and calculating column recovery rate. Accurately weighing the berberine hydrochloride PBS solution and an equivalent amount of blank liposome, mixing, eluting on a column, collecting free drug components, diluting with methanol to a constant volume, injecting a sample, measuring, and calculating the recovery rate.
And (3) determining the encapsulation efficiency of the BR-PL liposome. Taking 0.1mL of BR liposome, adding methanol solution into one part of BR liposome to destroy liposome, diluting mobile phase to 10mL, shaking, passing through 0.45 μm microporous membrane, performing high performance liquid chromatograph at 212nm for sample injection determination, and recording peak area as AtotSubstituting into standard curve to calculate concentration, and recording as Ctot. Loading the other part onto the top of the cation exchange fiber column, and centrifuging at 2000r/min for 4 min; continuously adding 0.4mL redistilled water to the top of the cation exchange fiber column, centrifuging at 2000r/min for 4min, continuously operating for 3 times, mixing eluates, performing sample injection measurement at 212nm of high performance liquid chromatograph, and recording peak area as A1Substituting into standard curve to calculate concentration, and recording as C1. The encapsulation efficiency was calculated according to the following formula: EE ═ 1-C1)/Ctot×100％。
In one embodiment of the invention, the determination method of the Lf connection percentage of the brain-targeted berberine long-circulating nanoliposome BR-Lf comprises the following steps: separating the liposome and the Lf by using a sephadex microcolumn based on the molecular sieve principle according to the difference of molecular weights of the liposome and the Lf, and measuring the connection rate of the Lf by using a Coomassie brilliant blue method.
The Lf ligation was determined by Coomassie Brilliant blue. Taking 2 parts of 100 mu l BR-Lf reaction solution, passing one part through a Sephadex G-100 micro-column to remove unreacted Lf, and passing the other part through the column, and specifically operating as follows: 0.1ml of Lf-TP-PL is added with 5ml of acid Coomassie brilliant blue G250 developing solution, mixed evenly and then the absorbance A is measured at 595nm of an ultraviolet-visible spectrophotometerafterAnd AbeforeSubstituting into standard curve to calculate concentration CafterAnd CbeforeThe Lf grafting rate was calculated according to the formula: lfgrafed＝Cafter/Cbefore×100％。
The embodiment of the invention provides brain targeting evaluation of brain-targeted berberine long-circulating nanoliposome BR-Lf, which adopts a mouse fluorescence imaging method and specifically comprises the following steps:
lateral ventricle injection of Abeta1-42And (5) establishing an AD mouse model. The modified ethanol injection method is used for preparing DiR common liposome DiR-CL, PEGylated DiR liposome DiR-PL and lactoferrin DiR liposome DiR-Lf. 45 AD mice successfully modeled were randomly divided into 3 groups of 15 mice each, at 2.0 mg.kg-1DiR dose of DiR-CL, DiR-PL and DiR-Lf were injected into the tail vein of each group of mice, observed in a small animal live imager at 2, 4, 8, 12 and 24h after injection, respectively, and fluorescence and white photographs were taken. After completion of photographing at each time point, 3 mice were sacrificed by removing neck, brain, heart, liver, spleen, lung and kidney were separated, washed with physiological saline and blotted with filter paper, and then fluorescence photographs were taken in a small animal living body imager. And (3) carrying out quantitative analysis on the near infrared fluorescence intensity of the isolated brain by using molecular imaging software carried by the system.
The embodiment of the invention provides a method for determining influence of brain-targeted berberine long-circulating nanoliposomes BR-Lf on the behavioural ability of an AD model mouse, which adopts a Y maze experiment and specifically comprises the following steps:
the Y-maze experimental device consists of three support arms with 120-degree included angles, which are A, B, C three arms respectively. After the adaptive training is finished, the animal is placed at the tail end of the arm A, the animal freely goes in and out of the three arms, the total times (N) of entering the three arms within 5min of the mouse and the arm entering sequence are recorded, the continuous entering of the three different arms is taken as one-time correct alternate reaction, and the correct alternate reaction times (N) are recordeds). The excrement is cleared up in time in the experimental process, and the remaining smell is eliminated. The spontaneous alternation response rate (alternation behavior%) is used for reflecting the spatial working memory capacity of the mouse, and the calculation is carried out according to the following formula: alternation behavior (%) ═ Ns/(N-2)×100％。
The embodiment of the invention provides a study on an AD (anti-AD) mechanism of brain-targeted berberine long-circulating nanoliposome BR-Lf, which comprises the steps of kit detection and Western blot method, and the influence of BR-Lf on acetylcholinesterase activity, Tau protein hyperphosphorylation and neuronal apoptosis is measured, and the study specifically comprises the following steps:
the kit detects the activity of acetylcholinesterase, takes out mouse hippocampal tissue frozen at-80 ℃, and carries out detection according to the ratio of 1: tissue lysate (5. mu.L PMSF per 1ml lysate) was added at a rate of 10 (10. mu.L/mg tissue) and homogenate was prepared at low temperature using an ultrasonic cell disruptor. Standing the homogenate in ice bath for 1h, then centrifuging at 12000 xr/min at 4 ℃ for 20min, taking the supernatant for content determination, and operating according to the kit steps. AChE viability in tissues was calculated.
Immunohistochemistry method observes the expression of mouse hippocampal tissue phosphorylated Tau protein at position 396. Dewaxing and hydrating paraffin sections; repairing citrate thermoantigen; hydrogen peroxide blocks endogenous peroxidase; BSA blocking, and blocking for 30min in a room-temperature wet box; and (5) throwing off redundant liquid without washing. Adding primary antibody and secondary antibody, respectively, and incubating in oven wet box at 37 deg.C for 30 min. PBS was shaken gently for 5min × 3 times. DAB color development; performing hematoxylin counterstaining; dehydrating and sealing; the target area was observed under a microscope and the positive expression area was analyzed using ImageJ software.
Western Blot assay (Western Blot). And (3) taking the mouse brain hippocampus tissue for standby from a refrigerator at the temperature of-80 ℃, extracting total protein, and then carrying out protein quantification according to the BCA protein concentration determination kit instruction. Primary and secondary antibodies were added sequentially. The membrane was washed 3 times with PBS for 10min each time. And (3) displaying a strip obtained by immunoreaction by using a hypersensitive luminescent liquid, transferring the strip into a gel imaging analyzer for exposure and development, analyzing by using IPP image analysis software, quantifying target protein in a sample, and determining difference and change of target protein expression among groups by using beta-actin as an internal reference. The application of the principles of the present invention will be further described with reference to the accompanying drawings and specific embodiments.
1 preparation and process optimization of stealth brain-targeted berberine nano-liposome (BR-Lf)
Materials and instruments:
berberine hydrochloride (content > 98%, Shanghai Hotan Biotechnology Co., Ltd.); hydrogenated soya lecithin (HSPC, Lucas Meyer, germany); cholesterol (CH, nanjing new drug industry ltd); polyethylene glycol monomethyl ether 2000-distearoyl phosphatidylethanolamine (mPEG2000-DSPE, Genzyme, USA); distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-carboxyl conjugate (DSPE-PEG2000-COOH, Avastin pharmaceutical science and technology Co., Ltd.); lactoferrin (Lf, japan and mitsunka limited); methanol (chromatographically pure, chemical division of Shandong Yuwang practice Co., Ltd.); 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl, Sigma, USA); n-hydroxysuccinimide (NHS, Sigma, USA); triethylamine (TEA, bodi chemical ltd, Tianjin); phosphoric acid (analytically pure, Tianjin, Daloco chemical reagent works); ammonium sulfate ((NH4)2SO4, analytically pure, available from Xiong chemical Co., Ltd.); acetonitrile (chromatographically pure, hanbang science and technology, jiangsu); potassium dihydrogen phosphate (KH2PO4, analytical grade, majoram chemical reagent works, tianjin); absolute ethanol (pharmaceutical, Anhuite Biochemical Co., Ltd.); redistilled water (Wahaha group, Inc.); coomassie Brilliant blue G250 (e.g., Giese Biotech, Inc.).
