CN111233979A

CN111233979A - Gemini-type amphiphilic short peptide and application thereof as hydrophobic drug carrier

Info

Publication number: CN111233979A
Application number: CN202010103267.9A
Authority: CN
Inventors: 邱峰; 彭飞; 张文胜
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-02-19
Filing date: 2020-02-19
Publication date: 2020-06-05

Abstract

The invention relates to a short peptide and application thereof as a carrier of a hydrophobic drug. The related short peptide has the amino acid sequence of (Y)_n‑Pro‑X‑X‑Pro‑(Y)_n. The short peptide can load various hydrophobic drugs through a molecular self-assembly mechanism, and forms nano spherical micelles with uniform particle sizes with the hydrophobic drugs, so that the short peptide can effectively transport the drugs into cells or tissues to exert the drug effect of the drugs, is safe, has no obvious cytotoxicity, has a simple preparation process, and is a hydrophobic drug carrier with great development prospect.

Description

Gemini-type amphiphilic short peptide and application thereof as hydrophobic drug carrier

Technical Field

The invention belongs to the field of drug carriers, and particularly relates to a Gemini amphiphilic short peptide and application thereof as a hydrophobic drug carrier.

Background

At present, many small molecule drugs in clinic are hydrophobic in nature and poor in water solubility, and are generally dispersed and dissolved in an aqueous solution or an aqueous solution by using a carrier in a preparation so as to be administrated by injection. The carriers of hydrophobic drugs widely used clinically at present mainly comprise materials such as lipid. Taking paclitaxel, the most common anticancer drug, as an example, the drug on the market

The Taxol injection Taxol contains about 527mg/mL polyoxyethylene castor oil and 49.7% absolute ethyl alcohol as solvents; the most commonly used general anesthetic drugs such as propofol are fat emulsion preparations with lipid molecules such as natural soybean oil, egg yolk lecithin, etc. as carriers; the vasodilator alprostadil adopts fat emulsion injection with lipid molecules such as soybean oil, lecithin, oleic acid and the like as carriers; the hypertensive drug clevidipine also contains an oil for injection and a phospholipid component. However, the clinical application of lipid components has some problems such as poor stability (AnesthAnalg 2003, 97: 769-. During the clinical use, Taxol injection shows more adverse reactions such as acute hypersensitivity (Allergy assay ImmunoRes 2016, 8: 174-; the propofol fat emulsion injection has the problem of propofol infusion syndrome (PRIS) (Crit Care2015, 19: 398); the clevidipine injection has serious limitationUse of patients with disorders of lipid metabolism (see clevidipine injection instructions), and the like. The above problems are substantially all associated with lipid compositions. The development of novel carrier materials for hydrophobic drugs which do not contain lipid components is therefore very promising.

At present, other non-lipid materials are used clinically, for example, a paclitaxel-encapsulated human albumin preparation Abraxane is marketed in the United states in 2005, and has the advantages of small side effect, short administration time and reduced adverse reaction (Int JANOMedicine 2009, 4: 99-105). However, this formulation is limited by the human blood source of the albumin carrier and the corresponding risk of microbial and viral contamination, which is expensive. The preparation Cynviloq of paclitaxel carried by polymer mPEG-PLLA material is marketed in Korea in 2007 (AdvDrug DeliverRev 2017, 122: 20-30), but the price of the polymer material is high, the synthesis process is complex, and the nano toxicity of the polymer needs to be paid continuous attention. In the aspect of biological safety, the artificially synthesized short peptide has unique advantages and is a potential drug carrier material. The chinese invention patent (ZL patent No. 00105625.5, granted publication No. CN1148227C, entitled "therapeutic compound and its use") discloses a therapeutic compound based on a short peptide carrier and its use. The invention discloses a method for preparing a therapeutic compound by chemically combining paclitaxel, glutamic acid and aspartic acid, and the drug is directly loaded by nanospheres formed by self-assembly of short peptides.

Artificially designed self-assembled short peptides are receiving increasing attention as a class of materials that are internationally emerging in recent years (Nano Today 2016, 11: 41-60). One class of amphiphilic short peptides which are designed by simulating the structure of the traditional surfactant and have typical hydrophilic heads and hydrophobic tails are important points of concern for many subject groups at home and abroad (Acc Chem Res2017, 50: 2440-2448). The material is artificially synthesized and mainly comprises natural amino acid, so the material has natural advantages in the aspects of controllability of quality and purity, biocompatibility, degradability and the like. The amphiphilic short peptide can be driven by hydrophobic effect to self-assemble to form a plurality of nano structures such as tube cavities or vesicles, rod-shaped or spherical micelles, monomolecular or bimolecular membranes and the like, and is an ideal drug carrier material. In addition, some amphiphilic short peptides can form a package on the membrane protein through the combination of the hydrophobic tail part and the hydrophobic region of the membrane protein, so that the stability of the membrane protein in an aqueous solution is improved, and the amphiphilic short peptides are also used for the research on the membrane protein (PLoS One 2011, 6: e25067), which also proves the potential of the amphiphilic short peptides to package hydrophobic molecules from the other side.

