CN107412162B - Method for improving drug loading capacity of camptothecin in block copolymer micelle - Google Patents
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Abstract
The invention discloses a method for improving the drug loading capacity of camptothecin in a segmented copolymer micelle, which comprises the steps of firstly modifying camptothecin by cystamine, then preparing the modified camptothecin and a prepared PC L-PEG-PC L triblock copolymer into drug-loaded micelles, improving the water solubility of hydrophobic camptothecin by the method, improving the encapsulation rate to 60-80% from 10% before modification, and improving the drug loading capacity to 7-8% from 1%.
Description
Technical Field
The invention relates to a method for improving the drug loading capacity of camptothecin in a segmented copolymer micelle and endowing the drug loading system with GSH response release performance, in particular to a method for modifying camptothecin by cystamine with disulfide bonds and encapsulating the camptothecin by a PC L-PEG-PC L triblock copolymer micelle.
Background
Camptothecin is a hydrophobic anticancer drug extracted from plants, has inhibiting effect on various animal tumors, has good effect on gastrointestinal tract and head and neck cancers, and has no cross drug resistance with common antitumor drugs. Disulfide bonds generally refer in the field of biochemistry to bonds in cysteine residues in peptide and protein molecules. The reducing substances Glutathione (GSH), dithiothreitol, etc. can reduce disulfide bond into sulfhydryl (-SH) group. Due to the high content of GSH in the tumor cells, the drug carrier containing disulfide bonds can respond to and release drugs in the tumor cells. The amphiphilic block copolymer refers to a special polymer which contains a hydrophilic chain segment and a hydrophobic chain segment in the same macromolecule and has affinity effect on both a water phase and an oil phase. The amphiphilic block copolymer can be self-assembled to form a micelle, and the hydrophobic drug is wrapped in the cavity of the micelle, so that the concentration of the hydrophobic drug in the solution can be improved. The size, structure, shape and aggregation number of the micelle can be regulated and controlled by changing parameters such as molecular weight of each chain segment of the block copolymer, concentration of a medicament and the like, so that the micelle can be widely applied to various fields such as biochemistry, material preparation, separation and purification and the like.
Wang et al (Biomacromolecules,2008,9,388-395) adopts PEG-PC L-PEG triblock copolymer with the molecular weight of 15000-4000-15000 to encapsulate unmodified camptothecin, when the ratio of the drug to the block copolymer is 15:200, the drug loading rate of the drug is 6.1 +/-0.18%, and the encapsulation rate of the drug is 85.7 +/-0.1%, and the drug loading rate of hydroxycamptothecin in PEG-PC L polymer micelle determined by a Bingche method in a graduation paper HP L C method is reported to be 3.63% and 36.3%, but the drug loading micelle prepared by the method is low in drug loading rate or has no response release function, so that the application of the micelle in the anti-tumor field is limited.
Disclosure of Invention
The invention aims to solve the defects in the background technology, the camptothecin is modified, and then the modified camptothecin and the block copolymer are prepared into the drug-loaded micelle, so that the method for improving the drug-loaded amount of the modified camptothecin in a drug delivery system is achieved, and the introduction of the disulfide bond in the drug delivery system endows the drug carrier with the effect of responding and releasing the drug to high-concentration GSH in tumor cells.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for improving the drug loading capacity of camptothecin in a block copolymer micelle comprises the steps of modifying camptothecin by using cystamine, and preparing the modified camptothecin and a block copolymer into a drug-loaded micelle.
The block copolymer is a PC L-PEG-PC L triblock copolymer.
