A Gemini Amphiphilic Short Peptide and Its Application as a Hydrophobic Drug Carrier

Abstract

This invention relates to a short peptide and its application as a carrier for hydrophobic drugs. The amino acid sequence of the short peptide is (Y). n -Pro-X-X-Pro-(Y) n The short peptide of this invention can load a variety of hydrophobic drugs through a molecular self-assembly mechanism, forming uniformly sized nanosphere micelles with the hydrophobic drugs. This enables the effective transport of drugs into cells or tissues to exert their therapeutic effects. It is safe with no significant cytotoxicity, and the formulation process is simple, making it a highly promising carrier for hydrophobic drugs.

Description

Technical Field

[0001] This invention belongs to the field of drug carriers, and more specifically, this invention relates to a bigenic amphiphilic short peptide and its application as a hydrophobic drug carrier. Background Technology

[0002] Many small-molecule drugs used in clinical practice are hydrophobic and poorly water-soluble. Therefore, they typically require carriers to disperse and dissolve them in aqueous or aqueous solutions before administration by injection. Currently, lipids and other materials are widely used carriers for hydrophobic drugs in clinical practice. Taking paclitaxel, one of the most commonly used anticancer drugs, as an example, the marketed drug... Taxol injection contains approximately 527 mg / mL of polyoxyethylene castor oil and 49.7% anhydrous ethanol as a solvent; the most commonly used general anesthetic drugs, such as propofol, are fat emulsion preparations with lipid molecules such as natural soybean oil and egg yolk lecithin as carriers; the vasodilator alprostadil uses fat emulsion injections with lipid molecules such as soybean oil, lecithin, and oleic acid as carriers; the antihypertensive drug clopidogrel also contains injectable oils and phospholipids. However, the clinical application of lipid components has some problems, such as poor stability (Anesth Analg 2003, 97: 769-771), causing injection pain (Acta Anaesthesiol Scand 2001, 45: 839-841), inducing hyperlipidemia (Lancet 2001, 357: 606-607), and easily causing rapid bacterial growth and inducing infection (Anesth Analg. 1999, 88: 209-212). Taxol injection has experienced numerous adverse reactions during clinical use, such as acute hypersensitivity reactions (Allergy Asthma Immunol Res 2016, 8:174-177) and neurotoxicity (Nanomedicine 2015, 11:1925-1938); propofol fat emulsion injection has the problem of propofol infusion syndrome (PRIS) (Crit Care 2015, 19:398); clovidilpine injection has limited use in patients with severe lipid metabolism disorders (see clovidilpine injection package insert), and so on. These problems are primarily related to lipid components. Therefore, the development of novel lipid-free hydrophobic drug carrier materials is highly promising.

[0003] Several other non-lipid materials are currently used clinically. For example, Abraxane, a drug carrier loaded with human serum albumin, was launched in the United States in 2005. It has fewer side effects, shorter dosing time, and reduced adverse reactions (Int J Nanomedicine 2009, 4:99-105). However, this dosage form is limited by the availability of human blood for the albumin carrier and the associated risks of microbial and viral contamination, making it expensive. Cynviloq, a drug carrier loaded with the polymer mPEG-PLLA, was launched in South Korea in 2007 (AdvDrug DeliverRev 2017, 122:20-30). However, polymer materials are expensive, the synthesis process is complex, and the toxicity of polymer nanoparticles needs further attention. In terms of biocompatibility, artificially synthesized short peptides have unique advantages and are also a promising drug carrier material. Chinese invention patent (patent number ZL 00105625.5, authorization announcement number CN1148227C, entitled "Therapeutic Compound and Its Application") discloses a therapeutic compound based on a short peptide carrier and its application. This invention patent uses a chemical combination of paclitaxel with glutamic acid and aspartic acid to form the therapeutic compound, rather than directly loading the drug onto nanospheres formed by the self-assembly of short peptides.

[0004] Artificially designed self-assembled short peptides have become an emerging class of materials internationally in recent years and have received increasing attention (Nano Today 2016, 11:41-60). Among them, amphiphilic short peptides, designed to mimic the structure of traditional surfactants and possessing typical hydrophilic heads and hydrophobic tails, are a focus of attention for many research groups both domestically and internationally (Acc Chem Res 2017, 50:2440-2448). Because these are artificially synthesized materials mainly composed of natural amino acids, they possess inherent advantages in terms of controllability of quality and purity, biocompatibility, and biodegradability. Driven by hydrophobic interactions, amphiphilic short peptides can self-assemble into various nanostructures such as luminous or vesicle-like structures, rod-shaped or spherical micelles, monolayers, or bilayers, making them ideal drug carrier materials. In addition, some amphiphilic short peptides can encapsulate membrane proteins by binding their hydrophobic tails to the hydrophobic regions of membrane proteins, thereby improving the stability of membrane proteins in aqueous solutions. This has also been used in the study of membrane proteins (PLoS One 2011, 6: e25067), which also confirms the potential of amphiphilic short peptides to encapsulate hydrophobic molecules from another perspective.

[0005] However, currently reported amphiphilic short peptides have low drug loading capacity, poor formulation stability, and significantly lower drug loading capacity than most natural lipid molecules. These problems are the reasons why amphiphilic short peptide carriers have not yet become widespread.

