阅读说明：本技术 靶向乳铁蛋白受体的多功能纳米药物载体及其制备方法与载药组合物 (Multifunctional nano-drug carrier targeting lactoferrin receptor, preparation method thereof and drug-loaded composition ) 是由 吴海强 许晨舒 李晨阳 欧阳娜 王亦男 熊炜 李霞 于 2021-09-01 设计创作，主要内容包括：本发明涉及靶向乳铁蛋白受体的多功能纳米药物载体及其制备方法与载药组合物。所述多功能纳米药物载体由下式所示结构的多聚硒代氨基酸两亲性嵌段共聚物自组装,继而通过乳铁蛋白修饰而成；其中,22≤n≤454、2≤x≤50、2≤y≤50,n、x、y均为整数。本发明药物载体能够负载药物分子,制成缓释控释的靶向药物输送系统,能够将药物分子定向的送到病变部位,降低给药频率、提高治疗效果等。该药物载体兼具一般氨基酸类药物载体优势的同时,既具备了硒的多种生物学功能,又具备了靶向乳铁蛋白受体的特异性,是一类新型、靶向、多功能药物载体,适用于乳铁蛋白受体异常相关多种疾病的各类药物的药物载体研究、开发及临床应用。(The invention relates to a multifunctional nano-drug carrier targeting a lactoferrin receptor, a preparation method thereof and a carrierA pharmaceutical composition. The multifunctional nano-drug carrier is formed by self-assembling polyseleno amino acid amphiphilic block copolymer with a structure shown in the following formula and then modifying the self-assembled polyseleno amino acid amphiphilic block copolymer by lactoferrin; 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. The drug carrier can load drug molecules to prepare a sustained-release controlled-release targeted drug delivery system, and can directionally deliver the drug molecules to a pathological change part, reduce the administration frequency, improve the treatment effect and the like. The drug carrier has the advantages of common amino acid drug carriers, has multiple biological functions of selenium, has the specificity of a targeted lactoferrin receptor, is a novel, targeted and multifunctional drug carrier, and is suitable for drug carrier research, development and clinical application of various drugs for various diseases related to lactoferrin receptor abnormality.)

1. The multifunctional nano-drug carrier for targeting the lactoferrin receptor is characterized by comprising nano-micelles and lactoferrin combined on the surfaces of the nano-micelles, wherein the nano-micelles are formed by self-assembling polyseleno amino acid amphiphilic block copolymers with structures shown in a formula (I), and the nano-micelles pass through the R₁(ii) binds to the lactoferrin;

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;

-R₁is selected from-OCH₃、-COOH、-NH₂or-MAL;

-R₂is selected from-CH (CH)₃)CH₃、-H、-CH₃、-CH₂CH(CH₃)CH₃、-CH(CH₃)CH₂CH₃、-CH₂OH、-CH₂SH、-CH₂CH₂SCH₃、-CH(OH)CH₃、

-R₃Is selected from-CH₂CH₂SeCH₃or-CH₂SeH。

2. The multifunctional nanomicelle carrier targeting lactoferrin receptor according to claim 1, wherein the nanomicelle exists in the form of nanospheres, nanocolumns or nanovesicles.

3. The preparation method of the multifunctional nano-drug carrier targeting the lactoferrin receptor of any one of claims 1 to 2, comprising the following steps:

providing a nanomicelle;

dispersing lactoferrin into a buffer solution, adding EDCI and NHS, and sequentially stirring and activating at room temperature in a dark place;

removing unreacted EDCI and NHS in the activated reaction solution, and then dispersing in a buffer solution to obtain an activated lactoferrin dispersion solution;

adding the nano micelle into the activated lactoferrin dispersion liquid, and reacting under stirring;

purifying the reacted system to obtain the multifunctional nano-drug carrier targeting the lactoferrin receptor;

wherein the nano-micelle is an amino-modified nano-micelle, and the lactoferrin is a carboxyl-modified lactoferrin;

or the nano-micelle is a carboxyl-modified nano-micelle, and the lactoferrin is amino-modified lactoferrin.

4. The method for preparing the multifunctional nano-drug carrier targeting the lactoferrin receptor of claim 3, wherein the lactoferrin is dispersed to 0.01mol/LKH with pH of 6₂PO₄In a buffer solution;

the dosage ratio of the lactoferrin to EDCI and NHS is 1 mg: 2-6 mg: 4-8 mg;

the time of the lucifugal room temperature activation treatment is 15min-12 h.

5. The preparation method of the multifunctional nano-drug carrier targeting the lactoferrin receptor of any one of claims 1 to 2, comprising the following steps:

providing a nanomicelle;

dissolving lactoferrin in sodium borate solution, adding Traut's reagent, and stirring;

enabling the stirred reaction solution to pass through a Zeba desalting column to remove impurities, and obtaining a thiolated lactoferrin solution;

subjecting the nanomicelle to NaH₂PO₄After dispersion, adding the thiolated lactoferrin solution, and stirring at room temperature for reaction;

and (3) carrying out purification treatment on the system after the room-temperature stirring reaction to obtain the multifunctional nano-drug carrier targeting the lactoferrin receptor.

6. The preparation method of the multifunctional nano-drug carrier targeting the lactoferrin receptor of claim 5, wherein the molar ratio of the lactoferrin to the Traut's reagent is 1: 30-50 parts of;

the dosage ratio of the thiolated lactoferrin to the nano micelle is 1 g: 2-100 mg;

the stirring reaction time at room temperature is 6-12 h.

