CN111686258A - T7 polypeptide modified targeting nano system and preparation method and application thereof - Google Patents

T7 polypeptide modified targeting nano system and preparation method and application thereof Download PDF

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CN111686258A
CN111686258A CN202010459844.8A CN202010459844A CN111686258A CN 111686258 A CN111686258 A CN 111686258A CN 202010459844 A CN202010459844 A CN 202010459844A CN 111686258 A CN111686258 A CN 111686258A
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cyclodextrin
polyethyleneimine
polypeptide
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郑燕芳
吕海
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Abstract

The invention discloses a T7 polypeptide modified targeting nano system and a preparation method and application thereof. The T7 polypeptide modified targeting nano system comprises a nano delivery carrier material, wherein the nano delivery carrier material is carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide. The nano system has high-affinity pH response to transferrin receptors over-expressed by tumor cells, can be used for loading various chemotherapeutic drugs, can quickly release the loaded drugs in a tumor acid microenvironment, and plays a synergistic anti-tumor role.

Description

T7 polypeptide modified targeting nano system and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a T7 polypeptide modified targeting nano system and a preparation method and application thereof.
Background
Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. Currently, over a hundred different types of cancer exist worldwide, which can develop by almost any cellular variation within the body. With the increasing incidence and mortality rates in China, cancer is becoming a major public health problem for society. A total of 368 ten thousand new cancer cases and 223 ten thousand cancer death cases were estimated nationally in 2013. Among them, esophageal cancer, breast cancer, gastric cancer, liver cancer, colorectal cancer and esophageal cancer are the most common cancers, accounting for about half of all new cases of cancer. Esophageal cancer is the seventh most common cancer in the world, ranking the sixth in terms of cancer-related mortality.
With the development of research in the biomedical field, a variety of drugs for treating esophageal cancer have been developed, such as paclitaxel, curcumin, and cisplatin. However, most chemotherapy drugs have no targeting property, and kill cancer cells and normal tissue cells at the same time, so that some normal tissues and organs of the body can generate obvious toxic reaction. In addition, drug resistance can be generated in long-term drug therapy, so that the chemotherapy effect is greatly reduced, and the treatment effect of the esophageal cancer is poor.
To improve therapeutic efficacy and reduce drug toxicity and enrichment, a variety of drug delivery systems are currently being developed. In the past decades, the application of nano materials (NPs) as carriers of chemotherapeutic drugs is receiving more and more attention, and research shows that the nano carriers can significantly improve the anti-tumor efficiency of various chemotherapeutic drugs.
Cyclodextrins (CD) are macrocyclic compounds made up of D-glucopyranose units joined end-to-end by alpha-1, 4 glycosidic linkages, the common alpha-, beta-and gamma-cyclodextrins having 6, 7 and 8 glucose units, respectively. The structure of the cyclodextrin is similar to that of a hollow round table, the surface of an inner cavity of the cyclodextrin is formed by hydrogen atoms on C3 and C5 and oxygen atoms on glycosidic bonds, and the cyclodextrin is in a hydrophobic environment; the outer side is hydrophilic due to the accumulation of hydroxyl groups. The unique amphiphilic structure enables cyclodextrin to be frequently used as a 'host' molecule to form an inclusion compound with a plurality of hydrophobic 'guest' small molecules through host-guest interaction, and the cyclodextrin has wide application in the fields of personal care, food chemistry, environmental chemistry, pharmaceutical chemistry and the like. At present, a type of supramolecular hydrogel which is formed by taking cyclodextrin as a main body and through the interaction of the main body and an object is widely used as a research of an injectable drug carrier due to the characteristics of simple preparation process, mild preparation condition, easy regulation and control of gel property, shear thinning, thixotropy and the like. However, natural cyclodextrin has limited its application in nanocarriers due to its low solubility and single functional group, and it needs to be chemically modified.
