CN107875399B - Telmisartan modified chitosan oligosaccharide fatty acid nanoparticle and preparation and application thereof - Google Patents
Telmisartan modified chitosan oligosaccharide fatty acid nanoparticle and preparation and application thereof Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
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Abstract
The invention provides telmisartan modified chitosan oligosaccharide fatty acid which is a high molecular substance, wherein amino groups on the chitosan oligosaccharide are substituted by fatty acid and telmisartan molecules. Wherein the molecular weight of the chitosan oligosaccharide fatty acid is 1-20 kDa, the deacetylation degree is 70-100%, the carbon chain length of the fatty acid is C12-C22, and the amino substitution degree is 1-50%. The invention further chemically modifies telmisartan with small molecular weight on the basis of chitosan oligosaccharide fatty acid structure which has stable structure, strong passive targeting tumor effect and high in-vivo stability, not only increases the in-vivo stability of the medicament, but also has the capability of actively targeting tumor cells through the interaction with angiotensin II receptor I highly expressed on the surface of the tumor cells, and can improve the curative effect of the antitumor medicament in tumor treatment. The representative structural general formula is as follows:
Description
Technical Field
The invention belongs to the field of pharmacy, and relates to telmisartan modified chitosan oligosaccharide fatty acid nanoparticles, a preparation method thereof and application of the synthesized telmisartan modified chitosan oligosaccharide fatty acid nanoparticles.
Background
Although chemotherapy, radiotherapy and other treatment methods can prolong the survival time of patients, the distribution of small-molecule antitumor drugs lacks specificity, and the small-molecule antitumor drugs can easily damage normal organs of human bodies, such as the existing doxorubicin hydrochloride injection, and can easily generate serious myocardial degenerative diseases, bone marrow suppression and other side effects, thereby limiting clinical application.
The nano drug delivery system for encapsulating the free drugs can passively diffuse to tumor tissues by enhancing the permeation and interception effects, so that the antitumor effect is improved. Although nano-drugs improve patient survival in terms of treatment, clinical treatment results are not as optimistic as preclinical experimental results. Therefore, researchers can realize more aggregation at the focus of tumor by modifying target heads such as monoclonal antibodies, polypeptides, nucleic acids, aptamers and the like on the surface of a nano drug delivery system by utilizing the difference of the expression level of receptors of normal cells and tumor cells.
Chitosan is a cationic polymer consisting of glucosamine, which can be obtained by deacetylating chitin with alkali or enzymes. The natural polycation natural polymer material has better biocompatibility, biodegradability and safety. The cell pairs have higher uptake capacity for fat-soluble substances, but the chitosan oligosaccharide is not easy to permeate cell membranes due to lack of lipophilicity. The amphiphilic polymer prepared by chemically grafting the chitosan oligosaccharide and the lipophilic fatty acid can form micelles in an aqueous medium in a self-aggregation mode. The nanoparticle can be rapidly taken up by tumor cells due to the secondary nucleus formed on the surface of the micelle by the modified fatty acid. After the micelle is coated with the anti-tumor drug in a physical coating mode, the therapeutic activity of the original anti-tumor drug can be obviously improved, and the function of reversing the drug resistance of tumor cells can be realized.
Prior studies have demonstrated that angiotensin II receptor I is a family of G protein-coupled receptors expressed on cell membranes and is highly expressed on the cell membranes of a variety of tumors, such as breast, pancreatic, ovarian, and the like. Angiotensin II receptor I may therefore be a potential receptor for achieving targeting of the tumor microenvironment.
Angiotensin II receptor I inhibitors such as telmisartan have been approved by the FDA to protect the visceral organs of hypertensive patients. Telmisartan has also been shown by researchers to be a potent intermediate in ARBs with AT1R affinity was strongest.
