CN110179993B - Ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response and preparation method thereof - Google Patents
Ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response and preparation method thereof Download PDFInfo
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- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
Abstract
The invention discloses a ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response, which is characterized in that a ganoderma lucidum polysaccharide-based conjugate is formed by chemically connecting rutin, phenylboronic acid, ganoderma lucidum polysaccharide, dithiodipropionic acid and dihydroartemisinin to form rutin-phenylboronic acid-ganoderma lucidum polysaccharide-dithiodipropionic acid-dihydroartemisinin, the ganoderma lucidum polysaccharide is used as a hydrophilic segment, the rutin and the dihydroartemisinin are used as a hydrophobic segment, and the rutin-phenylboronic acid-ganoderma lucidum polysaccharide-dithiodipropionic acid-dihydroartemisinin conjugate is self-assembled in an aqueous solution to encapsulate 10-hydroxycamptothecin to form the drug-loaded nanoparticle.
Description
Technical Field
The invention relates to a ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response and a preparation method thereof, belonging to the technical field of biological medicines and nano-medicines.
Background
The nano drug delivery system is widely concerned by researchers as an important mode for delivering cancer chemotherapy drugs, but the nonspecific release of the drugs can bring serious toxic and side effects to normal tissues. The stimulus-responsive drug release is a common drug delivery strategy of a nano system, can be released at a fixed point of diseased tissues or cells, and can be correspondingly and intelligently controlled to release under specific pathological changes and specific physiological environments, so that the distribution of the drug in the body is improved, the treatment effect is enhanced, the toxic and side effects are reduced, and the application prospect is wide.
The ganoderma lucidum polysaccharide GLP is a natural high-molecular polysaccharide, is one of the most effective components of ganoderma lucidum, is glucan which is formed by three strands of monosaccharide chains and has a spiral three-dimensional configuration (a three-level structure), is a macromolecular compound, and has a molecular weight of thousands to hundreds of thousands. In recent years, a great deal of research work at home and abroad is dedicated to the extraction, separation, purification and structural verification of ganoderma lucidum polysaccharide, and the pharmacological actions of the ganoderma lucidum polysaccharide in resisting tumor and enhancing immunity are researched, but the research of using the ganoderma lucidum polysaccharide as a nano-drug carrier material is almost not reported at present. Similar to most natural high molecular polysaccharides, the ganoderma lucidum polysaccharide has good safety and biocompatibility, and a large number of hydroxyl groups in the molecular structure of the ganoderma lucidum polysaccharide enable the ganoderma lucidum polysaccharide to have strong hydrophilic capacity and can be conveniently modified, so that the ganoderma lucidum polysaccharide can be used as a hydrophilic carrier of a nano-drug delivery system.
Research shows that the tumor cells have unique physiological microenvironment, low pH value, high temperature, high concentration reduced Glutathione (GSH) and other characteristics. Boric acid can be spontaneously combined with a compound containing an o-diol structure in a weakly alkaline environment to form a borate bond which can be broken in an acidic environment, and the pH responsiveness is realized. Disulfide bonds can be reduced into free sulfydryl under the stimulation of a reducing environment (such as high glutathione concentration), so that the structure of the cross-linked micelle is damaged, and the micelle has reduction responsiveness, and therefore, the disulfide bonds can be used for designing and preparing a reduction-responsive nano micelle carrier.
On the basis, the research uses the pH response of phenylboronic acid (PBA) and the redox response performance of disulfide bond to design and prepare a dual-response nano-drug carrier based on ganoderan, and simultaneously loads three anti-cancer drugs, namely Rutin (Rutin), Dihydroartemisinin (DHA) and 10-Hydroxycamptothecin (HCPT), so that the anti-cancer effect of the drug is improved. So far, no report on the research of a stimulus-response type nano-drug carrier taking ganoderma lucidum polysaccharide as a material exists.
Disclosure of Invention
The invention aims to establish lucid ganoderma polysaccharide-based conjugate drug-loaded nanoparticles with pH/redox dual response and a preparation method thereof. The preparation method of the drug-loaded nanoparticles prepared by taking ganoderma lucidum polysaccharide as a material is simple to operate, does not use a surfactant, and has targeting property and low toxicity.
