CN113730598B - Multifunctional nano-drug carrier targeting glucose transport protein 1, preparation method thereof and drug carrying composition - Google Patents

Multifunctional nano-drug carrier targeting glucose transport protein 1, preparation method thereof and drug carrying composition Download PDF

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CN113730598B
CN113730598B CN202111022895.5A CN202111022895A CN113730598B CN 113730598 B CN113730598 B CN 113730598B CN 202111022895 A CN202111022895 A CN 202111022895A CN 113730598 B CN113730598 B CN 113730598B
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nano
drug
drug carrier
targeted
glut1
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CN113730598A (en
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吴海强
欧阳娜
熊炜
王亦男
李晨阳
许晨舒
李霞
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Shenzhen University
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Abstract

The invention relates to a multifunctional nano-drug carrier targeting glucose transport protein 1, a preparation method thereof and a drug carrying composition. The nano-drug carrier is formed by self-assembly of a structural copolymer shown in a formula (I); a structure of formula (I):the copolymer can self-assemble into nano drug carriers (such as nano spherical micelle, nano rod micelle and nano vesicle) in different forms under different environments. The targeting GLUT1 and nano-drug carrier can be used for research and development of various drugs, can load drug molecules, can be prepared into a slow-release and controlled-release targeting drug delivery system, can directionally deliver the drug molecules to lesion sites, reduces the administration frequency, improves the treatment effect and the like. The targeted GLUT1 and multifunctional nano-drug carrier has the advantages of common amino acid nano-drug carriers, and simultaneously has various biological functions of selenium and targeted GLUT1The specificity is a novel, targeted and multifunctional drug carrier.

Description

Multifunctional nano-drug carrier targeting glucose transport protein 1, preparation method thereof and drug carrying composition
Technical Field
The invention relates to the technical field of biomedical drug carriers and slow-release materials, in particular to a multifunctional nano drug carrier targeting glucose transport protein 1, a preparation method thereof and a drug carrying composition.
Background
The human body has a very complex physiological environment, multiple barriers are needed to be passed from the taking of the medicine to the playing of the medicine, only a small part of the medicine can play the medicine effect at last, the treatment effect is seriously influenced, and meanwhile, the toxic and side effects are brought. How to enhance the utilization rate, safety and the like of the medicine has great significance for improving the treatment effect of diseases and human health. In recent years, research relating to different types of drug carriers has received great attention.
The drug carrier is mainly natural or synthetic polymer materials, and forms a drug control system with drug molecules in different forms through chemical bonding, physical adsorption or encapsulation, and can realize the timed, positioned and quantitative release of the drugs through a series of physical, chemical and biological control under the condition of not reducing the efficacy of the original drug molecules and inhibiting the side effects of the original drug molecules, thereby helping to enhance the curative effect of the drug. Drug carrier systems have been used for a variety of routes of administration including injection, oral administration, transdermal absorption, and the like. The nano-drug carrier is a novel carrier with the particle size of 10-1000 nm, has the advantages of reducing the toxic and side effects of the drug, improving the stability of the drug, slowly releasing the drug and targeted release of the drug and the like because the particle size of the nano-drug carrier is smaller than that of capillary passages, and is highly valued in recent years. The nano-drug carrier comprises polymer micelle, nanocapsule, nanosphere, nanoliposome, solid lipid nanoparticle, magnetic nanoparticle and the like. Various polymer materials can be used for research and development of nano drug carriers, but biocompatibility, biodegradability, safety and the like are important problems to be considered.
Amino acids are the basic constituent units of biologically functional macromolecular proteins, and are the basic substances of proteins required for the nutrition of the body. The polyamino acid prepared from aspartic acid, glutamic acid, lysine, alanine, phenylalanine and the like is a biological full-degradation high molecular material which has low toxicity, good biocompatibility and easy absorption and metabolism by organisms, and has great development potential in the field of drug carriers. However, due to strong hydrogen bonding action among amino acid molecules and the like, the drug carrier material has the defects of poor water solubility, difficult control of in vivo degradation rate and period and the like, and is difficult to realize targeted transmission and the like.
Accordingly, there is a need for improvement and development in the art.
Disclosure of Invention
Functionalization and intellectualization are strategic trends in the development of current nano-drug carriers. PEG (polyethylene glycol) has a flexible hydrophilic long chain, is nontoxic and non-immunogenic, has been approved by the FDA for clinical use, and is one of the most promising materials in the currently known hydrophilic carriers. PEG and polyamino acid are combined to form a block copolymer, so that the hydrophilicity of the polyamino acid block can be improved, the adsorption of protein on the surface of a material in a body and the adhesion of cells can be reduced, the polyamino acid can be protected from being damaged by an immune system, the circulation time of the material in the body can be prolonged, and the like. In addition, multiple functional groups can be introduced at two ends of the PEG, so that the comprehensive performance of the polyamino acid nano-drug carrier is obviously enhanced.
Researches show that the selenium serving as the essential trace element of the human body has important biological functions of resisting oxidization, regulating immunity, antagonizing harmful heavy metals, resisting aging and the like. Selenium deficiency is associated with the onset of many human diseases including diabetes, cancer, and neurodegenerative diseases, among others. Selenium intake by humans is mainly two ways: inorganic selenium has low utilization rate and high toxicity; organic selenium, such as seleno-amino acid, has better biocompatibility, high utilization rate, lower toxicity and higher safety, and is easier to be absorbed by human body. Therefore, the introduction of seleno-amino acid is hopeful to actively promote the research and development of the functionalized nano polyamino acid drug carrier.
Glucose transporter 1 (facilitative glucose transporterl, GLUT 1) is a major functional protein responsible for D-glucose and other hexoses transport, and its abnormalities play an important regulatory role in the pathogenesis of a variety of major diseases, including malignant tumors, neurodegenerative diseases, and the like. The consumption of glucose in malignant tumor cells is far higher than that of normal cells, and GLUT1 on the surfaces of tumor cells shows characteristic high expression. The glucose consumption of the brain accounts for 30% of the whole body, and GLUT1 is also highly expressed on the luminal side and the abluminal side of brain capillary endothelial cells, and is a main transportation path for glucose and other hexoses crossing the blood brain barrier, and the abnormality of the GLUT1 has important promotion effect on various central nervous system diseases. Therefore, GLUT1 can be used as an important target point for targeted drug delivery of the functional nano-drug carrier.
Therefore, in view of the defects that the current polyamino acid nano-carrier has single main function and can only be used as a drug carrier, lacks targeting property and the like, the invention provides the multifunctional nano-drug carrier targeting GLUT1, has the advantages of the common amino acid nano-drug carrier, simultaneously has multiple biological functions of selenium, specifically targets GLUT1, and is a novel, targeting and multifunctional nano-drug carrier.
Specifically, the technical scheme of the invention is as follows:
a targeted GLUT1 and multifunctional nano-drug carrier, wherein the targeted GLUT1 and polyseleno-amino acid amphiphilic block copolymer with a structure shown in a formula (I) is self-assembled;
wherein n is more than or equal to 22 and less than or equal to 454, x is more than or equal to 2 and less than or equal to 50, y is more than or equal to 2 and less than or equal to 50, and n, x and y are integers;
-R 1 selected from the group consisting ofOne of the following;
-R 2 selected from-CH (CH) 3 )CH 3 、-H、-CH 3 、-CH 2 CH(CH 3 )CH 3 、-CH(CH 3 )CH 2 CH 3 、/>-CH 2 OH、/>-CH 2 SH、-CH 2 CH 2 SCH 3 、/>-CH(OH)CH 3 、/>One of the following;
-R 3 selected from-CH 2 CH 2 SeCH 3 、-CH 2 One of SeH.
The invention discloses a preparation method of a targeted GLUT1 multifunctional nano-drug carrier, which comprises the following steps:
dissolving the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with the structure shown in the formula (I) in a first organic solvent, and oscillating until the two are uniformly mixed to obtain a polymer solution;
adding deionized water into the polymer solution, and stirring and reacting for 2-3h to obtain a mixed solution;
And dialyzing the obtained mixed solution by using a dialysis bag filled with deionized water, and filtering the solution in the dialysis bag to obtain the nano-drug carrier in the form of nano-rod micelle.
The invention discloses a preparation method of a targeted GLUT1 multifunctional nano-drug carrier, which comprises the following steps:
dissolving the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with the structure shown in the formula (I) in a second organic solvent, and removing the second organic solvent to prepare a film;
then adding the mixture into double distilled water for hydration, shaking and mixing uniformly, carrying out ultrasonic treatment, centrifuging, freezing and drying to obtain the nano-drug carrier in the form of nano-vesicles.
The invention discloses a preparation method of a targeted GLUT1 multifunctional nano-drug carrier, which comprises the following steps:
dissolving the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with the structure shown in the formula (I) in an oil phase solution to obtain a copolymer oil phase solution;
sucking the aqueous phase solution into the copolymer oil phase solution by using a syringe, performing ultrasonic treatment, sucking all the ultrasonic solution by using the syringe, injecting the solution into the external aqueous phase solution, and stirring;
and finally, centrifuging, washing, freezing and drying to obtain the nano drug carrier in the form of nano spherical micelle.
