Actively targeted branched polypeptide, nano drug carrier, anti-tumor nano drug, and preparation method and application thereof

Abstract

The present invention discloses an active targeting branched polypeptide, a nano drug carrier, an anti-tumor nano drug, and a preparation method and application thereof, belonging to the field of biomedicine technology. The active targeting branched polypeptide of the present invention is an amphiphilic branched polypeptide having an amino acid sequence of (FHF)2KRGD and branching at K, or an amino acid sequence having an equivalent drug-carrying function derived from the amphiphilic branched polypeptide by replacing, adding or deleting one or more amino acids, or a homologous polypeptide comprising the (FHF)2KRGD sequence; the polypeptide nano drug carrier is a nanoscale vesicle formed by self-assembly of the amphiphilic branched polypeptide; the amphiphilic branched polypeptide is prepared by a synthetic, semi-synthetic or recombinant synthetic method, and the amphiphilic branched polypeptide encapsulates the anti-tumor drug in a hydrophobic cavity through self-assembly to obtain an actively targeted anti-tumor nano drug; the branched polypeptide is non-toxic and has good biocompatibility and high stability, and the nano drug carrier has a high drug loading capacity, is used for anti-tumor drug loading, and has a good anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an active targeting branched polypeptide, a nano drug carrier, an anti-tumor nano drug, and a preparation method and application thereof. Background Art

[0002] Cancer, a serious threat to human life and health, is currently treated primarily with surgery, radiotherapy, and chemotherapy. However, these therapies are not ideal for improving patients' long-term prognosis or survival rates. Consequently, many researchers have shifted their focus to small molecules that offer high activity, minimal adverse reactions, and resistance to drug resistance. However, some highly active small molecules exhibit low stability, low selectivity, and water insolubility. Due to renal filtration and protease degradation, these small molecules exhibit poor stability, short half-lives, low blood concentrations, and low bioavailability. Furthermore, their poor selectivity can damage normal cells and tissues in the human body, resulting in toxic side effects.

[0003] To address these technical drawbacks, scientists have developed numerous drug carriers to help small molecules of anti-tumor drugs enter the human body, improve their stability, enhance their water solubility, and aid in targeted delivery. Similar drug carriers include liposomes, micelles, vesicles, and hydrogels. Each of these drug delivery systems has its own advantages and disadvantages, and selecting a stable and efficient drug delivery system is a crucial task.

[0004] Peptides are multifunctional molecules. Through molecular design, they can exhibit amphiphilic properties similar to phospholipids. They can also be enhanced with targeting sequences to improve their tumor targeting. Amphiphilic peptides encapsulate anti-tumor drugs, enabling sustained release and targeted drug delivery.

[0005] Chinese patent application number 201910571060.1 discloses an actively targeted amphiphilic polypeptide nanodrug carrier and its preparation and application. The nanocarrier consists of a hydrophobic alkyl chain coupled to the N-terminus of a polypeptide chain with active targeting functionality, and fluorescent functional molecules modified on the side chains of the polypeptide chain. The amphiphilic polypeptides self-assemble to form the nanocarrier. This nanocarrier can actively target tumor cells and enter them through receptor-mediated endocytosis. The amphiphilic polypeptides interact strongly with phospholipid molecules, promoting phagocytosis of the nanodrug by tumor cells. However, the encapsulation efficiency of this nanocarrier is only approximately 64%, which remains to be improved. Summary of the Invention

[0006] The technical purpose of the present invention is to provide an actively targeted branched polypeptide, an actively targeted nano-drug carrier formed by self-assembly of an amphiphilic branched polypeptide in an aqueous solution, an actively targeted anti-tumor nano-drug obtained by encapsulating an anti-tumor drug in an actively targeted nano-drug carrier, and a preparation method thereof, as well as the use of the actively targeted branched polypeptide and the actively targeted nano-drug carrier in the preparation of anti-tumor drugs.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0008] An active targeting branched polypeptide, the active targeting branched polypeptide is an amphiphilic branched polypeptide, or an amino acid sequence having equivalent drug-carrying function formed by replacing, adding or deleting one or more amino acids of the amphiphilic branched polypeptide, or a homologous polypeptide comprising the amino acid sequence of the amphiphilic branched polypeptide; the amino acid sequence of the amphiphilic branched polypeptide is (FHF)2KRGD, branched at K, and the structural formula from N-terminus to C-terminus is:

[0009] .

