CN107674210B - Triphenylphosphine-chitosan stearic acid graft drug-loaded micelle, preparation and application thereof - Google Patents
Triphenylphosphine-chitosan stearic acid graft drug-loaded micelle, preparation and application thereof Download PDFInfo
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- CN107674210B CN107674210B CN201710860315.7A CN201710860315A CN107674210B CN 107674210 B CN107674210 B CN 107674210B CN 201710860315 A CN201710860315 A CN 201710860315A CN 107674210 B CN107674210 B CN 107674210B
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- stearic acid
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- 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
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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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
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
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- 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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- A61K9/00—Medicinal preparations characterised by special physical form
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- 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
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- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
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Abstract
The invention provides a triphenylphosphine-chitosan stearic acid graft drug-loaded micelle, which is characterized in that a two-step amide method is adopted to graft tetracarboxylbutylphenylphosphine to a chitosan stearic acid graft to obtain the triphenylphosphine-chitosan stearic acid graft with a mitochondrion targeting function, and an anti-tumor drug adriamycin is encapsulated by a dialysis method to obtain the mitochondrion targeting triphenylphosphine-chitosan stearic acid graft drug-loaded micelle. The drug-loaded micelle provided by the invention has a function of efficient targeting of mitochondria, and the encapsulated drug adriamycin can reduce the leakage of adriamycin in normal tissues and non-target parts to the maximum extent, reduce the toxic and side effects of adriamycin, accurately deliver a large amount of adriamycin to the mitochondria of tumor cells in a targeted manner, increase the concentration of adriamycin in the mitochondria of the tumor cells, induce the apoptosis of the tumor cells and greatly improve the anti-tumor curative effect.
Description
Technical Field
The invention belongs to the field of pharmacy, relates to construction of a mitochondrion targeting drug delivery system, and particularly relates to construction of a mitochondrion targeting triphenylphosphine-chitosan stearic acid graft drug-loaded micelle and application of the mitochondrion targeting triphenylphosphine-chitosan stearic acid graft drug-loaded micelle in an anti-tumor drug.
Background
Cancer has six major characteristics which include: unlimited proliferation potential, insensitivity to growth inhibitory signals, inhibition of apoptosis, sustained proliferation signals, induction of angiogenesis, invasion and metastasis to other sites through the capillary wall and basement membrane. Since cancer cells are normal cell mutations, chemotherapeutic agents with the potential to kill cancer cells also produce non-specific toxicity to normal tissues.
The drug molecules act on the receptors, enzymes and ion channels of focus cells mainly through the space occupying effect. In recent years, the regulation of signal pathways associated with pharmacological effects has also begun to become a candidate site for drug action. The cell membrane of the focus cell, the subcellular structures of mitochondria, endoplasmic reticulum, nucleus, nuclear membrane and the like in cytoplasm are the main action sites of drug molecules. The number of drug molecules which can enter the action site is very limited due to the limitation of the self physicochemical properties of the drug molecules, and the drug molecules are the main cause of great toxic and side effects and low curative effect of the existing drugs.
The drug delivery system can deliver the drug directly to the molecular action site, thereby improving the drug concentration at the target site and further greatly improving the curative effect. Compared with the traditional drug delivery system, the targeting drug delivery system aiming at the subcellular structure containing the drug molecule action site becomes the key for solving the problems.
Mitochondrial dysfunction has a great correlation with cancer cells, including their unlimited proliferative potential, impaired apoptosis, insensitivity to anti-growth signals, enhanced anabolism, and reduced autophagy. Since mitochondria mediate apoptosis to play an important role in cell death, mitochondria are important targets for inducing apoptosis in tumor cells. The apoptosis-promoting medicine is delivered to tumor cell mitochondria by a molecular targeting technology, so that the curative effect and the safety of the medicine can be greatly improved. Designing a mitochondrial-targeted drug delivery system that triggers apoptosis of tumor cells may therefore be a promising cancer treatment strategy.
Sufficient lipophilicity coupled with delocalized positive charge is a prerequisite for mitochondrial targeting. Mitochondria maintain a constant membrane potential of about-180 to-200 mV in their lipid bilayer through the oxidative phosphorylation pathway. This high negative membrane potential is not present in any other organelle, which provides a selective accumulation condition for lipophilic cations. Various lipophilic cations, including tetracarboxybutyltriphenylphosphine, triphenylphosphine, rhodamine 123, fluoropyridines and anthracyclines, have been shown to selectively accumulate in the mitochondria when delivered intracellularly.
In order to reduce off-target effect and better inhibit tumor cell growth, it is important to target the mitochondrial induction of apoptosis of tumor cells. The mitochondrion targeting is combined with a micelle drug delivery system, so that on one hand, tumor cell apoptosis is induced, on the other hand, the side effect of the drug is reduced, and the anti-tumor curative effect can be greatly improved.
The polymer micelle formed by self-aggregation of amphiphilic block copolymer molecules in an aqueous medium is a novel nano drug delivery carrier for delivering drugs. The drug carrier has the characteristics of specific drug carrier properties, such as small particle size, good stability and biocompatibility in vivo and in vitro, controlled release of the drug, biological membrane permeability and the like, and is considered to be a novel targeted drug delivery system with broad prospects. The polymer micelle passively targets tumor parts through the functions of enhancing penetration and retention.
The chitosan stearic acid graft is obtained by modifying polycation natural polymer material chitosan with fatty acid, and the graft can form a graft micelle through self-assembly in an aqueous medium, and has a rapid tumor cell uptake function.
Disclosure of Invention
The invention aims to provide a preparation method of a triphenylphosphine-chitosan stearic acid graft, wherein the molecular weight of chitosan is 19kDa, the carbon chain length of fatty acid is eighteen carbons, the deacetylation degree of chitosan is 95%, the amino substitution degree is 18.0-20.7%, and the modification proportion of triphenylphosphine is 1.2-3.9%.
The representative chemical structural general formula is as follows:
the preparation method is realized by the following preparation steps:
(1) synthesizing a chitosan stearic acid graft according to the method provided by the invention patent ZL 200610051601.0:
taking chitosan with the molecular weight of 19kDa, adding distilled water for ultrasonic dissolution, and preparing a solution with the concentration of 20 mg/ml. And taking stearic acid according to the molar ratio of 25:1 of stearic acid to chitosan, and taking the stearic acid according to the molar ratio of carbodiimide to stearic acid of 6:1, mixing carbodiimide, and mixing according to the volume ratio of ethanol to distilled water of 1:2 adding ethanol, dissolving with ultrasonic wave in water bath, and stirring at 60 deg.C for 50 min. Slowly adding the mixture into a preheated chitosan aqueous solution at 60 ℃, stirring and reacting for 12 hours at 60 ℃, cooling to room temperature, placing the final reaction solution into a dialysis bag with the molecular cut-off of 7000Da, dialyzing for 72 hours with distilled water, and washing the dialyzate with absolute ethyl alcohol to remove residual stearic acid after freeze drying to obtain the chitosan stearic acid graft.
(2) Synthesis of triphenylphosphine-chitosan stearic acid graft
Dissolving tetracarboxybutyltriphenylphosphine in a small amount of anhydrous dimethyl sulfoxide, and dissolving the solution by ultrasonic treatment to prepare a 10mg/ml solution. And further according to carbodiimide: the molar ratio of the tetracarboxybutyl triphenylphosphine is 3: 1, taking carbodiimide, and mixing the carbodiimide: the molar ratio of the tetracarboxyl butyl triphenylphosphine is 3: 1, adding N-hydroxysuccinimide into the solution, and activating for 2 hours to obtain reaction liquid 1. According to the following amino polyethylene glycol 2000: the molar ratio of the tetracarboxybutyl triphenylphosphine is 1: 1, dissolving amino polyethylene glycol 2000 in a small amount of anhydrous dimethyl sulfoxide, adding the solution into the reaction solution 1, and stirring the solution at room temperature for 9 hours to obtain a reaction solution 2. Adding a mixture of amino polyethylene glycol 2000 and amino polyethylene glycol in a molar ratio of 1: 1 was stirred at room temperature for 12 hours to obtain reaction solution 3. According to the free amino group on the stearic acid of the chitosan: the molar ratio of the tetracarboxyl butyl triphenylphosphine is 5: 1-20: 1, dissolving chitosan stearic acid graft in a small amount of deionized water. The reaction solution 3 was added to the aqueous solution of chitosan stearic acid graft and stirred at room temperature for 24 hours. And (3) putting the final reaction solution into a dialysis bag with the molecular weight cutoff of 7000, dialyzing with pure water for 48 hours, and freeze-drying to obtain triphenylphosphine-chitosan stearic acid (solid powder).
The synthetic route is as follows:
in the synthetic route, 1) is the activation of carbodiimide (EDC) and N-hydroxysuccinimide (NHS), 2) is the structural formula of amino-polyethylene glycol 2000, 3) is the structural formula of disuccinimide, and 4) is the structural formula of chitosan stearic acid
The chitosan fatty acid graft used in the invention is a national invention patent of 'fluorescence labeling hydrophobic modified chitosan polymer and a preparation method and application' (patent number: ZL 2005100507981); and "surface-modified hydrophobically modified chitosan polymer micelle and method for preparing the same" (patent No. ZL 200610051601.0). The molecular weight of chitosan in the chitosan fatty acid graft is 19 kDa; the carbon chain length of the fatty acid is eighteen carbons; the deacetylation degree of chitosan is 95%; the degree of amino substitution was 16.8%.