[0006] A literature report describes a "gemini amphiphilic short peptide," which differs from traditional amphiphilic short peptides containing only one hydrophobic tail and one hydrophilic head. This amphiphilic short peptide contains two hydrophobic tails and two hydrophilic heads. The short peptide achieves its gemini structure by forming disulfide bonds between two cysteine ​​residues (Cys) under specific conditions. Compared with traditional amphiphilic short peptides, it has a higher drug loading capacity. However, its reliance on Cys to form disulfide bonds has certain limitations in design and poor stability (Colloid Surface A 2015, 469: 263-270). Summary of the Invention

[0007] The purpose of this invention is to provide a novel geminal amphiphilic short peptide with drug loading effects comparable to lipid carriers, which is independent of disulfide bonds. The technical solution is as follows:

[0008] A twin-type amphiphilic short peptide has the following general formula: (Y)n-Pro-XX-Pro-(Y)n, where X is a hydrophilic amino acid, Y is a hydrophobic amino acid, and n represents the number of hydrophobic amino acids, ranging from 4 to 8.

[0009] Hydrophilic and hydrophobic amino acids are defined according to their hydrophilicity index (JMolBiol 1982, 157: 105-132). Amino acids with a hydrophilicity index ≥ -0.4 are hydrophobic, and those with a hydrophilicity index ≤ -0.7 are hydrophilic. The hydrophilicity indices of amino acids are shown in the table below:

[0010] amino acid abbreviation Hydrophilicity Index amino acid abbreviation Hydrophilicity 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

[0011] As mentioned above, the short peptide has chemical modifications at its N-terminus and / or C-terminus;

[0012] N-terminal chemical modifications are selected from: alkyl acylation (e.g., acetylation, formylation, etc.), biotin labeling, fatty acid modification (e.g., Palm, Myr, Lauryl, etc.), benzoylation, 2-aminobenzoylation, maleimide, haloalkyl acylation (e.g., trifluoroacetylation, chloroacetylation, bromoacetylation, etc.), succinylation, hydrazinolacamide, fluorescent labeling (e.g., FAM, FITC, TAMRA, etc.).

[0013] C-terminal chemical modifications can be selected from: amidation, esterification, aldehydeation, alcoholation, succinylation, and fluorescent labeling (such as AMC, CMK, FMK, etc.).

[0014] As mentioned above, the short peptide (Y)n is any one or more of glycine, alanine, valine, leucine, isoleucine, or phenylalanine in any order.

[0015] As mentioned above, for short peptides, Y is any one or more of glycine, alanine, or valine in any order.

[0016] As mentioned earlier, in the short peptide, Y represents alanine.

[0017] For the aforementioned short peptides, n is 5 to 6.

[0018] As mentioned above, for short peptides, XX is any one or a combination of two of the following in any order: serine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0019] As mentioned above, for short peptides, X is selected from glutamic acid or lysine.

[0020] As mentioned earlier, in short peptides, X represents lysine.

[0021] The short peptides described above have amino acid sequences as shown in SEQ ID NO.1, 3, 4, 5 or 6; preferably, as shown in SEQ ID NO.1.

[0022] As with the aforementioned short peptides, the N-terminal chemical modification is acetylation;

[0023] And / or, the C-terminus is chemically modified to amidation (the C atom in the amide group can be the C atom that is naturally present in the C segment of the short peptide).

[0024] The aforementioned short peptides are used as carriers in the preparation of hydrophobic drugs.

[0025] As described above, the carrier is a micelle formed by the self-assembly of the short peptide; preferably, the micelle is a spherical micelle.

[0026] As described above, the hydrophobic drugs include, but are not limited to, paclitaxel, doxorubicin, curcumin, docetaxel, doxorubicin, vincristine, camptothecin, hydroxycamptothecin, etoposide, retinoic acid, fluorouracil, methotrexate, teniposide, daunorubicin, aclarubicin, sorafenib, methylprednisolone, minocycline, cisplatin, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, dapsone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, and raloxifene. The following are any one or a mixture of several of the following: clobiprofen, indomethacin, ibuprofen, tamoxifen, diclofenac, naproxen, piroxicam, raltegravir, efavirenz, nelfinavir, atazanavir, ritonavir, sirolimus, spironolactone, tacrolimus, talinolol, terfenadine, estradiol, vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, alprostadil, complex fat-soluble vitamins, dexamethasone, flurbiprofen ester, clovidipine, croton oil, cyclosporine, insulin, etc.

[0027] Preferably, the hydrophobic drug is paclitaxel, doxorubicin, etomidate, or propofol.

[0028] A hydrophobic drug carrier, wherein the carrier is a micelle formed by the self-assembly of the aforementioned short peptide; preferably, the micelle is a spherical micelle.

[0029] A nanocarrier formulation, wherein the formulation uses a hydrophobic drug as the active ingredient and micelles formed by the self-assembly of the aforementioned short peptides as the carrier.

[0030] As mentioned above, in nanocarrier formulations, the ratio of short peptides to active ingredients is 5 μmol: 1–100 mg.

