[go: up one dir, main page]

CN118831052B - Preparation method and application of puerarin-loaded nanocrystalline polymer micelles - Google Patents

Preparation method and application of puerarin-loaded nanocrystalline polymer micelles Download PDF

Info

Publication number
CN118831052B
CN118831052B CN202411311645.7A CN202411311645A CN118831052B CN 118831052 B CN118831052 B CN 118831052B CN 202411311645 A CN202411311645 A CN 202411311645A CN 118831052 B CN118831052 B CN 118831052B
Authority
CN
China
Prior art keywords
puerarin
mpeg
solution
reaction
liquid
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.)
Active
Application number
CN202411311645.7A
Other languages
Chinese (zh)
Other versions
CN118831052A (en
Inventor
程勐
涂亮星
邵益森
于晓明
金�一
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.)
Affiliated Hospital of Jiangxi University of TCM
Original Assignee
Affiliated Hospital of Jiangxi University of TCM
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 Affiliated Hospital of Jiangxi University of TCM filed Critical Affiliated Hospital of Jiangxi University of TCM
Priority to CN202411311645.7A priority Critical patent/CN118831052B/en
Publication of CN118831052A publication Critical patent/CN118831052A/en
Application granted granted Critical
Publication of CN118831052B publication Critical patent/CN118831052B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • 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
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
    • C08G65/3328—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof heterocyclic
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Polymers & Plastics (AREA)
  • Cardiology (AREA)
  • Vascular Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Inorganic Chemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to a preparation method and application of puerarin-loaded nanocrystalline polymer micelle, which comprises the steps of dissolving PVP-K30 in a beaker, adding puerarin bulk drug, shearing in a high-speed shearing machine to obtain crude suspension, homogenizing the crude suspension in a high-pressure homogenizer to obtain puerarin nanocrystalline liquid, dissolving carrier material mPEG-TK-GA in methanol, removing the methanol by reduced pressure rotary evaporation to obtain a transparent film, adding puerarin nanocrystalline liquid, ultrasonically dissolving the film, oscillating and hydrating by a constant-temperature oscillator, and finally filtering by a filter membrane to obtain the puerarin-loaded nanocrystalline polymer micelle. The polymer micelle reduces the liver metabolism level of puerarin by adopting 18-alpha glycyrrhizic acid, improves the drug release possibility of the drug at the atherosclerosis plaque position by adopting the polymer micelle responded by ROS, and improves the targeted enrichment of the drug plaque position.

