CN108264578B - Polysaccharide grafted folic acid copolymer and preparation method of nanoparticles thereof - Google Patents
Polysaccharide grafted folic acid copolymer and preparation method of nanoparticles thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of polysaccharide grafted folic acid copolymer and nanoparticles thereof, which comprises the steps of dissolving folic acid by using anhydrous dimethyl sulfoxide, then adding 1-hydroxy benzotriazole and N-N' -dicyclohexylcarbodiimide, carrying out carboxyl activation reaction, mixing the carboxyl activated folic acid solution with the dimethyl sulfoxide solution of polysaccharide, carrying out esterification reaction for 24-72 h under the conditions of nitrogen protection and temperature of 40-80 ℃, and purifying to obtain a copolymer mixture; freeze-drying to obtain the polysaccharide grafted folic acid copolymer freeze-dried powder; namely the polysaccharide grafted folic acid copolymer. The polysaccharide grafted folic acid copolymer is used as a carrier in the preparation of a nanoparticle preparation with a folic acid receptor targeting function, and the nanoparticle preparation is used for tumor targeted drug delivery.
Description
Technical Field
The invention relates to the field of biological targeting functional polymer sustained-release materials, in particular to a polysaccharide grafted folic acid copolymer and a preparation method of nanoparticles thereof.
Background
Cancer is one of the major diseases threatening human health at present, and chemotherapy currently occupies an important position in the clinical treatment of cancer. However, most chemotherapy drugs currently in use have the problems of poor water solubility and short circulation cycle in vivo. Although the treatment effect of most chemotherapy drugs is remarkable, the chemotherapy drugs are distributed in the whole body in a non-targeted manner, so that the bioavailability of the chemotherapy drugs is reduced on one hand, and the chemotherapy drugs cause great toxic and side effects on the other hand. With the development of science and technology, the nano drug delivery system is one of the most effective means for solving the above problems. At present, the preparation method of the nano drug delivery system mainly comprises an emulsification method, a dialysis method, a hydration method and a solvent volatilization method. The preparation of the nanoparticles by the emulsification method requires adding organic solvents such as trichloromethane, forming emulsion by an ultrasonic mode, and volatilizing the emulsion to form the nanoparticles. However, organic solvents such as chloroform are miscible with water in a certain ratio and are difficult to completely remove. The residual organic solvent is toxic to human body. The dialysis method comprises dissolving carrier material and medicine in organic solvent, and removing organic solvent by dialysis to obtain water solution of nanometer medicine carrying system. However, the dialysis time is often too long, and 1 to 3 days are needed to obtain the required nano drug-carrying system. The hydration method is that carrier material and medicine are dissolved in organic solvent, the organic solvent is evaporated and dried to obtain a layer of adherent film of carrier material and medicine, and then water phase is added and stirred. The adherent film gradually forms a nano drug-loading system in the water phase. This method requires co-solvent of the material and the drug in an organic solvent, which greatly limits the choice of material. Solvent evaporation also requires the use of large amounts of organic solvents, which are, however, miscible with water in certain proportions and difficult to remove completely. The residual organic solvent is harmful to human health.
How to seek a method which has simple process and can avoid the use of organic solvent to prepare the nano-drug carrier capable of treating the tumor is an urgent technical problem to be solved.
Disclosure of Invention
The invention aims to overcome the defects and the improvement requirements of the assembling method, and provides a polysaccharide grafted folic acid copolymer and a preparation method of nanoparticles thereof. The problems that the existing nano drug-carrying system is long in preparation period and toxic is easily caused by organic solvent residue are solved.
To achieve the above object, the present invention provides a polysaccharide-grafted folic acid copolymer, which has the following general formula:
wherein R is polysaccharide with molecular weight of 5000-500000 Da.
Further, R is selected from dextran, chitosan oligosaccharide, pullulan, hydroxyethyl cellulose and hyaluronic acid.
Still further, the polysaccharide-folate copolymer is a dextran-grafted folate copolymer having the general formula:
wherein n is 100-10000; preferably, n is 640.
Furthermore, the substitution degree of folic acid on polysaccharide is 20-50%; in order to ensure that the folic acid-polysaccharide graft polymer can be assembled into nanoparticles with the particle size of less than 100 nanometers; further preferably 42%.
The invention also provides a preparation method of the polysaccharide grafted folic acid copolymer, which comprises the following steps:
1) dissolving folic acid and activating its carboxyl group: dissolving folic acid by using anhydrous dimethyl sulfoxide, then adding 1-hydroxybenzotriazole and N-N' -dicyclohexylcarbodiimide to perform carboxyl activation reaction, and stirring at room temperature for 2-4 hours to obtain a terminal carboxyl activated folic acid solution;
2) dissolving polysaccharide: under the protection of helium, fully dissolving polysaccharide with the molecular weight of 5000-500000 Da in anhydrous dimethyl sulfoxide at the temperature of 50-70 ℃ to obtain a dimethyl sulfoxide solution of the polysaccharide:
3) esterification reaction: mixing the carboxyl activated folic acid solution obtained in the step 1) with the dimethyl sulfoxide solution of the polysaccharide obtained in the step 2), performing esterification reaction for 24-72 hours under the protection of nitrogen at the temperature of 40-80 ℃, and purifying to obtain a copolymer mixture;
4) and (3) purification: co-dialyzing the copolymer mixture for 1-5 days by using a PBS (phosphate buffer solution), removing unreacted 1-hydroxybenzotriazole, N-N' -dicyclohexylcarbodiimide, folic acid and a DMSO (dimethyl sulfoxide) solvent, freezing the liquid in a dialysis bag for 3-5 hours at the temperature of-20 to-25 ℃ after dialysis is finished, then freeze-drying at the temperature of-40 to-60 ℃, and obtaining the polysaccharide grafted folic acid copolymer freeze-dried powder after freeze-drying; namely the polysaccharide grafted folic acid copolymer.
Further, in the step 1), the feeding molar ratio of folic acid, 1-hydroxybenzotriazole and N-N' -dicyclohexylcarbodiimide is 1: 1-2;
in the step 1), in the carboxyl activation reaction, the reaction temperature is 40-80 ℃, and the reaction time is 30-60 min.
Still further, in the step 2), the polysaccharide is selected from dextran, chitosan oligosaccharide, pullulan, hydroxyethyl cellulose and hyaluronic acid.
