CN114230634B - Acid-responsive anticancer peptide and preparation method thereof - Google Patents

Abstract

The invention discloses an artificially synthesized anticancer peptide and an acid-responsive nanoparticle precursor thereof. The nanoparticle is formed by connecting anticancer peptide and amphiphilic monomethyl polyethylene glycol-polypropylene glycol polymer through acid-responsive chemical bonds. Experiments prove that the anticancer peptide kills tumor cells through membrane breaking activity and has the advantages of excellent broad-spectrum anticancer activity and drug resistance. The nanoparticle is selectively sensitive to tumor subacid environment, can completely release anticancer peptide, and plays an active role in targeting tumor cell lines. And has the advantages of hemolysis resistance, high plasma stability, low in vivo system toxicity, systemic administration and the like. The nanoparticle has obvious inhibition effect on various tumor cells including triple negative breast cancer, and particularly has good clinical application potential on drug-resistant triple negative breast cancer.

Description

Acid-responsive anticancer peptide and preparation method thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to an artificially synthesized anticancer peptide and an acid-responsive nanoparticle thereof.

Background

The easy drug resistance and poor selectivity are always the problems to be solved urgently by chemotherapeutic anti-cancer drugs. Taking the Breast Cancer subtype Triple Negative Breast Cancer (TNBC) as an example, chemotherapy remains the standard treatment for TNBC due to the low expression or deletion of estrogen receptors, progesterone receptors, and human epidermal growth factor (HER 2) receptors, resulting in endocrine and anti-HER 2 targeted monoclonal antibody therapy not being effective against TNBC. However, TNBC has high tumor heterogeneity, and the expression of drug efflux pumps such as ATP-binding cassette transporters is significantly excessive, so that drug resistance is easily caused, which leads to reduced chemotherapy sensitivity, and leads to the dilemma that TNBC with drug resistance can not be cured by drugs.

Research shows that some antibacterial peptides can exert membrane breaking activity and have broad-spectrum anticancer effect, and are also called as anticancer peptides. The anticancer peptide is a kind of amphiphilic polypeptide with cation and hydrophobic residue, and is adsorbed on the surface of tumor cell membrane through electrostatic interaction between the positive charge area and the negatively charged lipid on the surface of tumor cell membrane, so that the anticancer peptide is inserted into the lipid of cell membrane through the hydrophobic structure area to destroy the fluidity of cell membrane, and can directly pass through the cell membrane or generate holes to make the content leak out to kill tumor cells. The anticancer peptide kills tumor cells in a physical membrane breaking mode, directly causes irreversible damage to cell membranes, does not depend on the metabolic state of cells and does not need to enter the cells to play a role, and therefore the problem of drug resistance is not easy to cause. In view of the unique advantage of low drug resistance of anticancer peptides, it has become a hot spot in the industry, for example: melittin is proved to have good killing activity on liver cancer, lung cancer, breast cancer, gastric cancer and leukemia cell lines at a cell level, and a few anticancer peptides have entered a clinical test stage. Nevertheless, the antitumor therapy of anticancer peptides has not made a major breakthrough. The defects of low selectivity, normal tissue cytotoxicity, hemolytic toxicity, instability in serum, low bioavailability, need of intratumoral administration and the like are still the main bottlenecks preventing the application of the anticancer peptide in tumor treatment.

Disclosure of Invention

In order to overcome the technical defects, the invention discloses a synthesized anticancer peptide and prodrug acid-responsive anticancer peptide nanoparticles obtained by modifying the synthesized anticancer peptide based on acid-responsive groups; and further discloses a preparation method of the two.

The molecular structure of the anticancer peptide is shown as the following formula:

。

the preparation method comprises the following steps: the synthetic scheme is shown in figure 1, and is obtained by polymerizing L-glutamic acid as a raw material to form a polymer peptide and then performing cationic modification.

The acid-responsive anticancer peptide nanoparticles are formed by connecting the anticancer peptide and an amphiphilic monomethyl polyethylene glycol-polypropylene glycol polymer through a specific acid-responsive chemical bond. The synthetic scheme of the preparation method is shown in figure 2.

The molecular structure of the acid-responsive anticancer peptide nanoparticle is shown as the following formula:

。

the pH value of the acid response environment of the nanoparticle is 6.5-7.2, and the optimal pH value is 6.6-6.8.

The nano particle is an amphiphilic particle with the average particle size of 60-70 nm.

The invention further discloses application of the anticancer peptide and the acid-responsive anticancer peptide nanoparticles thereof in preparing medicaments for treating tumors such as breast cancer, colon cancer, pancreatic cancer and the like, in particular application in preparing medicaments for treating triple negative breast cancer, including triple negative breast protomer cancer and metastatic cancer.

The anticancer peptide of the invention is a radial amphiphilic anticancer peptide which is artificially synthesized for the first time by taking L-glutamic acid as a raw material. Experiments prove that the anticancer peptide directly and irreversibly kills tumor cells in a physical membrane rupture mode, has obvious killing effect on various breast cancer cell lines including triple negative breast cancer, colon cancer and pancreatic cancer cell lines, and has good broad-spectrum anticancer activity and drug resistance advantage.

In order to reduce the toxicity of the anticancer peptide on normal cells and improve the stability of the anticancer peptide in blood plasma, the anticancer peptide is bonded with an amphiphilic monomethyl polyethylene glycol-polypropylene glycol polymer through a specific acid response chemical bond by utilizing the characteristic that the pH value of a tumor microenvironment is lower than that of a normal tissue and is slightly acidic (pH is 6.5-7.2), and the acid response nanoparticle precursor is automatically assembled.

The acid-responsive anticancer peptide nanoparticles of the invention are proved by experiments to have the following characteristics and advantages:

1. the anticancer peptide is positioned in the nano-particles, so that the cytotoxicity of the anticancer peptide can be effectively shielded, the hemolytic activity can be obviously reduced, and the stability of the anticancer peptide can be improved.

2. The specific acid response chemical bond of the nanoparticle is selectively sensitive to a tumor subacidity (pH 6.5-7.2) microenvironment to break, and can completely release the anticancer peptide to play an active role in targeting a tumor cell system. Test results show that the nanoparticle has almost no membrane rupture activity under the condition of pH 7.4, but the membrane rupture activity under the slightly acidic environment of pH 6.8 is similar to that of free anticancer peptide, and the nanoparticle has a good targeting effect.

3. The nanoparticle has amphipathy, the average particle size is between 60 and 70nm, the particle size distribution is good (PDI is lower than 0.3), and the nanoparticle has good solubility. Researches prove that the nanoparticles with the particle size range have longer half-life period in vivo and are easy to accumulate at tumor parts, and the bioavailability of the anticancer peptide is further improved.

4. The detection of the maximum tolerated dose and the hepatotoxicity of the mice shows that the nanoparticles have no obvious damage to the hepatotoxicity and the renal functions of the mice, and have the condition of systemic administration without intratumoral administration.

5. The nanoparticles have obvious treatment effects on primary tumor and metastatic tumor of triple negative breast cancer through in vivo animal experiments, and particularly have good clinical application potential on drug-resistant triple negative breast cancer.