A drug-loaded composition, which comprises the nano-drug carrier and a drug wrapped in the nano-drug carrier.
The beneficial effects are that: the multifunctional nano-drug carrier targeting GLUT1 is obtained by self-assembling a targeting GLUT1 and a polyseleno amino acid amphiphilic block copolymer with a structure shown in a formula (I), and different forms comprise nano spherical micelle, nano rod micelle or nano vesicle and the like. The targeted GLUT1 and the multifunctional drug carrier can load drug molecules to prepare a slow-release controlled-release targeted drug delivery system, can directionally deliver the drug molecules to a lesion site, can be used for drug delivery in various modes, and can be used for slow-release controlled-release according to the needs, so that the drug delivery frequency is reduced, the treatment effect is improved, and the toxic and side effects of the drug are reduced. Compared with the prior art, the targeted GLUT1 and multifunctional nano-drug carrier provided by the invention has the advantages of common amino acid nano-drug carrier, has multiple biological functions of selenium and specificity of targeted GLUT1, is a novel, targeted and multifunctional drug carrier, and is suitable for research, development and clinical application of the drug carrier of various drugs of various diseases related to GLUT1 abnormality.
Drawings
FIG. 1 is a diagram of glycosylated polyethylene glycol amine Glu-PEG according to the invention 45 -NH 2 1H NMR spectrum of (C).
FIG. 2 is a schematic diagram of the amphiphilic block copolymer Glu-PEG of the targeted GLUT1 and polyseleno amino acid according to the present invention 45 -PBLA 2 -PMet(Se) 2 1H NMR spectrum of (C).
Fig. 3 is a TEM (transmission electron microscope) image of the targeted GLUT1, polyseleno-amino acid nanosphere micelle according to the present invention.
Fig. 4 is a TEM (transmission electron microscope) image of the targeted GLUT1, polyseleno-amino acid nanorod micelle according to the present invention.
Fig. 5 is a TEM (transmission electron microscope) image of the targeted GLUT1, polyseleno-amino acid nanovesicles according to the invention.
FIG. 6 is a cell targeted uptake laser confocal map of the glycosylation modified polyseleno amino acid nanosphere micelles of the invention.
Detailed Description
The invention provides a multifunctional nano-drug carrier targeting glucose transport protein 1, a preparation method thereof and a drug carrying composition, and aims to make the purposes, technical schemes and effects of the invention clearer and more definite, and the invention is further described in detail below. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The embodiment of the invention provides a targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer, which has a structure shown in a formula (I):
Wherein n is more than or equal to 22 and less than or equal to 454, x is more than or equal to 2 and less than or equal to 50, y is more than or equal to 2 and less than or equal to 50, and n, x and y are integers;
-R 1 selected from the group consisting ofOne of the following;
-R 2 selected from-CH (CH) 3 )CH 3 、-H、-CH 3 、-CH 2 CH(CH 3 )CH 3 、-CH(CH 3 )CH 2 CH 3 、/>-CH 2 OH、/>-CH 2 SH、-CH 2 CH 2 SCH 3 、/>-CH(OH)CH 3 、/>One of the following;
-R 3 selected from-CH 2 CH 2 SeCH 3 、-CH 2 SeH, etc.
The targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer provided by the embodiment of the invention has a structure shown in a formula (I), and is an amphiphilic triblock copolymer formed by glycosylation modified polyethylene glycol hydrophilic chain segments, ionic polyamino acid and polyseleno amino acid hydrophobic chain segments. The targeted GLUT1 and polyseleno-amino acid amphiphilic block copolymer can be self-assembled with the same amphiphilic block copolymer or different amphiphilic block copolymers, such as self-assembly between uncharged amphiphilic block copolymers, between uncharged amphiphilic block copolymers and charged amphiphilic block copolymers, between amphiphilic block copolymers with the same charge, or between amphiphilic block copolymers with opposite charges, and the like; can also self-assemble into nano-micelles with different forms under different environments, such as nano-spherical micelles, nano-rod micelles or nano-vesicles. The nano micelle formed by self-assembly of the targeted GLUT1 and the polyseleno amino acid amphiphilic block copolymer can be used as a drug carrier to load drug molecules to prepare a slow-release controlled-release targeted drug delivery system, the drug molecules can be directionally delivered to a lesion part and can be administrated in various modes, and the entrapped drug can be released from the delivery system in a slow-release controlled-release manner according to requirements, so that the administration frequency is reduced, the treatment effect is improved, and the toxic and side effects of the drug are reduced. Compared with the prior art, the targeted GLUT1 and polyseleno-amino acid amphiphilic block copolymer provided by the embodiment of the invention has the advantages of common amino acid polymers, simultaneously has multiple biological functions of selenium and specificity of targeted GLUT1, is a novel and multifunctional amphiphilic block copolymer, and is suitable for research, development and clinical application of various drug carriers of various drugs of GLUT1 abnormality related diseases.
Glycosylation (i.e., R) in embodiments of the invention 1 Group) modified polyethyleneglycol amine block is used for providing a hydrophilic end to form a shell of the nano micelle, so that the circulation time of the nano micelle in the body can be prolonged; on the other hand, the targeting group glycosyl can obviously improve the targeting property of drug delivery. The polyseleno amino acid block provides a hydrophobic end for carrying medicine to provide a stable nano medicine carrier which has the functions of medicine slow release and controlled release and various biological functions of selenium; the active ionic groups in the ionic polyamino acid can be used for crosslinking between block copolymers so as to provide a more stable and better-function drug sustained-release and controlled-release carrier.
The embodiment of the invention provides a preparation method of the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer, which comprises the following steps:
s10, dissolving tert-butyl acetate polyethylene glycol amine in an organic solvent to obtain a tert-butyl acetate polyethylene glycol amine solution;
s11, dissolving amino acid-N-internal carboxylic anhydride in an organic solvent to obtain an amino acid-N-internal carboxylic anhydride solution;
s12, dissolving seleno-amino acid-N-internal carboxylic anhydride in an organic solvent to obtain seleno-amino acid-N-internal carboxylic anhydride solution;
S13, mixing a tert-butyl acetate polyethylene glycol amine solution and an amino acid-N-internal carboxylic anhydride solution, and placing the mixture in an inert atmosphere for stirring reaction;
s14, after the reaction is finished, adding a seleno-amino acid-N-internal carboxylic anhydride solution, and continuing to stir and react to obtain an amphiphilic block copolymer;
s15, dissolving the amphiphilic block copolymer in a potassium carbonate aqueous solution to obtain an amphiphilic block copolymer aqueous solution, and preparing benzyl chloroformate (molecular formula ClCO 2 CH 2 C 6 H 5 Abbreviated as Cbz-Cl) is dissolved in dioxane and slowly dripped into the amphiphilic block copolymer aqueous solution under the ice bath condition, the ice bath is removed after the dripping is finished, the temperature is slowly raised to room temperature and the reaction is kept overnight, and the amphiphilic block copolymer with the end amino Cbz protection is obtained after the reaction is completed;
s16, dissolving the amphiphilic block copolymer protected by the terminal amino Cbz in excessive trifluoroacetic acid (TFA), and stirring at room temperature overnight to obtain a carboxyl terminal deprotected amphiphilic block copolymer;
s17, dissolving the carboxyl end deprotected amphiphilic block copolymer in an organic solvent, sequentially adding DCC and NHS, and stirring for reaction to obtain a carboxyl activated amphiphilic block copolymer solution;
s18, dissolving glucose (Glu) in an organic solvent, adding Triethylamine (TEA), and stirring to obtain a glucose solution;
S19, adding the glucose solution into a carboxyl activated amphiphilic block copolymer solution, and placing the solution in an inert atmosphere for stirring reaction to obtain a glycosylated amphiphilic block copolymer;
and S20, removing Cbz at the tail end of the glycosylated amphiphilic block copolymer to obtain the targeted GLUT1 and polyseleno-amino acid amphiphilic block copolymer with the deprotected tail end amino group. The synthetic reaction formula is shown as follows:
the tert-butyl ester at one end of the tert-butyl acetate polyethylene glycol block is removed to obtain a carboxyl polyethylene glycol block, and then amide condensation is carried out on the carboxyl and amino groups on glucose, so that the glucose is connected into the block polymer.
In one embodiment, the amino acid-N-internal carboxylic anhydride is L-valine-N-internal carboxylic anhydride, ε -benzyloxycarbonyl-L-lysine-N-internal carboxylic anhydride, γ -benzyl-L-aspartate-N-internal carboxylic anhydride, γ -benzyl-L-glutamate-N-internal carboxylic anhydride, γ -propynyl-L-glutamate-N-internal carboxylic anhydride, γ -2-chloroethyl-L-glutamate-N-internal carboxylic anhydride, glycine-N-internal carboxylic anhydride, L-alanine-N-internal carboxylic anhydride, L-leucine-N-internal carboxylic anhydride, L-isoleucine-N-internal carboxylic anhydride, L-phenylalanine-N-internal carboxylic anhydride, L-tryptophan-N-internal carboxylic anhydride, L-serine-N-internal carboxylic anhydride, L-tyrosine-N-internal carboxylic anhydride, ε -benzyloxycarbonyl-L-cysteine-N-internal carboxylic anhydride, L-asparagine-N-internal carboxylic anhydride, L-glutamine-N-internal carboxylic anhydride, L-leucine-N-internal carboxylic anhydride, arginine-N-internal carboxylic anhydride, or threonine-N-internal carboxylic anhydride.