However, the amphiphilic short peptide vectors reported at present have low drug loading rate, poor preparation stability and significantly lower drug loading capacity than most natural lipid molecules, and the problems are the reason why the amphiphilic short peptide vectors are not popularized yet.

There is a report in the literature on a "gemini type amphiphilic short peptide" which is different from a conventional amphiphilic short peptide having only one hydrophobic tail and one hydrophilic head and which comprises an amphiphilic short peptide having two hydrophobic tails and two hydrophilic heads; the short peptide relies on the formation of disulfide bonds between two cysteine (Cys) under specific conditions to realize a Gemini structure, and compared with the traditional amphiphilic short peptide, the short peptide relies on the formation of disulfide bonds between the two Cys to form disulfide bonds, has certain design limitation and poor stability (Colloid Surface A2015, 469: 263-ion 270).

Disclosure of Invention

The invention aims to provide a novel gemini amphiphilic short peptide which has a drug loading effect comparable to that of a lipid carrier and does not depend on disulfide bonds, and the technical scheme is as follows:

a gemini amphiphilic short peptide has the following general formula: (Y) n-Pro-X-X-Pro- (Y) n, wherein X is hydrophilic amino acid, Y is hydrophobic amino acid, and n represents the number of hydrophobic amino acid and has a value range of 4-8.

The hydrophilic amino acids and the hydrophobic amino acids are defined according to the height of a hydrophilic index (JMolBiol1982, 157: 105-132), the hydrophilic index is equal to or more than-0.4 and is hydrophobic amino acids, the hydrophilic index is equal to or less than-0.7 and is hydrophilic amino acids, and the hydrophilic index of the amino acids is shown in the following table:

amino acid abbreviations	Hydropathic index	Amino acid abbreviations	Hydropathic index
				R	-4.5	S	-0.8
K	-3.9	T	-0.7
				N	-3.5	G	-0.4
D	-3.5	A	1.8
				Q	-3.5	M	1.9
E	-3.5	C	2.5
				H	-3.2	F	2.8
P	-1.6	L	3.8
				Y	-1.3	V	4.2
W	-0.9	I	4.5

The short peptide as the above, wherein the N terminal and/or the C terminal of the short peptide are/is provided with chemical modification;

the N-terminal chemical modification is alternatively selected from: alkylacylation (e.g., acetylation, formylation, etc.), biotin labeling, fatty acid modification (e.g., Palm, Myr, Lauryl, etc.) benzoylation, 2-aminobenzoylation, maleimide, haloalkanoylation (e.g., trifluoroacetyl, chloroacetyl, bromoacetyl, etc.), succinylation, hydrazinenicotinamide, fluorophore labeling (e.g., FAM, FITC, TAMRA, etc.) fluorophore labeling.

The C-terminal chemical modification is alternatively selected from: amidation, esterification, aldehyde group, alcohol group, succinylation, fluorescent group labeling (such as AMC, CMK, FMK and the like).

The short peptide (Y) n is any one or combination of glycine, alanine, valine, leucine, isoleucine or phenylalanine in any sequence.

The short peptide is the short peptide, and Y is any one or combination of glycine, alanine or valine in any sequence.

As with the short peptides described above, Y is alanine.

The short peptide has n of 5-6.

The short peptide is any one or combination of two of serine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.

As for the short peptides, X is selected from glutamic acid or lysine.

As with the short peptides described above, X is lysine.

The amino acid sequence of the short peptide is shown as SEQ ID NO.1, 3, 4, 5 or 6; preferably, as shown in SEQ ID NO. 1.

The short peptide as described above, wherein the N-terminal chemical modification is acetylation;

and/or, the C terminal is chemically modified into amidation (the C atom in the amido group can be the C atom carried by the C segment of the short peptide).

The use of the aforementioned short peptides in the preparation of a carrier for hydrophobic drugs.

The carrier is a micelle formed by self-assembly of the short peptide; preferably, the micelles are spherical micelles.

As previously mentioned, the hydrophobic drugs include, but are not limited to, paclitaxel, doxorubicin, curcumin, docetaxel, doxorubicin, vincristine, camptothecin, hydroxycamptothecin, etoposide, tretinoin, fluorouracil, methotrexate, teniposide, daunorubicin, aclacinomycin, sorafenib, methylprednisolone, minocycline, cisplatin, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, chlorobenzenesulfone, azithromycin, amphotericin B, griseofulvin, celecoxib, raloxifene, cloroxifene, indomethacin, ibuprofen, tamoxifen, diclofenac, naproxen, piroxicam, lartirapavir, efavir, nelfinavir, atazanavir, ritonavir, sirolimus, ambroxeton, tacrolimus, talinolol, tacrolimus, and loperamide, One or a mixture of more of terfenadine, estradiol, vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, alprostadil, composite fat-soluble vitamin, dexamethasone, flurbiprofen ester, clevidipine, brucea javanica oil, cyclosporine, insulin and the like;

preferably, the hydrophobic drug is paclitaxel, doxorubicin, etomidate or propofol.