Still further, in the step 3), the mass ratio of folic acid in the carboxyl activated folic acid solution to polysaccharide in the dimethyl sulfoxide solution of the polysaccharide is 1: 1-4
Further, in the step 4), in the dialysis process, the molecular weight of the dialysis bag is 3500 Da-24000 Da, the freezing temperature is-20 ℃, and the freezing time is 4 h; the freeze-drying temperature was-50 ℃.
The invention also provides a preparation method of the polysaccharide grafted folic acid copolymer nanoparticles, which comprises the following steps:
1) dissolving the polysaccharide-grafted folic acid copolymer in water to obtain a solution of the polysaccharide-grafted folic acid copolymer with the concentration of 0.1-100 mg/ml;
2) adjusting the pH value of the solution of the polysaccharide grafted folic acid copolymer to 8-12, and stirring for 5-30 min under the state; and then adjusting the pH value of the solution to 7.2-7.4 within 0-10 s to obtain the polysaccharide-grafted folic acid copolymer nanoparticles, wherein the particle size of the polysaccharide-grafted folic acid copolymer nanoparticles is less than 100 nm.
The invention has the beneficial effects that:
1) the polysaccharide grafted folic acid copolymer provided by the invention has simple synthesis steps, and can obtain the required polymer by only one-step reaction. The polysaccharide (particularly glucan) and folic acid adopted by the polymer have good biological safety and biocompatibility, and have the value of in-vivo injection application.
2) Compared with other methods such as an emulsification method, a dialysis method and the like, the method for preparing the polysaccharide grafted folic acid copolymer nanoparticles by using the pH regulation method provided by the invention has the advantages of shorter operation time, simpler operation method and no need of adding an organic reagent. The nano-size of the material is below 100nm, the material has good stability in PBS buffer solution and Fetal Bovine Serum (FBS) (figure 2), and the material has wide application prospect;
3) the folic acid in the polysaccharide grafted folic acid copolymer nanoparticles prepared by the pH regulation method provided by the invention can be used for loading adriamycin or other electropositive drugs. The results of cell uptake experiments show (fig. 3) that folic acid still retains the active targeting property to folic acid receptors except for participating in the assembly and drug loading of nanoparticles. The result of mouse tissue distribution shows (figure 4) that the nanoparticle has passive targeting and active targeting bidirectional targeting characteristics.
Drawings
FIG. 1 is a synthetic scheme for the preparation of dextran grafted folate copolymer of example 1;
FIG. 2 is an infrared spectrum of the glucan-grafted folic acid copolymer prepared in example 1;
FIG. 3 is a NMR spectrum of a glucan-grafted folic acid copolymer prepared in example 1;
FIG. 4 is a DLS image (FIG. 4A) and a TEM image (FIG. 4B) of the glucan-grafted folate copolymer nanoparticle prepared in example 1;
FIG. 5 is a synthetic scheme of the chitooligosaccharide-grafted folate copolymer prepared in example 2;
FIG. 6 is an infrared spectrum of the chitosan oligosaccharide grafted folic acid copolymer prepared in example 2;
FIG. 7 is a nuclear magnetic resonance spectrum of the chitosan oligosaccharide grafted folic acid copolymer prepared in example 2;
FIG. 8 is a DLS image (FIG. 8A) and a TEM image (FIG. 8B) of the chitosan oligosaccharide grafted folate copolymer nanoparticle prepared in example 2;
FIG. 9 is a synthesis scheme of the pullulan grafted folate copolymer prepared in example 3;
FIG. 10 is an infrared spectrum of a pullulan-grafted folic acid copolymer prepared in example 3;
FIG. 11 is a NMR spectrum of a Pullulan polysaccharide grafted folate copolymer prepared in example 3;
FIG. 12 is a DLS image (FIG. 12A) and a TEM image (FIG. 12B) of the pullulan-grafted folate copolymer nanoparticles prepared in example 3;
FIG. 13 is a synthetic route diagram for the hyaluronic acid-grafted folate copolymer prepared in example 4;
FIG. 14 is an infrared spectrum of the hyaluronic acid-grafted folate copolymer prepared in example 4;
FIG. 15 is a NMR spectrum of the hyaluronic acid-grafted folate copolymer prepared in example 4;
fig. 16 is a DLS image (fig. 16A) and a transmission electron micrograph (fig. 16B) of the hyaluronic acid-grafted folate copolymer nanoparticle prepared in example 4;
FIG. 17 is a synthetic scheme of the hydroxyethylcellulose-grafted folate copolymer prepared in example 5;
FIG. 18 is an infrared spectrum of the hydroxyethylcellulose-grafted folate copolymer prepared in example 5;
FIG. 19 is the NMR spectrum of the hydroxyethylcellulose-grafted-folate copolymer prepared in example 5;
FIG. 20 is a DLS image (FIG. 20A) and a TEM image (FIG. 20B) of the hydroxyethylcellulose-grafted folate copolymer nanoparticles prepared in example 5;
FIG. 21 shows the results of experiments on the reaction of dextran grafted folate copolymer nanoparticles loaded with doxorubicin prepared according to the present invention with tetramethylazozole (MTT) trace enzyme;
FIG. 21A is a graph of doxorubicin @ dextran-folate (DOX @ DEX-FA) versus folate + doxorubicin @ dextran-folate (FA + DOX @ DEX-FA) for breast cancer cytotoxicity experiments in 4T1 mice;
FIG. 21B is a graph of Doxorubicin (DOX) and folate + doxorubicin (FA + DOX) for 4T1 mouse breast cancer cytotoxicity experiments;
FIG. 21C is a graph of doxorubicin @ dextran-folate (DOX @ DEX-FA) versus folate + doxorubicin @ dextran-folate (FA + DOX @ DEX-FA) for A549 mouse lung cancer cytotoxicity experiments;
FIG. 21D is a cytotoxicity assay of Doxorubicin (DOX) group versus folate + doxorubicin (FA + DOX) group in A549 mouse lung cancer;
FIG. 22 is a diagram showing the results of the cell uptake experiment of the doxorubicin-loaded dextran-grafted folate copolymer nanoparticles prepared according to the present invention;
in the figure, FIG. 22A is a graph showing the results of the-4T 1 cell uptake assay;
fig. 22B is a graph of a549 fine uptake assay results.
Detailed Description
In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples.
Example 1
A dextran-grafted folate copolymer having the general formula:
n is 640 and the molecular weight of dextran is 40k Da.