The telmisartan containing carboxyl is grafted with partial amino in the structure of chitosan oligosaccharide fatty acid, and the prepared product can form a micelle with an active targeting effect through self-aggregation in water. The hydrophobic core of the micelle can solubilize hydrophobic drug molecules. Due to the surface modified telmisartan, the drug delivery system can improve the drug uptake of cells and increase the drug effect of anti-tumor treatment. The application of the active targeting carrier material can greatly improve the uptake of antitumor drugs by tumor cells and increase the aggregation of the drugs at tumor parts, and finally improve the treatment efficacy of patients.
Disclosure of Invention
The invention aims to provide telmisartan modified chitosan oligosaccharide fatty acid nanoparticles, which have a representative structural general formula as follows:
wherein:
a is the percentage of sugar single rings of which the amino groups in the telmisartan modified chitosan oligosaccharide fatty acid nanoparticles are not substituted, the range is 40-90 percent,
b is the percentage of sugar single ring substituted by fatty acid in the telmisartan modified chitosan oligosaccharide fatty acid nanoparticles, the range is 1-50%,
c is the percentage of sugar single ring substituted by telmisartan in the telmisartan modified chitosan oligosaccharide fatty acid nanoparticles, and the range is 0.3-2.0%.
The second purpose of the invention is to provide a preparation method of telmisartan modified chitosan oligosaccharide fatty acid, which is realized by the following scheme:
1. preparing a reaction solution: weighing 15 mmol-30 mmol of telmisartan, dissolving in 1.5mL of N, N-dimethylformamide, adding 150mmol of N-hydroxysuccinimide and 150mmol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and activating at 60 ℃ for 1 hour;
2. weighing 2mmol of chitosan oligosaccharide fatty acid, dissolving in 4mL of water, performing ultrasonic treatment for 30 times by using a probe, adding the reaction solution, supplementing 4.0mL of N, N-dimethylformamide, and reacting for 12 hours at 60 ℃; and (3) putting the reaction solution into a dialysis bag, dialyzing for 2 days in acid solution with the pH value of 1.0, continuously dialyzing for 2 days with distilled water, and freeze-drying to obtain the target product telmisartan modified chitosan oligosaccharide fatty acid.
The chitosan fatty acid used in the invention is covered by the national invention patent of 'fluorescence labeling hydrophobic modified chitosan polymer and preparation method and application' (patent number: ZL2005100507981) and 'surface modification hydrophobic modified chitosan oligosaccharide polymer administration micelle and preparation method thereof' (patent number: ZL 200610051601.0). The substitution degree of the fatty acid amino group of the chitosan oligosaccharide is 1 to 50 percent; wherein the molecular weight of the chitosan oligosaccharide is 1-200 kDa; the carbon chain length of the fatty acid is: C12-C22; the deacetylation degree of the chitosan oligosaccharide is 70-100%.
The third purpose of the invention is to provide the application of telmisartan modified chitosan oligosaccharide fatty acid in the preparation of anti-tumor immunotherapy drugs, in particular the application in the preparation of anti-tumor immunotherapy drugs targeting breast cancer tumor cells. Research results show that telmisartan modified chitooligosaccharide fatty acid can actively target breast cancer tumor cells.
The telmisartan modified chitosan oligosaccharide fatty acid provided by the invention is a high molecular substance, wherein an amino group on the chitosan oligosaccharide is substituted by a fatty acid and telmisartan molecules. Wherein the molecular weight of the chitosan oligosaccharide fatty acid is 1-20 kDa, the deacetylation degree is 70-100%, the carbon chain length of the fatty acid is C12-C22, and the amino substitution degree is 1-50%. The invention further chemically modifies telmisartan with small molecular weight on the basis of chitosan oligosaccharide fatty acid structure which has stable structure, strong passive targeting tumor effect and high in-vivo stability, not only increases the in-vivo stability of the medicament, but also has the capability of actively targeting tumor cells through the interaction with angiotensin II receptor I highly expressed on the surface of the tumor cells, and can improve the curative effect of the antitumor medicament in tumor treatment. The invention has the advantages that: on the basis of the previous research work, abundant free amino groups on the chitosan oligosaccharide fatty acid react with carboxyl groups with reactivity on telmisartan molecules under the action of a catalyst to synthesize a telmisartan modified chitosan oligosaccharide fatty acid delivery system, so that the in-vivo stability of the drug is improved, and the drug uptake and anti-tumor effect of tumor cells can be improved.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of telmisartan, chitosan oligosaccharide fatty acid and telmisartan modified chitosan oligosaccharide fatty acid.