The second purpose of the invention is to provide a construction method of a triphenylphosphine-chitosan stearic acid graft drug-loaded micelle, wherein the loaded drug is adriamycin, and the construction method is realized by the following scheme:
(1) base adriamycin preparation: weighing 200mg of doxorubicin hydrochloride, dissolving the doxorubicin hydrochloride in 20mL of dimethyl sulfoxide, adding triethylamine, stirring the mixture overnight, placing the reaction solution in a dialysis bag, dialyzing the reaction solution with distilled water for 48 hours, collecting a product in the dialysis bag, centrifuging the product for 10min at 8000r, collecting a precipitate, dissolving the precipitate with water, repeating the steps for three times to remove residual doxorubicin hydrochloride, and freeze-drying the product to obtain basic group doxorubicin;
(2) the preparation of the triphenylphosphine-chitosan stearic acid graft drug-loaded micelle comprises the steps of weighing the triphenylphosphine-chitosan stearic acid graft, dissolving the triphenylphosphine-chitosan stearic acid graft in deionized water, carrying out ultrasonic treatment for 30 times by using a probe, wherein the power is 400w, the operation lasts for 2s and stops for 3s, and preparing a graft micelle solution of 2 mg/mL. And adding dimethyl sulfoxide into basic group adriamycin to prepare a solution with the concentration of 2 mg/mL. Adding dimethyl sulfoxide solution containing 2mg/mL adriamycin according to the dosage of 5-15% of adriamycin to the graft by mass, stirring for 2 hours at room temperature in a dark place, transferring into a dialysis bag with the molecular weight cutoff of 3500 after the reaction is finished, dialyzing for 24 hours by pure water, collecting the dialyzed product, centrifuging for 10 minutes at a low temperature of 8000r to remove the adriamycin which is not encapsulated by the graft micelle, and collecting the supernatant to obtain the triphenylphosphine-chitosan stearic acid graft drug-loaded micelle.
The third purpose of the invention is to provide the application of the triphenylphosphine-chitosan stearic acid grafted drug-loaded micelle in the preparation of mitochondrion-targeted antitumor drugs. Researches show that the triphenylphosphine-modified chitosan stearic acid graft has high mitochondrial targeting capability, and the triphenylphosphine-chitosan stearic acid graft drug-loaded micelle has obvious antitumor activity.
The invention provides a triphenylphosphine-chitosan stearic acid graft drug-loaded micelle, which is characterized in that a two-step amide method is adopted to graft tetracarboxylbutylphenylphosphine to a chitosan stearic acid graft to obtain the triphenylphosphine-chitosan stearic acid graft with a mitochondrion targeting function, and an anti-tumor drug adriamycin is encapsulated by a dialysis method to obtain the mitochondrion targeting triphenylphosphine-chitosan stearic acid graft drug-loaded micelle. The drug-loaded micelle provided by the invention has a function of efficient targeting of mitochondria, and the encapsulated drug adriamycin can reduce the leakage of adriamycin in normal tissues and non-target parts to the maximum extent, reduce the toxic and side effects of adriamycin, accurately deliver a large amount of adriamycin to the mitochondria of tumor cells in a targeted manner, increase the concentration of adriamycin in the mitochondria of the tumor cells, induce the apoptosis of the tumor cells and greatly improve the anti-tumor curative effect.
Drawings
FIG. 1 is the NMR spectrum of triphenylphosphine-chitosan stearic acid graft, wherein A is tetracarboxybutyl triphenylphosphine, B is amino polyethylene glycol, C is chitosan stearic acid graft, and D is triphenylphosphine-chitosan stearic acid graft.
FIG. 2 shows the quantitative uptake of triphenylphosphine-chitosan stearic acid grafts incubated on breast cancer MCF-7 cells for 4, 12, and 24 hours.
FIG. 3 shows the co-localization coefficient analysis of triphenylphosphine-chitosan stearic acid graft in MCF-7 cell mitochondria.
Fig. 4 is a release profile of triphenylphosphine-chitosan stearic acid drug-loaded micelles in a pH6.8 release medium.
FIG. 5 shows the antitumor effect of triphenylphosphine-chitosan stearic acid drug-loaded micelle on MCF-7 cells.
Detailed Description
The invention is further illustrated by means of examples and figures.
Example 1
(1) Preparation of low molecular weight chitosan
Adding 50g of chitosan with the molecular weight of 450kDa and the deacetylation degree of 95 percent into 1500mL of hydrochloric acid aqueous solution with the volume ratio of 1.2 percent, stirring for 2 hours at the temperature of 55 ℃ to fully swell the chitosan, slowly adding chitosan enzyme solution with the weight ratio of 2 percent, carrying out chitosan enzymolysis reaction at the temperature of 55 ℃, and controlling the degradation degree of the chitosan by gel permeation chromatography. After the reaction is finished, stirring for 0.5 hour at 80 ℃, adding activated carbon with the weight/volume ratio of 0.3 percent, diluting the reaction solution, filtering by using a Buchner funnel, treating the filtrate by using a 0.45 mu m microporous filter membrane, and freeze-drying to obtain the low-molecular-weight chitosan, wherein the weight-average molecular weight of the obtained chitosan is 19.0 kDa. The chitosan with molecular weight of 450kDa is degraded to 19kDa and is called low molecular weight.
(2) Synthesis of chitosan stearic acid graft
Dissolving the chitosan with the molecular weight of 19.0kDa in distilled water by ultrasonic treatment to prepare a solution with the concentration of 20 mg/ml. And mixing stearic acid according to the molar ratio of 25:1 of stearic acid to chitosan, carbodiimide according to the molar ratio of 6:1 of carbodiimide to stearic acid, and mixing ethanol and distilled water according to the volume ratio of 1:2 adding ethanol, dissolving with ultrasonic wave in water bath, and stirring at 60 deg.C for 50 min. Slowly adding the mixture into a preheated chitosan aqueous solution at 60 ℃, stirring and reacting for 12 hours at 60 ℃, cooling to room temperature, placing the final reaction solution into a dialysis bag with the molecular cut-off of 7000Da, dialyzing for 72 hours with distilled water, and washing the dialyzate with absolute ethyl alcohol to remove residual stearic acid after freeze drying to obtain the chitosan stearic acid graft.
(3) Synthesis of triphenylphosphine-chitosan stearic acid graft
Dissolving tetracarboxybutyltriphenylphosphine in a small amount of anhydrous dimethyl sulfoxide, and dissolving the solution by ultrasonic treatment to prepare a 10mg/ml solution. And further according to carbodiimide: the molar ratio of the tetracarboxybutyl triphenylphosphine is 3: 1, taking carbodiimide, and mixing the carbodiimide: the molar ratio of the tetracarboxyl butyl triphenylphosphine is 3: 1, adding N-hydroxysuccinimide into the solution, and activating for 2 hours to obtain reaction liquid 1. According to the following amino polyethylene glycol 2000: the molar ratio of the tetracarboxybutyl triphenylphosphine is 1: 1, dissolving amino polyethylene glycol 2000 in a small amount of anhydrous dimethyl sulfoxide, adding the solution into the reaction solution 1, and stirring the solution at room temperature for 9 hours to obtain a reaction solution 2. Adding a mixture of amino polyethylene glycol 2000 and amino polyethylene glycol in a molar ratio of 1: 1 was stirred at room temperature for 12 hours to obtain reaction solution 3. According to the free amino group on the stearic acid of the chitosan: the molar ratio of the tetracarboxyl butyl triphenylphosphine is 5:1, dissolving chitosan stearic acid graft in a small amount of deionized water. The reaction solution 3 was added to the aqueous solution of chitosan stearic acid graft and stirred at room temperature for 24 hours. And (3) placing the final reaction solution into a dialysis bag with the molecular weight cutoff of 7000, dialyzing with pure water for 48 hours, and freeze-drying to obtain triphenylphosphine-chitosan stearic acid solid powder.
And (3) determining the amino substitution degree of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid graft by adopting a trinitrobenzene sulfonic acid method. Dissolving 1-10 mg of chitosan with different weights in 2mL of distilled water respectively, adding 2mL of 4% sodium bicarbonate and 2mL of 0.1% trinitrobenzene sulfonic acid, incubating for 2 hours at 37 ℃, adding 2mL of 2mol/L hydrochloric acid, shaking up, measuring absorbance at 344nm, and preparing a standard curve. Respectively dissolving 6mg of each of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid in 3mL of distilled water, and calculating according to a standard curve by the same method, wherein the amino substitution degree of the triphenylphosphine-chitosan stearic acid graft is 18.0%, the amino substitution degree of the chitosan stearic acid graft is 16.8%, and the triphenylphosphine modification proportion is 1.2%.