Agilent 1260Infinity high performance liquid chromatograph (Agilent, USA); UV5100 type ultraviolet-visible spectrophotometer (anhui instrument science and technology ltd, anhui); DF-101S heat collection type constant temperature heating magnetic stirrer (Guyi City Yingyu Yuhua apparatus factory); an ake TDL80-2B centrifuge (Shanghai' an pavilion scientific instruments factory); BS124s electronic analytical balance (Sartorius, germany); JY92-II ultrasonic cell crusher (Ningbo science and technology Biotechnology Co., Ltd.); nicomp-380 Particle size Zeta potentiometers (Particle Sizing Systems, USA); type 732 cation exchange fibers (national chemical group chemical agents limited); type 717 anion exchange fibers (national pharmaceutical group chemical agents, ltd); polyvinylidene microporous filter membrane (Shanghai Movix scientific instruments Co., Ltd.).
1.1 Pre-prescription study of BR-PL nanoliposomes
1.1.1 establishment of HPLC detection method for Berberine encapsulation efficiency
The method comprises the following steps:
(1) preparation of blank liposomes
The blank liposome is prepared by using ammonium sulfate as a hydration medium and adopting an improved ethanol injection method. Accurately weighing the film material according to the prescription in a penicillin bottle, wherein the HSPC/CH ratio is 3:1, mixing with 10% (v/v) ethanol, heating in 65 deg.C water bath, volatilizing most ethanol, adding 200mM (NH) preheated to the same temperature4)2SO43mL of the solution is stirred in a water bath for 20min to obtain a blank liposome primary product. Treating with ultrasonic cell pulverizer, and sieving with 0.8 μm, 0.45 μm, and 0.22 μm microporous membrane to obtain blank liposome (PL) of hollow PEG.
(2) Preparation of BR-PL liposomes
Treatment of anion exchange fiber: preparing saturated sodium chloride solution, and performing ultrasonic treatment until the saturated sodium chloride solution is not dissolved, and taking supernatant. Taking the amount of the fiber to be treated to be 3 times of that of the fiber, placing the fiber in salt solution to be soaked for 18-20h, draining the salt solution, rinsing the fiber with clear water to remove water without yellow, then soaking the fiber in 5% hydrochloric acid for 4-8h, draining the acid solution, washing the fiber with water until the neutral pH value is 7, finally soaking the fiber in 2-4% sodium hydroxide solution for 4-8h, draining the alkali solution, washing the fiber with water until the neutral pH value is 7, then soaking the fiber in saturated salt solution for 18-20h, draining the salt solution, rinsing the fiber with clear water until the neutral pH value is 7, and adding 20% ethanol for storage for later use.
Treatment of cation exchange fibers: preparing saturated sodium chloride solution, and performing ultrasonic treatment until the saturated sodium chloride solution is not dissolved, and taking supernatant. Taking the amount of the fiber to be treated to be 3 times that of the fiber, placing the fiber in salt solution to be soaked for 18-20h, draining the salt solution, rinsing with clear water to remove water without yellow, soaking the fiber in 2-4% sodium hydroxide solution for 4-8h, draining the alkali solution, rinsing with water to neutral pH of 7, soaking the fiber in 5% hydrochloric acid for 4-8h, draining the acid solution, rinsing with water to neutral pH of 7, soaking the fiber in saturated salt solution for 18-20h, draining the salt solution, rinsing with clear water to neutral, and adding 20% ethanol for storage.
The fiber column was packed with about 2.5mL of anionic fiber, centrifuged at 2000r/min for 4min to obtain about 2mL of anionic column, then packed with about 1.5mL of cationic fiber, centrifuged at 2000r/min for 4min to obtain anionic: the cation is about 2: 1. taking 0.2mL blank liposome suspension, loading the blank liposome suspension on the top end of a centrifugal pretreated 3mL anion-cation mixed ion exchange fiber column [ anion: cation ═ 2:1(v/v)]Adding 0.2mL of redistilled water at the same time, staying for 10min, centrifuging at 2000r/min for 4min, adding 0.1mL of redistilled water at the top end of the column, centrifuging at 2000r/min for 4min for elution, merging the eluates, and uniformly mixing to obtain the product (NH) with the final phospholipid concentration of about 20mg/mL4)2SO4Liposomal suspensions of transmembrane ion gradients. Taking a proper amount of gradient liposome PL, adding 6.0mg/mL berberine hydrochloride solution according to the drug-lipid ratio of 1:10(w/w), incubating at 50 deg.C for 10min, and stopping drug loading in ice water bath to obtain BR-PL liposome.
(3) Chromatographic conditions
A chromatographic column: a C18 column (4.6X 250mm, 5 μm, Thermo Co.); mobile phase: acetonitrile-0.05 mol/L KH2PO4(23:77) (v/v), pH 3.0 with phosphoric acid; flow rate: 1.0 ml/min; column temperature: 30 ℃; sample introduction amount: 20 mu l of the mixture; detector wavelength: 212 nm.
(4) Investigation of linear relationships
Precisely measuring 0.5, 1.0, 2.0, 3.0 and 4.0mL of berberine hydrochloride reference solution 50.0 μ g/mL, respectively placing in a 10mL measuring flask, diluting to scale with double distilled water, shaking, and filtering with 0.45 μm microporous membrane to obtain standard solutions with concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 μ g/mL. 20 mu L of sample injection high performance liquid chromatograph is respectively taken for analysis, and the peak area A is recorded. Linear regression was performed on the concentration C (. mu.g/mL) as peak area A.
(5) Plotting of elution Curve
Taking l ml of the prepared BR-PL liposome to be loaded on a column, eluting with PBS (phosphate buffer solution) with the pH value of 7.4 at the speed of 1 ml/min; the eluate was collected in 2ml per tube. Taking 1ml and 10ml measuring bottles of each tube, breaking methanol into a fixed volume, filtering with a 0.45 mu m microporous filter membrane, determining with a liquid chromatograph, recording peak area, and drawing an elution curve.
(6) Analysis of recovery of free drug and liposomes by fiber column
Preparing 0.20mg/ml hydrochloric acid berberine PBS solution, ultrasonically reducing drug particles, and vortex vibrating to promote dissolution; eluting 0.2ml according to the above conditions, collecting free drug, determining with liquid chromatograph, recording peak area, and calculating column recovery rate.
Accurately weighing the berberine hydrochloride PBS solution and an equivalent amount of blank liposome, mixing, eluting on a column, collecting free drug components, diluting with methanol to a constant volume, injecting a sample, measuring, and calculating the recovery rate.
(7) Determination of BR-PL Liposome encapsulation efficiency
Taking 0.1mL of BR liposome, adding methanol solution into one part of BR liposome to destroy liposome, diluting mobile phase to 10mL, shaking, passing through 0.45 μm microporous membrane, performing high performance liquid chromatograph at 212nm for sample injection determination, and recording peak area as AtotSubstituting into standard curve to calculate concentration, and recording as Ctot. Loading the other part onto the top of the cation exchange fiber column, and centrifuging at 2000r/min for 4 min; continuously adding 0.4mL redistilled water to the top of the cation exchange fiber column, centrifuging at 2000r/min for 4min, continuously operating for 3 times, mixing eluates, performing sample injection measurement at 212nm of high performance liquid chromatograph, and recording peak area as A1Substituting into standard curve to calculate concentration, and recording as C1. The encapsulation efficiency was calculated according to the following formula: EE ═ 1-C1)/Ctot×100％。
As a result:
(1) investigation of linear relationships
The linear equation obtained by linear relation investigation is as follows: 94459x-22315, R20.9996. Shows that the berberine hydrochloride has good linear relation in the concentration range of 2.5-20.0 mug/mL as shown in figure 2.