Fig. 2 shows the release curves of adriamycin, chitosan oligosaccharide fatty acid drug-loaded micelle and telmisartan modified chitosan oligosaccharide fatty acid drug-loaded micelle in release media with different pH values.
FIG. 3 shows the result of the flow cytometry for detecting the uptake of telmisartan modified chitosan oligosaccharide fatty acid carriers on breast cancer cells in different bonding ratios.
Detailed Description
The invention is further illustrated by means of examples and figures.
Example 1
According to the national invention patent of 'fluorescence labeling hydrophobic modified chitosan polymer and preparation method and application' (patent number: ZL2005100507981) and 'surface modification hydrophobic modified chitosan oligosaccharide polymer administration micelle and preparation method thereof' (patent number: ZL200610051601.0), chitosan oligosaccharide fatty acid is synthesized and prepared.
Determining the amino substitution degree of the chitosan oligosaccharide stearic acid by adopting a trinitrobenzene sulfonic acid method, dissolving 1-10 mg of chitosan oligosaccharide with different weights in 2mL of distilled water respectively, adding 2mL of 4% sodium bicarbonate and 2mL of 0.1% trinitrobenzene sulfonic acid, incubating at 37 ℃ for 2 hours, adding 2mL of 2mol/L hydrochloric acid, shaking up, determining the absorbance at 344nm, preparing a standard curve, dissolving 4mg of chitosan oligosaccharide fatty acid in 2mL of distilled water, operating in the same way, and calculating the amino substitution degree of the chitosan oligosaccharide-stearic acid to be 6.71 according to the standard curve;
preparation of telmisartan modified chitosan oligosaccharide fatty acid:
dissolving telmisartan 7.71mg in 1.5mL of N, N-dimethylformamide, adding N-hydroxysuccinimide 17.25mg and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride 28.65mg, and activating at 60 ℃ for 1 hour;
weighing 40mg of chitosan oligosaccharide fatty acid, dissolving in 4mL of water, carrying out ultrasonic treatment for 30 times by using a probe, adding all the reaction solution, supplementing 4.0mL of N, N-dimethylformamide, and reacting for 12 hours at 60 ℃. And (3) putting the reaction solution into a dialysis bag, dialyzing for 2 days in acid solution with the pH value of 1.0, continuously dialyzing for 2 days with distilled water, and freeze-drying to obtain the telmisartan modified chitosan oligosaccharide fatty acid.
Respectively weighing 4mg of chitosan oligosaccharide fatty acid and telmisartan modified chitosan oligosaccharide fatty acid, adding 2mL of water, performing ultrasonic treatment for 30 times by using a probe, respectively taking 200 mu l of fluorescein isothiocyanate of 2mg/mL, and stirring for 12 hours at room temperature in a dark place. And (3) putting the reaction solution into a dialysis bag, dialyzing for 1 day, and centrifuging to obtain fluorescein isothiocyanate labeled chitosan oligosaccharide fatty acid and telmisartan modified chitosan oligosaccharide fatty acid.
In 24-well cell culture plates, 1mL of 1X 10-containing solution was added per well5An MCF-7 cellAt 37 ℃ and 5% CO2Culturing in an incubator for 12 hours, and adding 100 mul of telmisartan modified chitosan oligosaccharide fatty acid into each hole after the cells are completely attached to the wall. And continuously incubating for 12 hours, discarding the culture solution, washing three times by using an isotonic phosphate buffer solution, digesting the cells by using 0.25% pancreatin, centrifuging to obtain cell precipitates, re-suspending the cell precipitates by using 0.5mL of buffer solution, and detecting the fluorescence value of fluorescein isothiocyanate by using a flow cytometer. The detection result shows that the chitosan oligosaccharide fatty acid and the telmisartan modified chitosan oligosaccharide fatty acid are respectively 12.8 and 22.1.