Example 2
(1) Preparation of low molecular weight chitosan
Adding 50g of chitosan with the molecular weight of 450kDa and the deacetylation degree of 95 percent into 1500mL of hydrochloric acid aqueous solution with the volume ratio of 1.2 percent, stirring for 2 hours at the temperature of 55 ℃ to fully swell the chitosan, slowly adding chitosan enzyme solution with the weight ratio of 2 percent, carrying out chitosan enzymolysis reaction at the temperature of 55 ℃, and controlling the degradation degree of the chitosan by gel permeation chromatography. After the reaction is finished, stirring for 0.5 hour at 80 ℃, adding activated carbon with the weight/volume ratio of 0.3 percent, diluting the reaction solution, filtering by using a Buchner funnel, treating the filtrate by using a 0.45 mu m microporous filter membrane, and freeze-drying to obtain the low-molecular-weight chitosan, wherein the weight-average molecular weight of the obtained chitosan is 19.0 kDa.
(2) Synthesis of chitosan stearic acid graft
Dissolving the chitosan with the molecular weight of 19.0kDa in distilled water by ultrasonic treatment to prepare a solution with the concentration of 20 mg/ml. And mixing stearic acid according to the molar ratio of 25:1 of stearic acid to chitosan, carbodiimide according to the molar ratio of 6:1 of carbodiimide to stearic acid, and mixing ethanol and distilled water according to the volume ratio of 1:2 adding ethanol, dissolving with ultrasonic wave in water bath, and stirring at 60 deg.C for 50 min. Slowly adding the mixture into a preheated chitosan aqueous solution at 60 ℃, stirring and reacting for 12 hours at 60 ℃, cooling to room temperature, placing the final reaction solution into a dialysis bag with the molecular cut-off of 7000Da, dialyzing for 72 hours with distilled water, and washing the dialyzate with absolute ethyl alcohol to remove residual stearic acid after freeze drying to obtain the chitosan stearic acid graft.
(3) Synthesis of triphenylphosphine-chitosan stearic acid graft
Dissolving tetracarboxybutyltriphenylphosphine in a small amount of anhydrous dimethyl sulfoxide, and dissolving the solution by ultrasonic treatment to prepare a 10mg/ml solution. And further according to carbodiimide: the molar ratio of the tetracarboxybutyl triphenylphosphine is 3: 1, taking carbodiimide, and mixing the carbodiimide: the molar ratio of the tetracarboxyl butyl triphenylphosphine is 3: 1, adding N-hydroxysuccinimide into the solution, and activating for 2 hours to obtain reaction liquid 1. According to the following amino polyethylene glycol 2000: the molar ratio of the tetracarboxybutyl triphenylphosphine is 1: 1, dissolving amino polyethylene glycol 2000 in a small amount of anhydrous dimethyl sulfoxide, adding the solution into the reaction solution 1, and stirring the solution at room temperature for 9 hours to obtain a reaction solution 2. Adding a mixture of amino polyethylene glycol 2000 and amino polyethylene glycol in a molar ratio of 1: 1 was stirred at room temperature for 12 hours to obtain reaction solution 3. According to the free amino group on the stearic acid of the chitosan: the molar ratio of the tetracarboxybutyl triphenylphosphine is 10: 1, dissolving chitosan stearic acid graft in a small amount of deionized water. The reaction solution 3 was added to the aqueous solution of chitosan stearic acid graft and stirred at room temperature for 24 hours. And (3) placing the final reaction solution into a dialysis bag with the molecular weight cutoff of 7000, dialyzing with pure water for 48 hours, and freeze-drying to obtain triphenylphosphine-chitosan stearic acid solid powder.
And (3) determining the amino substitution degree of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid graft by adopting a trinitrobenzene sulfonic acid method. Dissolving 1-10 mg of chitosan with different weights in 2mL of distilled water respectively, adding 2mL of 4% sodium bicarbonate and 2mL of 0.1% trinitrobenzene sulfonic acid, incubating for 2 hours at 37 ℃, adding 2mL of 2mol/L hydrochloric acid, shaking up, measuring absorbance at 344nm, and preparing a standard curve. Respectively dissolving 6mg of each of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid in 3mL of distilled water, and calculating according to a standard curve by the same method, wherein the amino substitution degree of the triphenylphosphine-chitosan stearic acid graft is 19.2%, the amino substitution degree of the chitosan stearic acid graft is 16.8%, and the triphenylphosphine modification proportion is 2.4%.
Example 3
(1) Preparation of low molecular weight chitosan
Adding 50g of chitosan with the molecular weight of 450kDa and the deacetylation degree of 95 percent into 1500mL of hydrochloric acid aqueous solution with the volume ratio of 1.2 percent, stirring for 2 hours at the temperature of 55 ℃ to fully swell the chitosan, slowly adding chitosan enzyme solution with the weight ratio of 2 percent, carrying out chitosan enzymolysis reaction at the temperature of 55 ℃, and controlling the degradation degree of the chitosan by gel permeation chromatography. After the reaction is finished, stirring for 0.5 hour at 80 ℃, adding activated carbon with the weight/volume ratio of 0.3 percent, diluting the reaction solution, filtering by using a Buchner funnel, treating the filtrate by using a 0.45 mu m microporous filter membrane, and freeze-drying to obtain the low-molecular-weight chitosan, wherein the weight-average molecular weight of the obtained chitosan is 19.0 kDa.
(2) Synthesis of chitosan stearic acid graft
Dissolving the chitosan with the molecular weight of 19.0kDa in distilled water by ultrasonic treatment to prepare a solution with the concentration of 20 mg/ml. And mixing stearic acid according to the molar ratio of 25:1 of stearic acid to chitosan, carbodiimide according to the molar ratio of 6:1 of carbodiimide to stearic acid, and mixing ethanol and distilled water according to the volume ratio of 1:2 adding ethanol, dissolving with ultrasonic wave in water bath, and stirring at 60 deg.C for 50 min. Slowly adding the mixture into a preheated chitosan aqueous solution at 60 ℃, stirring and reacting for 12 hours at 60 ℃, cooling to room temperature, placing the final reaction solution into a dialysis bag with the molecular cut-off of 7000Da, dialyzing for 72 hours with distilled water, and washing the dialyzate with absolute ethyl alcohol to remove residual stearic acid after freeze drying to obtain the chitosan stearic acid graft.
(3) Synthesis of triphenylphosphine-chitosan stearic acid graft
Dissolving tetracarboxybutyltriphenylphosphine in a small amount of anhydrous dimethyl sulfoxide, and dissolving the solution by ultrasonic treatment to prepare a 10mg/ml solution. And further according to carbodiimide: the molar ratio of the tetracarboxybutyl triphenylphosphine is 3: 1, taking carbodiimide, and mixing the carbodiimide: the molar ratio of the tetracarboxyl butyl triphenylphosphine is 3: 1, adding N-hydroxysuccinimide into the solution, and activating for 2 hours to obtain reaction liquid 1. According to the following amino polyethylene glycol 2000: the molar ratio of the tetracarboxybutyl triphenylphosphine is 1: 1, dissolving amino polyethylene glycol 2000 in a small amount of anhydrous dimethyl sulfoxide, adding the solution into the reaction solution 1, and stirring the solution at room temperature for 9 hours to obtain a reaction solution 2. Adding a mixture of amino polyethylene glycol 2000 and amino polyethylene glycol in a molar ratio of 1: 1 was stirred at room temperature for 12 hours to obtain reaction solution 3. According to the free amino group on the stearic acid of the chitosan: the molar ratio of the tetracarboxybutyl triphenylphosphine is 20: 1, dissolving chitosan stearic acid graft in a small amount of deionized water. The reaction solution 3 was added to the aqueous solution of chitosan stearic acid graft and stirred at room temperature for 24 hours. And (3) placing the final reaction solution into a dialysis bag with the molecular weight cutoff of 7000, dialyzing with pure water for 48 hours, and freeze-drying to obtain triphenylphosphine-chitosan stearic acid solid powder.
And (3) determining the amino substitution degree of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid graft by adopting a trinitrobenzene sulfonic acid method. Dissolving 1-10 mg of chitosan with different weights in 2mL of distilled water respectively, adding 2mL of 4% sodium bicarbonate and 2mL of 0.1% trinitrobenzene sulfonic acid, incubating for 2 hours at 37 ℃, adding 2mL of 2mol/L hydrochloric acid, shaking up, measuring absorbance at 344nm, and preparing a standard curve. Respectively dissolving 6mg of each of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid in 3mL of distilled water, and calculating according to a standard curve by the same method, wherein the amino substitution degree of the triphenylphosphine-chitosan stearic acid graft is 20.7%, the amino substitution degree of the chitosan stearic acid graft is 16.8%, and the triphenylphosphine modification proportion is 3.9%.