Therefore, in view of the above situation, there is a need to develop a nano delivery vehicle with biological responsiveness, which can achieve the enrichment of drugs at the tumor site, and at the same time, due to the acidic environment at the tumor site, the drugs can be rapidly released, the damage to normal tissues is reduced, and the purpose of combined treatment of esophageal cancer is achieved.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a T7 polypeptide modified targeted nano system, a preparation method and application thereof, the nano system has high-affinity pH response to a transferrin receptor over-expressed by tumor cells, can simultaneously load a plurality of chemotherapeutic drugs and is used for treating esophageal cancer, and the nano system rapidly releases the loaded drugs in a tumor acidic microenvironment and plays a synergistic anti-tumor role.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a T7 polypeptide modified targeting nano system comprises a nano delivery carrier material, wherein the nano delivery carrier material is carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide.
The nano delivery carrier material is a carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide composite material, the carboxymethyl cyclodextrin is taken as a main body, the carboxymethyl cyclodextrin is conjugated with a target polypeptide T7 through polyethyleneimine and polyethylene glycol, the enrichment of a drug at a tumor part can be realized, and meanwhile, the drug can be rapidly released due to the acidic environment of the tumor part, so that the damage to normal tissues is reduced, and the purpose of jointly treating tumors is achieved.
Preferably, the nano delivery carrier material is also loaded with an esophageal cancer chemotherapeutic drug. The nano delivery carrier material has good biocompatibility, can load various esophagus cancer chemotherapy drugs, realizes the enrichment of the drugs at tumor parts, simultaneously can quickly release the drugs due to the acid environment of the tumor parts, and plays a role in synergistic antitumor, reduces the damage to normal tissues, improves the utilization rate of the drugs, and provides a good treatment platform for the combined treatment of esophagus cancer.
Preferably, the esophageal cancer chemotherapeutic drug comprises docetaxel and curcumin. Docetaxel (DTX) is a second generation drug of the paclitaxel family that shows good survival benefit in patients with esophageal cancer, but single chemotherapeutic drugs have limitations and side effects that lead to reduced patient compliance. Curcumin has anti-inflammatory and anti-tumor effects, and has chemosensitization effect. The docetaxel and curcumin can reduce the dosage of each component medicament by exerting a synergistic effect, improve the anti-tumor effect and further reduce the side effect.
The invention also provides a preparation method of the T7 polypeptide modified targeting nano system, which comprises the following steps:
(1) synthesis of carboxymethyl beta-cyclodextrin
Dissolving beta-cyclodextrin and sodium hydroxide in water, adding chloroacetic acid solution, heating and stirring for reaction, adjusting the pH of the solution to 6-7 after the reaction is finished, adding excessive ethanol to generate white precipitate, filtering the solid precipitate, and drying in vacuum to obtain carboxymethyl beta-cyclodextrin;
(2) synthesis of carboxymethyl beta-cyclodextrin-polyethyleneimine
Weighing carboxymethyl beta-cyclodextrin, dissolving the carboxymethyl beta-cyclodextrin in DMSO, adding N, N' -carbonyldiimidazole, stirring for reaction, then dropwise adding polyethyleneimine into an activated carboxymethyl beta-cyclodextrin solution, stirring for reaction at room temperature, and after the reaction is finished, dialyzing, and freeze-drying to obtain carboxymethyl beta-cyclodextrin-polyethyleneimine;
(3) synthesis of carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol
Mixing carboxymethyl β -cyclodextrin-polyethyleneimine with maleimideDissolving amine-polyethylene glycol-active ester in water, and adding saturated NaHCO3Regulating the pH value of the reaction mixture to 7.5-8.5 by using the solution, stirring the reaction mixture at room temperature for reaction, dialyzing, and freeze-drying to obtain carboxymethyl β -cyclodextrin-polyethyleneimine-polyethylene glycol;
(4) synthesis of carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide
Dissolving carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol in water, adding a T7 polypeptide solution, adjusting the pH of a reaction system to 6.0-6.5, stirring at room temperature for reaction, reacting the Mal group of the carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol with the thiol group of the T7 polypeptide, dialyzing after the reaction is finished, and freeze-drying to obtain the carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide.
