CN113616804B - Multifunctional nano-drug carrier targeting lactoferrin receptor, preparation method thereof and drug carrying composition - Google Patents

Multifunctional nano-drug carrier targeting lactoferrin receptor, preparation method thereof and drug carrying composition Download PDF

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CN113616804B
CN113616804B CN202111029111.1A CN202111029111A CN113616804B CN 113616804 B CN113616804 B CN 113616804B CN 202111029111 A CN202111029111 A CN 202111029111A CN 113616804 B CN113616804 B CN 113616804B
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lactoferrin
nano
drug
drug carrier
micelle
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CN113616804A (en
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吴海强
许晨舒
李晨阳
欧阳娜
王亦男
熊炜
李霞
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Shenzhen University
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
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    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals

Abstract

The invention relates to a multifunctional nano-drug carrier targeting lactoferrin receptor, a preparation method thereof and a drug carrying composition. The multifunctional nano-drug carrier is formed by self-assembling a polyseleno amino acid amphiphilic block copolymer with a structure shown in the following formula, and then modifying the 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 slow-release controlled-release targeted drug delivery system, and can directionally deliver the drug molecules to a lesion site, reduce the administration frequency, improve the treatment effect and the like. The drug carrier has the advantages of common amino acid drug carriers, simultaneously has multiple biological functions of selenium and specificity of targeting lactoferrin receptors, is a novel, targeted and multifunctional drug carrier, and is suitable for research, development and clinical application of various drugs of various diseases related to the abnormality of the lactoferrin receptors.

Description

Multifunctional nano-drug carrier targeting lactoferrin receptor, 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 lactoferrin receptors, 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 carriers have been used in 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 acids and the like, the drug carrier material has the defects of poor water solubility, difficult control of in vivo degradation rate and period, difficult realization of 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. The inventor researches that polyethylene glycol (PEG) has flexible hydrophilic long chain, no toxicity and no immunogenicity, and FDA has been approved for clinical use, and is one of the most development-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 protein on the surface of a material, the adhesion of cells and the like in a body can be reduced. Because PEG has the advantage of being difficult to be recognized by an immune system during in vivo circulation, polyamino acid can be protected from being damaged by the immune system, and the in vivo circulation time of materials is prolonged. 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.
Further researches show that the selenium serving as the essential trace element of the human body has important biological functions of resisting oxidation, regulating immunity, resisting 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.
Lactoferrin (Lf) is a multifunctional iron ion transport glycoprotein, belonging to the transferrin family, mainly expressed and secreted in glandular epithelial cells, mainly regulating the stabilization of internal environment ions, resisting microbial infection, regulating cell growth, inhibiting platelet aggregation, enhancing immunity, etc., while its biological functions are mainly mediated by Lactoferrin receptors. Recent researches have found that the brain neurons and microvascular epithelial cells of patients suffering from central nerve cell degenerative diseases such as Alzheimer's disease, parkinson's disease and the like have significantly increased expression level specificity of lactoferrin receptors, and the abnormality of the receptor directly induces and promotes the complex diseases. Therefore, the lactoferrin receptor can be used as an important target 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, can only be used as a medicine carrier, lacks targeting property and the like, the invention provides the multifunctional nano-medicine carrier targeting the lactoferrin receptor, has the advantages of the common amino acid nano-medicine carrier, simultaneously has multiple biological functions of selenium, specifically targets the lactoferrin receptor, and is a novel, targeting and multifunctional nano-medicine carrier.
Specifically, the technical scheme of the invention is as follows:
a multifunctional nano-drug carrier targeting a lactoferrin receptor, wherein the multifunctional nano-drug carrier comprises nano-micelles and ferrilactoprotein combined on the surfaces of the nano-micelles, the nano-micelles are formed by self-assembly of a polyseleno amino acid amphiphilic block copolymer with a structure shown in a formula (I), and the nano-micelles are combined with the ferrilactoprotein 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;
-R 1 selected from-OCH 3 、-COOH、-NH 2 or-MAL;
-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。
Optionally, the nanomicelles exist in the form of nanospheres, nanorods, or nanovesicles.