6. The anticancer peptide and the acid response nanoparticles thereof take L-glutamic acid as a raw material, the raw material is easy to obtain, the preparation method is simple, the cost is low, and the large-scale production can be realized.

Drawings

FIG. 1 is a schematic diagram of the synthesis of the anticancer peptide of the present invention;

FIG. 2 is a schematic diagram of the synthesis of acid-responsive anticancer peptide nanoparticles according to the present invention;

FIG. 3 is a GPC characterization map of the inventive intermediate polymer C12-PButLG;

FIG. 4 is a nuclear magnetic hydrogen spectrum of the inventive intermediate polymer C12-PButLG;

FIG. 5 is a nuclear magnetic hydrogen map of the anticancer peptide C12-PButLG-CA of the present invention;

FIG. 6 is a nuclear magnetic hydrogen spectrum of the acid-responsive anticancer peptide nanoparticle intermediate PEO-PPO-CDM of the present invention;

FIG. 7 is a nuclear magnetic hydrogen spectrum of the acid-responsive anticancer peptide nanoparticles of the present invention;

FIG. 8 is (PEO-PPO-CDM)₂-nuclear magnetic hydrogen map assembled in heavy water of C12-PButLG-CA;

FIG. 9 is (PEO-PPO-CDM) ₂-nuclear magnetic hydrogen spectrum of C12-PButLG-CA nanoparticles after addition of deuterated hydrochloric acid;

FIG. 10 is (PEO-PPO-CDM)₂-degradation profile of C12-PButLG-CA at pH = 6.8;

FIG. 11A is a graph showing the killing activity of anticancer peptides against different breast cancer cell lines, and B is a graph showing the killing activity of other tumor cell lines;

FIG. 12 shows different concentrations in examples^NRPeptide、^RGraph of the hemolytic activity of Peptide and Peptide;

FIG. 13 shows different concentrations of the compounds in the pH 7.4 and pH 6.8 environments of the examples^NRPeptide、^RPeptide and Peptide have a tumor cell inhibition effect curve chart;

FIG. 14 shows the pH values at different values in the examples^RGraph of inhibitory effect of Peptide on tumor cells;

FIG. 15 shows the pH values of the samples obtained by scanning electron microscopy in the examples under different pH conditions^RPictures of results of Peptide on EMT6 cells; a and B are blank control group pictures without treatment, and C is under the environment of pH 7.4^RGraph of tumor cells after Peptide treatment, D is pH 6.8 environment^RPepthe pictures of the tumor cells after tide treatment, wherein a-d are partial enlarged images of corresponding pictures on the left side, and arrows indicate the positions of holes on the surface of the cell membrane;

FIG. 16 shows that under the environment of pH 7.4 and pH 6.8 in the example,^Rdiagrammatic representation of the situation where Peptide causes leakage of cellular contents,

a is^NRPeptide and^R(iii) a condition in which Peptide causes leakage of intracellular LDH; b is silver staining detection^RA condition where Peptide causes leakage of intracellular protein;

FIG. 17 shows that after endocytosis inhibitors (2-deoxy-D-glucose, chlorpromazine, methyl-beta-cyclodextrin and wortmannin) of different pathways inhibit tumor cell endocytosis in examples,^Ra histogram of the inhibitory effect of Peptide on tumor cell viability in the environments of pH 7.4 and pH 6.8, data are shown as mean ± standard deviation, n.s. means no significant difference;

FIG. 18 is a graphical representation of the dosing regimen for the mouse model of triple negative breast tumor in the examples;

FIG. 19 is a drawing of an embodiment^RA graph of inhibition of tumor growth of EMT6 tumor-bearing mice by Peptide in vivo is shown, wherein A is a tumor growth curve, B is tumor weight, and C is a tumor picture;

FIG. 20 is a drawing of an embodiment^RA graph of inhibition of tumor growth of a 4T1 tumor-bearing mouse by Peptide in vivo is shown, wherein A is a tumor growth curve, B is tumor weight, and C is a tumor picture;

FIG. 21 shows an embodiment^RA histogram for the number of lung metastases of breast cancer cells of mice bearing 4T1 tumor in vivo by Peptide;

FIG. 22 shows an embodiment^RThe copy of the HE stained section of the lung and the liver of the breast cancer of a mouse with 4T1 tumor-bearing in vivo inhibited by Peptide, wherein the black dotted line is tumor tissue;

FIG. 23 shows an example of the present invention^RHistogram of in vivo systemic toxicity assay in Peptide mice;

FIG. 24 is a drawing of an embodiment^NRNuclear magnetic hydrogen spectrum of Peptide.

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

Name and corresponding abbreviations or code descriptions:

the anticancer Peptide of the embodiment is referred to as Peptide for short, and the code in the synthetic route is C12-PButLG-CA;

the acid-responsive anticancer peptide nanoparticles of the embodiment are simply called^RPeptide, the code number in the synthetic route is (PEO)

-PPO-CDM)₂-C12-PButLG-CA;

The non-acid-responsive anticancer peptide nanoparticles of the embodiment are simply called^NRPeptide, the code number in the synthetic route is (PEO-

PPO-SA)₂-C12-PButLG-CA;

1.Peptide、^RPeptide and^NRpreparation of Peptide

1.1 Synthesis and characterization of Peptide

The synthetic circuit diagram of Peptide is shown in FIG. 1, and the specific synthetic method comprises the following steps:

(1) synthesis of glutamic acid derivative ButLG

Weighing 10.0 g of L-glutamic acid into a round-bottom flask, adding 15 mL of 3-buten-1-ol, placing the mixture into an ice bath, fully stirring, slowly dropwise adding 4.0 mL of concentrated sulfuric acid, reacting at room temperature for 24 h, pumping out unreacted 3-buten-1-ol, adding saturated sodium bicarbonate to neutralize until the pH is =7, performing suction filtration and washing to obtain a solid, adding the solid into 4.0 mL of isopropanol and 4.0 mL of deionized water, dissolving at 80 ℃, cooling, recrystallizing, performing suction filtration and washing with diethyl ether to obtain a solid which is a glutamic acid derivative ButLG.

(2) Synthesis of the reactive monomer ButLG-NCA

Weighing 5.0 g of glutamic acid derivative ButLG into a round-bottom flask, adding about 150 mL of anhydrous Tetrahydrofuran (THF), placing the mixture in an ice bath for stirring, adding 7.2 g of triphosgene, reacting at 50 ℃ for 2.5 h, draining the solvent, transferring the mixture into a glove box, and purifying by a column to obtain the active monomer ButLG-NCA.

(3) Synthesis of Polymer C12-PButLG

Weighing 2.5 g of ButLG-NCA monomer into a round-bottom flask, adding 5.0 mL of anhydrous Dimethylformamide (DMF) and 200 mg of dodecylamine, stirring for reacting for 24 h, extracting the DMF, dissolving the DMF with dichloromethane, dropwise adding the mixture into a mixed solution of diethyl ether and n-hexane with a volume ratio of 1:1 for precipitation, removing a supernatant, draining to obtain a polymer C12-PButLG, and measuring GPC and nuclear magnetic hydrogen spectra, wherein the obtained spectra are shown in figure 3 and figure 4.