Firstly, synthesizing carboxymethyl chitosan with biological responsiveness and good biocompatibility by utilizing beta-cyclodextrin and monochloroacetic acid through a substitution reaction; and then the nano-carrier is conjugated with a targeting polypeptide T7, so that the enrichment of the nano-carrier at a tumor site is increased.
The room temperature in the invention is 20-25 ℃.
Preferably, in the step (1), the dosage ratio of the beta-cyclodextrin, the sodium hydroxide, the water and the chloroacetic acid is (1-4) g, (0.1-0.5) g, (10-50) mL: (1-10) mL, more preferably 2g:0.3g:30mL:5mL, the obtained carboxymethyl chitosan has good biological responsiveness and good biocompatibility.
Preferably, in the step (1), the reaction temperature is 40-60 ℃, more preferably 50 ℃; the reaction time is 2-6h, more preferably 4 h.
Preferably, in the step (2), the dosage ratio of the carboxymethyl beta-cyclodextrin, the DMSO, the N, N' -carbonyldiimidazole and the polyethyleneimine is (0.5-2) g (1-20) mL: (0.1-0.6) g: (50-200) mg, preferably 1.2g:10mL:0.36g:100 mg.
Parameters such as reaction temperature, reaction time and the like involved in the preparation process of the T7 polypeptide modified targeted nano system can be correspondingly adjusted according to the using amount of reaction components.
Preferably, in the step (2), polyethyleneimine is dropwise added to the activated carboxymethyl β -cyclodextrin solution, and the reaction is stirred at room temperature for 6 to 18 hours, more preferably 12 hours.
Preferably, in the step (3), the dosage ratio of the carboxymethyl beta-cyclodextrin-polyethyleneimine, the maleimide-polyethylene glycol-active ester and the water is (0.5-1.5) g, (0.5-2) mL, more preferably 1g:1g:10 mL;
preferably, in the step (3), the stirring reaction time is 2-4h, and more preferably 4 h.
Preferably, in the step (4), the dosage ratio of the carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol to the water to the T7 polypeptide is (0.1-0.5) g (1-10) mL: (0.05-0.1) g, preferably 0.25g:5mL:0.08 g;
preferably, in the step (4), the stirring reaction time is 10-36h, and more preferably 24 h.
Preferably, the method further comprises the step (5) of synthesizing the curcumin and docetaxel loaded carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide, and specifically comprises the following steps:
dissolving carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide in ethyl acetate, adding docetaxel and curcumin solution, and performing ultrasonic treatment to form a colloidal solution;
and adding the PVA solution into the colloidal solution, performing ultrasonic treatment to form W/O/W double emulsion, centrifuging and washing to obtain the curcumin and docetaxel loaded carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide.
The nano system also loads curcumin and docetaxel, can control and release curcumin and docetaxel, and can jointly treat esophageal cancer. The nano delivery carrier improves the treatment effect of the esophageal cancer and lays a foundation for the future development of the combined chemotherapy.
The invention also provides application of the T7 polypeptide modified targeting nano system in preparation of anti-esophageal cancer drugs.
Compared with the prior art, the invention has the beneficial effects that:
the nano system has good biocompatibility, can load various chemotherapeutic drugs, has tumor targeting property, reduces the damage of the chemotherapeutic drugs to normal tissues, improves the utilization rate of the drugs and improves the treatment effect of tumors.
The preparation process of the nano system is simple, the required raw materials are easy to obtain, and the nano system is expected to be widely applied to the field of biomedical materials.