(4) Physical and chemical properties of triphenylphosphine-chitosan stearic acid graft
And (3) measuring the tetracarboxybutyl triphenylphosphine, amino polyethylene glycol, chitosan stearic acid graft and triphenylphosphine-chitosan stearic acid graft by using a nuclear magnetic resonance spectrometry. 10mg of each of the four-carboxybutyltriphenylphosphine, aminopolyethylene glycol, chitosan stearic acid graft and triphenylphosphine-chitosan stearic acid graft was weighed, dissolved in 0.5mL of D2O, and measured by nuclear magnetic resonance 1H-NMR. Referring to fig. 1, a is tetracarboxylbutyl triphenylphosphine, B is aminopolyethylene glycol, C is chitosan stearic acid graft, and D is triphenylphosphine-chitosan stearic acid graft. The synthesis of triphenylphosphine-chitosan stearic acid graft can be determined from the figure.
Measuring the critical micelle concentration of the triphenylphosphine-chitosan stearic acid graft by adopting a pyrene fluorescence method, precisely weighing 12mg of pyrene, placing the pyrene into a 100mL volumetric flask, adding acetone to dissolve and fix the volume, transferring 1mL of the pyrene solution, placing the pyrene solution into the 100mL volumetric flask to dilute and fix the volume, transferring 0.5mL of the diluted pyrene solution, respectively placing the pyrene solution into 10mL glass test tubes, volatilizing the acetone at 50 ℃, respectively adding 5mL of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid graft with different concentrations, and controlling the final concentration of the pyrene to be 7 × 10-7And (3) carrying out ultrasonic treatment for 30min in a room-temperature water bath at mol/L. Scanning the excitation and emission spectra of pyrene, where Ex ═ 337nm, Em: the fluorescence intensity was measured at 374nm I1, 384nm I3, 2.5nm and 10nm slits and calculated to be 29.2 μ g/mL critical micelle concentration of chitosan stearic acid and 62.4 μ g/mL critical micelle concentration of triphenylphosphine-chitosan stearic acid.
Dissolving triphenylphosphine-chitosan stearic acid graft and chitosan stearic acid graft in distilled water, performing ultrasonic treatment for 30 times with power of 400w for 2s, and performing intermittent treatment for 3s to obtain 1mg/mL graft micelle solution. The particle size and surface potential of the particles are measured by an analyzer, the particle size of the chitosan stearic acid is 91.2 +/-0.93 nm, and the Zeta potential is 16.9 +/-0.40 mV. The particle size of the triphenylphosphine-chitosan stearic acid graft is 100.4 +/-23.1 nm, and the Zeta potential is 23.7 +/-0.95 mV.
(5) Cellular uptake and mitochondrial co-localization of triphenylphosphine-chitosan stearic acid grafts
The glycolipid graft fluorescence labeling micelle is adopted for cell uptake and mitochondria co-localization research. Marking chitosan stearic acid graft and triphenylphosphine-chitosan stearic acid graft with Fluorescein Isothiocyanate (FITC). Respectively taking the chitosan stearic acid graft or the triphenylphosphine-chitosan stearic acid graft carrier, dissolving in deionized water, and preparing into a glycolipid graft carrier solution of 2 mg/mL. And dissolving FITC in absolute ethyl alcohol to prepare a solution of 2 mg/mL. At 400rpm, 40 μ of the ethanol solution of LFITC was slowly dropped into the glycolipid graft carrier solution, and the solution was stirred for 4 hours in the dark. Then, the mixture was placed in a dialysis bag having a molecular weight cut-off of 3500, and dialyzed with deionized water for 8 hours. And (4) centrifuging the dialyzed and purified product at 8000rpm for 10min, and taking supernatant to obtain the FITC-labeled glycolipid graft carrier.
Taking MCF-7 cells with good growth state, and adding 2 × 105The cells were seeded in 6-well slide-coated cell culture plates at a density of 5% CO at 37 ℃%2Culturing until the fusion degree reaches 75%, and adding FITC marked chitosan stearic acid graft or triphenylphosphine-chitosan stearic acid graft carrier solution. Incubation was continued for 4, 12, and 24 hours, respectively, and cells were collected. And (3) quantitatively detecting the cell uptake conditions of the FITC-chitosan stearic acid graft and the FITC-triphenylphosphine-chitosan stearic acid graft carrier by using a flow cytometer. The results are shown in FIG. 2.
Taking MCF-7 cells with good growth state, and collecting the cells at 5 × 104The cells were seeded in 24-well glass slide-coated cell culture plates at a density of 5% CO at 37 ℃%2Culturing until the fusion degree reaches 75%, and adding FITC marked chitosan stearic acid graft or triphenylphosphine-chitosan stearic acid graft carrier solution. And continuously and respectively incubating for 24 hours, sucking out the culture medium, adding a phenol red-free DMEM culture solution containing the mitochondrial probe, incubating for 30min, and washing for three times by PBS. Fixing the cover glass with 4% paraformaldehyde in dark, taking out after 20min, embedding glycerol on the glass slide, and sealing. The cell uptake and mitochondria co-localization conditions of the FITC-chitosan stearic acid graft and the FITC-triphenylphosphine-chitosan stearic acid graft carrier are observed by a laser confocal scanning microscope, the co-localization coefficient is analyzed by Imaris software, and the result is shown in figure 3.
As shown in fig. 3, through analysis and calculation by Imaris software, the co-localization coefficient of triphenylphosphine-chitosan stearic acid graft and mitochondria was 0.49, which is significantly different from chitosan stearic acid. The result shows that the triphenylphosphine-chitosan stearic acid grafting has a high-efficiency mitochondrion targeting function.
(6) Preparation of triphenylphosphine-chitosan stearic acid graft drug-loaded micelle
Base adriamycin preparation: weighing 200mg of doxorubicin hydrochloride, dissolving the doxorubicin hydrochloride in 20mL of dimethyl sulfoxide, adding triethylamine, stirring the mixture overnight, placing the reaction solution in a dialysis bag, dialyzing the reaction solution with distilled water for 48 hours, collecting a product in the dialysis bag, centrifuging the product for 10min at 8000r, collecting a precipitate, dissolving the precipitate with water, repeating the steps for three times to remove residual doxorubicin hydrochloride, and freeze-drying to obtain the basic group doxorubicin.
Weighing triphenylphosphine-chitosan stearic acid graft, dissolving in deionized water, performing ultrasonic treatment for 30 times by using a probe, wherein the power is 400w, the work time is 2s, and the work time is 3s, and preparing graft micelle solution of 2 mg/mL. And adding dimethyl sulfoxide into basic group adriamycin to prepare a solution with the concentration of 2 mg/mL. Adding dimethyl sulfoxide solution containing 2mg/mL adriamycin according to the dose of 5% of adriamycin to the graft by mass, stirring for 2 hours at room temperature in a dark place, transferring to a dialysis bag with the molecular weight cutoff of 3500 after the reaction is finished, dialyzing for 24 hours by pure water, collecting the dialyzed product, centrifuging for 10 minutes at a low temperature of 8000r to remove the adriamycin which is not encapsulated by the graft micelle, and collecting the supernatant to obtain the triphenylphosphine-chitosan stearic acid graft drug-loaded micelle.
The content of adriamycin in the drug-loaded micelle of the triphenylphosphine-chitosan stearic acid graft is determined by a fluorescence spectrophotometry method. 0.5mL of a 1mg/mL doxorubicin-containing dimethyl sulfoxide solution was precisely measured in a 10mL volumetric flask using a measuring instrument containing dimethyl sulfoxide: diluting with 9:1 water as solvent to scale, mixing, and using as mother liquor. Taking appropriate amount of mother liquor, diluting with solvent to obtain adriamycin solution of 0.05, 0.08, 0.1, 0.3, 0.5, 0.8, 1, 2 mug/mL, measuring fluorescence intensity of each concentration solution by a fluorescence spectrophotometer, wherein Em is 565nm, Ex is 505nm, slit is 5.0nm, working voltage is 700V, and plotting the fluorescence intensity as ordinate and the concentration as abscissa to obtain a standard curve.
The drug-loading rate and the encapsulation rate of the adriamycin in the triphenylphosphine-chitosan stearic acid drug-loaded micelle and the chitosan stearic acid drug-loaded micelle are respectively determined by an organic solvent extraction-ultrafiltration centrifugation method. Taking 10 mu L of 1mg/mL adriamycin drug-loaded nanoparticle solution, adding a solvent containing dimethyl sulfoxide: diluting the solution with 9:1 water as solvent to 1000 μ L, performing water bath ultrasound for 30 minutes, measuring the fluorescence intensity of the sample by a fluorescence spectrophotometer, and calculating the concentration of the free drug in the drug-loaded nanoparticle solution according to a standard curve. And placing 500 mu L of adriamycin drug-loaded nanoparticle solution into an ultrafiltration centrifugal tube, centrifuging at 10000rpm for 20 minutes, and taking filtrate to measure the concentration of unencapsulated free drug.
Entrapment efficiency ═ mass of doxorubicin in the drug-loaded graft micelle sample-mass of unencapsulated free doxorubicin)/mass of doxorubicin dosed × 100%
The drug loading rate (doxorubicin mass in drug-loaded graft micelle sample-unencapsulated free doxorubicin mass)/(doxorubicin mass in drug-loaded graft micelle sample-unencapsulated free doxorubicin mass + graft micelle mass) x 100%
The drug-loading rate of the triphenylphosphine-chitosan stearic acid drug-loaded micelle is calculated to be 4.3%, and the encapsulation rate is 89.6%; the drug loading rate of the chitosan stearic acid drug-loaded micelle is 3.9 percent, and the encapsulation rate is 81.5 percent.