7. A drug-loaded composition comprising the lactoferrin receptor-targeting multifunctional nano-drug carrier of any one of claims 1 to 2 and a drug encapsulated in the multifunctional nano-drug carrier.

8. The drug-loaded composition of claim 7, wherein the drug is selected from one or more of anti-AD drugs, anti-tumor drugs, steroidal anti-inflammatory drugs, non-steroidal anti-inflammatory drugs, metabolic regulation drugs, antibiotics, cardiovascular drugs, antiviral drugs, antifungal drugs, and immunomodulators.

9. The drug-loaded composition of claim 7, wherein the mass ratio of the multifunctional nano-drug carrier to the drug is 1 mg: (0.1-10) mg.

Technical Field

The invention relates to the technical field of biomedical drug carriers and sustained-release materials, in particular to a multifunctional nano drug carrier targeting a lactoferrin receptor, a preparation method thereof and a drug-loaded composition.

Background

The human body has a very complex physiological environment, and the medicine needs multiple obstacles from the time of taking to exerting the effect, so that only a small part of the medicine can exert the effect, the treatment effect is seriously influenced, and meanwhile, toxic and side effects are brought. How to enhance the utilization rate and safety of the medicine and the like has great significance for improving the treatment effect of diseases and human health. In recent years, different types of drug carriers have received a great deal of attention.

The drug carrier is mainly natural or synthetic high molecular material, and forms a drug control system through chemical bonding, physical adsorption or wrapping with drug molecules in different forms, so that the timed, positioned and quantitative release of the drug can be realized through a series of physical, chemical and biological controls under the condition of not reducing the drug effect of the original drug molecules and inhibiting the side effects of the original drug molecules, and the curative effect of the drug can be enhanced. Pharmaceutical carriers have been used in a variety of routes of administration, including injection, oral, transdermal, and the like. The drug carriers are various, and the nano drug carriers are novel carriers with the particle size of 10-1000 nm, and have the advantages of reducing the toxic and side effects of the drugs, improving the stability of the drugs, slowly releasing and controlling the release of the drugs, targeting the release of the drugs and the like because the particle size of the nano drug carriers is smaller than that of capillary vessels. The nano-drug carrier comprises polymer micelle, nano-capsule and nano-sphere, nano-liposome, solid lipid nano-particle, magnetic nano-particle 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 issues which need to be considered.

Amino acids are the basic constituent units of biologically functional macromolecular proteins and are the basic substances of proteins required for body nutrition. The polyamino acid prepared by adopting aspartic acid, glutamic acid, lysine, alanine, phenylalanine and the like is a fully biodegradable high polymer material which has low toxicity and good biocompatibility and is easy to be absorbed and metabolized by organisms, and has great development potential in the field of drug carriers. However, due to strong hydrogen bond action among amino acids and the like, the drug carrier materials have the defects of poor water solubility, difficult control of degradation rate and period in vivo, difficult realization of targeted transmission and the like.

Accordingly, the prior art remains to be improved and developed.

Disclosure of Invention

Functionalization and intellectualization are strategic trends of the development of the current nano-drug carrier. The inventor researches to find that polyethylene glycol (PEG) has flexible hydrophilic long chain, is non-toxic and non-immunogenic, has been approved by 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 can be improved, and the adsorption of in vivo proteins on the surface of the material, the adhesion of cells and the like can be reduced. Because PEG has the advantage of being not easily identified by an immune system during in vivo circulation, the polyamino acid can be protected from being damaged by the immune system, the circulation time of the material in vivo is prolonged, and the like. In addition, various functional groups can be introduced into two ends of PEG, so that the comprehensive performance of the polyamino acid nano-drug carrier is obviously enhanced.

Further research shows that the trace element selenium essential to human body has important biological functions of resisting oxidation, regulating immunity, resisting harmful heavy metal, resisting senility, etc. Selenium deficiency is associated with the pathogenesis of many human diseases, including diabetes, cancer, and neurodegenerative diseases, among others. There are two main ways for the human body to take up selenium: inorganic selenium, low utilization rate and high toxicity; organic selenium, such as seleno-amino acid, has better biocompatibility, high utilization rate, easier absorption by human body, lower toxicity and higher safety. Therefore, the introduction of seleno-amino acid is expected to actively promote the research and development of the functionalized nano-polyamino acid drug carrier.

Lactoferrin (Lactoferrin, abbreviated as Lf) is a multifunctional iron ion transport glycoprotein, belongs to the transferrin family, is mainly expressed and secreted in glandular epithelial cells, mainly regulates internal environment ion stability, resists microbial infection, regulates cell growth, inhibits platelet aggregation, enhances immunity and the like, and the biological function of the Lactoferrin (Lactoferrin) is mainly realized by Lactoferrin receptor mediation. Recent researches show that the expression level specificity of lactoferrin receptors of epithelial cells of brain neurons and microvasculature of patients with central nerve cell degenerative diseases such as Alzheimer disease, Parkinson disease and the like is remarkably increased, and the abnormality directly induces and promotes the complex diseases. Therefore, the lactoferrin receptor can be used as an important target point of a functional nano-drug carrier for targeted drug delivery.