In one embodiment, the seleno-amino acid-N-internal carboxylic anhydride is seleno-L-methionine-N-internal carboxylic anhydride or seleno-L-cysteine-N-internal carboxylic anhydride.
In one embodiment, the glucose is 3, 5-0-benzylidene-1, 2-0-isopropylidene-alpha-D-glucopyranose, 4-aminophenyl-beta-D-glucopyranose, or 2-amino-2-deoxy-D-glucopyranose.
In one embodiment, the molar ratio of the tert-butyl acetate polyethylene glycol amine, the amino acid-N-internal carboxylic anhydride and the seleno amino acid-N-internal carboxylic anhydride is 1:2-50:2-50.
In one embodiment, the organic solvent comprises one or more of anhydrous N, N-dimethylformamide, anhydrous tetrahydrofuran, and anhydrous chloroform.
In one embodiment, the ratio of the amphiphilic block copolymer protected by the terminal amino group Cbz to the trifluoroacetic acid is 100mg to (5-10 mL), and the reaction time is 2-8 h.
In one embodiment, the molar ratio of the carboxyl end deprotected amphiphilic block copolymer to DCC and NHS is 1: (1.05-1.5): (1.2-1.6).
In one embodiment, the reaction time of the carboxyl end deprotected amphiphilic block copolymer with DCC and NHS is 6-24 h, and the reaction temperature is 20-30 ℃.
In one embodiment, the molar ratio of glucose to triethylamine is 1: (2-8).
In one embodiment, the molar ratio of the carboxyl end deprotected amphiphilic block copolymer to the glucose solution is 1:0.6-1.5, and the reaction time is 6-24 h.
In one embodiment, the carboxy-terminal deprotected amphiphilic block copolymer is dissolved in an organic solvent at a ratio of (1-5) g:100 mL of the carboxy-terminal deprotected amphiphilic block copolymer to the organic solvent.
In one embodiment, glucose is dissolved in an organic solvent at a dosage ratio of (1-5) g/100 mL of glucose to the organic solvent.
In one embodiment, the tert-butyl acetate polyethylene glycol amine is dissolved in an organic solvent at a dosage ratio of (1-5) g:100 mL.
In one embodiment, the amino acid-N-endocarboxylic anhydride is dissolved in an organic solvent at a dosage ratio of (1-5) g:100 mL.
In one embodiment, the seleno-amino acid-N-internal carboxylic anhydride is dissolved in an organic solvent at a dosage ratio of (1-5) g to 100 mL.
In one embodiment, the time for the reaction of the tert-butyl acetate polyethylene glycol amine and the amino acid-N-internal carboxylic anhydride is 24 to 72 hours; the temperature of the reaction is 25-30 ℃.
The embodiment of the invention discloses application of a targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer in a drug carrier.
The embodiment of the invention provides a multifunctional nano-drug carrier targeting GLUT1, which is formed by self-assembling a targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with a structure shown in a formula (I);
wherein n is more than or equal to 22 and less than or equal to 454, x is more than or equal to 2 and less than or equal to 50, and y is more than or equal to 2 and less than or equal to 50, wherein n, x and y are integers;
-R 1 selected from the group consisting ofEtc.;
-R 2 selected from-CH (CH) 3 )CH 3 、-H、-CH 3 、-CH 2 CH(CH 3 )CH 3 、-CH(CH 3 )CH 2 CH 3 、/>-CH 2 OH、/>-CH 2 SH、-CH 2 CH 2 SCH 3 、/>-CH(OH)CH 3 、/>
-R 3 Selected from-CH 2 CH 2 SeCH 3 or-CH 2 SeH。
The targeting nano-drug carrier provided by the embodiment of the invention is formed by self-assembling a targeting GLUT1 and polyseleno amino acid amphiphilic block copolymer with a structure shown in a formula (I). The targeted GLUT1 and the polyseleno-amino acid amphiphilic block copolymer can be self-assembled with the same amphiphilic block copolymer or different amphiphilic block copolymers, such as self-assembly between uncharged amphiphilic block copolymers, between uncharged amphiphilic block copolymers and charged amphiphilic block copolymers, between amphiphilic block copolymers with the same charge, or between amphiphilic block copolymers with opposite charges, and the like; can also self-assemble into nano-micelles with different forms under different environments, such as nano-spherical micelles, nano-rod micelles or nano-vesicles. The nano micelle formed by self-assembly of the targeted GLUT1 and the polyseleno amino acid amphiphilic block copolymer can be used as a drug carrier to load drug molecules to prepare a slow-release controlled-release targeted drug delivery system, the drug molecules can be directionally delivered to a lesion part and can be administrated in various modes, and the entrapped drug can be released from the delivery system in a slow-release controlled-release manner according to requirements, so that the administration frequency is reduced, the treatment effect is improved, and the toxic and side effects of the drug are reduced. Compared with the prior art, the targeted GLUT1 and multifunctional nano-drug carrier provided by the embodiment of the invention has the advantages of common amino acid nano-drug carriers, simultaneously has multiple biological functions of selenium and specificity of the targeted GLUT1, is a novel, targeted and multifunctional drug carrier, and is suitable for research, development and clinical application of the drug carrier of various drugs of various diseases related to GLUT1 abnormality.
In one embodiment, the targeted GLUT1, multifunctional nano-drug carrier exists in the form of nano-spherical micelles, nano-rod micelles, nano-vesicles, or the like.
In the embodiment of the invention, the difference of the structures of the nano drug carrier formed by self-assembly is determined by the difference of the molecular weight ratio P of the hydrophobic chain segment and the hydrophilic chain segment, specifically: when P is less than or equal to 1/3, forming nano spherical micelle; when P is more than or equal to 1/3 and less than or equal to 1/2, forming nano rod-shaped micelle; 1/And when P is more than or equal to 2 and less than or equal to 1, forming nano vesicles. Wherein the hydrophobic chain segment isThe hydrophilic segment is +.>
The embodiment of the invention provides a preparation method of the targeted GLUT1 multifunctional nano-drug carrier, which comprises the following steps:
s20, dissolving the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with the structure shown in the formula (I) in a first organic solvent, and oscillating until the two are uniformly mixed to obtain a polymer solution;
s21, adding deionized water into the polymer solution, and stirring and reacting for 2-3 hours to obtain a mixed solution;
and S22, dialyzing the obtained mixed solution by using a dialysis bag filled with deionized water, and filtering the solution in the dialysis bag to obtain the nano-drug carrier in the form of nano-rod micelle.
In one embodiment, the first organic solvent includes, but is not limited to, one or more of dimethyl sulfoxide, N-dimethylformamide, chloroform, and the like.
In one embodiment, the targeted GLUT1, the polyseleno-amino acid amphiphilic block copolymer and the first organic solvent are used in a dosage ratio of 10mg to (1-3) mL; the dosage ratio of the targeted and polyseleno amino acid amphiphilic block copolymer to deionized water is 10mg to (2-5) mL.
In one embodiment, the dialysis bag is dialyzed for 3 to 7 days with water being changed every other day.
In one embodiment, the dialysis bag has a molecular weight cut-off of 1000 to 5000Da.
The embodiment of the invention provides a preparation method of a targeting nano-drug carrier, which comprises the following steps:
s30, dissolving the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with the structure shown in the formula (I) in a second organic solvent, and removing the organic solvent to prepare a film;
s31, adding the mixture into double distilled water for hydration, vibrating and uniformly mixing, carrying out ultrasonic treatment, centrifuging, freezing and drying to obtain the targeting nano drug carrier in the form of nano vesicles.
In one embodiment, the step of removing the organic solvent to form a film specifically includes: rotary evaporation to form a uniform film and vacuum to remove residual organic solvent.
In one embodiment, the second organic solvent includes, but is not limited to, one or more of a volatile solvent such as methylene chloride, tetrahydrofuran, and the like.
In one embodiment, the targeted GLUT1, the polyseleno-amino acid amphiphilic block copolymer and the second organic solvent are used in a dosage ratio of 10mg to (1-2) mL; the dosage ratio of the targeted GLUT1 to the polyseleno amino acid amphiphilic block copolymer to the double distilled water is 10mg to (2-5) mL.
In one embodiment, the hydration temperature is in the range of 50 to 70 ℃.
In one embodiment, the hydration time is between 1 and 2 hours.
In one embodiment, the centrifugation speed is 2000-3000 r/min and the centrifugation time is 25-35 min.
The embodiment of the invention provides a preparation method of a targeting nano-drug carrier, which comprises the following steps:
s40, dissolving the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with the structure shown in the formula (I) in an oil phase solution to obtain a copolymer oil phase solution;
s41, sucking the aqueous phase solution into the copolymer oil phase solution by using a syringe, performing ultrasonic treatment, sucking all the ultrasonic solution by using the syringe, and then injecting the ultrasonic solution into the external aqueous phase solution for stirring;
S42, finally, centrifuging, washing, freezing and drying to obtain the targeting nano-drug carrier in the form of nano spherical micelle.
