CN112472815A

CN112472815A - Selenium peptide and preparation method and application thereof

Info

Publication number: CN112472815A
Application number: CN202011491968.0A
Authority: CN
Inventors: 王浩; 易宇; 魏子钰
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2020-12-16
Filing date: 2020-12-16
Publication date: 2021-03-12
Anticipated expiration: 2040-12-16
Also published as: CN112472815B

Abstract

The invention provides a selenium peptide and a preparation method and application thereof, wherein the selenium peptide comprises an oxidation response unit, an enzyme response fragmentation unit and a tumor targeting unit which are sequentially connected from an N end to a C end; wherein the oxidation response unit comprises oxidation response selenium amino acid which is fatty chain-L-selenocysteine. The invention also provides a self-assembly selenium peptide nano-medicament, which comprises the selenium peptide and an anti-tumor medicament. The oxidative response selenium amino acid has the capacity of sensitively responding to an oxidative environment, the selenium peptide has a tumor targeting function, the particle size is reduced through enzyme response fracture so as to increase the permeability and the capacity of accelerating the drug release in tumor cells, and the self-assembly selenium peptide nano-drug formed by the selenium peptide and an anti-tumor drug has an excellent tumor treatment effect, low toxic and side effects and a wide application prospect.

Description

Selenium peptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biotechnology and medicament, and particularly relates to selenium peptide and a preparation method and application thereof.

Background

Cancer is a disease that seriously harms people's life and health. Under the influence of living environment and unhealthy life style, in recent years, the number of people suffering from cancer and dying from cancer is rising, and how to effectively treat cancer is a great scientific problem for human beings and is one of the key points of modern medical research.

Chemotherapy plays an important role in cancer treatment, and can kill tumor cells in vivo to the maximum extent and inhibit the recurrence and metastasis of tumors as a systemic treatment means. Traditional small molecule anticancer agents such as platinum drugs, paclitaxel and doxorubicin, etc. have great harm to normal tissues and organs of patients while killing tumor cells. In recent years, the development of nano-medicine has brought new research directions for tumor treatment. Research shows that the antitumor drug with larger toxic and side effects is loaded into the nano particles to form the nano drug, so that the toxic and side effects of the drug can be reduced while the treatment effect is improved. However, the complex physiological barriers in the human body affect the therapeutic effect of the nano-drug, such as the rapid clearance of the nano-drug in blood, the poor enrichment efficiency in tumor parts, the difficult penetration in tumor tissues, and the low drug release efficiency in tumor cells, which hinder the further clinical development of the conventional nano-drug.

At present, the problems of faster metabolism, poor enrichment efficiency, difficult permeation and lower release efficiency generally exist in the nano-drugs for tumors. How to provide a nano-drug which can perform multi-level response aiming at the physiological barrier of a human body and has the advantages of good biocompatibility, small toxic and side effect and higher response sensitivity to a tumor microenvironment becomes a problem to be solved urgently.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides the selenium peptide and the preparation method and the application thereof, the oxidative response selenium amino acid can sensitively respond to tumors, and the hydrophobicity is increased by adding the fatty chain to help the drug to be deaggregated; the selenium peptide has a tumor targeting function, a matrix metalloproteinase II (MMP-2 enzyme) response fragmentation function and an active oxygen response function, and the self-assembled selenium peptide nano-drug formed by the selenium peptide and a tumor treatment drug has good permeability, higher drug release rate and stronger tumor inhibition capability, and has extremely wide application value.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a selenopeptide comprising an oxidation response unit, an enzyme response cleavage unit and a tumor targeting unit, which are sequentially connected from N-terminus to C-terminus;

wherein the oxidation response unit comprises oxidation response selenium amino acid which is fatty chain-L-selenocysteine.

In the invention, the tumor targeting unit can target tumor cells and integrin (alpha) on the surface of neovasculature of tumor tissues by intravenous injection_vβ₃) The enrichment of the medicine at the tumor part is obviously improved; after the medicine reaches the tumor tissue, the MMP-2 enzyme excessively expressed in the tumor tissue can cut off an enzyme response fragmentation unit in the selenium peptide, so that the hydration particle size of the medicine is reduced, and the medicine is beneficial to permeating into the deep part of the tumor tissue; after the medicine enters the tumor cells through endocytosis, the high-expression active oxygen in the tumor cells can oxidize the oxidation response selenium amino acid in the oxidation response unit, so that the hydrophobicity of the oxidation response selenium amino acid is changed, the internal medicine is disaggregated, the anti-tumor medicine is released, and the purpose of tumor treatment is achieved. Through the mutual matching of the tumor targeting unit, the enzyme response fragmentation unit and the oxidation response unit, the selenium peptide can help the drug to generate better treatment effect on the tumor, and the level of the drug taken in normal cells is greatly reduced compared with that of the tumor cells, so that the drug can be effectively reduced to normal tissues and organsThe toxic and side effects of (1).

Preferably, the fatty chain in the fatty chain-L-selenocysteine comprises a saturated hydrocarbyl chain and/or an unsaturated hydrocarbyl chain, preferably a saturated hydrocarbyl chain.

Preferably, the aliphatic chain includes any one of a linear aliphatic chain, a branched aliphatic chain or a cyclic aliphatic chain or a combination of at least two of them, and for example, may be a linear aliphatic chain or a combination of a branched aliphatic chain and a cyclic aliphatic chain, and is preferably a linear aliphatic chain.

Preferably, the number of carbon atoms in the aliphatic chain is an integer of 1 to 18, and may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

Preferably, the fatty chain is C₆、C₁₂Or C₁₈Any one of the saturated linear aliphatic chains.

Preferably, the aliphatic chain-L-selenocysteine comprises L-Sec (C)₆H₁₃)-OH、L-Sec(C₁₂H₂₅) -OH or L-Sec (C)₁₈H₃₇) Any one or a combination of at least two of-OH, for example, L-Sec (C)₆H₁₃) -OH or L-Sec (C)₁₂H₂₅) -OH and L-Sec (C)₁₈H₃₇) -a combination of-OH.

Preferably, the aliphatic chain-L-selenocysteine comprises L-Sec (C)₁₂H₂₅) -OH, said L-Sec (C)₁₂H₂₅) -OH has the formula shown in formula I:

preferably, the oxidation-responsive element comprises at least 1 oxidation-responsive selenoamino acid, the number of oxidation-responsive selenoamino acids can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and the oxidation-responsive selenoamino acids are linked by a linking amino acid.

Preferably, the oxidation responsive unit comprises 2 oxidation responsive seleno-amino acids.

Preferably, the number of said linking amino acids is 1.

Preferably, the linking amino acid is K (lysine).

Preferably, the enzyme responsive cleavage unit comprises a matrix metalloproteinase II cleaved polypeptide sequence.

Preferably, the polypeptide sequence of the enzyme responsive cleavage unit is PLGVR (proline-leucine-glycine-valine-arginine).

Preferably, the tumor targeting unit comprises a polypeptide sequence targeting tumor integrin.

Preferably, the polypeptide sequence of the tumor targeting unit is RGD (arginine-glycine-aspartic acid).