Preferably, the method for improving the drug loading of the camptothecin in the block copolymer micelle comprises the following steps:
s1 modification of cystamine of camptothecin
S1-1, adding excess triphosgene into cystamine dihydrochloride solution by taking N, N-diisopropylethylamine and 4-dimethylaminopyridine as catalysts, and connecting one end of the triphosgene to the cystamine dihydrochloride after reaction to generate a cystamine intermediate; then adding camptothecin into the cystamine intermediate, and connecting the camptothecin to two ends of the cystamine intermediate through a substitution reaction to generate a modified camptothecin-cystamine compound crude product;
s1-2, subjecting the obtained crude camptothecin-cystamine compound to silica gel column chromatography purification treatment to obtain a modified camptothecin purified product;
s2 preparation of Block copolymer
S2-1, adding polyethylene glycol (PEG) into anhydrous toluene, stirring for 1-1.5 hours at 80-90 ℃ to obtain a mixed solution, then adding caprolactone and stannous isooctanoate into the mixed solution, and stirring for 12-15 hours at 120-130 ℃ to obtain a crude product;
s2-2, distilling the crude product prepared in the step S2-2, pouring the crude product into a 50m L single-mouth bottle, carrying out reduced pressure distillation at the temperature of 30 ℃ and the rotating speed of 80r/min, removing toluene, and then precipitating the product by using cold ether to obtain a PC L-PEG-PC L triblock copolymer;
s3 preparation of drug-loaded micelle
And (3) mixing the camptothecin-cystamine compound prepared in the step S1 and the PC L-PEG-PC L triblock copolymer prepared in the step S2, dissolving the mixture in dichloromethane by ultrasonic, performing rotary evaporation for 1.5 to 2.5 hours to completely remove the solvent, then dropwise adding distilled water, and performing ultrasonic treatment to obtain the drug-loaded micelle.
The mass ratio of the N, N-diisopropylethylamine, the 4-dimethylaminopyridine, the triphosgene, the cystamine dihydrochloride and the camptothecin added in the step S1 is (0.5-2): (0.5-2): (0.2-1): (1-3): 1.
the cystamine dihydrochloride in the step S1 is cystamine dihydrochloride treated with sodium hydroxide.
The method for treating cystamine dihydrochloride with sodium hydroxide comprises the steps of accurately weighing 1.6g of sodium hydroxide and 4.5g of cystamine dihydrochloride, mixing, adding into a 100m L single-neck flask containing 50m L tetrahydrofuran, magnetically stirring for 48 hours at room temperature, filtering to remove a solid product after stirring is finished, and performing rotary evaporation to remove a solvent at 30 ℃ and 80r/min to obtain the required cystamine dihydrochloride.
The conditions for carrying out the silica gel column chromatography purification treatment on the crude product of the camptothecin-cystamine compound in the step S1-2 are as follows: the mobile phase is chloroform: methanol (6-8): (2-4) (volume ratio); the filler is column chromatography silica gel; the temperature was room temperature.
The mass ratio of the polyethylene glycol, the toluene, the caprolactone and the stannous isooctanoate added in the step S2-1 is (1-3): (20-40): 2: (0.01-0.03).
The distillation of the crude product obtained in step S2-2 is carried out by pouring the crude product into a 50m L single-neck flask, and distilling under reduced pressure at 30 deg.C and 80 r/min.
The molecular weight of the PC L-PEG-PC L triblock copolymer prepared in the step S2 is 5000-7000, wherein the ratio of the molecular weight of the PC L segment to the molecular weight of the PEG segment is 1.5-3.
The proportion of the camptothecin-cystamine compound, the PC L-PEG-PC L triblock copolymer and the dichloromethane added in the step S3 is (1-8) to 10: 250.
A drug-loaded micelle with a spherical structure and a particle size of 40-60 nm.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention takes cystamine as an intermediate, and the camptothecin is connected to two ends of the cystamine to improve the drug loading rate of the camptothecin in a PC L-PEG-PC L triblock copolymer micelle and improve the water solubility of the camptothecin, the encapsulation rate of the camptothecin can be improved to 60-80% from 10% before modification, and the drug loading rate is improved to 7-8% from 1%.