In one embodiment, the oil phase solution includes, but is not limited to, one or more of a methylene chloride solution, a tetrahydrofuran solution, and the like.
In one embodiment, the aqueous phase solution and the external aqueous phase solution are one or more of aqueous solutions of emulsifiers such as polyoxyethylene ether, polyoxypropylene ether, polyvinyl alcohol and the like.
In one embodiment, the aqueous phase solution has a mass fraction of 0.1% to 0.2%.
In one embodiment, the mass fraction of the external aqueous phase solution is 0.25% to 0.5%.
In one embodiment, the targeted, polyseleno-amino acid amphiphilic block copolymer and the oil phase solution are used in a ratio of (1-5) g to 100mL.
In one embodiment, the time of the sonication is 30 seconds/time, two times in total, 30 seconds apart.
In one embodiment, the stirring time is 2 to 4 hours; the stirring rate was 400r/min.
The embodiment of the invention provides a drug-loaded composition, which comprises the targeting nano drug carrier and a drug wrapped in the targeting nano drug carrier.
In one embodiment, the drug is selected from one or more of anti-AD drugs, anti-tumor drugs, steroidal or non-steroidal anti-inflammatory drugs, metabolic regulation drugs, antibiotics, cardiovascular drugs, antiviral drugs, antifungal drugs, immunomodulators, and the like, but is not limited thereto.
In one embodiment, the mass ratio of the targeting nano-drug carrier to the drug is 1mg to (0.1-10) mg.
The embodiment of the invention provides a preparation method of the drug-loaded composition, which comprises the following steps:
dissolving the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with the structure shown in the formula (I) in a first organic solvent, and oscillating until the two are uniformly mixed to obtain a polymer solution;
adding a medicine into the polymer solution, stirring, adding deionized water, and stirring for 2-3h to obtain a mixed solution;
dialyzing the obtained mixed solution with a dialysis bag filled with deionized water, and filtering the solution in the dialysis bag to obtain the drug-carrying composition.
In one embodiment, the mass ratio of the targeted GLUT1 and the polyseleno amino acid amphiphilic block copolymer to the drug is 1mg to (0.1-10) mg.
The embodiment of the invention provides a preparation method of the drug-loaded composition, which comprises the following steps:
dissolving the targeted GLUT1, the polyseleno amino acid amphiphilic block copolymer and the drug with the structure shown in the formula (I) in a second organic solvent, and removing the second organic solvent to prepare a film;
then adding into double distilled water for hydration, shaking and mixing uniformly, carrying out ultrasonic treatment, centrifuging, freezing and drying to obtain the medicine carrying composition.
In one embodiment, the mass ratio of the targeted GLUT1 and the polyseleno amino acid amphiphilic block copolymer to the drug is 1mg to (0.1-10) mg.
The embodiment of the invention provides a preparation method of the drug-loaded composition, which comprises the following steps:
dissolving the targeted GLUT1, the polyseleno amino acid amphiphilic block copolymer and the drug with the structure shown in the formula (I) in an oil phase solution, sucking the water phase solution into the oil phase solution by using a syringe, performing ultrasonic treatment, sucking all the ultrasonic solution by using the syringe, injecting the ultrasonic solution into an external water phase solution, and stirring;
finally, centrifuging, washing, freezing and drying to obtain the medicine carrying composition.
In one embodiment, the mass ratio of the targeted GLUT1 and the polyseleno amino acid amphiphilic block copolymer to the drug is 1mg to (0.1-10) mg.
The invention is further illustrated by the following specific examples.
Example 1:
glycosylated polyethylene glycol amine (GLU-PEG) n -NH 2 ) Is synthesized by (a)
100mg BocNH-PEG was taken n -COOH (0.05 mmol, mn=2000) was dissolved in 5mL DMF solution, 13mg DCC, 9mg NHS were added sequentially, stirred overnight at room temperature, and the by-product was removed to give carboxy-terminally activated BocNH-PEG n -COOH.15mg of glucamine (Glu-NH) 2 ) Dissolving in 1mL of super-dry DMF solution, stirring for 5min, adding triethylamine, stirring for 15min, and dripping into carboxyl end activated BocNH-PEG n -COOH solution, at room temperature for 12h. After the reaction is finished, dropwise adding the reaction solution into vigorously stirred absolute ethyl ether, centrifuging to obtain a yellow crude product, dissolving the crude product into THF again, precipitating with absolute ethyl ether, centrifuging, and finally drying in vacuum at room temperature overnight to obtain Glu-PEG n -NH-Boc. 1mg Glu-PEG n dissolving-NH-Boc in 5mL trifluoroacetic acid (TFA), stirring overnight at room temperature, removing Boc group on amino terminal of PEG to obtain glucamine polyglycol amine (Glu-PEG) n -NH 2 ). Nuclear magnetic resonance detection (the solvent is deuterated dimethyl sulfoxide) is carried out on the product of the embodiment, and the detection result is shown in figure 1. The specific synthetic route is as follows:
three glycosylated polyethylene glycol polyaspartic acid polyseleno methionine amphiphilic block copolymers (GLU-PEG) of different P values are listed below n -PASp x -PMet(Se) y ) (p=the ratio of the molecular weight of total polyamino acid to the molecular weight of glycosylated polyethyleneglycol amine) to prepare three multifunctional nano-drug carriers of different self-assembled morphology, see in particular examples 2-4. The specific synthetic route is as follows:
example 2:
glycosylated polyethylene glycol polyaspartic acid poly selenomethionine block copolymer (Glu-PEG) 45 -PAsp2-PMet(Se) 2 ) Is synthesized by (a)
Weigh 0.1g tBu-PEG 45 -NH 2 (0.05 mmol, mn=2000) was dried in vacuo in a vacuum oven for 4 hours and then dissolved in 3ml of dried DMF as macroinitiator. 25mg of freshly prepared benzyl L-aspartate NCA (BLA-NCA) (0.1 mmol) was weighed into 1ml of super-dry DMF and the two reaction solutions were then mixed by syringe and placed under nitrogen and stirred for 24h. After the completion of the reaction, 22.3mg of newly prepared seleno-L-methionine NCA (0.1 mmol) was weighed and dissolved in 1ml of super-dry DMF, and the mixture was added to the reaction mixture, followed by stirring. After 24 hours, the reaction was ended. Settling and centrifuging the triblock polymer in ice anhydrous isopropyl ether, and drying in a vacuum oven for 24 hours to obtain a product of tert-butyl acetate polyethylene glycol polyaspartate benzyl ester polyseleno methionine block copolymer tBu-PEG 45 -PBLA 2 -PMet(Se) 2 . The product was protected by a conventional amino protection strategy from the amino groups at the ends of the block copolymer by Cbz-Cl. The product is further stirred with trifluoroacetic acid to react for 2 hours to remove tert-butyl ester groups at the PEG end, the mixture is settled in ice anhydrous isopropyl ether, centrifuged and dried to obtain carboxyl polyethylene glycol poly benzyl aspartate poly selenomethionine segmented copolymer, the product is added into 1.2e.q.DCC (dicyclohexylcarbodiimide) and 1.5e.q.NHS (N-hydroxysuccinimide) to be stirred and activated at room temperature for overnight, and then Glu-NH of 1.2e.q. is added 2 And 1.5e.q. triethylamine are stirred overnight at room temperature to obtain glycosylated polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) block copolymer Glu-PEG 45 -PBLA 2 -PMet(Se) 2 . The product is then subjected to an alkaline hydrolysis reaction to remove benzyl ester from the block copolymer. The block copolymer was dissolved in an appropriate amount of 0.5mol/LNaOH solution and stirred for 1h, then the solution was dialyzed with deionized water (MWCO: 2000) and lyophilized to obtain the final product Glu-PEG 45 -PAsp 2 -PMet(Se) 2 . Nuclear magnetic resonance detection (the solvent is deuterated trifluoroacetic acid) is carried out on the product of the example, and the detection result is shown in figure 2.