Example 2
According to the national invention patent of 'fluorescence labeling hydrophobic modified chitosan polymer and preparation method and application' (patent number: ZL2005100507981) and 'surface modification hydrophobic modified chitosan oligosaccharide polymer administration micelle and preparation method thereof' (patent number: ZL200610051601.0), chitosan oligosaccharide fatty acid is synthesized and prepared.
Determining the amino substitution degree of the chitosan oligosaccharide stearic acid by adopting a trinitrobenzene sulfonic acid method, dissolving 1-10 mg of chitosan oligosaccharide with different weights in 2mL of distilled water respectively, adding 2mL of 4% sodium bicarbonate and 2mL of 0.1% trinitrobenzene sulfonic acid, incubating at 37 ℃ for 2 hours, adding 2mL of 2mol/L hydrochloric acid, shaking up, determining the absorbance at 344nm, preparing a standard curve, dissolving 4mg of the chitosan oligosaccharide fatty acid in 2mL of distilled water, operating the same method, and calculating the amino substitution degree of the chitosan oligosaccharide-stearic acid to be 6.71 according to the standard curve;
preparation of telmisartan modified chitosan oligosaccharide fatty acid:
dissolving telmisartan 11.56mg in 1.5mL of N, N-dimethylformamide, adding N-hydroxysuccinimide 17.25mg and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride 28.65mg, and activating at 60 ℃ for 1 hour;
weighing 40mg of chitosan oligosaccharide fatty acid, dissolving in 4mL of water, carrying out ultrasonic treatment for 30 times by using a probe, adding all the reaction solution, supplementing 4.0mL of N, N-dimethylformamide, and reacting for 12 hours at 60 ℃. And (3) putting the reaction solution into a dialysis bag, dialyzing for 2 days in acid solution with the pH value of 1.0, continuously dialyzing for 2 days with distilled water, and freeze-drying to obtain a telmisartan modified chitosan oligosaccharide fatty acid product.
Respectively weighing 4mg of chitosan oligosaccharide fatty acid and telmisartan modified chitosan oligosaccharide fatty acid, adding 2mL of water, performing ultrasonic treatment for 30 times by using a probe, respectively taking 200 mu l of fluorescein isothiocyanate of 2mg/mL, and stirring for 12 hours at room temperature in a dark place. And (3) putting the reaction solution into a dialysis bag, dialyzing for 1 day, and centrifuging to obtain fluorescein isothiocyanate labeled chitosan oligosaccharide fatty acid and telmisartan modified chitosan oligosaccharide fatty acid.
In 24-well cell culture plates, 1mL of 1X 10-containing solution was added per well5MCF-7 cells, at 37 ℃ with 5% CO2Culturing in an incubator for 12 hours, and adding 100 mul of telmisartan modified chitosan oligosaccharide fatty acid into each hole after the cells are completely attached to the wall. And continuously incubating for 12 hours, discarding the culture solution, washing three times by using an isotonic phosphate buffer solution, digesting the cells by using 0.25% pancreatin, centrifuging to obtain cell precipitates, re-suspending the cell precipitates by using 0.5mL of buffer solution, and detecting the fluorescence value of fluorescein isothiocyanate by using a flow cytometer. The detection result shows that the chitosan oligosaccharide fatty acid and the telmisartan modified chitosan oligosaccharide fatty acid are respectively 12.8 and 35.8.
Example 3
According to the national invention patent of 'fluorescence labeling hydrophobic modified chitosan polymer and preparation method and application' (patent number: ZL2005100507981) and 'surface modification hydrophobic modified chitosan oligosaccharide polymer administration micelle and preparation method thereof' (patent number: ZL200610051601.0), chitosan oligosaccharide fatty acid is synthesized and prepared.
