CN110200921B - Anti-inflammatory liposome with targeting effect and preparation and application thereof - Google Patents

Anti-inflammatory liposome with targeting effect and preparation and application thereof Download PDF

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CN110200921B
CN110200921B CN201910529237.1A CN201910529237A CN110200921B CN 110200921 B CN110200921 B CN 110200921B CN 201910529237 A CN201910529237 A CN 201910529237A CN 110200921 B CN110200921 B CN 110200921B
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liposome
inflammatory
nitric oxide
oxymatrine
parts
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谭松巍
张伟
唐庆
谭晨
张冲
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Huazhong University of Science and Technology
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    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • 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
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • 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
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • 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
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • 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
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Abstract

The invention discloses an anti-inflammatory liposome with a targeting effect, and preparation and application thereof, and belongs to the technical field of medicines. The liposome is of a phospholipid bilayer structure, and cholesterol, an amphiphilic molecule with negative charges and a nitric oxide donor molecule are embedded in the phospholipid bilayer structure; the cholesterol is used for enhancing the stability of the phospholipid bilayer structure, the negatively charged amphiphilic molecule is used for enabling the liposome to have a targeting effect, and the nitric oxide donor molecule is used for releasing nitric oxide; the hydrophilic cavity of the phospholipid bilayer structure is embedded with an anti-inflammatory active ingredient. The preparation method comprises the steps of firstly obtaining blank liposome by adopting a hydration method, and then carrying out entrapment of the anti-inflammatory active ingredients by adopting a pH gradient method. The liposome prepared by the invention can play a synergistic effect of an anti-inflammatory active ingredient and NO, improve the distribution of the medicament, target the colon part and improve the treatment effect of ulcerative colitis.

Description

Anti-inflammatory liposome with targeting effect and preparation and application thereof
Technical Field
The invention belongs to the technical field of medicines, relates to an anti-inflammatory liposome with a targeting effect, and preparation and application thereof, and particularly relates to an anti-inflammatory liposome which has an NO release function and can play a role in passively targeting an inflammatory colon part to play an anti-inflammatory role.
Background
Alkaloids exist in nature (mainly plant, but haveAlso present in animals) have base-like properties. Most of the Chinese herbal medicines have complex ring structures, nitrogen is mostly contained in the ring, and the Chinese herbal medicines have remarkable biological activity and are one of important effective components in the Chinese herbal medicines. Wherein the representative Oxymatrine (Oxymatrine) is quinolizidine alkaloid separated from radix Sophorae Flavescentis (Sophora flavescens Ait) belonging to Sophora of Phaseoluceae, and has wide biological activity, such as antiinflammatory, antitumor, analgesic, antiarrhythmic, antitussive, and antibacterial effects. The structural formula is
Figure GDA0002661176440000011
Molecular formula C15H24N2O2Molecular weight is 264.37, it is easily soluble in water, methanol, chloroform, benzene, and hardly soluble in diethyl ether, and it is transparent granular crystal. The oxymatrine as an immunomodulator and various matrine compound preparations prepared from other medicines are effectively used for treating clinical UC by inhibiting inflammation caused by lymphocytes and reducing secretion of inflammatory factors. The currently mainstream oxymatrine preparation types comprise injections, suppositories, capsules and tablets, and the preparation forms in the development state comprise targeted preparations, sustained and controlled release preparations and transdermal absorption preparations. However, oxymatrine has the disadvantages of short half-life period in vivo, fast elimination in vivo, half-life period in blood of about 1-2h, low bioavailability of oral preparations, and generally can reach expected effective blood concentration by intravenous drip, but intravenous administration of high dose has certain potential safety hazard and short half-life period of the medicament, which seriously hinders the development of clinical therapeutic medicines of oxymatrine. The selection of the medicament form has close relation with the effective utilization rate and the in vivo half-life of the medicament, scientific optimization and research and development of new medicament forms are carried out on the common clinical oxymatrine preparation, the bioavailability of the oxymatrine can be obviously improved, the research and development of new medicament forms and the application of new materials can be expanded, and the clinical application of the matrine preparation can be expanded, so that the clinical curative effect of the oxymatrine preparation is enhanced.
The liposomal drug delivery system, a new drug delivery system, has been relatively well studied. The liposome is a double-layer lipid molecule which can be metabolized normally and has good biocompatibility, and has great development potential as a drug carrier. Has the following characteristics: (1) consists of biodegradable substances (phospholipids), is nontoxic to human bodies and is stable in blood; (2) the affinity with cells is strong, and the capability of the encapsulated drug to permeate cell membranes can be increased; (3) the dosage can be reduced, the toxicity can be reduced, the allergic reaction can be alleviated, the drug release can be delayed, the elimination speed and degree of the drug in the body can be reduced, and the curative effect can be improved; (4) change the distribution of the drug in the tissue and enhance the selective action of the drug; (5) the surface properties of the liposome, such as particle size, surface charge and assembly specific antibody, are changed, so that the selectivity of the drug to certain targets can be improved.