A hydrophobic drug carrier, wherein the carrier is a micelle formed by self-assembly of the short peptide; preferably, the micelles are spherical micelles.

A nano-carrier preparation takes hydrophobic drugs as active ingredients, and takes micelles formed by self-assembly of the short peptides as carriers.

In the nano-carrier preparation, the content ratio of the short peptide to the active ingredient is 5 mu mol: 1-100 mg.

The nano-carrier preparation as described above, wherein the hydrophobic drug is paclitaxel, adriamycin, curcumin, docetaxel, doxorubicin, vincristine, camptothecin, hydroxycamptothecin, etoposide, tretinoin, fluorouracil, methotrexate, teniposide, daunorubicin, aclacinomycin, sorafenib, methylprednisolone, minocycline, cisplatin, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, chlorobenzenesulfone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, raloxifene, cloroxafine, indomethacin, ibuprofen, tamoxifen, diclofenac, naproxen, piroxicam, lativelavir, efavir, nelfinavir, atazanavir, ritonavir, sirolimus, acesulfame, tacrolimus, tacrine, and other, One or a mixture of more of terfenadine, estradiol, vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, alprostadil, composite fat-soluble vitamin, dexamethasone, flurbiprofen ester, clevidipine, brucea javanica oil, cyclosporine, insulin and the like;

The Gemini type amphiphilic short peptide is a brand new design, and has 2 hydrophobic tails under any condition, and is obtained by forming a corner structure through two Pro on the basis of the traditional single hydrophobic tail amphiphilic short peptide. Compared with the current oligopeptide carrier, the drug-loading capacity of the Gemini amphiphilic oligopeptide is obviously improved; the short peptide of the invention is used as a carrier of a hydrophobic drug to prepare a preparation, and the drug effect can reach the level close to that of the existing lipid carrier drug preparation.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a structural schematic diagram of gemini amphiphilic short peptides APK, APE, APKE, GAVPK and AVLFPK, and amphiphilic short peptide A6K with a single hydrophobic tail.

FIG. 2 is a schematic diagram of Gemini amphiphilic short peptide self-assembly and its mechanism for loading hydrophobic drugs.

FIG. 3 is an electron microscope image of an APK self-assembly nano-membrane structure and a loading mode thereof, wherein the APK self-assembly nano-membrane structure and the loading mode thereof are used for forming uniform nanospheres with hydrophobic drug molecules pyrene, and a contrast electron microscope image of an irregular nanosphere formed by loading pyrene with a traditional amphiphilic short peptide A6K with a single hydrophobic tail.

FIG. 4 is a graph of the effect of APK-loaded paclitaxel (APK-PTX) on inhibiting proliferation of ovarian cancer cells skov 3.

FIG. 5 is a graph showing the effect of APK-loaded doxorubicin (APK-DOX) on inhibiting the proliferation of ovarian cancer cell skov 3.

FIG. 6 is a graph showing the effect of APK on the ovarian cancer cells skov 3.

FIG. 7 is an electron micrograph of APK loaded paclitaxel.

FIG. 8 is an electron micrograph of APK loaded with doxorubicin.

Figure 9 is an electron micrograph of APK loaded etomidate.

FIG. 10 is an electron micrograph of APK loaded propofol.

FIG. 11 is a circular dichroism spectrum of gemini amphiphilic short peptides APK, APE, APKE, GAVPK and AVLFPK showing that they have similar random secondary structures.

FIG. 12 is the fluorescence spectra of the gemini amphiphilic short peptides APK, APE, APKE, GAVPK and AVLFPK in combination with ThT, showing that they have similar self-assembly behavior.

FIG. 13 is an electron microscope image of the gemini amphiphilic short peptide APE, APKE, GAVPK and AVLFPK loading mode hydrophobic drug pyrene forming nanospheres.

Detailed Description

Example 1: loading of short peptide on mode hydrophobic drug pyrene

The material of the invention is as follows: (Ala)₆-Pro-Lys-Lys-Pro-(Ala)₆I.e. Ac-Ala-Ala-Ala-Ala-Ala-Ala-Pro-Lys-Lys-Pro-Ala-Ala-Ala-Ala-NH₂Abbreviated as APK, Ac represents acetyl; APK is SEQ ID NO.1 in the amino acid sequence table; (Ala)₆-Lys, Ac-Ala-Ala-Ala-Ala-Ala-Ala-Lys-COOH, abbreviated A6K, SEQ ID No.2 of the amino acid sequence listing, having the structure shown in FIG. 1, and assigned to Shanghai Borate Biotech Co., Ltd for chemical synthesis.

Pyrene and DMSO were purchased from Sigma-Aldrich.

Preparing a short peptide mother solution: APK was dissolved in water at 0.5mM and sonicated for 5 min.