(2) The camptothecin modification method provided by the invention selects triphosgene as an intermediate, modifies the intermediate by cystamine, and firstly, excessive triphosgene reacts with cystamine, so that one end of the triphosgene is connected to the cystamine under the action of a catalyst, and the other end of the triphosgene is in a state to be reacted; reacting one end of the reaction with camptothecin under the action of a catalyst to obtain a crude product. And then separating and purifying the crude product by using a chromatographic silica gel column to obtain a purified product of the modified camptothecin. Because the functional group on the camptothecin is hydroxyl and the functional group on the cystamine is amino, two trichloromethyl groups on the triphosgene can simultaneously react with the hydroxyl and the amino, and the camptothecin is connected to two ends of the cystamine as an intermediate. The method is simple to operate and high in reaction efficiency.
(3) The drug-loaded micelle prepared by the fusion method has the nanoparticle with the particle size of 40-60nm, so that the drug-loaded micelle can be enriched in tumor tissues through passive targeting effect.
Drawings
FIG. 1 is a flow chart of the experimental principle used in example 1;
FIG. 2 is an optical photograph of the modified drug-loaded micelle obtained in example 1;
FIG. 3 is a transmission electron microscope image of the drug-loaded micelle obtained in example 1;
FIG. 4 is a D L S particle size distribution diagram of the drug-loaded micelle obtained in example 1;
FIG. 5 is a UV spectrum obtained in example 1;
FIG. 6 is a standard curve of high performance liquid chromatography obtained in example 1;
FIG. 7 is a graph of the high performance liquid chromatography loaded micelle sample obtained in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below. This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the invention to those skilled in the art, and the present invention will only be defined by the appended claims.
Example 1
The method for improving the drug loading of camptothecin in the PC L-PEG-PC L triblock copolymer comprises the following steps:
s1 modification of cystamine of camptothecin
S1-1, taking 0.5g of N, N-diisopropylethylamine and 0.5g of 4-dimethylaminopyridine as catalysts, adding 1g of triphosgene into a cystamine dihydrochloride solution containing 3g of cystamine dihydrochloride, and connecting one end of the triphosgene to the cystamine dihydrochloride after reaction to generate a cystamine intermediate; then adding 1g of camptothecin into the cystamine intermediate, and connecting the camptothecin to two ends of the cystamine intermediate through a substitution reaction to generate a modified camptothecin-cystamine compound crude product;
s1-2, subjecting the obtained crude camptothecin-cystamine compound to silica gel column chromatography purification treatment to obtain a modified camptothecin purified product;
the conditions of the purification treatment of the chromatographic silica gel column are as follows: the mobile phase is chloroform: methanol is 7:3 (volume ratio), the filler is column chromatography silica gel, and the temperature is room temperature;
s2 preparation of Block copolymer
S2-1, adding 2g of polyethylene glycol (PEG) into 20g of anhydrous toluene, stirring for 1-1.5 hours at 80-90 ℃ to obtain a mixed solution, then adding 2g of caprolactone and 0.03g of stannous isooctanoate into the mixed solution, and stirring for 12-15 hours at 120-130 ℃ to obtain a crude product;
s2-2, distilling the crude product prepared in the step S2-2, pouring the crude product into a 50m L single-mouth bottle, carrying out reduced pressure distillation at the temperature of 30 ℃ and the rotating speed of 80r/min, removing toluene, and then precipitating the product by using cold ether to obtain a PC L-PEG-PC L triblock copolymer;
s3 preparation of drug-loaded micelle
Adding 0.01g of camptothecin-cystamine compound prepared in the step S1, 0.1g of PC L-PEG-PC L triblock copolymer prepared in the step S2 and 3m L of dichloromethane into a 50m L single-neck flask, mixing, performing ultrasonic dissolution, performing rotary evaporation for 2 hours to completely remove the solvent, adding 10m L distilled water into the single-neck flask, performing ultrasonic dissolution to obtain a drug-loaded micelle, and performing freeze drying on the obtained micelle for later use;
s4 determination of drug loading and encapsulation efficiency
S4-1, preparing the camptothecin before modification into solutions with different concentrations by using methanol, taking a C18 commercial column as a sample column, and measuring an absorption peak at 265nm by using high performance liquid chromatography to prepare a standard curve of the camptothecin;
s4-2, taking 0.01g of micelle sample freeze-dried in the step S3, ultrasonically dissolving the micelle sample in 10m L DTT aqueous solution with the solubility of 0.05 mol/L, stirring the mixture for two hours, measuring an absorption peak of the micelle sample at 265nm by using high performance liquid chromatography to obtain a 0.3 mu L sample, comparing the absorption peak with a standard curve in S4-1, and calculating the encapsulation efficiency and the drug loading rate of the micelle.