Disclosure of Invention
The invention solves the technical problems that the liposome-entrapped anti-inflammatory drug can not target to an inflammation part, has low entrapment rate and low drug utilization rate in the prior art. According to the invention, the amphiphilic molecules with negative charges and the nitric oxide donor molecules are embedded on the bilayer of the liposome, so that the liposome can target inflammation parts, and the utilization rate of the medicine is improved.
According to a first aspect of the present invention, there is provided an anti-inflammatory liposome having a targeting effect, said liposome being a phospholipid bilayer structure in which cholesterol, a nitric oxide donor molecule and a negatively charged amphiphilic molecule are embedded; the cholesterol is used for enhancing the stability of the phospholipid bilayer structure, the negatively charged amphiphilic molecule is used for enabling the liposome to have a targeting effect, and the nitric oxide donor molecule is used for releasing nitric oxide; the hydrophilic cavity of the phospholipid bilayer structure is embedded with an anti-inflammatory active ingredient.
Preferably, the hydrophobic end of the negatively charged amphiphilic molecule is distearoylphosphatidylethanolamine and the hydrophilic end is polyethylene glycol; the anti-inflammatory active ingredient is oxymatrine, leonurus alkaloid or berberine.
Preferably, the nitric oxide donor molecule is a nitrate based compound capable of releasing NO.
Preferably, the liposomes have a diameter of 180nm to 220 nm.
Preferably, the liposome comprises 8-16 parts by mass of phospholipid, 2-4 parts by mass of cholesterol, 2-4 parts by mass of negatively charged amphiphilic molecules, 1-2 parts by mass of nitric oxide donor molecules and 1-2 parts by mass of anti-inflammatory active ingredients.
According to another aspect of the present invention, there is provided a method for preparing the anti-inflammatory liposome with targeting effect, comprising the steps of:
(1) dissolving phospholipid, cholesterol, nitric oxide donor molecules and amphiphilic molecules with negative charges in an organic solvent, and removing the organic solvent by spin drying or removing the organic solvent by reduced pressure evaporation to form a film;
(2) placing the film obtained in the step (1) in an acid buffer solution for hydration, and then carrying out ultrasonic treatment to enable phospholipid to form a liposome with a bilayer structure, wherein cholesterol, nitric oxide donor molecules and amphiphilic molecules with negative charges are embedded in the bilayer; then, redispersing by adopting a water-based filter membrane to obtain a solution containing blank liposome;
(3) adjusting the pH value of the solution containing the blank liposome obtained in the step (2) to 7.0-7.4, then adding an anti-inflammatory active ingredient, and incubating to enable the anti-inflammatory active ingredient to be entrapped in a hydrophilic cavity in the liposome under the internal and external pH gradients of the liposome;
(4) and (3) after the incubation in the step (3) is finished, dialyzing to remove the non-entrapped anti-inflammatory active ingredients, centrifuging, removing the precipitate, and taking the supernatant to obtain the anti-inflammatory liposome with the targeting effect.
Preferably, the hydrophilic end of the negatively charged amphiphilic molecule of step (1) is distearoyl phosphatidyl ethanolamine, and the hydrophobic end is polyethylene glycol; the nitric oxide donor molecule is a nitrate compound capable of releasing NO;
the liposome comprises 8-16 parts of phospholipid, 2-4 parts of cholesterol, 2-4 parts of negatively charged amphiphilic molecules, 1-2 parts of nitric oxide donor molecules and 1-2 parts of anti-inflammatory active ingredients.
Preferably, the anti-inflammatory active ingredient in the step (3) is oxymatrine, leonurus alkaloid or berberine, the incubation temperature is 50-60 ℃, and the incubation time is 1-2 h.
Preferably, the pore size of the aqueous filter membrane in the step (2) is 0.22um-0.45 um; the rotating speed of the centrifugation in the step (4) is 2000rpm-3000rpm, and the time of the centrifugation is 5min-15 min.
According to another aspect of the present invention, there is provided a use of any of the anti-inflammatory liposomes having a targeting effect for preparing an anti-inflammatory drug;
preferably, the anti-inflammatory drug is a drug for treatment of colon inflammation.
Generally, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) the invention aims to provide a preparation method of a liposome which has high encapsulation efficiency and can passively target a colonic inflammation part, improve the utilization rate of an anti-inflammatory active ingredient, prolong the action time of a medicament, and improve the treatment effect on ulcerative colitis by combining the mucosa protection effect of an NO release material.
(2) The anti-inflammatory active ingredient-NO liposome prepared by the invention has the effects of passively targeting inflammatory colon parts with the particle size of 200nm and the zeta potential of-15 mV, and simultaneously can improve the distribution of the medicament, prolong the half-life period of the medicament in vivo and improve the bioavailability of the medicament.
(3) According to the invention, amphipathic molecules with negative charges are embedded in the liposome, preferably distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000(DSPE-PEG2000) modified liposome, so that the surface of the liposome is negatively charged, and the inflammatory colon part is positively charged due to high expression of cationic protein, thus achieving the effect of passively targeting the inflammatory colon part to play a therapeutic role.