Preparing pyrene mother liquor: pyrene powder was dissolved in DMSO at 10 mM.

And (3) placing 10mL of the short peptide mother liquor into the room temperature magnetic stirring condition of 2000rpm/min, dropwise adding 100 mu L of pyrene mother liquor into the short peptide mother liquor by using a pipette, continuing to magnetically stir for 30min after the addition is finished, performing ultrasonic treatment for 10min, and storing at 4 ℃.

Morphological features were observed by high resolution transmission electron microscopy. mu.L of the sample solution was applied to a 400 mesh copper net for 5min and then blotted dry with a piece of filter paper. Then 10. mu.L of 2% phosphotungstic acid was added for staining for 3 min. The final staining solution was blotted dry with filter paper and air dried. And then imaging by adopting a transmission electron microscope.

As can be seen from FIG. 3, APK can independently self-assemble to form a nano-film structure, and after pyrene is loaded on APK, nano-spheres with particle size less than 50nm, uniform size and regular shape can be formed. In contrast, A6K loading pyrene with a single hydrophobic tail can only form nanoparticles with particle size over 200nm, and irregular shape and size.

The results of example 1 show that: APK enables efficient loading of hydrophobic drugs.

Example 2: loading of paclitaxel by short peptides

Materials: APK entrusts Shanghai Boratae Biotech limited company to carry out chemical synthesis; paclitaxel was purchased from Dalian Meiren Biotechnology Ltd; absolute ethyl alcohol was purchased from a chemical reagent plant of the metropolis department.

Preparing a paclitaxel mother solution: the paclitaxel powder was dissolved in anhydrous ethanol at 10 mg/mL.

Placing 10mL of the short peptide mother liquor at room temperature under the condition of magnetic stirring at 2000rpm/min, dropwise adding 100 mu L of paclitaxel mother liquor into the short peptide mother liquor by using a pipette (the dosage ratio of APK to paclitaxel is 5 mu mol: 1mg), continuing to magnetically stir for 30min after the addition is finished, performing ultrasonic treatment for 10min, and storing at 4 ℃.

Example 3: loading of paclitaxel by short peptides

APK entrusts Shanghai Boratae Biotech limited company to carry out chemical synthesis; paclitaxel was purchased from Dalian Meiren Biotechnology Ltd; absolute ethyl alcohol was purchased from a chemical reagent plant of the metropolis department.

Preparing a short peptide mother solution: APK was dissolved in water at 1mM and sonicated for 5 min.

Preparing a paclitaxel mother solution: the paclitaxel powder was dissolved in anhydrous ethanol at 20 mg/mL.

Placing 1mL of paclitaxel mother liquor at room temperature under the condition of magnetic stirring at 2000rpm/min, dropwise adding 100mL of short peptide mother liquor into the paclitaxel mother liquor by using a pipette (the dosage ratio of APK to paclitaxel is 5 mu mol: 1mg), continuing to magnetically stir for 30min after the addition is finished, performing ultrasound for 10min, and storing at 4 ℃.

Example 4: loading of paclitaxel by short peptides

Dissolving APK and 10mg/mL paclitaxel powder in 2mLDMSO (the dosage ratio of APK to paclitaxel is 5 μmol: 100mg) according to 0.5mM, and performing ultrasonic treatment for 5 min; removing organic solvent with vacuum drier, re-dissolving with 10mL water, ultrasonic treating for 10min, and storing at 4 deg.C

Example 5: inhibition of ovarian cancer cells by paclitaxel-short peptide formulations

Human ovarian cancer cells skov3 at 5X 10³Cells/well density were seeded in 96-well plates and incubated for 24 hours. The supernatant was removed, paclitaxel-short peptide preparations (prepared as in example 2) at various concentrations and taxol (a commercial paclitaxel drug preparation) at corresponding concentrations were added as controls, and after 48h incubation, the cell viability was determined using cck-8 reagent method. OD values reflecting the cell viability were measured at 490nm using a microplate fluorometer.

The comparison between the experimental group and the control group shows that the paclitaxel-short peptide preparation (APK-PTX) has significant inhibition effect on tumor cells, and is equivalent to the drug taxol in the control group (figure 4).

Example 6: loading of doxorubicin with short peptides

Reagent: doxorubicin hydrochloride was purchased from gangrenum biotechnology limited, and triethylamine was purchased from metropolis chemical reagent factory.

Desalting by using doxorubicin hydrochloride: dissolving doxorubicin hydrochloride powder 10mg in 10ml of methanol, adding 10 μ L of triethylamine, magnetically stirring overnight, and evaporating the organic solvent to dryness under a vacuum apparatus to obtain doxorubicin powder.

Preparing adriamycin mother liquor: doxorubicin powder was dissolved in anhydrous ethanol at 10 mg/mL.

Placing 10mL of short peptide mother liquor at room temperature under the condition of magnetic stirring at 2000rpm/min, dropwise adding 100 mu L of adriamycin mother liquor into the short peptide mother liquor by using a pipette (the dosage ratio of APK to adriamycin is 5 mu mol: 1mg), continuing to magnetically stir for 30min after the addition is finished, performing ultrasonic treatment for 10min, and storing at 4 ℃.