[0010] Furthermore, the homologous polypeptide has 85% to 100% amino acid sequence identity with the amino acid sequence (FHF)2KRGD.

[0011] The present invention also provides an active targeting nano drug carrier, which is a nanoscale vesicle formed by self-assembly of the above-mentioned amphiphilic branched polypeptide.

[0012] The present invention also provides an active targeting anti-tumor nano-drug, which is an anti-tumor drug encapsulated in the above-mentioned active targeting nano-drug carrier.

[0013] Furthermore, the anti-tumor drug is a fat-soluble natural active small molecule.

[0014] The present invention also provides a method for preparing the above-mentioned active targeted anti-tumor nanomedicine, comprising the following steps:

[0015] S1. preparing an amphiphilic branched polypeptide, wherein the amino acid sequence of the amphiphilic branched polypeptide is (FHF)2KRGD, and the polypeptide is branched at K;

[0016] S2. Dissolving the amphiphilic branched polypeptide prepared in step S1 and the anti-tumor drug simultaneously; the amphiphilic branched polypeptide encapsulates the anti-tumor drug in the hydrophobic cavity through self-assembly to obtain an actively targeted anti-tumor nanomedicine.

[0017] Furthermore, in step S1, the amphiphilic branched polypeptide is synthesized by solid phase synthesis, liquid phase synthesis or recombinant synthesis.

[0018] Furthermore, in step S2, the specific steps of preparing the active targeted anti-tumor nanomedicine by self-assembly are:

[0019] S21, dissolving the anti-tumor drug and the amphiphilic branched polypeptide in a water-miscible organic solvent, and stirring in a closed manner to obtain a mixed solution A;

[0020] S22, adding ddH2O dropwise to the mixed solution A, mixing and stirring to obtain a mixed solution B;

[0021] S23, removing the organic solvent from the stirred mixed solution B by rotary evaporation, wherein the amphiphilic branched polypeptide forms vesicles encapsulating the anti-tumor drug during the rotary evaporation process;

[0022] S24. Ultracentrifuging the vesicles encapsulating the anti-tumor drug to remove the unencapsulated anti-tumor drug, thereby obtaining an actively targeted anti-tumor nanomedicine.

[0023] Furthermore, in step S21, the organic solvent is methanol, the molar ratio of the anti-tumor drug and the amphiphilic branched polypeptide is 1:1, and the closed stirring time is 30 minutes;

[0024] In step S22, the mixing and stirring time is 2 hours;

[0025] In step S24, the vesicles encapsulating the anti-tumor drug are ultracentrifuged at 12,000 rpm for 5 minutes, fixed to volume with water, and then stored at 4° C. or freeze-dried and stored at −20° C. to obtain an actively targeted anti-tumor nanomedicine.

[0026] The present invention also provides the use of the above-mentioned active targeting branched polypeptide or active targeting nano drug carrier in the preparation of anti-tumor drugs.

[0027] Furthermore, the anti-tumor drug includes a therapeutic amount of the above-mentioned active targeting branched polypeptide or active targeting nano drug carrier and other pharmaceutically acceptable excipients.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention provides an active targeting branched polypeptide, which is an amphiphilic branched polypeptide, or an amino acid sequence having the same drug-carrying function formed by replacing, adding or deleting one or more amino acids of the amphiphilic branched polypeptide, or a homologous polypeptide comprising the amino acid sequence of the amphiphilic branched polypeptide. The amphiphilic branched polypeptide can be prepared by a commonly used solid-phase synthesis method, and the purity can reach more than 95%. The sequence of the amphiphilic branched polypeptide from the N-terminus to the C-terminus is (FHF)2KRGD, and it branches at K. It is a new polypeptide molecule that does not contain any other modifications. Its N-terminus is hydrophobic and its C-terminus is hydrophilic, which makes the polypeptide molecule amphiphilic and is conducive to self-assembly to form a vesicle structure. In addition, the amphiphilic branched polypeptide sequence is simple and easy to synthesize;

[0030] (2) The active targeted nano drug carrier of the present invention can encapsulate drugs through self-assembly, and can encapsulate a variety of anti-tumor drugs with an encapsulation rate of 60%-90%, meeting clinical needs; the active targeted nano drug carrier is formed by self-assembly of amphiphilic branched polypeptides. The material is non-toxic, has good biocompatibility, and has tumor site specific targeting, making it a biosafe and ideal drug carrier;