(2) Plotting of elution Curve
The number of the sample bottle was plotted as the abscissa and the peak area as the ordinate to plot the elution curve, and the results are shown in FIG. 3. The fiber column is proved to be capable of effectively separating the free medicament from the liposome.
(3) Analysis of free drug recovery by fiber column
The column recovery rate is observed in table 1, which shows that the fiber column has no adsorption to the free drug, and the liposome has no influence on the elution of the free drug.
TABLE 1 recovery rate test results of fiber column
Figure BDA0003207041130000151
(4) Determination of BR-PL Liposome encapsulation efficiency
Taking 0.1mL of BR liposome, adding methanol solution into one part of BR liposome to destroy liposome, loading the other part of BR liposome onto cation exchange fiber column, eluting to obtain eluate, measuring by high performance liquid chromatograph at 212nm, and recording peak area as AtotAnd A1Substituting into standard curve to calculate concentration, and recording as CtotAnd C1. And (3) calculating the encapsulation efficiency according to a formula: EE ═ 1-C1)/CtotX 100%. The encapsulation rate of the BR-PL liposome is 75.36 percent
1.1.2 preparation Process research of BR-PL nanoliposome
The method comprises the following steps:
(1) passive drug loading: thin film dispensing-ultrasonic method
Respectively and precisely weighing hydrogenated soybean lecithin, cholesterol, polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine (the mass ratio is HSPC: Ch: DSPE-PEG2000 ═ 3: 1: 1) as a membrane material for preparing liposome, and berberine hydrochloride (the mass ratio of medicine to fat is 1:10) and dissolving in ethanol; placing the solution in a l00ml ground round bottom flask, performing vacuum rotary evaporation (100rpm) in a constant-temperature water bath at 40 ℃, and volatilizing ethanol; forming a uniform lipid film on the wall of the round bottle, adding PBS (pH7.4) buffer solution, and performing rotary hydration on the round bottle in a constant-temperature water bath at 40 ℃ to enable a liposome membrane to fall off to obtain suspension; performing ultrasonic treatment on the probe in ice bath for 2 seconds and stopping for 3 seconds for 10 minutes; sieving with 0.8 μm, 0.45 μm, and 0.22 μm microporous filter membrane sequentially to obtain PEG-TP nanoliposome.
(2) Active drug loading: ammonium sulfate gradient method
Accurately weighing the membrane material in a vial, mixing with 10% (v/v) ethanol, heating in 65 deg.C water bath, volatilizing most ethanol, adding 200mM (NH4) preheated to the same temperature2SO43mL of the solution is stirred in a water bath for 20min to obtain a blank liposome primary product. Treating with ultrasonic cell pulverizer, and sieving with 0.8 μm, 0.45 μm, and 0.22 μm microporous membrane to obtain blank liposome (PL) of hollow PEG. Collecting 0.2mL PL suspension, loading onto the top of 3mL anion and cation mixed ion exchange fiber column subjected to centrifugal pretreatment, eluting, mixing eluates, and mixing to obtain (NH)4)2SO4Liposomal suspensions of transmembrane ion gradients. Taking a proper amount of gradient liposome PL, adding 6.0mg/mL berberine hydrochloride solution according to the drug-lipid ratio of 1:10(w/w), incubating at 50 deg.C for 10min, and stopping drug loading in ice water bath to obtain BR-PL liposome.
The BR-PL nano liposome prepared by the two methods takes the entrapment rate and the particle size distribution as the investigation indexes, and the preparation method is selected preferentially.
As a result:
respectively adopting a film dispersion method and an ammonium sulfate gradient method to prepare BR-PL nano liposome, adopting Nicomp-380 particle size based on a dynamic light scattering principle to determine the particle size of the liposome, and adopting an HPLC method to determine the BR entrapment rate. The BR-PL liposome prepared by the thin film dispersion method has the particle size of 112.6 +/-4.5 nm, the PI of 0.246 +/-0.032 and the BR entrapment rate of 50.4 +/-2.2%. The BR-PL liposome prepared by the ammonium sulfate gradient method has the particle size of 104.2 +/-3.6 nm, the PI value of 0.143 +/-0.012 and the BR entrapment rate of 76.4 +/-3.9 percent. In contrast, the BR liposome prepared by the ammonium sulfate gradient method is better, so the BR liposome is prepared by the active drug-loaded ammonium sulfate gradient method.
1.2 preparation condition optimization of BR-PL nanoliposome
The method comprises the following steps:
according to the screening result of the BR-PL liposome preparation method, the BR-PL liposome is prepared by adopting an ammonium sulfate gradient method, the particle size distribution and the entrapment rate of the liposome are taken as investigation indexes, the prescription is subjected to single factor investigation, one condition is changed while other conditions are fixed, and the influence of different drug-to-lipid ratios, drug loading time and drug loading temperature on the BR-PL liposome is investigated.
(1) Ratio of drug to fat
Excessive drug incorporation can severely affect lipid bilayer formation. Fixing other conditions (the drug loading time is 10min, the drug loading temperature is 50 ℃), changing the proportion of berberine hydrochloride and phospholipid, preparing BR-PL nano liposome by an ammonium sulfate gradient method, and investigating the influence of drug lipid ratio on the encapsulation efficiency.
(2) Time of drug loading
The drug loading time is too short, and the liposome can not fully encapsulate the drug; the long time easily affects the stability and the manufacturability of the liposome. Fixing other conditions (the drug-lipid ratio is 1:10, the drug loading temperature is 50 ℃), changing the drug loading time, preparing BR-PL nano liposome according to an ammonium sulfate gradient method, and investigating the influence of the drug loading time on the liposome encapsulation efficiency.
(3) Temperature of drug loading
During the preparation of liposome, the temperature must be above the phase transition temperature of phospholipid to complete the entrapment of the drug, however, too high temperature may easily destroy the stability of liposome. Fixing other conditions (the drug-lipid ratio is 1:20, the drug loading time is 15min), changing the drug loading temperature, preparing BR-PL nano liposome according to an ammonium sulfate gradient method, and investigating the influence of different drug loading temperatures on the liposome encapsulation efficiency.
As a result:
(1) ratio of drug to fat
Fixing other conditions, changing the ratio of BR to phospholipid, preparing BR-PL nano liposome by ammonium sulfate gradient method, and the relationship between liposome entrapment rate and drug-lipid ratio is shown in FIG. 4. As can be seen from the figure, the BR-PL liposome encapsulation rate is increased and then decreased along with the increase of the drug-to-lipid ratio; when the medicine-fat ratio is 1:20, the encapsulation efficiency of the liposome is the maximum. It can be seen that the encapsulation efficiency of the liposome is directly related to the drug-lipid ratio, too much dosage exceeds the loading capacity of phospholipid, so that stable liposome cannot be formed, and the higher encapsulation efficiency requires an optimal drug-lipid ratio.
(2) Time of drug loading
Other reaction conditions are unchanged, BR-PL nano liposome is prepared in different drug loading time, and the relation between the entrapment efficiency and the drug-lipid ratio is shown in figure 5. As can be seen from the figure, the entrapment rate of the BR-PL liposome is increased along with the prolonging of the drug loading time; when the drug loading time is 15min, the entrapment rate of the BR-PL liposome reaches the maximum value; the encapsulation efficiency then gradually decreases. The long drug loading time can affect the stability of the liposome, and the high entrapment rate requires a proper drug loading time.
(3) Temperature of drug loading
The relationship between the entrapment rate and the drug loading temperature of the BR-PL nano liposome prepared at different drug loading temperatures is shown in figure 6. It can be seen that the entrapment rate of the BR-PL liposome is increased and then slightly decreased with the increase of the drug loading temperature, which indicates that the optimum temperature, here 55 ℃, is required for liposome-entrapped BR.
Therefore, in the research, the preparation conditions for preparing the BR-PL nano liposome by the ammonium sulfate gradient method are determined as follows: 20. the drug loading temperature is 55 ℃, and the drug loading time is 15 min.