[0031] As described above in the nanocarrier formulation, the hydrophobic drug is paclitaxel, doxorubicin, curcumin, docetaxel, doxorubicin, vincristine, camptothecin, hydroxycamptothecin, etoposide, retinoic acid, fluorouracil, methotrexate, teniposide, daunorubicin, aclarubicin, sorafenib, methylprednisolone, minocycline, cisplatin, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, dapsone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, raloxifene. The following are any one or a mixture of several of the following: clobiprofen, indomethacin, ibuprofen, tamoxifen, diclofenac, naproxen, piroxicam, raltegravir, efavirenz, nelfinavir, atazanavir, ritonavir, sirolimus, spironolactone, tacrolimus, talinolol, terfenadine, estradiol, vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, alprostadil, complex fat-soluble vitamins, dexamethasone, flurbiprofen ester, clovidipine, croton oil, cyclosporine, insulin, etc.

[0032] Preferably, the hydrophobic drug is paclitaxel, doxorubicin, etomidate, or propofol.

[0033] The twin-type amphiphilic short peptide of this invention is a novel design that, based on the traditional single hydrophobic tail amphiphilic short peptide, forms a turn structure with two Pro groups, resulting in a twin-type amphiphilic short peptide with two hydrophobic tails under all conditions. The twin-type amphiphilic short peptide of this invention exhibits significantly improved drug loading capacity compared to current short peptide carriers; using the short peptide of this invention as a carrier for hydrophobic drugs in formulation preparation can achieve efficacy approaching that of existing lipid carrier drug formulations.

[0034] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0035] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the structures of the twin amphiphilic short peptides APK, APE, APKE, GAVPK and AVLFPK, as well as the amphiphilic short peptide A6K with a single hydrophobic tail.

[0037] Figure 2 This is a schematic diagram illustrating the mechanism of self-assembly of bigonal amphiphilic short peptides and their loading of hydrophobic drugs.

[0038] Figure 3 Electron microscopy images of the self-assembled nanofilm structure of APK and its loading mode, showing the formation of uniform nanospheres from the hydrophobic drug molecule pyrene, and contrasting electron microscopy images of irregular nanospheres formed by loading pyrene with the conventional amphiphilic short peptide A6K with a single hydrophobic tail.

[0039] Figure 4 The effect of paclitaxel (APK-PTX) loaded in the APK inhibiting the proliferation of ovarian cancer cells skov3.

[0040] Figure 5 A diagram showing the effect of doxorubicin (APK-DOX) loaded into an APK on inhibiting the proliferation of ovarian cancer cells skov3.

[0041] Figure 6 This image shows the effect of APK on ovarian cancer cells skov3.

[0042] Figure 7 Electron micrograph of paclitaxel loaded into the APK.

[0043] Figure 8 Electron micrograph of an APK loaded with doxorubicin.

[0044] Figure 9 Electron micrograph of the APK loaded with etomidate.

[0045] Figure 10 Electron micrograph of propofol loaded into the APK.

[0046] Figure 11 The circular dichroism spectra of the bigonal amphiphilic short peptides APK, APE, APKE, GAVPK, and AVLFPK show that they have similar random secondary structures.

[0047] Figure 12The fluorescence spectra of the binding of the bigonal amphiphilic short peptides APK, APE, APKE, GAVPK and AVLFPK to ThT show that they have similar self-assembly behaviors.

[0048] Figure 13 Electron microscopy images of pyrene nanospheres formed by loading the bigonal amphiphilic short peptides APE, APKE, GAVPK, and AVLFPK. Detailed Implementation

[0049] Example 1: Loading of short peptides with the model hydrophobic drug pyrene

[0050] The materials of this invention are: (Ala)6-Pro-Lys-Lys-Pro-(Ala)6, i.e., Ac-Ala-Ala-Ala-Ala-Ala-Ala-Pro-Lys-Lys-Pro-Ala-Ala-Ala-Ala-Ala-Ala-NH2, abbreviated as APK, where Ac represents acetyl; APK is SEQ ID NO.1 in the amino acid sequence listing; (Ala)6-Lys, i.e., Ac-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Lys-COOH, abbreviated as A6K, is SEQ ID NO.2 in the amino acid sequence listing, and its structure is as follows. Figure 1 As shown, the chemical synthesis was commissioned to Shanghai Botai Biotechnology Co., Ltd.

[0051] Pyrene and DMSO were purchased from Sigma-Aldrich.

[0052] Preparation of short peptide stock solution: Dissolve APK in water at 0.5mM and sonicate for 5 minutes.

[0053] Preparation of pyrene stock solution: Dissolve pyrene powder in DMSO at a concentration of 10 mM.

[0054] Place 10 mL of short peptide stock solution in a room temperature magnetic stirring chamber at 2000 rpm / min. Add 100 μL of pyrene stock solution dropwise to the short peptide stock solution using a pipette. After the addition is complete, continue magnetic stirring for 30 min, then sonicate for 10 min, and store at 4℃.

[0055] Morphological characteristics were observed using a high-resolution transmission electron microscope. 10 μL of sample solution was added to a 400-mesh copper grid for 5 min, then blotted dry with a piece of filter paper. Next, 10 μL of 2% phosphotungstic acid was added for staining for 3 min. The final staining solution was blotted dry with filter paper and allowed to air dry. Transmission electron microscopy was then used for imaging.

[0056] from Figure 3It can be seen that APK can independently self-assemble into a nanofilm structure, and when loaded with pyrene, APK can form nanospheres with a particle size of less than 50 nm, uniform size, and regular shape. In contrast, A6K loaded with pyrene with a single hydrophobic tail can only form nanoparticles with a particle size of more than 200 nm and irregular shape and size.