The preparation method of the glucan-grafted folic acid copolymer shown in figure 1 comprises the following steps:
1) dissolving folic acid and activating its carboxyl group: dissolving 0.5g folic acid in 5ml anhydrous dimethyl sulfoxide, adding 1-hydroxy benzotriazole and N-N' -dicyclohexyl carbodiimide, performing carboxyl activation reaction at 60 ℃ for 30min, and stirring at room temperature for 2-4 h to obtain a terminal carboxyl activated folic acid solution; wherein the feeding molar ratio of the folic acid, the 1-hydroxybenzotriazole and the N-N' -dicyclohexylcarbodiimide is 1:1: 1;
2) dissolving glucan: 0.5g of dextran of molecular weight 40k Da, fully dissolved in 5ml of anhydrous dimethylsulfoxide at 50 ℃ under helium protection to give a dimethylsulfoxide solution of dextran:
3) esterification reaction: mixing the carboxyl activated folic acid solution obtained in the step 1) with the dimethyl sulfoxide solution of the polysaccharide obtained in the step 2), carrying out esterification reaction for 48 hours under the conditions of nitrogen protection and 50 ℃, and purifying to obtain a copolymer mixture;
4) and (3) purification: dialyzing the copolymer mixture by using a PBS (phosphate buffer solution), dialyzing the copolymer mixture for 3 days with the molecular weight of a dialysis bag being 3500Da, removing unreacted 1-hydroxybenzotriazole, N-N' -dicyclohexylcarbodiimide, folic acid and DMSO solvents, transferring the liquid in the dialysis bag to a plastic culture dish after dialysis is finished, freezing for 4 hours at the temperature of-20 ℃, then freeze-drying at the temperature of-50 ℃, and obtaining the polysaccharide grafted folic acid copolymer freeze-dried powder after freeze-drying; namely the glucan grafted folic acid copolymer DEX-FA.
The chemical structure of the glucan-folic acid grafted polymer prepared by the invention is confirmed by infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (1H-NMR). As can be seen from FIG. 2, the infrared absorption of the glucan-grafted folic acid copolymer was 1730cm as compared with the infrared spectrum of glucan-1A new absorption peak appeared, which was attributed to the C ═ O stretching vibration peak of the ester bond formed by the reaction. The infrared absorption of dextran grafted folic acid copolymer is 1697cm-1And 1643cm-1A new absorption peak appears, attributed to the stretching vibration of the aromatic ring on folic acid. These newly appearing absorption peaks demonstrate the successful grafting of folic acid onto dextran.
As can be seen from FIG. 3, compared with the nuclear magnetic spectrum of glucan, a new set of peaks appears at 6.5-9.0ppm for glucan-folic acid, and the peaks are attributed to protons on aromatic rings on folic acid. The glucan-folate shows a new set of peaks at 1.5-3ppm, which are attributed to the proton peaks on the methylene group in folate. The correctness of the chemical structure of the glucan-folic acid graft polymer prepared in this example 1 was confirmed from the NMR chart. The results of the nuclear magnetic resonance further confirmed that the substitution degree of folic acid on dextran was 46.4 wt% as a result of DEX-FA.
Preparation of dextran grafted folic acid copolymer nanoparticles: the method comprises the following steps:
1) dissolving the glucan-grafted folic acid copolymer in water to obtain a solution of the glucan-grafted folic acid copolymer with the concentration of 0.1-100 mg/ml;
2) adding sodium hydroxide solution, adjusting pH of the solution of dextran grafted folic acid copolymer to 9, and stirring for 10min under the condition; and then, quickly adding a hydrochloric acid solution, and regulating the pH value of the solution to 7.2 within 5s to obtain the glucan-grafted folic acid copolymer nanoparticles, wherein the particle size of the glucan-grafted folic acid copolymer nanoparticles in the solution is less than 100 nm.
Microscopic observation of the dextran grafted folic acid copolymer nanoparticles:
and (3) taking 1mg/ml of glucan grafted folic acid copolymer nanoparticles, and carrying out ultrasonic treatment for 10min to obtain the glucan grafted folic acid copolymer nanoparticles for later use. Placing a copper net in a surface dish covered with filter paper, dripping 20 mu L of nanoparticle dispersion liquid of 1mg/ml on the copper net, dyeing with 0.2% phosphotungstic acid, naturally drying at room temperature, observing the morphology of the nanoparticle dispersion liquid by using a projection electron microscope, measuring the particle size and distribution of micelles by using a laser particle sizer, measuring the temperature to be 25 ℃, balancing for 2min, and using a laser power supply: He-Ne laser with a wavelength of 633 nm.
The preparation method of the nanoparticle preparation 1 (the adriamycin-coated glucan-grafted folic acid copolymer nanoparticle) comprises the following steps:
1) adjusting the pH value of the glucan grafted folic acid copolymer nanoparticles to 9; ultrasonic crushing under ice bath condition, and simultaneously adding adriamycin dimethyl sulfoxide solution without hydrochloric acid to obtain solution after ultrasonic treatment;
2) ultrasonically crushing the solution subjected to ultrasonic treatment for 5min under the ice bath condition, and then adjusting the pH value to 7.2; obtaining dextran grafted folic acid copolymer nanometer particle with adriamycin;
3) centrifuging and ultrafiltering the dextran grafted folic acid copolymer nanoparticles coated with the adriamycin obtained in the step 2), wherein the molecular weight cut-off of an ultrafiltration tube is 10000Da, and ultrafiltering for 10 times to obtain the dextran grafted folic acid copolymer nanoparticles coated with the adriamycin, namely the nanoparticle preparation 1. The mass ratio of adriamycin to glucan grafted folic acid copolymer nanoparticles is 1: 10.
as can be seen from the DLS results in FIG. 4, the diameter of the doxorubicin-loaded dextran-grafted folate copolymer nanoparticles was 90nm and the distribution was uniform. TEM results were consistent with DLS results.
Example 2
A chitosan oligosaccharide grafted folate copolymer having the general formula:
r is chitosan oligosaccharide with molecular weight of 5 kDa.