Description

Preparation method and application of puerarin-loaded nanocrystalline polymer micelle
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a preparation method and application of puerarin-loaded nanocrystalline polymer micelle.
Background
The etiology of cardiovascular and cerebrovascular diseases is that the high incidence, high disability rate and high death rate have become global important public health problems. Atherosclerosis (atherosclerosis, AS), i.e., an atherosclerotic lesion of arterial angiogenesis, causes stenosis or blockage of the lumen of a blood vessel, resulting in ischemia, hypoxia or necrosis of the tissue, and is a major cause of cardiovascular and cerebrovascular diseases. The medicine for treating AS has a plurality of traditional Chinese medicines and active ingredients thereof, such AS red sage root, notoginseng, szechuan lovage rhizome, kudzuvine root and the like, but the active ingredients of the traditional Chinese medicines are mostly indissolvable components, the oral bioavailability is low, and the administration can be carried out only by intravenous injection, and the traditional Chinese medicine injection generally has the problems of low solubility, wide distribution range, high elimination speed, so that the defects of short in-vivo residence time, lack of tissue specific distribution and the like exist, and the increase of the curative effect of the indissolvable traditional Chinese medicine active ingredient injection on AS is a serious difficulty in the current traditional Chinese medicine preparation research.
Modern pharmacological research shows that puerarin has the pharmacological actions of dilating blood vessel, increasing myocardial contractility, resisting oxidation, protecting liver and the like, and is mainly used for treating cardiovascular and cerebrovascular diseases, such as atherosclerosis heart disease (coronary heart disease), angina pectoris, hypertension and the like in clinic. In addition, puerarin can inhibit differentiation of mouse Vascular Smooth Muscle Cells (VSMCs) to osteoblasts by down regulating expression of osteogenic genes Runx2 and BMP2, inhibit vascular calcification, activate ERK5/KLF2 pathway by inhibiting NLRP3/Caspase1/IL-1 beta and NF-kappa B cell pathway, reduce generation of inflammatory factors IL-1 beta and ROS, secrete adhesion factors, inhibit inflammatory reaction, promote cholesterol efflux mediated by ABCA1 pathway, and reduce intracellular cholesterol level. Therefore, puerarin can be used for treating AS by acting on a plurality of cell passages, inhibiting inflammatory reaction and vascular calcification, reducing intracellular cholesterol level and the like.
Because puerarin has low solubility and poor film permeability, the oral bioavailability is low, and the treatment of diseases can only be carried out in an injection administration mode clinically at present. In puerarin injection, 50% (v/v) propylene glycol is added as a solubilizer to increase puerarin solubility. Because of the stimulation of the solubilizer and the high blood concentration easily caused by injection administration, the puerarin injection is easy to produce adverse reactions such as hemolysis, fever, skin itch, chest distress, short breath and the like in the use process. In addition, puerarin has short half-life (t 1/2α about 10 min) in human body, wide distribution range (about 0.3L/kg) and short elimination half-life (t 1/2β about 74 min), so that the puerarin has the defects of short residence time in vivo, lack of tissue specific distribution and the like, and severely restricts the clinical application of the puerarin. If the puerarin injection is modified, the aim of enhancing efficiency and reducing toxicity is achieved, the clinical application prospect is greatly improved, and meanwhile, the quality upgrading improvement of the traditional Chinese medicine injection which is represented by the puerarin injection and is widely used clinically but has application defects is provided for research experience.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method and application of puerarin-loaded nanocrystalline polymer micelle.
The invention adopts the following technical scheme that the preparation method of the puerarin-loaded nanocrystalline polymer micelle comprises the following steps:
PVP-K30 is stirred and dissolved by pure water in a beaker, puerarin raw material medicine is added into the beaker, and shearing is carried out in a high-speed shearing machine, so as to obtain crude suspension;
homogenizing the crude suspension in a high-pressure homogenizer, and cooling to room temperature to obtain transparent puerarin nanocrystalline liquid;
Carboxylation reaction is carried out on mPEG2000 to obtain mPEG-COOH, then activation reaction is carried out on the mPEG-COOH to obtain mPEG-COOH activation liquid, and the mPEG-COOH activation liquid is reacted with NH 2-TK-NH2 to synthesize mPEG-TK-NH 2;
For a pair of Performing an activation reaction to obtainAn activating solution for activating the saidThe activating solution reacts with the mPEG-TK-NH 2 to obtain a carrier material mPEG-TK-GA;
Dissolving the carrier material mPEG-TK-GA in methanol, removing the methanol by reduced pressure rotary evaporation to obtain a transparent film, adding the puerarin nanocrystalline liquid into the transparent film, ultrasonically dissolving the transparent film, oscillating and hydrating by a GAs bath constant temperature oscillator, and finally filtering by a filter membrane to obtain the puerarin-loaded nanocrystalline polymer micelle.
The preparation method of the puerarin-loaded nanocrystalline polymer micelle aims at the problem of rapid in-vivo metabolism of puerarin by adopting the effective components of liquorice() Aiming at the problem of poor targeting of puerarin disease sites, the polymer micelle adopting ROS response is adopted to improve the possibility of drug release at the atherosclerosis plaque sites, thereby improving the targeting enrichment of the drug plaque sites, and simultaneously, the prepared polymer micelle further improves the uptake of the drug by pathological cells (macrophages) at the atherosclerosis sites by entrapping puerarin nano crystals, thereby ensuring the utilization effect of puerarin.
Further, the step of carboxylating mPEG2000 to obtain mPEG-COOH specifically includes:
Adding mPEG2000 into acetone, heating in a water bath, stirring until mPEG2000 is completely dissolved, adding a Jones reagent for reaction until the reaction liquid is blue and has deep blue precipitate at the bottom, adding isopropanol for quenching, performing suction filtration to obtain light blue-green filtrate, adding pure water into the light blue-green filtrate, and then removing acetone by rotary evaporation to obtain acetone-free filtrate;
adding sodium bicarbonate into the acetone-free filtrate, stirring while adding until no bubbles are generated, standing, and performing rotary evaporation on the supernatant to remove water to obtain a bluish thick paste;
Adding chloroform into the sea blue thick paste for solid-liquid extraction, wherein the lower layer is a milky turbid liquid, the upper layer is a blue floccule, collecting the milky turbid liquid by adopting a separating funnel, extracting for a plurality of times by using chloroform, and then carrying out suction filtration and rotary evaporation to remove the chloroform to obtain mPEG-COONa;
Dissolving mPEG-COONa in pure water, adding hydrochloric acid solution to adjust pH to a preset value, removing water and hydrogen chloride by rotary evaporation to obtain a viscous semitransparent concentrated solution, adding chloroform for extraction, filtering, removing chloroform by rotary evaporation, and cooling to obtain white mPEG-COOH solid.
Further, the step of performing an activation reaction on the mPEG-COOH to obtain an mPEG-COOH activation solution specifically includes:
Dissolving mPEG-COOH, dicyclohexylcarbodiimide and N-hydroxysuccinimide in dichloromethane, and stirring and activating at room temperature to obtain milky mPEG-COOH activating solution.
Further, the preparation steps of the NH 2-TK-NH2 specifically include:
Adding cysteine hydrochloride and acetone into a reaction bottle, adding chloroform into the reaction bottle, and introducing dry hydrogen chloride gas for reaction to obtain thick paste-like reaction liquid;
Filtering the thick paste reaction liquid, adopting chloroform to clean and precipitate, then pumping to dry, placing filter residues into an equal volume of methanol/diethyl ether mixed solution for recrystallization, filtering, and drying the filter residues in vacuum to obtain a vacuum dried product;
And (3) dissolving the vacuum dried product in NaOH solution for reaction to obtain clear liquid, and adding dichloromethane for extraction to obtain NH 2-TK-NH2.
Further, the step of reacting the mPEG-COOH activating solution with NH 2-TK-NH2 to synthesize mPEG-TK-NH 2 specifically includes:
Dropwise adding the mPEG-COOH activating solution into a dichloromethane solution dissolved with NH 2-TK-NH2, reacting under the protection of nitrogen, filtering, and performing rotary evaporation on the obtained filtrate to remove dichloromethane to obtain a concentrated solution;
Precipitating the concentrated solution in diethyl ether/methanol mixed solution with volume ratio of 10:1 at low temperature, collecting filter residues, and finally vacuum drying to obtain mPEG-TK-NH 2.
Further, the saidThe preparation method comprises the following steps:
Slowly adding sodium hydroxide into pure water, stirring while adding 18 beta-H-diammonium glycyrrhizinate into the solution, heating and refluxing for reaction, slowly dripping H 2SO4 solution under ice bath condition, regulating pH value to a preset value, stirring while adding white solid, and stirring under ice bath condition to obtain white thick paste reaction solution;