The invention provides a ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response and a preparation method thereof, and is characterized in that Rutin, phenylboronic acid, ganoderma lucidum polysaccharide, dithiodipropionic acid and dihydroartemisinin are chemically connected to form a Rutin (Rutin) -phenylboronic acid (PBA) -Ganoderma Lucidum Polysaccharide (GLP) -dithiodipropionic acid (DPA) -Dihydroartemisinin (DHA) conjugate, the ganoderma lucidum polysaccharide is used as a hydrophilic segment, the Rutin and the dihydroartemisinin are used as a hydrophobic segment, and the Rutin-phenylboronic acid-ganoderma lucidum polysaccharide-dithiodipropionic acid-dihydroartemisinin conjugate is self-assembled in an aqueous solution to encapsulate 10-hydroxycamptothecin to form the drug-loaded nanoparticle (RCGDD/HCPT).
The rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate has a structure shown in a formula I:
the technical scheme of the invention is as follows:
a method for synthesizing Ganoderma lucidum polysaccharide conjugate with pH/redox dual response comprises:
the preparation method of the ganoderma lucidum polysaccharide-based conjugate nano-particles comprises the following steps:
(1) chemically linking rutin with phenylboronic acid to form rutin-phenylboronic acid conjugate, dissolving rutin in N, N-dimethylformamide, and reacting with tetracarboxyphenylboronic acid at room temperature2Reacting for 24 hours under protection; then dripping the reaction liquid into chloroform to separate out a precipitate, washing the precipitate with chloroform for three times, and drying in vacuum to obtain a rutin-phenylboronic acid conjugate;
(2) chemically linking dithiodipropionic acid and dihydroartemisinin to form dithiodipropionic acid-dihydroartemisinin conjugate, dissolving dithiodipropionic acid in dimethyl sulfoxide, adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) organic base to activate carboxyl for 3h, adding dihydroartemisinin and 4-Dimethylaminopyridine (DMAP), and reacting at room temperature under N-dimethyl condition2Reacting for 24 hours under protection, after the reaction is finished, dropping the reaction liquid into deionized water, separating out a precipitate, repeatedly washing with the deionized water, redissolving the solid with dimethyl sulfoxide, dialyzing for 48 hours with the deionized water, and freeze-drying to obtain a dithiodipropionic acid-dihydroartemisinin conjugate;
(3) reacting the rutin-phenylboronic acid conjugate and the dithiodipropionic acid-dihydroartemisinin conjugate obtained in the steps (1) and (2) with ganoderma lucidum polysaccharide, respectively dissolving the phenylboronic acid-rutin conjugate and the dithiodipropionic acid-dihydroartemisinin conjugate in dimethyl sulfoxide, adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) organic base to activate carboxyl for 3h, then mixing the solutions, adding ganoderma lucidum polysaccharide and 4-Dimethylaminopyridine (DMAP), and carrying out N-phase reaction at room temperature2Reacting for 24h under protection, dialyzing with deionized water for 48h after the reaction is finished, and freeze-drying to obtain rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate;
(4) the preparation method comprises the steps of dissolving Rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate and 10-Hydroxycamptothecin (HCPT) in dimethyl sulfoxide (DMSO), dropwise adding the obtained solution into a high-speed stirring aqueous solution for self-assembly for a period of time, dialyzing for 3 hours by Phosphate Buffer Solution (PBS), filtering by a filter membrane, and freeze-drying to obtain the Rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin/10-hydroxycamptothecin drug-loaded nanoparticles (Rutin-PBA-GLP-DPA-DHA/HCPT).