Preferably, the oxidation-responsive unit, the enzyme-responsive cleavage unit and the tumor targeting unit are separated by a spacer amino acid X₁And X₂Are connected.

Preferably, the selenium peptide is:

[L-Sec(alkyl)-OH]-K[L-Sec(alkyl)-OH]-X₁-PLGVR-X₂-RGD, wherein alkyl represents an aliphatic chain, said selenium peptide having the specific structural formula shown in formula II:

preferably, the spacer amino acids comprise natural amino acids and/or unnatural amino acids, preferably G.

Preferably, the selenium peptide is:

[L-Sec(C₁₂H₂₅)-OH]-K[L-Sec(C₁₂H₂₅)-OH]-GPLGVRGRGD, having a specific formula as shown in formula III:

in a second aspect, the present invention provides a method for preparing the selenium peptide of the first aspect, the method comprising:

preparing oxidation response selenium amino acid protected by Boc group, sequentially coupling the amino acid and the oxidation response selenium amino acid to resin according to the direction from C end to N end by solid phase synthesis, and eluting to obtain the selenium peptide.

The preparation method of the selenium peptide is a conventional technical means in the field, is mature in technology, simple to operate and high in yield, is easy to master by technical personnel in related fields, can realize production and processing in related laboratories or factories, has the potential of large-scale batch production, and creates conditions for application and popularization of related products.

Preferably, the method of preparing the Boc group-protected oxidation-responsive selenoamino acid comprises:

reacting selenium powder with sodium borohydride to obtain disodium diselenide, mixing the disodium diselenide with 1-halogenated alkane for reaction to prepare alkyl diselenide, and adding a reducing agent for reduction to obtain alkyl selenol;

and mixing the alkyl selenol and N-tert-butyloxycarbonyl-O-p-toluenesulfonyl serine methyl ester to obtain the aliphatic chain-L-selenocysteine methyl ester protected by the Boc group, and removing the methyl to obtain the aliphatic chain-L-selenocysteine protected by the Boc group.

Preferably, the 1-haloalkane comprises a 1-bromoalkane.

Preferably, the number of carbon atoms of the 1-bromoalkane is an integer of 1 to 18, and may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, preferably 6, 12 or 18.

Preferably, the mixing reaction is carried out under oil bath conditions.

Preferably, the temperature of the oil bath is 45 to 55 ℃, for example, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃ or 55 ℃, preferably 50 ℃.

Preferably, the mixing reaction time is 10-14 h, for example, 10h, 10.5h, 11h, 11.5h, 12h, 12.5h, 13h, 13.5h or 14h, preferably 12 h.

Preferably, the reducing agent comprises sodium borohydride.

Preferably, the step of cooling is further included before the reducing agent is added.

Preferably, the reduction time is 25-35 min, for example, 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min or 35min, preferably 30 min.

Preferably, the mixing time is 5-7 h, for example, 5h, 5.5h, 6h, 6.5h or 7h, preferably 6 h.

Preferably, the methyl group is removed, and the method further comprises the steps of ethyl acetate extraction and silica gel column chromatography purification.

Preferably, the method for removing the methyl group is to put the reaction product into a solution containing lithium hydroxide and stir the reaction product.

Preferably, the stirring time is 55-65 min, such as 55min, 56min, 57min, 58min, 59min, 60min, 61min, 62min, 63min, 64min or 65 min.

Preferably, the methyl group removal step further comprises an ethyl acetate extraction step.

Preferably, the synthesis step of the Boc group-protected aliphatic chain-L-selenocysteine can be represented by the following reaction formula:

preferably, the step of solid phase synthesis comprises:

swelling the resin, removing Fmoc group of resin amino acid, and sequentially coupling the amino acid and the oxidation response seleno-amino acid onto the resin.

Preferably, the step of swelling the resin comprises soaking the resin in anhydrous DMF and placing on a shaker.

Preferably, the amino acid comprises an amino acid protected with an Fmoc group.

Preferably, the step of removing the Fmoc group of the amino acid is further included before coupling the amino acid to the resin.

Preferably, the step of removing the Boc group of the oxidation-responsive selenoamino acid is further included before coupling the oxidation-responsive selenoamino acid to the resin.

As a preferred technical scheme, the preparation method of the selenium peptide specifically comprises the following steps:

(1) reacting selenium powder with sodium borohydride to obtain disodium diselenide, mixing the disodium diselenide with 1-bromoalkane with 1-18 carbon atoms at 45-55 ℃ in an oil bath for reaction for 10-14 hours to obtain alkyl diselenide, cooling, and adding sodium borohydride for reduction for 25-35 min to obtain alkyl selenol;

(2) mixing the alkyl selenol and N-tert-butyloxycarbonyl-O-p-toluenesulfonyl serine methyl ester for 5-7 h, and then carrying out ethyl acetate extraction and silica gel column chromatography purification to obtain aliphatic chain-L-selenocysteine methyl ester protected by Boc group;

(3) stirring the obtained aliphatic chain-L-selenocysteine methyl ester protected by the Boc group in a solution containing lithium hydroxide for 55-65 min, removing methyl, and extracting with ethyl acetate to obtain aliphatic chain-L-selenocysteine protected by the Boc group;

(4) soaking the resin in anhydrous DMF, placing the resin on a shaking table, swelling the resin, and removing Fmoc groups of amino acids of the resin;

(5) removing the Fmoc group of the amino acid protected by the Fmoc group, and coupling the obtained amino acid without the Fmoc group to resin;

(6) and removing the Boc group of the oxidation response selenium amino acid protected by the Boc group, coupling the obtained oxidation response selenium amino acid without the Boc group onto resin, and eluting to obtain the selenium peptide.

In a third aspect, the present invention provides a self-assembled selenium peptide nano-drug, which comprises the selenium peptide and an anti-tumor drug as described in the first aspect.

In the invention, the selenium peptide can help the nano-drug to enrich at the tumor cell part through the mutual matching of the tumor targeting unit, the enzyme response fragmentation unit and the oxidation response unit; after reaching the tumor tissue, the oxidation response unit is cut off by MMP-2 enzyme excessively expressed by the tumor, so that the hydration particle size of the nano-drug is reduced, and the nano-drug can be favorably permeated into the deep part of the tumor tissue; the high-expression active oxygen in the tumor cells oxidizes the oxidation response units, so that the hydrophobicity of the selenium peptide is changed, the nano-drugs are depolymerized, the anti-tumor drugs are released, the treatment effect on the tumors is better, the toxic and side effects on normal tissues and organs are smaller, and the application value is higher.

Preferably, the anti-tumor drug is a hydrophobic drug, preferably doxorubicin.

Preferably, the hydrated particle size of the self-assembled selenium peptide nano-drug is 200-250 nm, such as 200nm, 205nm, 210nm, 215nm, 220nm, 225nm, 230nm, 235nm, 240nm, 245nm or 250 nm.

Preferably, the hydrated particle size of the self-assembled selenium peptide nano-drug is reduced to 50-150 nm under the action of matrix metalloproteinase II, and the hydrated particle size can be 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm or 150 nm.