Therefore, in view of the defects that the current polyamino acid nano-carrier has single main function, can only be used as a drug carrier, lacks targeting property and the like, the invention provides the multifunctional nano-drug carrier for targeting the lactoferrin receptor, has the advantages of common amino acid nano-drug carriers, has multiple biological functions of selenium, specifically targets the lactoferrin receptor, and is a novel, targeted and multifunctional nano-drug carrier.

Specifically, the technical scheme of the invention is as follows:

a multifunctional nano-drug carrier targeting a lactoferrin receptor comprises nano-micelles and lactoferrin combined on the surfaces of the nano-micelles, wherein the nano-micelles are formed by self-assembling polyselenocarbamic acid amphiphilic block copolymers with a structure shown in a formula (I), and are combined with the lactoferrin through R1;

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;

-R₁is selected from-OCH₃、-COOH、-NH₂or-MAL;

-R₂is selected from-CH (CH)₃)CH₃、-H、-CH₃、-CH₂CH(CH₃)CH₃、-CH(CH₃)CH₂CH₃、-CH₂OH、-CH₂SH、-CH₂CH₂SCH₃、-CH(OH)CH₃、

-R₃Is selected from-CH₂CH₂SeCH₃or-CH₂SeH。

Optionally, the nanomicelle is in the form of nanospheres, nanorods or nanovesicles.

The invention relates to a preparation method of a multifunctional nano-drug carrier targeting a lactoferrin receptor, which comprises the following steps:

providing a nanomicelle;

dispersing lactoferrin into a buffer solution, adding EDCI and NHS, and sequentially stirring and activating at room temperature in a dark place;

removing unreacted EDCI and NHS in the activated reaction solution, and then dispersing in a buffer solution to obtain an activated lactoferrin dispersion solution;

adding the nano micelle into the activated lactoferrin dispersion liquid, and reacting overnight under stirring;

purifying the reacted system to obtain the multifunctional nano-drug carrier targeting the lactoferrin receptor;

wherein the nano-micelle is an amino-modified nano-micelle, and the lactoferrin is a carboxyl-modified lactoferrin;

or the nano-micelle is a carboxyl-modified nano-micelle, and the lactoferrin is amino-modified lactoferrin.

Optionally, the lactoferrin is dispersed in 0.01mol/L KH of pH 6₂PO₄In a buffer solution;

the dosage ratio of the lactoferrin to EDCI and NHS is 1 mg: 2-6 mg: 4-8 mg;

the time of the lucifugal room temperature activation treatment is 15min-12 h.

The invention relates to a preparation method of a multifunctional nano-drug carrier targeting a lactoferrin receptor, which comprises the following steps:

providing a nanomicelle;

dissolving lactoferrin in sodium borate solution, adding Traut's reagent, and stirring;

enabling the stirred reaction solution to pass through a Zeba desalting column to remove impurities, and obtaining a thiolated lactoferrin solution;

subjecting the nanomicelle to NaH₂PO₄After dispersion, the thiolated lactoferrin is addedStirring the solution at room temperature for reaction;

and (3) carrying out purification treatment on the system after the room-temperature stirring reaction to obtain the multifunctional nano-drug carrier targeting the lactoferrin receptor.

Optionally, the molar ratio of lactoferrin to Traut's reagent is 1: 30-50 parts of;

the dosage ratio of the thiolated lactoferrin to the nano micelle is 1 g: 2-100 mg;

the stirring reaction time at room temperature is 6-12 h.

The drug-carrying composition comprises the multifunctional nano-drug carrier targeting the lactoferrin receptor and a drug wrapped in the multifunctional nano-drug carrier.

Optionally, the drug is selected from one or more of anti-AD drugs, anti-tumor drugs, steroidal anti-inflammatory drugs, non-steroidal anti-inflammatory drugs, metabolic regulation drugs, antibiotics, cardiovascular drugs, antiviral drugs, antifungal drugs, and immunomodulators.

Optionally, the mass ratio of the multifunctional nano-drug carrier to the drug is 1 mg: (0.1-10) mg.

Has the advantages that: the multifunctional nano-drug carrier targeting the lactoferrin receptor is prepared by self-assembling polyselenocarbamic acid amphiphilic block copolymer with a structure shown in formula (I), and then performing lactoferrin modification through a functional group at the outer end of PEG (polyethylene glycol) to obtain the multifunctional nano-drug carrier targeting the lactoferrin receptor. The multifunctional nano-drug carrier can directionally deliver drug molecules to a pathological change part, improve drug effect, and simultaneously prepare a slow-release and controlled-release drug delivery system, and the encapsulated drug can be released in a slow-release and controlled-release manner from the delivery system 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 multifunctional nano-drug carrier provided by the invention has the advantages of common amino acid nano-drug carriers, has multiple biological functions of selenium, has the specificity of a targeted lactoferrin receptor, is a novel, targeted and multifunctional drug carrier, and is suitable for drug carrier research, development and clinical application of various drugs for various diseases related to lactoferrin receptor abnormality.

Drawings

FIG. 1 is a TEM (Transmission Electron microscope) image of the polyseleno amino acid nano spherical micelle in the embodiment of the present invention.

Fig. 2 is a TEM (transmission electron microscope) image of the lactoferrin modified polyseleno amino acid nano spherical micelle in the embodiment of the present invention.

FIG. 3 is a graph of the particle size of the polyselenocarbamic acid nano-spherical micelle according to the embodiment of the present invention.