Under ice bath condition, dissolving the white thick paste reaction solution in H 2SO4 solution, stirring, filtering, collecting filter residue, and drying to obtain white powder 。
Further, the pair ofPerforming an activation reaction to obtainThe step of activating the liquid specifically comprises:
Will be Dissolving in N, N-dimethylformamide until the solution is clear, slowly adding dichloromethane until the solution is clear, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide for activation reaction to obtainActivating liquid.
Further, said bringing saidThe step of reacting the activating solution with the mPEG-TK-NH 2 to obtain a carrier material mPEG-TK-GA specifically comprises the following steps:
The said Dropwise adding the activation solution into dichloromethane solution dissolved with mPEG-TK-NH 2, reacting under the protection of nitrogen, performing suction filtration, and removing dichloromethane from the obtained filtrate by rotary evaporation to obtain concentrated solution;
Precipitating the concentrated solution in diethyl ether at 0 ℃ while dropwise adding and stirring to obtain white precipitate, filtering, and vacuum drying the collected filter residue to obtain carrier material mPEG-TK-GA.
Furthermore, the puerarin-loaded nanocrystalline polymer micelle is in a regular round shape, and the particle size is 132.6nm plus or minus 2.55nm.
The invention also provides an application of the puerarin-loaded nanocrystalline polymer micelle prepared by the puerarin-loaded nanocrystalline polymer micelle preparation method in preparation of anti-atherosclerosis drugs.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a flow chart of a preparation method of puerarin-loaded nanocrystalline polymer micelle;
FIG. 2 is a schematic representation of the synthetic route of the carrier material mPEG-TK-GA of the invention;
FIG. 3 is an infrared signature of the carrier material mPEG-TK-GA and intermediates thereof of the invention;
FIG. 4 is a 1 H-NMR spectrum of the carrier material mPEG-TK-GA of the invention;
FIG. 5 shows the main active component of Glycyrrhrizae radix against puerarin in example 4 of the present invention Phase sumSchematic representation of the effect of phase metabolic stability;
FIG. 6 is a schematic representation of the effect of the carrier material mPEG-TK-GA on puerarin metabolic stability in example 4 of the present invention;
FIG. 7 is a schematic representation of the fluorescence distribution of DiR-labeled polymer micelles in ApoE-/-mouse ex vivo tissue according to example 5 of the invention;
FIG. 8 is a graph showing the fluorescence intensity statistics of 2h and 6h in isolated blood vessels in example 5 of the present invention;
FIG. 9 is a schematic diagram showing the uptake mechanism of various puerarin preparations in normal and inflammatory macrophages in example 7 of the present invention;
FIG. 10 is a graph showing the uptake levels of various puerarins in normal and inflammatory macrophages in example 7 of the present invention;
FIG. 11 is a graph showing the time profile of administration of puerarin and its formulations in SD rats according to comparative example 1 of the present invention;
FIG. 12 is an enlarged partial view of the 1h drug profile of puerarin and its preparation in SD rats according to comparative example 1 of the present invention;
FIG. 13 is an SEM image of puerarin and its preparation according to comparative example 2 of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are exemplary and intended to illustrate embodiments of the invention and should not be construed as limiting the invention.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless explicitly defined otherwise.
Example 1
Referring to fig. 1, in embodiment 1 of the present invention, a method for preparing puerarin-loaded nanocrystalline polymer micelle includes:
S1, PVP-K30 (polyvinylpyrrolidone-K30) is stirred and dissolved by pure water in a beaker, puerarin raw material medicine (Pue raw material medicine) is added into the beaker, and is sheared in a high-speed shearing machine to obtain crude suspension, in the embodiment, 0.8g of PVP-K30 is weighed and placed in a 50mL beaker, pure water 40 mL is added, stirring and dissolving are carried out, pue raw material medicine 0.4g is added into the beaker, and is sheared for 3min at 13000rpm, 3min at 16000rpm and 1min at 19000rpm in sequence in the high-speed shearing machine to obtain crude suspension.
S2, homogenizing the crude suspension in a high-pressure homogenizer until the temperature is reduced to room temperature to obtain clarified puerarin nanocrystalline (Pue (NCs) liquid), wherein in the embodiment, homogenizing the crude suspension in the high-pressure homogenizer for 2 times at 50bar, homogenizing the crude suspension for 2 times at 200bar and homogenizing the crude suspension for 10 times at 800bar in sequence, and recovering the temperature to room temperature to obtain clarified puerarin nanocrystalline (Pue (NCs) liquid, wherein the Pue (NCs) liquid contains 10mg/mL puerarin nanocrystalline.
S3, performing carboxylation reaction on the mPEG2000 to obtain mPEG-COOH, then performing activation reaction on the mPEG-COOH to obtain an mPEG-COOH activated liquid, and reacting the mPEG-COOH activated liquid with NH 2-TK-NH2 to synthesize mPEG-TK-NH 2;
s3.1, carrying out carboxylation reaction on mPEG2000 to obtain mPEG-COOH, wherein the step of obtaining the mPEG-COOH specifically comprises the following steps:
Adding mPEG2000 into acetone, heating in a water bath, stirring until mPEG2000 is completely dissolved, adding a Jones reagent for reaction until the reaction liquid is blue, adding isopropyl alcohol for quenching until the bottom is dark blue, carrying out suction filtration to obtain light blue green filtrate, adding pure water into the light blue green filtrate, and then removing acetone by rotary evaporation to obtain acetone-free filtrate, in the embodiment, weighing 50.0g of mPEG2000, adding 800mL of acetone, heating in a 30 ℃ water bath, stirring until mPEG2000 is completely dissolved, adding 45mL of Jones reagent, reacting at 25 ℃ for 24 hours, wherein the reaction liquid is blue, the bottom is dark blue precipitate, adding 10mL of isopropyl alcohol for quenching, carrying out suction filtration, adding 400mL of pure water into the filtrate, and carrying out rotary evaporation to remove acetone under the condition of 40 ℃ to 60 ℃ to obtain acetone-free filtrate;
Adding sodium bicarbonate into the acetone-free filtrate, stirring while adding until no bubbles are generated, standing, and performing rotary evaporation on the supernatant to remove water to obtain a bluish thick paste;
Adding chloroform into the sea blue thick paste for solid-liquid extraction, wherein the lower layer is in a milky turbid liquid, the upper layer is in a blue floccule, collecting the lower layer of the milky turbid liquid by adopting a separating funnel, extracting for 3 times by using chloroform, and then carrying out suction filtration, and removing the chloroform by rotary evaporation to obtain mPEG-COONa;
Dissolving mPEG-COONa in pure water, adding hydrochloric acid solution to adjust pH to a preset value, steaming to remove water and hydrogen chloride by rotary evaporation to obtain a viscous semitransparent concentrated solution, adding chloroform for extraction, filtering, steaming the filtrate by rotary evaporation to remove the chloroform, and cooling to obtain white mPEG-COOH solid, in the embodiment, dissolving mPEG-COONa in 100mL of pure water, adding 10% hydrochloric acid solution to adjust pH to 1, steaming to remove water and hydrogen chloride by rotary evaporation to obtain a viscous semitransparent concentrated solution, adding chloroform for extraction, filtering, steaming the filtrate by rotary evaporation to remove the chloroform, and cooling to obtain the white mPEG-COOH solid.
S3.2, carrying out an activation reaction on the mPEG-COOH to obtain an mPEG-COOH activated liquid, wherein the step of obtaining the mPEG-COOH activated liquid specifically comprises the following steps:
in this example, 100g of mPEG-COOH, 11.5g of Dicyclohexylcarbodiimide (DCC) and 6.5g of N-hydroxysuccinimide (NHS) are weighed, dissolved in 600mL of dichloromethane, stirred and activated at room temperature for 6 hours to obtain a milky activated mPEG-COOH liquid.
The preparation steps of S3.3 and NH 2-TK-NH2 specifically comprise:
adding cysteine hydrochloride and acetone into a reaction bottle, adding chloroform into the reaction bottle, and introducing dry hydrogen chloride gas to react to obtain thick paste-like reaction liquid, wherein 170.4g of cysteine hydrochloride and 234.5g of acetone are weighed, 120mL of chloroform is added into the reaction bottle, and the dry hydrogen chloride gas is introduced to react for 6 hours at 25 ℃ to obtain thick paste-like reaction liquid after the reaction is finished;
Filtering the thick paste reaction liquid, adopting chloroform to clean and precipitate for 2 times, then pumping to dry, placing filter residues into an equal volume of methanol/diethyl ether mixed liquid, recrystallizing for 2 times at-20 ℃, filtering, and drying the filter residues in vacuum at 40 ℃ to obtain a vacuum dried substance;
The dried product was dissolved in 600mL NaOH solution at a concentration of 6mol/L, reacted at 25℃for 12 hours to give a clear liquid, which was extracted with methylene chloride to give NH 2-TK-NH2.
S3.4, the step of reacting the mPEG-COOH activating solution with NH 2-TK-NH2 to synthesize mPEG-TK-NH 2 specifically comprises the following steps:
weighing 20g of NH 2-TK-NH2, dissolving in 350mL of dichloromethane, dropwise adding the mPEG-COOH activated liquid into the dichloromethane solution dissolved with NH 2-TK-NH2, reacting for 24 hours at 20 ℃ under the protection of nitrogen, filtering, and performing rotary evaporation on the obtained filtrate to remove the dichloromethane to obtain concentrated solution;
Precipitating the concentrated solution in diethyl ether/methanol mixed solution with volume ratio of 10:1 at low temperature, collecting filter residues, and finally vacuum drying to obtain mPEG-TK-NH 2.