Preferably, the self-assembled selenium peptide nano-drug is disassembled under oxidizing conditions.

Preferably, the working principle of the self-assembly selenium peptide nano-drug for treating tumor is shown in figure 1.

Through intravenous injection, the self-assembly selenium peptide nano-drug targets tumor cells and integrin on the surface of neovasculature of tumor tissues through a tumor targeting unit in the selenium peptide, so as to realize the enrichment of the drug in tissues of tumor sites; MMP-2 enzyme excessively expressed in the tumor tissue cuts an enzyme response fracture unit in the selenium peptide, the particle size of the self-assembled selenium peptide nano-drug is reduced, the self-assembled selenium peptide nano-drug permeates into the depth of the tumor tissue, and then enters into tumor cells through endocytosis; the expression level of active oxygen in tumor cells is remarkably improved, and the fatty chain-L-selenocysteine in the selenium oxide peptide changes the hydrophilicity, so that the medicament is depolymerized, and the anti-tumor medicament is released to kill the tumor cells. The MMP-2 enzyme expression amount on the surface of a normal cell is less, the content of active oxygen free radicals in the cell is less, and the self-assembled selenium peptide nano-drug also lacks the capability of targeted enrichment, so that the uptake capability of the drug and the release level of the drug in the cell are obviously reduced, and the high-efficiency and low-toxicity treatment effect is achieved.

In a fourth aspect, the present invention provides a method for preparing the self-assembled selenium peptide nano-drug according to the third aspect, wherein the method for preparing the self-assembled selenium peptide nano-drug comprises mixing the selenium peptide with an anti-tumor drug.

In the invention, the oxidation response selenium amino acid in the selenium peptide has better hydrophobicity, is beneficial to the self-assembly of the medicament, is convenient for the production and use of the medicament, and has smaller particle size and better permeability; the self-assembly process does not generate covalent bonds and reverse reaction, the yield is extremely high, and the production cost is also reduced; the self-assembled selenium peptide nano-drug prepared by the method has good stability and can be stably stored for at least 12 hours at room temperature; the preparation method is simple and efficient, energy-saving and environment-friendly, has low requirements on the technical level of operators, and promotes the popularization and use of the nano-drug.

Preferably, the selenium peptide is dissolved in a sodium phosphate buffer.

Preferably, the pH of the sodium phosphate buffer is 7.0 to 7.8, for example, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8, preferably 7.4.

Preferably, the antineoplastic drug is dissolved in dichloromethane.

Preferably, the mixing step further comprises a step of removing dichloromethane by distillation under reduced pressure.

Preferably, the method further comprises a step of centrifuging after removing the dichloromethane.

Preferably, the rotation speed of the centrifugation is 5000-6500 rpm, for example, 5000rpm, 5100rpm, 5200rpm, 5300rpm, 5400rpm, 5500rpm, 5600rpm, 5700rpm, 5800rpm, 5900rpm, 6000rpm, 6100rpm, 6200rpm, 6300rpm, 6400rpm or 6500rpm, preferably 6000 rpm.

As a preferred technical scheme, the preparation method of the self-assembled selenium peptide nano-drug specifically comprises the following steps:

dissolving selenium peptide in a sodium phosphate buffer solution with the pH value of 7.0-7.8, dissolving an anti-tumor drug in dichloromethane, violently stirring and mixing the solution, removing the dichloromethane through reduced pressure distillation, and centrifuging at 5000-6500 rpm to obtain a supernatant, namely the self-assembly selenium peptide nano-drug.

In a fifth aspect, the present invention provides the use of the selenium peptide of the first aspect and/or the self-assembled selenium peptide nano-drug of the third aspect in the preparation of a medicament for treating a tumor.

In the invention, the oxidation response selenium amino acid has better hydrophobicity and the ability of sensitively responding to the oxidation environment; the selenium peptide can help the medicine to be enriched at the tumor part and increase the permeability of the medicine to the tumor; the self-assembly selenium peptide nano-drug can effectively overcome multilayer physiological barriers of a human body through multi-level sensitive response, has better treatment effect and better compatibility with the human body, and has smaller toxic and side effects on normal tissues and organs and higher application value.

Compared with the prior art, the invention has the following beneficial effects:

(1) the aliphatic chain-L-selenocysteine has sensitive oxidation response capability, and can help the release of the drug by changing hydrophobicity in the environment containing active oxygen free radicals; the fatty chain-L-selenocysteine has good hydrophobicity, is beneficial to self-assembly of the medicine, and has smaller grain diameter and better permeability; the preparation method is simple and feasible, energy-saving and environment-friendly, and has the potential of large-scale production;

(2) the selenium peptide can be effectively enriched at a tumor part through mutual matching of the tumor targeting unit, the enzyme response fracture unit and the oxidation response unit, the particle size of the medicament is gradually reduced through sequential response of the enzyme response fracture unit and the oxidation response unit, the final release of the antitumor medicament is realized only when the selenium peptide enters the interior of a tumor tissue, the treatment effect of the medicament is improved, meanwhile, the toxic and side effects on normal tissues and organs are also reduced, and the application value is extremely high;

(3) the self-assembly selenium peptide nano-drug has a small particle size of only 240 +/-13 nm; the stability is good, and the stability can be maintained at least within 12h at room temperature; has good tumor targeting ability, can be cut by MMP-2 enzyme to reduce the particle size to 100-150 nm, improves the permeability, and further realizes depolymerization under the action of active oxygen free radicalsThe medicine is promoted to be released, the enrichment and the penetration into the deep part of the tumor tissue in the tumor tissue are realized, the controllable release of the medicine is realized, the obstruction of the physiological barrier of a human body to the tumor treatment is effectively overcome, and the high-efficiency and low-toxicity treatment of the tumor is realized; has good killing ability to tumor cells, and IC to MDA-MB-231 cells₅₀The value is 1.04 mu mol/L, the growth of tumor cells in a mouse body can be inhibited, and the effect is obvious; the retention time of the medicine in blood is prolonged, the retention amount of the nano particles after 1 hour of injection can reach 54.8 +/-7.8%, the treatment effect is better, and the bioavailability is higher; the nano-drug is self-assembled through hydrophilic and hydrophobic effects, the preparation method is simple and efficient, the permeability and the compatibility with human bodies of the nano-drug are excellent, and the application prospect is very wide.