Example 7: loading of doxorubicin with short peptides

1mL of adriamycin mother liquor is placed at room temperature under the condition of magnetic stirring at 2000rpm/min, 100mL of short peptide mother liquor is dropwise added into the adriamycin mother liquor by a pipette (the dosage ratio of APK to adriamycin is 5 mu mol: 1mg), magnetic stirring is continued for 30min after the addition is finished, ultrasonic treatment is carried out for 10min, and the mixture is stored at 4 ℃.

Example 8: loading of doxorubicin with short peptides

Dissolving APK in 2mLDMSO according to 0.5mM and 10mg/mL adriamycin powder (namely the dosage ratio of APK to adriamycin is 5 mu mol: 100mg), and performing ultrasonic treatment for 5 min; the organic solvent was removed by vacuum dryer, redissolved with 10mL water, sonicated for 10min, and stored at 4 ℃.

Example 9: inhibitory effect of Adriamycin-short peptide preparation on ovarian cancer cells

Human ovarian cancer cells skov3 at 5X 10³Cells/well density were seeded in 96-well plates and incubated for 24 hours. The supernatant was removed, and doxorubicin-short peptide preparations (prepared using the method of example 6) at various concentrations, as well as doxorubicin hydrochloride at the corresponding concentrations, were added as controls, and after incubation for 48h, the cell viability was determined using the cck-8 reagent method. OD values reflecting the cell viability were measured at 490nm using a microplate fluorometer.

The comparison between the experimental group and the control group shows that the adriamycin-short peptide preparation (APK-DOX) has obvious inhibition effect on the tumor cells, and is equivalent to the control group medicament adriamycin hydrochloride (figure 5).

Example 10: loading of etomidate with short peptide

Materials: (Ala)₆-Pro-Lys-Lys-Pro-(Ala)₆APK, for short, entrusted to Shanghai Boratae Biotech limited for chemical synthesis; etomidate was purchased from Dalian Meiren Biotechnology Ltd; absolute ethyl alcohol was purchased from a chemical reagent plant of the metropolis department.

Preparing a short peptide mother solution: APK was dissolved in 0.9% saline at 0.5mM, vortexed and sonicated for 10 min.

Weighing 20mg etomidate, adding into 10mL of short peptide mother liquor (the dosage ratio of APK to etomidate is 5 mu mol: 20mg), performing ultrasonic treatment for 20min after vortex, stirring for 40min under the condition of magnetic stirring at room temperature of 2000rpm/min, and storing at room temperature.

Example 11: anesthetic effect of intravenous single injection of etomidate-oligopeptide preparation of rat tail

Healthy adult male SD rats (body weight: 295. + -.14 g) were used. Rats were placed in a fixture, the tail was exposed, the lateral tail vein was found, and the needle was placed into the tube after alcohol wiping (etomidate-short peptide formulation was prepared using the method of example 10). 0.6mL of medicine is uniformly administered at the administration speed of 0.1mL/s, after the administration is finished, 0.05mL of air is pushed to ensure that the medicine completely enters the tail vein, the indwelling needle is pulled out, and the cotton swab is pressed to stop bleeding. Rats were quickly removed and placed in empty cage sites, and rat responses were observed for sedation scoring and adverse events were recorded (CFDA "guidelines for drug word administration toxicity" (prosecution 2013-05-26)). Sedation score (Psychopharmacology 1996, 125: 105-: the muscle tension of the four limbs is normal, the autonomous activity can be kept, and the response is sensitive for 0 minute; apparent thigmotaxis (rats tend to stay close to the cage rim) for 1 point; retreating and balancing disorder for 2 minutes; forelimb erection less than 60 degrees, ataxia 3 points; lying prostrate, unable to stand, can only support 4 minutes by the belly; the righting reflection disappears for 5 minutes. Observation of disappearance of righting reflex (Anesthesiology 2000,93 (3): 837-: the righting reflex disappeared and lasted for more than 30 seconds was marked as "+", otherwise as "-".

A ramp-up experiment was performed starting from the 1mg/kg dose and finding a "+" and "-" dose, respectively, as ED₅₀(half effective amount, amount of drug that elicits 50% of maximal response intensity) reference range for administration of the experiment. Determination of ED by sequential method₅₀The administration is started from a low dose, the sedative effect of the rat is observed, if the dosage is "-", the next administration dose is reduced (r is 1.5, equal ratio), and if the dosage is "+" the next administration dose is increased equal ratio; from "-" to "+" or "+" to "-" is a cross, 5 co-crossing experiments were terminated. By dixon-mood method (ED)₅₀ED of drug in rats was calculated as lg-1(Σ C/Σ t))₅₀. And a 95% confidence interval of 95% CI ═ lg-1(1 gED) was calculated₅₀±1.96slgED₅₀)，slgED₅₀＝{[∑M-(∑C)2/∑t]/(∑t·(∑t-1)}1/2。

According to the measured ED₅₀Single tail vein injection of 2ED in rats following the above administration method₅₀Drug, sedation of observed ratsAnd (5) effect. By comparison with the clinical etomidate fat emulsion (foley), it can be seen that etomidate-short peptide preparation has equivalent anesthetic effect to the commercial etomidate preparation (foley), and no obvious adverse reaction occurs (table 1).

