CN110664734B - Preparation method of microgel based on shear force sensitivity and CD44 receptor targeting - Google Patents
Preparation method of microgel based on shear force sensitivity and CD44 receptor targeting Download PDFInfo
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- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
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- 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
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- 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
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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Abstract
The invention discloses a preparation method of microgel based on shear force sensitivity and CD44 receptor targeting, belonging to the technical field of nano materials. The method comprises the following steps: the method comprises the following steps of synthesizing an amphiphilic polymer PAA-b-PEHA, synthesizing an amphiphilic graft copolymer P (AA-g-HEMA) -b-PEHA, synthesizing a hyaluronic acid hydrogel precursor HAGMA, synthesizing a shear response and CD44 receptor targeted dual-response microgel HACBC. The nanometer microgel prepared by the invention combines mechanical force sensitivity and CD44 receptor targeting, can be more accurately targeted to an affected part and release therapeutic drugs at the affected part, and the used raw materials are degradable materials with good biocompatibility and low toxic and side effects, and have extremely low side effects on a human body.
Description
Technical Field
The invention belongs to the technical field of nano materials, and particularly relates to a double-response intelligent microgel nano drug-loading system capable of performing shear force response and CD44 receptor targeting at a thrombus position in a blood vessel.
Background
Atherosclerosis is a chronic inflammatory disease, and the incidence of atherosclerosis is increasing year by year due to the ever-increasing living standard of people. The biggest threat to atherosclerosis is vascular rupture due to thrombosis and unstable plaque, leading to cardiovascular and cerebrovascular complications such as Acute Coronary Syndrome (ACS) and stroke. Over the past decade, the main drugs used to treat atherosclerosis are aspirin, statins and other potent antiplatelet drugs, as well as various drugs that lower the level of Low Density Lipoprotein (LDL). In the further research on the pathological mechanism of atherosclerosis, the pathological part is enriched with inflammatory macrophages caused by a large amount of Reactive Oxygen Species (ROS). Thus, biochemical targeted delivery of anti-inflammatory and antioxidant drugs has become the current mainstay of inhibition of Reactive Oxygen Species (ROS) and inflammatory macrophages. The specific symptoms of vascular stenosis caused by thrombus are not sufficiently valued by scientists.
Mechanical stress sensitive materials are continuously developed and innovated as one of the research hotspots. However, in the field of biomedical carriers, there are few studies and applications related to mechanical stress sensitive materials. There are few mechanically sensitive hydrogels that give solutions to vascular stenosis. Therefore, the design of the drug carrier which can release the drug at the thrombus through the shear force response and can accurately target the thrombus has important significance.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: overcomes the defects and problems in the background art, and provides a preparation method of double-response intelligent microgel which is applicable to atherosclerosis, sensitive to shearing force and targeted by a CD44 receptor.
The technical scheme of the invention is as follows:
a preparation method of microgel based on shear force sensitivity and CD44 receptor targeting comprises the following steps:
1) synthesis of amphiphilic Polymer PAA-b-PEHA:
under the anhydrous and oxygen-free conditions, CuCl (cuprous chloride) and 2, 2-bipyridine are added into a reaction bottle to be complexed to be brick red, then tBA (tert-butyl acrylate), 2-EHA (isooctyl acrylate) and DMF (N, N-dimethylformamide) are respectively added, the mixture is uniformly mixed with CuCl:2, 2-bipyridine: tBA:2-EHA: DMF 1: 2-5: 35-40: 60-80, the mixture is heated to 80 ℃ to react for 12 hours, the reaction is stopped by ice water bath, and then trichloromethane (CHCl) is used3) Filtering with neutral alumina, rotary steaming, and precipitating the concentrated solution in methanol to obtain PtBA-b-PEHA; completely dissolving PtBA-b-PEHA in CHCl3Adding TFA (trifluoroacetic acid) into a solution with the concentration of 50-100 mg/mL, reacting at normal temperature for 48h according to the mol ratio of TFA to PtBA-b-PEHA of 1: 15-20; adding CHCl into the reaction bottle after the reaction is finished3Performing rotary evaporation, and precipitating the obtained concentrated solution in excessive methanol to obtain PAA-b-PEHA;
2) synthesis of amphiphilic graft copolymer P (AA-g-HEMA) -b-PEHA:
completely dissolving the PAA-b-PEHA prepared in the step 1) in THF (tetrahydrofuran) to obtain a solution with the concentration of 10-25 mg/ml, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) respectively according to the molar ratio, continuously activating for 60min after feeding, finally adding hydroxyethyl methacrylate (HEMA) into the solution according to the molar ratio, wherein PAA-b-PEHA and HEMA are respectively 1: 70-100 in N2Reacting for 24 hours in a dark place in an atmosphere; after the reaction is finished, the mixture is concentrated by rotary evaporation and precipitated in excessive methanol to obtain P (AA-g-HEMA) -b-PEHA.
