CN116333049B

CN116333049B - Atypical hydrophobic amino acid-based self-assembled short peptide and application thereof

Info

Publication number: CN116333049B
Application number: CN202310186188.2A
Authority: CN
Inventors: 邱峰; 彭飞; 张文胜; 刘静
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2024-03-29
Anticipated expiration: 2040-07-03
Also published as: WO2022001701A1; CN116425833A; CN111748016B; CN111748016A; CN116333049A; CN116425833B

Abstract

The invention provides a self-assembled short peptide based on atypical hydrophobic amino acid and application thereof, belonging to the field of drug carriers. The amino acid sequence of the short peptide is shown as SEQ ID NO. 1. The short peptide can load a plurality of hydrophobic drugs through a molecular self-assembly mechanism to form nanospheres with uniform particle sizes, so that the drugs can be effectively transported into cells or tissues to exert the drug effect, the short peptide is safe and has no obvious cytotoxicity, the preparation process is simple, and the short peptide is a carrier of the hydrophobic drugs with great development prospect.

Description

Atypical hydrophobic amino acid-based self-assembled short peptide and application thereof

The present application is a divisional application filed on the invention patent application with the application number of 202010638716.X and the application date of 2020, 07 and 03.

Technical Field

The invention belongs to the field of drug carriers, and in particular relates to a self-assembled short peptide based on atypical hydrophobic amino acid and application of the self-assembled short peptide as a hydrophobic drug carrier.

Background

In the current drug development, many potential small molecule drugs are hydrophobic substances and have poor water solubility, and the preparation generally needs to be dispersed and dissolved in aqueous solution or aqueous solution by using a carrier to be administered by injection. The hydrophobic drug carriers widely used in clinic at present mainly comprise materials such as lipid and the like. Taking the most commonly used anticancer drug taxol as an example, the drugs are marketedTaxol injection Taxol contains about 527mg/mL polyoxyethylene castor oil and 49.7% absolute ethyl alcohol as solvent; the most commonly used general anesthetic drugs such as propofol are fat emulsion preparations taking lipid molecules such as natural soybean oil, egg yolk lecithin and the like as carriers; the vasodilator adopts lipid molecules such as soybean oil, lecithin, oleic acid and the like as a carrier of fat emulsion injection; the antihypertensive drug clevidipine butyrate also contains injectable oil and phospholipid components. However, there are still problems with the clinical use of lipid components such as poor stability (Anesth Analg 2003, 97:769-771), causing pain for injection (Acta Anaesthesiol Scand 2001, 45:839-841), inducing hyperlipidemia (Lancet 2001, 357:606-607), and susceptibility to infection by rapid bacterial growth (Anesth Analg 1999, 88:209-212). The Taxol injection Taxol also has more adverse reactions in the clinical use process, such as acute hypersensitivity (Allergy Asthma Immunol Res 2016, 8:174-177), neurotoxicity (Nanomedicine 2015, 11:1925-1938) and the like; the problem of propofol fat emulsion injection with propofol infusion syndrome (propofol infusion syndrome, PRIS) (Crit Care 2015, 19:398); clevidipine butyrate injection limits the use of patients with severe lipid metabolism disorders (see clevidipine butyrate injection instructions), and so on. The above problems are essentially all related to lipid components. Therefore, it is very promising to develop hydrophobic drug carrier materials that do not contain lipid components.

Currently, some non-lipid materials have been applied in clinic, such as a preparation Abraxane of human serum albumin-entrapped paclitaxel, which has been marketed in the United states in 2005, and has small side effects, short administration time and reduced adverse reactions (Int J Nanomedicine 2009, 4:99-105). However, this dosage form is limited by the human blood source of the albumin carrier and the corresponding risk of microbial and viral contamination, which is expensive. Paclitaxel formulations Cynviloq have been marketed in korea in 2007 (Adv Drug Deliver Rev 2017,122: 20-30) using polymer mPEG-PLLA materials, but polymer materials are expensive, synthesis processes are complicated, and polymer nanotoxicity still needs to be continued. In terms of biosafety, the artificially synthesized short peptide has unique advantages and is a very potential drug carrier material. For example, chinese patent No. 00105625.5 (ZL patent No. CN1148227C entitled "therapeutic compound and use thereof") discloses a therapeutic compound based on a short peptide carrier and use thereof. However, the invention is to form therapeutic compounds by chemical combination of paclitaxel, glutamic acid and aspartic acid, rather than directly load the drug by nanoparticles formed by self-assembly of short peptides.

Artificially designed self-assembled short peptides are receiving increasing attention as a material of the type which is internationally emerging in recent years (Nano Today 2016, 11:41-60). The polypeptide is a kind of short peptide molecules which are artificially designed and synthesized and are composed of natural amino acids, and can self-assemble in aqueous solution to form structures such as nanotubes, nanofibers, nanovesicles or nanoparticles. Because the components of the self-assembled short peptide are short peptides, degradation products are natural L-type amino acids, and the self-assembled short peptide has good biocompatibility and safety. The artificial design and synthesis mode can make the sources of the materials clear, the quality is controllable, and the functional modification is convenient.