The invention discloses a preparation method of a multifunctional nano-drug carrier targeting lactoferrin receptors, which comprises the following steps:
providing a nano micelle;
dispersing lactoferrin into a buffer solution, adding EDCI and NHS, and sequentially stirring and performing light-shielding room temperature activation treatment;
removing unreacted EDCI and NHS in the reaction liquid after the activation treatment, and dispersing in a buffer solution to obtain an activated lactoferrin dispersion liquid;
adding the nano micelle into the activated lactoferrin dispersion liquid, and reacting overnight under stirring;
purifying the reacted system to obtain a multifunctional nano-drug carrier targeting the lactoferrin receptor;
the nano micelle is an amino modified nano micelle, and the lactoferrin is carboxyl modified lactoferrin;
or the nano micelle is a carboxyl modified nano micelle, and the lactoferrin is amino modified lactoferrin.
Alternatively, the lactoferrin is dispersed to 0.01mol/L KH at ph=6 2 PO 4 Buffer solution;
the dosage ratio of lactoferrin to EDCI and NHS is 1mg:2-6mg:4-8mg;
the light-shielding room temperature activation treatment time is 15min-12h.
The invention discloses a preparation method of a multifunctional nano-drug carrier targeting lactoferrin receptors, which comprises the following steps:
providing a nano micelle;
dissolving lactoferrin in a sodium borate solution, adding Traut's reagent, and stirring;
passing the stirred reaction solution through a Zeba desalting column to remove impurities and obtain a thiolated lactoferrin solution;
the nano micelle is treated with NaH 2 PO 4 After dispersion, adding the sulfhydryl lactoferrin solution, and stirring at room temperature for reaction;
and (3) purifying 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;
the dosage ratio of the sulfhydryl lactoferrin to the nano micelle is 1g:2-100mg;
the stirring reaction time at room temperature is 6-12 h.
A drug-loaded composition, which comprises the multifunctional nano-drug carrier targeting lactoferrin receptors and a drug wrapped in the multifunctional nano-drug carrier.
Optionally, the drug is selected from one or more of an anti-AD drug, an anti-tumor drug, a steroidal anti-inflammatory drug, a non-steroidal anti-inflammatory drug, a metabolic regulation drug, an antibiotic, a cardiovascular drug, an antiviral drug, an antifungal drug, an immunomodulator.
Optionally, the mass ratio of the multifunctional nano-drug carrier to the drug is 1mg: (0.1-10) mg.
The beneficial effects are that: the multifunctional nano-drug carrier targeting the lactoferrin receptor is prepared by self-assembling a poly seleno-amino acid amphiphilic block copolymer with a structure shown in a formula (I), and then carrying out lactoferrin modification through a PEG outer end functional group. The multifunctional nano-drug carrier can directionally send drug molecules to a lesion site, improve the drug effect, and simultaneously prepare a slow-release and controlled-release drug delivery system, and the entrapped drug can be released from the delivery system in a slow-release and controlled-release manner according to the requirement, so that the drug 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, simultaneously has multiple biological functions of selenium and specificity of targeting lactoferrin receptor, is a novel, targeted and multifunctional drug carrier, and is suitable for research, development and clinical application of various drugs of various diseases related to the abnormality of lactoferrin receptor.
Drawings
Fig. 1 is a TEM (transmission electron microscope) image of a polyseleno-amino acid nanosphere micelle according to an embodiment of the present invention.
Fig. 2 is a TEM (transmission electron microscope) image of a lactoferrin modified polyseleno-amino acid nanosphere micelle according to an example of the present invention.
FIG. 3 is a graph of the nanometer laser particle size of the polyseleno-amino acid nanosphere micelle according to an embodiment of the present invention.