1.3 preparation process optimization of BR-Lf nano liposome
1.3.1. Examination of the Effect of removing and not excluding Water on encapsulation efficiency
The method comprises the following steps:
and respectively investigating the influence of removing and not excluding water on the encapsulation efficiency, and optimizing BR-Lf preparation conditions.
(1) Preparation of carboxyl terminal PL:
accurately weighing the membrane material according to the prescription in a penicillin bottle, wherein the phospholipid/cholesterol ratio is 3: 1; mPEG2000-DSPE/DSPE-PEG2000-COOH ratio of 1:2, mixing with 10% (v/v) ethanol, heating in 65 deg.C water bath, volatilizing most ethanol, adding 200mM (NH) preheated to the same temperature4)2SO4Stirring the solution in water bath for 20min to obtain blank liposome primary product. Treating with ultrasonic cell pulverizer, and grading sequentially with 0.8 μm, 0.45 μm, and 0.22 μm microporous filter membrane to obtain blank liposome (PL-COOH) of PEG at empty carboxyl terminal.
(2) Preparation of the desired solution
EDC preparation, EDC & HCl 80mg is precisely weighed into a 10mL measuring flask, and distilled water or PBS solution is added to dissolve and dilute to scale, thus obtaining EDC solution of 8 mg/mL.
Preparation of NHS: NHS 24mg is weighed accurately into a 10mL measuring flask, and distilled water or PBS solution is added to dissolve and dilute to the scale mark, so as to obtain NHS solution of 2.4 mg/mL.
(3) Preparation of BR-Lf
BR-Lf 1: precisely transferring 0.5mL of carboxyl-terminal PL-COOH liposome (phospholipid concentration is about 20mg/mL, 0.25 mu mol-COOH) without an external water phase, placing the liposome in a 10mL penicillin bottle, adding 335 mu L of LEDC aqueous solution (8mg/mL, 40 mu mol) and 335 mu L of NHS aqueous solution (2.4mg/mL, 20 mu mol) under the condition of ice-water bath, activating the liposome by magnetic stirring for 0.5h, and removing the ice-water bath to restore the reaction system to the room temperature. To this was added 12mg of Lf, and after Lf was sufficiently dissolved, 1.4. mu.L (0.4. mu. mol) of TEA was added. After 4h of reaction, the reaction was terminated by an ice-water bath. Taking a proper amount of liposome suspension, removing an external water phase by an anion-cation fiber column, adding 6.0mg/mL BR solution according to a drug-lipid ratio of 1:20(w/w), incubating at the constant temperature of 55 ℃ for 15min, and stopping drug loading in an ice-water bath to obtain BR-Lf 1.
BR-Lf 2: taking a proper amount of carboxyl terminal PL-COOH liposome suspension, removing an external water phase by an anion-cation fiber column, adding 6.0mg/mL of a drug solution according to a drug-lipid ratio of 1:20(w/w), incubating at the constant temperature of 55 ℃ for 15min, and stopping drug loading in an ice-water bath to obtain the carboxyl terminal BR-PL liposome. Precisely transferring 0.5mL of carboxyl-terminal BR-PL liposome, placing the carboxyl-terminal BR-PL liposome in a 10mL penicillin bottle, adding 335 mu L of LEDC aqueous solution (8mg/mL, 40 mu mol) and 335 mu L of NHS aqueous solution (2.4mg/mL, 20 mu mol) under the condition of ice-water bath, magnetically stirring and activating for 0.5h, and removing the ice-water bath to restore the reaction system to the room temperature. To this was added 12mg of Lf, and after Lf was sufficiently dissolved, 1.4. mu.L (0.4. mu. mol) of TEA was added. After 4h of reaction, the reaction was terminated by an ice-water bath. Thus obtaining BR-Lf 2.
(4) Comparison of encapsulation efficiencies of BR-Lf1 and BR-Lf2
Taking BR-Lf1 and BR-Lf2 liposome 0.1mL respectively, adding methanol solution into one part to destroy liposome, diluting mobile phase to 10mL, shaking, passing through 0.45 μm microporous membrane, injecting sample, measuring, and recordingRecording peak area AtotSubstituting into standard curve to calculate concentration, and recording as Ctot. Loading the other part onto the top of the cation exchange fiber column, and centrifuging at 2000r/min for 4 min; continuously adding 0.4mL redistilled water to the top of the cation exchange fiber column, centrifuging at 2000r/min for 4min, continuously operating for 3 times, combining the eluates, performing sample injection measurement, and recording the peak area as A1Substituting into standard curve to calculate concentration, and recording as C1. The encapsulation efficiency was calculated according to the following formula: EE ═ 1-C1)/Ctot×100％。
As a result:
after the lactoferrin is connected by adopting the PL-COOH liposome without the carboxyl end of the external water phase, the external water phase is removed, and BR-Lf1 is prepared by loading BR with a drug, wherein the entrapment rate is 90.5 +/-5.2%; the BR-Lf2 is prepared by removing external water phase carboxyl end PL-COOH liposome, carrying medicine and then connecting lactoferrin, and the entrapment rate is 75.9 +/-4.7%. Therefore, the study identified a protocol for linking lactoferrin first-removing the external aqueous phase-loading (see fig. 1).
1.3.2. establishment of method for determining Lf grafting ratio of BR-Lf nanoliposome
Method: separating the liposome and the Lf by using a sephadex microcolumn based on the molecular sieve principle according to the difference of molecular weights of the liposome and the Lf, and measuring the adsorption rate of the Lf by using a Coomassie brilliant blue method.
(1) Establishing a standard curve for measuring the Lf content by a Coomassie brilliant blue method
Acid staining solution: accurately weighing Coomassie brilliant blue G250100.0 mg in a 1000ml measuring flask, adding 50ml 95% ethanol for dissolving, adding 100ml phosphoric acid, diluting with redistilled water to scale, mixing, and filtering.
Control solution: precisely weighing Lf 10.0mg in a 10ml measuring flask, adding water to dissolve and diluting to scale, thus obtaining Lf mother liquor of 1 mg/ml.
Precisely transferring 0ml, 0.01 ml, 0.02 ml, 0.04 ml, 0.06 ml, 0.08 ml and 0.l ml of the reference substance solution, respectively placing in 10ml test tubes with plugs, respectively adding water to 0.1ml, respectively adding 5.0ml of acidic staining solution, uniformly mixing, and immediately measuring absorbance at 595nm of an ultraviolet-visible spectrophotometer; while tube 0 was used as a blank control. The absorbance was linearly regressed with the control solution concentration.
(2) Determination of Lf grafting rate of BR-Lf nano-liposome
Preparation of dextran gel microcolumn: sephadex G-100 was soaked in distilled water overnight and allowed to swell sufficiently. After degassing, the mixture was filled in a 2.5mL syringe, and after the water content naturally flowed down, the mixture was centrifuged at 2000rpm for 4min to remove excess water for use.
The Lf ligation was determined by Coomassie Brilliant blue. Taking 2 parts of 100 mu l BR-Lf reaction solution, passing one part through a Sephadex G-100 micro-column to remove unreacted Lf, and passing the other part through the column, and specifically operating as follows: 0.1ml of Lf-TP-PL is added with 5ml of acid Coomassie brilliant blue G250 developing solution, mixed evenly and then the absorbance A is measured at 595nm of an ultraviolet-visible spectrophotometerafterAnd AbeforeSubstituting into standard curve to calculate concentration CafterAnd CbeforeThe Lf grafting rate was calculated according to the formula: lfgrafed＝Cafter/Cbefore×100％。
As a result:
since the absorbance values were already lower than 0.2 at Lf concentrations lower than 40. mu.g/ml, the Lf standards of 100-1000. mu.g/ml and 400-1000. mu.g/ml were plotted, respectively, and the results are shown in FIG. 7. When the Lf concentration is 100-20.9987, when the Lf concentration is 400-20.9969. From the linear regression equation of Lf, R2>0.99, can be used for measuring the content of Lf. Preliminary determination of Lfgrafed53.0. + -. 3.1%.