Fig. 4 is a nano laser particle size diagram of the lactoferrin modified polyseleno amino acid nano spherical micelle in the embodiment of the present invention.

Fig. 5 is a laser confocal diagram of the cell-targeted uptake of the lactoferrin-modified polyseleno amino acid nano-spherical micelle in the embodiment of the present invention.

Detailed Description

The invention provides a multifunctional nano-drug carrier targeting a lactoferrin receptor, a preparation method thereof and a drug-loaded composition, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The embodiment of the invention provides a multifunctional nano-drug carrier targeting a lactoferrin receptor, wherein the multifunctional nano-drug carrier comprises a nano-micelle and a lactoferrin combined on the surface of the nano-micelle, the nano-micelle is formed by self-assembling polyseleno amino acid amphiphilic block copolymer with a structure shown in a formula (I), and the nano-micelle is formed by the R-type polymer through self-assembly₁(ii) binds to the lactoferrin;

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;

-R₁is selected from-OCH₃、-COOH、-NH₂or-MAL (maleimide group), etc.;

-R₂is selected from-CH (CH)₃)CH₃、-H、-CH₃、-CH₂CH(CH₃)CH₃、-CH(CH₃)CH₂CH₃、-CH₂OH、-CH₂SH、-CH₂CH₂SCH₃、-CH(OH)CH₃、

-R₃Is selected from-CH₂CH₂SeCH₃or-CH₂SeH。

The multifunctional nano-drug carrier targeting the lactoferrin receptor provided by the embodiment of the invention is prepared from-OCH at the outer end of polyethylene glycol in the nano-micelle₃、-COOH、-NH₂Or the functional groups such as-MAL and the like are chemically connected with the carboxyl or amino of the lactoferrin through various modes such as esterification or amide condensation, and the surface of the nano micelle is covered with a layer of protein coat to obtain the lactoferrin modified multifunctional nano-drug carrier targeting the lactoferrin receptor. The multifunctional nano-drug carrier is utilized to directionally deliver drug molecules to a pathological change part, the drug effect is improved, meanwhile, a slow-release and controlled-release drug delivery system is prepared, and the encapsulated drug can be released in a slow-release and controlled-release manner from the delivery system 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 multifunctional nano-drug carrier provided by the embodiment of the invention has one functionThe carrier has the advantages of the common amino acid nano-drug carrier, has multiple biological functions of selenium, has the specificity of a targeted lactoferrin receptor, is a novel, targeted and multifunctional drug carrier, and is suitable for drug carrier research, development and clinical application of various drugs for various diseases related to lactoferrin receptor abnormality.

Specifically, the polyethylene glycol amine in the structure shown in the formula (I) in the embodiment of the invention 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 vivo can be prolonged; on the other hand, the modified polypeptide is used for providing a targeted modified group, so that the treatment effect of the drug is improved, and the toxic and side effects of the drug are reduced. The polyseleno amino acid provides a hydrophobic end for carrying medicine, so as to provide a stable nano-medicine carrier which not only has the functions of slow release and controlled release of the medicine, but also has multiple biological functions of selenium. Active ion groups in the ionic polyamino acid can be used for cross-linking between block copolymers so as to provide a more stable nano-drug carrier with better drug slow-release and controlled-release functions.

The embodiment of the invention provides a preparation method of the multifunctional nano-drug carrier targeting the lactoferrin receptor, which comprises the following steps:

s10, providing nano-micelles;

s11, dispersing lactoferrin into a buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDCI) and N-hydroxysuccinimide (NHS), and sequentially stirring and activating at room temperature in a dark place;

s12, removing unreacted EDCI and NHS in the activated reaction solution, and then dispersing in a buffer solution to obtain an activated lactoferrin dispersion solution;

s13, adding the nano micelle into the activated lactoferrin dispersion liquid, and reacting under stirring;

s14, purifying the reacted system to obtain the multifunctional nano-drug carrier targeting the lactoferrin receptor;

the nano micelle is an amino-modified nano micelle (namely, a functional group at the outer end of PEG is an amino), and the lactoferrin is carboxyl-modified lactoferrin;

or the nano micelle is a carboxyl-modified nano micelle (namely, the functional group at the outer end of the PEG is a carboxyl), and the lactoferrin is amino-modified lactoferrin.

In one embodiment, the lactoferrin is present in a 1mg to EDCI, NHS ratio: 2-6 mg: 4-8 mg.

In one embodiment, the KH is₂PO₄The buffer pH can be suitably floated up and down by 1-2 units. The KH₂PO₄The buffer solution is 0.01 mol/L-0.1 mol/L.

In one embodiment, the lactoferrin is dispersed to 0.01mol/L KH at pH 6₂PO₄In a buffer.

In one embodiment, the lactoferrin is conjugated to KH₂PO₄The dosage ratio of the buffer solution is 1 mg: 1-5 mL.

In one embodiment, the dark room temperature activation treatment is carried out for 15min to 12h, preferably for 20 min.

In this embodiment, the nano-micelle is a polyselenocarbamic acid amphiphilic block copolymer having a structure represented by formula (I) (-R in formula (I))₁is-COOH or-NH₂) The nano micelle and the preparation method of the copolymer are disclosed in the patent document with the application number of CN202011344949.5 and the patent name of polyseleno amino acid amphiphilic block copolymer, the preparation method and the application, which are not described in detail herein.