S4, pairing() Performing an activation reaction to obtain() Activating liquid to be activated() The activation solution reacts with mPEG-TK-NH 2 to obtain a carrier material mPEG-TK-GA;
S4.1, further, the step of, () The preparation method comprises the following steps:
Slowly adding sodium hydroxide into pure water, stirring while adding 18 beta-H-diammonium glycyrrhizinate, heating and refluxing for reaction, slowly dropwise adding H 2SO4 solution under ice bath condition, regulating pH value to a preset value, stirring while adding white solid, continuing stirring under ice bath condition to obtain white thick paste reaction liquid, in the embodiment, weighing 40g sodium hydroxide, slowly adding 150mL pure water, stirring while adding to completely dissolve, adding 60g 18 beta-H-diammonium glycyrrhizinate, heating and refluxing for reaction for 6H, slowly dropwise adding 2.5mol/L H 2SO4 solution under ice bath condition, regulating pH value to 2.0, stirring while adding white solid, continuing stirring under ice bath condition for 0.5H until all reaction liquid is thick paste;
Under ice bath condition, dissolving the white thick paste reaction solution in 600mL ice H 2SO4 solution with pH value of 2.0, stirring for 10min, filtering, collecting the residue, and drying to obtain white powder ()。
S4.2, pair() Performing an activation reaction to obtain() The step of activating the liquid specifically comprises:
Will be () Dissolving in N, N-dimethylformamide until the solution is clear, slowly adding dichloromethane until the solution is clear, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide for activation reaction to obtain() In this example, 4.5g of the activating solution was weighedIn 20mL of N, N-Dimethylformamide (DMF), the solution was clarified, 80mL of methylene chloride was slowly added to the solution, the solution was clarified, dicyclohexylcarbodiimide (DCC) 3.72g and N-hydroxysuccinimide (NHS) 2.11g were added, and the mixture was activated at 20℃for 6 hours.
S4.3, will() The step of reacting the activation solution with mPEG-TK-NH 2 to obtain a carrier material mPEG-TK-GA specifically comprises the following steps:
11.3g of the compound mPEG-TK-NH 2 was weighed out and dissolved in 100mL of methylene chloride () Dropwise adding the activation solution into methylene dichloride solution dissolved with mPEG-TK-NH 2, reacting for 24 hours at 20 ℃ under the protection of nitrogen, and then carrying out suction filtration, and removing methylene dichloride from the obtained filtrate by rotary evaporation to obtain concentrated solution;
Precipitating the concentrated solution in diethyl ether at 0 ℃ while dropwise adding and stirring to obtain white precipitate, filtering, and vacuum drying the collected filter residue to obtain carrier material mPEG-TK-GA.
The synthesis route of the carrier material mPEG-TK-GA is shown in FIG. 2, specifically, the mPEG, mPEG-COOH, mPEG-TK-NH 2 and the carrier material mPEG-TK-GA are respectively characterized by using IR (infrared spectrum), the carrier material mPEG-TK-GA is characterized by using 1 H-NMR (nuclear magnetic resonance hydrogen spectrum) with DMSO as a solvent, and the successful synthesis of the carrier material mPEG-TK-GA is shown by referring to FIGS. 3 to 4.
Referring specifically to FIG. 3, in the mPEG infrared spectrum, the C-H stretching vibration peak is at 2887cm -1, the-OH stretching vibration peak is at 3480cm -1, the C-O-C stretching vibration peak is at 1111cm -1, compared with mPEG, the mPEG-COOH presents a carboxyl-COOH characteristic absorption peak at 1738cm -1, compared with mPEG-COOH, the-COOH characteristic absorption peak disappears in the mPEG-TK-NH 2 infrared spectrum, the amide characteristic peak appears at 1541cm -1, and compared with mPEG-TK-NH 2, the mPEG-TK-GA presents more obvious amide bond characteristic peaks at 1541cm -1 and 1454cm -1. From the infrared results, it was found that characteristic peaks of mPEG, TK and GA were reflected in the mPEG-TK-GA polymer, suggesting that it was successfully synthesized.
Referring to FIG. 4, 1 H-NMR spectrum characteristic peak analysis of mPEG-TK-GA: a peak (characteristic shift3.2) Corresponds to-OCH 3, peak b (characteristic shiftAbout 3.5 is corresponding to-OCH 2CH2, peak c (characteristic shift)About 4.0) corresponds to-CH 2 CON, d peak, e peak (characteristic shiftAll between 2.5 and 3.0) corresponds to-CH 2 and f peak (characteristic shift1.5) Corresponds to-CH 3, g peak (characteristic shift4.6) Corresponds to-CH, h peak (characteristic shift5.58) Bimodal, indicating that the GA in the polymer mPEG-TK-GA is。
S5, dissolving carrier material mPEG-TK-GA in methanol, removing the methanol by reduced pressure rotary evaporation to obtain a transparent film, adding puerarin nano-crystalline liquid into the transparent film, ultrasonically dissolving the transparent film, oscillating and hydrating by a GAs bath constant temperature oscillator, and finally filtering by a filter membrane to obtain puerarin-carrying nano-crystalline polymer micelle, in the embodiment, weighing 1.2g of carrier material mPEG-TK-GA, dissolving in 40mL of methanol, removing the methanol by reduced pressure rotary evaporation to obtain the transparent film, vacuum drying for 12h, removing residual methanol, adding 12mL of puerarin nano-crystalline liquid (Pue (NCs) liquid), 18mL of pure water, ultrasonically dissolving the film for 3min, oscillating and hydrating by the GAs bath constant temperature oscillator for 6h, and filtering by a 0.45 mu m filter membrane to obtain puerarin-carrying nano-crystalline polymer micelle (Pue (NCs) @ mPEG-TK-GA.
Furthermore, the puerarin-loaded nano-crystalline polymer micelle is in a regular round shape, and the particle size is 132.6nm plus or minus 2.55nm.
The invention also provides an application of the puerarin-loaded nanocrystalline polymer micelle in preparing an anti-atherosclerosis drug.
The functional material selected by the invention is mainly (1) effective component of licoriceOn the one hand, the preparation is used AS a metabolism inhibitor to inhibit metabolism of puerarin by liver and further improve in vivo residence time of puerarin, on the other hand, the preparation is used AS a forming material of a carrier material, a mode of' medicine assistance is adopted to improve medicine carrying capacity of a multi-component traditional Chinese medicine drug delivery system (preparation) (compared with the mode that glycyrrhizic acid and other medicines are loaded into a micelle together, the glycyrrhizic acid is used AS the forming material, a space can be reserved to increase other medicine carrying capacity while the preparation is loaded with enough glycyrrhizic acid), and (2) a small molecular compound ketal (thioketal, TK) serving AS a bridging segment to connect a hydrophilic segment and a hydrophobic segment in the carrier material is stable in TK under a normal tissue low-level ROS environment, a micelle structure is stable, TK is cracked under an AS lesion site high-level ROS environment, micelle structure is damaged, and a targeting medicine releasing function of the medicine carrying system is realized, and (3) the conventional hydrophilic end material mPEG of the carrier material can endow of the carrier material with certain hydrophilicity, so that hydrophilic and lipophilic property of the carrier material can be self-assembled into a polymer micelle.
In specific implementation, the model drug loaded in the micelle and the mode thereof can be puerarin original drug or puerarin nanocrystalline, wherein the puerarin nanocrystalline is easier to be absorbed by lesion cells (macrophages) of an atherosclerosis plaque part, so that the invention has better disease treatment effect.
The preparation method of the puerarin-loaded nanocrystalline polymer micelle aims at the problem of rapid in-vivo metabolism of puerarin by adopting the effective components of liquorice() Aiming at the problem of poor targeting of puerarin disease sites, polymer micelle adopting ROS response is adopted to improve the possibility of drug release at the atherosclerosis plaque sites, and further improve the targeting enrichment of the drug at the plaque sites, and meanwhile, the prepared polymer micelle further improves the uptake of the drug by pathological cells (macrophages) at the atherosclerosis sites by entrapping puerarin nano-crystals, thereby ensuring the utilization effect of puerarin.
Example 2
The embodiment 2 of the invention provides a puerarin-carrying nanocrystalline polymer micelle, which is prepared by adopting the puerarin-carrying nanocrystalline polymer micelle preparation method, taking 40ml puerarin-carrying nanocrystalline polymer micelle as an example, wherein the puerarin-carrying nanocrystalline polymer micelle comprises 0.4g-0.8g puerarin, 2.0g-4.0g mPEG-TK-GA, 0.4g-0.8g stabilizer, preferably PVP-K30 and the rest filler, wherein the filler is purified water, and the puerarin content can be 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, mPEG-TK-GA content can be 2.0g, 2.2g, 2.4g, 2.6g, 2.8g, 3.0g, 3.4g, 3.6g, 3.8g, 4.0g stabilizer can be 0.4g, 0.5g, 0.8g stabilizer, 0.7g, 0.8g and the like.
Example 3
The embodiment 3 of the invention provides a preparation method of puerarin-carrying or puerarin nano-crystalline polymer micelle, which comprises the steps of weighing a prescribed amount of carrier material mPEG-TK-GA, dissolving in methanol, removing the methanol by reduced pressure rotary evaporation to obtain a transparent film, adding prescribed amount of puerarin nano-crystalline liquid, adding pure water to the prescribed volume, ultrasonically dissolving the film, carrying out constant-temperature oscillation hydration for 6 hours, and filtering with a 0.45 mu m filter membrane to obtain the puerarin-carrying nano-crystalline polymer micelle (Pue (NCs) @ mPEG-TK-GA), wherein the mass ratio of the puerarin nano-crystalline liquid to the carrier material mPEG-TK-GA can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10, and the film-forming solvent methanol can also be ethanol, acetone and the like, and the constant-temperature oscillation hydration time can be 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours and 8 hours.