3) Synthesis of hyaluronic acid hydrogel precursor HAGMA:
completely dissolving sodium Hyaluronate (HA) in deionized water to obtain a solution with the concentration of 5-10 mg/ml, respectively adding 4-Dimethylaminopyridine (DMAP) and tetrabutylammonium bromide (TBAB) into the hyaluronic acid solution, and stirring at room temperature for 1h according to the mol ratio of HA to DMAP to TBAB being 1: 4-7: 6-8.5; adding Glycidyl Methacrylate (GMA) into the mixed solution according to a molar ratio of HA to GMA of 1: 15-25, and adding N2Reacting for 48 hours at room temperature under the protection condition; after the reaction is finished, adding NaCl into the reaction solution until the concentration reaches 5%, and then adding methanol and water in a volume ratio of 3:1 into the reaction solution to obtain a mixed precipitation solution; centrifugally separating white precipitate, dissolving the white precipitate in deionized water, and dialyzing with 1mol/L sodium chloride water solution and deionized water for 24 hr to obtain HAGMA;
4) synthetic shear response and CD44 receptor targeted dual response microgel HACBC:
taking the products P (AA-g-HEMA) -b-PEHA and simvastatin (simvastatin) prepared in the step 2) to be respectively dissolved in THF and DMF, wherein the concentrations are respectively 2-5 mg/ml and 10-15 mg/ml, uniformly mixing according to the volume ratio of 15-25: 1, dripping the mixed solution into deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30min after finishing, and then dialyzing the obtained solution for 24h to remove tetrahydrofuran; after dialysis is completed, a drug-loaded micelle is obtained;
mixing the obtained drug-loaded micelle solution with HAGMA prepared in the step 3) according to the mass ratio of 1: 0.75-1.5, wherein the final concentration of HAGMA is 0.5-2 mg/ml, adding an N-vinyl pyrrolidone (NVP) solution containing 2, 2-dimethoxy-2-phenyl acetophenone (DMPA) into the mixed solution, and irradiating with ultraviolet light to obtain the microgel with shear force response and CD44 receptor targeting according to the molar ratio of HAGMA to DMPA to NVP of 1: 3-5: 4.5-7.
In the dialysis described in step 3) and step 4), the cut-off molecular weight of the dialysis bag used is preferably 10000.
Irradiating the ultraviolet light in the step 4), preferably irradiating the ultraviolet light with 355nm ultraviolet light for 5 min.
Has the advantages that:
1. the nano microgel prepared by the invention is a mechanical force sensitive drug carrier, and can release therapeutic drugs in a targeted manner at a thrombus part.
2. The nano microgel prepared by the invention is a CD44 receptor targeted drug carrier, and can be used for targeting to an inflammation part where thrombus is located by biological recognition of a CD44 receptor on the surface of macrophage.
3. The nano microgel prepared by the invention combines mechanical force sensitivity and CD44 receptor targeting, can be more accurately targeted to an affected part and releases therapeutic drugs on the affected part.
4. The material used by the invention is HAGMA or P (AA-g-HEMA) -b-PEHA which is degradable material with good biocompatibility and low toxic and side effects, and has extremely low side effect on human body.