The embodiment of the invention provides a preparation method of the multifunctional nano-drug carrier targeting the lactoferrin receptor, which comprises the following steps:

s20, providing nano-micelles;

s21, dissolving lactoferrin in a sodium borate solution, adding a Traut' S reagent, and stirring;

s22, enabling the stirred reaction solution to pass through a Zeba desalting column, and removing impurities to obtain a thiolated lactoferrin solution;

s23, using NaH for the nano-micelle₂PO₄After dispersion, adding the thiolated lactoferrin solution (the sulfhydryl can react with-MAL of the nano micelle), and stirring at room temperature for reaction;

and S24, purifying the system after the room-temperature stirring reaction to obtain the multifunctional nano-drug carrier targeting the lactoferrin receptor.

It should be noted that room temperature herein means 16 to 25 ℃.

In this embodiment, the nano-micelle is a polyselenocarbamic acid amphiphilic block copolymer having a structure represented by formula (I) (-R in formula (I))₁is-OCH₃or-MAL), the preparation method of the nano-micelle and the copolymer is described in application number CN202011344949.5, and the patent name is polyseleno amino acid amphiphilic block copolymer, and the preparation method and the application thereof are not repeated herein.

In one embodiment, the molar ratio of lactoferrin to Traut's reagent is 1: 30-50, and the optimal molar ratio is 1: 40.

in one embodiment, the ratio of thiolated lactoferrin to nanomicelle is 1 g: 2-100mg, and the optimal dosage ratio is 1 g: 2 mg.

In one embodiment, the reaction time with stirring at room temperature is 6-12 h, and preferably 9 h.

The embodiment of the invention provides a medicine-carrying composition, which comprises a multifunctional nano-medicine carrier targeting a lactoferrin receptor and a medicine wrapped in the multifunctional nano-medicine 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 multifunctional nano-drug carrier to the drug is 1 mg: (0.1-10) mg.

The invention is further illustrated by the following specific examples.

Example 1:

preparation of nano spherical micelle

10mg of mPEG was taken₄₅-PV₂-PMet(Se)₂(P ═ 0.28) and 10mg NH₂-PEG₄₅-PV₂-PMet(Se)₂(P ═ 0.28) was completely dissolved in 2mL of DMSO, and the polymer was uniformly mixed by shaking to obtain a polymer solution; then dropwise adding 5mL of deionized water into the solution, and stirring for 2 hours to obtain a solution; directly adding the obtained solution into a dialysis bag with the molecular weight cutoff of 2000DA, dialyzing for 7 days at room temperature in 2L deionization, changing water once every day, filtering the solution in the dialysis bag (with the aperture of 450nm) by using a water phase filter head, and filtering out macromolecular precipitates and aggregated micelle particles to obtain the target product nano spherical micelle.

Example 2:

preparation of nanorod micelles

10mg of mPEG was taken₄₅-PV₂-PMet(Se)₄(P ═ 0.46) and 10mg NH₂-PEG₄₅-PV₂-PMet(Se)₄(P ═ 0.46) was completely dissolved in 2mL of methylene chloride, and the solution was rotary-evaporated in an eggplant-shaped bottle to prepare a uniform thin film, and the residual organic solvent was removed by vacuum overnight. Adding 5mL of double distilled water into an eggplant-shaped bottle, hydrating for 1h at 60 ℃, shaking and mixing uniformly, performing ultrasonic treatment in a water bath to obtain a nano rodlike micelle dispersion, centrifuging for 30min at 3000r/min, removing large aggregated particles, and freeze-drying clear liquid to obtain the target product nano rodlike micelle.

Example 3:

preparation of nanovesicles

16mL of a 0.25% aqueous solution of polyvinyl alcohol was weighed into a 50mL beaker and stirred. Weigh 10mg mPEG₄₅-PV₂-PMet(Se)₆(P ═ 0.64) and 10mg NH₂-PEG₄₅-PV₂-PMet(Se)₆(P ═ 0.64) in 4mL of CH₂Cl₂And (3) carrying out ultrasonic treatment on the solution twice by using an ultrasonic cell crusher, wherein the working time of one ultrasonic treatment is 4s, the intermittent time is 4s, the total working time is 30s, and the interval time of the two ultrasonic treatments is 30 s. Aspiration with syringe at the beginning of the first ultrasound0.1ml of a 0.1% aqueous solution of polyvinyl alcohol was poured into the solution, and emulsification was observed. After the ultrasonic treatment is finished, the solution is completely sucked out by using an injector, slowly and uniformly injected into the aqueous solution of polyvinyl alcohol with the mass fraction of 0.25%, and stirred for 2 hours at room temperature. And finally, repeatedly washing, freezing and drying to obtain the target product nano vesicle.

Example 4:

preparation of anion-cation composite nano spherical micelle

Take 5mg NH₂PEG45-PAsp2-pmet (se)2 (P0.30), 5mg mPEG45-PAsp2-pmet (se)2 (P0.30) and 10mg mPEG45-PLL2-pmet (se)2 (P0.30) were completely dissolved in 2mL DMSO, and the polymer was uniformly mixed by shaking to obtain a polymer solution; then dropwise adding 5mL of deionized water into the solution, and stirring for 2 hours to obtain a solution; directly adding the obtained solution into a dialysis bag with the molecular weight cutoff of 2000DA, dialyzing for 7 days at room temperature in 2L deionization, changing water once every day, filtering the solution in the dialysis bag (with the aperture of 450nm) by using a water phase filter head, and filtering out macromolecular precipitates and aggregated micelle particles to obtain the target product nano spherical micelle. The formed nano spherical micelle is observed by a transmission electron microscope, the micelle is in a regular spherical shape, the result is shown in figure 1, the measured particle size is about 100nm, and the result is shown in figure 2.