Fig. 4 is a nano-laser particle size diagram of the lactoferrin modified polyseleno-amino acid nano-spherical micelle according to an embodiment of the present invention.
FIG. 5 is a graph showing laser confocal cell targeted uptake of the lactoferrin modified polyseleno-amino acid nanosphere micelle according to the example of the present invention.
Detailed Description
The invention provides a multifunctional nano-drug carrier targeting lactoferrin receptor, a preparation method and a drug carrying composition thereof, and the invention is further described in detail below for making the purposes, technical schemes and effects of the invention clearer and more definite. 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 multifunctional nano-drug carrier targeting a lactoferrin receptor, wherein the multifunctional nano-drug carrier comprises nano-micelles and ferrilactoprotein combined on the surfaces of the nano-micelles, the nano-micelles are formed by self-assembly of a polyseleno amino acid amphiphilic block copolymer with a structure shown in a formula (I), and the nano-micelles pass through R 1 Binding to said ferritin;
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-OCH 3 、-COOH、-NH 2 or-MAL (maleimide group), etc.;
-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 multifunctional nano-drug carrier targeting lactoferrin receptor consists of the-OCH at the outer end of polyethylene glycol in the nano-micelle 3 、-COOH、-NH 2 or-MAL functional groups and carboxyl or amino of lactoferrin are chemically connected in various modes such as esterification or amide condensation, 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 multifunctional nano-drug carrier is utilized to directionally send drug molecules to a lesion site, so that the drug effect is improved, and a slow-release and controlled-release drug delivery system is prepared, and the entrapped drug can be released from the delivery system in a slow-release and controlled-release manner according to the requirement, so that the drug 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 the advantages of common amino acid nano-drug carriers, simultaneously has multiple biological functions of selenium and specificity of targeting lactoferrin receptor, 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 the abnormality of the lactoferrin receptor.
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 the body can be prolonged; on the other hand, the targeting modification group is used for providing a targeting modification group, so that the treatment effect of the medicine is improved, and the toxicity and side effects of the medicine are reduced. The polyseleno amino acid 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 simultaneously has 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 nano-drug carrier with more stability and better drug slow-release and controlled-release functions.
The embodiment of the invention provides a preparation method of a multifunctional nano-drug carrier targeting a lactoferrin receptor, which comprises the following steps:
s10, providing nano micelle;
s11, dispersing lactoferrin into a buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS), and sequentially stirring and performing light-shielding room temperature activation treatment;
s12, removing unreacted EDCI and NHS in the reaction solution after the activation treatment, and dispersing the reaction solution in a buffer solution to obtain an activated lactoferrin dispersion liquid;
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, the functional group at the outer end of PEG is 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 PEG is carboxyl), and the lactoferrin is amino modified lactoferrin.
In one embodiment, the lactoferrin to EDCI, NHS dosage ratio is 1mg:2-6mg:4-8mg.
In one embodiment, the KH 2 PO 4 The buffer pH may suitably be floated up and down by 1-2 units. The KH 2 PO 4 The buffer solution is 0.01mol/L to 0.1mol/L.
In one embodiment, the lactoferrin is dispersed to a ph=0.01 mol/L KH of 6 2 PO 4 In a buffer.
In one embodiment, the lactoferrin is mixed with KH 2 PO 4 The buffer dosage ratio was 1mg:1-5mL.
In one embodiment, the light-shielding room temperature activation treatment is performed for 15min to 12h, preferably for 20min.
In the embodiment, the nano micelle is formed by a polyseleno amino acid amphiphilic block copolymer with a structure shown in a formula (I), wherein-R 1 is-COOH or-NH 2 ) The self-assembly is carried out, the preparation methods of the nano micelle and the copolymer are shown in CN202011344949.5, and the patent name is a polyseleno amino acid amphiphilic block copolymer, and the preparation methods and application of the copolymer are not described in detail.