Example 4
(1) Preparation of low molecular weight chitosan
Adding 50g of chitosan with the molecular weight of 450kDa and the deacetylation degree of 95 percent into 1500mL of hydrochloric acid aqueous solution with the volume ratio of 1.2 percent, stirring for 2 hours at the temperature of 55 ℃ to fully swell the chitosan, slowly adding chitosan enzyme solution with the weight ratio of 2 percent, carrying out chitosan enzymolysis reaction at the temperature of 55 ℃, and controlling the degradation degree of the chitosan by gel permeation chromatography. After the reaction is finished, stirring for 0.5 hour at 80 ℃, adding activated carbon with the weight/volume ratio of 0.3 percent, diluting the reaction solution, filtering by using a Buchner funnel, treating the filtrate by using a 0.45 mu m microporous filter membrane, and freeze-drying to obtain the low-molecular-weight chitosan, wherein the weight-average molecular weight of the obtained chitosan is 19.0 kDa.
(2) Synthesis of chitosan stearic acid graft
Dissolving the chitosan with the molecular weight of 19.0kDa in distilled water by ultrasonic treatment to prepare a solution with the concentration of 20 mg/ml. And mixing stearic acid according to the molar ratio of 25:1 of stearic acid to chitosan, carbodiimide according to the molar ratio of 6:1 of carbodiimide to stearic acid, and mixing ethanol and distilled water according to the volume ratio of 1:2 adding ethanol, dissolving with ultrasonic wave in water bath, and stirring at 60 deg.C for 50 min. Slowly adding the mixture into a preheated chitosan aqueous solution at 60 ℃, stirring and reacting for 12 hours at 60 ℃, cooling to room temperature, placing the final reaction solution into a dialysis bag with the molecular cut-off of 7000Da, dialyzing for 72 hours with distilled water, and washing the dialyzate with absolute ethyl alcohol to remove residual stearic acid after freeze drying to obtain the chitosan stearic acid graft.
(3) Synthesis of triphenylphosphine-chitosan stearic acid graft
Dissolving tetracarboxybutyltriphenylphosphine in a small amount of anhydrous dimethyl sulfoxide, and dissolving the solution by ultrasonic treatment to prepare a 10mg/ml solution. And further according to carbodiimide: the molar ratio of the tetracarboxybutyl triphenylphosphine is 3: 1, taking carbodiimide, and mixing the carbodiimide: the molar ratio of the tetracarboxyl butyl triphenylphosphine is 3: 1, adding N-hydroxysuccinimide into the solution, and activating for 2 hours to obtain reaction liquid 1. According to the following amino polyethylene glycol 2000: the molar ratio of the tetracarboxybutyl triphenylphosphine is 1: 1, dissolving amino polyethylene glycol 2000 in a small amount of anhydrous dimethyl sulfoxide, adding the solution into the reaction solution 1, and stirring the solution at room temperature for 9 hours to obtain a reaction solution 2. Adding a mixture of amino polyethylene glycol 2000 and amino polyethylene glycol in a molar ratio of 1: 1 was stirred at room temperature for 12 hours to obtain reaction solution 3. According to the free amino group on the stearic acid of the chitosan: the molar ratio of the tetracarboxybutyl triphenylphosphine is 20: 1, dissolving chitosan stearic acid graft in a small amount of deionized water. The reaction solution 3 was added to the aqueous solution of chitosan stearic acid graft and stirred at room temperature for 24 hours. And (3) placing the final reaction solution into a dialysis bag with the molecular weight cutoff of 7000, dialyzing with pure water for 48 hours, and freeze-drying to obtain triphenylphosphine-chitosan stearic acid solid powder.
And (3) determining the amino substitution degree of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid graft by adopting a trinitrobenzene sulfonic acid method. Dissolving 1-10 mg of chitosan with different weights in 2mL of distilled water respectively, adding 2mL of 4% sodium bicarbonate and 2mL of 0.1% trinitrobenzene sulfonic acid, incubating for 2 hours at 37 ℃, adding 2mL of 2mol/L hydrochloric acid, shaking up, measuring absorbance at 344nm, and preparing a standard curve. Respectively dissolving 6mg of each of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid in 3mL of distilled water, and calculating according to a standard curve by the same method, wherein the amino substitution degree of the triphenylphosphine-chitosan stearic acid graft is 20.7%, the amino substitution degree of the chitosan stearic acid graft is 16.8%, and the triphenylphosphine modification proportion is 3.9%.
(4) Physical and chemical properties of triphenylphosphine-chitosan stearic acid graft
And (3) measuring the tetracarboxybutyl triphenylphosphine, amino polyethylene glycol, chitosan stearic acid graft and triphenylphosphine-chitosan stearic acid graft by using a nuclear magnetic resonance spectrometry. 10mg of each of the four-carboxybutyltriphenylphosphine, aminopolyethylene glycol, chitosan stearic acid graft and triphenylphosphine-chitosan stearic acid graft was weighed, dissolved in 0.5mL of D2O, and measured by nuclear magnetic resonance 1H-NMR. Referring to fig. 1, a is tetracarboxylbutyl triphenylphosphine, B is aminopolyethylene glycol, C is chitosan stearic acid graft, and D is triphenylphosphine-chitosan stearic acid graft. The synthesis of triphenylphosphine-chitosan stearic acid graft can be determined from the figure.
Measuring the critical micelle concentration of the triphenylphosphine-chitosan stearic acid graft by adopting a pyrene fluorescence method, precisely weighing 12mg of pyrene, placing the pyrene into a 100mL volumetric flask, adding acetone to dissolve and fix the volume, transferring 1mL of the pyrene solution, placing the pyrene solution into the 100mL volumetric flask to dilute and fix the volume, transferring 0.5mL of the diluted pyrene solution, respectively placing the pyrene solution into 10mL glass test tubes, volatilizing the acetone at 50 ℃, respectively adding 5mL of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid graft with different concentrations, and controlling the final concentration of the pyrene to be 7 × 10-7And (3) carrying out ultrasonic treatment for 30min in a room-temperature water bath at mol/L. Scanning the excitation and emission spectra of pyrene, where Ex ═ 337nm, Em: the fluorescence intensity was measured at 374nm I1, 384nm I3, 2.5nm and 10nm slits and calculated to be 29.2 μ g/mL critical micelle concentration of chitosan stearic acid and 62.4 μ g/mL critical micelle concentration of triphenylphosphine-chitosan stearic acid.
Dissolving triphenylphosphine-chitosan stearic acid graft and chitosan stearic acid graft in distilled water, performing ultrasonic treatment for 30 times with power of 400w for 2s, and performing intermittent treatment for 3s to obtain 1mg/mL graft micelle solution. The particle size and surface potential of the particles are measured by an analyzer, the particle size of the chitosan stearic acid is 91.2 +/-0.93 nm, and the Zeta potential is 16.9 +/-0.40 mV. The particle size of the triphenylphosphine-chitosan stearic acid graft is 100.4 +/-23.1 nm, and the Zeta potential is 23.7 +/-0.95 mV.
(5) Cellular uptake and mitochondrial co-localization of triphenylphosphine-chitosan stearic acid grafts
The glycolipid graft fluorescence labeling micelle is adopted for cell uptake and mitochondria co-localization research. Marking chitosan stearic acid graft and triphenylphosphine-chitosan stearic acid graft with Fluorescein Isothiocyanate (FITC). Respectively taking the chitosan stearic acid graft or the triphenylphosphine-chitosan stearic acid graft carrier, dissolving in deionized water, and preparing into a glycolipid graft carrier solution of 2 mg/mL. And dissolving FITC in absolute ethyl alcohol to prepare a solution of 2 mg/mL. At 400rpm, 40 μ of the ethanol solution of LFITC was slowly dropped into the glycolipid graft carrier solution, and the solution was stirred for 4 hours in the dark. Then, the mixture was placed in a dialysis bag having a molecular weight cut-off of 3500, and dialyzed with deionized water for 8 hours. And (4) centrifuging the dialyzed and purified product at 8000rpm for 10min, and taking supernatant to obtain the FITC-labeled glycolipid graft carrier.
Taking MCF-7 cells with good growth state, and adding 2 × 105The cells were seeded in 6-well slide-coated cell culture plates at a density of 5% CO at 37 ℃%2Culturing until the fusion degree reaches 75%, and adding FITC marked chitosan stearic acid graft or triphenylphosphine-chitosan stearic acid graft carrier solution. Incubation was continued for 4, 12, and 24 hours, respectively, and cells were collected. And (3) quantitatively detecting the cell uptake conditions of the FITC-chitosan stearic acid graft and the FITC-triphenylphosphine-chitosan stearic acid graft carrier by using a flow cytometer. The results are shown in FIG. 2.