The preparation method of the chitosan oligosaccharide grafted folic acid copolymer shown in figure 5 comprises the following steps:
1) dissolving folic acid and activating its carboxyl group: dissolving 0.5g folic acid in 5ml anhydrous dimethyl sulfoxide, adding 1-hydroxy benzotriazole and N-N' -dicyclohexyl carbodiimide, performing carboxyl activation reaction at 60 ℃ for 30min, and stirring at room temperature for 2-4 h to obtain a terminal carboxyl activated folic acid solution; wherein the feeding molar ratio of the folic acid, the 1-hydroxybenzotriazole and the N-N' -dicyclohexylcarbodiimide is 1:1: 1;
2) dissolving polysaccharide: under the protection of helium, 0.5g of chitosan oligosaccharide with molecular weight of 5kDa is fully dissolved in anhydrous dimethyl sulfoxide at the temperature of 50 ℃ to obtain a dimethyl sulfoxide solution of the chitosan oligosaccharide:
3) esterification reaction: mixing the carboxyl activated folic acid solution obtained in the step 1) with the dimethyl sulfoxide solution of the chitosan oligosaccharide obtained in the step 2), carrying out esterification reaction for 48 hours under the conditions of nitrogen protection and 48 ℃, and purifying to obtain a copolymer mixture;
4) and (3) purification: dialyzing the copolymer mixture by using a PBS (phosphate buffer solution), dialyzing the copolymer mixture for 35 days by using a dialysis bag with the molecular weight of 3500Da, removing unreacted 1-hydroxybenzotriazole, N-N' -dicyclohexylcarbodiimide, folic acid and a DMSO (dimethyl sulfoxide) solvent, transferring the liquid in the dialysis bag into a culture dish after dialysis is finished, freezing for 4 hours at the temperature of-20 ℃, then freeze-drying at the temperature of-50 ℃, and obtaining the chitosan oligosaccharide grafted folic acid copolymer freeze-dried powder after freeze-drying; namely the chitosan oligosaccharide grafted folic acid copolymer (CSO-FA).
The chemical structure of the prepared chitosan oligosaccharide grafted folic acid copolymer is confirmed by infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (1H-NMR). As can be seen from FIG. 6, the infrared absorption of the chitosan oligosaccharide-grafted folic acid copolymer was 1650cm as compared with that of the chitosan oligosaccharide-1A new absorption peak appears, which belongs to the-NH-CO-stretching vibration peak of amido bond generated by reaction. The infrared absorption of the chitosan oligosaccharide grafted folic acid copolymer is 1697cm-1And 1643cm-1A new absorption peak appears, attributed to the stretching vibration of the aromatic ring on folic acid. These newly appearing absorption peaks demonstrate the successful grafting of folic acid onto chitosan oligosaccharides.
As can be seen from FIG. 7, compared with the nuclear magnetic spectrum of chitosan oligosaccharide, a new set of peaks appears at 6.5-9.0ppm in glucan-folic acid, and belongs to the peaks of protons on aromatic rings on folic acid. Chitosan oligosaccharide-folic acid has a new group of peaks at 1.5-3ppm, which are attributed to the peak of proton on methylene in folic acid. The chemical structure of the chitosan oligosaccharide-grafted folic acid copolymer prepared in this example 2 was confirmed to be correct according to the nuclear magnetic resonance image. The results of nuclear magnetic resonance further confirmed that the substitution degree of folic acid on chitosan oligosaccharide was 36.2 wt% as a result of CSO-FA.
The preparation of the chitosan oligosaccharide grafted folic acid copolymer nanoparticle comprises the following steps: the method comprises the following steps:
1) dissolving the chitosan oligosaccharide grafted folic acid copolymer in water to obtain a solution of the chitosan oligosaccharide grafted folic acid copolymer with the concentration of 0.1-100 mg/ml;
2) adding sodium hydroxide solution, adjusting pH of the solution of chitosan oligosaccharide grafted folic acid copolymer to 9, and stirring for 5min under the condition; then, hydrochloric acid solution is quickly added, the pH value of the solution is adjusted to 7.2 within 5s, and the chitosan oligosaccharide-grafted folic acid copolymer nanoparticles are obtained, wherein the particle size of the chitosan oligosaccharide-grafted folic acid copolymer nanoparticles in the solution is less than 100 nm.
Microscopic observation of the chitosan oligosaccharide grafted folic acid copolymer nanoparticles:
taking 1mg/ml chitosan oligosaccharide grafted folic acid copolymer nanoparticles, and carrying out ultrasonic treatment for 10min for later use. Placing a copper net in a surface dish covered with filter paper, dripping 20 mu L of nanoparticle dispersion liquid of 1mg/ml on the copper net, dyeing with 0.2% phosphotungstic acid, naturally drying at room temperature, observing the morphology of the nanoparticle dispersion liquid by using a projection electron microscope, measuring the particle size and distribution of micelles by using a laser particle sizer, measuring the temperature at 25 ℃, balancing for 2min, and using a laser power supply: He-Ne laser with a wavelength of 633 nm.
The preparation method of the nanoparticle preparation 2 (chitosan oligosaccharide grafted folic acid copolymer nanoparticle coated with adriamycin) comprises the following steps:
1) adjusting the pH value of the chitosan oligosaccharide grafted folic acid copolymer nanoparticles to 8-12; ultrasonic crushing under ice bath condition, and simultaneously adding adriamycin dimethyl sulfoxide solution without hydrochloric acid to obtain solution after ultrasonic treatment;
2) ultrasonically crushing the solution subjected to ultrasonic treatment for 5-15 min under an ice bath condition, and then adjusting the pH value to 7.2-7.4; obtaining chitosan oligosaccharide grafted folic acid copolymer nanoparticles carrying adriamycin;
5) centrifuging and ultrafiltering the chitosan oligosaccharide grafted folic acid copolymer nanoparticles loaded with the adriamycin obtained in the step 4), wherein the molecular weight cut-off of an ultrafiltration tube is 3000-100000 Da, and ultrafiltering for 5-10 times to obtain the chitosan oligosaccharide grafted folic acid copolymer nanoparticles loaded with the adriamycin, namely the nanoparticle preparation 2. The mass ratio of adriamycin to chitosan oligosaccharide grafted folic acid copolymer nanoparticles is 1: 10.
as can be seen from the DLS results in FIG. 8, the particle size of the chitosan oligosaccharide grafted folic acid copolymer nanoparticles coated with adriamycin is 90nm, and the distribution is uniform. TEM results were consistent with DLS results.
Example 3
A pullulan-grafted folate copolymer having the general formula:
wherein R is pullulan; the molecular weight is 300 kDa.