However, existing self-assembled short peptides have their own disadvantages when used as hydrophobic drug carriers. The self-assembled short peptides for loading hydrophobic drugs are reported in the prior art in two major types, namely, a lamellar structure with a hydrophobic surface is formed to wrap drug particles, and the obtained drug-short peptide complex is mostly irregular micron-sized particles, and has poor uniformity and stability (Int J Nanomed 2011, 6:2143-2153); the other forms nano-scale micelle with compact hydrophobic inner core to load medicine, and has even particle size and better dispersivity, but very limited medicine loading capacity (Int J Nanomed 2015, 10:847-858).

The self-assembled short peptide reported in the literature has lower drug loading rate, poor preparation stability and significantly inferior drug loading capacity compared with most of natural lipid molecules, and the problems are the main reasons that the self-assembled short peptide carrier has not been popularized at present. In order to develop a self-assembled oligopeptide hydrophobic drug carrier with conversion value, the drug loading capacity of the self-assembled oligopeptide hydrophobic drug carrier needs to be improved as much as possible while ensuring the formation of uniform and stable drug loading nanoparticles.

The existing self-assembled short peptide contains a large amount of hydrophobic amino acids to drive the self-assembly and realize the loading of the hydrophobic drugs, and the existence of the large amount of hydrophobic amino acids leads to the limited water solubility of the short peptide, and is easy to aggregate and precipitate under high concentration. The solubility of the short peptide material itself and the loading capacity of the material to hydrophobic drugs appear to be a group of irreconcilable contradictions, greatly limiting the application of self-assembled short peptides as hydrophobic drug carriers.

Disclosure of Invention

The invention aims to provide a novel self-assembled short peptide based on atypical hydrophobic amino acid, which has a drug carrying effect comparable to that of a lipid carrier, and the technical scheme is as follows:

a short peptide has an amino acid sequence shown in SEQ ID NO. 1.

The short peptide as described above, wherein the N-terminal and/or C-terminal of the short peptide is chemically modified;

the N-terminal chemical modification is alternatively selected from: alkanoylation, biotin labeling, fatty acid modification, benzoylation, 2-aminobenzoylation, maleimide, haloalkylation, succinylation, dihydrazineimide, fluorescent group labeling;

the C-terminal chemical modification is alternatively selected from: amidation, esterification, hydroformylation, alcoholization, succinylation, and fluorescent group labeling.

The short peptide, wherein the fluorescent group chemically modified at the N-terminal is FAM, FITC or TAMRA;

the fluorescent group chemically modified at the C end is AMC, CMK and FMK.

The short peptide as described above, wherein the N-terminal is chemically modified to be acetylated; and/or, the C-terminus is chemically modified to amidate.

The use of the foregoing short peptides in the preparation of a hydrophobic drug carrier.

The use as described above, wherein the carrier is a nanosphere formed by self-assembly of the short peptide.

As in the case of the use described above, the hydrophobic drug is paclitaxel, doxorubicin, curcumin, docetaxel, vincristine, camptothecin, hydroxycamptothecin, etoposide, retinoic acid, fluorouracil, methotrexate, teniposide, daunorubicin, aclacin, sorafenib, methylprednisone, minocycline, cisplatin, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, chlorophenylsulfone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, raloxifene, flurbiprofen, indomethacin, ibuprofen, tamoxifen, simoxicillin, simvastatin, and other drugs any one or a mixture of several of diclofenac, naproxen, piroxicam, ralteravir, efavirenz, nelfinavir, atazanavir, ritonavir, sirolimus, busulfan, tacrolimus, ta Lin Luoer, terfenadine, estradiol, vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, alprostadil, complex liposoluble vitamins, dexamethasone, flurbiprofen ester, clevidipine butyrate, brucea oil, cyclosporine, insulin, adefovir, chloroquine, hydroxychloroquine, fampicvir, lopinavir, ritonavir and the like.

The use as described above, wherein the hydrophobic drug is paclitaxel, doxorubicin, etomidate or propofol.

A hydrophobic drug carrier, which is a nanosphere formed by self-assembly of the aforementioned short peptides.

A nano-carrier preparation takes a hydrophobic drug as an active ingredient and takes nanospheres formed by self-assembly of the short peptides as carriers.

The content ratio of short peptide to hydrophobic drug in the aforementioned nanocarrier formulation was 5 μmol: 1-100 mg.

As in the case of the nanocarrier formulations described previously, the hydrophobic drug is paclitaxel, doxorubicin, curcumin, docetaxel, vincristine, camptothecin, hydroxycamptothecin, etoposide, retinoic acid, fluorouracil, methotrexate, teniposide, daunorubicin, aclacinomycin, sorafenib, cisplatin, methylprednisone, minocycline, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, chlorophenylsulfone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, raloxifene, flurbiprofen, indomethacin, ibuprofen, tamoxifen, pravastatin, and other drugs any one or a mixture of several of diclofenac, naproxen, piroxicam, ralteravir, efavirenz, nelfinavir, atazanavir, ritonavir, sirolimus, busulfan, tacrolimus, ta Lin Luoer, terfenadine, estradiol, vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, alprostadil, complex liposoluble vitamins, dexamethasone, flurbiprofen ester, clevidipine butyrate, brucea oil, cyclosporine, insulin, adefovir, chloroquine, hydroxychloroquine, fampicvir, lopinavir, ritonavir and the like.

As in the previous nanocarrier formulations, the hydrophobic drug is paclitaxel, doxorubicin, etomidate, or propofol.