1.3.3. optimization of BR-Lf nanoliposome preparation process
Method: investigating the influence of different carboxyl end contents and different Lf dosage on the Lf grafting rate
PL-COOH was prepared using ammonium sulfate as the hydration medium by modified ethanol injection. Accurately weighing the components of each prescription in a penicillin bottle, wherein the ratio of phospholipid to cholesterol is 3: 1; mPEG2000-DSPE/DSPE-PEG2000-COOH ratios of 1:2 and 2:1, ethanol with volume fraction of 10 percent is used for water bath at 65 DEG CHeating to dissolve, volatilizing most ethanol, respectively adding 4mL of 200 mmol/200 (NH4)2SO4 solution preheated to the same temperature, and stirring in 65 deg.C water bath for 20min to obtain blank liposome primary product. Treating the obtained liposome with ultrasonic cell pulverizer, and sieving with microporous membrane to obtain PL-COOH1 and PL-COOH 2.
Precisely transferring 2ml of PL-COOH1 liposome (containing 3mg and 1 mu mol of COOH) into a 10ml penicillin bottle, adding 1ml of EDC aqueous solution (8mg/ml and 40 mu mol) and 1ml of NHS aqueous solution (2.4mg/ml and 20 mu mol) under the condition of an ice-water bath, magnetically stirring and activating for 0.5h, and removing the ice-water bath to recover the reaction system to the room temperature. 6mg and 12mg of Lf were further added thereto, respectively, and after Lf was sufficiently dissolved, 1.4. mu.l (0.4. mu. mol) of TEA was added thereto. After 4h of reaction, the reaction was terminated by ice-water bath to obtain PL-Lf1 and PL-Lf 2.
Precisely transferring 2ml of PL-COOH2 liposome into a 10ml penicillin bottle, adding 1ml of EDC aqueous solution and 1ml of NHS aqueous solution into the penicillin bottle under the condition of ice-water bath, magnetically stirring and activating for 0.5h, and removing the ice-water bath to restore the reaction system to the room temperature. 6mg and 12mg of Lf were added thereto, respectively, and after Lf was sufficiently dissolved, 1.4. mu.l of TEA was added. After 4h of reaction, the reaction was terminated by ice-water bath to obtain PL-Lf3 and PL-Lf 4.
As a result:
PL-Lf liposomes were prepared using different carboxy terminus contents and different Lf dosages, and the particle size and Lf grafting ratio were compared, with the results shown in Table 2. It can be seen that under the same carboxyl terminal content, the graft ratio is slightly increased by reducing the addition amount of Lf, and the particle size is not greatly changed. The PL-Lf liposome prepared by changing the content of carboxyl has increased particle size and reduced Lf grafting rate. Therefore, mPEG was selected for this study2000-DSPE/DSPE-PEG2000-COOH ratio of 1:2, Lf is used in an amount of 6 mg.
TABLE 2 particle diameter and Lf connection ratio for different connection amounts
Figure BDA0003207041130000211
Optimized preparation of BR-Lf nano liposome
The method comprises the following steps:
and preparing BR-Lf nano liposome according to the optimal preparation condition, characterizing and verifying the optimal preparation condition.
The particle size of the liposome is detected by a Nicomp-380 particle size analyzer based on the dynamic light scattering principle. Taking a small amount of liposome in a sample tube, adding sterilized injection water to dilute by a certain factor, enabling the optical density value to be between 250 and 300, and shaking up. Measuring an angle: 90 degrees; power: 75 mW; temperature: 25 ℃; light source: He-Ne laser.
Observing the microstructure of the liposome by a transmission electron microscope, sucking a small amount of sample, dripping the sample on a copper net covered with a carbon film, carrying out negative dyeing for 2min by using 1.0% phosphotungstic acid, sucking away excessive liquid by using filter paper, naturally airing at room temperature, and then placing under the transmission electron microscope for observation and photographing.
As a result:
the particle sizes of BR-CL, BR-PL and BR-Lf which are prepared by gradient optimization and take ammonium sulfate as a hydration medium are 129.5 +/-4.6, 100.4 +/-3.2 and 118.6 +/-4.5 nm respectively; PI is respectively 0.235 plus or minus 0.012, 0.216 plus or minus 0.015 and 0.246 plus or minus 0.012, and the particle size distribution is narrow; the BR encapsulation rates are respectively 92.1 +/-3.0%, 94.7 +/-2.4% and 90.4 +/-1.2%; the grafting ratio of Lf in BR-Lf is 65.9 +/-2.2%, and the modification of lactoferrin has little influence on the particle size and the entrapment rate of BR liposome.
The microstructure of BR-CL, BR-PL and BR-Lf is observed by a transmission electron microscope, and the result is shown in figure 8, and the three liposomes are all round-like single-chamber liposomes, have obvious double molecular layer membranes, are distributed uniformly, and have the particle size basically consistent with the data measured by a Nicomp-380 particle size analyzer.
2 research on AD resistance of stealth brain-targeted berberine nano-liposome (BR-Lf)
Materials and instruments:
berberine hydrochloride (content > 98%, Shanghai Hotan Biotechnology Co., Ltd.); hydrogenated soya lecithin (HSPC, Lucas Meyer, germany); cholesterol (CH, nanjing new drug industry ltd); polyethylene glycol monomethyl ether 2000-distearoyl phosphatidylethanolamine (mPEG2000-DSPE, Genzyme, USA); distearoyl phosphatidyl EtherAlcohol amine-polyethylene glycol 2000-carboxyl conjugate (DSPE-PEG2000-COOH, Avention pharmaceutical science and technology Co., Ltd.); lactoferrin (Lf, japan and mitsunka limited); 1,1 '-dioctadecyl-3, 3,3',3 '-tetramethylindotricarbocyanine iodide (1, 1' -dioctadecyl-3,3, 3',3' -tetramethylindocarbocyanine iodide, DiR, ATT Bioquest, usa); absolute ethanol (analytically pure, department of Tianjin, Mimi European chemical reagent development center); sterilized water for injection (Beijing Shuanghe pharmaceutical technology Co., Ltd.); redistilled water (Hangzhou child haha group ltd); ammonium sulfate ((NH)4)2SO4(analytically pure, chemical corporation of Shingan); citric acid (C)6H8O7·H2O, tianjin mao chemical reagent plant); sodium phosphate (Na)3PO3Tianjin Damao chemical reagent plant); EDTA (analytical purity, Tianjin, Dalochi chemical reagent factory); ammonia (Tianjin Fuyu Fine chemical Co., Ltd.).
Agilent 1260Infinity high performance liquid chromatograph (Agilent, USA); UV5100 type ultraviolet-visible spectrophotometer (anhui instrument science and technology ltd, anhui); DF-101S heat collection type constant temperature heating magnetic stirrer (Guyi City Yingyu Yuhua apparatus factory); an ake TDL80-2B centrifuge (Shanghai' an pavilion scientific instruments factory); BS124s electronic analytical balance (Sartorius, germany); JY92-II ultrasonic cell crusher (Ningbo science and technology Biotechnology Co., Ltd.); a full-wavelength multifunctional microplate reader (Thermo Scientific, usa); small animal Living body imager (Bruker, USA).