FIG. 1 shows the flow chart of the experimental principle used in example 1, including modification of the diazo resin of camptothecin, synthesis of PC L-PEG-PC L triblock copolymer, and preparation of drug-loaded micelle.A dihydrochloride of cystamine activated by DIEA is slowly dropped into triphosgene activated by DMAP, the trichloromethyl group of the triphosgene reacts with the amino groups at both ends of the cystamine under the action of the Nu of nucleophilic reagent DMAP, and since the triphosgene is always in an excess state, the triphosgene is grafted to both ends of the cystamine.
The synthesis of the PC L-PEG-PC L triblock copolymer adopts caprolactone to open-loop polymerize under the condition that PEG is used as an initiator and stannous isooctanoate is used as a catalyst to obtain the amphiphilic PC L-PEG-PC L triblock copolymer.
Fig. 2 is an optical photograph of the drug-loaded micelle before and after modification obtained in example 1, wherein a is an effect diagram of the block copolymer encapsulating unmodified camptothecin, and b is an effect diagram of the block copolymer encapsulating modified camptothecin. As can be seen from the figure, the unmodified micelle is in a turbid state and will settle when standing, while the modified camptothecin is in an emulsion state and is relatively stable and will not settle when standing, i.e., the encapsulation rate of the unmodified camptothecin in the block copolymer is significantly lower than that of the modified camptothecin.
Fig. 3 is a transmission electron microscope image of the drug-loaded micelle obtained in example 1, and it can be seen from the image that the particle size of the drug-loaded micelle is about 40-60nm, and the particle size distribution is relatively uniform.
Fig. 4 is a D L S particle size distribution diagram of the drug-loaded micelle obtained in example 1, and it can be seen that the particle size of the drug-loaded micelle is about 40-60nm and the particle size distribution is relatively uniform.
FIG. 5 is the UV spectrum of the drug-loaded micelle obtained in example 1, from which it can be seen that the block copolymer has absorption peaks at 220 and 280, and no absorption peak at 360. The modified camptothecin had absorption peaks at 220, 280 and 360. The absorption peaks of the drug-loaded micelle at 220 and 280 are enhanced, and the absorption peak at 360 is also enhanced, so that the modified camptothecin is successfully encapsulated in the micelle.
Fig. 6 is a standard curve of the high performance liquid chromatography obtained in example 1, fig. 7 is a curve of a sample of the drug-loaded micelle of the high performance liquid chromatography obtained in example 1, peak areas are obtained by integrating the peaks, and the content of the drug in the sample is obtained by comparing the peak areas with the integration result of the standard curve, so that the encapsulation efficiency and the drug-loaded rate of the micelle are calculated, wherein the encapsulation efficiency is 83% and the drug-loaded rate is 8.4%.