Taking MCF-7 cells with good growth state, and collecting the cells at 5 × 104The cells were seeded in 24-well glass slide-coated cell culture plates at a density of 5% CO at 37 ℃%2Culturing until the fusion degree reaches 75%, and adding FITC marked chitosan stearic acid graft or triphenylphosphine-chitosan stearic acid graft carrier solution. And continuously and respectively incubating for 24 hours, sucking out the culture medium, adding a phenol red-free DMEM culture solution containing the mitochondrial probe, incubating for 30min, and washing for three times by PBS. Fixing the cover glass with 4% paraformaldehyde in dark, taking out after 20min, embedding glycerol on the glass slide, and sealing. FITC-chitosan stearic acid grafting observed by laser confocal scanning microscope andthe cellular uptake and mitochondrial co-localization of FITC-triphenylphosphine-chitosan stearic acid graft vector were analyzed by Imaris software for co-localization coefficient, and the results are shown in FIG. 3.
As shown in fig. 3, through analysis and calculation by Imaris software, the co-localization coefficient of triphenylphosphine-chitosan stearic acid graft and mitochondria was 0.49, which is significantly different from chitosan stearic acid. The result shows that the triphenylphosphine-chitosan stearic acid grafting has a high-efficiency mitochondrion targeting function.
(6) Preparation of triphenylphosphine-chitosan stearic acid graft drug-loaded micelle
Base adriamycin preparation: weighing 200mg of doxorubicin hydrochloride, dissolving the doxorubicin hydrochloride in 20mL of dimethyl sulfoxide, adding triethylamine, stirring the mixture overnight, placing the reaction solution in a dialysis bag, dialyzing the reaction solution with distilled water for 48 hours, collecting a product in the dialysis bag, centrifuging the product for 10min at 8000r, collecting a precipitate, dissolving the precipitate with water, repeating the steps for three times to remove residual doxorubicin hydrochloride, and freeze-drying to obtain the basic group doxorubicin.
Weighing triphenylphosphine-chitosan stearic acid graft, dissolving in deionized water, performing ultrasonic treatment for 30 times by using a probe, wherein the power is 400w, the work time is 2s, and the work time is 3s, and preparing graft micelle solution of 2 mg/mL. And adding dimethyl sulfoxide into basic group adriamycin to prepare a solution with the concentration of 2 mg/mL. Adding dimethyl sulfoxide solution containing 2mg/mL adriamycin according to the dose of 10% of adriamycin to the graft by mass, stirring for 2 hours at room temperature in a dark place, transferring to a dialysis bag with the molecular weight cutoff of 3500 after the reaction is finished, dialyzing for 24 hours by pure water, collecting the dialyzed product, centrifuging for 10 minutes at a low temperature of 8000r to remove the adriamycin which is not encapsulated by the graft micelle, and collecting the supernatant to obtain the triphenylphosphine-chitosan stearic acid graft drug-loaded micelle.
The content of adriamycin in the drug-loaded micelle of the triphenylphosphine-chitosan stearic acid graft is determined by a fluorescence spectrophotometry method. 0.5mL of a 1mg/mL doxorubicin-containing dimethyl sulfoxide solution was precisely measured in a 10mL volumetric flask using a measuring instrument containing dimethyl sulfoxide: diluting with 9:1 water as solvent to scale, mixing, and using as mother liquor. Taking appropriate amount of mother liquor, diluting with solvent to obtain adriamycin solution of 0.05, 0.08, 0.1, 0.3, 0.5, 0.8, 1, 2 mug/mL, measuring fluorescence intensity of each concentration solution by a fluorescence spectrophotometer, wherein Em is 565nm, Ex is 505nm, slit is 5.0nm, working voltage is 700V, and plotting the fluorescence intensity as ordinate and the concentration as abscissa to obtain a standard curve.
The drug-loading rate and the encapsulation rate of the adriamycin in the triphenylphosphine-chitosan stearic acid drug-loaded micelle and the chitosan stearic acid drug-loaded micelle are respectively determined by an organic solvent extraction-ultrafiltration centrifugation method. Taking 10 mu L of 1mg/mL adriamycin drug-loaded nanoparticle solution, adding a solvent containing dimethyl sulfoxide: diluting the solution with 9:1 water as solvent to 1000 μ L, performing water bath ultrasound for 30 minutes, measuring the fluorescence intensity of the sample by a fluorescence spectrophotometer, and calculating the concentration of the free drug in the drug-loaded nanoparticle solution according to a standard curve. And placing 500 mu L of adriamycin drug-loaded nanoparticle solution into an ultrafiltration centrifugal tube, centrifuging at 10000rpm for 20 minutes, and taking filtrate to measure the concentration of unencapsulated free drug.
Entrapment efficiency ═ mass of doxorubicin in the drug-loaded graft micelle sample-mass of unencapsulated free doxorubicin)/mass of doxorubicin dosed × 100%
The drug loading rate (doxorubicin mass in drug-loaded graft micelle sample-unencapsulated free doxorubicin mass)/(doxorubicin mass in drug-loaded graft micelle sample-unencapsulated free doxorubicin mass + graft micelle mass) x 100%.
The drug-loading rate of the triphenylphosphine-chitosan stearic acid drug-loaded micelle is calculated to be 8.1%, and the encapsulation rate is 87.9%; the drug loading rate of the chitosan stearic acid drug-loaded micelle is 7.4 percent, and the encapsulation rate is 79.5 percent.
Example 5
(1) Preparation of low molecular weight chitosan
Adding 50g of chitosan with the molecular weight of 450kDa and the deacetylation degree of 95 percent into 1500mL of hydrochloric acid aqueous solution with the volume ratio of 1.2 percent, stirring for 2 hours at the temperature of 55 ℃ to fully swell the chitosan, slowly adding chitosan enzyme solution with the weight ratio of 2 percent, carrying out chitosan enzymolysis reaction at the temperature of 55 ℃, and controlling the degradation degree of the chitosan by gel permeation chromatography. After the reaction is finished, stirring for 0.5 hour at 80 ℃, adding activated carbon with the weight/volume ratio of 0.3 percent, diluting the reaction solution, filtering by using a Buchner funnel, treating the filtrate by using a 0.45 mu m microporous filter membrane, and freeze-drying to obtain the low-molecular-weight chitosan, wherein the weight-average molecular weight of the obtained chitosan is 19.0 kDa.
(2) Synthesis of chitosan stearic acid graft
Dissolving the chitosan with the molecular weight of 19.0kDa in distilled water by ultrasonic treatment to prepare a solution with the concentration of 20 mg/ml. And mixing stearic acid according to the molar ratio of 25:1 of stearic acid to chitosan, carbodiimide according to the molar ratio of 6:1 of carbodiimide to stearic acid, and mixing ethanol and distilled water according to the volume ratio of 1:2 adding ethanol, dissolving with ultrasonic wave in water bath, and stirring at 60 deg.C for 50 min. Slowly adding the mixture into a preheated chitosan aqueous solution at 60 ℃, stirring and reacting for 12 hours at 60 ℃, cooling to room temperature, placing the final reaction solution into a dialysis bag with the molecular cut-off of 7000Da, dialyzing for 72 hours with distilled water, and washing the dialyzate with absolute ethyl alcohol to remove residual stearic acid after freeze drying to obtain the chitosan stearic acid graft.
(3) Synthesis of triphenylphosphine-chitosan stearic acid graft
Dissolving tetracarboxybutyltriphenylphosphine in a small amount of anhydrous dimethyl sulfoxide, and dissolving the solution by ultrasonic treatment to prepare a 10mg/ml solution. And further according to carbodiimide: the molar ratio of the tetracarboxybutyl triphenylphosphine is 3: 1, taking carbodiimide, and mixing the carbodiimide: the molar ratio of the tetracarboxyl butyl triphenylphosphine is 3: 1, adding N-hydroxysuccinimide into the solution, and activating for 2 hours to obtain reaction liquid 1. According to the following amino polyethylene glycol 2000: the molar ratio of the tetracarboxybutyl triphenylphosphine is 1: 1, dissolving amino polyethylene glycol 2000 in a small amount of anhydrous dimethyl sulfoxide, adding the solution into the reaction solution 1, and stirring the solution at room temperature for 9 hours to obtain a reaction solution 2. Adding a mixture of amino polyethylene glycol 2000 and amino polyethylene glycol in a molar ratio of 1: 1 was stirred at room temperature for 12 hours to obtain reaction solution 3. According to the free amino group on the stearic acid of the chitosan: the molar ratio of the tetracarboxybutyl triphenylphosphine is 20: 1, dissolving chitosan stearic acid graft in a small amount of deionized water. The reaction solution 3 was added to the aqueous solution of chitosan stearic acid graft and stirred at room temperature for 24 hours. And (3) placing the final reaction solution into a dialysis bag with the molecular weight cutoff of 7000, dialyzing with pure water for 48 hours, and freeze-drying to obtain triphenylphosphine-chitosan stearic acid solid powder.