[0031] (3) The active targeted anti-tumor nanomedicine of the present invention is a nanoscale vesicle with a diameter of about 200 nm. It can be retained at the tumor site through the EPR effect of the tumor, which helps the tumor cells to internalize and achieve better tumor cell killing effect;

[0032] (4) The amphiphilic branched polypeptide of the present invention contains an active targeting functional sequence Arg-Gly-Asp (RGD) in its sequence, targeting the integrin receptors overexpressed on the surface of tumor cells. The polypeptide nanodrug carrier prepared from the amphiphilic branched polypeptide can specifically recognize the integrin receptors and enter the tumor cells through receptor-mediated endocytosis. Compared with traditional small molecule drugs, the uptake of small molecule drugs by tumor cells is improved, thereby increasing the cytotoxicity of small molecule drugs.

[0033] (5) The hydrophobic end of the amphiphilic branched polypeptide sequence of the present invention contains the basic amino acid His (histidine). Histidine can be charged under acidic conditions, causing the vesicles (PepV polypeptide-loaded nanovesicles) to disintegrate and release the drug. Therefore, the PepV polypeptide-loaded nanovesicles based on the amphiphilic branched polypeptide are responsive to acidic pH, and the drug is released faster in the acidic microenvironment of the tumor.

[0034] (6) Compared with conventional anti-tumor drugs, the active targeted anti-tumor nanomedicine prepared by the present invention has a more gradual drug release rate. The drug release rate of PepV polypeptide-loaded nanovesicles containing different drugs is 50%-65% within 12 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a liquid chromatogram of the PepV polypeptide prepared in Example 1 of the present invention;

[0036] Figure 2 is a mass spectrum of the PepV polypeptide prepared in Example 1 of the present invention;

[0037] Figure 3 is a TEM image of the self-assembled morphology of the PepV polypeptide of Example 2 of the present invention;

[0038] Figure 4 3. These are photos of PepV polypeptide-loaded nanovesicles loaded with different drugs in Example 3 of the present invention, wherein (a) is a PepV polypeptide-loaded nanovesicle loaded with doxorubicin, (b) is a PepV polypeptide-loaded nanovesicle loaded with curcumin, (c) is a PepV polypeptide-loaded nanovesicle loaded with camptothecin, and (d) is a PepV polypeptide-loaded nanovesicle loaded with quercetin.

[0039] Figure 5 TEM images of PepV polypeptide drug-loaded nanovesicles encapsulating different drugs in Example 3 of the present invention, wherein (a) is a TEM image of PepV polypeptide drug-loaded nanovesicles encapsulating doxorubicin, (b) is a TEM image of PepV polypeptide drug-loaded nanovesicles encapsulating curcumin, (c) is a TEM image of PepV polypeptide drug-loaded nanovesicles encapsulating camptothecin, and (d) is a TEM image of PepV polypeptide drug-loaded nanovesicles encapsulating quercetin;

[0040] Figure 6 1 are drug release curves of the PepV polypeptide-loaded nanovesicles loaded with different drugs in Example 4 of the present invention, wherein (a) is a drug release curve of the PepV polypeptide-loaded nanovesicles loaded with doxorubicin, (b) is a drug release curve of the PepV polypeptide-loaded nanovesicles loaded with curcumin, (c) is a drug release curve of the PepV polypeptide-loaded nanovesicles loaded with camptothecin, and (d) is a drug release curve of the PepV polypeptide-loaded nanovesicles loaded with quercetin;

[0041] Figure 7 Graphs showing drug release profiles of PepV polypeptide-loaded nanovesicles encapsulating different drugs at different pH values ​​in Example 4 of the present invention, wherein (a) shows drug release profiles of PepV polypeptide-loaded nanovesicles encapsulating doxorubicin at different pH values, (b) shows drug release profiles of PepV polypeptide-loaded nanovesicles encapsulating curcumin at different pH values, (c) shows drug release profiles of PepV polypeptide-loaded nanovesicles encapsulating camptothecin at different pH values, and (d) shows drug release profiles of PepV polypeptide-loaded nanovesicles encapsulating quercetin at different pH values.