Example 2
The method for improving the drug loading of camptothecin in the PC L-PEG-PC L triblock copolymer comprises the following steps:
s1 modification of cystamine of camptothecin
S1-1, taking 1g of N, N-diisopropylethylamine and 1g of 4-dimethylaminopyridine as catalysts, adding 0.5g of triphosgene into a cystamine dihydrochloride solution containing 1g of cystamine dihydrochloride, and connecting one end of the triphosgene to the cystamine dihydrochloride after reaction to generate a cystamine intermediate; then adding 1g of camptothecin into the cystamine intermediate, and connecting the camptothecin to two ends of the cystamine intermediate through a substitution reaction to generate a modified camptothecin-cystamine compound crude product;
s1-2, subjecting the obtained crude camptothecin-cystamine compound to silica gel column chromatography purification treatment to obtain a modified camptothecin purified product;
the conditions of the purification treatment of the chromatographic silica gel column are as follows: the mobile phase is chloroform: methanol is 7:3 (volume ratio), the filler is column chromatography silica gel, and the temperature is room temperature;
s2 preparation of Block copolymer
S2-1, adding 1g of polyethylene glycol (PEG) into 30g of anhydrous toluene, stirring for 1-1.5 hours at 80-90 ℃ to obtain a mixed solution, then adding 2g of caprolactone and 0.02g of stannous isooctanoate into the mixed solution, and stirring for 12-15 hours at 120-130 ℃ to obtain a crude product;
s2-2, distilling the crude product prepared in the step S2-2, pouring the crude product into a 50m L single-mouth bottle, carrying out reduced pressure distillation at the temperature of 30 ℃ and the rotating speed of 80r/min, removing toluene, and then precipitating the product by using cold ether to obtain a PC L-PEG-PC L triblock copolymer;
s3 preparation of drug-loaded micelle
Adding 0.02g of camptothecin-cystamine compound prepared in the step S1, 0.1g of PC L-PEG-PC L triblock copolymer prepared in the step S2 and 3m L of dichloromethane into a 50m L single-neck flask, mixing, performing ultrasonic dissolution, performing rotary evaporation for 2 hours to completely remove the solvent, adding 10m L distilled water into the single-neck flask, performing ultrasonic dissolution to obtain a drug-loaded micelle, and performing freeze drying on the obtained micelle for later use;
s4 determination of drug loading and encapsulation efficiency
And (3) ultrasonically dissolving 0.01g of micelle sample freeze-dried in the step S3 in a DTT (diethylene glycol terephthalate) aqueous solution with the solubility of 10m L of 0.05 mol/L, stirring for two hours, measuring an absorption peak at 265nm of the micelle sample 0.3 mu L by using high performance liquid chromatography, and comparing the absorption peak with a standard curve to calculate the drug loading of the micelle, wherein the encapsulation rate is 73 percent and the drug loading is 7.6 percent.
Example 3
The method for improving the drug loading of camptothecin in the PC L-PEG-PC L triblock copolymer comprises the following steps:
s1 modification of cystamine of camptothecin
S1-1, taking 2g of N, N-diisopropylethylamine and 2g of 4-dimethylaminopyridine as catalysts, adding 1g of triphosgene into a cystamine dihydrochloride solution containing 2g of cystamine dihydrochloride, and connecting one end of the triphosgene to the cystamine dihydrochloride after reaction to generate a cystamine intermediate; then adding 1g of camptothecin into the cystamine intermediate, and connecting the camptothecin to two ends of the cystamine intermediate through a substitution reaction to generate a modified camptothecin-cystamine compound crude product;
s1-2, subjecting the obtained crude camptothecin-cystamine compound to silica gel column chromatography purification treatment to obtain a modified camptothecin purified product;
the conditions of the purification treatment of the chromatographic silica gel column are as follows: the mobile phase is chloroform: methanol is 7:3 (volume ratio), the filler is column chromatography silica gel, and the temperature is room temperature;
s2 preparation of Block copolymer
S2-1, adding 2g of polyethylene glycol (PEG) into 40g of anhydrous toluene, stirring for 1-1.5 hours at 80-90 ℃ to obtain a mixed solution, then adding 2g of caprolactone and 0.03g of stannous isooctanoate into the mixed solution, and stirring for 12-15 hours at 120-130 ℃ to obtain a crude product;