Example 5:

preparation of anion-cation composite nano spherical micelle

Completely dissolving 5mg of MAL-PEG45-PAsp2-pmet (se)2(P ═ 0.30), 5mg of mPEG45-PAsp2-pmet (se)2(P ═ 0.30) and 10mg of mPEG45-PLL2-pmet (se)2(P ═ 0.30) in 2mL of DMSO, and shaking to mix the polymer uniformly to obtain a polymer solution; then dropwise adding 5mL of deionized water into the solution, and stirring for 2 hours to obtain a solution; directly adding the obtained solution into a dialysis bag with the molecular weight cutoff of 2000DA, dialyzing for 7 days at room temperature in 2L deionization, changing water once every day, filtering the solution in the dialysis bag (with the aperture of 450nm) by using a water phase filter head, and filtering out macromolecular precipitates and aggregated micelle particles to obtain the target product nano spherical micelle. The formed nano spherical micelle is observed by a transmission electron microscope, and the micelle is in a regular spherical shape.

Example 6:

preparation of anion-cation composite nano rod-like micelle

5mg of mPEG is taken₄₅-PAsp₂-PMet(Se)₄(P＝0.47)、5mg NH₂-PEG₄₅-PAsp₂-PMet(Se)₄(P ═ 0.47) and 10mg mPEG₄₅-PLL₂-PMet(Se)₄(P ═ 0.49) was completely dissolved in 2mL of methylene chloride, and the solution was rotary-evaporated in an eggplant-shaped bottle to prepare a uniform thin film, and the residual organic solvent was removed by vacuum overnight. Adding 5mL of double distilled water into an eggplant-shaped bottle, hydrating for 1h at 60 ℃, shaking and mixing uniformly, performing ultrasonic treatment in a water bath to obtain a nano rodlike micelle dispersion, centrifuging for 30min at 3000r/min, removing large aggregated particles, and freeze-drying clear liquid to obtain the target product nano rodlike micelle. The formed nano rod-like micelle is observed by a transmission electron microscope, and the nanocapsule is in a baseball shape.

Example 7:

preparation of anion-cation composite nano vesicle

16mL of a 0.25% aqueous solution of polyvinyl alcohol was weighed into a 50mL beaker and stirred. Weigh 5mg mPEG₄₅-PAsp₂-PMet(Se)₆(P＝0.65)、5mg NH₂-PEG₄₅-PAsp₂-PMet(Se)₆(P ═ 0.65) and 10mg mPEG₄₅-PLL₂-PMet(Se)₆(P ═ 0.67) in 4mL of CH₂Cl₂And (3) carrying out ultrasonic treatment on the solution twice by using an ultrasonic cell crusher, wherein the working time of one ultrasonic treatment is 4s, the intermittent time is 4s, the total working time is 30s, and the interval time of the two ultrasonic treatments is 30 s. 0.1ml of a 0.1% aqueous solution of polyvinyl alcohol was injected into the solution by aspiration with a syringe at the start of the first sonication, and the emulsification was observed. After the ultrasonic treatment is finished, the solution is completely sucked out by using an injector, slowly and uniformly injected into the aqueous solution of polyvinyl alcohol with the mass fraction of 0.25%, and stirred for 2 hours at room temperature. And finally, repeatedly washing, freezing and drying to obtain the target product nano vesicle. The formed nano vesicles are observed by a transmission electron microscope, and the nano spheres are spherical.

Example 8:

preparation of anion-cation composite adriamycin-loaded nano spherical micelle

5mg of mPEG is taken₄₅-PAsp₂-PMet(Se)₂(P＝0.30)、5mg NH₂PEG₄₅-PAsp₂-PMet(Se)₂(P ═ 0.30) and 10mg mPEG₄₅-PLL₂-PMet(Se)₂(P ═ 0.30) and 2.7mg of doxorubicin were each completely dissolved in an appropriate amount of DMSO. Oscillating the former to uniformly mix the polymer to obtain a polymer solution, then slowly dropwise adding the adriamycin solution under rapid stirring, adding 2.7mg of adriamycin into the polymer solution, and slightly stirring to uniformly mix the polymer solution and the adriamycin solution; then dropwise adding 5mL of deionized water into the solution, and stirring for 2 hours to obtain a solution; directly adding the obtained solution into a dialysis bag with the molecular weight cutoff of 2000DA, dialyzing for 7 days at room temperature in 1L deionized water, changing water once every day, filtering the solution in the dialysis bag by using a water phase filter head (with the aperture of 450nm), and filtering out macromolecular precipitates and aggregated micelle particles to obtain the target product, namely the adriamycin-loaded nano spherical micelle.