5. The nano microgel prepared by the invention can efficiently and stably release medicines in a long-term circulation process, and provides great convenience for diagnosis and treatment of atherosclerosis.
Drawings
FIG. 1 is a nuclear magnetic map of PAA-b-PEHA prepared in example 1.
FIG. 2 is a nuclear magnetic map of P (AA-g-HEMA) -b-PEHA prepared in example 2.
FIG. 3 is a nuclear magnetic map of hyaluronic acid hydrogel precursor (HAGMA) prepared in example 3.
FIG. 4 is a TEM image of the morphology of the dual response intelligent microgel (HACBC) prepared in example 4 before and after shearing.
FIG. 5 is a graph showing drug release rate profiles of dual response smart microgel (HACBC) prepared in example 4 in vessels of different degrees of occlusion.
FIG. 6 is an in vitro cytographic image of the dual response intelligent microgel (HACBC) prepared in example 4.
Detailed Description
The present invention is described in detail by the following examples, but does not limit the scope of the invention as claimed.
Example 1: synthesis of amphiphilic Polymer PAA-b-PEHA
Under the anhydrous and oxygen-free conditions, 20mg of CuCl and 36mg of 2, 2-bipyridine are added into a reaction bottle to be complexed to brick red, 3mltBA, 2ml of 2-EHA and 2mlDMF are respectively added to be uniformly mixed, the mixture is heated to 80 ℃ to react for 12 hours, and the reaction is stopped by ice-water bath. 50ml of CHCl was added3Filtering with neutral alumina, rotary evaporating to concentrate to 4ml, precipitating in 1000ml methanol to obtain PtBA-b-PEHA; completely dissolving PtBA-b-PEHA in 10ml CHCl3Adding 1.5ml of TFA into the polymer solution, and reacting for 48 hours at normal temperature; after the reaction was completed, 100ml of CHCl was added to the reaction flask3Rotary evaporating to 5ml, repeating the above operation three times; precipitating the concentrated solution in 1000ml of methanol to obtain PAA-b-PEHA; the assignment of the nuclear magnetic spectrum H is clear from FIG. 1.
Example 2: synthesis of amphiphilic graft copolymer P (AA-g-HEMA) -b-PEHA
Dissolving 0.25g of PAA-b-PEHA in 50ml of THF, adding 80mgEDC and 155mgNHS respectively, and continuously activating for 60 min; finally, 3ml of HEMA was added to the reaction mixture in N2Reacting for 24 hours in a dark place in an atmosphere; inverse directionAfter the reaction is finished, performing rotary evaporation and concentration to 3-5 ml, and precipitating in 1000ml of methanol to obtain (PAA-g-HEMA) -b-PEHA; the assignment of the nuclear magnetic spectrum H is clear from FIG. 2.
Example 3: synthesis of hyaluronic acid hydrogel precursors (HAGMA)
Dissolving 0.5g of sodium hyaluronate in 50-100 ml of deionized water, mixing 0.977g of DMAP and 1.093g of TBAB, adding the mixture into a hyaluronic acid solution, and stirring at room temperature for 1 hour; adding 3-5 ml of GMA into the mixed solution, and reacting for 48 hours at room temperature under the protection of N2; after the reaction, NaCl was added to the reaction solution until the concentration reached 5%, and methanol was added thereto (methanol: water: 3:1, volume ratio); separating white precipitate with high speed centrifuge (rotation speed of 7000r/min), and repeating the above operation three times; dissolving the obtained white precipitate in 300ml deionized water, dialyzing with 1mol/L NaCl water solution and deionized water respectively for 24h, and obtaining HAGMA with cut-off molecular weight of 10000 in dialysis bag; the product was lyophilized and it can be seen from figure 3 that the nmr spectrum H was correctly assigned and GMA was successfully bound to hyaluronic acid.