As can be seen by GPC characterization of FIG. 3, the prepared C12-PButLG has monodispersity.

As can be seen from the nuclear magnetic hydrogen spectrum chart 4, the polypeptide C12-PButLG is successfully synthesized, the characteristic peaks of double bonds are shown at 5.75 ppm and 5.1 ppm, and the polymerization degree is 10 by calculation.

(4) Synthesis of cationic polypeptide C12-PButLG-CA

Dissolving 1.0 g of polymer C12-PButLG in 4 mL of Dimethylformamide (DMF), adding 1.2 g of mercaptoethylamine hydrochloride, introducing nitrogen for 15 min, exhausting air, adding 15 mg of catalyst 2, 2-dimethoxy-2-phenylacetophenone, keeping out of the sun, continuously introducing nitrogen for 15 min, performing reaction for 1 h by using 365 nm laser light, dialyzing in deionized water for 24 h, freeze-drying to obtain cationic polypeptide C12-PButLG-CA, measuring nuclear magnetic hydrogen spectrum (the map is shown in figure 5), and determining the target product as the anticancer peptide.

Compared with the polymer C12-PButLG, the nuclear magnetic hydrogen spectrum of the Peptide has the characteristic peak of double bonds disappeared, which indicates that the click is complete.

1.2 ^RSynthesis and characterization of Peptide

^RThe synthetic circuit diagram of Peptide is shown in FIG. 2, and the specific synthetic method comprises the following steps:

(1) synthesis of PEO-PPO-CDM

Weighing 30 mg of 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furylpropionic acid (CDM) and dissolving in 2mL of dichloromethane, placing on an ice bath and stirring, adding 100 μ L of oxalyl chloride, adding 10 μ L of Dimethylformamide (DMF) for catalytic reaction for 30min, reacting at room temperature for 2h, and pumping off the Dimethylformamide (DMF) and excess oxalyl chloride to obtain 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furylpropionyl chloride (CDM-Cl); weighing 1.0 g of polyethylene oxide-polypropylene oxide (PEO-PPO) to be dissolved in dichloromethane, placing the mixture on an ice bath to be stirred, dissolving 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionyl chloride (CDM-Cl) in dichloromethane, adding the solution into a dichloromethane solution of the polyethylene oxide-polypropylene oxide (PEO-PPO), adding 20 mu L of pyridine, reacting for 30min, reacting overnight at room temperature, concentrating, precipitating in diethyl ether, and centrifuging at low temperature to obtain the PEO-PPO-CDM;

the nuclear magnetic hydrogen spectrum characterization map is shown in FIG. 6.

（2）(PEO-PPO-CDM) ₂ Synthesis of-C12-PButLG-CA

Dissolving 1.0 g PEO-PPO-CDM in 6 mL dichloromethane, dissolving 80 mg C12-PButLG-CA in 2mL methanol, adding into PEO-PPO-CDM, adding 20 μ L triethylamine, reacting for 24 h, concentrating, precipitating into anhydrous ether, centrifuging at low temperature, removing supernatant, and draining to obtain (PEO-PPO-CDM) ₂ -C12-PButLG-CA, nuclear magnetic hydrogen spectrum, as shown in figure 7, determined as the target product^RPeptide。

1.3 ^NRPeptide（PEO-PPO-SA)₂-C12-PButLG-CA) synthesis and characterization

^NRThe molecular structural formula of Peptide is as follows:

the synthesis method comprises the following steps:

(1) synthesis of PEO-PPO-SA

Weighing 1.0 g of polyethylene oxide-polypropylene oxide (PEO-PPO) to be dissolved in dichloromethane, weighing 20 mg of maleic anhydride (SA) to be dissolved in 2mL of dichloromethane, adding the dichloromethane into the reaction, stirring the mixture for reaction, weighing 10 mg of 4-Dimethylaminopyridine (DMAP) to be added into the reaction, after reacting for 12 h, concentrating the mixture, precipitating the mixture in diethyl ether, centrifuging the mixture at a low temperature to obtain PEO-PPO-SA, and measuring a nuclear magnetic hydrogen spectrum to prove that the maleic anhydride (SA) is successfully bonded to the polyethylene oxide-polypropylene oxide (PEO-PPO).

(2) Synthesis of (PEO-PPO-SA)2-C12-PButLG-CA

Dissolving 1.0 g of PEO-PPO-SA in 6 mL of dichloromethane, adding 100 μ L of oxalyl chloride, adding 10 μ L of Dimethylformamide (DMF), catalyzing and reacting for 30min at room temperature for 2h,pumping off Dimethylformamide (DMF) and excess oxalyl chloride to obtain PEO-PPO-SA-Cl, dissolving 80 mg of C12-PButLG-CA in 2mL of methanol, adding into PEO-PPO-SA-Cl, adding 20 μ L of triethylamine, reacting for 24 h, concentrating, precipitating into anhydrous ether, centrifuging at low temperature, removing supernatant, and draining to obtain PEO-PPO modified non-responsive polypeptide (PEO-PPO-SA) ₂ -C12-PButLG-CA, nuclear magnetic hydrogen spectrum as shown in FIG. 24, demonstrating^NRThe Peptide is successfully prepared.

1.4 responsiveness verification

1.4.1 Nuclear magnetism proves that C12-PButLG-CA escapes in acid environment

20 mg of (PEO-PPO-CDM)₂-C12-PButLG-CA dissolved in 200. mu.L of dimethyl sulfoxide (DMSO), added dropwise to 2mL of heavy water, stirred and assembled into nanoparticles, washed 8 times with heavy water by ultrafiltration, and concentrated to give about 600. mu.L of heavy water solution, and the nuclear magnetism was measured, and as a result, as shown in FIG. 8, the signal of the polypeptide was masked and only a part of the signal of polyethylene oxide-polypropylene oxide (PEO-PPO) was observed. The nuclear magnetic hydrogen spectrum was continued after one hour by adding DCl in an amount of 10. mu.L to the sample, and the results are shown in FIG. 9. The presence of a signal for the polypeptide in the nuclear magnetic field was found, indicating that the polypeptide had been able to interact with the heavy water, possibly reaching the particle surface and even escaping into the heavy water.

1.4.2HPLC demonstration of degradation of the polypeptide in an acid Environment

Will (PEO-PPO-CDM) ₂ -C12-PButLG-CA dissolved in methanol (0.1 mg/mL), methanol solution was mixed with PB pH =6.8 at 1:1 and HPLC tested for 0, 30, 60 min degradation as shown in FIG. 10. It was suggested that the bonded polypeptide could be degraded to give free polypeptide C12-PButLG-CA substantially at pH =6.8 for 1 h.

Peptide and ^Rdetection of physical and chemical properties and pharmacological activity test of Peptide

2.1 materials of the experiment

2.1.1 Experimental animals and cell lines

Female ICR mice, 5 weeks old, were purchased from Silikedada laboratory animals, Inc. Breast cancer cell lines 4T1, EMT6, MDA-MB-231 and MCF-7, colon cancer cell line CT26 and pancreatic cancer cell line Panc02 were purchased from ATCC in the United states, MCF-7 Adriamycin resistant (MCF-7/ADR) cells provided by university of southern China. Breast cancer leaf tumor (PTB) cells were isolated from clinical specimens and cultured at the grand university Sun-fugan commemorative Hospital.