Example 3:
glycosylated polyethylene glycol polyaspartic acid poly selenomethionine block copolymer (Glu-PEG) 45 -PAsp 2 -PMet(Se) 4 ) Is combined with (a)Finished products
Weigh 0.1g tBu-PEG-NH 2 (0.05 mmol, mn=2000) was dried in vacuum in a vacuum oven for 4 hours and then dissolved in 3ml of DMF after drying to serve as a macroinitiator. 25mg of freshly prepared benzyl L-aspartate NCA (0.1 mmol) was weighed into 1ml of ultra-dry DMF and the two reaction solutions were then mixed by syringe and placed under nitrogen and reacted under stirring for 24h. After the completion of the reaction, 44.6mg of newly prepared seleno-L-methionine NCA (0.2 mmol) was dissolved in 1ml of super-dry DMF, and the mixture was added to the reaction mixture, followed by stirring. After 24 hours, the reaction was ended. Settling and centrifuging the triblock polymer in ice anhydrous isopropyl ether, and drying in a vacuum oven for 24 hours to obtain a product of tert-butyl acetate polyethylene glycol polyaspartate benzyl ester polyseleno methionine block copolymer tBu-PEG 45 -PBLA 2 -PMet(Se) 4 . The product was protected by a conventional amino protection strategy from the amino groups at the ends of the block copolymer by Cbz-Cl. The product is further stirred and reacted for 2 hours by a trifluoroacetic acid dissolving ice bath to remove tert-butyl ester groups at the PEG end, the mixture is settled in ice anhydrous isopropyl ether, centrifuged and dried to obtain carboxyl polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) segmented copolymer, the product is added into 1.2e.q.DCC and 1.5e.q.NHS, stirred and activated at room temperature for overnight, and then Glu-NH of 1.2 e.q.is added 2 And 1.5e.q. triethylamine are stirred overnight at room temperature to obtain glycosylated polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) block copolymer Glu-PEG 45 -PBLA 2 -PMet(Se) 4 . The product is then subjected to an alkaline hydrolysis reaction to remove benzyl ester from the block copolymer. The block copolymer was dissolved in an appropriate amount of 0.5mol/LNaOH solution and stirred for 1h, then the solution was dialyzed with deionized water (MWCO: 2000) and lyophilized to obtain the final product Glu-PEG 45 -PAsp 2 -PMet(Se) 4 。
Example 4:
glycosylated polyethylene glycol polyaspartic acid poly selenomethionine block copolymer (Glu-PEG) 45 -PAsp 2 -PMet(Se) 6 ) Is synthesized by (a)
Weigh 0.1g tBu-PEG-NH 2 (0.05 mmol, mn=2000) was dried in a vacuum oven for 4 hours and then dissolvedWas used as macroinitiator in 3ml of dried DMF. 25mg of freshly prepared benzyl L-aspartate NCA (0.1 mmol) was weighed into 1ml of ultra-dry DMF and the two reaction solutions were then mixed by syringe and placed under nitrogen and reacted under stirring for 24h. After the completion of the reaction, 66.9g of newly prepared seleno-L-methionine NCA (0.3 mmol) was weighed and dissolved in 1ml of super-dry DMF, and the mixture was added to the reaction mixture, followed by stirring. After 24 hours, the reaction was ended. Settling the triblock polymer in ice anhydrous isopropyl ether, centrifuging, and drying in a vacuum oven for 24 hours to obtain the product tert-butyl acetate polyethylene glycol polyaspartate benzyl polysilseduction methionine block copolymer tBu-PEG 45 -PBLA 2 -PMet(Se) 6 . The product was protected by a conventional amino protection strategy from the amino groups at the ends of the block copolymer by Cbz-Cl. The product is further stirred and reacted for 2 hours by a trifluoroacetic acid dissolving ice bath to remove tert-butyl ester groups at the PEG end, the mixture is settled in ice anhydrous isopropyl ether, centrifuged and dried to obtain carboxyl polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) segmented copolymer, the product is added into 1.2e.q.DCC and 1.5e.q.NHS, stirred and activated at room temperature for overnight, and then Glu-NH of 1.2 e.q.is added 2 And 1.5e.q. triethylamine are stirred overnight at room temperature to obtain glycosylated polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) block copolymer Glu-PEG 45 -PBLA 2 -PMet(Se) 6 . The product is then subjected to an alkaline hydrolysis reaction to remove benzyl ester from the block copolymer. The block copolymer was dissolved in an appropriate amount of 0.5mol/L NaOH solution and stirred for 1h, and then the solution was dialyzed with deionized water (MWCO: 2000) and lyophilized to obtain the final product Glu-PEG 45 -PAsp 2 -PMet(Se) 6 。
Three glycosylated polyethylene glycol polylysine polyseleno methionine amphiphilic cationic triblock copolymers (Glu-PEG) of different P values are also listed below n -PLL x -PMet(Se) y ) (p=the ratio of the molecular weight of total polyamino acid to the molecular weight of glycosylated polyethyleneglycol amine) to prepare three multifunctional nano-drug carriers of different self-assembled morphology, see in particular examples 5-7. The specific synthetic route is as follows:
Example 5:
glycosylated polyethylene glycol polylysine polyseleno methionine block copolymer Glu-mPEG 45 -PLL 2 -PMet(Se) 2 ) Is synthesized by (a)
Weigh 0.1g tBu-PEG-NH 2 (0.05 mmol, mn=2000) was dried in vacuum in a vacuum oven for 4 hours and then dissolved in 3ml of DMF after drying to serve as a macroinitiator. 31mg of freshly prepared ε -benzyloxycarbonyl-L-lysine NCA (Lys) (Z) NCA) (0.1 mmol) was dissolved in 1ml of ultra-dry DMF, then the two reaction solutions were mixed by syringe and placed under nitrogen atmosphere and stirred for reaction for 24h. After the completion of the reaction, 22.3mg of newly prepared seleno-L-methionine NCA (0.1 mmol) was weighed and dissolved in 1ml of super-dry DMF, and the mixture was added to the reaction mixture, followed by stirring. After 24 hours, the reaction was ended. Settling the triblock polymer in ice anhydrous isopropyl ether, centrifuging, and drying in a vacuum oven for 24 hours to obtain a product tert-butyl acetate polyethylene glycol poly benzyloxycarbonyl lysine poly selenomethionine block copolymer tBu-PEG 45 -PLL 2 -PMet(Se) 2 . The product was protected by a conventional amino protection strategy from the amino groups at the ends of the block copolymer by Cbz-Cl. The product is further stirred and reacted for 2 hours by a trifluoroacetic acid dissolving ice bath to remove tert-butyl ester groups at the PEG end, the mixture is settled in ice anhydrous isopropyl ether, centrifuged and dried to obtain carboxyl polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) segmented copolymer, the product is added into 1.2e.q.DCC and 1.5e.q.NHS, stirred and activated at room temperature for overnight, and then Glu-NH of 1.2 e.q.is added 2 And 1.5e.q. triethylamine are stirred overnight at room temperature to obtain glycosylated polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) block copolymer Glu-PEG 45 -PBLA 2 -PMet(Se) 2 . And removing the carbobenzoxy in the block copolymer by acidolysis reaction. The block copolymer was dissolved in an appropriate amount of trifluoroacetic acid, and 5 times of 33% HBr/AcOH solution was added under stirring in an ice bath, and after 4 hours of reaction, the crude product was obtained by precipitation and centrifugation in anhydrous diethyl ether. The crude product was then completely dissolved in DMSO and the solution was then deionized waterDialyzing (MWCO: 2000), dialyzing in deionized water for 24 hr, transferring into ammonia water with pH of 9.0, dialyzing in HCl solution with pH of 5.0 for 24 hr, dialyzing in deionized water for 24 hr, and lyophilizing to obtain Glu-PEG 45 -PLL 2 -PMet(Se) 2 。
Example 6:
glycosylated polyethylene glycol polylysine polyseleno methionine block copolymer (Glu-PEG) 45 -PLL 2 -PMet(Se) 4 ) Is synthesized by (a)
Weigh 0.1g tBu-PEG-NH 2 (0.05 mmol, mn=2000) was dried in vacuum in a vacuum oven for 4 hours and then dissolved in 3ml of DMF after drying to serve as a macroinitiator. 31mg of freshly prepared ε -benzyloxycarbonyl-L-lysine NCA (Lys) (Z) NCA) (0.1 mmol) was dissolved in 1ml of ultra-dry DMF, then the two reaction solutions were mixed by syringe and placed under nitrogen atmosphere and stirred for reaction for 24h. After the completion of the reaction, 44.6mg of newly prepared seleno-L-methionine NCA (0.2 mmol) was dissolved in 1ml of super-dry DMF, and the mixture was added to the reaction mixture, followed by stirring. After 24 hours, the reaction was ended. Settling the triblock polymer in ice anhydrous isopropyl ether, centrifuging, and drying in a vacuum oven for 24 hours to obtain a product tert-butyl acetate polyethylene glycol poly benzyloxycarbonyl lysine poly selenomethionine block copolymer tBu-PEG 45 -PLL 2 -PMet(Se) 4 . The product was protected by a conventional amino protection strategy from the amino groups at the ends of the block copolymer by Cbz-Cl. The product is further stirred and reacted for 2 hours by a trifluoroacetic acid dissolving ice bath to remove tert-butyl ester groups at the PEG end, the mixture is settled in ice anhydrous isopropyl ether, centrifuged and dried to obtain carboxyl polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) segmented copolymer, the product is added into 1.2e.q.DCC and 1.5e.q.NHS, stirred and activated at room temperature for overnight, and then Glu-NH of 1.2 e.q.is added 2 And 1.5e.q. triethylamine are stirred overnight at room temperature to obtain glycosylated polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) block copolymer Glu-PEG 45 -PBLA 2 -PMet(Se) 4 . And removing the carbobenzoxy in the block copolymer by acidolysis reaction. The block copolymer is dissolved in proper amount of trifluoroacetic acid and stirred in ice bath5 times of 33% HBr/AcOH solution was added, and after 4 hours of reaction, the crude product was obtained by precipitation and centrifugation in anhydrous diethyl ether. Dissolving the crude product in DMSO, dialyzing the solution with deionized water (MWCO: 2000) for 24 hr, dialyzing in ammonia water with pH of 9.0 for 24 hr, dialyzing in HCl solution with pH of 5.0 for 24 hr, dialyzing in deionized water for 24 hr, and lyophilizing to obtain Glu-PEG 45 -PLL 2 -PMet(Se) 4 。