As the nano-carrier preparation, the hydrophobic drug is paclitaxel, and the content ratio of the short peptide to the hydrophobic drug is 1 mu mol:1mg;

or the hydrophobic drug is doxorubicin, and the content ratio of the short peptide to the hydrophobic drug is 1 mu mol:1mg;

or the hydrophobic drug is etomidate, and the content ratio of the short peptide to the hydrophobic drug is 5 mu mol:20mg;

or the hydrophobic drug is propofol, and the content ratio of the short peptide to the hydrophobic drug is 9.9 mu mol/100 mg.

Although three amino acids of glutamine (Q), tyrosine (Y) and threonine (T) are generally classified as polar amino acids (hydrophilic amino acids) in textbooks, they have certain hydrophobicity in theory because their side chains contain hydrophobic groups such as dimethylene, benzene ring and methyl, respectively, and can provide hydrophobic effects for self-assembly as an atypical hydrophobic amino acid. Meanwhile, the amide groups at the ends of the glutamine (Q) and asparagine (N) side chains can also form hydrogen bonds to promote self-assembly. On the other hand, the amino acid side chain groups of glutamine (Q), tyrosine (Y), threonine (T), asparagine (N), serine (S) and glycine (G) are uncharged, and thus do not generate electrostatic repulsion, which is also very favorable for self-assembly. The following table summarizes the characteristics of these amino acids that are not traditional hydrophobic amino acids, but may be advantageous for short peptide self-assembly to occur.

Unlike traditional self-assembled short peptide with typical hydrophobic amino acids (alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F), tryptophan (W), methionine (M) and proline (P)) as main components, the short peptide of the invention is self-assembled short peptide mainly composed of atypical hydrophobic amino acids Q, Y, T, and is a brand new design. Compared with the current short peptide carrier, the short peptide has obviously improved drug carrying capacity, and can have better dispersibility and stability in aqueous solution; the short peptide of the invention is used as a carrier of a hydrophobic drug to prepare a preparation, so that the drug effect can reach the level close to the existing lipid carrier drug preparation.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a TEM image of a nanosphere formed after loading of Q5Y (a), Q6TY (b) and Q7GY (c) with pyrene. Scale bar = 100nm.

FIG. 2 is a graph showing fluorescence spectra of Q5Y-loaded pyrene-forming nanoparticle suspensions within 14 days.

Fig. 3 is a TEM image of A5Y loaded with pyrene to form non-uniform nanoparticles. Scale bar = 100nm.

FIG. 4 is a graph showing fluorescence spectra of A5Y-loaded pyrene-forming nanoparticle suspensions within 14 days.

FIG. 5 is a graph showing the proliferation inhibition effect of Q5Y-loaded paclitaxel (Q5Y-PTX) on ovarian cancer cells skov 3.

FIG. 6 is a graph showing the proliferation inhibition effect of Q6 TY-loaded doxorubicin (Q6 TY-DOX) on ovarian cancer cells skov 3.

FIG. 7 is a graph showing the effect of Q5Y, Q TY and Q7GY on ovarian cancer cell skov 3.

Fig. 8 is a TEM image of Q5Y, Q TY and Q7GY loaded paclitaxel forming nanospheres.

Fig. 9 is a TEM image of doxorubicin loaded Q5Y, Q TY and Q7GY to form nanospheres.

Fig. 10 is a TEM image of Q7GY loaded etomidate forming nanospheres.

Fig. 11 is a TEM image of Q7GY loaded propofol forming nanospheres.

FIG. 12 is the fluorescence spectra of short peptides Q5Y, Q3TY2, Q4GY2, Q4NY2, Q5A2SG combined with thioflavin T, showing that they have similar self-assembly behavior.

FIG. 13 is a fluorescence spectrum of the short peptides N5Y, S5Y, D5Y, E5Y, K5Y, R5Y and H5Y combined with thioflavin T, showing that they do not have self-assembly behavior.

Fig. 14 is a TEM image of the short peptides Q3TY2, Q4GY2, Q4NY2, Q5A2SG loaded with the model hydrophobic drug pyrene forming nanospheres.

Detailed Description

Example 1: loading of the short peptides with the model hydrophobic Compound pyrene

The material comprises the following components: ac-Gln-Gln-Gln-Gln-Gln-Tyr-NH ₂ Abbreviated as Q5Y, ac represents acetyl and is shown as SEQ ID NO.1 in the amino acid sequence table; ac-Thr-Gln-Gln-Gln-Gln-Gln-Gln-Tyr-NH ₂ Abbreviated as Q6TY,SEQ ID NO.2 in the amino acid sequence table; ac-Gly-Gln-Gln-Gln-Gln-Gln-Gln-Gln-Tyr-NH ₂ Abbreviated as Q7GY, SEQ ID NO.3 in the amino acid sequence Listing, was delegated to the company Shandong Katsuwonshi Biotechnology Co.

Pyrene was purchased from Sigma-Aldrich.

Q5Y, Q6TY, Q7GY were each dissolved in water at 5mM, and sonicated for 5min. 20mg of pyrene is added into 10mL of short peptide mother liquor, and the mixture is placed at room temperature and magnetically stirred at 2000rpm/min for 4 hours, and then is subjected to ultrasonic treatment for 30 minutes, so that stable milky suspension is formed, and the suspension is placed at 4 ℃ for storage.