[0042] Figure 8 The IC values ​​of the PepV polypeptide drug-loaded nanovesicles loaded with different drugs on HeLa cells in Example 5 of the present invention are 50 ;

[0043] Figure 9 This is a laser confocal microscopy image of the uptake effect of HeLa cells on PepV polypeptide nanovesicles loaded with doxorubicin in Example 6 of the present invention. DETAILED DESCRIPTION

[0044] The present invention provides an active targeting branched polypeptide, which is an amphiphilic branched polypeptide, or an amino acid sequence having equivalent drug-carrying function formed by replacing, adding or deleting one or more amino acids of the amphiphilic branched polypeptide, or a homologous polypeptide comprising the amino acid sequence of the amphiphilic branched polypeptide, wherein the homologous polypeptide comprises an amino acid sequence (FHF)2KRGD with at least 85% homology, more preferably an amino acid sequence with 100% homology (sequence identity); the structural formula of the amphiphilic branched polypeptide from N-terminus to C-terminus is:

[0045] ;

[0046] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods, and equipment used are conventional reagents, methods, and equipment in the art unless otherwise specified.

[0047] Example 1: Preparation of an amphiphilic branched polypeptide (hereinafter referred to as "PepV polypeptide")

[0048] The PepV polypeptides of the present invention may be synthetic, semisynthetic or recombinantly produced, for example, by solid phase synthesis, liquid phase synthesis or recombinant synthesis of the polypeptide.

[0049] This example uses a solid-phase synthesis method with Fmoc-Wang resin as the solid matrix. Different amino acids are added sequentially from the C-terminus to the N-terminus according to the peptide sequence to synthesize the PepV peptide (at a concentration of 100 μM). The structural formula of the PepV peptide is:

[0050] ;

[0051] The specific synthesis steps are as follows:

[0052] (1) Activation of the resin: Weigh 100 μM (39.44 mg) of Wang resin (purchased from Jier Biochemical (Shanghai) Co., Ltd.) linked to Fmoc-protected aspartic acid, pour it into a clean, anhydrous solid phase reactor, add 5 mL of DCM (dichloromethane) to dissolve and activate it overnight;

[0053] (2) Cleaning the resin: drain the liquid in the solid phase reactor, add 4 mL of DMF (N, N-dimethylformamide), shake thoroughly for 1 min, drain, and repeat this operation 8 times; collect a small amount of activated resin for Kaiser test;

[0054] (3) Fmoc removal: After draining the solvent, add 4 mL of DMF solution containing 20% ​​piperidine, place on a shaker, shake for 5 min, and drain the solvent; then add 4 mL of DMF solution containing 20% ​​piperidine, place on a shaker, and shake for 20 min;

[0055] (4) Cleaning the resin and removing piperidine: After draining the solvent, add 4 mL of DMF solution, place on a shaker, shake for 1 min, and then drain; repeat this process 8 times until the piperidine is completely removed;

[0056] (5) Weigh the amino acid to be connected (here, Fmoc-protected glycine) and the coupling reagent using an electronic balance: dissolve 4 times the molar amount of the amino acid (i.e., Fmoc-protected glycine, 400 μM), 4 times the molar amount of HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 400 μM), and 4 times the molar amount of HOBT (1-hydroxybenzotriazole, 400 μM) in 4 mL of DMF, mix until completely dissolved, add the mixture to the solid phase reactor, mix thoroughly with the resin from which piperidine was removed in step (4), and shake on a shaker for 5 minutes;

[0057] (6) After the shaking time is up, add DIEA (N,N-diisopropylethylamine) in an amount 8 times the molar amount of the resin prepared in step (5), mix thoroughly, place on a shaker, and time the reaction for 2 hours;

[0058] (7) Repeat steps (2) to (6) to sequentially insert Fmoc-protected arginine and double-Fmoc-protected lysine;

[0059] (8) Weigh out 2 times the molar amount (i.e., 800 μM) of phenylalanine in step (5) and 2 times the molar amount (i.e., 800 μM) of other reagents (HBTU, HOBT, DIEA), mix until completely dissolved, add to the solid phase reactor and mix thoroughly with the resin, and shake on a shaker for 5 minutes;

[0060] (9) Repeat steps (2) to (6) to sequentially insert histidine and phenylalanine, and then proceed to step (8);

[0061] (10) Kaiser test: Ninhydrin reacts with ammonia or primary amine to produce a purple-red complex. Kaiser reagent includes: 6% ninhydrin ethanol solution; 80% phenol ethanol solution; 2% 0.001M KCN ​​pyridine solution. Take a small amount of the resin after the reaction in step (6) and the resin in step (2), add 2 to 3 drops of each of the three components in the Kaiser reagent, and heat at 100°C for 1 to 2 minutes. If it turns blue or reddish brown, it indicates that there are free amino groups. Otherwise, it indicates that the peptide chain is completely connected.