(4) The invention adopts a pH gradient method to entrap the anti-inflammatory active ingredients, thereby greatly improving the drug encapsulation efficiency. A pH gradient method is adopted to encapsulate anti-inflammatory active ingredients, the method belongs to an active drug loading mechanism, and the encapsulation rate of oxymatrine drug is up to 70%.
(5) The particle size of the particles is reduced, the residence time of the preparation in colon can be prolonged, and the nano-scale particles can improve the permeability and residence time of the preparation in inflammation parts. The liposome prepared by the invention has the advantages that the particle size is about 200 nm.
(6) NO has the function of protecting cells in different body systems, and the mechanism of NO is to enhance the protection function by increasing the blood flow of intestinal mucosa and supplying sufficient oxygen and nutrient substances to the mucosa. In addition, NO may also act as a mucosal protective effect by reducing inflammatory factor interactions, increasing mucus secretion, and other mechanisms. Can exert combined treatment effect with anti-inflammatory active ingredients through the vasodilation and mucosa protection effects. The invention adopts the liposome structure as the carrier structure of the anti-inflammatory active ingredient, and combines the NO donor material, thereby achieving the purposes of improving the utilization rate of the medicament, targeting the inflammatory colon part and improving the treatment effect of the ulcerative colitis by combining the anti-inflammatory effect, and providing a new thought for the clinical treatment of the ulcerative colitis.
(7) The invention utilizes NO donor material and the NO donor material to prepare liposome by rotating a membrane together with lipid material, and utilizes a pH gradient method to entrap anti-inflammatory active ingredients, so as to obtain the liposome which can improve the distribution of the medicament, improve the bioavailability of the medicament and passively target inflammation parts to play a role in anti-inflammatory treatment. The NO liposome is a novel drug-carrying system, and has important academic significance and potential clinical application value.
Drawings
FIG. 1 and FIG. 2 are the electron microscope image and the hydrated particle size distribution diagram of oxymatrine-NO liposome, respectively.
FIG. 3 and FIG. 4 are the stability chart of the particle size and potential size of oxymatrine-NO liposome, respectively.
FIG. 5 is a graph showing the in vitro release results of oxymatrine-NO liposomes.
Fig. 6 and 7 are bar graphs of in vivo imaging mean fluorescence values and ex vivo colon mean fluorescence values, respectively.
FIG. 8 and FIG. 9 are the length and weight of colon tissue of experimental mice treated by different administration groups.
FIG. 10, FIG. 11, FIG. 12, FIG. 13 and FIG. 14 are the pathological section diagrams of the colon of mice in the blank control group, the inflammation model group, the free oxymatrine group, the oxymatrine liposome group and the oxymatrine-NO liposome group, respectively.
FIG. 15 is a graph showing the results of the CRP content in the serum of mice in different treatment groups.
FIG. 16 is a graph showing the results of the treatment of the MPO content in the colon tissue of the mice in the experimental groups with different doses.
FIG. 17, FIG. 18, FIG. 19, FIG. 20 and FIG. 21 are the pathological section diagrams of the colon of mice in the blank control group, the inflammation model group, the free berberine group, the berberine liposome group and the berberine-NO liposome group, respectively.
Fig. 22, 23, 24, 25 and 26 are pathological section views of the colon of mice in a blank control group, an inflammation model group, a free leonurus alkaloid group, a leonurus alkaloid liposome group and a leonurus alkaloid-NO liposome group, respectively.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and 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. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The present invention utilizes the NO donor material TNO3And an anti-inflammatory active ingredient such as oxymatrine, TNO3The nitric acid ester compound can release NO, and simultaneously, a liposome carrier system is adopted to carry the medicine, so that the medicine can be passively and targetedly conveyed, the release time of the medicine is maintained, the bioavailability of the medicine is improved, the clinical treatment administration mode can be improved, and the administration pain of a patient is reduced.
The first step of determining the anti-inflammatory active ingredients, such as an oxymatrine entrapment method: at present, liposome medicaments are coated in various methods, and the pH gradient method is used for coating the medicaments. The drug loading mode of the pH gradient method belongs to active drug loading, which is realized by utilizing the principle that some alkaline drugs have stronger lipid solubility in a non-ionic state and can pass through a lipid bilayer in an electrically neutral form, but ions after ionization cannot pass through the lipid bilayer. The acidic buffer salt is usually wrapped by liposome, and then the external water phase is adjusted to be neutral by alkali, so as to establish the pH gradient inside and outside the liposome, and then the medicine exists in a lipophilic neutral form under the pH environment of the external water phase and can permeate through a lipid bilayer membrane. In the liposome internal water phase, the medicine is converted into ion form by protonation of the acid encapsulated in the liposome internal water phase, and can not pass through the lipid bilayer to return to the external water phase and is encapsulated in the liposome. The OMT oil-water distribution coefficient is greatly influenced by the pH value and the ionic strength of a medium, the liposome prepared by a passive drug loading method has low encapsulation efficiency, and active drug loading is carried out by different pH values of an internal water phase and an external water phase, so that the encapsulation efficiency can be greatly improved.