Morphological features were observed by high resolution transmission electron microscopy. After diluting the above suspension with water 5 times, 10. Mu.L of the sample solution was applied to a 400 mesh copper wire for 5 minutes, and then blotted with a piece of filter paper. Then, 10. Mu.L of 2% phosphotungstic acid was added thereto for dyeing for 3min. The final staining solution was blotted with filter paper and dried. And then imaging by adopting a transmission electron microscope.

The suspension of the above Q5Y-loaded pyrene was allowed to stand at room temperature, 50. Mu.L of the suspension was taken out from the top layer on days 0, 7 and 14, respectively, diluted to 500. Mu.L with water, and then its fluorescence spectrum at 336nm wavelength excitation was measured with a fluorescence spectrophotometer.

Results:

from FIG. 1, it can be seen that Q5Y, Q6TY and Q7GY can form nanospheres with particle diameters smaller than 100nm, uniform sizes and regular shapes after pyrene is loaded.

As can be seen from FIG. 2, the fluorescence spectrum of the suspension obtained by loading Q5Y with pyrene, whose fluorescence at 480nm from pyrene nanoparticles remained substantially unchanged for 14 days, shows that the nanoparticle suspension formed by loading Q5Y with pyrene remained stable and uniform over a longer period of time.

The results of example 1 show that: Q5Y, Q6TY and Q7GY can load indissolvable model hydrophobic compounds pyrene to form nanospheres with uniform size, so that stable emulsion can be formed at the concentration of up to 2mg/mL, and the short peptides can be used for effectively loading the hydrophobic compounds.

Example 2: loading of the short peptides with the model hydrophobic Compound pyrene

Short peptide Ac-Ala-Ala-Ala-Tyr-NH ₂ Abbreviated as A5Y, ac represents acetyl, SEQ ID NO.4 in the amino acid sequence Listing, and was delegated to chemical synthesis by Shandong Haibotai Biotechnology Co.

Pyrene was purchased from Sigma-Aldrich.

A5Y was dissolved in water according to its maximum solubility (about 0.5 mM) and sonicated for 5min. 20mg of pyrene is added into 10mL of short peptide mother liquor, and the mixture is placed at room temperature and magnetically stirred at 2000rpm/min for 4 hours, and then is subjected to ultrasonic treatment for 30 minutes to form milky suspension, and the milky suspension is placed at 4 ℃ for storage.

The suspension of A5Y-loaded pyrene described above was left to stand at room temperature, 50. Mu.L of the suspension was taken out from the top layer on days 0, 7 and 14, respectively, diluted to 500. Mu.L with water, and then its fluorescence spectrum at 336nm wavelength excitation was measured with a fluorescence spectrophotometer.

Results:

as can be seen from FIG. 3, the particle size of the particles formed after A5Y is loaded with pyrene is larger than 100nm, and the particles are uneven in size and irregular in shape.

As can be seen from FIG. 4, the fluorescence spectrum of the suspension obtained by A5Y-loading pyrene, whose fluorescence peak at 480nm from pyrene nanoparticles was rapidly decreased, shows that the suspension formed by A5Y-loading pyrene was not very stable, and precipitation rapidly occurred during the standing process.

The results of example 2 show that: although the A5Y short peptide mainly composed of typical hydrophobic amino acids can disperse pyrene which is a model hydrophobic compound to form nano-scale particles, the drug carrying capacity is poor due to the limited solubility of the short peptide. The formed drug particles are larger, uneven in size and irregular in shape, so that the formed emulsion is extremely unstable in character and is precipitated quickly. The above results demonstrate that short peptides composed of typical hydrophobic amino acids do not load hydrophobic drugs to form good formulations compared to the materials of the present invention.

Example 3: loading of paclitaxel with short peptides

Materials: Q5Y entrusts the chemical synthesis of Shandong Botai Biotechnology Co., ltd; paclitaxel was purchased from calico biotechnology limited; the absolute ethyl alcohol is purchased in a chemical reagent factory of the Chemielong of the City department.

Preparing a short peptide mother solution: Q5Y was dissolved in water at 1mM and sonicated for 5min.

Preparing paclitaxel mother liquor: paclitaxel powder was dissolved in absolute ethanol at 20 mg/mL.

10mL of short peptide mother liquor is placed under the condition of magnetic stirring at room temperature at 2000rpm/min, 500 mu L of taxol mother liquor is added into the short peptide mother liquor drop by a liquid transfer device (the dosage ratio of Q5Y to taxol is 1 mu mol:1 mg), after the addition is finished, the magnetic stirring is continued for 30min, the ultrasonic treatment is carried out for 10min, and the short peptide mother liquor is preserved at 4 ℃.

Example 4: loading of paclitaxel with short peptides

Q6TY entrusts the chemical synthesis of Shandong Botai Biotechnology Co., ltd; paclitaxel was purchased from calico biotechnology limited; the absolute ethyl alcohol is purchased in a chemical reagent factory of the Chemielong of the City department.

Preparing a short peptide mother solution: q6TY was dissolved in water at 1mM and sonicated for 15min.

500 mu L of taxol mother solution is placed under the condition of magnetic stirring at room temperature of 2000rpm/min, 10mL of short peptide mother solution is added into taxol mother solution drop by a liquid transfer device (the dosage ratio of Q6TY to taxol is 1 mu mol:1 mg), after the addition is finished, the magnetic stirring is continued for 30min, the ultrasonic treatment is carried out for 10min, and the solution is preserved at the temperature of 4 ℃.