Example 7:
glycosylated polyethylene glycol polylysine polyseleno methionine block copolymer (Glu-PEG) 45 -PLL 2 -PMet(Se) 6 ) Is synthesized by (a)
Weigh 0.1g tBu-PEG-NH 2 (0.05 mmol, mn=2000) was dried in vacuum in a vacuum oven for 4 hours and then dissolved in 3ml of DMF after drying to serve as a macroinitiator. 31mg of freshly prepared ε -benzyloxycarbonyl-L-lysine NCA (Lys) (Z) NCA) (0.1 mmol) was dissolved in 1ml of ultra-dry DMF, then the two reaction solutions were mixed by syringe and placed under nitrogen atmosphere and stirred for reaction for 24h. After the completion of the reaction, 66.9mg of newly prepared seleno-L-methionine NCA (0.3 mmol) was weighed and dissolved in 1ml of super-dry DMF, and the mixture was added to the reaction mixture, followed by stirring. After 24 hours, the reaction was ended. Settling the triblock polymer in ice anhydrous isopropyl ether, centrifuging, and drying in a vacuum oven for 24 hours to obtain a product tert-butyl acetate polyethylene glycol poly benzyloxycarbonyl lysine poly selenomethionine block copolymer tBu-PEG 45 -PLL 2 -PMet(Se) 6 . The product was protected by a conventional amino protection strategy from the amino groups at the ends of the block copolymer by Cbz-Cl. The product is further stirred and reacted for 2 hours by a trifluoroacetic acid dissolving ice bath to remove tert-butyl ester groups at the PEG end, the mixture is settled in ice anhydrous isopropyl ether, centrifuged and dried to obtain carboxyl polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) segmented copolymer, the product is added into 1.2e.q.DCC and 1.5e.q.NHS, stirred and activated at room temperature for overnight, and then Glu-NH of 1.2 e.q.is added 2 And 1.5e.q. triethylamine are stirred overnight at room temperature to obtain glycosylated polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) block copolymer Glu-PEG 45 -PBLA 2 -PMet(Se) 6 . And removing the carbobenzoxy in the block copolymer by acidolysis reaction. The block copolymer was dissolved in an appropriate amount of trifluoroacetic acid, and 5 times of 33% HBr/AcOH solution was added under stirring in an ice bath, and after 4 hours of reaction, the crude product was obtained by precipitation and centrifugation in anhydrous diethyl ether. Dissolving the crude product in DMSO, dialyzing the solution with deionized water (MWCO: 2000) for 24 hr, dialyzing in ammonia water with pH of 9.0 for 24 hr, dialyzing in HCl solution with pH of 5.0 for 24 hr, dialyzing in deionized water for 24 hr, and lyophilizing to obtain Glu-PEG 45 -PLL 2 -PMet(Se) 6 。
The following also lists an amphiphilic triblock copolymer of glycosylated polyethylene glycol polyvaline and polyseleno methionine (Glu-PEG) n -PV x -PMet(Se) y ) To prepare a multifunctional nano-drug carrier without charges, see in particular examples 8-10.
The specific synthetic route is as follows:
example 8:
glycosylated polyethylene glycol poly valine poly selenomethionine block copolymer (Glu-PEG) 45 -PV 2 -PMet(Se) 2 ) Is synthesized by (a)
Weigh 0.1g tBu-PEG-NH 2 (0.05 mmol, mn=2000) was dried in vacuum in a vacuum oven for 4 hours and then dissolved in 3ml of DMF after drying to serve as a macroinitiator. 15mg of freshly prepared L-valine NCA (0.1 mmol) was weighed into 1ml of ultra-dry DMF and the two reaction solutions were then mixed by syringe and placed under nitrogen and stirred for 24h. After the completion of the reaction, 23mg of newly prepared seleno-L-methionine NCA (0.1 mmol) was weighed and dissolved in 1ml of super-dry DMF, and the mixture was added to the reaction mixture, followed by stirring. After 24 hours, the reaction was ended. Settling the triblock polymer in ice anhydrous isopropyl ether, centrifuging, and drying in a vacuum oven for 24 hours to obtain the product tert-butyl acetate polyethylene glycol poly (benzyloxycarbonyl) lysine poly (selenomethionine) Block copolymer tBu-PEG 45 -PV 2 -PMet(Se) 2 . The product was protected by a conventional amino protection strategy from the amino groups at the ends of the block copolymer by Cbz-Cl. The product is further stirred and reacted for 2 hours by a trifluoroacetic acid dissolving ice bath to remove tert-butyl ester groups at the PEG end, the mixture is settled in ice anhydrous isopropyl ether, centrifuged and dried to obtain carboxyl polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) segmented copolymer, the product is added into 1.2e.q.DCC and 1.5e.q.NHS, stirred and activated at room temperature for overnight, and then Glu-NH of 1.2 e.q.is added 2 And 1.5e.q. triethylamine are stirred overnight at room temperature to obtain glycosylated polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) block copolymer Glu-PEG 45 -PV 2 -PMet(Se) 2 。
Example 9:
glycosylated polyethylene glycol poly valine poly selenomethionine block copolymer (Glu-PEG) 45 -PV 2 -PMet(Se) 4 ) Is synthesized by (a)
Weigh 0.1g tBu-PEG-NH 2 (0.05 mmol, mn=2000) was dried in vacuum in a vacuum oven for 4 hours and then dissolved in 3ml of DMF after drying to serve as a macroinitiator. 15mg of freshly prepared L-valine NCA (0.1 mmol) was weighed into 1ml of ultra-dry DMF and the two reaction solutions were then mixed by syringe and placed under nitrogen and stirred for 24h. After the reaction, 46mg of newly prepared seleno-L-methionine NCA (0.2 mmol) was weighed and dissolved in 1ml of super-dry DMF, and the mixture was added to the reaction mixture, followed by stirring. After 24 hours, the reaction was ended. Settling the triblock polymer in ice anhydrous isopropyl ether, centrifuging, and drying in a vacuum oven for 24 hours to obtain a product tert-butyl acetate polyethylene glycol poly benzyloxycarbonyl lysine poly selenomethionine block copolymer tBu-PEG 45 -PV 2 -PMet(Se) 4 . The product was protected by a conventional amino protection strategy from the amino groups at the ends of the block copolymer by Cbz-Cl. The product is further stirred with trifluoroacetic acid to react for 2 hours to remove tert-butyl ester groups at the PEG end, the mixture is settled, centrifuged and dried in ice anhydrous isopropyl ether to obtain carboxyl polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) block copolymer, and then the product is added into a middle chamber of 1.2e.q.DCC and 1.5e.q.NHSActivated overnight with gentle stirring, then 1.2e.q. Glu-NH was added 2 And 1.5e.q. triethylamine are stirred overnight at room temperature to obtain glycosylated polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) block copolymer Glu-PEG 45 -PV 2 -PMet(Se) 4 。
Example 10:
glycosylated polyethylene glycol poly valine poly selenomethionine block copolymer (Glu-PEG) 45 -PV 2 -PMet(Se) 6 ) Is synthesized by (a)
Weigh 0.1g tBu-PEG-NH 2 (0.05 mmol, mn=2000) was dried in vacuum in a vacuum oven for 4 hours and then dissolved in 3ml of DMF after drying to serve as a macroinitiator. 15mg of freshly prepared L-valine NCA (0.1 mmol) was weighed into 1ml of ultra-dry DMF and the two reaction solutions were then mixed by syringe and placed under nitrogen and stirred for 24h. After the completion of the reaction, 69mg of newly prepared seleno-L-methionine NCA (0.3 mmol) was dissolved in 1ml of super-dry DMF, and the mixture was added to the reaction mixture, followed by stirring. After 24 hours, the reaction was ended. Settling the triblock polymer in ice anhydrous isopropyl ether, centrifuging, and drying in a vacuum oven for 24 hours to obtain a product tert-butyl acetate polyethylene glycol poly benzyloxycarbonyl lysine poly selenomethionine block copolymer tBu-PEG 45 -PV 2 -PMet(Se) 6 . The product was protected by a conventional amino protection strategy from the amino groups at the ends of the block copolymer by Cbz-Cl. The product is further stirred and reacted for 2 hours by a trifluoroacetic acid dissolving ice bath to remove tert-butyl ester groups at the PEG end, the mixture is settled in ice anhydrous isopropyl ether, centrifuged and dried to obtain carboxyl polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) segmented copolymer, the product is added into 1.2e.q.DCC and 1.5e.q.NHS, stirred and activated at room temperature for overnight, and then Glu-NH of 1.2 e.q.is added 2 And 1.5e.q. triethylamine are stirred overnight at room temperature to obtain glycosylated polyethylene glycol poly (benzyl aspartate) poly (selenomethionine) block copolymer Glu-PEG 45 -PV 2 -PMet(Se) 6 。
Example 11:
preparation of nanosphere micelles
20mg Glu-PEG is taken 45 -PV 2 -PMet(Se) 2 (p=0.28) was completely dissolved in 2mL DMSO, and the polymer was uniformly mixed by shaking to obtain a polymer solution; dropwise adding 5mL of deionized water into the mixture, and stirring the mixture for 2 hours to obtain a solution; directly adding the obtained solution into a dialysis bag with molecular weight cut-off of 2000DA, dialyzing at room temperature in 2L deionized water for 7 days, changing water once a day, filtering the inner solution of the dialysis bag with a water phase filter head (aperture 450 nm), and filtering out macromolecule precipitate and aggregated micelle particles to obtain the target product Glu-PEG 45 -PV ＝ -PMet(Se) 2 Nano spherical micelle.