The second step of preparing anti-inflammatory active ingredient-NO liposome, comprising the following steps:
1) film forming: weighing soybean lecithin, cholesterol, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000(DSPE-PEG2000) and nitric oxide donor molecule (TNO) according to proportion3) Dissolving the materials in 4mL of dichloromethane, placing the materials in a round-bottom flask, spin-drying the organic solvent under the condition of 25 ℃, and then placing the materials in a vacuum drying oven for drying for 30 min;
2) hydration: adding 3mL of sodium citrate buffer solution with pH of 5.0 into the drained film, carrying out 100-120W power ultrasonic treatment for 30min until the solution is completely hydrated, and filtering the solution for 3 times by using an aqueous filter membrane with the pore diameter of 0.22-0.45um to obtain an empty liposome;
3) carrying out medicine loading: adjusting pH of blank liposome solution to 7.0-7.4 with saturated dipotassium hydrogen phosphate solution, and adding 5mg of antiinflammatory active ingredient into blank liposome;
4) and (3) incubation: putting the solution in a shaking table at 50-60 ℃ to incubate for 1-2 h;
5) and (3) dialysis: after incubation, dialyzing with PBS as external phase to remove free antiinflammatory active ingredient, and dialyzing for 3 times by changing dialysate every half an hour;
6) centrifuging: after dialysis, the obtained solution is centrifuged at low speed for 5-15min, and the bottom precipitate is discarded to obtain the liposome carrying the anti-inflammatory active ingredient.
And thirdly, the anti-inflammatory active ingredient NO liposome prepared by the invention has better treatment effect on experimental acute enteritis of mice through treatment experiments of the ulcerative colitis of the mice.
Example 1: preparation of oxymatrine-NO liposome
Comparing different liposome preparation methods, determining the optimal matrine oxide entrapment method as pH gradient method by drug encapsulation efficiency, and the optimal drug-lipid ratio is 1:10 (ratio of antiinflammatory drug to sum of phospholipid, cholesterol, negatively charged amphiphilic molecule and nitric oxide donor molecule), and the optimal formulation ratio is soybean lecithin, cholesterol, DSPE-PEG and nitric oxide donor molecule (TNO)3) The mass ratio is 4:1:1: 0.5.
1) Preparation of oxymatrine-NO liposome
33.2mg of soybean lecithin, 8.3mg of cholesterol, 8.3mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000) and TNO were weighed respectively34.1mg，TNO3The molecular formula of (A) is as follows:
Figure GDA0002661176440000081
dissolving the materials in 4mL of dichloromethane, placing the dichloromethane in a round-bottom flask, performing reduced pressure spin-drying on the solvent at the temperature of 25 ℃, and then placing the obtained product in a vacuum drying oven for drying for 30 min; adding 3mL of sodium citrate buffer solution with pH of 5.0 into the drained film, performing ultrasonic treatment at 100W power for 30min until the buffer solution is completely hydrated, and dispersing with an aqueous filter membrane with pore diameter of 0.22um for 3 times to redisperse the liposome to obtain blank liposome; regulating pH of blank liposome solution to 7.0 with saturated dipotassium hydrogen phosphate solution, and adding 5mg oxymatrine medicine into blank liposome; placing the solution in a shaking table at 50 ℃ to incubate for 1 h; after the incubation is finished, dialyzing with PBS as external phase to remove free oxymatrine medicine, changing dialysate every half hour, and dialyzing for 3 times; after dialysis, the obtained solution is centrifuged at 2000r/min for 5min, and sediment at the bottom is removed to obtain oxymatrine-NO liposome.
Example 2: preparation of oxymatrine-NO liposome
Weighing soybean lecithin 66.4mg, cholesterol 8.3mg, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000(DSPE-PEG2000)8.3mg and TNO34.1mg, dissolving the materials in 4mL of dichloromethane, placing the materials in a round-bottom flask, performing reduced pressure spin-drying on the solvent at the temperature of 25 ℃, and then placing the materials in a vacuum drying oven for vacuum drying for 30 min; adding 3mL of sodium citrate buffer solution with pH of 5.0 into the drained film, performing ultrasonic treatment at 110W power for 30min until the solution is completely hydrated, and dispersing the solution for 3 times by using an aqueous filter membrane with the pore diameter of 0.35um to obtain a blank liposome; regulating pH of blank liposome solution to 7.2 with saturated dipotassium hydrogen phosphate solution, and adding 5mg oxymatrine medicine into blank liposome; placing the solution in a shaking table at 55 ℃ to incubate for 1.5 h; after the incubation is finished, dialyzing with PBS as external phase to remove free oxymatrine medicine, changing dialysate every half hour, and dialyzing for 3 times; after dialysis, the obtained solution is centrifuged at 2500r/min for 10min, and sediment at the bottom is removed to obtain oxymatrine-NO liposome.