[0057] The results of Example 1 show that the APK can effectively load hydrophobic drugs.

[0058] Example 2: Loading of paclitaxel with short peptides

[0059] Materials: APK was chemically synthesized by Shanghai Botai Biotechnology Co., Ltd.; paclitaxel was purchased from Dalian Meilun Biotechnology Co., Ltd.; anhydrous ethanol was purchased from Chengdu Kelong Chemical Reagent Factory.

[0060] Preparation of short peptide stock solution: Dissolve APK in water at 0.5mM and sonicate for 5 minutes.

[0061] Preparation of paclitaxel stock solution: Dissolve paclitaxel powder in anhydrous ethanol at a concentration of 10 mg / mL.

[0062] Place 10 mL of the short peptide stock solution at room temperature and magnetically stir at 2000 rpm / min. Then, add 100 μL of paclitaxel stock solution dropwise to the short peptide stock solution using a pipette (APK to paclitaxel ratio is 5 μmol: 1 mg). After the addition is complete, continue magnetic stirring for 30 min, then sonicate for 10 min, and store at 4℃.

[0063] Example 3: Loading of paclitaxel with short peptides

[0064] APK was chemically synthesized by Shanghai Botai Biotechnology Co., Ltd.; paclitaxel was purchased from Dalian Meilun Biotechnology Co., Ltd.; and anhydrous ethanol was purchased from Chengdu Kelong Chemical Reagent Factory.

[0065] Preparation of short peptide stock solution: Dissolve APK in water at 1mM and sonicate for 5 minutes.

[0066] Preparation of paclitaxel stock solution: Dissolve paclitaxel powder in anhydrous ethanol at a concentration of 20 mg / mL.

[0067] Place 1 mL of paclitaxel stock solution at room temperature and magnetically stir at 2000 rpm / min. Then, add 100 mL of short peptide stock solution dropwise to the paclitaxel stock solution using a pipette (APK to paclitaxel ratio is 5 μmol: 1 mg). After the addition is complete, continue magnetic stirring for 30 min, then sonicate for 10 min, and store at 4℃.

[0068] Example 4: Loading of paclitaxel with short peptides

[0069] APK was chemically synthesized by Shanghai Botai Biotechnology Co., Ltd.; paclitaxel was purchased from Dalian Meilun Biotechnology Co., Ltd.; and anhydrous ethanol was purchased from Chengdu Kelong Chemical Reagent Factory.

[0070] Preparation of short peptide stock solution: Dissolve APK in water at 1mM and sonicate for 5 minutes.

[0071] Preparation of paclitaxel stock solution: Dissolve paclitaxel powder in anhydrous ethanol at a concentration of 20 mg / mL.

[0072] Dissolve APK (0.5 mM) and paclitaxel powder (10 mg / mL) in 2 mL of DMSO (APK to paclitaxel ratio: 5 μmol: 100 mg), and sonicate for 5 min. Remove the organic solvent using a vacuum desiccator, reconstitute with 10 mL of water, sonicate again for 10 min, and store at 4 °C.

[0073] Example 5: Inhibitory effect of paclitaxel-short peptide preparation on ovarian cancer cells

[0074] Human ovarian cancer cells Skov3 were injected at a rate of 5 × 10⁻⁶. 3 Cells were seeded at a density of 1 / 2 well in 96-well plates and incubated for 24 hours. The supernatant was removed, and different concentrations of paclitaxel-peptide formulations (prepared according to the method in Example 2) and corresponding concentrations of paclitaxel (a commercially available paclitaxel formulation) were added as controls. After incubation for 48 hours, cell viability was determined using the CCK-8 assay. The OD value, reflecting cell viability, was measured at 490 nm using a microplate fluorescence spectrometer.

[0075] The comparison between the experimental and control groups showed that paclitaxel-peptide formulation (APK-PTX) had a significant inhibitory effect on tumor cells, comparable to that of the control group drug, Taxol. Figure 4 ).

[0076] Example 6: Loading of doxorubicin with short peptides

[0077] Reagents: Doxorubicin hydrochloride was purchased from Dalian Meilun Biotechnology Co., Ltd., and triethylamine was purchased from Chengdu Kelong Chemical Reagent Factory.

[0078] Preparation of short peptide stock solution: Dissolve APK in water at 0.5mM and sonicate for 5 minutes.

[0079] Desalting of doxorubicin hydrochloride: Dissolve 10 mg of doxorubicin hydrochloride powder in 10 ml of methanol, add 10 μL of triethylamine, stir magnetically overnight, and evaporate the organic solvent under vacuum to obtain doxorubicin powder.

[0080] Doxorubicin stock solution preparation: Dissolve doxorubicin powder in anhydrous ethanol at a concentration of 10 mg / mL.

[0081] Place 10 mL of short peptide stock solution in a room temperature magnetic stirring chamber at 2000 rpm / min. Add 100 μL of doxorubicin stock solution dropwise to the short peptide stock solution using a pipette (APK to doxorubicin ratio is 5 μmol: 1 mg). After the addition is complete, continue magnetic stirring for 30 min, then sonicate for 10 min, and store at 4 °C.

[0082] Example 7: Loading of doxorubicin with short peptides

[0083] Reagents: Doxorubicin hydrochloride was purchased from Dalian Meilun Biotechnology Co., Ltd., and triethylamine was purchased from Chengdu Kelong Chemical Reagent Factory.