2.1.2 Experimental reagents

Experimental reagent: fetal bovine serum, 0.25% trypsin, 1640 medium, DMEM medium and DMEM/F12 medium were purchased from Gibco (usa); anti-penicillin-streptomycin was purchased from shanghai bi yunnan (china); MTT, doxorubicin hydrochloride were purchased from gangrenum (china); PBS phosphate buffer powder packets (2L/bag) were purchased from Wuhan Boston (China); fresh sheep blood was purchased from Guangzhou future (China); triton X-100 was purchased from Sigma (USA); DMSO was purchased from shanghai life man (china); a Peptide, ^RPeptide and^NRpeptide was synthesized by university of southern China.

All material concentrations in the test refer to the amount of Peptide in the material. For example: 5 mg/mL ^RPeptide: weighing 25 mg ^RThe Peptide is added with 400 mu L DMSO for full dissolution, namely the Peptide containing 5 mg/mL^RAnd (3) a Peptide mother liquor.

2.2 Experimental methods

2.2.1 cell culture

EMT6 cells, MDA-MB-231 cells, MCF-7 cells, Panc02 cells and CT26 cells were all cultured in 1640 medium containing 10% fetal bovine serum and 1% double antibody, and 4T1 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody. MCF-7/ADR cells were cultured in 1640 medium containing 10% fetal bovine serum, 1% diabody and 1. mu.M doxorubicin to maintain the doxorubicin-resistance of the cells. PTB cells were cultured in DMEM/F12 medium containing 15% fetal bovine serum and 1% double antibody. All media used in the experiment were not changed unless the experiment specifically required. The cells were cultured in an incubator at 37 ℃ and 5% CO2 and 95% humidity.

2.2.2 cell viability assay

(1) Tumor cells were seeded at a density of 1 × 104 cells per 100 μ L per well in 96-well plates and cultured overnight in cell culture incubator cultures.

(2) The 10 mg/mL Peptide mother liquor was diluted to 40. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL, and 5. mu.g/mL with 1640-Flat medium, respectively.

(3) Old culture medium in a 96-well plate is aspirated, 100 mu L of culture solution containing different concentrations of Peptide is added into each well, and the mixture is placed in a cell culture box for incubation for 1 h.

(4) MTT stock (5 mg/mL) was diluted to 1 mg/mL with 1640 medium, and after 1 h the medium containing material was aspirated, 100. mu.L of MTT-containing medium was added to each well, and wells containing no cells were used as a blank. The 96-well plate was placed in an incubator and incubation continued for 3 h. The supernatant was then discarded, 100 μ L DMSO was added to each well and shaken on a shaker for 10min to dissolve formazan sufficiently, and absorbance at 490 nm was detected for each well by a multifunctional microplate reader.

2.2.3 preparation and characterization of anticancer peptide nanoparticles

2 10mL sample bottles were taken, 3 mL PBS was added, and the mixture was placed on a magnetic stirrer at 500 rpm. Taking 5 mg/mL^RPeptide and^NRand respectively dripping 500 mu L of the Peptide mother liquor into a sample bottle, and stirring for 15 min by adjusting the rotation speed to 400 rpm. The prepared particles were then transferred to 14000 Da dialysis bags and dialyzed in PBS, PBS was changed every 1 h, dialyzed for 4 h, and then the particles were transferred to centrifuge tubes and made to 625. mu.g/mL.

50 μ L of each prepared particle was diluted 10 times with PBS and the particle size was measured. Respectively taking 10 μ L of the extract with a concentration of 5 mg/mL^RPeptide and^NRand (4) detecting a Zeta potential in the Peptide mother liquor. The particle size and the zeta potential of the nanoparticles were measured using a dynamic light scattering particle sizer (Malvern Zetasizer Nano ZS 90).

2.2.4 nanoparticle size stability test

Respectively mixing 625 mu g/mL^RPeptide and^NRthe Peptide particles are diluted by 10 times by using a 1640 culture medium and fetal bovine serum with the final concentration of 10%, and the particle sizes of the diluted Peptide particles are measured by using a dynamic light scattering instrument immediately and after incubation for 0.5 h, 1 h, 2h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h and 12 h respectively.

2.2.5 hemolysis assay

(1) Taking a proper amount of fresh sheep blood in a centrifuge tube, centrifuging for 3min at 4 ℃ and 3000 rpm, and discarding the supernatant. The erythrocyte pellet was resuspended in a 10-fold volume of PBS, and then centrifuged at 3000 rpm for 3min at 4 ℃ and the supernatant was discarded. The washing with PBS was repeated three times, and the washed sheep red blood cells were diluted to 4% (v/v) by adding PBS to resuspend.

(2) Respectively taking the mixture with a proper concentration of 5 mg/mL^RPeptide and^NRthe Peptide stock solution and 10 mg/mL Peptide solution were diluted to 400. mu.g/mL with PBS pH 7.4, and then released with PBS to the following concentration gradients: 400 mg/mL, 200 mg/mL, 100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL.

(3) 200 μ L of diluted erythrocyte suspension is taken and put into a centrifuge tube, 200 μ L of the diluents with different concentrations are respectively added, 200 μ L of PBS is added into a negative control, and 200 μ L of 0.2% Triton X-100 is added into a positive control. After incubation at 37 ℃ for 1 h, centrifugation at 3000 rpm was carried out for 3 min. 100 μ L of the supernatant was transferred to a 96-well plate, and absorbance was measured at 576 nm for each well. The hemolysis rate is calculated as follows: hemolysis rate (%) = (sample absorbance-negative control absorbance)/(positive control absorbance-negative control absorbance) × 100%.

2.2.6 maximum tolerated dose detection

(1) Preparing nano particles: 1 10mL sample vial was taken, 6 mL PBS was added, and the mixture was placed on a magnetic stirrer and adjusted to 500 rpm. Taking 6 mg/mL^RDropwise adding 2mL of Peptide mother solution into a sample bottle, adjusting the rotating speed to 400 rpm, stirring for 15 min, transferring the particles into a 14000 Da dialysis bag, putting the dialysis bag into PBS, dialyzing, replacing PBS every 1 h, dialyzing for 4 h, transferring the particles into a centrifuge tube, and fixing the volume to 1.4 mg/mL.

(2) The Peptide stock solution (10 mg/mL) was diluted to 1.4mg/mL with PBS.

(3) 64 ICR mice were divided randomly into 8 groups of 8 mice each. Group 1, group 2, group 3, group 4 and group 5 were administered tail vein injections of 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 17.5 mg/kg and 20 mg/kg, respectively^RA Peptide nanoparticle. Group 6, group 7 and group 8 were injected tail vein with 1.25 mg/kg, 2.5 mg/kg and 5 mg/kg Peptide, respectively. Mice were observed for three consecutive days after dosing.

2.3 test results (data were analyzed using Graph prism 6.0 software.)