The amino substitution degree of the chitosan oligosaccharide stearic acid is determined by adopting a trinitrobenzene sulfonic acid method, 1-10 mg of chitosan oligosaccharide with different weights are respectively dissolved in 2mL of distilled water, 2mL of 4% sodium bicarbonate and 2mL of 0.1% trinitrobenzene sulfonic acid are added, incubation is carried out for 2 hours at 37 ℃, 2mL of 2mol/L hydrochloric acid is added, shaking is carried out, absorbance is determined at 34nm, a standard curve is prepared, 4mg of the chitosan oligosaccharide fatty acid is dissolved in 2mL of distilled water, the operation is carried out by the same method, and the amino substitution degree of the chitosan oligosaccharide-stearic acid is calculated to be 6.71 according to the standard curve.
Preparation of telmisartan modified chitosan oligosaccharide fatty acid:
dissolving telmisartan 15.42mg in 1.5mL of N, N-dimethylformamide, adding N-hydroxysuccinimide 17.25mg and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride 28.65mg, and activating at 60 ℃ for 1 hour;
weighing 40mg of chitosan oligosaccharide fatty acid, dissolving in 4mL of water, carrying out ultrasonic treatment for 30 times by using a probe, adding all the reaction solution, supplementing 4.0mL of N, N-dimethylformamide, and reacting for 12 hours at 60 ℃. And (3) putting the reaction solution into a dialysis bag, dialyzing for 2 days in acid solution with the pH value of 1.0, continuously dialyzing for 2 days with distilled water, and freeze-drying to obtain a telmisartan modified chitosan oligosaccharide fatty acid product.
And (3) measuring telmisartan, chitosan oligosaccharide stearic acid and telmisartan modified chitosan oligosaccharide fatty acid by using nuclear magnetic resonance spectroscopy. Weighing 5mg of each of chitosan oligosaccharide fatty acid and telmisartan modified chitosan oligosaccharide fatty acid, and respectively using 0.5mLD2Dissolving telmisartan 5mg in telmisartan 0.5ml of solution of telmisartan D6 by nuclear magnetic resonance1And H-NMR determination results are shown in figure 1, wherein A is telmisartan, B is chitosan oligosaccharide fatty acid, and C is telmisartan modified chitosan oligosaccharide fatty acid.
And (3) determining the critical micelle concentration of the chitosan oligosaccharide fatty acid and the telmisartan modified chitosan oligosaccharide fatty acid by adopting a pyrene fluorescence method. Accurately weighing 12mg of pyrene, placing the pyrene in a 100mL volumetric flask, adding acetone to dissolve and fixing the volume. Transferring 1mL of the pyrene solution, placing the pyrene solution in a 100mL volumetric flask for dilution and constant volume. 0.5mL of the diluted pyrene solution was transferred to 10mL glass tubes, and acetone was evaporated at 50 ℃.5mL of chitosan oligosaccharide fatty acid solution with different concentrations and telmisartan modified chitosan oligosaccharide fatty acid solution are respectively added, and the final concentration of pyrene is controlled to be 7 x 10-7And (3) carrying out ultrasonic treatment for 30min in a room-temperature water bath at mol/l. Scanning excitation and emission spectra of pyrene (EX 337nm, EM: I)1＝374nm，I3384nm, slit 2.5nm and 10nm), the fluorescence intensity was measured, and the critical micelle concentration was calculated to be 48.3 μ g/mL and 16.1 μ g/mL, respectively.
4mg of chitosan oligosaccharide fatty acid and telmisartan modified chitosan oligosaccharide fatty acid are precisely weighed respectively, 2mL of water is added, ultrasonic treatment is carried out for 30 times by a probe, and 200 mul of 2mg/mL adriamycin dimethyl sulfoxide solution is added respectively. Magnetically stirring for 2 hours, and dialyzing in a dialysis bag for 2 days to obtain the chitosan oligosaccharide fatty acid drug-loaded micelle and the telmisartan modified chitosan oligosaccharide fatty acid drug-loaded micelle.