The preparation method of the glucan-grafted folic acid copolymer shown in fig. 9 comprises the following steps:
1) dissolving folic acid and activating its carboxyl group: dissolving 0.5g folic acid in 5ml anhydrous dimethyl sulfoxide, adding 1-hydroxy benzotriazole and N-N' -dicyclohexyl carbodiimide, performing carboxyl activation reaction at 60 ℃ for 30min, and stirring at room temperature for 2-4 h to obtain a terminal carboxyl activated folic acid solution; wherein the feeding molar ratio of the folic acid, the 1-hydroxybenzotriazole and the N-N' -dicyclohexylcarbodiimide is 1:1: 1;
2) dissolving polysaccharide: under the protection of helium, 0.5g of pullulan with the molecular weight of 300kDa is fully dissolved in anhydrous dimethyl sulfoxide at the temperature of 50 ℃ to obtain a dimethyl sulfoxide solution of the pullulan:
3) esterification reaction: mixing the carboxyl activated folic acid solution obtained in the step 1) with the dimethyl sulfoxide solution of the pullulan obtained in the step 2), carrying out esterification reaction for 48 hours under the conditions of nitrogen protection and 50 ℃, and purifying to obtain a copolymer mixture;
4) and (3) purification: dialyzing the copolymer mixture by using a PBS solution, dialyzing for 3 days by using a dialysis bag with the molecular weight of 3500Da, removing unreacted 1-hydroxybenzotriazole, N-N' -dicyclohexylcarbodiimide, folic acid and a DMSO solvent, transferring the liquid in the dialysis bag into a plastic culture dish after dialysis is finished, freezing for 4 hours at the temperature of-20 ℃, then freeze-drying at the temperature of-50 ℃, and obtaining the pullulan polysaccharide grafted folic acid copolymer freeze-dried powder after freeze-drying; namely Pullulan grafted folic acid copolymer (Pullulan-FA).
The chemical structure of the prepared pullulan grafted folic acid copolymer is confirmed by infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (1H-NMR). As can be seen from FIG. 10, the infrared absorption of the pullulan-folic acid graft polymer was 1730cm, compared to the infrared spectrum of pullulan-1A new absorption peak appeared, which was attributed to the C ═ O stretching vibration peak of the ester bond formed by the reaction. The infrared absorption of the pullulan-folic acid graft polymer is 1697cm-1And 1643cm-1A new absorption peak appears, attributed to the stretching vibration of the aromatic ring on folic acid. These newly appearing absorption peaks demonstrate the successful grafting of folic acid onto pullulan.
As can be seen from FIG. 11, compared with the nuclear magnetic spectrum of dextran, a new set of peaks appears at 6.5-9.0ppm for pullulan-folic acid, and belongs to the peaks of protons on aromatic rings on folic acid. A group of new peaks appear in 1.5-3ppm of pullulan-folic acid, and are attributed to peaks of protons on methylene in folic acid. The chemical structure of the pullulan-grafted folic acid copolymer prepared in this example 3 was confirmed to be correct according to the nuclear magnetic resonance image. The results of NMR confirmed that the substitution degree of folic acid in pullulan was 46.4 wt% as a result of pullulan-FA.
Preparation of pullulan grafted folic acid copolymer nanoparticles: the method comprises the following steps:
1) dissolving the pullulan grafted folic acid copolymer in water to obtain a solution of the pullulan grafted folic acid copolymer with the concentration of 0.1-100 mg/ml;
2) adding sodium hydroxide solution, adjusting pH of the solution of pullulan grafted folic acid copolymer to 9, and stirring for 5min under the condition; and then, quickly adding a hydrochloric acid solution, and regulating the pH value of the solution to 7.2 within 5s to obtain the pullulan grafted folic acid copolymer nanoparticles, wherein the particle size of the pullulan grafted folic acid copolymer nanoparticles in the solution is less than 100 nm.
Microscopic observation of the pullulan grafted folic acid copolymer nanoparticles:
taking 1mg/ml pullulan-folic acid nanoparticles, and carrying out ultrasonic treatment for 10min for later use. Placing a copper net in a surface dish covered with filter paper, dripping 20 mu L of nanoparticle dispersion liquid of 1mg/ml on the copper net, dyeing with 0.2% phosphotungstic acid, naturally drying at room temperature, observing the morphology of the nanoparticle dispersion liquid by using a projection electron microscope, measuring the particle size and distribution of micelles by using a laser particle sizer, measuring the temperature to be 25 ℃, balancing for 2min, and using a laser power supply: He-Ne laser with a wavelength of 633 nm.
The preparation method of the nanoparticle preparation 3 (pullulan grafted folic acid copolymer nanoparticles coated with adriamycin) comprises the following steps:
1) adjusting the pH value of the pullulan grafted folic acid copolymer nanoparticles to 8-12; ultrasonic crushing under ice bath condition, and simultaneously adding adriamycin dimethyl sulfoxide solution without hydrochloric acid to obtain solution after ultrasonic treatment;
2) ultrasonically crushing the solution subjected to ultrasonic treatment for 5-15 min under an ice bath condition, and then adjusting the pH value to 7.2-7.4; obtaining pullulan grafted folic acid copolymer nanoparticles carrying adriamycin;
3) centrifuging and ultrafiltering the pullulan polysaccharide grafted folic acid copolymer nanoparticles coated with the adriamycin obtained in the step 2), wherein the cut-off molecular weight of an ultrafiltration tube is 3000-100000 Da, and ultrafiltering for 5-10 times to obtain the pullulan polysaccharide grafted folic acid copolymer nanoparticles coated with the adriamycin, namely the nanoparticle preparation 3. The mass ratio of adriamycin to pullulan grafted folic acid copolymer nanoparticles is 1: 5 to 100.
As can be seen from the DLS results in fig. 12, the particle size of the doxorubicin-entrapped pullulan-folic acid was about 90nm, and the distribution was uniform. TEM results were consistent with DLS results.
Example 4
A hyaluronic acid-grafted folate copolymer having the general formula:
wherein R is hyaluronic acid with molecular weight of 50 kDa.