Example 3: preparation of oxymatrine-NO liposome
33.2mg of soybean lecithin, 16.6mg of cholesterol, 16.6mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000) and TNO were weighed respectively34.1mg, dissolving the materials in 4mL of dichloromethane, placing the materials in a round-bottom flask, performing reduced pressure spin-drying on the solvent at the temperature of 25 ℃, and then placing the materials in a vacuum drying oven for vacuum drying for 30 min; adding 3mL of sodium citrate buffer solution with pH of 5.0 into the drained film, performing ultrasonic treatment at 120W for 30min until the solution is completely hydrated, and dispersing the solution for 3 times by using an aqueous filter membrane with the pore diameter of 0.45um to obtain a blank liposome; regulating pH of blank liposome solution to 7.4 with saturated dipotassium hydrogen phosphate solution, and adding 5mg oxymatrine medicine into blank liposome; placing the solution in a shaking table at 60 ℃ for incubation for 2 h; after the incubation is finished, dialyzing with PBS as external phase to remove free oxymatrine medicine, changing dialysate every half hour, and dialyzing for 3 times; after dialysis, centrifuging the obtained solution at 3000r/min for 15min, and removing bottom precipitate to obtain oxymatrine-NO liposome.
Example 4:
1) measurement of particle diameter and Zeta potential
Measurement of particle size:
the oxymatrine-NO liposome prepared in example 1 was diluted 20-fold with PBS, 2mL of the diluted liposome was added to a sample cell, the particle size of the liposome was measured using a laser scattering particle size meter, the sample equilibrium time was 120s, 20 cycle times were measured, and the measurement temperature was set to 25 ℃. The final measurement of the sample is the average of 20 measurements. The hydrated particle size distribution is shown in FIG. 2, where the degree of uniformity of particle size is indicated by polydispersity, and the particles are more uniform with smaller polydispersity.
Measurement of Zeta potential
After the particle size is measured, the oxymatrine-NO liposome is transferred to a potential cup, the Zeta potential measuring condition is set to be the equilibrium time of 120s, the minimum 20 circulation time is measured each time, and the measuring temperature is set to be 25 ℃. The final measurement of the sample is the average of 20 measurements.
Table 1 shows the particle size and potential of 5 batches of oxymatrine-NO liposomes, wherein the average particle size is within 200nm, the polydispersity index (PDI) is between 0.184 and 0.297, and the potential is about-15 mV.
TABLE 1
Figure GDA0002661176440000101
2) Morphological observation
Observed with a Transmission Electron Microscope (TEM). The oxymatrine-NO liposomes were diluted to 1 μ g/mL (lipid concentration) using deionized water as a dispersion medium and filtered with a 0.2 μm microfiltration membrane. Then, the copper mesh with the carbon film laid thereon was placed on the liposome solution, taken out after 1min, blotted dry with filter paper, and then the copper mesh with the liposome particles trapped therein was floated on a 1% uranyl acetate aqueous solution, taken out after 1min and blotted dry with filter paper. After being left at room temperature overnight for air drying, the film was observed and recorded at 120kV using a transmission electron microscope. Fig. 1 and fig. 2 are an electron microscope image and a hydrated particle size distribution diagram of the anti-inflammatory liposome with targeting effect, respectively. Under a transmission electron microscope, the oxymatrine-NO liposome is in a sphere-like shape, and the particle diameter is about 200nm and is consistent with the particle diameter result.
Example 5: determination of encapsulation efficiency of oxymatrine-NO liposome
1) Method for measuring oxymatrine content
The chromatographic conditions were as follows
A chromatographic column: agilent SB-C18Columns (4.6X 150mm, 5 um); mobile phase: acetonitrile KH2PO4 (pH adjusted to 2 with 0.01%, H3PO 4) 5: 95; detection wavelength: 220 nm; flow rate: 1.0 mL/min;
column temperature: 20 ℃; sample introduction amount: 20 ul;
2) drawing of standard curve
Precisely weighing 1mg of oxymatrine, adding a proper amount of methanol, and placing in a 10ml volumetric flask to prepare oxymatrine mother liquor with the concentration of 100 ug/ml. Diluting into series of standard solutions with concentrations of 50, 25, 12.5, 6.25 and 3.12ug/mL respectively. And (4) determining chromatographic peak area A values of the standard substances with different concentrations according to the chromatographic conditions. Taking the concentration C of the oxymatrine as an independent variable, performing linear regression on the corresponding chromatographic peak area A value to obtain a standard curve linear regression equation of the oxymatrine: Y4492X-839, R20.9993; the result shows that the oxymatrine is in a linear relationship within the range of 3.12-100 ug/mL.
3) Determination of encapsulation efficiency
Taking oxymatrine-NO liposome 100ul, destroying liposome structure with DMF solvent 100ul and acetonitrile solvent 400ul, and measuring with the high performance liquid chromatography. The encapsulation rate of oxymatrine in the liposome is calculated by the following formula: encapsulation efficiency% is the amount of oxymatrine entrapped in liposomes/total amount of oxymatrine put into liposomes × 100%. The drug concentration is calculated by a reference method (one point comparison method), and the encapsulation rate of the oxymatrine in the liposome is about 70%.