And (3) measuring the content of adriamycin in the chitosan oligosaccharide fatty acid drug-loaded micelles and the telmisartan modified chitosan oligosaccharide fatty acid drug-loaded micelles by using a fluorescence spectrophotometry, dissolving the adriamycin in dimethyl sulfoxide, diluting the dimethyl sulfoxide to prepare solutions with different concentrations, and preparing a standard curve. A certain amount of chitosan oligosaccharide fatty acid drug-loaded micelle and telmisartan modified chitosan oligosaccharide fatty acid drug-loaded micelle are dissolved in dimethyl sulfoxide, the fluorescence value is measured, and the content (weight percentage) of adriamycin in the carrier is calculated to be 8.0% and 7.5% respectively through a standard curve.
Weighing 10mg of each chitosan oligosaccharide fatty acid modified by telmisartan, precisely weighing, and dissolving in 10mL of distilled water to obtain a solution of 1 mg/mL. The probe was sonicated 30 times (400w, work 2s, pause 3 s). The particle size and surface potential analyzer measures the particle size of the chitosan oligosaccharide fatty acid micelle and the particle size of the telmisartan modified chitosan oligosaccharide fatty acid micelle to be 62.3nm and 76.3nm, and the Zeta potential is 16.7mV and 35.1 mV.
And weighing 10mg of each chitosan oligosaccharide fatty acid drug-loaded micelle and telmisartan modified chitosan oligosaccharide fatty acid drug-loaded micelle, precisely weighing, and dissolving in 10mL of distilled water to obtain a solution of 1 mg/mL. The particle size and the particle size of the chitosan oligosaccharide fatty acid loaded micelle and telmisartan modified chitosan oligosaccharide fatty acid loaded particle size measured by a surface potential analyzer are 34.6nm and 30.3 nm.
The in vitro release behavior of the chitosan oligosaccharide fatty acid medicament carrying micelle and telmisartan modified chitosan oligosaccharide fatty acid is investigated:
the method comprises the steps of taking 1mL of chitosan oligosaccharide fatty acid drug-loaded micelle with the adriamycin concentration of 30 mu g/mL and telmisartan modified chitosan oligosaccharide fatty acid drug-loaded micelle, putting the chitosan oligosaccharide fatty acid drug-loaded micelle and telmisartan modified chitosan oligosaccharide fatty acid drug-loaded micelle into a dialysis bag (MWCO ═ 7000), putting the dialysis bag into 20mL of phosphate buffer solutions with different pH values (pH 5.0 and pH 7.4 respectively), and oscillating the solutions in a shaking table at 37 ℃. 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 72 hours. The concentration of the drug in the sample was measured by fluorescence spectrophotometry (EX ═ 505nm, EM ═ 565nm, slit ═ 5 nm). The release profile is shown in figure 2.
The chitosan oligosaccharide fatty acid drug-loaded micelle and the telmisartan modified chitosan oligosaccharide fatty acid drug-loaded micelle have the following antitumor effects:
the invention uses tumor cell inhibition rate to evaluate the survival rate of telmisartan modified chitooligosaccharide fatty acid cells, and the cell survival rate test adopts a tetrazolium salt colorimetric method to measure. Thin breast cancerCell MCF-7 cells as a model, 200. mu.l of 2X 10 cells were added to each well of a 96-well cell culture plate3The culture solution of MCF-7 cells is placed at 37 ℃ and 5% CO2Culturing in an incubator for 12 hours, after the cells are completely attached to the wall, respectively adding adriamycin solution, chitosan oligosaccharide fatty acid drug-loaded micelles and telmisartan modified chitosan oligosaccharide fatty acid drug-loaded micelles with different concentrations into cell holes, and taking untreated blank cells as a reference, wherein each hole is provided with a plurality of holes; 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, the absorbance at 570nm was measured with an enzyme-linked detector, and the cell viability was calculated according to the following formula:
cell viability (%). test absorbance/control absorbance. times.100%
Through calculation, the IC50 of the adriamycin, the chitosan oligosaccharide fatty acid drug-loaded micelle and the telmisartan modified chitosan oligosaccharide fatty acid drug-loaded micelle on MCF-7 cells is 1.80 mug/mL, 4.39 mug/mL and 2.49 mug/mL respectively.