[0062] (11) After the peptide chain is connected, the resin is washed and deprotected twice with piperidine;

[0063] (12) Wash the resin 10 times with DMF (4 mL each time); then wash the resin 10 times with DCM (4 mL each time);

[0064] (13) Vacuum drying of samples;

[0065] (14) After the sample is dried, transfer the resin to a heart-shaped bottle, install a magnetic stirrer, fix the heart-shaped bottle, slowly add the mixed cutting reagent (trifluoroacetic acid: ultrapure water: thioanisole: phenol: ethanedithiol = 82.5:5:5:5:2.5), add a magnetic bar for thorough stirring, and react at room temperature for 12 hours;

[0066] (15) After the reaction is completed, the reactants are transferred to a solid phase reactor to react the untransferred resin in the solid phase reactor. The reaction is allowed to stand for 10 min. The chicken heart flask is rinsed with TFA (trifluoroacetic acid). All the resin and solution are poured into a new solid phase reactor. The mixture is then filtered under a nitrogen flow. The filtrate is placed in a round-bottom flask and blown dry under a nitrogen flow.

[0067] (16) When the sample in the round-bottom flask is blown until it becomes viscous, remove the nitrogen and pour about 20 mL of icy ether into the round-bottom flask to precipitate the polypeptide. Disperse the insoluble matter thoroughly, then balance it and place it in a refrigerated centrifuge. Centrifuge it at 8000 rpm for 15 min at 4°C. Discard the supernatant and dissolve it in 20 mL of icy ether to disperse it and centrifuge it. Repeat this operation and centrifuge three times. Vacuum dry the precipitate to obtain the crude PepV polypeptide.

[0068] (17) The crude PepV polypeptide was purified by preparative HPLC and then analyzed for purity by analytical HPLC. The results are as follows: Figure 1 As shown;

[0069] (18) The purified target peptide was identified using ESI high-resolution mass spectrometry, and its mass spectrum is shown in the following figure: Figure 2 shown by Figure 2The molecular weight of the synthesized PepV polypeptide was measured to be 1337.49, which is consistent with the molecular weight of the polypeptide with the sequence (FHF)2KRGD. This indicates that the polypeptide prepared in this example is the target polypeptide, i.e., the PepV polypeptide, whose amino acid sequence is (FHF)2KRGD, with a branch at K, and the structural formula from N-terminus to C-terminus is:

[0070] .

[0071] Example 2: Preparation and Observation of Self-Assembled Nanovesicles (Actively Targeted Nanodrug Carriers) of PepV Peptides

[0072] Taking the PepV polypeptide prepared in Example 1 as an example, the PepV polypeptide self-assembled nanovesicles were prepared according to the following steps:

[0073] (1) Prepare a 250 μM PepV peptide solution and allow it to self-assemble at room temperature overnight;

[0074] (2) Prepare 20 mg / mL phosphotungstic acid (PTA) and adjust the pH to 6.0-7.0 with 0.1 M sodium hydroxide;

[0075] (3) Take a TEM copper grid, drop a drop of self-assembled PepV polypeptide sample on the copper grid to form a water droplet, let it stand for 1 minute, and then use filter paper to absorb the solvent at the edge of the copper grid to make the copper grid semi-wet; then add a drop of phosphotungstic acid dye solution (pH 6.0-7.0), dye for about 1.5 minutes, and dry it with filter paper; then add a drop of ultrapure water, let it stand for 10 seconds, and absorb the ultrapure water; repeat the washing with ultrapure water twice; finally, place the copper grid in a culture dish to dry;

[0076] (4) Observation of the morphology of PepV polypeptide self-assembled nanovesicles using transmission electron microscopy;

[0077] like Figure 3 As shown, the PepV polypeptide of the present invention can self-assemble into nano-sized vesicles after incubation at room temperature overnight, and the particle size of the blank PepV polypeptide nanovesicles is relatively uniform, about 30-60 nm.