2.1. In vivo imaging research BR-Lf nano liposome brain targeting
The method comprises the following steps:
lateral ventricle injection of Abeta1-42And (5) establishing an AD mouse model. The modified ethanol injection method is used for preparing DiR common liposome DiR-CL, PEGylated DiR liposome DiR-PL and lactoferrin DiR liposome DiR-Lf. 45 AD mice successfully modeled were randomly divided into 3 groups of 15 mice each, at 2.0 mg.kg-1DiR dose of DiR-CL, DiR-PL and DiR-Lf were injected into the tail vein of each group of mice, observed in a small animal live imager at 2, 4, 8, 12 and 24h after injection, respectively, and fluorescence and white photographs were taken. Each timeAfter the photographing at each time point was completed, 3 mice were sacrificed by removing necks, brains, hearts, livers, spleens, lungs and kidneys were separated, washed with physiological saline and blotted with filter paper, and then fluorescent photographs were taken in a small animal living body imager under the conditions: lambda [ alpha ]ex＝720nm，λem790nm, exposure time 10 s. And (3) carrying out quantitative analysis on the near infrared fluorescence intensity of the isolated brain by using molecular imaging software carried by the system.
As a result:
in the experiment, living fluorescence imaging is adopted to investigate the influence of Lf modification on BR liposome brain targeting capability. As can be seen from FIG. 9, the DiR-Lf group in (A) has a significantly higher fluorescence signal at the head than DiR-CL and DiR-PL, and a large amount of fluorescence signal has appeared at the brain site 2h after injection, indicating that the drug has reached the brain; the signal increases first and then slowly decreases, lasting up to 24h in the brain. The results of in vitro organ imaging experiments and quantitative analysis (see B and C) of the mice show that at the same time point, the fluorescence intensity is DiR-Lf > DiR-PL > DiR-CL in sequence, and the results are consistent with the results of in vivo imaging. The fluorescence intensity of the DiR-Lf brain is obviously higher than that of the DiR-PL, and the fluorescence intensity is 2.0 times of that of the DiR-PL 2h after injection; when the time is 4h, the fluorescence intensity of the brain of the mice in the DiR-Lf group is the maximum and is 2.3 times of that of the DiR-PL; at 8h, the fluorescence intensity of the brains of mice in the DiR-Lf group is reduced, and the fluorescence intensity of the DiR-PL group reaches the maximum value of the group but is still lower than that of the DiR-Lf group. Lf is modified to enable the carried drug to be accumulated in brain tissue, and in the research of a living body imaging system, the brain shows higher fluorescence intensity and longer retention time, which indicates that the brain has good brain targeting property. In the invention, the fluorescence imaging result shows that the DiR-Lf has obviously enhanced fluorescence intensity compared with DiR-CL and DiR-PL brain, probably because the DiR-Lf passes through BBB through Lf receptor mediated transfer, and the brain targeting capability of the liposome is obviously improved.
Influence of BR-Lf nanoliposomes on behavioral competence of AD model mice
The method comprises the following steps:
and a Y maze experiment detects the influence of the BR-Lf nano liposome on the behavioral ability of the AD model mouse. The mice were divided into 6 groups, i.e., sham surgery group, model group, BR-S, BR-CL, BR-PL andBR-Lf groups of 9 individuals each (n-9). Lateral ventricle injection of Abeta1-42After the AD mouse model is successfully established, the groups begin tail vein injection administration, the BR concentration is 10mg/Kg, 1 time every other day, and 7 times in total.
The Y-maze experimental device consists of three support arms with 120-degree included angles, which are A, B, C three arms respectively. After the adaptive training was completed, the animals were placed at the end of arm A and allowed to freely pass through the three arms, the total number of times of entry into the three arms (N) and the order of entry into the arms within 5min of the mice were recorded, and the number of times of correct alternate reaction (N) was recorded with the continuous entry into three different arms as one correct alternate reactions). The excrement is cleared up in time in the experimental process, and the remaining smell is eliminated. The spontaneous alternation response rate (alternation behavior%) is used for reflecting the spatial working memory capacity of the mouse, and the calculation is carried out according to the following formula: alternation behavior (%) ═ Ns/(N-2)×100％。
As a result:
the invention adopts Y maze experiment to detect the influence of different BR preparations on the working memory capacity of AD model mice, and the result shows that compared with a pseudo-operation group, the A beta-peptide injection has the advantages that1-42Injection significantly reduced spontaneous alternation response rate in mice: (##P<0.01), suggesting us a β1-42Inducing the mice to have the impairment of the working memory and the spatial memory. Compared with the model group, the spontaneous alternation reaction rates of the BR-S group and the three different BR liposome groups are improved, wherein the differences of the BR-S group and the BR-CL group are not obvious, and the BR-PL (B, B and C)*P<0.05) and BR-Lf groups (**P<0.01) the difference was significant. And the spontaneous alternation reaction rate of the BR-Lf group is obviously higher than that of BR-S (**P<0.01) group and BR-PL (*P<0.05) group.
LfR in the brain of neurodegenerative diseases is increased, and Lf modified PEG liposome is combined with LfR at the BBB on the basis of prolonging the circulation time in vivo, is converted into a positively charged group, and is combined with the negatively charged BBB under physiological conditions. Lf carries the carried drug to enter differentiated BBB endothelial cells (BBCEC), and specific unidirectional transport occurs through LfR mediation, and no obvious endothelial degradation occurs, so that the drug concentration at the focus part is increased. The BR-Lf of the invention has the most obvious improvement on the behavioral ability of AD model mice, and the brain targeting property of the liposome is improved by the Lf, which is consistent with the result of in vivo imaging experiments.
Discussion of anti-AD action mechanism of BR-Lf nanoliposome
2.3.1. Taking materials
All mice were divided into two parts, one part was perfused to take brains, after fixation in 4% paraformaldehyde in a refrigerator at 4 ℃ for 48 hours, different concentrations of alcohol were dehydrated, xylene was transparent, wax was soaked, and embedded for use. The other part was taken directly from the brain: cutting off head of mouse, taking out brain tissue, sucking off blood stain on ice box with filter paper, separating hippocampus and cortex rapidly in EP tube, quick freezing with liquid nitrogen, and storing in-80 deg.C refrigerator.
2.3.2. Determination of acetylcholinesterase Activity
The method comprises the following steps:
the kit detects the activity of acetylcholinesterase, takes out mouse hippocampal tissue frozen at-80 ℃, and carries out detection according to the ratio of 1: tissue lysate (5. mu.L PMSF per 1ml lysate) was added at a rate of 10 (10. mu.L/mg tissue) and homogenate was prepared at low temperature using an ultrasonic cell disruptor. Standing the homogenate in ice bath for 1h, then centrifuging at 12000 xr/min at 4 ℃ for 20min, taking the supernatant for content determination, and operating according to the kit steps. AChE viability in tissues was calculated.
As a result:
the effect of different BR formulations on AChE activity in hippocampal tissues of AD model mice is shown in figure 11. Compared with a sham operation group, the activity of the model group mouse hippocampal tissue AChE is obviously improved (P is less than 0.01); compared with the model group, the BR-S group and different BR liposome groups can reduce AChE activity to different degrees; the groups BR-PL and BR-LF were statistically different (P < 0.01). The effect of decreasing AChE activity was more pronounced in the BR-Lf group compared to the BR-PL group (P < 0.01). The Lf modified PEG liposome entraps BR, so that the concentration of BR reaching the brain is effectively improved, and the inhibition of BR-Lf on AChE is most obvious under the same administration dose.
2.3.3. determination of phosphorylation of Tau protein
The method comprises the following steps:
immunohistochemistry method observes the expression of mouse hippocampal tissue phosphorylated Tau protein at position 396. Dewaxing and hydrating paraffin sections; repairing citrate thermoantigen; hydrogen peroxide blocks endogenous peroxidase; BSA blocking, and blocking for 30min in a room-temperature wet box; and (5) throwing off redundant liquid without washing. Adding primary antibody and secondary antibody, respectively, and incubating in oven wet box at 37 deg.C for 30 min. PBS was shaken gently for 5min × 3 times. DAB color development; performing hematoxylin counterstaining; dehydrating and sealing; the target area was observed under a microscope and the positive expression area was analyzed using ImageJ software.