Respectively weighing 4mg of chitosan oligosaccharide fatty acid and telmisartan modified chitosan oligosaccharide fatty acid, adding 2mL of water, performing ultrasonic treatment for 30 times by using a probe, respectively taking 200 mu l of fluorescein isothiocyanate of 2mg/mL, and stirring for 12 hours at room temperature in a dark place. And (3) putting the reaction solution into a dialysis bag, dialyzing for 1 day, and centrifuging to obtain fluorescein isothiocyanate labeled chitosan oligosaccharide fatty acid and telmisartan modified chitosan oligosaccharide fatty acid.
In 24-well cell culture plates, 1mL of 1X 10-containing solution was added per well5MCF-7 cells, at 37 ℃ with 5% CO2Culturing in an incubator for 12 hours, and adding 100 mul of telmisartan modified chitosan oligosaccharide fatty acid into each hole after the cells are completely attached to the wall. And continuously incubating for 12 hours, discarding the culture solution, washing three times by using an isotonic phosphate buffer solution, digesting the cells by using 0.25% pancreatin, centrifuging to obtain cell precipitates, re-suspending the cell precipitates by using 0.5mL of buffer solution, and detecting the fluorescence value of fluorescein isothiocyanate by using a flow cytometer. The detection results are shown in fig. 3, in which the chitosan oligosaccharide fatty acid and telmisartan modified chitosan oligosaccharide fatty acid are 12.8 and 53.4, respectively.
Claims (2)
1. A telmisartan modified chitosan oligosaccharide fatty acid nanoparticle is characterized in that the representative structural general formula of the telmisartan modified chitosan oligosaccharide fatty acid nanoparticle is as follows:
wherein:
a is the percentage of sugar single rings of which amino groups in the telmisartan modified chitosan oligosaccharide fatty acid nanoparticles are not substituted, and the range is 40% -90%,
b is the percentage of sugar single ring substituted by fatty acid in the telmisartan modified chitosan oligosaccharide fatty acid nanoparticles, the range is 1% -50%,
c is the percentage of sugar single ring substituted by telmisartan in the telmisartan modified chitosan oligosaccharide fatty acid nanoparticles, and the range is 0.3% -2.0%;
the telmisartan modified chitosan oligosaccharide fatty acid is obtained by the following preparation method:
(1) preparing a reaction solution: dissolving telmisartan of 15 mmol-30 mmol in 1.5mL of N, N-dimethylformamide, adding 150mmol of N-hydroxysuccinimide and 150mmol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and activating at 60 ℃ for 1 hour;
(2) weighing 2mmol of chitosan oligosaccharide fatty acid, dissolving in 4mL of water, performing ultrasonic treatment for 30 times by using a probe, adding the reaction solution, supplementing 4.0mL of N, N-dimethylformamide, and reacting for 12 hours at 60 ℃; and (3) putting the reaction solution into a dialysis bag, dialyzing for 2 days in acid solution with the pH value of 1.0, continuously dialyzing for 2 days with distilled water, and freeze-drying to obtain the target product telmisartan modified chitosan oligosaccharide fatty acid.
2. The application of telmisartan modified chitosan oligosaccharide fatty acid nanoparticles in the preparation of anti-tumor immunotherapeutic drugs according to claim 1, characterized by being applied in the preparation of anti-tumor immunotherapeutic drugs targeting breast cancer tumor cells.
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| CN101148483A (en) * | 2007-11-08 | 2008-03-26 | 浙江大学 | Chitosan oligosaccharide-stearic acid grafting compound modified by folic acid, preparation method and application thereof |
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