2.3.1 killing Effect of Peptide on tumor cells

The killing effect of Peptide on tumor cells was detected by MTT assay, and the results are shown in FIG. 11, which shows that Peptide has significant killing effect on various breast cancer cell lines including triple negative breast cancer, colon cancer and pancreatic cancer cell lines.

2.3.2 ^RPeptide and^NRparticle size and potential detection of Peptide

Prepared by a dialysis method^RPeptide and^NRpeptide, the particle size and the potential of the Peptide are detected, and the result is displayed^RPeptide and^NRthe average particle size of the Peptide is 60-70nm, the particle size distribution is good (PDI is lower than 0.3), the particle size of the nanoparticle is not obviously changed in the environment of pH 7.4 and pH 6.8, and the potential of the nanoparticle are close to 0 mV.

2.3.3 ^RPeptide and^NRstability of Peptide in serum

Detection of^RPeptide and^NRthe results of the particle sizes of the Peptide in the culture medium containing 10% FBS at different time points show that the particle sizes of the two particles are not obviously changed in 12 h along with the time lapse, and the nanoparticles can stably exist in serum without obvious aggregation or disintegration.

2.3.4 ^RPeptide and^NRshielding effect of Peptide on Peptide cytotoxicity

Detection by hemolysis assay^RPeptide and^NRtoxicity of Peptide and free Peptide to normal cells. The results are shown in figure 12 of the drawings,^Rpeptide and^NRthe haemolytic activity of Peptide was significantly lower than that of free Peptide.

Injection into mice^RPeptide and free Peptide the maximum tolerated dose in mice was determined. The results show that the mouse pairs^RThe maximum tolerated dose of the Peptide nanoparticles was 12.5 mg/kg, significantly higher than the maximum tolerated dose for Peptide of 2.5 mg/mL.

3. ^RPeptide micro-acid environment selective damage tumor cell membrane test

3.1 Experimental materials

3.1.1 Experimental cell lines: experimental animals and cell lines as above.

3.1.2 Experimental reagents

Experimental reagent: CCK8 kits were purchased from GLPBIO (usa); cisplatin, Propidium Iodide (PI) was purchased from zonemet (china); cellmask green, Protein marker from Thermo (USA); the lactate dehydrogenase cytotoxicity detection kit, the rapid silver staining kit and the 5 Xprotein loading buffer solution are purchased from Shanghai Biyun (China); hepes buffer (100 ×) was purchased from Corning (usa); Sulfo-Cy5-NHS lipid was purchased from AAT Bioquest (USA); Tris/glycine/SDS electrophoresis buffer (10X), PAGE gel rapid preparation kit purchased from Shanghai Yazyme (China); the Annexin V-FITC/PI apoptosis detection kit is purchased from Wuhan Irelette (China); 25% glutaraldehyde purchased from shanghai redun (china); absolute ethanol was purchased from shanghai bio-workers (china); 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Sephadex G-50 was purchased from Sigma (USA); 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG) was purchased from corencharma (switzerland); chlorpromazine, wortmannin, 2-deoxy-D-glucose and methyl- β -cyclodextrin were purchased from shanghai maculin (china); 8-amino-1, 3, 6-naphthalenetrisulfonic acid disodium salt hydrate (ANTS), 1' - [1, 4-phenylenebis (methylene) ] bis (1-pyridinium) Dibromide (DPX) was purchased from TCI (Japan); na2HPO4 was purchased from Aladdin (China). The other reagents were as above.

3.1.3 solutions required for the experiments

10 mg/mL chlorpromazine: 10 mg of chlorpromazine was weighed, dissolved thoroughly in 1 mL of DMSO, and stored at-20 ℃.

50 μ M wortmannin: adding 654 mu L DMSO into 14 mg of wortmannin, fully dissolving, and storing at-20 ℃.

0.9M 2-deoxy-D-glucose: 74 mg of 2-deoxy-D-glucose was dissolved in 501. mu.L of purified water and stored at-20 ℃.

50 mM methyl- β -cyclodextrin: dissolving 65 mg of methyl-beta-cyclodextrin in 998 mu L of DMSO, and storing at-20 ℃.

1 mg/mL Sulfo-Cy 5-NHS: 10 mg of Sulfo-Cy5-NHS was dissolved in 10mL of DMSO to obtain a 1 mg/mL solution of Sulfo-Cy5-NHS, which was stored at-20 ℃.

Buffer 1: 710 mg of Na2HPO4 powder was taken, dissolved in 450 mL of ultrapure water, the pH was adjusted to 7.0, and the volume was made 500 mL with ultrapure water, i.e., 10 mM Na2HPO 4.

Buffer 2: 710 mg of Na2HPO4 powder and 5.26 g of NaCl powder were weighed out, dissolved in 450 mL of ultrapure water, the pH was adjusted to 7.4, and the solution was made up to 500 mL, i.e., 10 mM Na2HPO4+90 mM NaCl, with ultrapure water.

ANTS mother liquor: 15 mg of ANTS was weighed and 2.81 mL of Buffer1 was added to dissolve well, i.e., 5.34 mg/mL of ANTS.

DPX mother liquor: 40 mg of DPX was weighed and added to 2.11 mL of Buffer1 to dissolve well, i.e., 18.99 mg/mL of DPX.

20 mg/mL DOPE: 20 mg of POPE was weighed out and dissolved thoroughly by adding 1 mL of chloroform.

20 mg/mL POPG: 20 mg of POPG was weighed and dissolved thoroughly by adding 1 mL of chloroform.

3.2 Experimental methods

3.2.1 CCK8 method for detecting killing effect of acid-responsive anticancer peptide nanoparticles on tumor cells

(1) Tumor cells were seeded at a density of 1 × 105 cells per 100 μ L per well in 96-well plates and placed in a cell culture incubator overnight.

(2) Mixing 5 mg/mL^RPeptide and^NRthe Peptide mother liquor and 10 mg/mL Peptide mother liquor were diluted to 40. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL, 5. mu.g/mL (DMSO has been equilibrated) with serum-containing 1640 medium at pH 6.8 and pH 7.4, respectively.

(3) The 96-well plate was removed, the old medium was discarded, and 100. mu.L of the above dilution was added to each well, three wells each. And blanks of the pH 6.8 medium and the pH 7.4 medium were set, respectively.

(4) The CCK8 stock solution was diluted 10-fold with medium, the old medium was discarded, and 100 μ L of CCK 8-containing medium was added to each well, and the wells without cells were used as a blank. The 96-well plate was placed in an incubator and incubation continued for 3 h. The absorbance of each well at 490 nm was measured by a multifunctional microplate reader.

3.2.2 Cy5 labeled anticancer peptide nanoparticle preparation

Taking 5 mg/mL ^R500. mu.L of Peptide mother liquor, 265. mu.L of 1 mg/mL Sulfo-Cy5-NHS (molar ratio of Peptide to Cy 5: 10: 4), mixing, and incubating at room temperature for 8 h. And (3) adding 1 mL of PBS into 1 sample bottle, placing on a magnetic stirrer at the rotating speed of 500 rpm, slowly dripping the mixed solution into the PBS, and stirring at the rotating speed of 400 rpm in a dark place for 1 h. Transferring the solution into an ultrafiltration tube, centrifuging at 1000 rpm for 20 min until the bottom liquid is light blue, adding 500 μ L PBS, centrifuging at 1000 rpm for 20 min until the bottom liquid is still light blue, and repeatedly adding PBS and centrifuging until the bottom liquid is colorless. After centrifugation, the particles were collected and the volume was adjusted to 4 mL, i.e., 625. mu.g/mL.