And (3) determining the amino substitution degree of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid graft by adopting a trinitrobenzene sulfonic acid method. Dissolving 1-10 mg of chitosan with different weights in 2mL of distilled water respectively, adding 2mL of 4% sodium bicarbonate and 2mL of 0.1% trinitrobenzene sulfonic acid, incubating for 2 hours at 37 ℃, adding 2mL of 2mol/L hydrochloric acid, shaking up, measuring absorbance at 344nm, and preparing a standard curve. Respectively dissolving 6mg of each of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid in 3mL of distilled water, and calculating according to a standard curve by the same method, wherein the amino substitution degree of the triphenylphosphine-chitosan stearic acid graft is 20.7%, the amino substitution degree of the chitosan stearic acid graft is 16.8%, and the triphenylphosphine modification proportion is 3.9%.
(4) Physical and chemical properties of triphenylphosphine-chitosan stearic acid graft
And (3) measuring the tetracarboxybutyl triphenylphosphine, amino polyethylene glycol, chitosan stearic acid graft and triphenylphosphine-chitosan stearic acid graft by using a nuclear magnetic resonance spectrometry. 10mg of each of the four-carboxybutyltriphenylphosphine, aminopolyethylene glycol, chitosan stearic acid graft and triphenylphosphine-chitosan stearic acid graft was weighed, dissolved in 0.5mL of D2O, and measured by nuclear magnetic resonance 1H-NMR. Referring to fig. 1, a is tetracarboxylbutyl triphenylphosphine, B is aminopolyethylene glycol, C is chitosan stearic acid graft, and D is triphenylphosphine-chitosan stearic acid graft. The synthesis of triphenylphosphine-chitosan stearic acid graft can be determined from the figure.
Measuring the critical micelle concentration of the triphenylphosphine-chitosan stearic acid graft by adopting a pyrene fluorescence method, precisely weighing 12mg of pyrene, placing the pyrene into a 100mL volumetric flask, adding acetone to dissolve and fix the volume, transferring 1mL of the pyrene solution, placing the pyrene solution into the 100mL volumetric flask to dilute and fix the volume, transferring 0.5mL of the diluted pyrene solution, respectively placing the pyrene solution into 10mL glass test tubes, volatilizing the acetone at 50 ℃, respectively adding 5mL of the triphenylphosphine-chitosan stearic acid graft and the chitosan stearic acid graft with different concentrations, and controlling the final concentration of the pyrene to be 7 × 10-7And (3) carrying out ultrasonic treatment for 30min in a room-temperature water bath at mol/L. Scanning excitation and emission spectra of pyreneSpectrum, where Ex ═ 337nm, Em: the fluorescence intensity was measured at 374nm I1, 384nm I3, 2.5nm and 10nm slits and calculated to be 29.2 μ g/mL critical micelle concentration of chitosan stearic acid and 62.4 μ g/mL critical micelle concentration of triphenylphosphine-chitosan stearic acid. Dissolving triphenylphosphine-chitosan stearic acid graft and chitosan stearic acid graft in distilled water, performing ultrasonic treatment for 30 times with power of 400w for 2s, and performing intermittent treatment for 3s to obtain 1mg/mL graft micelle solution. The particle size and surface potential of the particles are measured by an analyzer, the particle size of the chitosan stearic acid is 91.2 +/-0.93 nm, and the Zeta potential is 16.9 +/-0.40 mV. The particle size of the triphenylphosphine-chitosan stearic acid graft is 100.4 +/-23.1 nm, and the Zeta potential is 23.7 +/-0.95 mV.
(5) Cellular uptake and mitochondrial co-localization of triphenylphosphine-chitosan stearic acid grafts
The glycolipid graft fluorescence labeling micelle is adopted for cell uptake and mitochondria co-localization research. Marking chitosan stearic acid graft and triphenylphosphine-chitosan stearic acid graft with Fluorescein Isothiocyanate (FITC). Respectively taking the chitosan stearic acid graft or the triphenylphosphine-chitosan stearic acid graft carrier, dissolving in deionized water, and preparing into a glycolipid graft carrier solution of 2 mg/mL. And dissolving FITC in absolute ethyl alcohol to prepare a solution of 2 mg/mL. At 400rpm, 40 μ of the ethanol solution of LFITC was slowly dropped into the glycolipid graft carrier solution, and the solution was stirred for 4 hours in the dark. Then, the mixture was placed in a dialysis bag having a molecular weight cut-off of 3500, and dialyzed with deionized water for 8 hours. And (4) centrifuging the dialyzed and purified product at 8000rpm for 10min, and taking supernatant to obtain the FITC-labeled glycolipid graft carrier.
Taking MCF-7 cells with good growth state, and adding 2 × 105The cells were seeded in 6-well slide-coated cell culture plates at a density of 5% CO at 37 ℃%2Culturing until the fusion degree reaches 75%, and adding FITC marked chitosan stearic acid graft or triphenylphosphine-chitosan stearic acid graft carrier solution. Incubation was continued for 4, 12, and 24 hours, respectively, and cells were collected. And (3) quantitatively detecting the cell uptake conditions of the FITC-chitosan stearic acid graft and the FITC-triphenylphosphine-chitosan stearic acid graft carrier by using a flow cytometer. The results are shown in FIG. 2.
Taking MCF-7 cells with good growth state, and collecting the cells at 5 × 104The cells were seeded in 24-well glass slide-coated cell culture plates at a density of 5% CO at 37 ℃%2Culturing until the fusion degree reaches 75%, and adding FITC marked chitosan stearic acid graft or triphenylphosphine-chitosan stearic acid graft carrier solution. And continuously and respectively incubating for 24 hours, sucking out the culture medium, adding a phenol red-free DMEM culture solution containing the mitochondrial probe, incubating for 30min, and washing for three times by PBS. Fixing the cover glass with 4% paraformaldehyde in dark, taking out after 20min, embedding glycerol on the glass slide, and sealing. The cell uptake and mitochondria co-localization conditions of the FITC-chitosan stearic acid graft and the FITC-triphenylphosphine-chitosan stearic acid graft carrier are observed by a laser confocal scanning microscope, the co-localization coefficient is analyzed by Imaris software, and the result is shown in figure 3.
As shown in fig. 3, through analysis and calculation by Imaris software, the co-localization coefficient of triphenylphosphine-chitosan stearic acid graft and mitochondria was 0.49, which is significantly different from chitosan stearic acid. The result shows that the triphenylphosphine-chitosan stearic acid grafting has a high-efficiency mitochondrion targeting function.
(6) Preparation of triphenylphosphine-chitosan stearic acid graft drug-loaded micelle
Base adriamycin preparation: weighing 200mg of doxorubicin hydrochloride, dissolving the doxorubicin hydrochloride in 20mL of dimethyl sulfoxide, adding triethylamine, stirring the mixture overnight, placing the reaction solution in a dialysis bag, dialyzing the reaction solution with distilled water for 48 hours, collecting a product in the dialysis bag, centrifuging the product for 10min at 8000r, collecting a precipitate, dissolving the precipitate with water, repeating the steps for three times to remove residual doxorubicin hydrochloride, and freeze-drying to obtain the basic group doxorubicin.
Weighing triphenylphosphine-chitosan stearic acid graft, dissolving in deionized water, performing ultrasonic treatment for 30 times by using a probe, wherein the power is 400w, the work time is 2s, and the work time is 3s, and preparing graft micelle solution of 2 mg/mL. And adding dimethyl sulfoxide into basic group adriamycin to prepare a solution with the concentration of 2 mg/mL. Adding dimethyl sulfoxide solution containing 2mg/mL adriamycin according to the dose of 15% of adriamycin to the graft by mass, stirring for 2 hours at room temperature in a dark place, transferring to a dialysis bag with the molecular weight cutoff of 3500 after the reaction is finished, dialyzing for 24 hours by pure water, collecting the dialyzed product, centrifuging for 10 minutes at a low temperature of 8000r to remove the adriamycin which is not encapsulated by the graft micelle, and collecting the supernatant to obtain the triphenylphosphine-chitosan stearic acid graft drug-loaded micelle.
The content of adriamycin in the drug-loaded micelle of the triphenylphosphine-chitosan stearic acid graft is determined by a fluorescence spectrophotometry method. 0.5mL of a 1mg/mL doxorubicin-containing dimethyl sulfoxide solution was precisely measured in a 10mL volumetric flask using a measuring instrument containing dimethyl sulfoxide: diluting with 9:1 water as solvent to scale, mixing, and using as mother liquor. Taking appropriate amount of mother liquor, diluting with solvent to obtain adriamycin solution of 0.05, 0.08, 0.1, 0.3, 0.5, 0.8, 1, 2 mug/mL, measuring fluorescence intensity of each concentration solution by a fluorescence spectrophotometer, wherein Em is 565nm, Ex is 505nm, slit is 5.0nm, working voltage is 700V, and plotting the fluorescence intensity as ordinate and the concentration as abscissa to obtain a standard curve.
The drug-loading rate and the encapsulation rate of the adriamycin in the triphenylphosphine-chitosan stearic acid drug-loaded micelle and the chitosan stearic acid drug-loaded micelle are respectively determined by an organic solvent extraction-ultrafiltration centrifugation method. Taking 10 mu L of 1mg/mL adriamycin drug-loaded nanoparticle solution, adding a solvent containing dimethyl sulfoxide: diluting the solution with 9:1 water as solvent to 1000 μ L, performing water bath ultrasound for 30 minutes, measuring the fluorescence intensity of the sample by a fluorescence spectrophotometer, and calculating the concentration of the free drug in the drug-loaded nanoparticle solution according to a standard curve. And placing 500 mu L of adriamycin drug-loaded nanoparticle solution into an ultrafiltration centrifugal tube, centrifuging at 10000rpm for 20 minutes, and taking filtrate to measure the concentration of unencapsulated free drug.