Or weighing the puerarin with the prescription amount and the carrier material mPEG-TK-GA with the prescription amount, dissolving in methanol, removing the methanol by reduced pressure rotary evaporation to obtain a transparent film, adding purified water to the prescription volume, ultrasonically dissolving the film, oscillating and hydrating for 6h at constant temperature, and filtering with a 0.45 mu m filter membrane to obtain the puerarin-carrying polymer micelle (Pue@mPEG-TK-GA), wherein the mass ratio of puerarin to the carrier material mPEG-TK-GA can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10, the film forming solvent methanol can also be ethanol, acetone and the like, and the constant temperature oscillating and hydrating time can be 2h, 3h, 4h, 5h, 6h, 7h and 8h.
Example 4
To examine toThe effect of mPEG-TK-GA as a carrier material synthesized by key materials on liver metabolism puerarin.
Firstly, examining two major active components of triterpenoid saponins in liquorice、) And the effect of licoflavone (liquiritin (LR), isoliquiritigenin (iLR)) on Pue (puerarin) metabolic stability, to explore the active ingredients in licorice which can prolong Pue in systemic circulation time. Preparation of concentration levels of 600. Mu.M, 150. Mu.M, 50. Mu.M, respectivelySolution(s),Solution, LR solution, iLR solution, adding toPhase or phaseIn the phase reaction system, each reaction system is added withSolution(s),The concentration of the solution, LR solution and iLR mu L of Pue solution is 2.5 mu M, the concentration of liver microsome is 1.0g/L, and the incubation time is 120min.
As shown in (A) of FIG. 5, glycyrrhrizae radix as main active component contains puerarinThe effect of the phase metabolism stability is schematically shown, and the result shows that in the concentration range of 5 mu M-60 mu M,、And LR are all to PuePhase metabolism has an inhibitory effect, whereas iLR has an inhibitory effect on Pue in this concentration rangePhase metabolism shows inhibition followed by induction.At a concentration of PueNo significant difference in phase metabolism inhibition; at a concentration of Pue Phase metabolism inhibition is reduced at high concentration, LR is reduced to Pue in the concentration rangePhase metabolism inhibition is gradually enhanced, but the inhibition degree isAndWeaker than the above, as shown in (B) of FIG. 5, the main active ingredient of Glycyrrhrizae radix is puerarinSchematic of the influence of phase metabolic stability, in the concentration range of 5 mu M-60 mu M,LR and iLR are all to PuePhase metabolism has an inhibitory effect onAt a concentration of PuePhase metabolism shows inhibition followed by induction.At a concentration of PuePhase metabolism inhibition increases and decreases with increasing concentration, LR increases Pue in the concentration rangePhase metabolism inhibition tends to decrease with increasing concentration, iLR for Pue in this concentration rangeThe phase metabolism inhibition effect is gradually enhanced with the increase of the concentration. In conclusion, the method comprises the steps of,And LR pair PuePhase sumThe phase metabolism inhibition effect is more stable, has the possibility of increasing Pue in the systemic circulation time, andHas stronger inhibition effect. Therefore, the preferred active ingredients of licorice are。
Then, byAs shown in FIG. 6, the effect of the polymer mPEG-TK-GA synthesized by the key material on the puerarin metabolism is shown, and the result shows that the mPEG-TK-GA has an effect on the puerarin metabolism stability (n=3) in the concentration range of 5 mu M-60 mu M on puerarin in vitroPhase sumThe phase metabolism has inhibiting effect, and the inhibiting effect has no dependence on concentration, which indicates effective components of Glycyrrhrizae radixCan inhibit metabolism of puerarin by liver, and further increase retention time of puerarin in vivo.
Example 5
DiR probe labeling was used to study the in vivo targeting of Pue (NCs) @ mPEG-TK-GA micelles.
DiR probe is adopted to label Pue (NCs) @ mPEG-TK-GA micelle, and the micelle is respectively given to a normal mouse and an atherosclerosis mouse by intravenous injection and is respectively marked as a C57 control group and an ApoE -/- group, and the fluorescence intensity of main in vitro tissues (heart, liver, spleen, lung, kidney and blood vessel) is measured 2h and 6h after the administration. Referring to fig. 7 and 8, the results show that compared with the C57 control group, the fluorescence intensity in the ApoE -/- group full-length aorta is significantly enhanced, the fluorescence intensity at 2h is 1.8 times that of the C57 control group, the fluorescence intensity at 6h is 4.7 times that of the ApoE -/- group, and the fluorescence intensity at 6h has no significant difference, which indicates that the Pue (NCs) @ mPEG-TK-GA micelle can be targeted to the plaque part distributed in an AS mouse (atherosclerosis mouse), and the drug concentration of the plaque part is improved.
Example 6
SD rats were used as model animals to study the pharmacokinetic properties of puerarin-loaded nanocrystalline polymeric micelles.
SD rats weighing 250 g+ -20 g were taken, and after 1 week of adaptive feeding, they were randomly divided into 4 groups, pue (NCs) @ mPEG-TK-GA micelle group, pue @ mPEG-TK-GA micelle group, pue injection group, pue (NCs) group. Pre-administration 12h, no water forbidden during fasting, and tail vein injection, wherein the administration dosage is 25mg/kg according to Pue. Blood was collected from the retroorbital venous plexus of SD rats 1min, 5min, 15min, 30 min and 1h, 2h, 3h, 4h, 6h, 8h, 12h post-orbital venous plexus of SD rats, and the blood was collected in a centrifuge tube containing heparin sodium in an amount of about 0.3mL. Blood samples were centrifuged at 4000 rpm at 4 ℃ for 10: 10 min, and plasma was collected and stored at-20 ℃ for later use. The content of Pue in the plasma was determined by HPLC and the pharmacokinetic data were counted using DAS3.3.0 pharmacokinetic data analysis software, all data being expressed as mean.+ -. SD. One-way ANOVA analysis of pharmacokinetic data using SPSS22.0 data analysis software, p <0.05 represents significant differences.
As shown in Table 1, the average residence time (MRT), AUC and half-life (t 1/2) of the mPEG-TK-GA micelle directly carrying puerarin or carrying puerarin nanocrystals in SD rats are all obviously higher than those of Pue injection group, the clearance rate is obviously lower than those of Pue injection group (Pue injection), and the drug delivery system of the mPEG-TK-GA micelle carrying puerarin or carrying puerarin nanocrystals can obviously prolong the systemic circulation time of puerarin, wherein AUC (0-t) is the area under a drug time curve within 12h, AUC (0-∞) is the area under a drug time curve after 12h, CL is the clearance rate, and C max is the maximum blood drug concentration or peak concentration.
Table 1 puerarin and its formulations pharmacokinetic parameters in SD rats (mean±sd, n=5)
Note that due to the table space limitation, mPEG-TK-GA is represented by the abbreviation mPTG.
Example 7
Uptake of Pue (NCs) @ mPEG-TK-GA polymer micelles by cells.
By examining the uptake and uptake mechanisms of different puerarin preparations in normal Raw264.7 cells and high ROS level Raw264.7 cells. The Raw264.7 cells are inoculated into a 24-well plate at the concentration of 8 multiplied by 10 4/well, a complete culture solution is added, the mixture is cultured for 24 hours in a cell culture box, part of normal Raw264.7 cells are replaced by fresh complete culture solution to serve as a control group, the rest normal Raw264.7 cells are discarded by old culture solution, the complete culture solution with the LPS concentration of 2 mug/mL is replaced to serve as an LPS group, and the control group and the LPS group are continuously cultured in the cell culture box for 24 hours at the culture temperature of 37 ℃.
The old culture solution is discarded from the control group and the LPS group, and the control group and the LPS group are equally divided into Pue injection groups, pue (NCs) groups, pue@mPTG groups, pue (NCs) @ mPTG groups, pue injection groups, pue (NCs) groups, pue@mPTG groups and Pue (NCs) @ mPTG groups which are respectively provided with four groups, wherein puerarin injection (Pue injection) with the concentration of 0.05mg/mL, 0.2mg/mL, 1mg/mL and 3mg/mL is added into each group of Pue injection groups; pue (NCs) groups each containing puerarin nanocrystalline (Pue (NCs)) at a concentration of 0.05mg/mL, 0.2mg/mL, 1mg/mL, 3mg/mL, pue@mPTG groups each containing puerarin-TK-GA micelles (Pue@mPTG) at a concentration of 0.05mg/mL, 0.2mg/mL, 3mg/mL, pue@mPTG) at a concentration of 0.05mg/mL, 0.2mg/mL, 1mg/mL, 3mg/mL, puerarin nanocrystalline mPEG-TK-GA (Pue@mPEG) at a concentration of 0.2mg/mL, 3mg/mL, 2h, blank DMEM for 2 washing, 200. Mu.L/well 0.1M in a PBS bottom wall, scraping the cells in a PBS wall, scraping the same vortex for 10 mu.L in a PBS wall, and collecting the supernatant after shaking for 10min, and measuring the same by HPLC. Protein pellet was resuspended in 100. Mu.L of 0.01M PBS and assayed for protein content in accordance with the BCA protein assay kit, wherein the control group had a Raw264.7 cell uptake level as shown in FIG. 10 (C) and the LPS group had a high ROS level, raw264.7 cell uptake level as shown in FIG. 10 (D).