The invention relates to a preparation method of ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticles with pH/redox dual response, which is characterized in that rutin: phenylboronic acid ═ 2: 1-5: 1, preferably 3.5: 1.
the invention relates to a preparation method of ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticles with pH/redox dual response, which is characterized in that dihydroartemisinin: dithiodipropionic acid ═ 0.5: 1-2: 1, preferably 1.4: 1.
the invention relates to a preparation method of ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticles with pH/redox dual response, which is characterized in that rutin-phenylboronic acid: dithiodipropionic acid-dihydroartemisinin: ganoderma lucidum polysaccharide 0.5: 0.5: 1-2: 2: 1, preferably 2: 1.5: 0.7: 1.
the invention relates to a preparation method of ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticles with pH/redox dual response, which is characterized in that EDC (ethylene diamine tetraacetic acid) is added in the formula: NHS: DMAP ═ 2: 2: 1-5: 5: 1, preferably 3: 2: 1.
the invention prepares the ganoderma lucidum polysaccharide-based conjugate drug-loaded nano particle with pH/redox dual response, which takes natural high molecular ganoderma lucidum polysaccharide with good biocompatibility and anticancer effect as a drug carrier, improves the circulation time of anticancer drugs in vivo, simultaneously leads the ganoderma lucidum polysaccharide-based conjugate drug-loaded nano particle to have pH and redox dual response performance by accessing phenylboronic acid and a disulfide bond, can target cancer cells and reduces the damage of the drugs to normal cells.
Description of the figures (samples taken from example 1)
FIG. 1 is a schematic diagram of a nuclear magnetic resonance of rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate;
FIG. 2 is a TEM schematic diagram of rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin/10-hydroxycamptothecin nano-particle;
FIG. 3 is a schematic view of the sustained release of rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin/10-hydroxycamptothecin nanoparticles in glutathione phosphate buffers of different pH values and different concentrations;
FIG. 4 is a schematic diagram showing the toxicity of pure drugs and rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin/10-hydroxycamptothecin nanoparticles on MCF-7 cells;
FIG. 5 is a schematic diagram showing the relative tumor inhibition rate of pure drugs and rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin/10-hydroxycamptothecin nanoparticles on mice with breast cancer;
Detailed Description
Example 1:
1. weighing 350mg of rutin, putting the rutin into a 10mL dry three-necked bottle, adding 5mL of N, N-dimethylformamide DMF, and continuously introducing N into the three-necked bottle2Adding 100mg of APBA, and reacting for 24h at room temperature; after the reaction is finished, dropping the reaction liquid into chloroform, separating out a precipitate, washing the precipitate for three times by using chloroform, filtering to obtain a solid, and drying the solid in vacuum for 12 hours to obtain a rutin-phenylboronic acid conjugate;
2. weighing 100mg of dithiodipropionic acid, dissolving the dithiodipropionic acid in 5mL of DMSO, respectively adding 140mg of carbodiimide (EDC) and 90mg of N-hydroxysuccinimide (NHS), and activating carboxyl for 3h at room temperature; 140mg of dihydroartemisinin and 45mg of 4-Dimethylaminopyridine (DMAP) were added and the mixture was cooled to room temperature N2Reacting for 24 hours under protection, dropping the reaction solution into deionized water after the reaction is finished, separating out a precipitate, repeatedly washing with deionized water, redissolving the solid in 2mL DMSO, dialyzing with deionized water for 48 hours, and freeze-drying to obtain a dithiodipropionic acid-dihydroartemisinin conjugate;
3. weighing 180mg of rutin-phenylboronic acid conjugate, dissolving in 3mL of DMSO, adding 72mg of EDC and 40mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution A; weighing 130mg of dithiodipropionic acid-dihydroartemisinin conjugate, dissolving in 3mL of DMSO, adding 78mg of EDC and 40mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution B; mixing solution A and solution B, and adding 90mg purified ganoderanSugar and 50mg DMAP, in N2Under the protection of (1), stirring for 24h at room temperature, dialyzing with deionized water for 48h after the reaction is finished, and freeze-drying to obtain a rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate with the purity of more than 95%;
4. weighing 16mg of rutin-phenylboronic acid-ganoderma lucidum polysaccharide-dithiodipropionic acid-dihydroartemisinin conjugate and 8mg of 10-hydroxycamptothecin, dissolving in 0.5mL of sodium dodecyl benzene sulfonate (MSO), fully dissolving, dropwise adding into 3.5mL of PBS (phosphate buffer solution) solution with high-speed stirring and pH of 7.4, and stirring at the rotating speed of 650r/min for 15 min; and transferring the reaction solution into a 3500D dialysis bag for dialysis for 24h, and then freeze-drying for 48h to obtain the drug-loaded nanoparticles.