Example 6: stability research and in-vitro release of oxymatrine-NO liposome
1) Stability study of oxymatrine-NO liposome
The results of the stability graphs of the particle size and the potential size are shown in fig. 3 and fig. 4, wherein the hydration particle size and the potential change of the oxymatrine-NO liposome (OM @ TN-lip) under the storage condition of 4 ℃ within 7 days are respectively and continuously measured by utilizing dynamic light scattering. The result shows that the OM @ TN-lip has small particle size and potential change when being stored at 4 ℃ for 7 days, the particle size is kept at about 200nm, the potential is about-15 mV, and the storage at 4 ℃ is relatively stable.
2) In vitro release of oxymatrine-NO liposome
The in vitro release of oxymatrine-NO liposome is determined by dialysis: the in vitro release of the drug at 37 ℃ was determined by high performance liquid chromatography. Taking 10mL of newly prepared oxymatrine-NO liposome, firstly measuring the total amount of oxymatrine entrapped in the liposome by using HPLC, then placing the liposome in a 3500MW dialysis bag for fastening, taking 20mL of PBS as a release phase, taking 200uL of samples to be measured at specified time points of 1, 3, 6, 12, 24, 48, 72 and 96h, simultaneously supplementing 200uL of PBS, and immediately detecting the concentration of oxymatrine in an external phase by using an HPLC method. The release curve of oxymatrine is plotted by using the time point as the abscissa and the oxymatrine release amount corresponding to the time point as the ordinate, and as shown in fig. 5, the cumulative release amount of oxymatrine gradually increases with time, the release rate is faster before 24 hours, and thereafter, although the release rate gradually increases, the release rate is slower, and the cumulative release amount of oxymatrine reaches 52.8% at 96 hours.
Example 7: liposome mouse in vivo imaging study
1) Preparation of DiR liposomes
The preparation method of the DiR-labeled liposome comprises the following steps: the method comprises the steps of precisely weighing DiR and lipid materials in a ratio of 1:100 by mass, dissolving the DiR and the lipid materials in dichloromethane with a proper volume, placing the mixture in a round-bottom flask, removing dichloromethane solvent through rotary evaporation and vacuum drying, and forming a thin uniform film on the bottom and the inner wall of the round-bottom flask. And adding a proper amount of PBS buffer solution into the round-bottom flask, carrying out hydration and ultrasonic treatment for 10min, and filtering and removing excessive DiR through a 0.2-micron polycarbonate membrane to obtain the DiR-labeled targeting liposome.
2) In vivo and ex vivo imaging in inflammatory models and normal mice
The model mice and normal mice are divided into 2 groups respectively, 3 mice in each group are respectively administered by intraperitoneal injection for in vivo imaging, 8 mice in the other group are used for in vitro imaging, the DIR administration amount of each mouse is 50ug, and the administration scheme is as follows:
group A is normal mice intraperitoneal injection free Dir, group B is colitis model mice intraperitoneal injection free Dir, group C is normal mice intraperitoneal injection DiR marked liposome, and group D is colitis model mice intraperitoneal injection DiR marked liposome. After the mice are anesthetized at the set time points of 2, 8, 24 and 36h after the intraperitoneal administration, the mice are subjected to live imaging to shoot fluorescence images, the distribution change of the DiR-marked liposome in the mice along with the time is investigated according to the fluorescence position and intensity, and at each time point, 2 mice are dissected and immediately taken out of the heart, the liver, the spleen, the lung, the kidney and the colon for in vitro imaging. The bar graph of the mean fluorescence values of the in vivo colon tissue is shown in FIG. 6, and the bar graph of the mean fluorescence values of the ex vivo colon tissue is shown in FIG. 7. The result shows that under the same Dir dosage, the histogram result of the average fluorescence value of in-vivo imaging shows that the fluorescence intensity of A, B two groups is very weak, which indicates that no colon targeting effect exists in a normal mouse or an enteritis model mouse by free Dir, the C group and the D group have stronger fluorescence values, and the fluorescence value of the D group is higher than that of the C group, which indicates that the liposome has certain targeting at the colon part and has more obvious targeting effect at the inflammation part; the mean fluorescence value histogram result of the isolated colon tissue A, B, C shows that the fluorescence values of the three groups are lower, the fluorescence value of the group D is higher and is highest at 36h, which indicates that the free Dir has no accumulation at the colon part in normal mice or ulcerated mice, the liposome has a certain accumulation but is weak at the colon part of the normal mice, but has better accumulation at the colon part of the ulcerated mice, and the liposome has better targeting property at the inflammatory colon part.
Example 8: pharmacodynamic study of oxymatrine-NO liposome
After the mouse acute enteritis model is successfully established, the mice are randomly divided into 5 groups of 8 mice, and the groups are respectively as follows: blank control group, inflammation model group (UC), free Oxymatrine (OM), oxymatrine liposome group (OM @ lip), oxymatrine-NO liposome group (OM @ TN-lip). The blank control group and inflammation model group are injected with normal saline, the other experimental groups are respectively administered with free oxymatrine, oxymatrine liposome and oxymatrine-NO liposome in the abdominal cavity for 7 days, and the administration amount is 20mg/kg of oxymatrine.