The embodiment of the invention provides a preparation method of a multifunctional nano-drug carrier targeting a lactoferrin receptor, which comprises the following steps:
s20, providing nano micelle;
s21, dissolving lactoferrin in a sodium borate solution, adding Traut' S reagent, and stirring;
s22, passing the stirred reaction solution through a Zeba desalting column to remove impurities and obtain a thiolated lactoferrin solution;
s23, using NaH for the nano micelle 2 PO 4 After dispersion, adding the sulfhydryl lactoferrin solution (sulfhydryl can react with-MAL of nano micelle), and stirring at room temperature for reaction;
s24, purifying the system after the room-temperature stirring reaction to obtain the multifunctional nano-drug carrier targeting the lactoferrin receptor.
The room temperature is 16 to 25 ℃.
In the embodiment, the nano micelle is formed by a polyseleno amino acid amphiphilic block copolymer with a structure shown in a formula (I), wherein-R 1 is-OCH 3 or-MAL), the preparation method of the nano micelle and the copolymer is shown in CN202011344949.5 with the patent name in detailThe patent documents called polyseleno amino acid amphiphilic block copolymer, its preparation method and application are not described here in detail.
In one embodiment, the molar ratio of lactoferrin to Traut's reagent is 1:30-50, the optimal molar ratio is 1:40.
in one embodiment, the ratio of the amount of thiolated lactoferrin to the amount of nanomicelle is 1g:2-100mg, the optimal dosage ratio is 1g:2mg.
In one embodiment, the stirring reaction at room temperature is carried out for a period of time ranging from 6 to 12 hours, preferably for a period of time ranging from 9 hours.
The embodiment of the invention provides a medicine carrying composition, which comprises the multifunctional nano medicine carrier targeting lactoferrin receptors and medicines 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 1mg: (0.1-10) mg.
The invention is further illustrated by the following specific examples.
Example 1:
preparation of nanosphere micelles
Taking 10mg mPEG 45 -PV 2 -PMet(Se) 2 (p=0.28) and 10mg NH 2 -PEG 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 dialysis bag with molecular weight cut-off of 2000DA, dialyzing at room temperature for 7 days in 2L deionized water, changing water once daily, filtering (pore diameter 450 nm) solution in the dialysis bag with water phase filter head, and filtering out macromolecular precipitate and polymerThe collected micelle particles are used for obtaining the target product nano spherical micelle.
Example 2:
preparation of nanorod-like micelles
Taking 10mg mPEG 45 -PV 2 -PMet(Se) 4 (p=0.46) and 10mg NH 2 -PEG 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 3:
preparation of nanovesicles
16mL of a 0.25% aqueous solution of polyvinyl alcohol was measured in a 50mL beaker and stirred. 10mg mPEG was weighed 45 -PV 2 -PMet(Se) 6 (p=0.64) and 10mg NH 2 -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 4:
preparation of anion-cation composite nano spherical micelle
Taking 5mg NH 2 -PEG45-PAsp2-PMet (Se) 2 (p=0.30), 5mg mpeg45-PAsp2-PMet (Se) 2 (p=0.30) and 10mg mpeg45-PLL2-PMet (Se) 2 (p=0.30) were completely dissolved in 2mL DMSO, and the polymer was uniformly mixed by shaking to obtain a polymer solution; then drop by drop into it5mL of ionized water, and stirring 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 result is shown in figure 1, the particle size is about 100nm, and the result is shown in figure 2.
Example 5:
preparation of anion-cation composite nano spherical micelle
Taking 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) to be completely dissolved in 2mL of DMSO, and oscillating to uniformly mix the polymers 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. And observing the formed nano spherical micelle by using a transmission electron microscope, wherein the micelle is in a regular spherical shape.