Example 2:
1. weighing 300mg of rutin, placing into a 10mL dry three-necked bottle, adding into 5mL of N, N-dimethylformamide DMF, and continuously introducing N into the three-necked bottle2Adding 150mg of APBA, and reacting for 24 hours at room temperature; after the reaction is finished, dropping the reaction liquid into chloroform, separating out a precipitate, washing the precipitate for three times by using chloroform, filtering to obtain a solid, and drying the solid in vacuum for 12 hours to obtain a rutin-phenylboronic acid conjugate;
2. weighing 150mg of dithiodipropionic acid, dissolving the dithiodipropionic acid in 5mL of DMSO, respectively adding 210mg of carbodiimide (EDC) and 150mg of N-hydroxysuccinimide (NHS), and activating carboxyl for 3h at room temperature; 300mg of dihydroartemisinin and 80mg of 4-Dimethylaminopyridine (DMAP) were added thereto, and the mixture was cooled to room temperature under N2Reacting for 24 hours under protection, after the reaction is finished, dropping the reaction liquid into deionized water, separating out a precipitate, repeatedly washing with the deionized water, redissolving the solid in 2mL of DMSO, dialyzing with the deionized water for 48 hours, and freeze-drying to obtain a dithiodipropionic acid-dihydroartemisinin conjugate;
3. weighing 50mg of rutin-phenylboronic acid conjugate, dissolving in 3mL of DMSO, adding 20mg of EDC and 20mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution A; weighing 45mg of dithiodipropionic acid-dihydroartemisinin conjugate, dissolving in 3mL of DMSO, adding 25mg of EDC and 20mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution B; mixing solution A and solution B, adding 90mg purified ganoderan and 10mg DMAP, and adding into the mixture under N2Stirring for 24h at room temperature under the protection of (1), and after the reaction is finished, using deionized waterDialyzing for 48h, and freeze-drying to obtain rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate with purity of more than 95%;
4. weighing 20mg of rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate and 8mg of 10-hydroxycamptothecin, dissolving in 0.5mL of DMSO, fully dissolving, dropwise adding into 4.0mL of PBS solution with high-speed stirring and pH of 7.4, and stirring at the rotation speed of 550r/min for 15 min; and transferring the reaction solution into a 3500D dialysis bag for dialysis for 24h, and then freeze-drying for 48h to obtain the drug-loaded nanoparticles.
Example 3:
1. weighing 450mg of rutin, putting the rutin into a 10mL dry three-necked bottle, adding 3mL of N, N-dimethylformamide DMF, and continuously introducing N into the three-necked bottle2Adding 150mg of APBA, and reacting for 24h at room temperature; after the reaction is finished, dropping the reaction liquid into chloroform, separating out a precipitate, washing the precipitate for three times by using chloroform, filtering to obtain a solid, and drying the solid in vacuum for 12 hours to obtain a rutin-phenylboronic acid conjugate;
2. weighing 120mg of dithiodipropionic acid, dissolving in 5mL of DMSO, respectively adding 170mg of carbodiimide (EDC) and 150mg of N-hydroxysuccinimide (NHS), and activating carboxyl for 3h at room temperature; adding 120mg of dihydroartemisinin and 90mg of 4-Dimethylaminopyridine (DMAP) at room temperature N2Reacting for 24 hours under protection, after the reaction is finished, dropping the reaction liquid into deionized water, separating out a precipitate, repeatedly washing with the deionized water, redissolving the solid in 2mL of DMSO, dialyzing with the deionized water for 48 hours, and freeze-drying to obtain a dithiodipropionic acid-dihydroartemisinin conjugate;
3. weighing 100mg of rutin-phenylboronic acid conjugate, dissolving in 3mL of DMSO, adding 55mg of EDC and 40mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution A; weighing 100mg of dithiodipropionic acid-dihydroartemisinin conjugate, dissolving the dithiodipropionic acid-dihydroartemisinin conjugate in 3mL of DMSO, adding 40mg of EDC and 35mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution B; mixing solution A and solution B, adding 80mg purified ganoderan and 50mg DMAP, and adding into the mixture under N2Under the protection of (1), stirring at room temperature for 24h, dialyzing with deionized water for 48h after the reaction is finished, and freeze-drying to obtain rutin-phenylboronic acid-ganoderan-dithiodipropyl ether with the purity of more than 95%Acid-dihydroartemisinin conjugates;
4. weighing 15mg of rutin-phenylboronic acid-ganoderma lucidum polysaccharide-dithiodipropionic acid-dihydroartemisinin conjugate and 8mg of 10-hydroxycamptothecin, dissolving in 0.5mL of DMSO, fully dissolving, dropwise adding into 4.0mL of PBS solution with high-speed stirring and pH of 7.4, and stirring at the rotation speed of 550r/min for 15 min; and transferring the reaction solution into a 1000D dialysis bag for dialysis for 24h, and then freeze-drying for 48h to obtain the drug-loaded nanoparticles.