The first administration was taken as day 1, mice were fasted on day 7, and eyeballs of each group of mice were bled on day 8, and after standing, serum was centrifuged and stored in a refrigerator at-80 ℃ for measurement of serum C-reactive protein (CRP) content. The mouse is sacrificed after blood is taken, colon tissues are dissected and taken out, the colon tissues are cleaned by physiological saline, weighed and measured for length, then 4 percent paraformaldehyde of part of colon is taken and fixed for HE staining, and the other part of colon tissues is taken and used for detecting the content of Myeloperoxidase (MPO).
FIG. 8 and FIG. 9 are graphs of colon tissue length and weight of mice in different treatment groups. Fig. 10, fig. 11, fig. 12, fig. 13 and fig. 14 are pathological section views of the colon of a blank control group, an inflammation model group, a free oxymatrine group, an oxymatrine liposome group and an oxymatrine-NO liposome group) mouse, respectively. . The results of CRP content in serum of mice are shown in FIG. 15, and the results of MPO content in colon of mice are shown in FIG. 16. The results show that the colon of the model group is obviously shortened, the weight is reduced, the colon HE shows that the defect of crypt is obvious, the arrangement of glands is disordered, a large amount of goblet cells are lost, most epithelial cells are necrotic, a large amount of inflammatory cells are infiltrated, the CRP content of serum is increased, the MPO content of colon tissues is increased, and the inflammation degree is higher. Compared with the model group, the length and the weight of the colon of the free drug group have no obvious difference, the damage to the crypt is relatively serious, the form is changed, partial epithelium necrosis is caused, and a large amount of inflammatory cells are infiltrated, which indicates that the free oxymatrine has no obvious treatment effect on the DSS-induced colitis. Compared with the normal group, the colon length and the weight of the oxymatrine liposome group and the oxymatrine-NO liposome group have NO significant difference, and the HE result shows that the tissue structure is complete, the arrangement of glands is complete, crypts and goblet cells are basically normal, and NO obvious inflammatory cell infiltration exists, so that the treatment effect on UC is better, in comparison, the goblet cells of the oxymatrine-NO liposome group are more, the crypt form is more complete, the serum CRP content is lowest, and the colon tissue MPO content is lowest, so that the treatment effect of the oxymatrine liposome combined with NO donor material (TNO3) on UC is obvious, and the treatment effect of the oxymatrine-NO liposome on experimental enteritis of mice is best.
In conclusion, the oxymatrine-NO liposome prepared by the invention has uniform particle size and good stability, can realize high-efficiency encapsulation of oxymatrine, has an encapsulation rate of 70%, can passively target inflammatory colon parts, can prolong the drug action time, improve the drug bioavailability, and has a combined anti-inflammatory effect in combination with NO donor materials, thereby improving the treatment effect on ulcerative colitis.
Example 9: pharmacodynamic study of berberine-NO liposome
Berberine-NO liposomes were prepared in the same liposome preparation manner as in example 1, and their therapeutic effects on colitis in mice were examined. The experiments were divided into 5 groups, which were: a blank control group, an inflammation model group, a free berberine group, a berberine liposome group and a berberine-NO liposome group. Injecting normal saline into abdominal cavity of blank control group and inflammation model group, respectively administering free berberine, berberine liposome and berberine-NO liposome to abdominal cavity of other experimental groups, continuously administering for 7 days, fixing mouse colon 4% paraformaldehyde for HE staining, and administering berberine at 30mg/kg dose.
FIG. 17, FIG. 18, FIG. 19, FIG. 20 and FIG. 21 are the pathological section diagrams of the mouse colon of the blank control group, the inflammation model group, the free berberine group, the berberine liposome group and the berberine-NO liposome group, respectively. The results show that the defect of the crypt of the model group is obvious, a large amount of goblet cells are lost, most epithelial cells are necrotic, a large amount of inflammatory cells are infiltrated, and the degree of inflammation is high. The crypt destruction of the free berberine group is relatively severe, the morphology is changed, and partial epithelium necrosis is caused. The crypt structure of the berberine liposome group is relatively complete and has no obvious inflammatory cell infiltration. The berberine-NO liposome group has complete colon tissue structure, complete gland arrangement, basically normal crypt and goblet cells and NO inflammatory cell infiltration, and shows that the berberine-NO liposome has better treatment effect on UC.
Example 10: pharmacodynamic study of leonurus alkaloid-NO liposome
The leonurus alkaloid-NO liposome is prepared by adopting the preparation method of the liposome in the example 1, and the treatment effect on the mouse colitis is examined. The experiments were divided into 5 groups, which were: blank control group, inflammation model group (UC), free motherwort alkaloid group, motherwort alkaloid liposome group and motherwort alkaloid-NO liposome group. Injecting normal saline into abdominal cavity of blank control group and inflammation model group, respectively administering free herba Leonuri alkaloid, herba Leonuri alkaloid liposome, and herba Leonuri alkaloid-NO liposome to abdominal cavity of other experimental groups, continuously administering for 7 days, fixing mouse colon 4% paraformaldehyde for HE staining, wherein the administration amount is herba Leonuri alkaloid 30 mg/kg.