Drawings
FIG. 1 is a nuclear magnetic carbon spectrum of CM- β -CD-PEI-PEG of example 3 and CM- β -CD-PEI-PEG-T7 of example 4;
FIG. 2 is a transmission electron micrograph of CM- β -CD-PEI-PEG-T7(A) of example 4 and CM- β -CD-PEI-PEG-T7/DTX/CUR (B) of example 5;
FIG. 3 is the release profile of CUR (A) and DTX (B) in CM- β -CD-PEI-PEG-T7/DTX/CUR of example 5;
FIG. 4 is a graph of the effect of CM- β -CD-PEI-PEG-T7 of example 4 on KYSE150 activity in esophageal cancer cells;
FIG. 5 shows the results of inhibiting the proliferation of esophageal cancer KYSE150 in vitro.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.
Example 1: synthesis of carboxymethyl beta-cyclodextrin (CM-beta-CD)
Firstly, 2.14g of beta-cyclodextrin and 0.3g of sodium hydroxide are dissolved in 30mL of distilled water, then 5mL of chloroacetic acid solution with the mass fraction of 1% is added, and the mixture is stirred and reacted for 4 hours at 50 ℃; after the reaction is finished, regulating the pH value of the mixture to 7 by using hydrochloric acid; finally, an excess of ethanol was added to the neutral solution obtained, resulting in a white precipitate; the solid precipitate was filtered and dried in vacuo to give CM- β -CD.
Example 2: synthesis of carboxymethyl beta-cyclodextrin-polyethyleneimine (CM-beta-CD-PEI)
Weighing 0.12g of CM-beta-CD, dissolving the CM-beta-CD in 5mL of DMSO, adding 0.36g of N, N' -carbonyldiimidazole, and stirring for reacting for 2 hours; then, 100mg of polyethyleneimine is dropwise added into the activated CM-beta-CD solution, and the mixture is stirred and reacts for 12 hours at room temperature; after the reaction is finished, dialyzing the product in pure water for several days by using a dialysis bag with the molecular weight cutoff of 1000, and freeze-drying the dialyzate to obtain the product CM-beta-CD-PEI.
Example 3: synthesis of carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol (CM-beta-CD-PEI-PEG)
First 1g of CM- β -CD-PEI and 1g of MAL-PEG-NHS were dissolved in 10mL of water and the mixture was poured into a flask, then saturated NaHCO was added3Adjusting pH of the reaction mixture to 8, stirring the mixture at room temperature for 4 hr, dialyzing the mixture in pure water with dialysis bag with molecular weight cut-off of 1000, and freeze drying the dialyzate to obtain CM- β -CD-PEI-PEG.
Example 4: synthesis and characterization of carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide (CM-beta-CD-PEI-PEG-T7)
To conjugate CM- β -CD-PEI-PEG to the T7 polypeptide, 0.25g of CM- β -CD-PEI-PEG was dissolved in water; then 0.08g of T7 polypeptide solution is added, and the pH of the solution is adjusted to 6.5; stirring the reaction system at room temperature for 24 hours to react the Mal group of the CM-beta-CD-PEI-PEG with the thiol group of the Cys-T7; finally, the targeting nano-carrier CM-beta-CD-PEI-PEG-T7 is obtained through dialysis purification and freeze drying.
5mg of CM- β -CD-PEI-PEG-T7 prepared in example 4 and 5mg of CM- β -CD-PEI-PEG prepared in example 3 were respectively dissolved in heavy water and the chemical structures thereof were characterized by using a nuclear magnetic resonance hydrogen spectrometer, the results are shown in FIG. 1, T7 and CM- β -CD-PEI construct a drug nanocarrier (CM- β -CD-PEI-PEG-T7) by NHS-PEG-MAL, D of NMR spectrum2The O solvent peak is located at 4.7ppm, the chemical shifts of the proton absorption peaks are 3.724 and 3.822ppm due to T7 peptide in CM- β -CD-PEI-PEG-T7, and the proton absorption peaks of β -CD appear at the chemical shifts of 5.0 and 3.2-3.6ppm, which proves that CM- β -CD-PEI-PEG-T7 is successfully synthesized.