As a result:
and observing the expression condition of phosphorylated Tau protein of mouse hippocampal tissues at 396 site by Western blot. As shown in FIG. 12, compared with the sham-operated group, the phosphorylation level of the serine 396 site of the Tau protein of the mouse in the model group is obviously increased (P < 0.01); the BR-S group and the different BR liposome groups both reduced P-Tau levels to different extents (P <0.05) compared to the model group. Compared with the BR-PL group, the BR-LF group has more remarkable P-Tau reducing effect (P < 0.05). The BR-Lf obviously reduces the over-phosphorylation of the Ser396 site, and improves the working memory capacity of an AD model mouse.
2.3.4. Effect of apoptosis
The method comprises the following steps:
the expression of Bax and Bcl-2 proteins in the mouse hippocampal region is detected by Western blotting (Western Blot). And (3) taking the mouse brain hippocampus tissue for standby from a refrigerator at the temperature of-80 ℃, extracting total protein, and then carrying out protein quantification according to the BCA protein concentration determination kit instruction. Primary and secondary antibodies were added sequentially. The membrane was washed 3 times with PBS for 10min each time. And (3) displaying a strip obtained by immunoreaction by using a hypersensitive luminescent liquid, transferring the strip into a gel imaging analyzer for exposure and development, analyzing by using IPP image analysis software, quantifying target protein in a sample, and determining difference and change of target protein expression among groups by using beta-actin as an internal reference.
As a result:
the expression of mouse hippocampal cell apoptosis proteins Bax and Bcl-2 is detected by a Western blot method, and the result is shown in figure 13. Compared with a sham operation group, the expression of Bcl-2 protein in the hippocampal tissue of the mouse in the model group is obviously reduced, and the expression of Bax protein is obviously increased (P is less than 0.01), which indicates that Abeta 1-42 can promote apoptosis. Compared with the model group, the mouse hippocampal tissue Bcl-2 protein expression of different BR liposome groups is increased, and the Bax protein expression is reduced (P < 0.05); compared with the BR-PL group, the BR-LF has more obvious improvement on Bcl-2 expression (P < 0.05). The BR-Lf can reverse Bcl-2 and Bax protein expression and Bcl-2/Bax ratio change, and the BR-Lf can reduce Bax protein expression by increasing Bcl-2 protein expression, inhibit abnormal apoptosis of hippocampal neurons of AD mice and relieve neuron damage. The apoptosis inhibition effect of BR-Lf is superior to that of BR-S, BR-CL and BR-PL, which shows that the Lf modified PEG lipid carrier improves the neuroprotective effect of BR, thereby improving the cognitive ability of mice.
2.4. Statistical analysis
All experimental results are statistically analyzed by SPSS 21.0 software and are analyzed by independent sample T test. Experimental data are expressed as Mean ± SD, with P <0.05 indicating statistically significant differences.
2.5. Conclusion
The Lf-modified PEG-modified BR liposome is successfully prepared, and the synergistic attenuation of BR against AD is preliminarily realized. The related results are not reported in the literature.
The invention determines a berberine encapsulation rate detection method and a lactoferrin connection rate detection method.
The invention optimizes the Lf modified PEG BR-Lf preparation process.
The invention verifies that the Lf modification improves the brain targeting property of the BR liposome; the BR-Lf anti-AD mechanism is explored: the model mouse model AD model behavior induced by Abeta 1-42 can be improved by inhibiting AChE activity, antagonizing Tau protein hyperphosphorylation and inhibiting neuronal apoptosis, and is expected to become a potential anti-AD drug.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. A preparation method of berberine long-circulating nanoliposome BR-Lf with brain targeting function is characterized by comprising the following steps:
preparing a blank liposome PL-COOH containing an antelope base end by using ammonium sulfate as a hydration medium and adopting an improved ethanol injection method;
taking 2 parts by mass of PL-COOH liposome, adding 1 part by mass of EDC aqueous solution and 1 part by mass of NHS aqueous solution under the condition of an ice-water bath, magnetically stirring and activating for 0.5h, recovering to room temperature, adding 6 parts by mass of lactoferrin Lf, fully dissolving, adding 1.4 parts by mass of TEA, reacting for at least 4h, and terminating the reaction in the ice-water bath to obtain the lactoferrin modified PEG blank liposome PL-Lf;
and removing an outer water phase from the PL-Lf suspension through an anion-cation fiber column, adding berberine hydrochloride BR solution according to the medicine-lipid ratio of 1: 15-30, incubating at the constant temperature of 40-55 ℃ for at least 15min, and stopping carrying the medicine in an ice-water bath to obtain the lactoferrin modified PEG berberine long-circulating nanoliposome BR-Lf.
2. The preparation method according to claim 1, wherein the external aqueous phase is removed by anion and cation fiber column according to the ratio of drug to lipid of 1:20 adding small chai alkali BR solution.
3. The method according to claim 1, wherein 6.0mg/mL of a solution of Chaetomium hydrochloride is added in a drug-to-lipid ratio of 1:20 (w/w).
4. The method of claim 1, comprising:
weighing oxidized soybean phospholipid HSPC and cholesterol CH according to the mass ratio of 2-4: 1, and weighing the oxidized soybean phospholipid HSPC and the cholesterol CH according to the mass ratio of 1: 1-3 weighing mPEG2000-DSPE/DSPE-PEG2000-COOH, and mixing with 5-15% ethanol by volume, heating in 65 deg.C water bath for at least 10min to volatilize ethanol, adding (NH) preheated to the same temperature4)2SO4Stirring the solution in water bath for at least 20min to obtain empty liposome primary product PL;
treating PL with ultrasonic cell pulverizer, and grading sequentially with 0.8um, 0.45 μm, and 0.22 μm microporous filter membrane to obtain blank liposome PL-COOH containing antelope base end.
5. The method of claim 4, wherein the ultrasonic cell disruptor is operated according to the following operating parameters: 200 Wx 2min, 400 Wx 6min, 1s of work and 1s of pause.
6. The method according to claim 4, wherein the oxidized soybean phospholipids HSPC and cholesterol CH are weighed according to a mass ratio of 3:1, and the mPEG is weighed according to a mass ratio of 1:22000-DSPE/DSPE-PEG2000-COOH, in admixture with 10% by volume of ethanol, heating in a water bath at 65 ℃ for at least 10min to evaporate the ethanol, adding 200mM (NH) preheated to the same temperature4)2SO43mL of the solution is stirred in a water bath for at least 20mm to obtain an empty liposome primary product PL.
7. The method of claim 1, wherein 1 μmol-COOH, EDC 8mg/ml, and NHS aqueous solution 2.4 mg/ml.
8. The method according to claim 1, wherein the step of removing the external aqueous phase from the PL-Lf suspension by using an anion and cation fiber column comprises:
taking 0.2 part by mass of PL-Lf suspension, loading the PL-Lf suspension onto the top end of a centrifugally pretreated anion-cation exchange fiber column, simultaneously adding 0.2 part by mass of redistilled water, staying for at least 10min, centrifuging for at least 4min at a rotating speed of 1000-2000 r/min, adding 0.1 part by mass of redistilled water at the top end of the column, centrifuging for at least 4min at a rotating speed of 1000-2000 r/min, eluting, merging eluents, and uniformly mixing to obtain the compound (NH) with the structure (NH)4)2SO4Liposomal suspensions of transmembrane ion gradients.