3.2.3 dynamic Observation of the interaction of anticancer peptide nanoparticles with tumor cell membranes

Panc02 cells were plated at a density of 5X 105 cells per 500. mu.L per well in 35mm multi-well glass plates and cultured overnight. Old medium was removed, 500 μ L1640 medium was added, and the mixture was incubated at 1: PI and Cellmask green were added at a ratio of 2000, respectively, and incubated for ten minutes. RPeptide-Cy5 pre-treated for 1 h in a pH 6.8 or pH 7.4 environment was added to a final concentration of 10.5. mu.g/mL. Fixing visual field by confocal microscope, collecting pictures every 7 s for 40 min continuously, observing dynamic change of cells in real time, and maintaining the temperature in the culture dish at 37 deg.C during observation. And (4) after the collection is finished, analyzing the result by ZEISS ZEN software.

3.2.4 scanning electron microscope observation of the destruction of anticancer peptide nanoparticles to cell membrane

(1) EMT6 cells were seeded at a density of 1X 105 cells per 1 mL per well in 12-well plates (containing sterile slide glass) and placed in a cell culture incubator overnight.

(2) Mixing 5 mg/mL^RThe Peptide mother liquor was diluted to 20. mu.g/mL (DMSO was equilibrated) with serum 1640 medium at pH 6.8 and pH 7.4, respectively.

(3) The 12-well plate was removed, the old medium was discarded, 500. mu.L of the above dilution was added to each well, and blank controls of pH 6.8 medium and pH 7.4 medium were set, respectively, and incubated in an incubator for 30 min.

(4) After 30min, the culture medium is discarded, PBS is added for washing for 3 times, and 2.5% glutaraldehyde is added for room temperature fixation for 1 min. The appropriate size centrifuge tube was sampled, filled with 2.5% glutaraldehyde, and the cell slide was transferred into the centrifuge tube.

(5) Taking cell slide, and gradient dehydrating in 30%, 50%, 70%, 90%, and 100% ethanol successively. And then placing the cell slide into a supercritical dryer for critical point drying, taking out a sample, spraying gold, and observing by a scanning electron microscope.

3.2.5 LDH Release test

(1) Tumor cells were seeded at a density of 1 × 105 cells per 100 μ L per well in 96-well plates and placed in a cell culture incubator overnight.

(2) Mixing 5 mg/mL^RPeptide and^NRthe Peptide mother liquor was diluted to 40. mu.g/mL (DMSO-equilibrated) with serum-free 1640 medium at pH 6.8 and pH 7.4, respectively.

(3) 200. mu.L of the above dilution was added to each well of the 96-well plate, 3 wells each. At the same time, 200. mu.L of the culture medium was added to the control wells, and 200. mu.L of the culture medium and 20. mu.L of the LDH-releasing reagent were added to the "sample maximum enzyme activity control wells" and mixed well, and the mixture was incubated in an incubator for 1 hour.

(4) After a predetermined time, the 96-well plate was placed in a centrifuge at 400 g and centrifuged for 5 min. 120. mu.L of the supernatant from each well was added to the corresponding well of a new 96-well plate.

(5) Working solution is prepared according to the kit method, 60 mu L of LDH detection working solution is respectively added into each hole, the mixture is uniformly mixed, and the mixture is incubated for 30min at room temperature in a dark place (the mixture is wrapped by aluminum foil and then placed on a horizontal shaking table to be slowly shaken). After the incubation, the absorbance of each well was measured at 490 nm. The equation for LDH release was: LDH release rate (%) = (treated sample absorbance-sample control well absorbance)/(absorbance for maximum enzyme activity of cells-sample control well absorbance) × 100%.

3.2.6 silver staining to detect intracellular protein Release

Sample preparation

(1) Tumor cells were seeded at a density of 2 × 105 cells per 2mL per well in 6-well plates and cultured overnight.

(2) Mixing 5 mg/mL^RPeptide mother liquorDiluted to 30. mu.g/mL and 15. mu.g/mL (DMSO-equilibrated) with pH 6.8 and pH 7.4 serum-free 1640 medium, respectively.

(3) The 6-well plate was removed, the old medium was discarded, and washed three times with PBS. 500. mu.L of the above dilution was added to each well, and 500. mu.L of serum-free 1640 medium (DMSO-balanced) at pH 7.4 and pH 6.8 was added to the blank. After 1 h, 300. mu.L of culture medium was collected from each dish, centrifuged at 5000 rpm for 5min, and the supernatant was collected and placed on ice.

(4) Respectively taking 40 μ L of the supernatant obtained in the step 3 and 40 μ L of 1640 medium containing 0.05% FBS, adding 10 μ L of 5 × Loading Buffer, mixing uniformly, performing metal bath at 95 ℃ for 10min, cooling to room temperature, and centrifuging at 8000 rpm for 1.5 min.

SDS-PAGE electrophoresis

(1) Checking the instruments needed by electrophoresis, cleaning the glass plate, aligning the glass plate after drying, and vertically clamping the glass plate on a frame.

(2) Preparing glue: the 10% PAGE gel of the kit is rapidly prepared by the PAGE gel of Yazyme company, a liquid-transferring gun is used for adding the lower layer gel to about 1 cm below a comb to observe whether the gel leaks or not, the lower layer gel is solidified and then the upper layer gel is added to a low-plate glass line by the liquid-transferring gun, and the comb is inserted.

Glue at the lower layer: 2.7 mL of lower layer glue solution; the lower layer gel buffer was 2.7 mL, and the modified coagulant was 60. mu.L.

Gluing the upper layer: 0.75 mL of the supernatant solution; 0.75 mL of upper layer glue buffer solution; 15 μ L of improved coagulant.

(3) The gel was mounted on the electrophoresis tank together with the glass plate, and the electrophoresis buffer was added. Each well was loaded with 20. mu.L of Marker 4. mu.L. Buffer solution is added continuously to 700 mL until the inner tank is full, and the rest is added to the outer tank.

(4) Electrophoresis: connecting an electrophoresis tank and an electrophoresis apparatus according to the sequence of the electrodes, selecting a constant voltage mode, setting the voltage to be 90V, carrying out electrophoresis for 15 min, then increasing the voltage to be 100V, carrying out electrophoresis for about 60 min, and stopping the electrophoresis until bromophenol blue just runs out.

Dyeing by adopting a silver staining kit, comprising the following steps:

preparing a reagent:

(1) fixing liquid: 50 mL of ethanol, 10mL of acetic acid and 40 mL of Milli-Q grade pure water are uniformly mixed to obtain a stationary liquid.

(2) 30% ethanol: 30 mL of absolute ethyl alcohol is added into 70 mL of Milli-Q grade pure water and mixed evenly.