Example 5: loading of paclitaxel with short peptides

Q7GY entrusts the chemical synthesis of Shanghai Biotechnology Co., ltd; paclitaxel was purchased from calico biotechnology limited; the absolute ethyl alcohol is purchased in a chemical reagent factory of the Chemielong of the City department.

Q7GY is dissolved in 2mL DMSO together with 5mM and 5mg/mL paclitaxel powder (the dosage ratio of Q7GY to paclitaxel is 1 mu mol:1 mg), and the solution is sonicated for 5min; removing the organic solvent by adopting a vacuum dryer, redissolving with 10mL of water, performing ultrasonic treatment for 10min, and storing at 4 ℃.

Example 6: inhibition of ovarian cancer cells by paclitaxel-oligopeptide formulations

Human ovarian cancer cells skov3 at 5×10 ³ Cell/well density was seeded in 96-well plates and incubated for 24 hours. The supernatants were removed, paclitaxel-oligopeptide formulations (prepared as in example 3) were added at various concentrations, and paclitaxel (commercially available paclitaxel drug) at corresponding concentrations were used as controls, and after 48h incubation, cell viability was determined using cck-8 kit. OD values reflecting cell viability were measured using a microplate fluorometer at 490nm wavelength.

Results:

as shown in FIG. 5, the paclitaxel-short peptide preparation (Q5Y-PTX) of the present invention has more remarkable inhibitory effect on tumor cells than the paclitaxel preparation Taxol (Taxol) on the market at PTX concentration of 0.01. Mu.g/mL; with the increase of the concentration, the inhibition effect of the medicine tends to be saturated, but the preparation still has stronger inhibition effect on ovarian cancer cells.

Example 7: loading of doxorubicin with short peptides

Reagent: doxorubicin hydrochloride was purchased from calico biotechnology limited and triethylamine was purchased from the metropolitan chemical reagent plant.

Desalting doxorubicin hydrochloride: 10mg of doxorubicin hydrochloride powder was dissolved in 10ml of methanol, 10. Mu.L of triethylamine was added thereto, and the mixture was magnetically stirred overnight, and the organic solvent was evaporated under vacuum to obtain doxorubicin powder.

Preparing doxorubicin mother solution: doxorubicin powder was dissolved in absolute ethanol at 20 mg/mL.

10mL of short peptide mother liquor is placed under the condition of magnetic stirring at room temperature at 2000rpm/min, 500 mu L of doxorubicin mother liquor is added into the short peptide mother liquor drop by a liquid transfer device (the dosage ratio of Q5Y to doxorubicin is 1 mu mol:1 mg), after the addition is finished, the magnetic stirring is continued for 30min, the ultrasonic treatment is carried out for 10min, and the short peptide mother liquor is preserved at 4 ℃.

Example 8: loading of doxorubicin with short peptides

Preparing a short peptide mother solution: q6TY was dissolved in water at 1mM and sonicated for 5min.

500 mu L of doxorubicin mother solution is placed under the condition of magnetic stirring at room temperature of 2000rpm/min, 10mL of short peptide mother solution is added into the doxorubicin mother solution drop by a pipettor (the dosage ratio of Q6TY to doxorubicin is 1 mu mol:1 mg), after the addition is finished, the magnetic stirring is continued for 30min, the ultrasonic treatment is carried out for 10min, and the solution is preserved at 4 ℃.

Example 9: loading of doxorubicin with short peptides

Q7GY is dissolved in 2mL DMSO according to 5mM and doxorubicin powder of 5mg/mL (namely, the dosage ratio of Q7GY to doxorubicin is 1 mu mol:1 mg), and the solution is sonicated for 5min; removing the organic solvent by adopting a vacuum dryer, redissolving with 10mL of water, performing ultrasonic treatment for 10min, and storing at 4 ℃.

Example 10: inhibition of ovarian cancer cells by doxorubicin-short peptide formulations

Human ovarian cancer cells skov3 at 5×10 ³ Cell/well density was seeded in 96-well plates and incubated for 24 hours. The supernatant was removed, doxorubicin-short peptide preparations (prepared using the method of example 8) at various concentrations were added, and doxorubicin hydrochloride at corresponding concentrations were used as a control, and after incubation for 48 hours, the cell viability was determined using cck-8 kit. Reflecting the cell viabilityThe OD of (C) was measured at 490nm using a microplate fluorometer.

Results:

the comparison of the experimental group and the control group shows that the doxorubicin-short peptide preparation (Q6 TY-DOX) has remarkable inhibition effect on tumor cells, which is equivalent to that of the control group drug doxorubicin hydrochloride (figure 6).

Example 11: loading of etomidate with short peptides

Q7GY entrusts the chemical synthesis of Shanghai Biotechnology Co., ltd; etomidate was purchased from dalteparin biotechnology limited; the absolute ethyl alcohol is purchased in a chemical reagent factory of the Chemielong of the City department.

Preparing a short peptide mother solution: q7GY was dissolved in 0.9% physiological saline at 0.5mM, and sonicated for 10min after vortexing.