Fig. 22, 23, 24, 25 and 26 are graphs of HE colon of mice of a blank control group, an inflammation model group, a free leonurus alkaloid group, a leonurus alkaloid liposome group and a leonurus alkaloid-NO liposome group, respectively. The results show that the crypt structure of the model group almost completely disappears, most epithelial cells are necrotic, and the inflammation degree is higher. The crypt morphological structure of the free leonurus alkaloid group is more severely damaged. The structure of the crypt of the leonurus alkaloid liposome group is relatively complete, and no obvious inflammatory cell infiltration exists. The structure of the crypt of the leonurus alkaloid-NO liposome group is complete, and inflammatory cells are not infiltrated, which shows that the leonurus alkaloid-NO liposome has better treatment effect on UC.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (11)

1. An anti-inflammatory liposome with targeting effect, which is characterized in that the liposome is a phospholipid bilayer structure, and cholesterol, a nitric oxide donor molecule and a negatively charged amphiphilic molecule are inlaid in the phospholipid bilayer structure; the cholesterol is used for enhancing the stability of the phospholipid bilayer structure, the negatively charged amphiphilic molecule is used for enabling the liposome to have a targeting effect, and the nitric oxide donor molecule is used for releasing nitric oxide; an anti-inflammatory active ingredient is embedded in a hydrophilic cavity of the phospholipid bilayer structure; the mass parts of the nitric oxide donor molecules are 1-2 parts, and the mass parts of the anti-inflammatory active ingredients are 1-2 parts.
2. The targeted anti-inflammatory liposome of claim 1, wherein the negatively charged amphiphilic molecule has a hydrophobic end of distearoylphosphatidylethanolamine and a hydrophilic end of polyethylene glycol; the anti-inflammatory active ingredient is oxymatrine, leonurus alkaloid or berberine.
3. The targeted anti-inflammatory liposome of claim 1, wherein the nitric oxide donor molecule is a nitrate-based compound capable of releasing NO.
4. The targeted anti-inflammatory liposome of claim 1, wherein the liposome has a diameter of 180nm to 220 nm.
5. The anti-inflammatory liposome with targeting effect of claim 1, wherein the liposome comprises 8-16 parts by mass of phospholipid, 2-4 parts by mass of cholesterol, and 2-4 parts by mass of negatively charged amphiphilic molecule.
6. The method for preparing anti-inflammatory liposome with targeting effect as claimed in any of claims 1-5, comprising the steps of:
(1) dissolving phospholipid, cholesterol, nitric oxide donor molecules and amphiphilic molecules with negative charges in an organic solvent, and removing the organic solvent by spin drying or removing the organic solvent by reduced pressure evaporation to form a film;
(2) placing the film obtained in the step (1) in an acid buffer solution for hydration, and then carrying out ultrasonic treatment to enable phospholipid to form a liposome with a bilayer structure, wherein cholesterol, nitric oxide donor molecules and amphiphilic molecules with negative charges are embedded in the bilayer; then, redispersing by adopting a water-based filter membrane to obtain a solution containing blank liposome;
(3) adjusting the pH value of the solution containing the blank liposome obtained in the step (2) to 7.0-7.4, then adding an anti-inflammatory active ingredient, and incubating to enable the anti-inflammatory active ingredient to be entrapped in a hydrophilic cavity in the liposome under the internal and external pH gradients of the liposome;
(4) and (3) after the incubation in the step (3) is finished, dialyzing to remove the non-entrapped anti-inflammatory active ingredients, centrifuging, removing the precipitate, and taking the supernatant to obtain the anti-inflammatory liposome with the targeting effect.
7. The method for preparing anti-inflammatory liposome with targeting effect as claimed in claim 6, wherein the hydrophilic end of the amphiphilic molecule with negative charge of step (1) is distearoylphosphatidylethanolamine, and the hydrophobic end is polyethylene glycol; the nitric oxide donor molecule is a nitrate compound capable of releasing NO;
the liposome comprises 8-16 parts of phospholipid, 2-4 parts of cholesterol, 2-4 parts of negatively charged amphiphilic molecules, 1-2 parts of nitric oxide donor molecules and 1-2 parts of anti-inflammatory active ingredients.
8. The method for preparing anti-inflammatory liposome with targeting effect as claimed in claim 6, wherein the anti-inflammatory active ingredient in step (3) is oxymatrine, leonurus alkaloid or berberine, the incubation temperature is 50-60 ℃, and the incubation time is 1-2 h.
9. The method for preparing anti-inflammatory liposome with targeting effect as claimed in claim 6, wherein the pore size of the aqueous membrane of step (2) is 0.22um-0.45 um; the rotating speed of the centrifugation in the step (4) is 2000rpm-3000rpm, and the time of the centrifugation is 5min-15 min.
10. Use of the targeted anti-inflammatory liposomes of any one of claims 1 to 5 for the preparation of anti-inflammatory drugs.
11. The use of claim 10, wherein the anti-inflammatory agent is an agent for treating inflammation of the colon.
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