s2-2, distilling the crude product prepared in the step S2-2, pouring the crude product into a 50m L single-mouth bottle, carrying out reduced pressure distillation at the temperature of 30 ℃ and the rotating speed of 80r/min, removing toluene, and then precipitating the product by using cold ether to obtain a PC L-PEG-PC L triblock copolymer;
s3 preparation of drug-loaded micelle
Adding 0.03g of camptothecin-cystamine compound prepared in the step S1, 0.1g of PC L-PEG-PC L triblock copolymer prepared in the step S2 and 3m L of dichloromethane into a 50m L single-neck flask, mixing, performing ultrasonic dissolution, performing rotary evaporation for 2 hours to completely remove the solvent, adding 10m L distilled water into the single-neck flask, performing ultrasonic dissolution to obtain a drug-loaded micelle, and performing freeze drying on the obtained micelle for later use;
s4 determination of drug loading and encapsulation efficiency
And (3) ultrasonically dissolving 0.01g of micelle sample freeze-dried in the step S3 in a DTT (diethylene glycol terephthalate) aqueous solution with the solubility of 10m L of 0.05 mol/L, stirring for two hours, measuring an absorption peak at 265nm of the micelle sample 0.3 mu L by using high performance liquid chromatography, and comparing the absorption peak with a standard curve to calculate the drug loading of the micelle, wherein the encapsulation rate is 69% and the drug loading is 7.3%.
Example 4
The method for improving the drug loading of camptothecin in the PC L-PEG-PC L triblock copolymer comprises the following steps:
s1 modification of cystamine of camptothecin
S1-1, taking 1g of N, N-diisopropylethylamine and 2g of 4-dimethylaminopyridine as catalysts, adding 0.5g of triphosgene into a cystamine dihydrochloride solution containing 1g of cystamine dihydrochloride, and connecting one end of the triphosgene to the cystamine dihydrochloride after reaction to generate a cystamine intermediate; then adding 1g of camptothecin into the cystamine intermediate, and connecting the camptothecin to two ends of the cystamine intermediate through a substitution reaction to generate a modified camptothecin-cystamine compound crude product;
s1-2, subjecting the obtained crude camptothecin-cystamine compound to silica gel column chromatography purification treatment to obtain a modified camptothecin purified product;
the conditions of the purification treatment of the chromatographic silica gel column are as follows: the mobile phase is chloroform: methanol is 7:3 (volume ratio), the filler is column chromatography silica gel, and the temperature is room temperature;
s2 preparation of Block copolymer
S2-1, adding 1g of polyethylene glycol (PEG) into 35g of anhydrous toluene, stirring for 1-1.5 hours at 80-90 ℃ to obtain a mixed solution, then adding 2g of caprolactone and 0.02g of stannous isooctanoate into the mixed solution, and stirring for 12-15 hours at 120-130 ℃ to obtain a crude product;
s2-2, distilling the crude product prepared in the step S2-2, pouring the crude product into a 50m L single-mouth bottle, carrying out reduced pressure distillation at the temperature of 30 ℃ and the rotating speed of 80r/min, removing toluene, and then precipitating the product by using cold ether to obtain a PC L-PEG-PC L triblock copolymer;
s3 preparation of drug-loaded micelle
Adding 0.04g of camptothecin-cystamine compound prepared in the step S1, 0.1g of PC L-PEG-PC L triblock copolymer prepared in the step S2 and 3m L of dichloromethane into a 50m L single-neck flask, mixing, performing ultrasonic dissolution, performing rotary evaporation for 2 hours to completely remove the solvent, adding 10m L distilled water into the single-neck flask, performing ultrasonic dissolution to obtain a drug-loaded micelle, and performing freeze drying on the obtained micelle for later use;
s4 determination of drug loading and encapsulation efficiency
And (3) ultrasonically dissolving 0.01g of micelle sample freeze-dried in the step S3 in a DTT (diethylene glycol terephthalate) aqueous solution with the solubility of 10m L of 0.05 mol/L, stirring for two hours, measuring an absorption peak at 265nm of the micelle sample 0.3 mu L by using high performance liquid chromatography, and comparing the absorption peak with a standard curve to calculate the drug loading of the micelle, wherein the encapsulation rate is 61 percent and the drug loading is 7 percent.