The preparation method of the hyaluronic acid grafted folic acid copolymer shown in figure 13 comprises the following steps:
1) dissolving folic acid and activating its carboxyl group: dissolving 0.5g folic acid in 5ml anhydrous dimethyl sulfoxide, adding 1-hydroxy benzotriazole and N-N' -dicyclohexyl carbodiimide, performing carboxyl activation reaction at 60 ℃ for 30min, and stirring at room temperature for 2-4 h to obtain a terminal carboxyl activated folic acid solution; wherein the feeding molar ratio of the folic acid, the 1-hydroxybenzotriazole and the N-N' -dicyclohexylcarbodiimide is 1:1: 1;
2) dissolving polysaccharide: 0.5g of hyaluronic acid with a molecular weight of 50kDa is dissolved in anhydrous dimethylsulfoxide at 500 ℃ under helium protection to obtain a dimethylsulfoxide solution of hyaluronic acid:
3) esterification reaction: mixing the carboxyl activated folic acid solution obtained in the step 1) with the dimethyl sulfoxide solution of hyaluronic acid obtained in the step 2), performing esterification reaction for 24-72 hours under the protection of nitrogen at the temperature of 40-80 ℃, and purifying to obtain a copolymer mixture;
4) and (3) purification: dialyzing the copolymer mixture by using a PBS solution, dialyzing for 3 days by using a dialysis bag with the molecular weight of 3500Da, removing unreacted 1-hydroxybenzotriazole, N-N' -dicyclohexylcarbodiimide, folic acid and a DMSO solvent, transferring the liquid in the dialysis bag into a culture dish after dialysis is finished, freezing for 4 hours at the temperature of-20 ℃, then freeze-drying at the temperature of-50 ℃, and obtaining the hyaluronic acid grafted folic acid copolymer freeze-dried powder after freeze-drying; namely hyaluronic acid grafted folic acid copolymer (HA-FA).
The chemical structure of the prepared hyaluronic acid-grafted folate copolymer is confirmed by infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (1H-NMR). As can be seen from FIG. 11, the infrared absorption of the hyaluronic acid-grafted folic acid copolymer was 1730cm as compared with the infrared spectrum of hyaluronic acid-1A new absorption peak appeared, which was attributed to the C ═ O stretching vibration peak of the ester bond formed by the reaction. The infrared absorption of the hyaluronic acid grafted folic acid copolymer is 1697cm-1And 1643cm-1A new absorption peak appears, attributed to the stretching vibration of the aromatic ring on folic acid. These newly appearing absorption peaks demonstrate the successful grafting of folic acid onto hyaluronic acid.
As can be seen from FIG. 15, a new set of peaks was observed at 6.5-9.0ppm for hyaluronic acid-folic acid, which are attributed to the proton on the aromatic ring of folic acid, compared to the nuclear magnetic spectrum of dextran. Hyaluronic acid-folic acid showed a new set of peaks at 1.5-3ppm, which were assigned to the proton peak on the methylene group in folic acid. The correctness of the chemical structure of the hyaluronic acid-folic acid graft polymer prepared in this example 4 was confirmed from the NMR chart. The results of nuclear magnetic resonance further confirm that the substitution degree of folic acid on pullulan is 37.5 wt% as a result of HA-FA.
Preparation of hyaluronic acid grafted folic acid copolymer nanoparticles: the method comprises the following steps:
1) dissolving a hyaluronic acid-grafted folic acid copolymer in water to obtain a solution of the hyaluronic acid-grafted folic acid copolymer with the concentration of 0.1-100 mg/ml;
2) adding sodium hydroxide solution, adjusting the pH value of the solution of the hyaluronic acid grafted folic acid copolymer to 9, and stirring for 5min under the state; then, hydrochloric acid solution is quickly added, the pH value of the solution is adjusted to 7.2 within 5s, and the hyaluronic acid grafted folic acid copolymer nanoparticles are obtained, wherein the particle size of the hyaluronic acid grafted folic acid copolymer nanoparticles in the solution is less than 100 nm.
Microscopic observation of the hyaluronic acid grafted folic acid copolymer nanoparticles:
taking 1mg/ml hyaluronic acid grafted folic acid copolymer nanoparticles, and carrying out ultrasonic treatment for 10min for later use. Placing a copper net in a surface dish covered with filter paper, dripping 20 mu L of nanoparticle dispersion liquid of 1mg/ml on the copper net, dyeing with 0.2% phosphotungstic acid, naturally drying at room temperature, observing the morphology of the nanoparticle dispersion liquid by using a projection electron microscope, measuring the particle size and distribution of micelles by using a laser particle sizer, measuring the temperature to be 25 ℃, balancing for 2min, and using a laser power supply: He-Ne laser with a wavelength of 633 nm.
The preparation method of the nanoparticle preparation 4 (hyaluronic acid grafted folic acid copolymer nanoparticles coated with adriamycin) comprises the following steps:
1) dissolving a hyaluronic acid-grafted folic acid copolymer in water to obtain a solution of the hyaluronic acid-grafted folic acid copolymer with the concentration of 0.1-100 mg/ml;
2) adding a proper amount of sodium hydroxide solution, adjusting the pH value of the solution of the hyaluronic acid grafted folic acid copolymer to 9, and stirring for 5min under the state; then quickly adding a hydrochloric acid solution, and regulating the pH value of the solution to 7.2 within 5s to obtain hyaluronic acid grafted folate copolymer nanoparticles, wherein the particle size of the hyaluronic acid grafted folate copolymer nanoparticles in the solution is less than 100 nm;
3) adjusting the pH value of the hyaluronic acid grafted folic acid copolymer nanoparticles to 8-12; ultrasonic crushing under ice bath condition, and simultaneously adding adriamycin dimethyl sulfoxide solution without hydrochloric acid to obtain solution after ultrasonic treatment;
4) ultrasonically crushing the solution subjected to ultrasonic treatment for 5-15 min under an ice bath condition, and then adjusting the pH value to 7.2-7.4; obtaining hyaluronic acid grafted folic acid copolymer nanoparticles carrying adriamycin;
5) centrifuging and ultrafiltering the hyaluronic acid grafted folate copolymer nanoparticle coated with the adriamycin obtained in the step 4), wherein the molecular weight cut-off of an ultrafiltration tube is 3000-100000 Da, and ultrafiltering for 5-10 times to obtain a hyaluronic acid grafted folate copolymer nanoparticle solution coated with the adriamycin, namely a nanoparticle preparation 4, wherein the mass ratio of the adriamycin to the hyaluronic acid grafted folate copolymer nanoparticle is 1: 10.
as can be seen from the DLS results in fig. 16, the doxorubicin-loaded hyaluronic acid-grafted folic acid copolymer nanoparticles were about 90nm and were uniformly distributed. TEM results were consistent with DLS results.
Example 5
A hydroxyethylcellulose-grafted folate copolymer having the general formula:
wherein R is hydroxyethyl cellulose with the molecular weight of 50 kDa.