Example 12:
preparation of nanorod-like micelles
20mg Glu-PEG is taken 45 -PV 2 -PMet(Se) 4 (p=0.46) was completely dissolved in 2mL of dichloromethane, and evaporated in a solvent bottle in a round-robin fashion to give a uniform film, and the residual organic solvent was removed by vacuum overnight. Adding 5mL double distilled water into a eggplant-shaped bottle, hydrating for 1h at 60 ℃, vibrating and uniformly mixing, performing ultrasonic treatment under water bath to obtain nanorod micelle dispersion liquid, centrifuging for 30min at 3000r/min, removing large aggregate particles, and performing freeze drying on clear liquid to obtain the target product nanorod micelle.
Example 13:
preparation of nanovesicles
16mL of a 0.25% aqueous solution of polyvinyl alcohol was measured in a 50mL beaker and stirred. Weigh 20mg Glu-PEG 45 -PV 2 -PMet(Se) 6 (p=0.64) in 4mL of CH 2 Cl 2 And (3) performing ultrasonic treatment on the solution twice by using an ultrasonic cell grinder, wherein the ultrasonic treatment time is 4s, the intermittent time is 4s, the total treatment time is 30s, and the ultrasonic treatment interval time is 30s. An aqueous solution of 0.1ml of 0.1% polyvinyl alcohol was sucked by a syringe at the beginning of the first ultrasonic treatment, and the emulsification was observed. After the ultrasonic treatment, the solution is completely sucked out by a syringe, slowly and uniformly injected into the aqueous solution of the polyvinyl alcohol with the mass fraction of 0.25 percent, and stirred for 2 hours at room temperature. And finally, repeatedly washing, freezing and drying to obtain the target product nano vesicle.
Example 14:
preparation of anion-cation composite nano spherical micelle
10mg Glu-PEG is taken 45 -PAsp 2 -PMet(Se) 2 (p=0.30) and 10mg Glu-PEG 45 -PLL 2 -PMet(Se) 2 (p=0.30) was completely dissolved in 2mL DMSO, and the polymer was uniformly mixed by shaking to obtain a polymer solution; dropwise adding 5mL of deionized water into the mixture, and stirring the mixture for 2 hours to obtain a solution; directly adding the obtained solution into a dialysis bag with the molecular weight cut-off of 2000DA, dialyzing at room temperature for 7 days in 2L deionization, changing water once a day, and then filtering (pore diameter of 450 nm) the inner liquid of the dialysis bag by using a water phase filter head, and filtering out macromolecule sediment and aggregated micelle particles to obtain the target product nanometer spherical micelle. The formed nano spherical micelle is observed by a transmission electron microscope, the micelle is in a regular spherical shape, the particle size is about 150nm, and the result is shown in figure 3.
Example 15:
preparation of anion-cation composite nanorod micelle
10mg Glu-PEG is taken 45 -PAsp 2 -PMet(Se) 4 (p=0.47) and 10mg Glu-PEG 45 -PLL 2 -PMet(Se) 4 (p=0.49) was completely dissolved in 2mL of dichloromethane, and evaporated in a solvent bottle in a round-robin fashion to give a uniform film, and the residual organic solvent was removed by vacuum overnight. Adding 5mL double distilled water into a eggplant-shaped bottle, hydrating for 1h at 60 ℃, vibrating and uniformly mixing, performing ultrasonic treatment under water bath to obtain nanorod micelle dispersion liquid, centrifuging for 30min at 3000r/min, removing large aggregate particles, and performing freeze drying on clear liquid to obtain the target product nanorod micelle. The nanocapsules were formed in a baseball shape by observation with a transmission electron microscope, and the particle size was about 100nm, and the result was shown in fig. 4.
Example 16:
preparation of anion-cation composite nano vesicle
16mL of a 0.25% aqueous solution of polyvinyl alcohol was measured in a 50mL beaker and stirred. 10mg Glu-PEG was weighed 45 -PAsp 2 -PMet(Se) 6 (p=0.65) and 10mg Glu-PEG 45 -PLL 2 -PMet(Se) 6 (p=0.67) in 4mL of CH 2 Cl 2 The solution is then treated with ultrasoundThe wave cell grinder carries out two times of ultrasonic, the working time of one time of ultrasonic is 4s, the intermittent time is 4s, the total working time is 30s, and the interval time of two times of ultrasonic is 30s. An aqueous solution of 0.1ml of 0.1% polyvinyl alcohol was sucked by a syringe at the beginning of the first ultrasonic treatment, and the emulsification was observed. After the ultrasonic treatment, the solution is completely sucked out by a syringe, slowly and uniformly injected into the aqueous solution of the polyvinyl alcohol with the mass fraction of 0.25 percent, and stirred for 2 hours at room temperature. And finally, repeatedly washing, freezing and drying to obtain the target product nano vesicle. The formed nanovesicles were observed by a transmission electron microscope, and the nanospheres were spherical, and the particle size was measured to be about 120nm, as shown in FIG. 5.
Example 17:
preparation of anion-cation composite doxorubicin-loaded nano spherical micelle
10mg Glu-PEG is taken 45 -PAsp 2 -PMet(Se) 2 (p=0.30) and 10mg Glu-PEG 45 -PLL 2 -PMet(Se) 2 (p=0.30) and 2.7mg of doxorubicin were completely dissolved in an appropriate amount of DMSO, respectively. Oscillating the former to uniformly mix the polymers to obtain a polymer solution, slowly dropwise adding an adriamycin solution under rapid stirring, adding 2.7mg of adriamycin into the polymer solution, and slightly stirring to uniformly mix the two; dropwise adding 5mL of deionized water into the mixture, and stirring the mixture for 2 hours to obtain a solution; directly adding the obtained solution into a dialysis bag with molecular weight cut-off of 2000DA, dialyzing at room temperature for 7 days in 1L deionized water, changing water once a day, filtering the inner liquid of the dialysis bag with a water phase filter head (aperture 450 nm), and filtering out macromolecule precipitate and aggregated micelle particles to obtain the target product Glu-PEG 45 -PAsp 2 -PMet(Se) 2 -doxorubicin nanospheres micelles.
Example 18:
preparation of anion-cation composite donepezil-carrying nano vesicle
16mL of an aqueous solution of 0.25% by mass polyvinyl alcohol was measured in a 50mL beaker and stirred. 10mg Glu-PEG was weighed 45 -PAsp 2 -PMet(Se) 6 (p=0.65) and 10mg Glu-PEG 45 -PLL 2 -PMet(Se) 6 (p=0.67) and 2.7mg of donepezil is completely dissolved in 4mL of CH 2 Cl 2 And (3) performing ultrasonic treatment on the solution twice by using an ultrasonic cell grinder, wherein the ultrasonic treatment time is 4s, the intermittent time is 4s, the total treatment time is 30s, and the ultrasonic treatment interval time is 30s. An aqueous solution of 0.1ml of 0.1% polyvinyl alcohol was sucked by a syringe at the beginning of the first ultrasonic treatment, and the emulsification was observed. After the ultrasonic treatment, the solution was completely sucked out by a syringe, slowly and uniformly poured into a 0.25% aqueous solution of polyvinyl alcohol, and stirred at room temperature for 2 hours. Finally, repeatedly washing, freezing and drying to obtain the target product Glu-PEG 45 -PAsp 2 -PMet(Se) 6 -donepezil nanovesicles.