Example 5
The method for improving the drug loading of camptothecin in the PC L-PEG-PC L triblock copolymer comprises the following steps:
s1 modification of cystamine of camptothecin
S1-1, taking 2g of N, N-diisopropylethylamine and 1g of 4-dimethylaminopyridine as catalysts, adding 1g of triphosgene into 1g of cystamine dihydrochloride solution containing 1g of cystamine dihydrochloride, and connecting one end of the triphosgene to the cystamine dihydrochloride after reaction to generate a cystamine intermediate; then adding 1g of camptothecin into the cystamine intermediate, and connecting the camptothecin to two ends of the cystamine intermediate through a substitution reaction to generate a modified camptothecin-cystamine compound crude product;
s1-2, subjecting the obtained crude camptothecin-cystamine compound to silica gel column chromatography purification treatment to obtain a modified camptothecin purified product;
the conditions of the purification treatment of the chromatographic silica gel column are as follows: the mobile phase is chloroform: methanol is 7:3 (volume ratio), the filler is column chromatography silica gel, and the temperature is room temperature;
s2 preparation of Block copolymer
S2-1, adding 2g of polyethylene glycol (PEG) into 20g of anhydrous toluene, stirring for 1-1.5 hours at 80-90 ℃ to obtain a mixed solution, then adding 2g of caprolactone and 0.01g of stannous isooctanoate into the mixed solution, and stirring for 12-15 hours at 120-130 ℃ to obtain a crude product;
s2-2, distilling the crude product prepared in the step S2-2, pouring the crude product into a 50m L single-mouth bottle, carrying out reduced pressure distillation at the temperature of 30 ℃ and the rotating speed of 80r/min, removing toluene, and then precipitating the product by using cold ether to obtain a PC L-PEG-PC L triblock copolymer;
s3 preparation of drug-loaded micelle
Adding 0.05g of camptothecin-cystamine compound prepared in the step S1, 0.1g of PC L-PEG-PC L triblock copolymer prepared in the step S2 and 3m L of dichloromethane into a 50m L single-neck flask, mixing, performing ultrasonic dissolution, performing rotary evaporation for 2 hours to completely remove the solvent, adding 10m L distilled water into the single-neck flask, performing ultrasonic dissolution to obtain a drug-loaded micelle, and performing freeze drying on the obtained micelle for later use;
s4 determination of drug loading and encapsulation efficiency
And (3) ultrasonically dissolving 0.01g of micelle sample freeze-dried in the step S3 in a DTT (diethylene glycol terephthalate) aqueous solution with the solubility of 10m L of 0.05 mol/L, stirring for two hours, measuring an absorption peak at 265nm of the micelle sample 0.3 mu L by using high performance liquid chromatography, and comparing the absorption peak with a standard curve to calculate the drug loading of the micelle, wherein the encapsulation rate is 60 percent and the drug loading is 7.2 percent.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are intended to be within the scope of the invention.
Claims (6)
1. A method for improving drug loading capacity of camptothecin in a block copolymer micelle is characterized in that the camptothecin is modified by cystamine, and then the modified camptothecin and a PC L-PEG-PC L triblock copolymer are prepared into drug loading micelles.