(3) Silver-staining sensitizing solution (1 ×): and (3) adding 99 mL of Milli-Q grade pure water into 1 mL of silver staining sensitization liquid (100 x), and mixing uniformly to obtain the silver staining sensitization liquid (1 x) (used within 2h after preparation).

(4) Silver solution (1 ×): and adding 99 mL of Milli-Q grade pure water into 1 mL of silver solution (100 x) and mixing uniformly to obtain the silver solution (1 x). (used within 2h after preparation).

(5) Silver staining color developing solution: and adding 20 mL of silver staining basic color development liquid (5 x) and 0.05 mL of silver staining color development accelerating liquid (2000 x) into 80 mL of Milli-Q grade pure water, and uniformly mixing to obtain the silver staining color development liquid (used within 20 min after preparation).

(6) Silver staining stop solution (1 ×): and (3) adding 95 mL of Milli-Q grade pure water into 5mL of silver staining stop solution (20 x), and mixing uniformly to obtain the silver staining stop solution (1 x) (used on the same day after preparation).

The experimental steps are as follows:

(1) fixing: after the electrophoresis is finished, the glass plate is taken down, the gel is taken out and put into 100 mL of stationary liquid, and the gel is placed in a shaking table and shaken at the room temperature of 60-70 rpm for 20 min.

(2) Washing with 30% ethanol: the stationary solution was discarded, 100 mL of 30% ethanol was added, and the mixture was shaken in a shaker at 60-70 rpm for 10min at room temperature.

(3) Water washing: 30% ethanol is discarded, 200 mL Milli-Q grade pure water is added, and shaking is carried out on a shaker at 60-70 rpm for 10min at room temperature.

(4) Sensitization: after washing, 100 mL of silver-stained sensitization solution (1X) was added and shaken by a shaker at 60-70 rpm for 2 min at room temperature.

(5) Water wash (2 total times): the silver-staining sensitization solution was discarded, 200 mL Milli-Q grade pure water was added, and the mixture was placed on a shaker at 60-70 rpm and shaken at room temperature for 1 min. The washing was repeated once.

(6) Silver staining: after washing, 100 mL of silver solution (1X) was added and shaken by 60-70 rpm in a shaker at room temperature for 10 min.

(7) Water washing: the silver solution was discarded, 100 mL Milli-Q grade pure water was added, and shaking was carried out at 60-70 rpm for 1min at room temperature.

(8) Color development: after washing, adding 100 mL of silver staining solution, placing the mixture in a shaking table, shaking at 60-70 rpm at room temperature for 2-5min until protein bands appear.

(9) And (4) terminating: discard the silver staining solution, add 100 mL silver staining stop solution (1X), shake with 60-70 rpm room temperature shaking table for 10 min.

(10) Water washing: the stop solution was discarded, 100 mL of Milli-Q grade pure water was added at 60-70 rpm, and shaking was carried out on a shaker at room temperature for 3 min.

(11) And (3) storage: the gel was stored in Milli-Q grade pure water.

3.2.7 Engulp inhibition assay

(1) 4T1 cells were plated in 96-well plates at a density of 1X 105 cells per 100. mu.L per well and cultured overnight.

(2) Taking 10 mg/mL chlorpromazine mother liquor, diluting to 10 mu g/mL by using a culture medium, and adding 100 mu L of chlorpromazine mother liquor into each hole of an experimental group; diluting the wortmannin mother solution with 50 mu M to 50 nM by using the culture medium, and adding 100 mu L of the wortmannin mother solution into each hole of the experimental group; diluting 0.9M 2-deoxy-D-glucose mother liquor to 50 mM with culture medium, and adding 100 μ L per well of experimental group; 50 mM methyl-beta-cyclodextrin stock solution was diluted to 50. mu.M with medium and 100. mu.L was added to each well of the experimental group. Placing in a middle culture box and incubating for 30 min.

(3) Mixing 5 mg/mL^RThe Peptide mother liquor was diluted to 40. mu.g/mL (DMSO was equilibrated) with 1640 medium at pH 6.8 and pH 7.4, respectively, while each inhibitor was added again (at a concentration consistent with that at the time of pre-incubation).

(4) After 30min of treatment, the 96-well plate was removed and the inhibitor-containing medium was discarded, and 100. mu.L of the dilution from step 3 was added to each well. And control groups of a pH 6.8 medium and a pH 7.4 medium were set, respectively.

(5) MTT stock (5 mg/mL) was diluted to 1 mg/mL with 1640 medium, and after 1 h the medium containing material was aspirated, 100. mu.L of MTT-containing medium was added to each well, and wells containing no cells were used as a blank. The 96-well plate was placed in an incubator and incubation continued for 3 h. The supernatant was then discarded, 100. mu.L of dimethyl sulfoxide was added to each well and shaken on a shaker for 10min to dissolve formazan sufficiently, and absorbance at 490 nm was detected for each well by a multifunctional microplate reader.

3.2.8 flow detection of cell death pattern

(1) 4T1 cells were plated in 6-well plates at a density of 5X 105 cells per 100. mu.L per well, respectively, and cultured overnight.

(2) A5 mM cisplatin stock solution was diluted to 20. mu.M and 10. mu.M in 1640 medium, respectively. 10 mg/mL of Peptide mother liquor was taken and diluted to 10. mu.g/mL and 5. mu.g/mL respectively with 1640 medium.

(3) And taking out the 6-well plate, discarding the old culture medium, adding 2mL of the diluent in the step 2 into each well, adding 2mL of the culture medium into a blank control well, and placing in an incubator for incubation for 24 h.

(4) The 6-well plate was removed, the cells were digested with EDTA-free trypsin, and resuspended in old medium at 1000 rpm for 3min, the supernatant was discarded, the cells were resuspended in PBS, filtered 1 time with 400 mesh nylon mesh, centrifuged at 1000 rpm for 3min, and the cells were collected in 1.5 mL EP tubes.

(5) Add 600. mu.L Binding Buffer to each tube to resuspend the cells, then take three EP tubes, label NA, FITC single staining, PI single staining, take 50. mu.L each from 6 tube cell suspension to these three EP tubes. The treated and blank cell suspensions were transferred to the corresponding new EP tubes at 300. mu.L/tube.

(6) The treatment group and blank control were mixed by adding 3. mu.L Annexin v-FITC to each tube and then 3. mu.L PI. 3 mul of Annexin v-FITC is added into a FITC single staining tube and mixed evenly, and 3 mul of PI is added into a PI single staining tube and mixed evenly. The reaction was carried out at room temperature for 15 min in the absence of light.

(7) The fluorescence of the cells is detected by a flow cytometer, necrotic cells are FITC +/PI +, and apoptotic cells are FITC +/PI-.

3.3 results of the experiment

And (3) data analysis: data were analyzed using Graph prism 6.0 and SPSS25.0 software, with P < 0.05 indicating that the differences were statistically significant.