[0078] Example 3: Preparation of PepV polypeptide-loaded nanovesicles (actively targeted anti-tumor nanomedicine)

[0079] Taking the PepV polypeptide prepared in Example 1 as an example, the steps for preparing PepV polypeptide-loaded nanovesicles are as follows:

[0080] (1) Weigh an appropriate amount of drug and PepV peptide and add them to methanol, seal and stir for 30 minutes;

[0081] (2) Add ddH2O dropwise and stir again for 2 h;

[0082] (3) The product stirred in step (2) is subjected to rotary evaporation to remove the organic solvent methanol. During this process, the PepV polypeptide forms vesicles that encapsulate the drug;

[0083] (4) Ultracentrifuge at 12000 rpm for 5 min to remove unencapsulated drugs, add ddH2O to make up to 5 mL, and store at 4°C, or freeze-dry and store at -20°C.

[0084] The actual picture of PepV polypeptide drug-loaded nanovesicles encapsulating drugs is as follows Figure 4 As shown, Figure 4 The left and right images in (a) to (d) show the directly dissolved drug and the drug encapsulated in PepV peptide-loaded nanovesicles, respectively;

[0085] (5) TEM was used to observe the morphology of the prepared PepV polypeptide drug-loaded nanovesicles, such as Figure 5 As shown, the diameter of PepV polypeptide-loaded nanovesicles is 200-400 nm;

[0086] (6) The lyophilized powder from step (4) was redissolved in methanol, and the content of the encapsulated drug was determined by high performance liquid chromatography. The drug loading (DE%) and encapsulation efficiency (EE%) were calculated using the following formulas. The results are shown in Table 1.

[0087] The formulas for calculating drug loading and encapsulation efficiency are as follows:

[0088]

[0089] As shown in Table 1, the drug loading capacity and encapsulation efficiency of the PepV polypeptide for different drugs are different. The drug loading range is 15%-22%, and the encapsulation efficiency can reach 60%-92%. This shows that the amphiphilic branched polypeptide designed in the present invention can encapsulate a variety of hydrophobic drug small molecules and has a strong encapsulation capacity.

[0090] Example 4: In vitro drug release experiment of PepV polypeptide loaded nanovesicles

[0091] The dialysis method was used to study the in vitro drug release of PepV peptide-loaded nanovesicles. The specific steps are as follows:

[0092] (1) The freeze-dried PepV peptide-loaded nanovesicles were prepared into 3 mL of dispersion using deionized water, placed in a 14 kD dialysis bag, and 50 mL of PBS containing 1% Tween 80 (pH 7.4) was used as the release medium.

[0093] (2) Place the dialysis bag in a 37°C constant temperature shaking incubator, draw 300 μL of release medium for detection at the designated time point, and replenish 300 μL of release medium at the same time;

[0094] (3) The released drug content was calculated by measuring the absorbance and the drug release rate-time curve was drawn; the curves of different drugs released from PepV-loaded nanovesicles are shown in Figure 2. Figure 6 As shown;

[0095] (4) Using PBS containing 1% Tween 80 as the release medium (pH 5.5), repeat steps (1) to (3) to draw the drug release rate-time curve of PepV peptide-loaded nanovesicles under acidic conditions. The results are as follows: Figure 7 shown.

[0096] Depend on Figure 6 It can be seen that compared with conventional anti-tumor drugs, PepV polypeptide-loaded nanovesicles can continuously release drugs within 12 hours. The drug release rate of PepV polypeptide-loaded nanovesicles containing different drugs is 50%-65% within 12 hours, which has a sustained release effect.

[0097] Depend on Figure 7 It can be seen that in the acidic solution environment of pH 5.5, the drug release rate of PepV polypeptide-loaded nanovesicles is faster than that in the solution environment of pH 7.4, indicating that PepV polypeptide-loaded nanovesicles are pH responsive.

[0098] Example 5: Cellular activity experiment of PepV polypeptide-loaded nanovesicles

[0099] The cell activity of PepV peptide-loaded nanovesicles was detected using the MTT assay. The specific steps are as follows:

[0100] (1) Preparation of reagents and culture medium

[0101] Preparation of MTT solution: Prepare 5 mg mL in PBS -1 MTT (Thiazolyl Blue, purchased from Sigma, catalog number: M5655) was wrapped in tin foil and stirred at room temperature for 30 min. After complete dissolution, it was filtered through a 0.22 μM filter membrane and stored at 4°C in the dark.