2. The method for increasing the drug loading of camptothecin in block copolymer micelles as claimed in claim 1, wherein the method comprises the following steps:
s1 modification of cystamine of camptothecin
S1-1, adding excess triphosgene into sodium hydroxide-treated cystamine dihydrochloride solution by taking N, N-diisopropylethylamine and 4-dimethylaminopyridine as catalysts, and reacting to generate a cystamine intermediate; then adding camptothecin into the cystamine intermediate, and generating a modified camptothecin-cystamine compound crude product after reaction;
s1-2, subjecting the obtained crude camptothecin-cystamine compound to silica gel column chromatography purification treatment to obtain a modified camptothecin purified product;
s2 preparation of Block copolymer
S2-1, adding polyethylene glycol (PEG) into anhydrous toluene, stirring for 1-1.5 hours at 80-90 ℃ to obtain a mixed solution, then adding caprolactone and stannous isooctanoate into the mixed solution, and stirring for 12-15 hours at 120-130 ℃ to obtain a crude product;
s2-2, distilling the crude product prepared in the step S2-1 to remove toluene, and then precipitating the product by using cold ether to obtain a PC L-PEG-PC L triblock copolymer, wherein the molecular weight of the prepared PC L-PEG-PC L triblock copolymer is 5000-7000, and the ratio of the molecular weight of the PC L chain segment to the molecular weight of the PEG chain segment is 1.5-3;
s3 preparation of drug-loaded micelle
And (2) mixing the camptothecin-cystamine compound prepared in the step S1 and the PC L-PEG-PC L triblock copolymer prepared in the step S2, dissolving the mixture in dichloromethane by ultrasonic, carrying out rotary evaporation for 1.5 to 2.5 hours to completely remove the solvent, then dropwise adding distilled water, and carrying out ultrasonic treatment to obtain a drug-loaded micelle, wherein the mass ratio of the added camptothecin-cystamine compound, the PC L-PEG-PC L triblock copolymer and the dichloromethane is (1-8) to 10: 250.
3. The method for improving the drug loading capacity of camptothecin in the block copolymer micelle as claimed in claim 2, wherein the mass ratio of the N, N-diisopropylethylamine, 4-dimethylaminopyridine, triphosgene, cystamine dihydrochloride and camptothecin added in step S1 is (0.5-2): (0.5-2): (0.2-1): (1-3): 1; the mass ratio of the polyethylene glycol, the toluene, the caprolactone and the stannous isooctanoate added in the step S2-1 is (1-3): (20-40): 2: (0.01-0.03).
4. The method for improving the drug loading of camptothecin in block copolymer micelle as claimed in claim 2, wherein the method for treating cystamine dihydrochloride with sodium hydroxide is as follows:
accurately weighing 1.6g of sodium hydroxide and 4.5g of cystamine dihydrochloride, mixing, adding into a 100m L single-neck flask containing 50m L tetrahydrofuran, magnetically stirring for 48 hours at room temperature, filtering to remove solid products after stirring, and performing rotary evaporation to remove the solvent at 30 ℃ and 80r/min to obtain the required cystamine dihydrochloride.
5. The method for increasing the drug loading of camptothecin in block copolymer micelle as claimed in claim 2, wherein the step S1-2 is carried out by subjecting the crude camptothecin-cystamine compound to silica gel column chromatography purification treatment under the following conditions:
the volume ratio of the mobile phase is chloroform: methanol (6-8) and (2-4); the filler is column chromatography silica gel; the temperature was room temperature.
6. The drug-loaded micelle with the spherical structure and the particle size of 40-60nm, which is prepared by the method of any one of claims 1-5.
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| CN105395483A (en) * | 2015-12-21 | 2016-03-16 | 江南大学 | Reducible and degradable hyperbranched polymer nano-micelle and preparation method thereof |
| CN106831805A (en) * | 2017-03-21 | 2017-06-13 | 莎穆(上海)生物科技有限公司 | A kind of camptothecine adriamycin prodrug and its preparation method and application |
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| CN101940792A (en) * | 2010-09-30 | 2011-01-12 | 中国医学科学院生物医学工程研究所 | PCL-b-PEG-b-PCL carried hydrophobic medicine polymer vesica as well as preparation method and application thereof |
| CN105395483A (en) * | 2015-12-21 | 2016-03-16 | 江南大学 | Reducible and degradable hyperbranched polymer nano-micelle and preparation method thereof |
| CN106831805A (en) * | 2017-03-21 | 2017-06-13 | 莎穆(上海)生物科技有限公司 | A kind of camptothecine adriamycin prodrug and its preparation method and application |
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