[0084] Preparation of short peptide stock solution: Dissolve APK in water at 0.5mM and sonicate for 5 minutes.

[0085] Desalting of doxorubicin hydrochloride: Dissolve 10 mg of doxorubicin hydrochloride powder in 10 ml of methanol, add 10 μL of triethylamine, stir magnetically overnight, and evaporate the organic solvent under vacuum to obtain doxorubicin powder.

[0086] Doxorubicin stock solution preparation: Dissolve doxorubicin powder in anhydrous ethanol at a concentration of 10 mg / mL.

[0087] Place 1 mL of doxorubicin stock solution at room temperature and magnetically stir at 2000 rpm / min. Then, add 100 mL of short peptide stock solution dropwise to the doxorubicin stock solution using a pipette (APK to doxorubicin ratio is 5 μmol: 1 mg). After the addition is complete, continue magnetic stirring for 30 min, then sonicate for 10 min, and store at 4℃.

[0088] Example 8: Loading of doxorubicin with short peptides

[0089] Reagents: Doxorubicin hydrochloride was purchased from Dalian Meilun Biotechnology Co., Ltd., and triethylamine was purchased from Chengdu Kelong Chemical Reagent Factory.

[0090] Preparation of short peptide stock solution: Dissolve APK in water at 0.5mM and sonicate for 5 minutes.

[0091] Desalting of doxorubicin hydrochloride: Dissolve 10 mg of doxorubicin hydrochloride powder in 10 ml of methanol, add 10 μL of triethylamine, stir magnetically overnight, and evaporate the organic solvent under vacuum to obtain doxorubicin powder.

[0092] Doxorubicin stock solution preparation: Dissolve doxorubicin powder in anhydrous ethanol at a concentration of 10 mg / mL.

[0093] Dissolve APK at 0.5 mM and doxorubicin powder at 10 mg / mL (i.e., the ratio of APK to doxorubicin is 5 μmol: 100 mg) in 2 mL of DMSO and sonicate for 5 min. Remove the organic solvent using a vacuum desiccator, reconstitute with 10 mL of water, sonicate again for 10 min, and store at 4 °C.

[0094] Example 9: Inhibitory effect of doxorubicin-short peptide preparation on ovarian cancer cells

[0095] Human ovarian cancer cells Skov3 were injected at a rate of 5 × 10⁻⁶. 3 Cells were seeded at a density of 1 / 2 well in 96-well plates and incubated for 24 hours. The supernatant was removed, and different concentrations of doxorubicin-peptide formulations (prepared using the method in Example 6) and corresponding concentrations of doxorubicin hydrochloride were added as controls. After incubation for 48 hours, cell viability was determined using the CCK-8 assay. The OD value, reflecting cell viability, was measured at 490 nm using a microplate fluorescence spectrometer.

[0096] The comparison between the experimental and control groups showed that the doxorubicin-short peptide preparation (APK-DOX) had a significant inhibitory effect on tumor cells, comparable to that of the control group drug, doxorubicin hydrochloride. Figure 5 ).

[0097] Example 10: Loading of short peptides with etomidate

[0098] Materials: (Ala)6-Pro-Lys-Lys-Pro-(Ala)6, abbreviated as APK, was chemically synthesized by Shanghai Botai Biotechnology Co., Ltd.; etomidate was purchased from Dalian Meilun Biotechnology Co., Ltd.; anhydrous ethanol was purchased from Chengdu Kelong Chemical Reagent Factory.

[0099] Preparation of short peptide stock solution: Dissolve APK in 0.9% physiological saline at 0.5mM, vortex and sonicate for 10min.

[0100] Weigh 20 mg of etomidate and add it to 10 mL of short peptide stock solution (the ratio of APK to etomidate is 5 μmol: 20 mg). Vortex and sonicate for 20 min. Then place it in a room temperature magnetic stirring environment at 2000 rpm / min and stir for 40 min. Store at room temperature.

[0101] Example 11: Anesthesia effect of a single intravenous injection of etomidate-short peptide formulation into rat tail vein

[0102] Healthy adult male SD rats (weight: 295±14g) were used. The rats were placed in a restraint device with their tails exposed. The lateral tail vein was located, wiped with alcohol, and an indwelling needle was inserted for drug administration (etomidate-short peptide formulation prepared using the method in Example 10). 0.6 mL of the drug was administered at a uniform rate of 0.1 mL / s. After administration, 0.05 mL of air was pushed in to ensure complete drug entry into the tail vein before removing the indwelling needle. Hemostasis was achieved by applying pressure with a cotton swab. The rats were quickly removed and placed in an empty cage. The rats' reactions were observed, sedation scores were recorded, and adverse reactions were documented (CFDA "Guidelines for Drug Administration Toxicity" (Draft for Comments, 2013-05-26)). Sedation score (Psychopharmacology 1996, 125: 105-112): Normal muscle tone in all four limbs, able to maintain voluntary activity and responsiveness 0 points; obvious tactile movement (rat tends to stay near the edge of the cage) 1 point; backward balance disorder 2 points; forelimbs less than 60 degrees, ataxia 3 points; prone, unable to stand, can only support itself with its abdomen 4 points; righting reflex absent 5 points. Observation of the absence of the righting reflex (Anesthesiology 2000, 93(3): 837-843): The absence of the righting reflex and its duration of more than 30 seconds is marked as "+", otherwise it is marked as "-".