Entrapment efficiency ═ mass of doxorubicin in the drug-loaded graft micelle sample-mass of unencapsulated free doxorubicin)/mass of doxorubicin dosed × 100%
The drug loading rate (doxorubicin mass in drug-loaded graft micelle sample-unencapsulated free doxorubicin mass)/(doxorubicin mass in drug-loaded graft micelle sample-unencapsulated free doxorubicin mass + graft micelle mass) x 100%.
The drug-loading rate of the triphenylphosphine-chitosan stearic acid drug-loaded micelle is calculated to be 10.9%, and the encapsulation rate is 81.3%; the drug loading rate of the chitosan stearic acid drug-loaded micelle is 10.2 percent, and the encapsulation rate is 75.8 percent.
Preparing 1mg/mL triphenylphosphine-chitosan stearic acid graft drug-loaded micelle and chitosan stearic acid graft drug-loaded micelle solution, and respectively measuring the particle size and surface potential of the drug-loaded micelle by using a particle size and surface potential measuring instrument. The particle size of the chitosan stearic acid drug-loaded micelle is measured to be 64.2 +/-2.80 nm, and the Zeta potential is 11.6 +/-1.48 mV. The particle size of the triphenylphosphine-chitosan stearic acid drug-loaded micelle is 67.2 +/-2.82 nm, and the Zeta potential is 18.7 +/-1.78 mV.
(7) Examination of in vitro release behavior of triphenylphosphine-chitosan stearic acid graft drug-loaded micelle takes 1mg/mL triphenylphosphine-chitosan stearic acid drug-loaded micelle and 1mL chitosan stearic acid drug-loaded micelle solution respectively, puts the triphenylphosphine-chitosan stearic acid drug-loaded micelle and the chitosan stearic acid drug-loaded micelle solution into a dialysis bag with the molecular cut-off of 7000, puts the dialysis bag into a centrifuge tube filled with 20mL phosphate buffer solution with pH of 6.8, and puts the centrifuge tube into a shaking table with the temperature of 37 ℃ for oscillation. Samples were taken at various time points, all release medium was discarded after sampling, 20mL of fresh medium was added and sampling was continued for 3 days. The concentration of the drug in the sample was measured by fluorescence spectrophotometry, where Ex is 505nm, Em is 565nm, slit is 5nm, and voltage is 700 v. The release profile is shown in fig. 4, and the results show that both the triphenylphosphine-chitosan stearic acid drug-loaded micelles and the chitosan stearic acid drug-loaded micelles released faster within the first 12 hours, and the subsequent drug release was slower and continued for up to 72 hours. The cumulative release of the two drug-loaded micelles is 41.9 percent and 32.96 percent respectively.
(8) Evaluation of antitumor effect of triphenylphosphine-chitosan stearic acid graft drug-loaded micelle
The invention uses tumor cell inhibition rate IC50The cell survival rate test adopts tetrazolium salt colorimetric method to determine, MCF-7 cell is taken as a model, 200 mu L of cell culture plate containing 4 × 10 is added into each hole3The culture solution of MCF-7 cells is placed at 37 ℃ and 5% CO2Culturing in incubator for 24 hr, adding free medicinal solution, chitosan stearic acid drug-loaded micelle, and triphenylphosphine-chitosan stearic acid drug-loaded micelle with different concentrations into cell hole after cell adherenceA bundle, using untreated blank cells as a control, and arranging multiple wells in each well; after 48 hours of incubation, 20. mu.L of 5mg/mL thiazole blue solution was added to each well, the incubation was continued for 4 hours and the supernatant was discarded, 200. mu.L of dimethyl sulfoxide was added to each well, absorbance was measured with an enzyme-linked detector, and the cell survival rate was calculated according to the following formula:
cell viability (%). test absorbance/control absorbance. times.100%
The cell survival rates of the free drug, the chitosan stearic acid drug-loaded micelle and the triphenylphosphine-chitosan stearic acid drug-loaded micelle on MCF-7 cells are shown in figure 5. Through calculation, the free drug, the chitosan stearic acid drug-loaded micelle and the triphenylphosphine-chitosan stearic acid drug-loaded micelle have IC on MCF-7 cells50The results were 4.61. mu.g/mL, 3.93. mu.g/mL, and 1.51. mu.g/mL, respectively. Research results show that the triphenylphosphine-chitosan stearic acid drug-loaded micelle has the most obvious inhibition effect on MCF-7 cells and has a better anti-tumor effect.
Claims (3)
1. A preparation method of triphenylphosphine-chitosan stearic acid graft, wherein the molecular weight of chitosan is 19kDa, the carbon chain length of fatty acid is eighteen carbons, the deacetylation degree of chitosan is 95%, the amino substitution degree is 18.0-20.7%, the modification proportion of triphenylphosphine is 1.2-3.9%, and the typical chemical structural general formula is as follows:
the method is characterized by comprising the following steps:
(1) synthesizing a chitosan stearic acid graft:
taking chitosan with the molecular weight of 19kDa, adding distilled water for ultrasonic dissolution, preparing a solution with the molecular weight of 20mg/ml, taking stearic acid according to the molar ratio of 25:1 of stearic acid to chitosan, and taking the mixture of carbodiimide and stearic acid according to the molar ratio of 6:1, mixing carbodiimide, and mixing according to the volume ratio of ethanol to distilled water of 1:2, adding ethanol, ultrasonically dissolving in a water bath, stirring for 50min at 60 ℃, then slowly adding the mixture into a chitosan aqueous solution preheated at 60 ℃, stirring and reacting for 12 hours at 60 ℃, cooling to room temperature, placing the final reaction solution into a dialysis bag with the molecular cut-off of 7000Da, dialyzing for 72 hours with distilled water, and washing with absolute ethanol to remove residual stearic acid after freeze drying of the dialysate to obtain a chitosan stearic acid graft;
(2) synthesis of triphenylphosphine-chitosan stearic acid graft
Dissolving tetracarboxybutyl triphenylphosphine in a small amount of anhydrous dimethyl sulfoxide, and dissolving the solution by ultrasonic treatment to prepare a 10mg/ml solution, wherein the ratio of carbodiimide: the molar ratio of the tetracarboxybutyl triphenylphosphine is 3: 1, taking carbodiimide, and mixing the carbodiimide: the molar ratio of the tetracarboxyl butyl triphenylphosphine is 3: 1, adding N-hydroxysuccinimide into the solution, activating for 2 hours to obtain reaction liquid 1, and adding amino polyethylene glycol 2000: the molar ratio of the tetracarboxybutyl triphenylphosphine is 1: 1, dissolving amino polyethylene glycol 2000 in a small amount of anhydrous dimethyl sulfoxide, adding the solution into the reaction solution 1, stirring the solution at room temperature for 9 hours to obtain a reaction solution 2, and adding a solvent into the reaction solution 2, wherein the molar ratio of the solvent to the amino polyethylene glycol 2000 is 1: 1, stirring for 12 hours at room temperature to obtain reaction liquid 3, and reacting according to the ratio of free amino on chitosan stearic acid: the molar ratio of the tetracarboxyl butyl triphenylphosphine is 5: 1-20: 1, dissolving chitosan stearic acid graft in deionized water, adding reaction liquid 3 into a chitosan stearic acid graft aqueous solution, stirring at room temperature for 24 hours, placing final reaction liquid into a dialysis bag with the cut-off molecular weight of 7000, dialyzing with the pure water for 48 hours, and freeze-drying to obtain triphenylphosphine-chitosan stearic acid; the synthetic route is as follows:
2. a preparation method of a triphenylphosphine-chitosan stearic acid graft drug-loaded micelle is characterized in that a drug encapsulated by the graft is adriamycin, and the preparation method is realized by the following steps:
taking 2mg/mL of the triphenylphosphine-chitosan stearic acid graft micelle solution of claim 1, adding a dimethyl sulfoxide solution containing 2mg/mL of basic group doxorubicin according to the mass ratio of the doxorubicin to the graft of 5-15%, stirring at room temperature in a dark place for 2 hours, transferring into a dialysis bag with the cut-off molecular weight of 3500, dialyzing for 24 hours by pure water, collecting the dialyzed product, centrifuging at 8000rpm for 10min at low temperature, removing the doxorubicin which is not encapsulated by the graft micelle, and collecting the supernatant to obtain a target triphenylphosphine-chitosan stearic acid graft drug-loaded micelle; in the triphenylphosphine-chitosan stearic acid graft drug-loaded micelle, the adriamycin comprises the following components in percentage by weight: 4.3 to 10.9 percent.
3. The application of the triphenylphosphine-chitosan stearic acid grafted drug-loaded micelle prepared according to the method of claim 2 in preparing a mitochondrion-targeted antitumor drug.
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