Example 4:
1. weighing 250mg of rutin, putting the rutin into a 10mL dry three-necked bottle, adding 3mL of N, N-dimethylformamide DMF, and continuously introducing N into the three-necked bottle2Adding 150mg of APBA, and reacting for 24h at room temperature; after the reaction is finished, dropping the reaction liquid into chloroform, separating out a precipitate, washing the precipitate for three times by using chloroform, filtering to obtain a solid, and drying the solid in vacuum for 12 hours to obtain a rutin-phenylboronic acid conjugate;
2. weighing 80mg of dithiodipropionic acid, dissolving in 5mL of DMSO, respectively adding 110mg of carbodiimide (EDC) and 100mg of N-hydroxysuccinimide (NHS), and activating carboxyl for 3h at room temperature; adding 120mg of dihydroartemisinin and 30mg of 4-Dimethylaminopyridine (DMAP) at room temperature N2Reacting for 24 hours under protection, after the reaction is finished, dropping the reaction liquid into deionized water, separating out a precipitate, repeatedly washing with the deionized water, redissolving the solid in 2mL of DMSO, dialyzing with the deionized water for 48 hours, and freeze-drying to obtain a dithiodipropionic acid-dihydroartemisinin conjugate;
3. weighing 120mg of rutin-phenylboronic acid conjugate, dissolving in 3mL of DMSO, adding 60mg of EDC and 40mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution A; weighing 100mg of dithiodipropionic acid-dihydroartemisinin conjugate, dissolving in 3mL of DMSO, adding 40mg of EDC and 35mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution B; mixing solution A and solution B, adding 80mg purified ganoderan and 40mg DMAP, and adding into the mixture under N2Under the protection of (1), stirring for 24h at room temperature, dialyzing with deionized water for 48h after the reaction is finished, and freeze-drying to obtain a rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate with the purity of more than 95%;
4. weighing 15mg of rutin-phenylboronic acid-ganoderma lucidum polysaccharide-dithiodipropionic acid-dihydroartemisinin conjugate and 6mg of 10-hydroxycamptothecin, dissolving in 0.5mL of DMSO, fully dissolving, dropwise adding into 4.0mL of PBS solution with high-speed stirring and pH of 7.4, and stirring at the rotation speed of 550r/min for 10 min; and transferring the reaction solution into a 3500D dialysis bag for dialysis for 24h, and then freeze-drying for 48h to obtain the drug-loaded nanoparticles.