[0103] A dose escalation experiment was conducted starting from 1 mg / kg, and a "+" and "-" dose were identified as the ED. 50 (The half-maximal effective dose, the amount that elicits 50% of the maximum response intensity) Dosage reference range for experiments. Sequential method for determining ED. 50 The sedative effect in rats was observed by starting with a low dose. If the effect was "-", the dose was decreased for the next rat (r = 1.5, in a proportional manner); if the effect was "+", the dose was increased proportionally for the next rat. A crossover occurred from "-" to "+" or "+" to "-", and the experiment terminated after five such crossovers. The dixon-mood method (ED) was used. 50 =lg-1(∑C / ∑t)) calculates the ED of the drug in rats. 50 And calculate the 95% confidence interval, 95% CI = lg-1(1gED). 50 ±1.96slgED 50 ), slgED 50 ={[∑M-(∑C)2 / ∑t] / (∑t·(∑t-1)}1 / 2.

[0104] Based on the measured ED 50 Rats were administered a single tail vein injection of 2 ED according to the above administration method. 50The drug was used to observe its sedative effect in rats. A comparison with the clinically used etomidate fat emulsion formulation (Folli) showed that the etomidate-short peptide formulation had comparable anesthetic effects to the commercially available etomidate formulation (Folli), with no significant adverse reactions (Table 1).

[0105] Table 1 Comparison of the anesthetic effects of etomidate-short peptide preparation and folic acid.

[0106]

[0107] Example 12: Loading propofol with short peptides

[0108] Materials: (Ala)6-Pro-Lys-Lys-Pro-(Ala)6, abbreviated as APK, was chemically synthesized by Shanghai Botai Biotechnology Co., Ltd.; propofol was purchased from Sigma-Aldrich.

[0109] Preparation of short peptide stock solution: Dissolve APK in 0.9% physiological saline at 1 mM, vortex and sonicate for 10 min.

[0110] Take 0.1 mL of propofol and add it dropwise to 9.9 mL of short peptide stock solution. Vortex and sonicate for 20 min. Then place it at room temperature and stir magnetically at 2000 rpm / min for 40 min. Store at room temperature.

[0111] Example 13: Anesthesia effect of a single intravenous injection of propofol-short peptide formulation into the tail vein of rats

[0112] Healthy adult male SD rats (weight: 295±14g) were used. The rats were placed in a restraint device with their tails exposed. The lateral tail vein was located, wiped with alcohol, and an indwelling needle was inserted for drug administration (propofol-short peptide formulation prepared using the method in Example 11). 0.6 mL of the drug was administered at a uniform rate of 0.1 mL / s. After administration, 0.05 mL of air was pushed in to ensure complete drug entry into the tail vein before removing the indwelling needle. Hemostasis was achieved by applying pressure with a cotton swab. The rats were quickly removed and placed in an empty cage. The rats' reactions were observed, sedation scores were recorded, and adverse reactions were documented (CFDA "Guidelines for Drug Administration Toxicity" (Draft for Comments, 2013-05-26)). Sedation score (Psychopharmacology 1996, 125: 105-112): Normal muscle tone in all four limbs, able to maintain voluntary activity and responsiveness 0 points; obvious tactile movement (rat tends to stay near the edge of the cage) 1 point; backward balance disorder 2 points; forelimbs less than 60 degrees, ataxia 3 points; prone, unable to stand, can only support itself with its abdomen 4 points; righting reflex absent 5 points. Observation of the absence of the righting reflex (Anesthesiology 2000, 93(3): 837-843): The absence of the righting reflex and its duration of more than 30 seconds is marked as "+", otherwise it is marked as "-".

[0113] A dose escalation experiment was conducted starting from 1 mg / kg, and a "+" and "-" dose were identified as the ED. 50 Dosage reference range for the experiment. Sequential method for determining ED. 50 The sedative effect in rats was observed by starting with a low dose. If the effect was "-", the dose was decreased for the next rat (r = 1.5, in a proportional manner); if the effect was "+", the dose was increased proportionally for the next rat. A crossover occurred from "-" to "+" or "+" to "-", and the experiment terminated after five such crossovers. The dixon-mood method (ED) was used. 50 =lg-1(∑C / ∑t)) calculates the ED of the drug in rats. 50 And calculate the 95% confidence interval: 95% CI = lg-1(lgED) 50 ±1.96slgED 50 ), slgED 50 ={[∑M-(∑C)2 / ∑t] / (∑t·(∑t-1)}1 / 2.

[0114] ED was compared with clinically used propofol fat emulsion formulation (Dapoxetine). 50 It can be seen that the anesthetic effect of propofol-short peptide preparation is comparable to that of commercially available clinical propofol preparation (Dapoxetine) (Table 2).

[0115] Table 2. Edema rates of propofol-short peptide formulations and propofol (Dapoxetine). 50contrast

[0116]

[0117] The results of Examples 5, 9, 11, and 13 show that the short peptide APK of the present invention can load various hydrophobic drugs, enabling the drugs to exert efficacy comparable to that of commercially available (fat emulsion) formulations at equivalent doses.