Drawings

FIG. 1 is the working principle of self-assembled selenium peptide nano-drug for treating tumor;

FIG. 2 shows detection C in example 3 of the present invention₁₂-picture of the results of MMP-2 enzyme response ability of selenium peptide;

FIG. 3 shows detection C in example 5 of the present invention₁₂-pictures of the results of the selenium peptide critical micelle concentration experiments;

FIG. 4 is a graph showing the results of measuring SeP/DOX hydrated particle size in example 6 of the present invention;

FIG. 5 is a transmission electron microscope photograph (scale 100nm) of SeP/DOX in example 6 of the present invention;

FIG. 6 is a photograph showing the results of detecting SeP/DOX targeting to tumors in example 9 of the present invention;

FIG. 7 is a graph (scale 100 μm) showing the results of detecting SeP/DOX permeability to tumor cell clusters in example 10 of the present invention;

FIG. 8 is a graph showing the results of detecting the killing effect of SeP/DOX on tumor cells in example 11 of the present invention;

FIG. 9 is a graph showing the results of detecting SeP/DOX for its anti-tumor effect on a mouse tumor-bearing model of orthotopic triple negative breast cancer in example 12 of the present invention.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Materials:

selenium powder, lauric acid, hydrogen peroxide, triethylamine, sodium borohydride, potassium bisulfate, anhydrous sodium sulfate, and piperidine were purchased from alatin (shanghai) ltd;

1-bromododecane and N-t-butoxycarbonyl-O-p-toluenesulfonylserine methyl ester were purchased from Shanghai Bigdi pharmaceutical science and technology Co., Ltd;

dichloromethane, petroleum ether, methanol and ethyl acetate were purchased from Beijing, modern Oriental Fine chemical Co., Ltd;

chromatographic grade acetonitrile and N, N-Dimethylformamide (DMF) were purchased from tianjin concider science and technology ltd;

deuterated chloroform (CDCl)₃) Purchased from Beijing Yinaoka technologies, Inc.;

doxorubicin hcl was purchased from shanghai meirui technologies ltd;

rink amide AM resin, Fmoc-amino acid, 1-Hydroxybenzotriazole (HOBT), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU) commercially available from Gill Biochemical (Shanghai) Inc.;

triisopropylsilane (TIS) and trifluoroacetic acid (TFA) were purchased from Michelin (Shanghai) Inc.;

DMEM cell culture medium, Fetal Bovine Serum (FBS), trypsin were purchased from semer feishel technologies (china) ltd;

CCK-8 cell counting reagents were purchased from Biyuntian biotechnological institute;

MMP-2 enzyme was purchased from near-shore protein science, Inc.;

the MDA-MB-231 cells are from the cell culture center of the institute of basic medicine of Chinese academy of medical sciences (Beijing, China);

female BALB/c mice and nude mice were purchased from Experimental animals technologies, Inc., Viton, Beijing.

Example 1

This example provides a Boc group-protected C₁₂Aliphatic chain-L Selenocysteine Boc-L-Sec (C)₁₂H₂₅) -OH, said Boc-L-Sec (C)₁₂H₂₅) The preparation method of the-OH comprises the following steps:

and (3) putting 0.64g of selenium powder and 0.60g of sodium borohydride into a 100mL round-bottom flask, adding 2mL of water, and violently reacting the selenium powder with the sodium borohydride to generate disodium diselenide. The round bottom flask was sealed with a rubber stopper and placed in a 50 ℃ oil bath with stirring. After 20min, 0.95mL of 1-bromododecane was mixed with 10mL of tetrahydrofuran, injected into the reaction flask via syringe, and the reaction flask was allowed to continue to react for 12h in a 50 ℃ oil bath. The reaction flask was cooled in an ice-water bath, and 2mL of an aqueous solution containing 0.3g of sodium borohydride was added to the round-bottom flask with a syringe and stirred at room temperature for 30 min.

2.74 t-Butoxycarbonyl-O-p-toluenesulfonylserine methyl ester 2.74g N was dissolved in 10mL of tetrahydrofuran, and added to the flask together with 1.1mL of triethylamine, and stirred at room temperature for 6 hours. After the reaction was completed, tetrahydrofuran in the reaction solution was removed by distillation under the reduced pressure, and the product was extracted with ethyl acetate. Drying the obtained ethyl acetate extract by using anhydrous sodium sulfate, concentrating under reduced pressure, and purifying by using silica gel column chromatography, wherein the eluent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 4: 1. Purifying to obtain light yellow oily liquid, namely Boc-L-Sec (C)₁₂H₂₅) OMe, product mass 2.44g, yield 67%.

Boc-L-Sec(C₁₂H₂₅) Nuclear magnetic and mass spectral characterization of OMe as follows:

1H NMR(400MHz,CDCl3):δ5.36(d,J＝8.1Hz,1H),4.70-4.50(m,1H),3.76(s,3H),2.98(d,J＝5.2Hz,2H),2.57(t,J＝7.5Hz,2H),1.70-1.55(m,2H),1.45(s,9H),1.26(s,18H),0.88(t,J＝6.7Hz,3H).13C NMR(101MHz,CDCl3):δ171.7,155.2,80.1,53.6,52.5,32.0,30.5,29.9,29.7(2),29.7(0),29.6(7),29.5(9),29.4,29.2,28.4,25.9,25.2,22.8,14.2.MS(ESI):C21H41NO4Se,m/z(％):calcd.451.2(100),found 474.2[M+Na]+(100),472.2(66),475.2(32).

the mass spectrum revealed that the prepared product was indeed Boc-L-Sec (C)₁₂H₂₅) OMe, for Boc-L-Sec (C)₁₂H₂₅) -OH synthesis.

The Boc-L-Sec (C) obtained above was used₁₂H₂₅) OMe was added to 30mL of a 1:1 volume mixture of water containing 0.5mol/L lithium hydroxide and 1, 4-dioxane, and the mixture was stirred at room temperature for 1 hour to remove the methyl group protection of carboxyl groups. After the reaction is finished, adjusting the pH value of the reaction solution to 4-5 by using 1mol/L potassium bisulfate, and extracting by using ethyl acetate. The obtained ethyl acetate extract is dried by anhydrous sodium sulfate and concentrated under reduced pressure, and then purified by silica gel column chromatography, wherein the eluent is ethyl acetate. The light yellow oily liquid obtained after purification is Boc-L-Sec (C)₁₂H₂₅) -OH, product mass 1.14g, yield 48%.

Boc-L-Sec(C₁₂H₂₅) Nuclear magnetic and mass spectral characterization of-OH as follows:

1H NMR(400MHz,CDCl3):δ5.46-5.22(m,1H),4.64-4.42(m,1H),3.02(d,J＝5.2Hz,2H),2.61(t,J＝7.4Hz,2H),1.64(p,J＝7.2Hz,2H),1.46(s,9H),1.40-1.30(m,2H),1.26(s,16H),0.88(t,J＝6.7Hz,3H).13C NMR(101MHz,CDCl3):δ175.2,155.6,80.8,53.5,32.1,30.6,30.0,29.8(1),29.7(9),29.7(6),29.6(8),29.5,29.3,28.4,25.5,22.8,14.3.77Se NMR(76MHz,CDCl3,dimethyl selenide(δ0ppm)as reference):δ131.3.MS(ESI):C20H39NO4Se,m/z(％):calcd.437.2(100),found 436.2[M-H]-(100),434.2(49),437.2(22).13C NMR(101MHz,CDCl3)δ175.2,155.6,80.8,77.5,77.2,76.8,53.5,32.1,30.6,30.0,29.8(1),29.7(9),29.7(6),29.6(8),29.5,29.3,28.4,25.6,25.4,22.8,14.3.

it was confirmed by mass spectrometry that the prepared substance was indeed Boc-L-Sec (C)₁₂H₂₅) -OH, useful in the preparation of selenium peptide.