20mg of etomidate is weighed, added into 10mL of short peptide mother liquor (the ratio of Q7GY to etomidate dosage is 5 mu mol:20 mg), vortexed and then subjected to ultrasonic treatment for 20min, and then placed under the condition of magnetic stirring at room temperature at 2000rpm/min, stirred for 40min and stored at room temperature.

Example 12: anesthetic effect of single injection of etomidate-oligopeptide preparation into tail vein of rat

Healthy adult male SD rats (body weight: 295.+ -.14 g) were used. Rats were placed in a holder, tail exposed, lateral tail vein found, and left needle cannulated for administration after alcohol swabbing (etomidate-oligopeptide formulation was prepared using the procedure of example 11). And 0.6mL of medicine is uniformly administered at a constant administration speed of 0.1mL/s, and after the administration is finished, 0.05mL of air is pushed to ensure that the medicine completely enters the tail vein and then the indwelling needle is pulled out, and the cotton swab presses for hemostasis. Rats were quickly removed and placed in empty cages, and the rat responses were observed for sedation scoring and adverse effects were recorded. Sedation score (Psychopharmacology 1996, 125:105-112): the tension of the limb muscles is normal, the autonomous activity can be kept, and the response sensitivity is 0 minutes; apparent thigmotaxis (rats tend to stay in a position near the cage rim) for 1 minute; withdrawal imbalance for 2 minutes; the forelimbs are erected to be less than 60 degrees, and ataxia is 3 minutes; prone, unable to stand, can only lean on abdomen to support 4 minutes; the specular reflection disappeared for 5 minutes. Observation of disappearance of the specular reflection (Anesthesiology 2000,93 (3): 837-843): the specular reflection disappears and continues for more than 30 seconds as "+", otherwise as "-".

Starting from a dose of 1mg/kg, a climbing experiment was performed to find out the dose of "+" and "-" respectively, as ED ₅₀ (half of the effective amount, the amount that causes 50% of the maximum response intensity). Sequential determination of ED ₅₀ Starting from the low dose, the sedative effect of the rats was observed, with the next dose decreasing (r=1.5, equal ratio) if "-", and with the next dose increasing equal ratio if "+"; from "-" to "+" or "+" to "-" is a crossover, 5 crossover experiments were terminated. By the dixon-mood method (ED ₅₀ =lg-1 (Σc/Σt) to calculate ED of the drug in rat body ₅₀ . And 95% confidence interval 95% ci=lg-1 (lgED) was calculated ₅₀ ±1.96slgED ₅₀ )，slgED ₅₀ ＝{[ΣM-(ΣC)2/Σt]/(Σt·(Σt-1)}1/2。

Based on measured ED ₅₀ A single tail vein injection of 2ED was performed on rats according to the above administration method ₅₀ Drug, the sedative effect of rats was observed.

Results:

by comparison with the clinically used etomidate fat emulsion formulation (felide), it can be seen that the etomidate-short peptide formulation was comparable to the etomidate commercial formulation (felide) in anesthetic effect, and no significant adverse reactions occurred (table 1).

TABLE 1 comparison of Etomidate-short peptide formulations and Etomidate (Furling) efficacy

Example 13: loading of Propofol with short peptides

Q7GY entrusts the chemical synthesis of Shanghai Biotechnology Co., ltd; propofol was purchased from Sigma-Aldrich.

Preparing a short peptide mother solution: q7GY was dissolved in 0.9% physiological saline at 1mM, and sonicated for 10min after vortexing.

100mg of propofol is taken and added into 9.9mL of short peptide mother liquor in a dropwise manner, the mixture is subjected to ultrasonic treatment for 20min after vortex, and then the mixture is placed under the condition of magnetic stirring at room temperature of 2000rpm/min, stirred for 40min and stored at room temperature.

Example 14: anesthetic effect of single injection of propofol-short peptide formulation into rat tail vein

Healthy adult male SD rats (body weight: 295.+ -.14 g) were used. Rats were placed in a holder, tail exposed, lateral tail vein found, and left for needle catheterization after alcohol swabbing (propofol-short peptide formulation was prepared using the procedure of example 13). And 0.6mL of medicine is uniformly administered at a constant administration speed of 0.1mL/s, and after the administration is finished, 0.05mL of air is pushed to ensure that the medicine completely enters the tail vein and then the indwelling needle is pulled out, and the cotton swab presses for hemostasis. Rats were quickly removed and placed in empty cages, and rat responses were observed for sedation scoring and adverse effects were recorded (CFDA "guidelines for drug word dosing toxicity" (solicitation opinion manuscript 2013-05-26)). Sedation score (Psychopharmacology 1996, 125:105-112): the tension of the limb muscles is normal, the autonomous activity can be kept, and the response sensitivity is 0 minutes; apparent thigmotaxis (rats tend to stay in a position near the cage rim) for 1 minute; withdrawal imbalance for 2 minutes; the forelimbs are erected to be less than 60 degrees, and ataxia is 3 minutes; prone, unable to stand, can only lean on abdomen to support 4 minutes; the specular reflection disappeared for 5 minutes. Observation of disappearance of the specular reflection (Anesthesiology 2000,93 (3): 837-843): the specular reflection disappears and continues for more than 30 seconds as "+", otherwise as "-".