[0118] Example 14: Effects of APK short peptide on ovarian cancer cells

[0119] Human ovarian cancer cells Skov3 were injected at a rate of 5 × 10⁻⁶. 3 Cells were seeded at a density of 1:1 in 96-well plates and incubated for 24 hours. The supernatant was removed, and different concentrations of short peptides were added. After incubation for 48 hours, cell viability was determined using the CCK-8 assay. The OD value, reflecting cell viability, was measured at 490 nm using a microplate fluorescence spectrometer.

[0120] The results show that the APK short peptide did not significantly inhibit tumor cells, proving that it has no obvious cytotoxicity. Figure 6 ).

[0121] Example 15: Nanostructure Characterization

[0122] Morphological characteristics were observed using a high-resolution transmission electron microscope. 10 μL of sample solution was added to a 400-mesh copper grid for 5 min, then blotted dry with a piece of filter paper. Next, 10 μL of 2% phosphotungstic acid was added for staining for 3 min. The final staining solution was blotted dry with filter paper and allowed to air dry. Transmission electron microscopy was then used for imaging.

[0123] from Figures 7-10 It can be seen that short peptides encapsulate drugs into nanoscale spherical micelles.

[0124] In traditional studies of amphiphilic short peptides with single hydrophobic tails, numerous studies have shown that using different hydrophobic amino acids as the hydrophobic tail and / or different hydrophilic amino acids as the hydrophilic head can result in amphiphilic short peptides that can form similar nanocarriers (ProcNatlAcadSci USA 2002, 99: 5355-5360; Langmuir 2003, 19: 4332). The length of the hydrophobic tail, i.e., the number of hydrophobic amino acids, can also be arbitrarily selected from 4 to 8 without fundamentally changing the ability of the short peptide to self-assemble into a nanocarrier (Nano Lett 2002, 2: 687; JPeptSci 2018, 24: e3062).

[0125] It is reasonable to infer that replacing the hydrophilic head amino acid Glu of the APK with two hydrophobic tails with other hydrophilic amino acids, and / or replacing the hydrophobic tail amino acid Ala of the APK with other hydrophobic amino acids, and / or changing the length of the hydrophobic tail (the single tail can be any value among 4-8 amino acids), will not fundamentally change the ability of the short peptides of the present invention to self-assemble into nanocarriers.

[0126] The following example illustrates this:

[0127] Example 16: Characterization of secondary structures of different short peptide geminis

[0128] APK, APE, APKE, GAVPK, and AVLFPK—the aforementioned short peptides were all chemically synthesized by Shanghai Botai Biotechnology Co., Ltd.

[0129] The sequences APE, APKE, GAVPK, and AVLFPK are shown in Table 3.

[0130] Table 3. APE, APKE, GAVPK, and AVLFPK sequences

[0131]

[0132] Note: Ac represents acetyl group.

[0133] The circular dichroism spectra of each short peptide were measured using a circular dichroism spectrometer to determine that they have similar secondary structures. Each short peptide was dissolved in water to prepare a 0.1 mM solution, and 800 μL of the solution was placed in a quartz cuvette with an optical path length of 2 mm. The circular dichroism spectra in the range of 185–260 nm were measured at 25 °C.

[0134] like Figure 11 As shown, all short peptides exhibit a negative peak around 195 nm, indicating that all designed short peptides have a random, freely extending secondary structure similar to APK. Therefore, it can be determined that, like APK, they can form a typical genomic structure by means of the turn formed by Pro.

[0135] Example 17: Characterization of the self-assembly ability of different short peptides

[0136] The short peptides APK, APE, APKE, GAVPK, and AVLFPK were chemically synthesized by Shanghai Botai Biotechnology Co., Ltd. Each short peptide was prepared into a 0.5 mM aqueous solution. Thioflavin T (ThT, purchased from Sigma-Aldrich) was added to every 500 μL of the short peptide solution to a final concentration of 10 μM. The fluorescence spectra in the range of 460–600 nm (excitation wavelength set to 450 nm) were measured using a fluorescence spectrophotometer to determine whether they exhibited self-assembly behavior similar to APK. Figure 12 As shown, all short peptides exhibited a typical ThT fluorescence peak around 490 nm, indicating that all designed short peptides have similar self-assembly behavior to APK.

[0137] Example 18: Characterization of pyrene nanospheres formed by hydrophobic drugs with different short peptide loading modes

[0138] The short peptides APK, APE, APKE, GAVPK, and AVLFPK were chemically synthesized by Shanghai Botai Biotechnology Co., Ltd.; pyrene was purchased from Sigma-Aldrich.

[0139] Preparation of short peptide stock solution: Dissolve each short peptide in water at 0.5 mM and sonicate for 5 min.

[0140] Preparation of pyrene stock solution: Dissolve pyrene powder in DMSO at a concentration of 10 mM.

[0141] Place 10 mL of short peptide stock solution in a room temperature magnetic stirring chamber at 2000 rpm / min. Add 100 μL of pyrene stock solution dropwise to the short peptide stock solution using a pipette. After the addition is complete, continue magnetic stirring for 30 min, then sonicate for 10 min, and store at 4℃.

[0142] Morphological characteristics were observed using a high-resolution transmission electron microscope. 10 μL of sample solution was added to a 400-mesh copper grid for 5 min, then blotted dry with a piece of filter paper. Next, 10 μL of 2% phosphotungstic acid was added for staining for 3 min. The final staining solution was blotted dry with filter paper and allowed to air dry. Transmission electron microscopy was then used for imaging.