Meanwhile, the same preparation method is adopted to replace the selenium powder with sulfur powder to prepare a reference molecule Boc-L-Cys (C)₁₂H₂₅) OH as a control for subsequent experiments.

Boc-L-Cys(C₁₂H₂₅) Nuclear magnetic and mass spectral characterization of-OH as follows:

1H NMR(400MHz,CDCl3):δ5.37(d,J＝7.2Hz 1H),4.50(s,1H),3.00(d,J＝5.1Hz,2H),2.56(t,J＝7.3Hz,2H),1.65-1.51(m,2H),1.46(s,9H),1.40-1.30(m,2H),1.26(s,16H),0.88(t,J＝6.6Hz,3H).13C NMR(101MHz,CDCl3):δ175.9,155.6,80.6,53.3,34.2,33.0,32.0,29.7(9),29.7(7),29.7(4),29.6(6),29.5,29.4,28.9,28.4,22.8,14.3.MS(ESI):C20H39NO4S,m/z(％):calcd.389.3,found 388.2[M-H]^-.

from the mass spectrum, it was confirmed that the prepared material was indeed Boc-L-Cys (C)₁₂H₂₅) -OH, which can be used in the preparation of subsequent control thiopeptides.

Example 2

This example provides a selenium peptide (C)₁₂-a selenium peptide) of the formula: [ L-Sec (C)₁₂H₂₅)-OH]-K[L-Sec(C₁₂H₂₅)-OH]GPLGVRGRGD, wherein the selenium peptide is synthesized by a polypeptide solid phase synthesis method, which comprises the following steps:

300mg of Rink amide AM resin (amino acid loading 0.35mmol/g) was weighed into a 10mL polypeptide synthesis tube, 8mL of anhydrous DMF was added thereto, the tube was placed on a shaker overnight to allow the resin to swell sufficiently, and DMF was removed by suction filtration pump.

8mL of Fmoc deprotecting agent (i.e., a mixture of DMF and piperidine in a volume ratio of 4: 1) was added to the polypeptide synthesis tube, and the Fmoc was removed from the amino acid on the resin by reaction for 10min, followed by 3-time alternate washing with dichloromethane and DMF.

A small amount of resin was put into a 1mL plastic centrifuge tube, and ninhydrin test solution (a mixture of 0.5g of ninhydrin, 0.1g of vitamin C, and 20g of phenol dissolved in 20mL of ethanol) was added and boiled for 1 min. Observing the color of the resin, and if the resin is changed into dark blue or dark purple, indicating that Fmoc protection removal is successful; the deprotection step is repeated if there is no change in color. DMF was removed from the polypeptide synthesis tube by suction filtration pump and the resin was washed 3 times with dichloromethane and DMF alternately.

216mg of Fmoc-L-aspartic acid-1-tert-butyl ester (Fmoc-Asp (Otbu) -OH), 199mg of HBTU and 70mg of HOBT were weighed in a test tube, 8mL of a coupling agent (i.e., a mixed solution of DMF and N-methylmorpholine at a volume ratio of 95: 5) was added to react for 10min for activation, and the solution was added to a polypeptide synthesis tube and reacted on a shaker at room temperature for 1 h. Removing DMF from the polypeptide synthesis tube with suction filtration pump, washing resin with dichloromethane and DMF for 3 times alternately, placing a small amount of resin in a 1mL plastic centrifuge tube, adding ninhydrin test solution, and boiling for 1 min. If the color of the resin is not changed, the amino acid coupling is successful; if the color becomes blue or purple, indicating incomplete coupling of the amino acid, the coupling step of the amino acid is repeated.

Repeating the steps of Fmoc-removing protection and amino acid coupling, and continuing to prepare Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, N' -bifluorenylmethoxycarbonyl-L-lysine (Fmoc-L-Lys (Fmoc) -OH), Boc-L-Sec (C)₁₂H₂₅) Coupling of-OH to the resin one by one.

After the coupling of the amino acid, the resin was washed alternately 3 times with dichloromethane and DMF and 2 times with methanol, the liquid was removed, and the resin was taken out and placed in a glass flask. 5mL of a lysate (i.e., a mixture of 4.75mL of trifluoroacetic acid, 125. mu.L of water, and 125. mu.L of triisopropylsilane) was added to the glass flask under ice-bath conditions, and the mixture was reacted for 2 hours with stirring to effect peptide cleavage. After the resin is filtered, the filtrate is blown to 1/10 with the original volume by nitrogen, and the crude selenium peptide is obtained by sedimentation with ice-cold anhydrous ether.

And purifying the crude selenium peptide by adopting a semi-preparative liquid phase, and detecting the purity of the selenium peptide by using HPLC (high performance liquid chromatography) to be higher than 95%. The molecular weight of the obtained selenium peptide is detected by a matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF-MS) and a quadrupole high-resolution mass spectrum, and the result is (C)₇₆H₁₄₁N₂₁O₁₅Se₂,m/z:calcd.1748,found1749[M+H]⁺) In addition, the selenium element has unique isotope distribution, and the isotope peak shape of the selenium peptide mass spectrum obtained by quadrupole high-resolution mass spectrum test is completely consistent with the simulation result of ChemDraw software, thereby further proving the successful synthesis of the selenium peptide.

Boc-L-Sec (C) was synthesized by the same polypeptide solid phase synthesis method₁₂H₂₅) Replacement of-OH by Boc-L-Cys (C)₁₂H₂₅) -OH, Synthesis of thiopeptide (C)₁₂-a thiopeptide) of the formula: [ L-Cys (C)₁₂H₂₅)-OH]-K[L-Cys(C₁₂H₂₅)-OH]GPLGVRGRGD, Simultaneous Synthesis of a control peptide (C) free of selenium and Sulfur₁₂-a control peptide) of formula C₁₂H₂₅-K(C₁₂H₂₅)-GPLGVRGRGD，C₁₂-thiopeptides and C₁₂Control peptides were used as control groups for subsequent experiments.

Example 3

This example examines C prepared in example 2₁₂-MMP-2 enzyme response ability of selenium peptide, the specific steps are as follows:

the MMP-2 enzyme was reacted with C₁₂Selenium peptide was mixed in PBS buffer at pH 7.4, and the concentrations were 50. mu.g/mL and 120. mu. mol/L, respectively. The two were reacted at 37 ℃ for 2 h. The solution after the reaction was tested by HPLC, where different peak-out times in the HPLC profile correspond to different substances and the peak area corresponds to the content of the substance, and the results are shown in fig. 2.

As can be seen from the figure, 80% of C was obtained after 2 hours of the reaction with the MMP-2 enzyme₁₂The selenium peptide is cut off by MMP-2 enzyme, and a new peak appears at the same time, which proves that a new substance is generated. The new substance was confirmed to be [ L-Sec (C) by MALDI-TOF-MS₁₂H₂₅)-OH]-K[L-Sec(C₁₂H₂₅)-OH]GPLG, identical to the cleavage site reported in the literature for the PLGVR peptide. The above results show that C₁₂The selenium peptide can be cut and shortened by MMP-2 enzyme, and has response capability of MMP-2 enzyme.