Example 6:
preparation of anion-cation composite nanorod micelle
Taking 5mg mPEG 45 -PAsp 2 -PMet(Se) 4 (P＝0.47)、5mg NH 2 -PEG 45 -PAsp 2 -PMet(Se) 4 (p=0.47) and 10mg mPEG 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 at 60 ℃ for 1h, vibrating and mixing uniformly, performing ultrasonic treatment under water bath to obtain nanorod micelle dispersion liquid, centrifuging at 3000r/min for 30min, removing large aggregate particles, and freeze-drying the clear liquid to obtain the target product nanorod gumA bundle. The formed nano rod-shaped micelle is observed by a transmission electron microscope, and the nanocapsule is baseball-shaped.
Example 7:
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. Weigh 5mg mPEG 45 -PAsp 2 -PMet(Se) 6 (P＝0.65)、5mg NH 2 -PEG 45 -PAsp 2 -PMet(Se) 6 (p=0.65) and 10mg mPEG 45 -PLL 2 -PMet(Se) 6 (p=0.67) 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. And observing the formed nano vesicles by using a transmission electron microscope, wherein the nanospheres are spherical.
Example 8:
preparation of anion-cation composite doxorubicin-loaded nano spherical micelle
Taking 5mg mPEG 45 -PAsp 2 -PMet(Se) 2 (P＝0.30)、5mg NH 2 PEG 45 -PAsp 2 -PMet(Se) 2 (p=0.30) and 10mg mPEG 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 in 1L deionized water for 7 days, and changing water once a dayAnd then filtering (pore diameter 450 nm) the dialysis bag inner liquid by using a water phase filter head, and filtering out macromolecule sediment and aggregated micelle particles to obtain the target product of the doxorubicin-loaded nano spherical micelle.
Example 9:
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. Weigh 5mg mPEG 45 -PAsp 2 -PMet(Se) 6 (P＝0.65)、5mg OH-PEG 45 -PAsp 2 -PMet(Se) 6 (p=0.65) and 10mg mPEG 45 -PLL 2 -PMet(Se) 6 (p=0.67) and 2.7mg donepezil were 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 mPEG 45 -PAsp 2 -PMet(Se) 6 -donepezil nanovesicles.
Example 10:
preparation of anion-cation composite CYP-loaded nano spherical micelle
Taking 5mg mPEG 45 -PAsp 2 -PMet(Se) 2 (P＝0.30)、5mg NH 2 -PEG 45 -PAsp 2 -PMet(Se) 2 (p=0.30) and 10mg mPEG 45 -PLL 2 -PMet(Se) 2 (p=0.30) and 1.0mgCYP 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 the CYP solution under rapid stirring, and slightly stirring to uniformly mix the two solutions; dropwise adding 5mL of deionized water into the mixture, and stirring the mixture for 2 hours to obtain a solution; the resulting solution was directly added to a dialysis bag with a molecular weight cut-off of 2000DA, in 1L deionizationAnd (3) dialyzing at room temperature for 7 days, changing water once a day, filtering (with the aperture of 450 nm) the dialysis bag inner liquid by using a water phase filter head, and filtering out macromolecule precipitation and aggregated micelle particles to obtain the target product CYP-carried nano spherical micelle.
Example 11:
preparation of anion-cation composite CYP-loaded nano spherical micelle
5mg of mPEG45-PAsp2-PMet (Se) 2 (P=0.30), 5mg of MAL-PEG45-PAsp2-PMet (Se) 2 (P=0.30) and 10mg of mPEG45-PLL2-PMet (Se) 2 (P=0.30) were taken and completely dissolved in a proper amount of DMSO, respectively, with 1.0mg of CYP. Oscillating the former to uniformly mix the polymers to obtain a polymer solution, slowly dropwise adding the CYP solution under rapid stirring, and slightly stirring to uniformly mix the two solutions; 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, filtering the inner liquid of the dialysis bag by using a water phase filter head (with the aperture of 450 nm), and filtering out macromolecule sediment and aggregated micelle particles to obtain the target product CYP-loaded nanosphere micelle.