Discarding old culture solution from control group and LPS group, and dividing control group and LPS group into Pue injection group, pue (NCs) group, pue@mPTG group, pue (NCs) @ mPTG group, pue injection group, pue (NCs) group, pue@mPTG group, pue (NCs) @ mPTG group each having seven small groups, pue injection group each having 1mg/mL puerarin injection (Pue injection), and incubating each small group under inhibition conditions of no inhibitor, colchicine, sodium azide, indomethacin, chlorpromazine, 4 ℃ for 2h, pue@mPTG group, pue (NCs) each having 1mg/mL puerarin Nanocrystalline Solution (NCs)), and incubating each small group under inhibition conditions of no inhibitor, colchicine, sodium azide, indomethacin, chlorpromazine, 4 ℃ under inhibition conditions of quercetin, GA-1 mg/mL of Pue, pue-5 mg/mL of PEG-15, and incubating each small group under inhibition conditions of no inhibitor, pue-4 ℃ under inhibition conditions of PEG-4 ℃ respectively, and incubating each small group under inhibition conditions of no inhibition of Pue (NCs), GA-4 ℃ under inhibition conditions of four PEG-g; discarding the culture solution containing the medicine, cleaning with DMEM for 2 times, adding 200 μl/well of 0.1M PBS, scraping off the cells adhered to the bottom, collecting in EP tube, adding 200 μl/hole methanol, scraping and washing bottom adherent cells, collecting in the same EP tube, swirling for 3min, mixing, centrifuging at 10000rpm for 10min, and determining puerarin content by supernatant HPLC. Protein pellet was resuspended in 100. Mu.L of 0.01M PBS and the protein content of the sample was determined in accordance with the BCA protein assay kit, wherein the relative cellular uptake of normal Raw264.7 cells of the control group is shown in FIG. 9 (A) and the relative cellular uptake of high ROS-level Raw264.7 cells of the LPS group is shown in FIG. 9 (B).
Referring to FIGS. 9 and 10, the results show that puerarin nanocrystals have higher cellular uptake efficiency than puerarin and that inflammatory macrophages after LPS induction uptake more than normal macrophages. The reason is that inflammatory macrophages after LPS induction take more drugs in the clathrin-mediated endocytosis pathway, and the nano-crystalline phase is easier to take through the pathway than the bulk drug.
In conclusion, based on the results of cell and animal experiments, the average residence time (MRT), AUC and half-life (t 1/2) of the polymer micelle carrying puerarin or puerarin nanocrystals in SD rats are obviously higher than those of puerarin injection groups, the clearance is obviously lower than those of the puerarin injection groups, the prepared micelle can prolong the in vivo residence time of puerarin, the targeting research shows that the distribution amount of the polymer micelle at the atherosclerosis plaque position is about 4.7 times that of normal animals, the prepared micelle has better lesion position targeting, and the cell uptake test shows that the puerarin or puerarin nanocrystals are mainly absorbed by macrophages at the atherosclerosis plaque position in an endocytosis mode, wherein the uptake amount of the puerarin nanocrystals by the cells is obviously higher than that of puerarin.
Comparative example 1
Loading puerarin nanocrystals prepared by PVP-K30, SDS, HPMC, P and chitosan serving as stabilizers into mPEG-TK-GA micelle, and then examining the characteristics of puerarin-loaded nanocrystal polymer micelle preparation and in-vivo pharmacokinetic behaviors.
Respectively weighing PVP-K30 mg, SDS 800mg, HPMC 400mg, P188 800mg and chitosan 400mg, dissolving in 40mL pure water, stirring at room temperature until completely dissolving, respectively weighing 5 parts of 400mg puerarin crude drugs, adding into the stabilizer solution, shearing by a high-speed shearing machine to obtain crude suspension, standing until bubble ablation, homogenizing the crude suspension in a high-pressure homogenizer to obtain puerarin nanocrystalline liquid, respectively marking the puerarin crude drugs as prescriptions F1-F5, weighing 400mg puerarin crude drugs, directly dispersing with 40mL pure water, preparing puerarin nanocrystalline liquid by high-speed shearing and high-pressure homogenization, marking as prescriptions F6, respectively weighing 6 parts of 300mg carrier material mPEG-TK-GA in a pear-shaped bottle, dissolving by adding methanol, and rotationally evaporating under reduced pressure to remove methanol to obtain transparent films. 6mL of the puerarin nanocrystalline liquid in the prescription F1-F6 is taken respectively, the film is dissolved by ultrasonic, the air bath constant temperature oscillator is hydrated for 6h at 25 ℃, and the puerarin nanocrystalline polymer micelle solution carried with F1-F6 is obtained by filtering with a 0.45 mu m filter membrane. 60mg of puerarin crude drug is weighed, 300mg of carrier material mPEG-TK-GA is added into the puerarin crude drug to be dissolved, methanol is removed by rotary evaporation under reduced pressure to obtain a transparent film, 6mL of pure water is added into the transparent film to be ultrasonically dissolved, a 25 ℃ air bath constant temperature oscillator is used for hydration for 6 hours, a 0.45 mu m filter membrane is used for filtering to obtain Pue@mPEG-TK-GA micelle solution, and the solution is marked as a prescription F7. F1-F7 sample solutions were prepared in parallel in 3 portions for detection analysis.
Referring to table 2, the effect of different stabilizers on drug loading, encapsulation efficiency and particle size of puerarin-loaded nanocrystalline mPEG-TK-GA was different. F1-F4 are clear liquid, F5 is gel state, and F6 is slightly turbid. Wherein the prescription F5 puerarin-chitosan nano-crystalline micelle is placed at room temperature for 24h for solution layering, and is not suitable for continuous investigation due to the large particle size (1000 nm). The encapsulation rate of puerarin nanocrystalline micelle without the stabilizer in the prescription F6 is lower than that of other prescriptions, and sediment is generated at the bottom of the puerarin nanocrystalline micelle after the puerarin nanocrystalline micelle is placed for 24 hours at room temperature, so that the puerarin nanocrystalline micelle is not suitable for continuous investigation. In terms of drug loading, the drug loading rate (DL) of puerarin molecules directly loaded in the prescription F7 is the highest, 18.0%, but is 83.6% higher than that of other prescriptions. The encapsulation efficiency of the prescription F2 Pue (SDS) NCs@mPEG-TK-GA micelle is highest and is 98.9%, and is 12.3% lower than that of other prescriptions, so that the prescriptions F1, F2, F3, F4 and F7 are subjected to in vitro release and in vivo pharmacokinetic behaviors of SD rats to further preferably prepare a stabilizer for nano crystallization by comprehensively examining the encapsulation efficiency, the drug loading and the particle size distribution, wherein Ave is the average particle size, and PDI is a polydisperse coefficient.
TABLE 2 influence of different stabilizers on puerarin-loaded nanocrystalline mPEG-TK-GA micelles (mean+ -SD, n=3)
Table 3 pharmacokinetic parameters of puerarin and its formulations in SD rats (mean±sd, n=5)
The statistics of the pharmacokinetic parameters of puerarin and the puerarin preparations in SD rats are shown in Table 3, wherein the prescription F8 is puerarin nano-crystalline liquid taking PVP-K30 as a stabilizer, the prescription F9 is puerarin injection (Pue injection), the drug time curves are shown in fig. 11 and 12, the average residence time (MRT), AUC and half-life (t 1/2) of the mPEG-TK-GA micelle directly coated with puerarin or coated with puerarin nano-crystalline in SD rats are obviously higher than those of Pue injection groups, the clearance is obviously lower than those of Pue injection groups, and the puerarin-carrying mPEG-TK-GA drug delivery system can obviously prolong the systemic circulation time. F8 The Pue (PVP) NCs group has no obvious difference in various pharmacokinetic parameters compared with the F9 Pue injection group, which indicates that the Pue (PVP) NCs group can not improve the pharmacokinetic behavior of puerarin obviously, the F2 Pue (SDS) @ mPEG-TK-GA group is found to have the phenomena of haematuria and lassitude of rats, the F3 Pue (HPMC) @ mPEG-TK-GA group is not suitable for intravenous injection, and the F4 Pue (P188) @ mPEG-TK-GA group has no obvious difference in the pharmacokinetic behavior of puerarin, and the drug loading rates (15.6% ) and encapsulation rates (87.1% and 89.7%) of the two groups of preparations are also not obviously different. F1 Pue (PVP) @ mPEG-TK-GA group and F9 Pue injection group, the AUC (0- ≡) of puerarin is improved by 2.18 times, MRT (0- ≡) is increased by 2.18 times, t 1/2 is increased by 3.5 times, the CL value is reduced by 2.14 times, and the degree of improvement of the pharmacokinetic behavior of puerarin is greatest compared with that of the F2, F3, F4 and F7 groups, so that the stabilizer is PVP-K30.
Comparative example 2
Respectively adopt the components comprisingPolymer mPEG-TK-GA and no contentThe polymer mPEG-PLA of (C) is used for preparing drug-containing polymer micelle and researching the appearance and the pharmacokinetic behavior of the micelle to evaluate the existence of drug-containing polymer micelleThe pharmacokinetic effects on the polymer micelles are shown in FIG. 13 and Table 4, and it is shown that the polymer micelles containThe pharmacokinetic property of the drug-containing polymer micelle prepared by the polymer mPEG-TK-GA is better than that of the drug-containing polymer micelle without the polymer mPEG-TK-GAWherein (a) in FIG. 13 is an SEM image of Pue raw materials, (b) in FIG. 13 is an SEM image of Pue nanometer crystals, (c) in FIG. 13 is an SEM image of Pue@mPEG-PLA micelles, (d) in FIG. 13 is an SEM image of Pue (Nc) @mPEG-PLA micelles, (e) in FIG. 13 is an SEM image of Pue@mPEG-TK-GA micelles, and (f) in FIG. 13 is an SEM image of Pue (Nc) @mPEG-TK-GA micelles.
Table 4 Pue in vivo pharmacokinetic parameters of SD rats injected via tail vein (mean+ -SD, n=5) for each formulation
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (10)