9. The method of claim 1, further comprising:
adopting anion and cation fiber column filtration and high performance liquid chromatography to measure BR encapsulation efficiency in BR-PL, which comprises the following steps:
chromatographic conditions are as follows: a chromatographic column: a C18 column (4.6X 250mm, 5 μm, Thermo Co.); mobile phase: acetonitrile-0.05 mol/L KH2PO4(23:77) (v/v), pH 3.0 with phosphoric acid; flow rate: 1.0 ml/min; column temperature: 30 ℃; sample introduction amount: 20 mu l of the mixture; detector wavelength: 212 nm;
linear investigation: precisely measuring 0.5, 1.0, 2.0, 3.0 and 4.0mL of berberine hydrochloride reference solution 50.0 μ g/mL, respectively placing in a 10mL measuring flask, diluting to scale with double distilled water, shaking, and filtering with 0.45 μm microporous membrane to obtain standard solutions with concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 μ g/mL. Respectively taking 20 mu L of sample injection high performance liquid chromatograph for analysis, recording the peak area A, and performing linear regression on the concentration C (mu g/mL) by using the peak area A;
and (3) drawing an elution curve: taking the prepared BR-PL liposome lml to be loaded on a column, eluting with PBS (phosphate buffer solution) with the pH value of 7.4 at the rate of 1 ml/min; the eluate was collected in 2ml per tube. Taking 1ml and 10ml measuring flasks of each tube, breaking methanol into a fixed volume, filtering with a 0.45 μm microporous filter membrane, determining with a liquid chromatograph, recording peak area, and drawing an elution curve;
analysis of fiber column for recovery of free drug and liposomes: preparing 0.20mg/ml hydrochloric acid berberine PBS solution, ultrasonically reducing drug particles, and vortex vibrating to promote dissolution; eluting 0.2ml according to the above conditions, collecting free drug, determining with liquid chromatograph, recording peak area, and calculating column recovery rate; accurately weighing the berberine hydrochloride PBS solution and an equivalent amount of blank liposome, mixing, eluting on a column, collecting free drug components, diluting with methanol to a constant volume, carrying out sample injection measurement, and calculating the recovery rate;
determination of BR-PL Liposome encapsulation efficiency: taking 0.1mL of BR liposome, adding methanol solution into one part of BR liposome to destroy liposome, diluting mobile phase to 10mL, shaking, passing through 0.45 μm microporous membrane, performing high performance liquid chromatograph at 212nm for sample injection determination, and recording peak area as AtotSubstituting into standard curve to calculate concentration, and recording as Ctot. Loading the other part onto the top of the cation exchange fiber column, and centrifuging at 2000r/min for 4 min; continuously adding 0.4mL of the mixtureEvaporating water to the top of cation exchange fiber column, centrifuging at 2000r/min for 4min, continuously operating for 3 times, mixing eluates, determining by high performance liquid chromatograph at 212nm, and recording peak area as A1Substituting into standard curve to calculate concentration, and recording as C1(ii) a The encapsulation efficiency was calculated according to the following formula: EE ═ 1-C1)/Ctot×100％。
10. The method of claim 1, further comprising:
the Lf connectivity was determined by Coomassie Brilliant blue: taking 2 parts of 100 mu l BR-Lf reaction solution, passing one part through a Sephadex G-100 micro-column to remove unreacted Lf, and passing the other part through the column, and specifically operating as follows: 0.1ml of Lf-TP-PL is added with 5ml of acid Coomassie brilliant blue G250 developing solution, mixed evenly and then the absorbance A is measured at 595nm of an ultraviolet-visible spectrophotometerafterAnd AbeforeSubstituting into standard curve to calculate concentration CafterAnd CbeforeThe Lf grafting rate was calculated according to the formula: lfgrafed＝Cafter/Cbefore×100％。
CN202110919767.4A 2021-08-11 2021-08-11 Berberine long-circulating nano-liposome with brain targeting function and preparation method thereof Withdrawn CN113694026A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110919767.4A CN113694026A (en) 2021-08-11 2021-08-11 Berberine long-circulating nano-liposome with brain targeting function and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110919767.4A CN113694026A (en) 2021-08-11 2021-08-11 Berberine long-circulating nano-liposome with brain targeting function and preparation method thereof

Publications (1)

Publication Number Publication Date
CN113694026A true CN113694026A (en) 2021-11-26

Family

ID=78652534

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110919767.4A Withdrawn CN113694026A (en) 2021-08-11 2021-08-11 Berberine long-circulating nano-liposome with brain targeting function and preparation method thereof

Country Status (1)

Country Link
CN (1) CN113694026A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114272252A (en) * 2021-11-28 2022-04-05 深圳市第二人民医院(深圳市转化医学研究院) Preparation method of triptolide and berberine co-loaded nano-liposome with brain targeting function
CN117338725A (en) * 2023-09-18 2024-01-05 山东泰合医药科技有限公司 Pretreatment method and analysis method of drug microsphere preparation

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114272252A (en) * 2021-11-28 2022-04-05 深圳市第二人民医院(深圳市转化医学研究院) Preparation method of triptolide and berberine co-loaded nano-liposome with brain targeting function
CN117338725A (en) * 2023-09-18 2024-01-05 山东泰合医药科技有限公司 Pretreatment method and analysis method of drug microsphere preparation
CN117338725B (en) * 2023-09-18 2024-04-09 山东泰合医药科技有限公司 Pretreatment method and analysis method of drug microsphere preparation

Similar Documents

Publication Publication Date Title
Wang et al. Curcumin-primed exosomes potently ameliorate cognitive function in AD mice by inhibiting hyperphosphorylation of the Tau protein through the AKT/GSK-3β pathway
Kong et al. Transferrin-modified osthole PEGylated liposomes travel the blood-brain barrier and mitigate Alzheimer’s disease-related pathology in APP/PS-1 mice
Lala et al. Harmine: evaluation of its antileishmanial properties in various vesicular delivery systems
CN106137967B (en) Target the preparation and application of the dual modified liposome drug delivery system of glioma
JP2001510451A (en) Ion carrier carrying weakly basic drug-liposome in the middle
CN108685875B (en) Natural nanoparticle-pharmaceutical composition for resisting Alzheimer disease and preparation method and application thereof
Geng et al. Binding to amyloid‐β protein by photothermal blood‐brain barrier‐penetrating nanoparticles for inhibition and disaggregation of fibrillation
CN103479578B (en) The Liposomal formulation of a kind of maleic acid Pixantrone and preparation technology thereof
CN113694026A (en) Berberine long-circulating nano-liposome with brain targeting function and preparation method thereof
KR20110042314A (en) Use of a chinese traditional medicinal composition for preparing medicine for promoting bone marrow-derived mesenchymal stem cell survival in vivo and cardiac muscle differentiation
CN105396122A (en) A Reconstituted High Density Lipoprotein Formulation And Production Method Thereof
US20230110087A1 (en) Ipa-3-loaded liposomes and methods of use thereof
US20210252168A1 (en) Amyloid b short peptide mediated brain targeted delivery system, preparation method therefor and use thereof
CN111920768A (en) Entrapped molecular targeted drug liposome and application thereof in preparation of tumor treatment drug
He et al. Preparation and evaluation of stearylamine-bearing pemetrexed disodium-loaded cationic liposomes in vitro and in vivo
Mu et al. Ligustrazine nanoparticle hitchhiking on neutrophils for enhanced therapy of cerebral ischemia‐reperfusion injury
CN109432049B (en) Rhein lipid vesicle nanoparticles with kidney targeting distribution characteristic and application thereof
RU2411935C1 (en) Pharmaceutical composition based on doxorubicine and phospholipid nanoparticles for treatment of oncologic diseases
Yang et al. Protection against neurodegeneration in the hippocampus using sialic acid-and 5-HT-moduline-conjugated lipopolymer nanoparticles
CN109453117A (en) Cardiac glycosides active ingredient composite lipidosome and preparation method thereof
CN106456624B (en) Use of FLAP inhibitors to reduce neuroinflammation-mediated injury in the central nervous system
CN105726520B (en) Lactone dimethylamine liposome atomized inhalation and its application with a smile
CN108159401B (en) Apelin liposome and preparation method thereof
CN115957348A (en) Targeted rheumatoid arthritis liposome drug delivery system, preparation method and application
CN113521035B (en) Preparation method and application of chemical immune combined therapeutic nano-drug

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication

Application publication date: 20211126

WW01 Invention patent application withdrawn after publication