Example 5:
1. weighing 200mg of rutin, placing into a 10mL dry three-necked bottle, adding into 3mL of N, N-dimethylformamide DMF, and continuously introducing N into the three-necked bottle2Adding 50mg of APBA, and reacting for 24h at room temperature; after the reaction is finished, dropping the reaction liquid into chloroform, separating out a precipitate, washing the precipitate for three times by using chloroform, filtering to obtain a solid, and drying the solid in vacuum for 12 hours to obtain a rutin-phenylboronic acid conjugate;
2. weighing 150mg of dithiodipropionic acid, dissolving the dithiodipropionic acid in 5mL of DMSO, respectively adding 210mg of carbodiimide (EDC) and 150mg of N-hydroxysuccinimide (NHS), and activating carboxyl for 3h at room temperature; 200mg of dihydroartemisinin and 40mg of 4-Dimethylaminopyridine (DMAP) were added thereto, and the mixture was cooled to room temperature under N2Reacting for 24 hours under protection, after the reaction is finished, dropping the reaction liquid into deionized water, separating out a precipitate, repeatedly washing with the deionized water, redissolving the solid in 2mL of DMSO, dialyzing with the deionized water for 48 hours, and freeze-drying to obtain a dithiodipropionic acid-dihydroartemisinin conjugate;
3. weighing 150mg of rutin-phenylboronic acid conjugate, dissolving in 3mL of DMSO, adding 80mg of EDC and 80mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution A; weighing 150mg of dithiodipropionic acid-dihydroartemisinin conjugate, dissolving in 3mL of DMSO, adding 60mg of EDC and 60mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution B; mixing solution A and solution B, adding 80mg purified ganoderan and 50mg DMAP, and adding into the mixture under N2Under the protection of (1), stirring for 24h at room temperature, dialyzing with deionized water for 48h after the reaction is finished, and freeze-drying to obtain a rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate with the purity of more than 95%;
4. weighing 15mg of rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate and 8mg of 10-hydroxycamptothecin, dissolving in 0.5mL of DMSO, fully dissolving, dropwise adding into 4.0mL of PBS solution with high-speed stirring and pH of 7.4, and stirring at the rotation speed of 500r/min for 15 min; and transferring the reaction solution into a 3500D dialysis bag for dialysis for 24h, and then freeze-drying for 48h to obtain the drug-loaded nanoparticles.
Example 6:
1. weighing 350mg of rutin, putting the rutin into a 10mL dry three-necked bottle, adding 5mL of N, N-dimethylformamide DMF, and continuously introducing N into the three-necked bottle2Adding 200mg of APBA, and reacting for 24h at room temperature; after the reaction is finished, dropping the reaction liquid into chloroform, separating out a precipitate, washing the precipitate for three times by using chloroform, filtering to obtain a solid, and drying the solid in vacuum for 12 hours to obtain a rutin-phenylboronic acid conjugate;
2. weighing 300mg of dithiodipropionic acid, dissolving the dithiodipropionic acid in 5mL of DMSO, respectively adding 420mg of carbodiimide (EDC) and 400mg of N-hydroxysuccinimide (NHS), and activating carboxyl for 3h at room temperature; 200mg of dihydroartemisinin and 150mg of 4-Dimethylaminopyridine (DMAP) were added thereto, and the mixture was cooled to room temperature under N2Reacting for 24 hours under protection, after the reaction is finished, dropping the reaction liquid into deionized water, separating out a precipitate, repeatedly washing with the deionized water, redissolving the solid in 3mL of DMSO, dialyzing with the deionized water for 48 hours, and freeze-drying to obtain a dithiodipropionic acid-dihydroartemisinin conjugate;
3. weighing 100mg of rutin-phenylboronic acid conjugate, dissolving in 3mL of DMSO, adding 55mg of EDC and 40mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution A; weighing 100mg of dithiodipropionic acid-dihydroartemisinin conjugate, dissolving in 3mL of DMSO, adding 40mg of EDC and 35mg of NHS, and activating carboxyl for 3h at room temperature to obtain a solution B; mixing solution A and solution B, adding 80mg purified ganoderan and 50mg DMAP, and adding into the mixture under N2Under the protection of (1), stirring for 24h at room temperature, dialyzing with deionized water for 48h after the reaction is finished, and freeze-drying to obtain a rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate with the purity of more than 95%;
4. weighing 50mg of rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate and 20mg of 10-hydroxycamptothecin, dissolving in 1mL of DMSO, fully dissolving, dropwise adding into 9mL of PBS solution with high-speed stirring and pH of 7.4, and stirring at the rotation speed of 650r/min for 10 min; and transferring the reaction solution into a 3500D dialysis bag for dialysis for 24h, and then freeze-drying for 48h to obtain the drug-loaded nanoparticles.