Example 4

This example examines C prepared in example 2₁₂-the oxidative response capacity of selenium peptide, the specific steps are as follows:

c is to be₁₂Selenium peptide, C₁₂Thio-peptides independently of H₂O₂Mixing, C after mixing₁₂Selenium peptide and C₁₂The concentration of thiopeptide is 60. mu. mol/L, H₂O₂The concentration of (2) is 100. mu. mol/L. Wherein H₂O₂The concentration of (A) is equivalent to the active oxygen concentration in tumor cells reported in the literature. The reaction was carried out at 37 ℃ and HPLC was used 30min after the start of the reactionReaction products of 1h and 2h were tested.

By calculation, C₁₂The oxidation degree of selenium peptide at 30min, 1h and 2h was 76%, 87% and 91%, respectively, while C was₁₂The oxidation degree of thiopeptide at 30min, 1h and 2h was 40%, 43% and 45%, respectively, which indicates that C prepared according to the invention₁₂The selenium peptide has sensitive and quick response capability to active oxygen with lower concentration in a tumor microenvironment, can accelerate the release of substances, and has wide application value in the preparation of tumor drugs.

Example 5

This example provides a self-assembled selenopeptide nano-drug consisting of C₁₂The selenium peptide and the doxorubicin are prepared by the following specific preparation process:

(1) preparation of hydrophobic doxorubicin:

20mg of doxorubicin hydrochloride was dissolved in 1mL of dimethyl sulfoxide, 50. mu.L of triethylamine was added, and mixed with shaking for 1 hour. The reaction solution was dialyzed for 24h in a dark condition using a dialysis bag with a cut-off molecular weight of 1000, and water was changed 5 times during dialysis. The dialyzed turbid solution was centrifuged, and the precipitate was collected and lyophilized.

(2) Determination of the critical micelle concentration:

PBS buffer with pyrene concentration of 24. mu.g/L, pH of 7.4 was used to prepare C concentrations of 0.5. mu. mol/L, 1. mu. mol/L, 3. mu. mol/L, 6. mu. mol/L, 10. mu. mol/L, 15. mu. mol/L, 20. mu. mol/L, 30. mu. mol/L, 40. mu. mol/L and 60. mu. mol/L, respectively₁₂Selenium peptide solution, measuring the fluorescence spectrum of the solution under excitation light with a wavelength of 330nm, calculating the ratio of the intensities of the emission peaks of each group of solutions at 384nm and 373nm, and plotting the ratio as the ordinate and the logarithm of the concentration as the abscissa, wherein the concentration point where the turn appears is the critical micelle concentration, and the result is shown in fig. 3.

As can be seen from the figure, C₁₂The critical micelle concentration of the selenium peptide is 3.9. mu. mol/L.

In the same manner, C is determined₁₂-thiopeptides and C₁₂The critical micelle concentration of the control peptide was 4.4. mu. mol/L and 2.5. mu. mol/L, respectively.

(3)C₁₂Selenium peptide/PolyRow ratioPreparation of Star nano-drug (SeP/DOX):

c with a concentration of 120. mu. mol/L₁₂Mixing PBS buffer solution (pH 7.4) of selenium peptide with 0.8g/mL hydrophobic doxorubicin dichloromethane solution, vigorously stirring to emulsify the solution, distilling under reduced pressure to remove dichloromethane, and centrifuging at 6000rpm to obtain supernatant which is the self-assembled selenium peptide nano-drug SeP/DOX.

By the same preparation method, mixing₁₂Replacement of selenium peptide by C₁₂Preparation of C-Thiopeptide₁₂Thiopeptide/doxorubicin nanomedicines (SP/DOX); c is to be₁₂Replacement of selenium peptide by C₁₂Control peptide preparation C₁₂Control peptide/doxorubicin nanomedicine (CtrP/DOX), used as control for subsequent experiments.

(4) Determination of drug encapsulation efficiency and loading rate:

the content of doxorubicin in the selenium peptide nano-drug solution is determined by adopting an ultraviolet-visible spectrophotometer, a standard curve of the concentration and the absorbance of hydrophobic doxorubicin is constructed, then the absorbance of the drug is measured, calculation is carried out according to the Lambert-beer law, the drug encapsulation rate of SeP/DOX is calculated to be 16.1%, the drug loading rate is calculated to be 32.8%, the drug encapsulation rates of SP/DOX and CtrP/DOX are calculated to be 12.5% and 10.9% respectively, and the drug loading rates are calculated to be 28.3% and 30.5% respectively.

Example 6

This example measured SeP/DOX hydrated particle sizes and SeP/DOX water and particle size measurements by DLS dynamic light scattering, the results of which are shown in FIG. 4.

As can be seen from the figure, the SeP/DOX hydrate particle size is 240 + -13 nm, the polydispersity PDI is 0.18 + -0.05, the particle size is small, and the drug permeation is facilitated.

The hydrated particle size of the thiopeptide nano-drug SP/DOX is measured to be 237 +/-18 nm and the PDI is measured to be 0.21 +/-0.09 by the same measuring method; the hydrated particle size of the control peptide nano-drug CtrP/DOX is 207 +/-13 nm, and the PDI is 0.26 +/-0.03.

This example also performed transmission electron microscopy on SeP/DOX.

The nano-drug was dropped on a copper mesh, incubated for 5min, after the water was absorbed by filter paper, uranyl acetate was dropped and stained for 5min, the liquid was absorbed by filter paper, washed with water for 5min, and after the liquid was absorbed by filter paper, observation was performed at 200kv, and the results are shown in fig. 5.

As can be seen from the figure, the nano-drug particles are uniform in size and have no oversized or undersized drug particles; the polydispersity coefficient shows that SeP/DOX particle size distribution is uniform, which shows that the prepared nano-drug has better uniformity and is beneficial to the permeation, depolymerization and release of the subsequent drug.

Example 7

This example tests SeP/DOX stability by the following steps:

and testing the hydrated particle size change of the selenium peptide nano-drug SeP/DOX within 12h at room temperature by adopting a DLS dynamic light scattering instrument.

The observation result shows that the hydrated particle size of SeP/DOX is not changed greatly after the self-assembled selenium peptide nano-drug SeP/DOX is placed at room temperature for 12 hours, which indicates that the self-assembled selenium peptide nano-drug SeP/DOX can maintain stability at least within 12 hours under the condition of being placed at room temperature.

Example 8

This example examines SeP/DOX responsiveness to MMP-2 enzyme and H₂O₂The oxidation response capability of the method comprises the following specific steps:

SeP/DOX was mixed with MMP-2 enzyme at a concentration of 50. mu.g/mL for MMP-2 enzyme and 120. mu. mol/L for selenium peptide, incubated at 37 ℃ for 2h, and then the particle size was measured using a DLS dynamic light scattering instrument.

The result of DLS particle size test shows that the particle size of the selenium peptide nanoparticles is reduced to 100-150 nm after incubation, which indicates that MMP-2 enzyme cuts selenium peptide, and the particle size of the nanoparticles is reduced.