Example 4: shear-responsive and CD44 receptor-targeted dual-responsive microgel (HACBC) synthesis
Taking 20mg of the final product P (AA-g-HEMA) -b-PEHA and 10mg of simvastatin in example 3, respectively dissolving in 5ml of THF and 1ml of DMF, mixing the two solutions to form a uniform solution, slowly dripping the mixed solution into deionized water at the speed of 0.5/min under the ultrasonic condition, continuing ultrasonic treatment for 30min after dripping is finished, and then putting the solution into a dialysis bag with the Da 10000 for dialysis with the deionized water for 24h to remove the THF and the DMF. And after the dialysis is finished, obtaining the drug-loaded micelle.
And (3) mixing 9ml of the drug-loaded micelle solution with 1ml of HAGMA aqueous solution (the concentration is 10mg/ml), adding 10 microliters of NVP photoinitiator (the molar ratio is 1:1.5) containing DMPA into the mixed solution, and irradiating for 5 minutes by 355nm ultraviolet light to obtain the double-response intelligent microgel with shear force response and CD44 receptor targeting.
Example 5: morphology and size, drug release rate and cell targeting test of HACBC microgel
Respectively dripping HACBC microgel which is not stimulated by mechanical stress and HACBC microgel which circulates for 36 hours in a narrow blood vessel simulator onto different carbon supporting membranes, wherein the dripping speed is 0.2m/h, and dripping samples for three times; drying the carbon-supported membrane at room temperature for 12h, and observing the morphology and the size of the micelle by using a Transmission Electron Microscope (TEM); fig. 4a and 4b are transmission electron microscope images of HACBC microgel before and after mechanical stress stimulation, and it can be clearly found that the microgel is not uniform in size, is broken and recombined, and is partially strained after mechanical stress stimulation.
Adding the HACBC microgel into a narrow blood vessel simulator, sampling once every hour for the first 4h, sampling once every two hours for 4-12 h, and sampling once every 12h after 12 h; collecting 2ml of sample each time, dialyzing in dialysis bag (MWCO:10000) for 6h, detecting the characteristic absorption peak intensity of the drug in the dialysate by using ultraviolet visible spectrometer, and calculating the drug release rate; FIG. 5 shows the drug release of HACBC microgel in simulated blood vessels with different degrees of obstruction, and it can be found that the drug release rate is obviously improved along with the increase of the degree of obstruction.
Mixing HACBC microgel wrapped with Nile red dye with culture medium (HACBC: culture medium: 2:8, volume ratio) and adding into culture dish of macrophage and endothelial cell activated by Lipopolysaccharide (LPS), and incubating for 10 h; washing the incubated cells with PBS buffer solution, fixing the cells with 4% paraformaldehyde solution for 15min, and staining the cell nucleus with 1 × DAPI working solution for 15 min; taking a picture of the cell sample by using a laser inverted confocal microscope at room temperature; it can be seen from FIG. 6 that HACBC microgel has significant targeting to inflammatory macrophages.