[0143] from Figure 13 It can be seen that each short peptide can encapsulate pyrene to form a uniformly sized nanosphere structure.

[0144] The results of Examples 16-18 show that the present invention has the general formula (Y). n -Pro-XX-Pro-(Y) n(Where Y is a hydrophobic amino acid, X is a hydrophilic amino acid, and n is 4–8) Various short peptides with the structure described above can form typical twin structures by means of the turn formed by Pro, and have similar self-assembly behavior, and can encapsulate hydrophobic drugs to form nanosphere structures. Therefore, it can be reasonably inferred that the general formula of this invention has the general formula (Y). n -Pro-XX-Pro-(Y) n The short peptides of the aforementioned structure that encapsulate hydrophobic drugs to form complexes can produce pharmacological effects similar to those of complexes formed by APKs encapsulating hydrophobic drugs.

[0145] In summary, the short peptides of this invention can effectively encapsulate hydrophobic drugs to form nanospheres, achieving efficacy comparable to commercially available fat emulsion formulations. SEQUENCE LISTING <110> West China Hospital of Sichuan University <120> A Gemini Amphiphilic Short Peptide and Its Application as a Hydrophobic Drug Carrier <130> GYKH1533-2019P018601CC <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> PRT <213> 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 <400> 2 Ala Ala Ala Ala Ala Ala Lys 1 5 <210> 3 <211> 16 <212> PRT <213> Artificial sequence <400> 3 Ala Ala Ala Ala Ala Ala For Glu Glu For Ala Ala Ala Ala Ala Ala 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> 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 <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 <400> 6 Leu Val Phe Phe Ala Pro Lys Lys Pro Ala Phe Phe Val Leu 1 5 10

Claims

1. A twin-type amphiphilic short peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1, 3, 4, 5 or 6.

2. The twin-type amphiphilic short peptide according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

3. Use of the gemini amphiphilic short peptide according to any one of claims 1-2 in the preparation of a carrier for a hydrophobic drug.

4. The use according to claim 3, characterized in that: The carrier is a micelle formed by the self-assembly of the twin amphiphilic short peptides.

5. The use according to claim 4, characterized in that: The micelles are spherical micelles.

6. The use according to any one of claims 3-5, characterized in that: The hydrophobic drugs mentioned are paclitaxel, doxorubicin, curcumin, docetaxel, doxorubicin, vincristine, camptothecin, hydroxycamptothecin, etoposide, retinoic acid, fluorouracil, methotrexate, teniposide, daunorubicin, aclarubicin, sorafenib, methylprednisolone, minocycline, cisplatin, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, dapsone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, raloxifene, and clobiprofen. Indomethacin, ibuprofen, tamoxifen, diclofenac, naproxen, piroxicam, raltegravir, efavirenz, nelfinavir, atazanavir, ritonavir, sirolimus, spironolactone, tacrolimus, talinolol, terfenadine, estradiol, vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, alprostadil, complex fat-soluble vitamins, dexamethasone, flurbiprofen ester, clovidipine, croton oil, cyclosporine, and insulin are all or a mixture of several of these.

7. The use according to claim 6, characterized in that, The hydrophobic drug is paclitaxel, doxorubicin, etomidate, or propofol.

8. A hydrophobic drug carrier, characterized in that: The carrier is a micelle formed by the self-assembly of a twin-type amphiphilic short peptide as described in any one of claims 1-2.

9. The hydrophobic drug carrier according to claim 8, characterized in that: The micelles are spherical micelles.

10. A nanocarrier formulation, characterized in that: The formulation uses a hydrophobic drug as the active ingredient and micelles formed by the self-assembly of the bisexual short peptides as described in any one of claims 1-2 as the carrier.

11. The nanocarrier formulation according to claim 10, characterized in that: The ratio of the content of the twin-type amphiphilic short peptide to the active ingredient is 5 μmol: 1–100 mg.

12. The nanocarrier formulation according to claim 10 or 11, characterized in that: The hydrophobic drugs mentioned are paclitaxel, doxorubicin, curcumin, docetaxel, doxorubicin, vincristine, camptothecin, hydroxycamptothecin, etoposide, retinoic acid, fluorouracil, methotrexate, teniposide, daunorubicin, aclarubicin, sorafenib, methylprednisolone, minocycline, cisplatin, atorvastatin, simvastatin, lovastatin, amiodarone, carbamazepine, carvedilol, chlorpromazine, cisapride, dapsone, azithromycin, neomycin, amphotericin B, griseofulvin, celecoxib, raloxifene, and clobiprofen. Indomethacin, ibuprofen, tamoxifen, diclofenac, naproxen, piroxicam, raltegravir, efavirenz, nelfinavir, atazanavir, ritonavir, sirolimus, spironolactone, tacrolimus, talinolol, terfenadine, estradiol, vitamin A, vitamin D, vitamin E, vitamin K, propofol, etomidate, perfluorocarbon, diazepam, alprostadil, complex fat-soluble vitamins, dexamethasone, flurbiprofen ester, clovidipine, croton oil, cyclosporine, and insulin are all or a mixture of several of these.

13. The nanocarrier formulation according to claim 12, characterized in that: The hydrophobic drug is paclitaxel, doxorubicin, etomidate, or propofol.

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