Starting from a dose of 1mg/kg, a climbing experiment was performed to find out the dose of "+" and "-" respectively, as ED ₅₀ Experimental dosing reference range. Sequential determination of ED ₅₀ Starting from the low dose, the sedative effect of the rats was observed, with the next dose decreasing (r=1.5, equal ratio) if "-", and with the next dose increasing equal ratio if "+"; from "-" to "+" or "+" to "-" is a crossover, 5 crossover experiments were terminated. By the dixon-mood method (ED ₅₀ =lg-1 (Σc/Σt) to calculate ED of the drug in rat body ₅₀ . And 95% confidence interval 95% ci=lg-1 (lgED) was calculated ₅₀ ±1.96slgED ₅₀ )，slgED ₅₀ ＝{[ΣM-(ΣC)2/Σt]/(Σt·(Σt-1)}1/2。

Results:

ED by comparison with clinically used Propofol fat emulsion formulations (Depu Li Ma) ₅₀ It can be seen that the anesthetic effect of the propofol-short peptide formulation was slightly better than that of the clinically marketed propofol formulation (Depu Li Ma) (Table 2).

TABLE 2 Propofol-short peptide formulations and ED of Propofol (Depu Li Ma) ₅₀ Comparison

Example 15: effect of short peptides on ovarian cancer cells

Human ovarian cancer cells skov3 at 5×10 ³ Cell/well density was seeded in 96-well plates and incubated for 24 hours. The supernatant was removed, and after incubation for 48h, the cell viability was determined using the cck-8 reagent method with different concentrations of Q5Y, Q TY and Q7GY short peptides. OD values reflecting cell viability were measured using a microplate fluorometer at 490 nm.

From the results, it can be seen that the Q5Y, Q6TY and Q7GY short peptides do not significantly inhibit tumor cells, proving that they have no significant cytotoxicity (fig. 7).

Example 16: nanostructure characterization

The morphological characteristics of nanospheres formed by hydrophobic drugs such as paclitaxel, doxorubicin, etomidate, propofol and the like loaded on Q5Y, Q TY and Q7GY are observed through a high-resolution Transmission Electron Microscope (TEM).

10. Mu.L of the sample solution was applied to a 400 mesh copper mesh for 5min, and then blotted dry with a piece of filter paper. Then, 10. Mu.L of 2% phosphotungstic acid was added thereto for dyeing for 3min. The final staining solution was blotted with filter paper and dried. And then imaging by adopting a transmission electron microscope.

From fig. 8 to 11, it can be seen that the short peptide entrapped drug becomes a nano-scale spherical micelle.

The results show that: the number of hydrophobic amino acids can vary within a range without materially altering the ability of the short peptides to self-assemble into nanocarriers.

In addition, the polypeptide composed of the atypical hydrophobic amino acid Q as a main component and Q, Y, T according to different arrangements and combinations is supplemented with N, S, G and other uncharged amino acids and A, V, L, I, F and other traditional hydrophobic amino acids, and the self-assembly capacity of the short peptide of the invention to form the nano-carrier is not changed essentially. This will be exemplified below:

example 17: characterization of the self-Assembly Capacity of different short peptides

The short peptides Q5Y, Q TY2, Q4GY2, Q4NY2, Q5A2SG, N5Y, S5Y, D5Y, E5Y, K5Y, R Y and H5Y were submitted to chemical synthesis by the company Heterotai Biotechnology Co.

The sequences of Q3TY2, Q4GY2, Q4NY2, Q5A2SG, N5Y, S5Y, D5Y, E5Y, K5Y, R5Y and H5Y are shown in Table 3.

TABLE 3 amino acid sequences of Q3TY2, Q4GY2, Q4NY2 and Q5A2SG

Note that: ac represents acetyl.

Each of the short peptides was prepared as a 0.5mM aqueous solution, thioflavin T (ThT, available from Sigma-Aldrich Co.) was added to each 500. Mu.L of the short peptide solution at a final concentration of 10. Mu.M, and the fluorescence spectrum (excitation wavelength set at 450 nm) in the range of 460 to 600nm was measured by a fluorescence spectrophotometer to determine the presence or absence of self-assembly behavior similar to Q5Y.

Results:

as shown in FIG. 12, all of the short peptides containing Q, Y, T as the main component exhibited typical THT fluorescence peaks around 490nm, indicating that these short peptides all have similar self-assembly behavior as Q5Y. When all Q in Q5Y was replaced with N, S, D, E, K, R or H which is also a polar amino acid but does not contain a hydrophobic group, as shown in FIG. 13, none of the resulting N5Y, S5Y, D5Y, E5Y, K5Y, R Y and H5Y short peptides exhibited typical ThT fluorescence peaks, indicating that the short peptides composed of these polar amino acids which do not contain a hydrophobic group do not have self-assembly behavior.

Example 18: characterization of formation of nanospheres from different short peptide loading modes of hydrophobic drug pyrene

The Q3TY2, Q4GY2, Q4NY2 and Q5A2SG short peptides are entrusted to chemical synthesis by the company of Haibotai biotechnology; pyrene was purchased from Sigma-Aldrich.

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

And (3) placing 10mL of the short peptide mother solution under the condition of magnetic stirring at room temperature at 2000rpm/min, adding 20mg of pyrene into the short peptide mother solution, continuing magnetic stirring for 30min after the addition, performing ultrasonic treatment for 10min, and storing at 4 ℃.