Claims (3)
1. A preparation method of microgel based on shear force sensitivity and CD44 receptor targeting comprises the following steps:
1) synthesis of amphiphilic Polymer PAA-b-PEHA:
under the anhydrous and oxygen-free conditions, CuCl and 2, 2-bipyridyl are added into a reaction bottle to be complexed to brick red, and then tBA and 2 are respectively addedEHA and DMF, according to the molar ratio of CuCl to 2, 2-bipyridine to tBA to 2-EHA to DMF to 1: 2-5: 35-40: 60-80), uniformly mixing, heating to 80 ℃, reacting for 12 hours, stopping the reaction in an ice-water bath, filtering and rotary evaporating by using chloroform and neutral alumina, precipitating the concentrated solution in methanol, and recording the obtained precipitate as PtBA-b-PEHA; completely dissolving PtBA-b-PEHA in CHCl3Adding TFA into a solution with the concentration of 50-100 mg/mL, reacting at normal temperature for 48h according to the mol ratio of TFA to PtBA-b-PEHA of 1: 15-20; adding CHCl into the reaction bottle after the reaction is finished3Performing rotary evaporation, and precipitating the obtained concentrated solution in excessive methanol to obtain PAA-b-PEHA;
2) synthesis of amphiphilic graft copolymer P (AA-g-HEMA) -b-PEHA:
completely dissolving the PAA-b-PEHA prepared in the step 1) in tetrahydrofuran to obtain a solution with the concentration of 10-25 mg/ml, then respectively adding EDC and NHS according to the molar ratio, wherein the ratio of PAA-b-PEHA to EDC to NHS is 1: 25-40: 70-85, continuously activating for 60min after feeding, finally adding HEMA into the solution according to the molar ratio of PAA-b-PEHA to HEMA is 1: 70-100, and adding N2Reacting for 24 hours in a dark place in an atmosphere; after the reaction is finished, performing rotary evaporation and concentration, and precipitating in excessive methanol to obtain P (AA-g-HEMA) -b-PEHA;
3) synthesis of hyaluronic acid hydrogel precursor HAGMA:
completely dissolving hyaluronic acid HA in deionized water to obtain a solution with the concentration of 5-10 mg/ml, respectively adding DMAP and TBAB into the hyaluronic acid solution, stirring at room temperature for 1h, wherein the molar ratio of HA to DMAP to TBAB is 1: 4-7: 6-8.5; adding GMA into the mixed solution according to the molar ratio of HA to GMA being 1: 15-25, and adding N2Reacting for 48 hours at room temperature under the protection condition; after the reaction is finished, adding NaCl into the reaction solution until the concentration reaches 5%, and then adding methanol and water in a volume ratio of 3:1 into the reaction solution to obtain a mixed precipitation solution; centrifugally separating white precipitate, dissolving the white precipitate in deionized water, and dialyzing with 1mol/L sodium chloride water solution and deionized water for 24 hr to obtain HAGMA;
4) synthetic shear response and CD44 receptor targeted dual response microgel HACBC:
taking the products P (AA-g-HEMA) -b-PEHA and simvastatin prepared in the step 2) to be respectively dissolved in THF and DMF, wherein the concentrations are respectively 2-5 mg/ml and 10-15 mg/ml, uniformly mixing according to the volume ratio of 15-25: 1, then dripping the mixed solution into deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30min after finishing, and then dialyzing the obtained solution for 24h to remove tetrahydrofuran; after dialysis is completed, a drug-loaded micelle is obtained;
mixing the obtained drug-loaded micelle solution with HAGMA prepared in the step 3) according to the mass ratio of 1: 0.75-1.5, wherein the final concentration of HAGMA is 0.5-2 mg/ml, adding NVP solution containing DMPA into the mixed solution, irradiating with ultraviolet light to obtain the microgel with shear force response and CD44 receptor targeting according to the molar ratio of HAGMA to DMPA to NVP of 1: 3-5: 4.5-7;
wherein, PAA-b-PEHA means an amphiphilic polymer, tBA means t-butyl acrylate, 2-EHA means isooctyl acrylate, P (AA-g-HEMA) -b-PEHA means an amphiphilic graft copolymer, HEMA means hydroxyethyl methacrylate, HAGMA means hyaluronic acid hydrogel precursor, GMA means glycidyl methacrylate, HACBC means a shear response and CD44 receptor targeted dual response microgel, DMF means N, N-dimethylformamide, TFA means trifluoroacetic acid, THF means tetrahydrofuran, EDC means 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, NHS means N-hydroxysuccinimide, HA means sodium hyaluronate, DMAP means 4-dimethylaminopyridine, TBAB means tetrabutylammonium bromide, DMPA means 2, 2-dimethoxy-2-phenylacetophenone, NVP means N-vinylpyrrolidone.
2. The method for preparing microgel based on shear force sensitivity and targeting of CD44 receptor according to claim 1, wherein the cut-off molecular weight of the dialysis bag used in the dialysis in step 3) and step 4) is 10000.
3. The method for preparing a microgel based on shear force sensitivity and targeting of CD44 receptors in claim 1, wherein the UV irradiation in step 4) is 355nm UV irradiation for 5 min.
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