Example 5: synthesis and characterization of curcumin and docetaxel loaded carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide (CM-beta-CD-PEI-PEG-T7/DTX/CUR)
Dissolving 40mg of CM-beta-CD-PEI-PEG-T7 in 1mL of ethyl acetate, and then adding 1mL of a mixed solution of docetaxel (docetaxel) and curcumin; emulsifying the mixture by sonication at a power of 150W for 60s to form a colloidal solution;
adding 2mL of PVA solution with the mass fraction of 4% into the colloidal solution, and carrying out ultrasonic treatment on the mixture for 90s at the power of 150W to form W/O/W double emulsion;
after 4 hours, CM- β -CD-PEI-PEG-T7/DTX/CUR was obtained by centrifugation at 5600 Xg for 10 minutes at room temperature and washed twice with distilled water.
Under the same condition, the mixed solution of docetaxel and curcumin in the method is replaced by docetaxel solution to prepare the docetaxel loaded carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide (CM-beta-CD-PEI-PEG-T7/DTX);
under the same condition, the mixed solution of docetaxel and curcumin in the method is replaced by curcumin solution to prepare the curcumin-loaded carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide CM-beta-CD-PEI-PEG-T7/CUR.
The concentrations of DTX and CUR were measured by uv spectrophotometry; the synthesized CM-beta-CD-PEI-PEG-T7/DTX/CUR was characterized by transmission electron microscopy, and the results are shown in FIG. 2. The transmission electron microscope shows that CM-beta-CD-PEI-PEG-T7/and CM-beta-CD-PEI-PEG-T7/DTX/CUR are both in a regular spherical shape with the size of about 200 nm.
Example 6: drug delivery
In vitro release studies of DTX and CUR of CM- β -CD-PEI-PEG-T7/DTX/CUR of example 5 were performed using dialysis membrane diffusion technique. Briefly, 3mL, 1mg/mL of CM- β -CD-PEI-PEG-T7/DTX/CUR solution was placed in dialysis bags, then placed in 10mL phosphate buffer containing 10% tween 80 (pH 5.5 or 7.4), incubated at 37 ℃, at a predetermined time point, 1mL of buffer was removed for uv-vis spectroscopy, and the solution was supplemented with an equal volume of fresh buffer. The cumulative release amount of DTX and CUR is calculated as follows:
percent released (%) -% -M1/M0X 100%
Where M1 is the mass of drug released and M0 is the total drug in the nanosystem.
As a result, as shown in fig. 3, the release amounts of DTX and CUR were higher than the release amount at pH 7.4 under pH 5.5, which proves that the nanomaterial had pH responsiveness.
Example 7: cytotoxicity
The cytotoxicity of CM-beta-CD-PEI-PEG-T7 (NP-T7) on the pre-esophageal cancer cell KYSE150 in example 4 is evaluated by using a method for detecting cell activity by MTT, and the specific operation steps are as follows: KYSE150 was first seeded at 5000/well density in 96-well plates and then placed in a carbon dioxide incubator overnight for anchorage. Subsequently, the original medium was aspirated and replaced with fresh complete medium containing various concentrations of CM- β -CD-PEI-PEG-T7, selected at a concentration range of 0-800 μ g/mL of CM- β -CD-PEI-PEG-T7, with 5 replicates per concentration. Then cultured in an incubator for 24, 48 and 72 hours, respectively. Then, the culture solution was carefully removed, 50. mu.L of MTT solution was added to each well, the solution in each well was discarded after culturing for 2 hours in a cell incubator, 100. mu.L of isopropanol was added to each well, after shaking the plate, the absorbance at a wavelength of 570nm was measured with a microplate reader, and the cell survival rate was calculated by the following formula:
cell survival (%) × (experimental absorbance-blank absorbance)/(negative control absorbance-blank absorbance) × 100%.
As shown in the figure 4, the CM-beta-CD-PEI-PEG-T7 has high cell survival rate under various concentration conditions, does not generate any cytotoxicity on KYSE150 cells, and reaches 90% when the concentration reaches 800 mu g/mL, so that the CM-beta-CD-PEI-PEG-T7 material is considered to have excellent biocompatibility and can be used as a safe drug carrier for tumor treatment research.