The preparation method of the glucan-grafted folic acid copolymer shown in fig. 17 comprises the following steps:
1) dissolving folic acid and activating its carboxyl group: dissolving 0.5g folic acid in 5ml anhydrous dimethyl sulfoxide, adding 1-hydroxy benzotriazole and N-N' -dicyclohexyl carbodiimide, performing carboxyl activation reaction at 60 ℃ for 30min, and stirring at room temperature for 2-4 h to obtain a terminal carboxyl activated folic acid solution; wherein the feeding molar ratio of the folic acid, the 1-hydroxybenzotriazole and the N-N' -dicyclohexylcarbodiimide is 1:1: 1;
2) dissolving polysaccharide: under the protection of helium, completely dissolving hydroxyethyl cellulose with molecular weight of 50kDa in anhydrous dimethyl sulfoxide at the temperature of 500 ℃ to obtain a dimethyl sulfoxide solution of the hydroxyethyl cellulose:
3) esterification reaction: mixing the carboxyl activated folic acid solution obtained in the step 1) with the dimethyl sulfoxide solution of hydroxyethyl cellulose obtained in the step 2), performing esterification reaction for 24-72 h under the protection of nitrogen and at the temperature of 40-80 ℃, and purifying to obtain a copolymer mixture;
4) and (3) purification: dialyzing the copolymer mixture by using a PBS (phosphate buffer solution), dialyzing the mixture for 3 days by using a dialysis bag with the molecular weight of 3500Da, removing unreacted 1-hydroxybenzotriazole, N-N' -dicyclohexylcarbodiimide, folic acid and a DMSO (dimethyl sulfoxide) solvent, transferring the liquid in the dialysis bag into a culture dish after dialysis is finished, freezing for 4 hours at the temperature of-20 ℃, then freeze-drying at the temperature of-50 ℃, and obtaining the hydroxyethyl cellulose grafted folic acid copolymer freeze-dried powder after freeze-drying; namely the hydroxyethyl cellulose grafted folic acid copolymer (HA-FA).
The chemical structure of the prepared hydroxyethyl cellulose-folic acid grafted polymer is confirmed by infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (1H-NMR). As can be seen from FIG. 19, the IR absorption of the hydroxyethylcellulose-folic acid graft polymer was 1730cm as compared to the IR spectrum of hydroxyethylcellulose-1A new absorption peak appeared, which was attributed to the C ═ O stretching vibration peak of the ester bond formed by the reaction. The infrared absorption of the hydroxyethyl cellulose-folic acid graft polymer is 1697cm-1And 1643cm-1A new absorption peak appears, attributed to the stretching vibration of the aromatic ring on folic acid. These newly appearing absorption peaks demonstrate the successful grafting of folic acid onto hydroxyethyl cellulose.
As can be seen from FIG. 20, a new set of peaks, attributed to protons on aromatic rings on folic acid, appeared at 6.5-9.0ppm for hydroxyethylcellulose-folic acid as compared with the nuclear magnetic spectrum of hyaluronic acid. Hydroxyethyl cellulose-folic acid showed a new set of peaks at 1.5-3ppm, which are attributed to the proton on methylene in folic acid. The correctness of the chemical structure of the hydroxyethylcellulose-grafted folate copolymer prepared in this example 6 was confirmed by NMR. The results of the nuclear magnetic resonance further confirmed that the substitution degree of folic acid on hydroxyethylcellulose was 46.4 wt% as a result of HEC-FA.
Preparation of hydroxyethyl cellulose grafted folic acid copolymer nanoparticles: the method comprises the following steps:
1) dissolving the hydroxyethyl cellulose grafted folic acid copolymer in water to obtain a solution of the hydroxyethyl cellulose grafted folic acid copolymer with the concentration of 0.1-100 mg/ml;
2) adding sodium hydroxide solution, adjusting the pH value of the solution of the hydroxyethyl cellulose grafted folic acid copolymer to 9, and stirring for 5min under the state; then, quickly adding a hydrochloric acid solution, and regulating the pH value of the solution to 7.2 within 5s to obtain the hydroxyethyl cellulose grafted folic acid copolymer nanoparticles, wherein the particle size of the hydroxyethyl cellulose grafted folic acid copolymer nanoparticles in the solution is less than 100 nm.
Observation of hydroxyethyl cellulose grafted folic acid copolymer nanoparticles by a microscope:
taking 1mg/ml nanometer particles of hydroxyethyl cellulose-folic acid, and performing ultrasonic treatment for 10 min. Placing a copper net in a surface dish covered with filter paper, dripping 20 mu L of nanoparticle dispersion liquid of 1mg/ml on the copper net, dyeing with 0.2% phosphotungstic acid, naturally drying at room temperature, observing the morphology of the nanoparticle dispersion liquid by using a projection electron microscope, measuring the particle size and distribution of micelles by using a laser particle sizer, measuring the temperature to be 25 ℃, balancing for 2min, and using a laser power supply: He-Ne laser with a wavelength of 633 nm.
The preparation method of the nanoparticle preparation 5 (the hydroxyethyl cellulose grafted folic acid copolymer nanoparticle coated with adriamycin) comprises the following steps:
1) adjusting the pH value of the hydroxyethyl cellulose grafted folic acid copolymer nanoparticles to 8-12; ultrasonically crushing under ice bath condition, and simultaneously adding dimethyl sulfoxide solution of the antitumor drug without hydrochloric acid to obtain solution after ultrasonic treatment;
2) ultrasonically crushing the solution subjected to ultrasonic treatment for 5-15 min under an ice bath condition, and then adjusting the pH value to 7.2-7.4; obtaining hydroxyethyl cellulose grafted folic acid copolymer nanoparticles carrying adriamycin;
3) centrifuging and ultrafiltering the hydroxyethyl cellulose grafted folic acid copolymer nanoparticle coated with the adriamycin obtained in the step 4), wherein the molecular weight cut-off of an ultrafiltration tube is 3000-100000 Da, and ultrafiltering for 5-10 times to obtain the hydroxyethyl cellulose grafted folic acid copolymer nanoparticle coated with the adriamycin, namely the nanoparticle preparation 5. The mass ratio of adriamycin to hydroxyethyl cellulose grafted folic acid copolymer nanoparticles is 1:10
As is clear from the DLS results in FIG. 20, the distribution of the particle size of the hydroxyethyl cellulose-folic acid entrapping adriamycin was uniform and about 90 nm. TEM results were consistent with DLS results.