[0102] Preparation of triple reagent: Dissolve 10 g of SDS (sodium dodecyl sulfate), 5 mL of isobutanol, and 0.12 mL of 10 M hydrochloric acid in double distilled water to make a 100 mL solution;

[0103] Preparation of cell culture medium: 90% DMEM medium (purchased from Gibco, catalog number: GMS12052.3.1) and 10% FBS;

[0104] Cell culture medium was used to culture HeLa cells (human cervical cancer cell line, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences);

[0105] (2) When the HeLa cells in the culture flask have grown to more than 80% confluence, add 1 mL of 0.25% trypsin (purchased from Sigma) for digestion and place incubator at 37°C, 5% CO2. When the cell digestion is complete, add 4 mL of cell culture medium and centrifuge at 1000 rpm for 4 minutes. Discard the supernatant, add cell culture medium and gently pipette to evenly disperse the cells in the culture medium.

[0106] (3) Count the number of cells using a cell counter and add cell culture medium to adjust the cell concentration to 5×10 4 / mL;

[0107] (4) Take the cell suspension from step (3) and plate it, add 100 μL to each well, and add 5000 cells per well, then place it in a 5% CO2, 37℃ incubator and incubate overnight to allow the cells to adhere;

[0108] (5) After the incubation, different concentrations of PepV peptide-loaded nanovesicles were prepared using serum-free culture medium (DMEM, purchased from Gibco, catalog number: GMS12052.3.2), added to a 96-well plate, and incubated with the HeLa cells in step (4) for 24 hours;

[0109] Among them, the blank group: only cell culture medium was added; the drug group: HeLa cells were added with cell culture medium containing drugs (using culture medium containing 0.5% DMSO to dissolve drugs, such as doxorubicin, curcumin, camptothecin or quercetin); PepV drug-loaded nanovesicle group: HeLa cells were added with PepV polypeptide drug-loaded nanovesicles loaded with different anti-tumor drugs;

[0110] (6) After incubation for 24 hours, aspirate the culture medium; dilute the previously prepared MTT solution ten-fold with phenol red-free DMEM complete culture medium, and then add 100 μL to each well; then place the 96-well plate in a 5% CO2, 37°C cell culture incubator;

[0111] (7) After 4 hours of reaction, 100 μL of triple reagent (10% SDS + 5% isobutanol + 0.012 mol / L HCl) was added to each well. The mixture was placed on a shaker at room temperature for 12 to 24 hours. The OD value of the solution at 570 nm was detected by a microplate reader. The IC values ​​of various drugs and PepV peptide-loaded nanovesicles on HeLa cells were calculated using SPSS software. 50 Value, see the result Figure 8 .

[0112] IC 50 The value can be used to measure the toxicity of drugs to tumor cells, that is, the stronger the cytotoxicity, the lower the value; Figure 8The results show that PepV polypeptide-loaded nanovesicles containing different drugs have different toxicities to HeLa cells, and their toxicity to HeLa cells is significantly higher than that of their corresponding anti-tumor drugs. This further illustrates that the PepV polypeptide-loaded nanovesicles prepared by the present invention can efficiently deliver drugs to tumor cells, thereby improving the anti-tumor effect.

[0113] Example 6: Laser confocal microscopy observation of PepV polypeptide-loaded nanovesicles uptake experiment

[0114] (1) When the HeLa cells in the culture flask have grown to more than 80% confluence, add 1 mL of 0.25% trypsin (purchased from Sigma) for digestion and place in a 37°C, 5% CO2 incubator for incubation. When the cell digestion is complete under an inverted microscope, add 4 mL of cell culture medium to terminate the digestion and centrifuge at 1000 rpm for 4 min. Discard the supernatant, add cell culture medium, and gently pipette to evenly disperse the cells in the culture medium.

[0115] (2) Count the number of cells using a cell counter and add cell culture medium to adjust the cell concentration to 10 6 / mL;

[0116] (3) Take the cell suspension from step (2) and add it to a culture dish specially designed for laser confocal microscopy, with a volume of 400 μL added to each well, that is, 400,000 cells per well, and then place it in a 5% CO2, 37°C incubator and incubate overnight to allow the cells to adhere;

[0117] (4) Aspirate the culture medium and add 400 μL of PepV polypeptide nanovesicle solution loaded with doxorubicin diluted in incomplete culture medium at a drug concentration of 5 μg / mL; doxorubicin served as the control group;

[0118] (5) After incubating the cells with drugs for 4 h, the culture medium was removed and the cells were rinsed 2-3 times with PBS to remove the unabsorbed drugs. Hoechst 33342 dye was then added and the cells were stained in the dark for 30 min.