Example 8: in vitro inhibition of cell proliferation assay
The synergistic effect of docetaxel and curcumin at different concentrations was evaluated by MTT analysis. KYSE150 cells were seeded at 5000 cells/well in 96-well plates and incubated overnight. Medium w was replaced with fresh medium (100. mu.L) containing either a single drug (docetaxel or curcumin) or a mixture of the two drugs at different ratios (DTX: CUR 2: 1, 1: 1 or 1: 2 w/w). DTX concentrations in KYSE150 cells were 0, 0.25, 0.5, 1, 2, 4, 8, 16 and 32ng/mL in this example assay, and cell viability was assessed by MTT assay after 48 hours of incubation. The medium was removed and the plates were treated with MTT solution without FBS (20. mu.L, 5mg/mL) and 1640 medium (200. mu.L) at 37 ℃ for 4 h. The MTT solution was discarded and DMSO (150. mu.L/well) was added to dissolve the dye. The absorbance was measured by spectrophotometry at 570 nm. Cell viability and inhibition were determined from the OD values. As shown in FIG. 5, the CM- β -CD-PEI-PEG-T7/DTX/CUR (T7-NP-DTX/DUR) group showed the lowest cell survival rate compared to the other groups, demonstrating that the targeted and combination therapy was able to inhibit the growth of esophageal cancer cells with high efficiency.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. The T7 polypeptide modified targeting nano system is characterized by comprising a nano delivery carrier material, wherein the nano delivery carrier material is carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide.
2. The T7 polypeptide modified targeted nanosystem of claim 1, wherein the nano delivery carrier material is further loaded with an esophageal cancer chemotherapeutic drug, preferably the esophageal cancer chemotherapeutic drug comprises docetaxel and curcumin.
3. The method for preparing the T7 polypeptide modified targeted nano system according to claim 1 or 2, wherein the method comprises the following steps:
(1) synthesis of carboxymethyl beta-cyclodextrin
Dissolving beta-cyclodextrin and sodium hydroxide in water, adding monochloroacetic acid solution, heating and stirring for reaction, adjusting the pH value of the solution to 6-7 after the reaction is finished, adding excessive ethanol to generate white precipitate, filtering the solid precipitate, and drying in vacuum to obtain carboxymethyl beta-cyclodextrin;
(2) synthesis of carboxymethyl beta-cyclodextrin-polyethyleneimine
Weighing carboxymethyl beta-cyclodextrin, dissolving the carboxymethyl beta-cyclodextrin in DMSO, adding N, N' -carbonyldiimidazole, stirring for reaction, then dropwise adding polyethyleneimine into an activated carboxymethyl beta-cyclodextrin solution, stirring for reaction at room temperature, and after the reaction is finished, dialyzing, and freeze-drying to obtain carboxymethyl beta-cyclodextrin-polyethyleneimine;
(3) synthesis of carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol
Dissolving carboxymethyl β -cyclodextrin-polyethyleneimine and maleimide-polyethylene glycol-active ester in water, and adding saturated NaHCO3Regulating the pH value of the reaction mixture to 7.5-8.5 by using the solution, stirring the reaction mixture at room temperature for reaction, dialyzing, and freeze-drying to obtain carboxymethyl β -cyclodextrin-polyethyleneimine-polyethylene glycol;
(4) synthesis of carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide
Dissolving carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol in water, adding a T7 polypeptide solution, adjusting the pH of a reaction system to 6.0-6.5, stirring at room temperature for reaction, reacting the Mal group of the carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol with the thiol group of the T7 polypeptide, dialyzing after the reaction is finished, and freeze-drying to obtain the carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide.