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

Results:

from fig. 14, it can be seen that each of the short peptides can encapsulate pyrene to form a nanosphere structure with uniform size.

The results of examples 17 to 18 show that the compounds of the general formula Q according to the invention _m X _n (m is 2-10, n is 0-5) the various short peptides of the structure have similar self-assembly behavior, and can wrap hydrophobic drugs to form a nanosphere structure. It can be reasonably deduced that: the general formula of the invention is shown as general formula Q _m X _n The compound formed by wrapping the hydrophobic drug with the short peptide with the structure in the formula (m is 2-10, n is 0-5) can generate similar drug effects as the compound formed by wrapping the hydrophobic drug with Q5Y.

In conclusion, the short peptide can effectively wrap the hydrophobic drugs to form nanospheres, and the drug effect of the short peptide is equivalent to that of a commercially available preparation taking lipid as a carrier. Meanwhile, the preparation developed by taking the short peptide of the invention as a carrier does not contain traditional lipid materials, so that side effects such as hyperlipidemia, lipid metabolism abnormality and the like caused by the lipid carrier can be avoided, and the preparation has better safety.

Claims

1. A short peptide, characterized in that: the amino acid sequence of the short peptide is shown as SEQ ID NO. 1.

2. A chemically modified oligopeptide, characterized in that: the N end and/or the C end of the short peptide with the amino acid sequence shown as SEQ ID NO.1 are/is provided with chemical modification;

the N-terminal chemical modification is alternatively selected from: acetylation, biotin labeling, fluorescent group labeling;

the C-terminal chemical modification is alternatively selected from: amidation, fluorescent group labeling.

3. The chemically modified short peptide of claim 2, wherein: the N-terminal is chemically modified into a fluorescent group mark, and the fluorescent group mark is FAM, FITC or TAMRA;

the C-terminal chemical modification is a fluorescent group mark, and the fluorescent group mark is AMC, CMK and FMK.

4. The chemically modified short peptide of claim 2, wherein: the N-terminal is chemically modified into acetylation; and/or, the C-terminus is chemically modified to amidate.

5. Use of the short peptide of claim 1 or the chemically modified short peptide of any one of claims 2-4 in the preparation of a hydrophobic drug carrier.

6. Use according to claim 5, characterized in that: the carrier is a nanosphere formed by self-assembly of the short peptide.

7. Use according to claim 5 or 6, characterized in that:

the hydrophobic drug is paclitaxel, doxorubicin, curcumin, docetaxel, vincristine, camptothecin, hydroxycamptothecin, etoposide, retinoic acid, fluorouracil, methotrexate, teniposide, daunorubicin, aclacin, sorafenib, methylprednisone, minocycline, cisplatin, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, chlorophenylsulfone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, raloxifene, flurbiprofen, indomethacin, ibuprofen, tamoxifen, simoxicillin, simvastatin, and other drugs diclofenac, naproxen, piroxicam, raltegravir, efavirenz, nelfinavir, atazanavir, ritonavir, sirolimus, busulfan, tacrolimus, ta Lin Luoer, terfenadine, estradiol, vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, prostadil, complex liposoluble vitamins, dexamethasone, flurbiprofen ester, clevidipine butyrate, brucea oil, cyclosporine, insulin, adefovir, chloroquine, hydroxychloroquine, fampicvir, lopinavir, ritonavir.

8. Use according to claim 7, characterized in that: the hydrophobic drug is paclitaxel, doxorubicin, etomidate or propofol.

9. A hydrophobic drug carrier, characterized by: the carrier is a nanosphere formed by self-assembly of the short peptide of claim 1 or the chemically modified short peptide of any one of claims 2-4.

10. A nanocarrier formulation characterized by: the preparation takes a hydrophobic drug as an active ingredient, and takes the nanospheres formed by self-assembly of the short peptide of claim 1 or the chemically modified short peptide of any one of claims 2-4 as a carrier.

11. The nanocarrier formulation of claim 10, wherein: the content ratio of short peptide to hydrophobic drug was 5. Mu. Mol: (1-100) mg.

12. The nanocarrier formulation of claim 10 or 11, wherein:

the hydrophobic drug is paclitaxel, doxorubicin, curcumin, docetaxel, vincristine, camptothecin, hydroxycamptothecin, etoposide, retinoic acid, fluorouracil, methotrexate, teniposide, daunorubicin, aclacinomycin, sorafenib, cisplatin, methylprednisone, minocycline, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, chlorophenylsulfone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, raloxifene, flurbiprofen, indomethacin, ibuprofen, tamoxifen, pravastatin, and other drugs diclofenac, naproxen, piroxicam, raltefravir, efavirenz, nelfinavir, atazanavir, ritonavir, sirolimus, busulfan, tacrolimus, ta Lin Luoer, terfenadine, estradiol, vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, prostadil, complex liposoluble vitamins, dexamethasone, flurbiprofen ester, clevidipine butyrate, brucea oil, cyclosporine, insulin, adefovir, chloroquine, hydroxychloroquine, fampicvir, lopinavir, ritonavir.

13. The nanocarrier formulation of claim 12, wherein: the hydrophobic drug is paclitaxel, doxorubicin, etomidate or propofol.

14. The nanocarrier formulation of claim 13, wherein: the hydrophobic drug is paclitaxel, and the content ratio of the short peptide to the hydrophobic drug is 1 mu mol:1mg;