Example 9:

preparation of anion-cation composite donepezil-loaded nano vesicle

16mL of a 0.25% polyvinyl alcohol aqueous solution was weighed into a 50mL beaker and stirred. Weigh 5mg mPEG₄₅-PAsp₂-PMet(Se)₆(P＝0.65)、5mg OH-PEG₄₅-PAsp₂-PMet(Se)₆(P ═ 0.65) and 10mg mPEG₄₅-PLL₂-PMet(Se)₆(P-0.67) and 2.7mg donepezil were completely dissolved in 4mL CH₂Cl₂And (3) carrying out ultrasonic treatment on the solution twice by using an ultrasonic cell crusher, wherein the working time of one ultrasonic treatment is 4s, the intermittent time is 4s, the total working time is 30s, and the interval time of the two ultrasonic treatments is 30 s. 0.1ml of a 0.1% aqueous solution of polyvinyl alcohol was injected into the solution by aspiration with a syringe at the start of the first sonication, and the emulsification was observed. After the ultrasonic treatment, the solution was completely sucked out by a syringe, slowly and uniformly injected 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 mPEG₄₅-PAsp₂-PMet(Se)₆-donepezil nanovesicles.

Example 10:

preparation of anion-cation composite CYP-loaded nano spherical micelle

5mg of mPEG is taken₄₅-PAsp₂-PMet(Se)₂(P＝0.30)、5mg NH₂-PEG₄₅-PAsp₂-PMet(Se)₂(P ═ 0.30) and 10mg mPEG₄₅-PLL₂-PMet(Se)₂(P ═ 0.30) and 1.0mg cyp were completely dissolved in an appropriate amount of DMSO, respectively. Oscillating the former to uniformly mix the polymer to obtain a polymer solution, slowly dropwise adding the CYP solution under rapid stirring, and slightly stirring to uniformly mix the CYP solution and the CYP solution; then dropwise adding 5mL of deionized water into the solution, and stirring for 2 hours to obtain a solution; directly adding the obtained solution into a dialysis bag with the molecular weight cutoff of 2000DA, dialyzing for 7 days at room temperature in 1L deionization, changing water once every day, filtering the solution in the dialysis bag by using an aqueous phase filter head (with the aperture of 450nm), and filtering out macromolecular precipitate and aggregated micelle particles to obtain the target product CYP-loaded nano spherical micelle.

Example 11:

preparation of anion-cation composite CYP-loaded nano spherical micelle

5mg of mPEG45-PASp2-PMet (Se)2 (P0.30), 5mg of MAL-PEG45-PASp2-PMet (Se)2 (P0.30) and 10mg of mPEG45-PLL2-PMet (Se)2 (P0.30) and 1.0mg of CYP were completely dissolved in a proper amount of DMSO, respectively. Oscillating the former to uniformly mix the polymer to obtain a polymer solution, slowly dropwise adding the CYP solution under rapid stirring, and slightly stirring to uniformly mix the CYP solution and the CYP solution; then dropwise adding 5mL of deionized water into the solution, and stirring for 2 hours to obtain a solution; directly adding the obtained solution into a dialysis bag with the molecular weight cutoff of 2000DA, dialyzing for 7 days at room temperature in 1L deionization, changing water once every day, filtering the solution in the dialysis bag by using an aqueous phase filter head (with the aperture of 450nm), and filtering out macromolecular precipitate and aggregated micelle particles to obtain the target product CYP-loaded nano spherical micelle.

Example 12:

in-vitro release test of anion-cation composite drug-loaded nano spherical micelle

4mg of the anion-cation composite adriamycin-loaded nano spherical micelle prepared in example 8 is accurately weighed and placed into a dialysis bag (molecular weight cut-off is 2000DA), and 5mL of phosphate buffer solution (lmol/L, pH7.4) is added into the dialysis bag for dispersion. The dialysis bag was placed in a triangular flask and 20mL of PBS (lmol/L, pH7.4) was added as a release medium. Placing the triangular flask in a constant temperature shaking table at 37 ℃ and 100r/min for release experiment, sampling 2mL every 1h, and supplementing fresh PBS with the same volume after sampling to ensure constant volume of the external liquid for releasing the drug. Measuring the ultraviolet absorbance of the sample at lambda-483 nm by an ultraviolet-visible spectrophotometer, and drawing an in-vitro drug release curve of the adriamycin nano micelle. And drawing an in-vitro drug release standard curve of the pure adriamycin product according to the operation. According to the in vitro drug release curve of the adriamycin nano micelle, the drug-loading rate of the anion-cation composite nano micelle is about 10.0 percent, and the encapsulation rate is 50.5 percent. Compared with an adriamycin in-vitro drug release standard curve, the drug-loaded nano-micelle provided by the invention is found to have no burst release phenomenon in the in-vitro release process, which shows that the drug adsorbed on the surface of the micelle is less, and most of the drug is wrapped in the micelle, so that the drug-loaded nano-micelle is proved to have a better controlled release effect, and is beneficial to reducing the toxic and side effects of the drug.