4. The method for preparing the T7 polypeptide modified targeted nano system of claim 3, wherein in the step (1), the dosage ratio of the beta-cyclodextrin, the sodium hydroxide, the water and the monochloroacetic acid is (1-4) g, (0.1-0.5) g, (10-50) mL: (1-10) mL, preferably 2g:0.3g:30mL:5 mL;
in the step (1), the reaction temperature is 40-60 ℃, preferably 50 ℃; the reaction time is 2-6h, preferably 4 h.
5. The method for preparing the T7 polypeptide modified targeted nano system of claim 3, wherein in the step (2), the dosage ratio of carboxymethyl β -cyclodextrin, DMSO, N' -carbonyldiimidazole and polyethyleneimine is (0.5-2) g (1-20) mL: (0.1-0.6) g: (50-200) mg, preferably 1.2g:10mL:0.36g:100 mg.
6. The method for preparing the T7 polypeptide modified targeted nano system according to claim 3 or 5, wherein in the step (2), polyethyleneimine is dropwise added into the activated carboxymethyl β -cyclodextrin solution, and the reaction is stirred at room temperature for 6-18h, preferably 12 h.
7. The method for preparing the T7 polypeptide modified targeted nano system according to claim 3, wherein in the step (3), the dosage ratio of carboxymethyl β -cyclodextrin-polyethyleneimine, maleimide-polyethylene glycol-active ester and water is (0.5-1.5) g, (0.5-2) g (5-15) mL, preferably 1g:1g:10 mL;
in the step (3), the stirring reaction time is 2-4h, preferably 4 h.
8. The method for preparing the T7 polypeptide modified targeted nano system of claim 3, wherein in the step (4), the ratio of the amount of carboxymethyl β -cyclodextrin-polyethyleneimine-polyethylene glycol to the amount of water and T7 polypeptide is (0.1-0.5) g (1-10) mL: (0.05-0.1) g, preferably 0.25g:5mL:0.08 g;
in the step (4), the stirring reaction time is 10-36h, preferably 24 h.
9. The method for preparing the T7 polypeptide modified targeted nano system of claim 3, further comprising the step of (5) synthesizing a carboxymethyl β -cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide loaded with curcumin and docetaxel, specifically:
dissolving carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide in ethyl acetate, adding docetaxel and curcumin solution, and performing ultrasonic treatment to form a colloidal solution;
adding the PVA solution into the colloidal solution, performing ultrasonic treatment to form W/O/W double emulsion, centrifuging and washing to obtain the carboxymethyl beta-cyclodextrin-polyethyleneimine-polyethylene glycol-T7 polypeptide loaded with the docetaxel and the paclitaxel.
10. Use of the T7 polypeptide-modified targeting nanosystem of claim 1 or 2 in the preparation of an anti-esophageal cancer medicament.
CN202010459844.8A 2020-05-26 2020-05-26 T7 polypeptide modified targeting nano system and preparation method and application thereof Pending CN111686258A (en)

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CN112843240A (en) * 2021-03-24 2021-05-28 苏州大学 Beta-cyclodextrin modified PAMAM targeted nano drug delivery carrier and preparation method thereof
CN113456830A (en) * 2021-06-02 2021-10-01 暨南大学 Infrared remote control protein nanogel and preparation method and application thereof
CN115414494A (en) * 2022-08-17 2022-12-02 南京中医药大学 Polypeptide nano vaccine and preparation method and application thereof

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112843240A (en) * 2021-03-24 2021-05-28 苏州大学 Beta-cyclodextrin modified PAMAM targeted nano drug delivery carrier and preparation method thereof
CN113456830A (en) * 2021-06-02 2021-10-01 暨南大学 Infrared remote control protein nanogel and preparation method and application thereof
CN115414494A (en) * 2022-08-17 2022-12-02 南京中医药大学 Polypeptide nano vaccine and preparation method and application thereof
CN115414494B (en) * 2022-08-17 2024-04-26 南京中医药大学 Polypeptide nanometer vaccine and preparation method and application thereof

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Application publication date: 20200922