TABLE 1 comparison of Etomidate-oligopeptide formulations with Forley anesthesia

Example 12: loading of propofol with short peptides

Materials: (Ala)₆-Pro-Lys-Lys-Pro-(Ala)₆APK, for short, entrusted to Shanghai Boratae Biotech limited for chemical synthesis; propofol was purchased from Sigma-Aldrich.

Preparing a short peptide mother solution: APK was dissolved in 0.9% saline at 1mM, vortexed and sonicated for 10 min.

0.1mL of propofol is taken and is dripped into 9.9mL of short peptide mother liquor, ultrasonic treatment is carried out for 20min after vortex, and then stirring is carried out for 40min under the condition of magnetic stirring at room temperature of 2000rpm/min, and the propofol is stored at room temperature.

Example 13: anesthetic effect of single injection of propofol-oligopeptide preparation into rat tail vein

Healthy adult male SD rats (body weight: 295. + -.14 g) were used. Rats were placed in a fixture, the tail was exposed, the lateral tail vein was found, and the needle was placed into the tube after alcohol wiping (propofol-short peptide formulation was prepared using the method of example 11). 0.6mL of medicine is uniformly administered at the administration speed of 0.1mL/s, after the administration is finished, 0.05mL of air is pushed to ensure that the medicine completely enters the tail vein, the indwelling needle is pulled out, and the cotton swab is pressed to stop bleeding. Rats were quickly removed and placed in empty cage sites, and rat responses were observed for sedation scoring and adverse events were recorded (CFDA "guidelines for drug word administration toxicity" (prosecution 2013-05-26)). Sedation score (Psychopharmacology 1996, 125: 105-: the muscle tension of the four limbs is normal, the autonomous activity can be kept, and the response is sensitive for 0 minute; apparent thigmotaxis (rats tend to stay close to the cage rim) for 1 point; retreating and balancing disorder for 2 minutes; forelimb erection less than 60 degrees, ataxia 3 points; lying prostrate, unable to stand, can only support 4 minutes by the belly; the righting reflection disappears for 5 minutes. Observation of disappearance of righting reflex (Anesthesiology 2000,93 (3): 837-: the righting reflex disappeared and lasted for more than 30 seconds was marked as "+", otherwise as "-".

A ramp-up experiment was performed starting from the 1mg/kg dose and finding a "+" and "-" dose, respectively, as ED₅₀Reference range for administration of the experiment. Determination of ED by sequential method₅₀The administration is started from a low dose, the sedative effect of the rat is observed, if the dosage is "-", the next administration dose is reduced (r is 1.5, equal ratio), and if the dosage is "+" the next administration dose is increased equal ratio; from "-" to "+" or "+" to "-" is a cross, 5 co-crossing experiments were terminated. By dixon-mood method (ED)₅₀ED of drug in rats was calculated as lg-1(Σ C/Σ t))₅₀. And calculating 95% confidence interval 95% CI ═ lg-1 (lgED)₅₀±1.96slgED₅₀)，slgED₅₀＝{[∑M-(∑C)2/∑t]/(∑t·(∑t-1)}1/2。

By comparison with clinically used propofol fat emulsion formulation (diprenia) ED₅₀It can be seen that the propofol-short peptide formulation is comparable to the clinical commercial formulation of propofol (diprenia) in its anesthetic effect (table 2).

TABLE 2ED of Propofol-short peptide formulations with Propofol (Diptillol)₅₀Comparison of

The results of examples 5, 9, 11 and 13 show that the short peptide APK of the present invention can be loaded with various hydrophobic drugs and that the drugs exhibit the equivalent efficacy as commercially available (fat emulsion) preparations at equivalent doses.

Example 14: effect of APK short peptide on ovarian cancer cells

Human ovarian cancer cells skov3 at 5X 10³Cells/well density were seeded in 96-well plates and incubated for 24 hours. Removing supernatant, adding short peptides with different concentrations, incubating for 48h, and determining the cell viability by adopting a cck-8 reagent method. Reflecting the rate of cell viabilityThe OD value of (b) was measured at a wavelength of 490nm using a microplate fluorometer.

The result shows that the APK short peptide has no obvious inhibition on tumor cells, and no obvious cytotoxicity is proved (figure 6).

Example 15: nanostructure characterization

From FIGS. 7 to 10, it can be seen that the drug encapsulated by the short peptide is a nano-sized spherical micelle.