Example 6
The active targeting property of folic acid in the polysaccharide grafted folate copolymer nanoparticle provided by the invention is investigated through a tetramethylazozole salt (MTT) trace enzyme reaction experiment. Dextran-grafted folate copolymer nanoparticles prepared according to the method of example 1 were examined.
First, MTT toxicity of the material on a folate receptor high-expression cell-4T 1 group and a folate receptor low-expression cell-A549 group is examined. MTT cytotoxicity assay procedure was as follows, 4T1 cells and A549 cells were cultured in a medium (PRMI1640) containing 10% Fetal Bovine Serum (FBS), 100units/mL penicillin and 100. mu.g/mL streptomycin at 37 ℃ with saturation humidity and 5% CO2The culture is carried out. The 4T1 cells and A549 cells in logarithmic growth phase were digested with 0.25% trypsin and beaten into cell suspensions, respectively, seeded at 4000cells/well density in 96-well plates at 37 ℃ and 5% CO2Culturing for 24h until the cell reaches 80-90% fusion degree, and sucking out the original culture solution.
Under different conditions (2mg/mL folic acid PRMI1640 culture solution is saturated for 2 hours and unsaturated), prepared adriamycin-coated dextran graft folate copolymer nanoparticles (adriamycin @ dextran-folic acid (DOX @ DEX-FA)) with the loading concentration of 0.0625. mu.g/mL, 0.125. mu.g/mL, 0.25. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 5. mu.g/mL and 10. mu.g/mL adriamycin (DOX)) and free adriamycin (DOX) solution are respectively added into the cultured cells, 20. mu.l MTT (5mg/mL) is added into each well after 24 hours of culture, and 5 percent CO is continuously added at 37 ℃ and 5 percent2Culturing for 4h in an incubator, carefully absorbing liquid in a plate hole, adding 150 mu l DMSO into each hole, and shaking for 15min by a shaking table to fully dissolve residual MTT-formazan crystals. Both blank (i.e., without cells and drug solution) and control (i.e., cells without drug treatment) groups were established. The blank set is zeroed and the absorbance OD at 490nm is determined on a microplate reader. Cell viability was calculated using the following formula, wherein ODtest,ODcontrol,ODbackgroundThe absorbance of the drug addition group, the control group and the blank group is respectively referred.
As can be seen from FIG. 21, the cytotoxicity test results of MTT showed that in 4T1 cells with high folate receptor expression, adriamycin @ glucan-folic acid (DOX @ DEX-FA) has higher toxicity than the group of folic acid + adriamycin @ glucan-folic acid (FA + DOX @ DEX-FA) at different adriamycin (DOX) concentrations, and that in A549 cells with low folate receptor expression, adriamycin @ glucan-folic acid @ DOX @ DEX-FA has similar toxicity to folic acid + adriamycin @ glucan-folic acid FA + DOX @ DEX-FA at different adriamycin (DOX) concentrations. The active targeting property of folic acid in the adriamycin @ glucan-folic acid DOX @ DEX-FA nanoparticles is shown in 4T1 cells with high expression of folic acid receptors, while folic acid in the adriamycin @ glucan-folic acid DOX @ DEX-FA nanoparticles does not show the active targeting property in A549 cells with low expression of folic acid receptors. Both results demonstrate that folic acid in adriamycin @ glucan-folic acid DOX @ DEX-FA still has active targeting property.
Example 7
The active targeting of folic acid in the polysaccharide grafted folic acid copolymer nanoparticles is further investigated through the experiment of taking the material by cells. Dextran-grafted folate copolymer nanoparticles prepared according to the method of example 1 were examined. First, cellular uptake of the material into the folate receptor high-expression cell-4T 1 group was examined.
The cell uptake assay procedure was as follows:
4T1 cell culture in PRMI1640 medium containing 10% fetal calf serum, 100U/mL penicillin and 100. mu.g/mL streptomycin, ECV304 cell culture in RPMI 1640 complete medium containing 10% fetal calf serum, 100U/mL penicillin and 100. mu.g/mL streptomycin, all at 37 ℃ in 5% CO2Culturing in a saturated humidity incubator. The cells are subjected to digestion passage, and the cells in logarithmic growth phase are taken for experiment.
4T1 cells and A549 cells in logarithmic growth phase were seeded in 6-well plates (2X 106/well) in parallel to 3 wells at 37 ℃ with 5% CO2The mixture was allowed to stand overnight under saturated humidity conditions, and DOX concentrations were added under different conditions (2mg/mL folic acid in PRMI1640 medium pre-saturated for 2 hours and unsaturated)DOX at 10. mu.g/mL and DOX @ DEX-FA nanoparticles were incubated in an incubator at 37 ℃ for 1h, the cells were collected, washed 3 times with PBS, and counted. Breaking cells by a cell breaker, adding methanol to extract DOX in the cells after breaking the cells, centrifuging, removing a methanol solution of DOX in a supernatant, and measuring the content of DOX in the supernatant by a fluorescence spectrophotometer. As can be seen from FIG. 22, DOX @ DEX-FA has higher cellular uptake relative to FA + DOX @ DEX-FA for fine 4T1 cells with high FA expression, while DOX @ DEX-FA has no significant difference relative to FA + DOX @ DEX-FA for Are49 cells with low FA expression. Doxorubicin (DOX) and folate + Doxorubicin @ dextran-folate (FA + DOX @ DEX-FA) showed no significant difference in uptake by both cells. These results indicate that folic acid in doxorubicin @ dextran-folic acid (DOX @ DEX-FA) still retained active targeting.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (1)
1. A preparation method of glucan grafted folic acid copolymer nanoparticles is characterized by comprising the following steps: the method comprises the following steps:
1) dissolving the glucan-grafted folic acid copolymer in water to obtain a solution of the glucan-grafted folic acid copolymer with the concentration of 0.1-100 mg/ml; wherein the general formula of the glucan grafted folic acid copolymer is as follows:
and n is 640;
2) adjusting the pH value of the solution of the glucan grafted folic acid copolymer to 8-12, and stirring for 5-30 min under the state; and then adjusting the pH value of the solution to 7.2-7.4 within 0-10 s to obtain the glucan grafted folic acid copolymer nanoparticles, wherein the particle size of the glucan grafted folic acid copolymer nanoparticles is less than 100 nm.
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