Then adding H with the final concentration of 0.1 percent into the selenium peptide nano particles after reaction₂O₂The solution was incubated at 37 ℃ for 1h and the particle size was measured using a DLS dynamic light scattering instrument.

The result of DLS particle size test shows that the particle size signal peak of the particles in the system is about 3 μm, which indicates that the oxidative environment causes the deaggregation of the nanoparticles and promotes the release of the drug.

The above results indicate that SeP/DOX has response ability to MMP-2 enzyme and active oxygen free radical, increases permeability to tumor tissue by reducing particle size, promotes drug release by oxidation, and improves therapeutic effect.

Example 9

The present example detects SeP/DOX targeting to tumors, and the specific steps are as follows:

MDA-MB-231 cells were cultured at an initial density of 1X 10⁵The cells were inoculated per well in 12-well plates and divided into 6 groups, untreated group (Non-treated), DOX group, CtrP/DOX group, SeP/DOX group, SeP/DOX and RGD inhibitor (cRGD) group, and CtrP/DOX and RGD inhibitor (cRGD) group. Cells at 37 ℃ with 5% CO₂Culturing for 24h under the environment of (1). The culture medium was removed and incubated with DOX, CtrP/DOX, SeP/DOX and cRGD, and CtrP/DOX and cRGD, respectively, for 2h, and the untreated group was subjected to the same experiment using PBS solution. After washing with PBS buffer, the cells were trypsinized and collected, and after washing with PBS buffer, the fluorescence intensity of doxorubicin in the cells was measured using BD Accuri C6 flow cytometer, and the results are shown in fig. 6.

As can be seen from the figure, compared with the untreated group, the doxorubicin (DOX group) and the CtrP/DOX group, the fluorescence intensity of the doxorubicin in the SeP/DOX group cells is remarkably enhanced, which indicates that SeP/DOX has the tumor targeting function; when incubated with the cRGD inhibitor, the fluorescence intensity of doxorubicin in the cells was significantly reduced, indicating that SeP/DOX achieved a tumor targeting function through the RGD domain. The above results indicate that SeP/DOX has excellent tumor targeting ability and mainly exerts targeting function through the tumor targeting unit in the selenium peptide.

Example 10

This example detects SeP/DOX permeability to tumor cell clusters by the following steps:

MDA-MB-231 cells were cultured at an initial density of 1X 10⁵And inoculating the cells in a 96-well plate paved with agarose, and after culturing for 14 days, aggregating the cells into spheres. The spherical tumor cell clusters are sucked out by a pipette gun, divided into 4 parallel groups and incubated with CtrP/DOX, SeP/DOX + MMP-2 enzyme and DOX for 2h respectively. Cell clusters were observed at 1/4, 1/2 using the Z-Stack mode in a CarlZeiss LSM710 laser scanning confocal microscopeAnd 3/4, the results are shown in FIG. 7.

As can be seen from the figure, the fluorescence intensity of doxorubicin in the central focal plane of the cell cluster is highest in the cell cluster incubated by SeP/DOX and MMP-2 enzyme, and is next to DOX and SeP/DOX, and CtrP/DOX is weaker. This shows that after MMP-2 enzyme cuts the self-assembly selenium peptide nano-drug, the particle size of the drug can be reduced, the infiltration capacity of the drug to tumor tissues is improved, and the treatment effect of the drug is improved.

Example 11

In this example, the killing effect of SeP/DOX on tumor cells is detected by the following specific steps:

inoculating MDA-MB-231 cells into a 96-well plate with the initial density of 5000/well, removing culture solution after culturing for 24h, dividing the cells into 30 groups in parallel, respectively adding CtrP/DOX, SeP/DOX and DOX with the concentrations of 0, 0.1 mu mol/L, 0.25 mu mol/L, 0.5 mu mol/L, 1 mu mol/L, 2 mu mol/L, 4 mu mol/L, 8 mu mol/L, 16 mu mol/L and 32 mu mol/L, incubating for 24h, removing culture medium with drugs, adding serum-free culture medium containing 10% CCK-8, incubating for 2h, detecting the absorbance at 450nm by using a microplate reader, and calculating half inhibitory concentration IC according to the absorbance value mapping₅₀The results are shown in FIG. 8.

As can be seen from the figure, IC's of CtrP/DOX, SeP/DOX and DOX₅₀The values were 16.7. mu. mol/L, 1.04. mu. mol/L and 0.08. mu. mol/L, respectively. SeP/DOX treated IC compared to CtrP/DOX₅₀And is small, which indicates that the compound has better inhibition effect on tumor cells. Although the effect of directly adding DOX on killing tumors is better, SeP/DOX is more suitable for being applied to the preparation of antitumor drugs considering that DOX has no selectivity on killing cells and has larger toxic and side effects on normal tissues.

Example 12

The method for detecting the anti-tumor effect of SeP/DOX on the tumor-bearing mouse model of the in-situ triple-negative breast cancer comprises the following specific steps:

establishment of tumor-bearing mouse model of in situ triple negative breast cancer: the number of the grooves is 5 multiplied by 10⁷The MDA-MB-231 cells are injected into the mammary gland fat pad of a BALB/c nude mouse for 2-3 weeksThe posterior tumor volume is up to about 150mm³. Tumor volumes were calculated as follows: tumor volume is 0.5 × length of long side x (length of short side)²。

30 tumor-bearing mice were randomly divided into 5 groups, and were injected with physiological Saline (Saline), DOX, CtrP/DOX, SP/DOX, and SeP/DOX, respectively, to give 6 tumor-bearing mice per group. The specific administration protocol was as follows, by tail vein injection, 4 times in total at 1d, 3d, 5d and 7d, the dose of each 5mg/kg of DOX was administered, the administration volume was 200. mu.L, and the same amount of physiological saline was injected into the physiological saline group. Tumor volumes were recorded 1 time per 2d and the results were counted. The significance test of each experimental group data was performed by using T-test (two-tailed) function in Excel software, representing p <0.01 and p <0.001, and the statistical results are shown in fig. 9.

As can be seen, SeP/DOX has an excellent effect of inhibiting tumor growth, and after 4 times of administration, the tumor volume of SeP/DOX group mice has almost no change, while the tumor volume of the other groups of mice has a significant increase. Combining the results of examples 11 and 12, it is shown that SeP/DOX not only has excellent antitumor effect at the cellular level, but also can inhibit tumor growth at the animal level, with significant effect, excellent performance, and extremely high application value.

Example 13

This example test C₁₂The clearance time of the selenium peptide nanoparticles in blood is specifically as follows:

preparation of C by substituting doxorubicin with TRITC using Methylisothiocyanate rhodamine (TRITC) as a fluorescent tracer with reference to the preparation method in example 5₁₂-selenopeptide/TRITC nanoparticles.

Respectively adding 200 μ L of TRITC and C₁₂The selenopeptide/TRITC nanoparticles were infused into BALB/C mice by tail vein injection, with the same concentration of TRITC in both samples. Collecting tail vein blood of the mouse at different time points to test the fluorescence intensity of TRITC, comparing the fluorescence intensity of the initial TRITC, and calculating the retention condition of the drug in the blood.