In the conventional research of single hydrophobic tail amphiphilic short peptide, a great deal of literature indicates that the amphiphilic short peptide obtained can form similar nano-carriers by using different hydrophobic amino acids as hydrophobic tails and/or different hydrophilic amino acids as hydrophilic heads (ProcNatlAcadsi USA 2002, 99: 5355-5360; Langmuir2003, 19: 4332.); the length of the hydrophobic tail, i.e. the number of hydrophobic amino acids, can also be arbitrarily chosen from 4-8 without substantially altering the ability of the short peptide to self-assemble into nanocarriers (Nano Lett 2002, 2: 687; JPeptSci2018, 24: e 3062.).

It can be reasonably speculated that the substitution of the hydrophilic head amino acid Glu of the APK with 2 hydrophobic tails for other hydrophilic amino acids, and/or the substitution of the hydrophobic tail amino acid Ala of the APK for other hydrophobic amino acids, and/or the change of the length of the hydrophobic tail (the value of the single-side tail is any in 4-8 amino acids) does not substantially change the self-assembly ability of the short peptide of the invention to form nanocarriers.

This will be illustrated below:

example 16: characterization of Gemini secondary structures of different short peptides

APK, APE, APKE, GAVPK and AVLFPK, and the short peptides are chemically synthesized by Shanghai Boratae Biotech limited.

The APE, APKE, GAVPK, AVLFPK sequences are shown in Table 3.

TABLE 3 APE, APKE, GAVPK, AVLFPK sequences

Note: ac represents an acetyl group.

The circular dichroism spectrum of each short peptide is measured by a circular dichroism spectrometer, so that the short peptides are judged to have similar secondary structures. Dissolving each short peptide in water to prepare a 0.1mM solution, placing 800 mu L of the solution in a quartz cuvette with an optical path length of 2mM, and measuring the circular dichroism spectrum of the solution within the range of 185-260 nm at the temperature of 25 ℃.

As shown in FIG. 11, all the short peptides showed a negative peak around 195nm, indicating that all the designed short peptides have random freely extended secondary structures similar to APK, and thus they were judged to have a typical gemini structure via the turn formed by Pro as in APK.

Example 17: characterization of self-assembly Capacity of different short peptides

APK, APE, APKE, GAVPK and AVLFPK short peptides were chemically synthesized by Shanghai Bordetella Biotech Co. Each short peptide was prepared into a 0.5mM aqueous solution, thioflavin T (ThT, available from Sigma-Aldrich) was added to 500. mu.L of the short peptide solution at a final concentration of 10. mu.M, and the fluorescence spectrum (excitation wavelength set at 450nm) was measured with a fluorescence spectrophotometer within the range of 460 to 600nm to determine whether or not APK-like self-assembly behavior existed. As shown in FIG. 12, all short peptides showed typical ThT fluorescence peaks around 490nm, indicating that all designed short peptides have similar self-assembly behavior as APK.

Example 18: characterization of different oligopeptide loading modes for forming nanospheres by hydrophobic drug pyrene

APK, APE, APKE, GAVPK and AVLFPK short peptides entrust Shanghai Boratae Biotech limited company to carry out chemical synthesis; pyrene was purchased from Sigma-Aldrich.

Preparing a short peptide mother solution: each short peptide was dissolved in water at 0.5mM and sonicated for 5 min.

Preparing pyrene mother liquor: pyrene powder was dissolved in DMSO at 10 mM.

From FIG. 13, it can be seen that each short peptide can entrap pyrene to form a nanosphere structure with uniform size.

The results of examples 16 to 18 show that the compounds of the present invention have the general formula (Y)_n-Pro-X-X-Pro-(Y)_n(wherein Y is hydrophobic amino acid, X is hydrophilic amino acid, and n is 4-8) the various short peptides of the structure can form a typical Gemini structure by virtue of a corner formed by Pro, have similar self-assembly behaviors, and can wrap hydrophobic drugs to form a nanosphere structure. It can therefore be reasonably concluded that: having the general formula of the invention having the general formula (Y)_n-Pro-X-X-Pro-(Y)_nThe compound formed by the short peptide with the structure wrapping the hydrophobic drug can generate the drug effect similar to that of the compound formed by APK wrapping the hydrophobic drug.

In conclusion, the short peptide can effectively wrap hydrophobic drugs to form nanospheres, and can play a drug effect equivalent to that of a commercially available fat emulsion preparation.