[0119] (6) Observe the imaging situation with a laser confocal microscope; set the channel-1 excitation wavelength (λ ex ) is 488nm, the emission wavelength (λ em ) was set to 575-585 nm to observe the fluorescence of doxorubicin (DOX group in the figure); set the excitation wavelength of channel 2 (λ ex ) is 350nm, the emission wavelength (λ em) is 450-470nm, and the fluorescence of Hoechst33342 dye is observed (Hoechst group in the figure); photos are taken and saved, and images of channel 1, channel 2, and the superposition of the two are automatically generated (Merge group in the figure).

[0120] Depend on Figure 9 It can be seen that after the cells were treated with PepV polypeptide nanovesicles loaded with doxorubicin, the cell uptake efficiency of doxorubicin was significantly higher than that of pure doxorubicin, and there was also obvious doxorubicin fluorescence in the cell nucleus; this shows that PepV polypeptide nanovesicles can improve the cell uptake of drugs.

[0121] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions based on the principles of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. An active targeting branched polypeptide, characterized in that: The active targeting branched polypeptide is an amphiphilic branched polypeptide; the amino acid sequence of the amphiphilic branched polypeptide is (FHF)2KRGD, branching at K, and the structural formula from N-terminus to C-terminus is: 。 2. An active targeted nano drug carrier, characterized in that: The active targeting nano drug carrier is a nanoscale vesicle formed by self-assembly of the active targeting branched polypeptide described in claim 1.

3. An active targeted anti-tumor nanomedicine, characterized in that: The actively targeted anti-tumor nanomedicine is an anti-tumor drug encapsulated in the actively targeted nanomedicine carrier according to claim 2; the anti-tumor drug is a fat-soluble natural active small molecule.

4. The method for preparing the active targeted anti-tumor nanomedicine according to claim 3, characterized in that: The following steps are involved: S1. preparing an amphiphilic branched polypeptide, wherein the amino acid sequence of the amphiphilic branched polypeptide is (FHF) 2KRGD, branched at K; S2. Dissolving the amphiphilic branched polypeptide prepared in step S1 and the anti-tumor drug simultaneously; the amphiphilic branched polypeptide encapsulates the anti-tumor drug in the hydrophobic cavity through self-assembly to obtain an actively targeted anti-tumor nanomedicine.

5. The method for preparing the active targeted anti-tumor nanomedicine according to claim 4, characterized in that: In step S2, the specific steps of preparing active targeted anti-tumor nanomedicine by self-assembly are: S21. The anti-tumor drug and the amphiphilic branched polypeptide are dissolved in a water-miscible organic solvent and stirred to obtain a mixture A; S22. ddH2O was added dropwise to the mixed solution A, and the mixture was stirred to obtain a mixed solution B; S23. The stirred mixture B is subjected to rotary evaporation to remove the organic solvent, and the amphiphilic branched polypeptide forms vesicles encapsulating the anti-tumor drug during the rotary evaporation process; S24. Ultracentrifuge the vesicles encapsulating the anti-tumor drug to remove the unencapsulated anti-tumor drug, thereby obtaining an actively targeted anti-tumor nanomedicine.

6. The method for preparing the active targeted anti-tumor nanomedicine according to claim 5, characterized in that: In step S21, the organic solvent is methanol, the molar ratio of the anti-tumor drug and the amphiphilic branched polypeptide is 1:1, and the closed stirring time is 30 minutes; In step S22, the mixing and stirring time is 2 hours; In step S24, the vesicles encapsulating the anti-tumor drug are ultracentrifuged at 12,000 rpm for 5 minutes, fixed to volume with water, and then stored at 4° C. or freeze-dried and stored at −20° C. to obtain an actively targeted anti-tumor nanomedicine.

7. Use of the actively targeted branched polypeptide according to claim 1 or the actively targeted nano drug carrier according to claim 2 in the preparation of an anti-cervical cancer drug carrier.

8. The use according to claim 7, characterized in that: The anti-cervical cancer drug includes the above-mentioned active targeting branched polypeptide or active targeting nano drug carrier and other pharmaceutically acceptable excipients.

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