The results show that TRITC and C were obtained 1h after the injection of the drug₁₂-retention of selenopeptide/TRITC nanoparticles in bloodThe amounts were 29.6. + -. 10.2% and 54.8. + -. 7.8%, respectively, indicating C₁₂Selenium peptide nanoparticles can prolong the retention time of molecules in blood. Compared with the direct injection of the medicine, the nano-particle medicine assembled by the selenium peptide has longer-acting blood retention effect, the medicine has long retention time in vivo, can obtain longer-acting treatment effect, and has high bioavailability.

In conclusion, the invention provides an oxidation response selenoamino acid, which has sensitive oxidation response capability, can change hydrophobicity in an environment containing active oxygen free radicals and helps the release of a medicament; the selenium peptide comprises an oxidation response unit, an enzyme response fragmentation unit and a tumor targeting unit which are connected in sequence, can be fragmented by MMP-2 enzyme and has the capability of oxidation response; the self-assembled selenium peptide nano-drug consists of C₁₂The selenium peptide and the antitumor drug are prepared, can be cut by MMP-2 enzyme to reduce the particle size, can be deaggregated in an oxidative environment to promote the release of the drug, has targeting property to tumors, has good permeability to tumor cell masses, can effectively kill tumor cells, inhibit the growth of tumors in vivo, prolong the detention time of the drug in vivo, has better stability, can be stably stored for more than 12 hours at room temperature, and has extremely wide application in the preparation of the drug for treating tumors.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A selenopeptide, which is characterized by comprising an oxidation response unit, an enzyme response fragmentation unit and a tumor targeting unit which are sequentially connected from N end to C end;

2. The selenium peptide of claim 1, wherein the fatty chain in the fatty chain-L-selenocysteine comprises a saturated hydrocarbyl chain and/or an unsaturated hydrocarbyl chain, preferably a saturated hydrocarbyl chain;

preferably, the aliphatic chain comprises any one of a linear aliphatic chain, a branched aliphatic chain or a cyclic aliphatic chain or a combination of at least two of the same, preferably a linear aliphatic chain;

preferably, the number of carbon atoms in the aliphatic chain is an integer of 1 to 18;

preferably, the fatty chain is C₆、C₁₂Or C₁₈Any one of saturated linear aliphatic chains;

preferably, the aliphatic chain-L-selenocysteine comprises L-Sec (C)₆H₁₃)-OH、L-Sec(C₁₂H₂₅) -OH or L-Sec (C)₁₈H₃₇) -any one or a combination of at least two of OH;

preferably, the aliphatic chain-L-selenocysteine is L-Sec (C)₁₂H₂₅) -OH, said L-Sec (C)₁₂H₂₅) -OH has the formula shown in formula I:

preferably, the oxidation response unit comprises at least 1 oxidation response selenium amino acid, and the oxidation response selenium amino acids are connected through a connecting amino acid;

preferably, the oxidation responsive unit comprises 2 oxidation responsive seleno-amino acids;

preferably, the number of said linking amino acids is 1;

preferably, the linking amino acid is K.

3. The selenium peptide of claim 1 or 2, wherein the enzyme responsive cleavage unit comprises a matrix metalloproteinase II cleaved polypeptide sequence;

preferably, the polypeptide sequence of the enzyme responsive cleavage unit is PLGVR;

preferably, the tumor targeting unit comprises a polypeptide sequence targeting tumor integrin;

preferably, the polypeptide sequence of the tumor targeting unit is RGD;

4. The selenium peptide of any one of claims 1 to 3, wherein the selenium peptide is:

preferably, the spacer amino acids comprise natural amino acids and/or unnatural amino acids, preferably G;

preferably, the selenium peptide is:

5. a method of preparing the selenium peptide of any of claims 1 to 4, comprising:

6. The method of claim 5, wherein said method of preparing said Boc group-protected oxidation-responsive seleno-amino acid comprises:

mixing the alkyl selenol and N-tert-butyloxycarbonyl-O-p-toluenesulfonyl serine methyl ester to obtain aliphatic chain-L-selenocysteine methyl ester protected by Boc group, and removing methyl to obtain aliphatic chain-L-selenocysteine protected by Boc group;

preferably, the 1-haloalkane comprises a 1-bromoalkane;

preferably, the number of carbon atoms of the 1-bromoalkane is an integer of 1-18, preferably 6, 12 or 18;

preferably, the mixing reaction is carried out under oil bath conditions;

preferably, the temperature of the oil bath is 45-55 ℃, and preferably 50 ℃;

preferably, the mixing reaction time is 10-14 h, preferably 12 h;

preferably, the reducing agent comprises sodium borohydride;

preferably, the method further comprises the step of cooling before adding the reducing agent;

preferably, the reduction time is 25-35 min, preferably 30 min;

preferably, the mixing time is 5-7 h, preferably 6 h;

preferably, the methyl group is removed, and the method further comprises the steps of ethyl acetate extraction and silica gel column chromatography purification;

preferably, the methyl group is removed by placing the reaction product in a solution containing lithium hydroxide and stirring;

preferably, the stirring time is 55-65 min;

preferably, the methyl group is removed, and then a step of ethyl acetate extraction is included;

preferably, the step of solid phase synthesis comprises:

swelling the resin, removing Fmoc groups of amino acids of the resin, and sequentially coupling the amino acids and the selenium amino acids with oxidation response onto the resin;

preferably, the step of swelling the resin comprises soaking the resin in anhydrous DMF and placing on a shaker;

preferably, the amino acids include amino acids protected by Fmoc group;

preferably, the step of coupling the amino acid to the resin further comprises the step of removing the Fmoc group of the amino acid;

7. The method of claim 5 or 6, wherein the method comprises the steps of:

8. A self-assembled selenopeptide nano-drug, which comprises the selenopeptide according to any one of claims 1 to 4 and an anti-tumor drug;

preferably, the anti-tumor drug is a hydrophobic drug, preferably doxorubicin;

preferably, the hydration particle size of the self-assembly selenium peptide nano-drug is 200-250 nm;

preferably, the hydrated particle size of the self-assembled selenium peptide nano-drug is reduced to 50-150 nm under the action of matrix metalloproteinase II;

9. A method of preparing the self-assembled selenopeptide nano-drug of claim 8, comprising mixing the selenopeptide with an anti-tumor drug;

preferably, the selenium peptide is dissolved in a sodium phosphate buffer;

preferably, the pH value of the sodium phosphate buffer solution is 7.0-7.8, preferably 7.4;

preferably, the antineoplastic drug is dissolved in dichloromethane;

preferably, the method also comprises the step of removing the dichloromethane by distillation under reduced pressure after the mixing;

preferably, the method also comprises a step of centrifuging after removing the dichloromethane;

preferably, the rotating speed of the centrifugation is 5000-6500 rpm, preferably 6000 rpm;

preferably, the preparation method of the self-assembled selenium peptide nano-drug comprises the following steps:

10. Use of the selenium peptide of any one of claims 1 to 4 and/or the self-assembled selenium peptide nano-drug of claim 8 for the preparation of a medicament for the treatment of a tumor.