Example 12:
in vitro release test of anion-cation composite drug-loaded nano spherical micelle
4mg of the anion-cation composite doxorubicin-loaded nano spherical micelle prepared in example 8 is precisely weighed and placed into a dialysis bag (the molecular weight cut-off is 2000 DA), and 5mL of phosphate buffer solution (lmol/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 (lmol/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. The in vitro drug release curve of the doxorubicin nano-micelle shows that 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 in-vitro drug release standard curve of doxorubicin, the drug-loaded nano-micelle provided by the invention has no burst release phenomenon in the in-vitro release process, which proves 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 13:
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 14:
preparation of targeted anion-cation composite CYP-loaded nano spherical micelle
Dispersing proper amount of carboxyl modified lactoferrin into 10mL of 0.01mol/L KH with PH=6 by ultrasonic 2 PO 4 Adding 80mg EDCI,120mg NHS into the buffer solution, magnetically stirring and uniformly mixing, and activating for 20min at room temperature in dark place. Conversion of reaction solutionRemove the unreacted EDCI and NHS by ultrafiltration in an ultrafiltration tube (molecular weight cut-off 10K). Then 0.01mol/L KH with pH=7.4 2 PO 4 Dispersing the buffer solution, adding a proper amount of the anion-cation composite CYP-carrying nano spherical micelle prepared in the example 10, and reacting overnight under magnetic stirring. Transferring into ultrafiltration tube (molecular weight cut-off 100K), ultrafiltering to remove unbound lactoferrin to obtain lactoferrin modified targeting multifunctional nano-drug carrier, observing with transmission electron microscope to form nano spherical micelle, and measuring particle diameter about 200nm as shown in figure 3 and figure 4.
Example 15:
preparation of targeted anion-cation composite CYP-loaded nano spherical micelle
A proper amount of lactoferrin is taken and dissolved in 0.15mol/L sodium borate solution (PH=8.5), 1mg/mL Traut's reagent is added, the mixture is stirred at a low speed for 1h at room temperature, and the mixture is passed through a Zeba (7 KMW,10 mL) desalting column to remove small molecular impurities, and the thiolated lactoferrin component is collected. Proper amount of NaH (0.2 mol/L) for the anion-cation composite CYP-loaded nano spherical micelle prepared in example 10 is taken 2 PO 4 After dispersion, adding calculated amount of thiolated lactoferrin solution, stirring at room temperature for reaction for 9 hours, transferring the reaction solution into an ultrafiltration tube, and ultrafiltering to remove unbound lactoferrin, thereby obtaining the lactoferrin modified targeted multifunctional nano-drug carrier.
Example 16:
qualitative observation of cell ingestion lactoferrin modified yin-yang ion composite CYP-loaded nano spherical micelle
231 cells at 2X 10 4 The individual/hole density is inoculated in a 48-hole culture plate for culture, after 24 hours, a DMEM solution carrying CYP nano spherical micelle modified by lactoferrin is incubated 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 with PBS for 3 times, 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 with PBS for 3 times, and observing the cell-to-lactoferrin modified CY under different time conditions under a fluorescence microscopeThe uptake of P nanosphere micelles is shown in figure 5. From fig. 5, it can be seen that the nuclei are stained blue with DAPI to locate the cell positions, and red fluorescent dye-loaded nano-micelles are distributed around the nuclei and filled in the cytoplasm, which proves that lactoferrin-modified CYP-loaded nano-spherical micelles smoothly enter the cells and are taken up by the cells.