Claims (6)
1. A ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response is characterized in that rutin, phenylboronic acid, ganoderma lucidum polysaccharide, dithiodipropionic acid and dihydroartemisinin are chemically connected to form a rutin-phenylboronic acid-ganoderma lucidum polysaccharide-dithiodipropionic acid-dihydroartemisinin conjugate, the ganoderma lucidum polysaccharide is used as a hydrophilic segment, the rutin and the dihydroartemisinin are used as hydrophobic segments, and the rutin-phenylboronic acid-ganoderma lucidum polysaccharide-dithiodipropionic acid-dihydroartemisinin conjugate is self-assembled in an aqueous solution to encapsulate 10-hydroxycamptothecin to form the drug-loaded nanoparticle.
2. The preparation method of the ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response according to claim 1, comprising the following steps:
(1) chemically linking rutin with phenylboronic acid to form rutin-phenylboronic acid conjugate, dissolving rutin in N, N-dimethylformamide, adding tetra-carboxyphenylboronic acid, and reacting at room temperature2Reacting for 24 hours under protection, then dripping the reaction liquid into chloroform, separating out a precipitate, washing the precipitate with chloroform for three times, and drying in vacuum to obtain a rutin-phenylboronic acid conjugate;
(2) chemically linking dithiodipropionic acid and dihydroartemisinin to form dithiodipropionic acid-dihydroartemisinin conjugate, dissolving dithiodipropionic acid in dimethyl sulfoxide, adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) organic base to activate carboxyl for 3h, adding dihydroartemisinin and 4-Dimethylaminopyridine (DMAP), and reacting at room temperature under N-dimethyl condition2Reacting for 24 hours under protection, after the reaction is finished, dropping the reaction liquid into deionized water, separating out a precipitate, repeatedly washing with the deionized water, redissolving the solid with dimethyl sulfoxide, dialyzing for 48 hours with the deionized water, and freeze-drying to obtain a dithiodipropionic acid-dihydroartemisinin conjugate;
(3) the rutin-phenylboronic acid conjugate obtained in the steps (1) and (2) and dithiodipropionic acid-dihydroReacting artemisinin conjugate with ganoderan, dissolving phenylboronic acid-rutin conjugate and dithiodipropionic acid-dihydroartemisinin conjugate in dimethyl sulfoxide, adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) organic base to activate carboxyl for 3h, mixing the solutions, adding ganoderan and 4-Dimethylaminopyridine (DMAP), and allowing reaction under room temperature under N-dimethyl sulfoxide (DMF) to obtain solution2Reacting for 24h under protection, dialyzing with deionized water for 48h after the reaction is finished, and freeze-drying to obtain rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate;
(4) the preparation method comprises the steps of dissolving Rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin conjugate and 10-Hydroxycamptothecin (HCPT) in dimethyl sulfoxide (DMSO), then dropwise adding the obtained solution into a high-speed stirring aqueous solution for self-assembly for a period of time, dialyzing for 24 hours by Phosphate Buffer Solution (PBS), and freeze-drying to obtain Rutin-phenylboronic acid-ganoderan-dithiodipropionic acid-dihydroartemisinin/10-hydroxycamptothecin drug-loaded nanoparticles (Rutin-PBA-GLP-DPA-DHA/HCPT).
3. The preparation method of the ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with the pH/redox dual response function according to claim 2, wherein the weight ratio of rutin: phenylboronic acid ═ 2: 1-5: 1.
4. the preparation method of the ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response as claimed in claim 3, wherein the weight ratio of dihydroartemisinin: dithiodipropionic acid ═ 0.5: 1-2: 1.
5. the preparation method of the ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response, according to claim 4, is characterized in that the ratio by mass of rutin-phenylboronic acid: dithiodipropionic acid-dihydroartemisinin: ganoderma lucidum polysaccharide 0.5: 0.5: 1-2: 2: 1.
6. the preparation method of the ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response according to claim 5, wherein the weight ratio of EDC: NHS: DMAP ═ 2: 2: 1-5: 5: 1.
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