SEQUENCE LISTING

<110> Sichuan university Hospital in western China

<120> Gemini amphiphilic short peptide and application thereof as hydrophobic drug carrier

<130>GYKH1533-2019P018601CC

<160>6

<170>PatentIn version 3.5

<210>1

<211>16

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>1

Ala Ala Ala Ala Ala Ala Pro Lys Lys Pro Ala Ala Ala Ala Ala Ala

1 5 10 15

<210>2

<211>7

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>2

Ala Ala Ala Ala Ala Ala Lys

1 5

<210>3

<211>16

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>3

Ala Ala Ala Ala Ala Ala Pro Glu Glu Pro Ala Ala Ala Ala Ala Ala

1 5 10 15

<210>4

<211>16

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>4

Ala Ala Ala Ala Ala Ala Pro Lys Glu Pro Ala Ala Ala Ala Ala Ala

1 5 10 15

<210>5

<211>16

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>5

Gly Val Val Ala Ala Ala Pro Lys Lys Pro Val Gly Gly Ala Val Val

1 5 10 15

<210>6

<211>14

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>6

Leu Val Phe Phe Ala Pro Lys Lys Pro Ala Phe Phe Val Leu

1 5 10

Claims

1. A gemini amphiphilic short peptide is characterized by having the following general formula: (Y) n-Pro-X-X-Pro- (Y) n, wherein X is hydrophilic amino acid, Y is hydrophobic amino acid, and n represents the number of hydrophobic amino acid and has a value range of 4-8.

2. The short peptide of claim 1, wherein: the N end and/or the C end of the short peptide are/is provided with chemical modification;

the N-terminal chemical modification is alternatively selected from: alkylacylation, biotin labeling, fatty acid modification, benzoylation, 2-aminobenzoylation, maleimide, haloalkanoylation, succinylation, hydrazinenicotinamide, fluorophore labeling (such as FAM, FITC, TAMRA, etc.);

the C-terminal chemical modification is alternatively selected from: amidation, esterification, hydroformylation, alcohol alkylation, succinylation, and fluorescent group labeling (such as AMC, CMK, FMK, etc.).

3. The short peptide of claim 1, wherein: (Y) n is any one or combination of glycine, alanine, valine, leucine, isoleucine or phenylalanine according to any sequence.

4. The short peptide of claim 3, wherein: (Y) n is any one or combination of several of glycine, alanine and valine in any sequence; preferably, Y is alanine.

5. The short peptide of claim 1, wherein: n is 5 to 6.

6. The short peptide according to claims 1-5, characterized in that: X-X is any one or the combination of two of serine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine according to any sequence.

7. The short peptide of claim 6, wherein: x is selected from glutamic acid or lysine; preferably, X is lysine.

8. The short peptide of claim 1, wherein: the amino acid sequence of the short peptide is shown as SEQ ID NO.1, 3, 4, 5 or 6; preferably, as shown in SEQ ID NO. 1.

9. The short peptide according to any one of claims 2 to 8, wherein: the N-terminal is chemically modified into acetylation;

and/or, the C-terminal chemical modification is amidation.

10. Use of a short peptide according to any one of claims 1 to 9 for the preparation of a carrier for a hydrophobic drug.

11. Use according to claim 10, characterized in that: the carrier is a micelle formed by self-assembly of the short peptide; preferably, the micelles are spherical micelles.

12. Use according to claim 10 or 11, characterized in that: the hydrophobic drug is paclitaxel, adriamycin, curcumin, docetaxel, doxorubicin, vincristine, camptothecin, hydroxycamptothecin, etoposide, tretinoin, fluorouracil, methotrexate, teniposide, daunorubicin, aclacinomycin, sorafenib, methylprednisolone, minocycline, cisplatin, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, chlorobenzenesulfone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, raloxifene, clopiroxicam, indomethacin, ibuprofen, tamoxifen, diclofenac, naproxen, piroxicam, lativir, efavirenz, nelfinavir, atazanavir, ritonavir, sirolimus, ambroxol, tacrolimus, terfenadine, estradiol, Any one or a mixture of more of vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, alprostadil, composite fat-soluble vitamin, dexamethasone, flurbiprofen axetil, clevidipine, brucea javanica oil, cyclosporine, insulin and the like;

13. A hydrophobic drug carrier characterized by: the carrier is a micelle formed by self-assembly of the short peptide according to any one of claims 1 to 9; preferably, the micelles are spherical micelles.

14. A nanocarrier formulation, comprising: the preparation uses hydrophobic drugs as active ingredients and micelles formed by self-assembly of the short peptides of any one of claims 1 to 9 as carriers.

15. The nanocarrier formulation of claim 14, wherein: the content ratio of the short peptide to the active ingredient is 5 mu mol: 1-100 mg.

16. The nanocarrier formulation of claim 14 or 15, wherein: the hydrophobic drug is paclitaxel, adriamycin, curcumin, docetaxel, doxorubicin, vincristine, camptothecin, hydroxycamptothecin, etoposide, tretinoin, fluorouracil, methotrexate, teniposide, daunorubicin, aclacinomycin, sorafenib, methylprednisolone, minocycline, cisplatin, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, chlorobenzenesulfone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, raloxifene, clopiroxicam, indomethacin, ibuprofen, tamoxifen, diclofenac, naproxen, piroxicam, lativir, efavirenz, nelfinavir, atazanavir, ritonavir, sirolimus, ambroxol, tacrolimus, terfenadine, estradiol, Any one or a mixture of more of vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, alprostadil, composite fat-soluble vitamin, dexamethasone, flurbiprofen axetil, clevidipine, brucea javanica oil, cyclosporine, insulin and the like;