In conclusion, the multifunctional nano-drug carrier targeting lactoferrin receptor provided by the invention comprises polyethylene glycol-containing external end functional groups (-COOH, -NH) in the nano-micelle 2 or-MAL and the like) and lactoferrin are chemically connected, and a layer of protein 'coat' is covered on the surface of the nano micelle to obtain the multifunctional nano-drug carrier of the lactoferrin modified targeting lactoferrin receptor. The medicine molecules are directionally delivered to the lesion site, the medicine effect is improved, and meanwhile, a slow-release and controlled-release medicine delivery system is prepared, and the entrapped medicine can be released from the delivery system in a slow-release and controlled-release manner according to the requirement, 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, simultaneously has multiple biological functions of selenium and specificity of targeting lactoferrin receptor, is a novel, targeted and multifunctional drug carrier, and is suitable for research, development and clinical application of various drugs of various diseases related to the abnormality of the lactoferrin receptor.
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 (8)

1. The multifunctional nano-drug carrier 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-assembly of a poly-selenoamino acid amphiphilic block copolymer with a structure shown in a formula (I), and the nano-micelles are prepared by the following steps ofThe R is 1 Binding to the lactoferrin;
formula (I);
wherein n=45, x=2, y=2;
-R 1 selected from the group consisting of-NH 2 ；
-R 2 Selected from the group consisting of、/>；
-R 3 Selected from-CH 2 CH 2 SeCH 3 。
2. The multifunctional nano-drug carrier targeting lactoferrin receptors of claim 1, wherein the nano-micelles exist in the form of nanospheres, nanorods or nanovesicles.
3. A method for preparing a multifunctional nano-drug carrier targeting lactoferrin receptors according to any one of claims 1-2, comprising the steps of:
providing a nano micelle;
dispersing lactoferrin into a buffer solution, adding EDCI and NHS, and sequentially stirring and performing light-shielding room temperature activation treatment;
removing unreacted EDCI and NHS in the reaction liquid after the activation treatment, and dispersing in a buffer solution to obtain an activated lactoferrin dispersion liquid;
adding the nano micelle into the activated lactoferrin dispersion liquid, and reacting under stirring;
purifying the reacted system to obtain a multifunctional nano-drug carrier targeting the lactoferrin receptor;
the nano micelle is an amino modified nano micelle, and the lactoferrin is carboxyl modified lactoferrin.
4. A method for preparing a multifunctional nano-drug carrier targeting lactoferrin receptors as claimed in claim 3, characterized in that, lactoferrin is dispersed to 0.01mol/L KH with ph=6 2 PO 4 Buffer solution;
the dosage ratio of lactoferrin to EDCI and NHS is 1mg:2-6mg:4-8mg;
the light-shielding room temperature activation treatment time is 15min-12h.
5. A method for preparing a multifunctional nano-drug carrier targeting lactoferrin receptors according to any one of claims 1-2, comprising the steps of:
providing a nano micelle;
dissolving lactoferrin in a sodium borate solution, adding Traut's reagent, and stirring;
passing the stirred reaction solution through a Zeba desalting column to remove impurities and obtain a thiolated lactoferrin solution;
the nano micelle is treated with NaH 2 PO 4 After dispersion, adding the sulfhydryl lactoferrin solution, and stirring at room temperature for reaction;
and (3) purifying the system after the room-temperature stirring reaction to obtain the multifunctional nano-drug carrier targeting the lactoferrin receptor.
6. The method for preparing a multifunctional nano-drug carrier targeting a lactoferrin receptor as set forth in claim 5, wherein the molar ratio of lactoferrin to Traut's reagent is 1:30-50;
the dosage ratio of the sulfhydryl lactoferrin to the nano micelle is 1g:2-100mg;
the stirring reaction time at room temperature is 6-12 h.
7. A drug-loaded composition comprising the multifunctional nano-drug carrier targeting lactoferrin receptors according to any one of claims 1-2 and a drug encapsulated in the multifunctional nano-drug carrier, wherein the drug is doxorubicin.
8. The drug-loaded composition of claim 7, wherein the mass ratio of the multifunctional nano-drug carrier to the drug is 1mg: (0.1-10) mg.
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