1. The preparation method of the puerarin-loaded nano-crystalline polymer micelle is characterized by comprising the following steps of:
PVP-K30 is stirred and dissolved by pure water in a beaker, puerarin raw material medicine is added into the beaker, and shearing is carried out in a high-speed shearing machine, so as to obtain crude suspension;
homogenizing the crude suspension in a high-pressure homogenizer, and cooling to room temperature to obtain transparent puerarin nanocrystalline liquid;
Carboxylation reaction is carried out on mPEG2000 to obtain mPEG-COOH, then activation reaction is carried out on the mPEG-COOH to obtain mPEG-COOH activation liquid, and the mPEG-COOH activation liquid is reacted with NH 2-TK-NH2 to synthesize mPEG-TK-NH 2;
For a pair of Performing an activation reaction to obtainAn activating solution for activating the saidThe activating solution reacts with the mPEG-TK-NH 2 to obtain a carrier material mPEG-TK-GA;
Dissolving the carrier material mPEG-TK-GA in methanol, removing the methanol by reduced pressure rotary evaporation to obtain a transparent film, adding the puerarin nanocrystalline liquid into the transparent film, ultrasonically dissolving the transparent film, oscillating and hydrating by a GAs bath constant temperature oscillator, and finally filtering by a filter membrane to obtain the puerarin-loaded nanocrystalline polymer micelle.
2. The method for preparing puerarin-loaded nanocrystalline polymer micelle according to claim 1, wherein the step of carboxylating mPEG2000 to obtain mPEG-COOH specifically comprises:
Adding mPEG2000 into acetone, heating in a water bath, stirring until mPEG2000 is completely dissolved, adding a Jones reagent for reaction until the reaction liquid is blue and has deep blue precipitate at the bottom, adding isopropanol for quenching, performing suction filtration to obtain light blue-green filtrate, adding pure water into the light blue-green filtrate, and then removing acetone by rotary evaporation to obtain acetone-free filtrate;
adding sodium bicarbonate into the acetone-free filtrate, stirring while adding until no bubbles are generated, standing, and performing rotary evaporation on the supernatant to remove water to obtain a bluish thick paste;
Adding chloroform into the sea blue thick paste for solid-liquid extraction, wherein the lower layer is a milky turbid liquid, the upper layer is a blue floccule, collecting the milky turbid liquid by adopting a separating funnel, extracting for a plurality of times by using chloroform, and then carrying out suction filtration and rotary evaporation to remove the chloroform to obtain mPEG-COONa;
Dissolving mPEG-COONa in pure water, adding hydrochloric acid solution to adjust pH to a preset value, removing water and hydrogen chloride by rotary evaporation to obtain a viscous semitransparent concentrated solution, adding chloroform for extraction, filtering, removing chloroform by rotary evaporation, and cooling to obtain white mPEG-COOH solid.
3. The method for preparing puerarin-loaded nanocrystalline polymer micelle according to claim 1, wherein the step of performing an activation reaction on mPEG-COOH to obtain mPEG-COOH activation solution specifically comprises:
Dissolving mPEG-COOH, dicyclohexylcarbodiimide and N-hydroxysuccinimide in dichloromethane, and stirring and activating at room temperature to obtain milky mPEG-COOH activating solution.
4. The method for preparing puerarin-loaded nanocrystalline polymer micelles of claim 1, wherein the preparation steps of NH 2-TK-NH2 specifically comprise:
Adding cysteine hydrochloride and acetone into a reaction bottle, adding chloroform into the reaction bottle, and introducing dry hydrogen chloride gas for reaction to obtain thick paste-like reaction liquid;
Filtering the thick paste reaction liquid, adopting chloroform to clean and precipitate, then pumping to dry, placing filter residues into an equal volume of methanol/diethyl ether mixed solution for recrystallization, filtering, and drying the filter residues in vacuum to obtain a vacuum dried product;
And (3) dissolving the vacuum dried product in NaOH solution for reaction to obtain clear liquid, and adding dichloromethane for extraction to obtain NH 2-TK-NH2.
5. The method for preparing puerarin-loaded nanocrystalline polymer micelle according to claim 1, wherein the step of reacting the mPEG-COOH activating solution with NH 2-TK-NH2 to synthesize mPEG-TK-NH 2 specifically comprises:
Dropwise adding the mPEG-COOH activating solution into a dichloromethane solution dissolved with NH 2-TK-NH2, reacting under the protection of nitrogen, filtering, and performing rotary evaporation on the obtained filtrate to remove dichloromethane to obtain a concentrated solution;
Precipitating the concentrated solution in diethyl ether/methanol mixed solution with volume ratio of 10:1 at low temperature, collecting filter residues, and finally vacuum drying to obtain mPEG-TK-NH 2.
6. The method for preparing puerarin-loaded nanocrystalline polymer micelle according to claim 1, wherein the method comprises the steps ofThe preparation method comprises the following steps:
Slowly adding sodium hydroxide into pure water, stirring while adding 18 beta-H-diammonium glycyrrhizinate into the solution, heating and refluxing for reaction, slowly dripping H 2SO4 solution under ice bath condition, regulating pH value to a preset value, stirring while adding white solid, and stirring under ice bath condition to obtain white thick paste reaction solution;
Under ice bath condition, dissolving the white thick paste reaction solution in H 2SO4 solution, stirring, filtering, collecting filter residue, and drying to obtain white powder 。
7. The method for preparing puerarin-loaded nanocrystalline polymer micelle according to claim 1, wherein the pair of puerarin-loaded nanocrystalline polymer micellesPerforming an activation reaction to obtainThe step of activating the liquid specifically comprises:
Will be Dissolving in N, N-dimethylformamide until the solution is clear, slowly adding dichloromethane until the solution is clear, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide for activation reaction to obtainActivating liquid.
8. The method for preparing puerarin-loaded nanocrystalline polymer micelle according to claim 1, wherein the step of subjecting the puerarin-loaded nanocrystalline polymer micelleThe step of reacting the activating solution with the mPEG-TK-NH 2 to obtain a carrier material mPEG-TK-GA specifically comprises the following steps:
The said Dropwise adding the activation solution into dichloromethane solution dissolved with mPEG-TK-NH 2, reacting under the protection of nitrogen, performing suction filtration, and removing dichloromethane from the obtained filtrate by rotary evaporation to obtain concentrated solution;
Precipitating the concentrated solution in diethyl ether at 0 ℃ while dropwise adding and stirring to obtain white precipitate, filtering, and vacuum drying the collected filter residue to obtain carrier material mPEG-TK-GA.
9. The method for preparing puerarin-loaded nanocrystalline polymer micelles of claim 1, wherein the puerarin-loaded nanocrystalline polymer micelles have a regular round shape and a particle size of 132.6nm ±2.55nm.
10. Use of a puerarin-loaded nanocrystalline polymer micelle prepared by the preparation method of the puerarin-loaded nanocrystalline polymer micelle according to any one of claims 1 to 9 in the preparation of an anti-atherosclerosis drug.
CN202411311645.7A 2024-09-20 2024-09-20 Preparation method and application of puerarin-loaded nanocrystalline polymer micelles Active CN118831052B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202411311645.7A CN118831052B (en) 2024-09-20 2024-09-20 Preparation method and application of puerarin-loaded nanocrystalline polymer micelles