Example 13:

detection of selenium antioxidation function in nano-drug carrier

The breast cancer MDA-MB-231 cell line (231 cells) was cultured in DMEM medium containing 10% fetal bovine serum and then incubated in 5% CO₂And culturing in an incubator at 37 ℃, carrying out passage once for 1-2 d, and taking the cells in logarithmic growth phase for experiment. 231 cells in logarithmic growth phase were taken at 8X 10³The cells/well were inoculated in 96-well plates and cultured for 24h before drug treatment. The experiment was divided into four groups: the dosage of the control group, the nano micelle group, the adriamycin group and the nano micelle and adriamycin group is nano micelle (100 mu mol/L) and adriamycin (2.0 mu mol/L), 100 mu L of the nano micelle and adriamycin are added into each hole, 6 repeated holes are arranged, and the same amount of DMEM culture medium without medicine is added into the control holes. And collecting the cells of each group after 24 hours, adding cell lysate for cracking on ice, and centrifuging to collect cell holoprotein. The BCA method is used for determining the protein concentration, the WST-1 method is used for determining the total SOD activity of cells, the TBA method is used for determining the MDA content of the cells, and the operation steps are strictly according to the requirements of the kit. The treatment was performed by measuring 231 intracellular SOD and MDA levelsCompared with the control group, the activity of cell SOD in each group is reduced, and the MDA level is increased (P is less than 0.05); wherein the total SOD activity of the cells of the nano-micelle and adriamycin combination group is reduced, and the MDA level is increased most obviously. Therefore, the nano-drug carrier has the antioxidant function of selenium, and the combination of the nano-drug carrier and the adriamycin has an additive effect.

Example 14:

preparation of targeted anion-cation composite CYP-loaded nano spherical micelle

Ultrasonically dispersing appropriate amount of carboxyl modified lactoferrin into 10mL of 0.01mol/L KH with pH of 6₂PO₄To the buffer, 80mg EDCI and 120mg NHS were added, mixed well by magnetic stirring, and activated for 20min at room temperature in the dark. The reaction solution was transferred to an ultrafiltration tube (cut-off molecular weight: 10K) and subjected to ultrafiltration to remove unreacted EDCI and NHS. Then the pH value is 7.4 and the concentration is 0.01mol/L KH₂PO₄Dispersing the buffer solution, adding a proper amount of the anion-cation composite CYP loaded nano spherical micelle prepared in the example 10, and reacting overnight under magnetic stirring. Transferring the carrier into an ultrafiltration tube (with the molecular weight cutoff of 100K), removing the unbound lactoferrin by ultrafiltration to obtain a lactoferrin-modified targeting and multifunctional nano-drug carrier, observing the formed nano-spherical micelle by using a transmission electron microscope, wherein the micelle is in a regular spherical shape, and the result is shown in figure 3, and the measured particle size is about 200nm, and the result is shown in figure 4.

Example 15:

preparation of targeted anion-cation composite CYP-loaded nano spherical micelle

Dissolving a proper amount of lactoferrin in 0.15mol/L sodium borate solution (pH 8.5), adding 1mg/mL Traut's reagent, stirring at low speed for 1h at room temperature, passing through a Zeba (7KMW, 10mL) desalting column, removing small molecular impurities, and collecting a thiolated lactoferrin component. Taking a proper amount of 0.2mol/L NaH for the anion-cation composite CYP-loaded nano spherical micelle prepared in example 10₂PO₄After dispersion, adding a calculated amount of thiolated lactoferrin solution, stirring at room temperature for reaction for 9 hours, transferring the reaction solution into an ultrafiltration tube, and removing unbound lactoferrin through ultrafiltration to obtain the lactoferrin-modified targeted and multifunctional nano-drug carrier.

Example 16:

qualitative observation of lactoferrin-modified anion-cation composite CYP-loaded nano spherical micelle taken by cell

231 cells at 2X 10⁴Inoculating the strain/well density into a 48-well culture plate for culture, and after 24 hours, using a lactoferrin modified DMEM solution carrying CYP nano spherical micelles to incubate for 1 hour at the temperature of 37 ℃, wherein the concentrations of the nano spherical micelles are respectively 50, 100, 200,400 and 600 mu g/mL. Discarding the nano spherical micelle solution, washing the cells for 3 times by PBS, washing the nano spherical micelles adsorbed on the surfaces of the cells, adding 3.7 percent formaldehyde solution for fixing for 10min, then dyeing nuclei for 10min by 100ng/mL DAPI solution, then rinsing for 3 times by PBS, and observing the uptake condition of the cells to the lactoferrin modified CYP loaded nano spherical micelles under different time conditions under a fluorescence microscope, wherein the result is shown in figure 5. As can be seen from FIG. 5, the nucleus was stained blue by DAPI to locate the cell position, and the nano-micelles carrying the red fluorescent dye were distributed around the nucleus and filled with cytoplasm, demonstrating that the lactoferrin-modified CYP-carrying nano-spherical micelles smoothly entered the cell and were taken up by the cell.

In conclusion, the multifunctional nano-drug carrier targeting the lactoferrin receptor provided by the invention is formed by that the nano-micelle contains polyethylene glycol with external end functionalized (-COOH, -NH)₂or-MAL, etc.) is chemically connected with lactoferrin, and a layer of protein coat is covered on the surface of the nano micelle to obtain the lactoferrin modified multifunctional nano-drug carrier targeting the lactoferrin receptor. The medicine molecules are directionally delivered to the pathological change part, the medicine effect is improved, meanwhile, a slow-release and controlled-release medicine delivery system is prepared, and the entrapped medicine can be released in a slow-release and controlled-release mode from the delivery system according to the needs, so that the administration frequency is reduced, the treatment effect is improved, and the toxic and side effects of the medicine are reduced. Compared with the prior art, the nano-drug carrier provided by the invention has the advantages of common amino acid nano-drug carriers, has multiple biological functions of selenium, has the specificity of a targeted lactoferrin receptor, is a novel, targeted and multifunctional drug carrier, and is suitable for drug carrier research, development and clinical application of various drugs for various diseases related to lactoferrin receptor abnormalityBed application.

It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.