Example 19:
in vitro release test of anion-cation composite drug-loaded nano micelle
4mg of the anion-cation composite doxorubicin-loaded nano spherical micelle prepared in example 17 is precisely weighed and placed into a dialysis bag (the molecular weight cut-off is 2000 DA), and 5mL of phosphate buffer solution (1 mol/L, pH 7.4) is added into the dialysis bag for dispersion. The dialysis bag was placed in a triangular flask, and 20mL of PBS (1 mol/L, pH 7.4) was added as a release medium. The triangular flask is placed in a constant temperature shaking table at 37 ℃ and 100r/min for release experiments, 2mL is sampled every 1h, and fresh PBS with the same volume is supplemented after sampling to ensure constant volume of the drug release external liquid. And measuring ultraviolet absorbance of the sample at lambda=483 nm by an ultraviolet-visible spectrophotometer, and drawing an in-vitro drug release curve of the doxorubicin nano micelle. And drawing an in-vitro drug release standard curve of the pure doxorubicin product according to the operation. Glu-PEG can be known from the in-vitro drug release curve of doxorubicin nano micelle 45 -PAsp 2 -PMet(Se) 2 The drug loading rate of the nano micelle is about 10.0%, and the encapsulation rate is 50.5%. And compared with an doxorubicin in-vitro drug release standard curve, the drug-loaded nano-micelle provided by the embodiment is found to have no burst release phenomenon in the in-vitro release process, so that the drugs adsorbed on the surface of the micelle are relatively less, most of the drugs are wrapped in the micelle, and the drug-loaded nano-micelle has a relatively good controlled release effect, thereby being beneficial to reducing the toxic and side effects of the drugs.
Example 20:
detection of selenium antioxidation function in nano-drug carrier
The breast cancer MDA-MB-231 cell strain (hereinafter referred to as 231 cells) was placed in 5% CO in DMEM medium containing 10% fetal bovine serum 2 Culturing in a 37 ℃ incubator, carrying out passage for 1-2 d, and taking cells in a logarithmic growth phase for experiment. 231 cells in logarithmic growth phase were taken at 8X 10 3 The cells were inoculated in 96-well plates and incubated for 24h before drug treatment. The experiments were divided into four groups: the administration dose of the control group, the nano micelle group, the doxorubicin group and the nano micelle plus doxorubicin group is nano micelle (100 mu mol/L), doxorubicin (2.0 mu mol/L), 100 mu L of each well is added, 6 compound wells are arranged, and the same amount of DMEM culture medium without medicines is added into the control wells. After 24 hours, the cells of each group are collected, added into cell lysate for lysis on ice, and centrifuged to collect the whole cell protein. The BCA method is used for measuring the protein concentration, the WST-1 method is used for measuring the total SOD activity of cells, the TBA method is used for measuring the MDA content of cells, and the operation steps are strictly according to the requirements of a kit. As can be seen from the measurement of the levels of SOD and MDA in 231 cells, the activity of the SOD in each treated group is reduced compared with that of the control group, and the level of MDA is increased (P is less than 0.05); wherein the total SOD activity of the nano micelle combined doxorubicin group cells is reduced, and the MDA level is increased most obviously. Therefore, the nano-drug carrier has the antioxidation function of selenium, and the combination of the nano-drug carrier and doxorubicin has additive effect.
Example 21:
preparation of anion-cation composite rhodamine B-loaded nano spherical micelle
10mg of Glu-PEG45-PAsp2-PMet (Se) 2 (P=0.30) and 10mg of Glu-PEG45-PLL2-PMet (Se) 2 (P=0.30) were taken and 1.0mg of rhodamine B were completely dissolved in an appropriate amount of DMSO, respectively. Oscillating the former to uniformly mix the polymers to obtain a polymer solution, slowly dropwise adding rhodamine B solution under rapid stirring, and slightly stirring to uniformly mix the rhodamine B solution and the rhodamine B solution; dropwise adding 5mL of deionized water into the mixture, and stirring the mixture for 2 hours to obtain a solution; directly adding the obtained solution into a dialysis bag with the molecular weight cut-off of 2000DA, dialyzing at room temperature for 7 days in 1L of deionized water, changing water once a day, and filtering (pore diameter of 450 nm) the inner liquid of the dialysis bag by using a water phase filter head to filter out macromolecule sediment and aggregated micelle particles, thereby obtaining the target product rhodamine B nano spherical micelle.
Example 22:
qualitative observation of cell uptake glycosylation modified anion-cation composite rhodamine B-loaded nano spherical micelle
231 cells were grown at 2X 10 4 The individual/hole density is inoculated in a 48-hole culture plate for culture, and after 24 hours, a DMEM solution of glycosylated rhodamine B nano spherical micelle is used for incubation for 1 hour at 37 ℃, and the concentration of the nano spherical micelle is respectively 50, 100, 200, 400 and 600 mug/mL. Discarding the nanosphere micelle solution, washing the cells for 3 times by using PBS, washing off the nanosphere micelle adsorbed on the cell surface, adding 3.7% formaldehyde solution for fixation for 10min, then using 100ng/mL DAPI solution for nuclear dyeing for 10min, rinsing for 3 times by using PBS, and observing the uptake condition of the cells on the glycosylated rhodamine B nanosphere micelle under a fluorescence microscope, wherein the result shows that: the nuclei were stained blue with DAPI to locate the cell sites, red fluorescent dye-loaded nanomicelle distributed around the nuclei, and filled in the cytoplasm, demonstrating that the uptake of the glycosylation modified rhodamine B-loaded nanosphere micelle by 231 cells was evident (fig. 6).
In summary, the targeted GLUT1 and polyseleno-amino acid amphiphilic block copolymer provided by the invention has a structure shown in formula (I), can be self-assembled into nano-drug carriers (such as nano spherical micelles, nano rod-shaped micelles and nano vesicles) in different forms under different environments, is loaded with drug molecules, and is prepared into a slow-release controlled-release targeted drug delivery system, the drug molecules can be directionally delivered to a lesion site and can be delivered in various modes, and the entrapped drug can be released from the delivery system in a slow-release controlled manner as required, so that the administration frequency is reduced, the treatment effect is improved, and the toxic and side effects of the drug are reduced. Compared with the prior art, the targeted GLUT1 and polyseleno-amino acid amphiphilic block copolymer provided by the embodiment of the invention has the advantages of common amino acids, simultaneously has multiple biological functions of selenium, has the specificity of the targeted GLUT1, is a novel and multifunctional amphiphilic block copolymer, and is suitable for research, development and clinical application of drug carriers of various drugs of various diseases related to GLUT1 abnormality.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.

Claims (7)

1. The targeted GLUT1 and multifunctional nano-drug carrier is characterized by being formed by self-assembling a targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with a structure shown in a formula (I);
wherein n=45, x=2, y=2, 4 or 6, n, x, y are integers;
-R 1 selected from the group consisting of
-R 2 Selected from-CH (CH) 3 )CH 3 、One of the following;
-R 3 selected from-CH 2 CH 2 SeCH 3 ；
The nano-drug carrier exists in the form of nano spherical micelle, nano rod-shaped micelle or nano vesicle;
the ratio of the molecular weight of the hydrophobic segment to the molecular weight of the hydrophilic segment is denoted as P,
when P is less than or equal to 1/3, the nano-drug carrier is in the form of nano-spherical micelle;
when 1/3<P is less than or equal to 1/2, the nano-drug carrier is a nano-drug carrier in the form of nano rod-shaped micelle;
when 1/2<P is less than or equal to 1, the nano-drug carrier is in a nano vesicle form;
the hydrophobic chain segment is
The hydrophilic chain segment is
2. A method for preparing the targeted GLUT1 multifunctional nano-drug carrier according to claim 1, which is characterized by comprising the following steps:
dissolving the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with the structure shown in the formula (I) in a first organic solvent, and oscillating until the mixture is uniformly mixed to obtain a polymer solution;
Adding deionized water into the polymer solution, and stirring and reacting for 2-3h to obtain a mixed solution;
and dialyzing the obtained mixed solution by using a dialysis bag filled with deionized water, and filtering the solution in the dialysis bag to obtain the nano-drug carrier in the form of nano-rod micelle.
3. A method for preparing the targeted GLUT1 multifunctional nano-drug carrier according to claim 1, which is characterized by comprising the following steps:
dissolving the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with the structure shown in the formula (I) in a second organic solvent, and removing the second organic solvent to prepare a film;
then adding the mixture into double distilled water for hydration, shaking and mixing uniformly, carrying out ultrasonic treatment, centrifuging, freezing and drying to obtain the nano-drug carrier in the form of nano-vesicles.
4. A method for preparing the targeted GLUT1 multifunctional nano-drug carrier according to claim 1, which is characterized by comprising the following steps:
dissolving the targeted GLUT1 and polyseleno amino acid amphiphilic block copolymer with the structure shown in the formula (I) in an oil phase solution to obtain a copolymer oil phase solution;
sucking the aqueous phase solution into the copolymer oil phase solution by using a syringe, performing ultrasonic treatment, sucking all the ultrasonic solution by using the syringe, injecting the solution into the external aqueous phase solution, and stirring;
And finally, centrifuging, washing, freezing and drying to obtain the nano drug carrier in the form of nano spherical micelle.
5. A drug-loaded composition comprising the nano-drug carrier of claim 1 and a drug entrapped within the nano-drug carrier.
6. The drug-loaded composition of claim 5, wherein the drug is selected from one or more of an anti-AD drug, an anti-tumor drug, a steroidal or non-steroidal anti-inflammatory drug, a metabolic regulation drug, an antibiotic, a cardiovascular drug, an antiviral drug, an antifungal drug, an immunomodulator.
7. The drug-loaded composition of claim 5, wherein the mass ratio of the nano-drug carrier to the drug is 1mg: (0.1-10) mg.
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