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202411311645.7A CN118831052B (en) 2024-09-20 2024-09-20 Preparation method and application of puerarin-loaded nanocrystalline polymer micelles

Publications (2)

Publication Number Publication Date
CN118831052A CN118831052A (en) 2024-10-25
CN118831052B true CN118831052B (en) 2024-11-29

Family

ID=93148494

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202411311645.7A Active CN118831052B (en) 2024-09-20 2024-09-20 Preparation method and application of puerarin-loaded nanocrystalline polymer micelles

Country Status (1)

Country Link
CN (1) CN118831052B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108567741A (en) * 2017-03-07 2018-09-25 武汉圣朗药物技术有限公司 Puerarin Liposomal formulation and preparation method thereof for treating atherosclerosis
CN108969484A (en) * 2018-09-18 2018-12-11 烟台大学 A kind of preparation method targeting atherosclerotic plaque nano material

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2016309847B2 (en) * 2015-08-19 2019-10-31 Xiamen Ginposome Pharmaceutical Co., Ltd. Liposomes with ginsenoside as membrane material and preparations and use thereof
WO2021142336A1 (en) * 2020-01-08 2021-07-15 Puretech Lyt, Inc. Vesicle compositions for oral delivery
CN112107689A (en) * 2020-10-21 2020-12-22 江西中医药大学 Preparation method and application of small-particle-size polyhydroxy traditional Chinese medicine active ingredient nanocrystal

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108567741A (en) * 2017-03-07 2018-09-25 武汉圣朗药物技术有限公司 Puerarin Liposomal formulation and preparation method thereof for treating atherosclerosis
CN108969484A (en) * 2018-09-18 2018-12-11 烟台大学 A kind of preparation method targeting atherosclerotic plaque nano material

Also Published As

Publication number Publication date
CN118831052A (en) 2024-10-25

Similar Documents

Publication Publication Date Title
US10960078B2 (en) Amphiphilic drug-drug conjugates for cancer therapy, compositions and methods of preparation and uses thereof
CN107669632A (en) A kind of pharmaceutical carrier, micella, antitumor and antitumor cell diversion medicaments preparation, and its production and use
CN110408047B (en) Nano coordination polymer and its preparation method and application
WO2022199408A1 (en) Preparation method for and application of novel injection abiraterone derivative
JP2019507190A (en) Specifically targeted biodegradable amphiphilic polymers for ovarian cancer, polymer vesicles produced therefrom and uses thereof
WO2014079377A1 (en) Antitumor prodrugs with function of p-glycoprotein inhibition
Yi et al. Synthesis, characterization, and formulation of poly-puerarin as a biodegradable and biosafe drug delivery platform for anti-cancer therapy
Jie et al. Superparamagnetic iron oxide nanoparticles/doxorubicin-loaded starch-octanoic micelles for targeted tumor therapy
KR20230044423A (en) Lapatinib Particles and Uses Thereof
CN104116711A (en) pH-sensitive polymeric micelle composition resisting tumor drug resistance
CN109432049B (en) Rhein lipid vesicle nanoparticles with kidney targeting distribution characteristic and application thereof
CN118831052B (en) Preparation method and application of puerarin-loaded nanocrystalline polymer micelles
CN107441043B (en) pH-sensitive mixed micelle and preparation method and application thereof
KR20120126356A (en) Nanoparticles comprising amphiphilic low molecular weight hyaluronic acid complex and a process for the preparation thereof
CN104045823A (en) Glycyrrhetinic acid derivative, and preparation method and application thereof
Guan et al. Design, synthesis, and characterization of glycyrrhetinic acid-mediated multifunctional liver-targeting polymeric carrier materials
CN110772644A (en) Polyethylene glycol modified cardiac glycoside compound prodrug and its antitumor use
CN109464674A (en) Carboxylated polyethylene glycol monomethyl ether-icariin nanoparticles, preparation method and application
CN109172524A (en) A kind of Puerarin micella and preparation method thereof, puerarin preparation
CN105030730A (en) Multi-targeting antitumor composite preparation and preparation method thereof
CN1857734B (en) Phytoagglutinin modified lactic acid-hydroxacetic acid copolymer and its preparing method
CN110615891B (en) A class of solanesyl thiosalicylic acid compounds, preparation method and application thereof
CN108578363B (en) Cantharidin-loaded polymer micelles and their preparation and application
Wang et al. iRGD-mediated reduction-responsive DSPE–PEG/LA–PLGA–TPGS mixed micelles used in the targeted delivery and triggered release of docetaxel in cancer
CN112121010A (en) A reduction-responsive vitamin E polyethylene glycol succinate micelle containing folic acid molecule and preparation method and application thereof

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
GR01 Patent grant
GR01 Patent grant