CCK8 experiment for detecting different concentrations under acidic and neutral conditions^RKilling effect of Peptide on tumor cells. Results display^RThe killing power of the Peptide on triple negative breast cancer cells under the condition of pH 6.8 is obviously stronger than that under the condition of pH 7.4,has no obvious difference with the killing effect of free Peptide^NRThe Peptide nanoparticles had little killing effect on tumor cells at both pH 7.4 and 6.8 (as shown in fig. 13).^RPeptide also showed the same effect in breast cancer phyllodes. The results of figure 14 suggest that,^Rthe killing effect of the Peptide on the tumor cells is gradually enhanced along with the reduction of the pH, and the influence of the reduction of the pH of the environment on the killing effect of the material is little when the pH reaches 6.8. CCK8 test was performed at different temperatures, and it was observed that various metabolic enzymes in cells were reduced in the low temperature environment^RWhether the effect of Peptide is affected or not. The results show that both at pH 7.4 and pH 6.8,^Rthere was no difference in the killing effect of Peptide at both 4 ℃ and 37 ℃.

To further verify^RThe Peptide can regulate the interaction between the anticancer Peptide and a tumor cell membrane, and the inventor bonds Cy5 fluorescent dye to the anticancer Peptide to form a fluorescent label^RPeptide-Cy5, and through green membrane dye labeling tumor cell membrane, dynamically observing pH 7.4 and pH 6.8 under confocal microscope^RInteraction of Peptide-Cy5 with tumor cell membrane. The results show that after pretreatment at pH 6.8^RPeptide-Cy5 can release anticancer Peptide, and Peptide-Cy5 with red fluorescence rapidly gathers on tumor cell membrane and presents a bubble spitting phenomenon, thereby leading PI to enter tumor cell nucleus. And pH 7.4 pretreated^RSince Peptide-Cy5 can not release anticancer Peptide, no red fluorescence is accumulated on tumor cell membrane.

At pH 7.4 and pH 6.8, by observing with scanning electron microscope^RTumor cell membrane morphology after Peptide treatment. The results are shown in FIG. 15, at pH 7.4^RThe morphology of the Peptide-treated EMT6 cells is similar to that of untreated control group normal cells, and the cell surfaces have more microvilli, are in a fusiform or polygonal shape and have no holes; at pH 6.8^RThe surface microvilli of the Peptide-treated tumor cells was reduced, pores of varying sizes were formed, and the cells were swollen.

At pH 7.4 and pH 6.8^RPeptide treatment of tumor cells followed by cell culture harvestingThe liquid detects the release conditions of LDH and protein in tumor cells through an LDH release experiment, SDS-PAGE gel electrophoresis and silver staining, and the result shows that^RPeptide caused the release of LDH and protein in tumor cells at pH 6.8, but hardly caused the release of LDH and protein in cells at pH 7.4 (results are shown in fig. 16).

To further prove^RPeptide does not need to enter cells to play a role, and cells are treated by different endocytosis inhibitors and observed^RKilling effect of Peptide on tumor cells. The result shows that under the condition that endocytosis inhibitors of different mechanisms such as the macropinocytic inhibitor wortmannin, the energy-dependent endocytosis inhibitor 2-deoxy-D-glucose, the clathrin-dependent endocytosis inhibitor chlorpromazine, the caveolin-dependent endocytosis inhibitor methyl-beta-cyclodextrin and the like inhibit endocytosis, the effect of the anticancer peptide nanoparticles on killing tumor cells in the slightly acidic environment is hardly influenced, and the result is shown in fig. 17.

The mode of tumor cell death caused by the anticancer peptide is researched by Annexin V/PI staining and flow cytometry detection, and the result shows that the traditional chemotherapeutic drug cis-platinum can induce the tumor cell apoptosis, and the anticancer peptide directly causes the tumor cell necrosis.

4.^RInhibitory Effect of Peptide on triple negative Breast cancer cell

4.1 Experimental materials

4.1.1 Experimental animals and cell lines

5 week old female Balb/c mice, purchased from Schlay Jingda laboratory animals Ltd. The rest are the same as the experimental animals and cell lines.

4.1.2 Experimental reagents

4% paraformaldehyde from Guangzhou Yongjin (China), xylene from Aladdin (China), hematoxylin and eosin from Wuhan Seville (China), neutral gum from Fuzhou Mexin (China), India ink from Dalian Melam (China). The rest is the same as above.

4.2 Experimental methods

EMT6 in-situ tumor model establishment and treatment effect evaluation

Establishing a tumor model: EMT6 cells were subcultured for expansion one day in advance, well-cultured tumor cells were starved for 4 h with serum-free medium the following day, then the cells were digested with 0.25% trypsin, collected, resuspended in sterile PBS and counted, the cell suspension was diluted to 4X 106/mL and placed on ice. Subsequently, 50. mu.L of cell suspension was injected with an insulin syringe into the second fat pad on the left side of each BALB/c mouse, taking care to avoid leakage.

Treatment and detection: when the average size of the tumor reached 40 mm3, the mice were randomly divided into 3 groups, and the tail vein of the treatment group was injected with 6 mg/kg^RPeptide, 6 mg/kg for tail vein injection of control group^NRPeptide, blank control group was injected tail vein with an equal volume of PBS and dosing was started on day 6 post-tumor implantation, with a dosing schedule of 18-1 in figure 18. Meanwhile, the body weight of the mouse is regularly measured by an electronic balance, the length and the width of the tumor are measured by a vernier caliper, and the calculation formula of the tumor volume is as follows: volume =0.5 × length × width 2. On day 18, the mice were sacrificed by cervical dislocation, and tumor tissues were photographed and weighed.

4T1 in-situ tumor model establishment and treatment effect evaluation

Establishing a tumor model: 4T1 cells were subcultured for expansion one day in advance, well-cultured tumor cells were starved for 4 h with serum-free medium the following day, then the cells were digested with 0.25% trypsin, collected, resuspended and counted in sterile PBS, and the cell suspension was diluted to 4X 106 cells/mL and placed on ice. Subsequently 50 μ L of cell suspension was injected in the second left fat pad of each BALB/c mouse with an insulin syringe, taking care to avoid leakage. After treatment, mice were sacrificed by cervical dislocation, and tumor tissues were photographed and weighed.

Treatment and detection: when the average size of the tumor reached 40 mm3, the mice were randomly divided into 2 groups, and the tail vein of the treatment group was injected with 6 mg/kg ^RPeptide, blank control group was injected tail vein with an equal volume of PBS and dosing schedule was 18-2 in figure 18. Meanwhile, the body weight of the mouse is regularly measured by an electronic balance, the length and the width of the tumor are measured by a vernier caliper, and the calculation formula of the tumor volume is as follows: volume =0.5 × length × width 2. On day 13, the mice were sacrificed by cervical dislocation, and tumor tissues were photographed and weighed.

4T1 metastatic tumor model establishment and treatment effect evaluation

Establishing a tumor model: the 4T1 cells were subcultured for expansion one day in advance, the 4T1 cells in good culture were starved for 4 h with serum-free medium the following day, followed by digestion of the cells with 0.25% trypsin, collection of the cells, resuspension and counting with sterile PBS, and the cell suspension was diluted to 1 × 105 cells/mL and placed on ice. Subsequently 100. mu.L of the cell suspension was injected intravenously at the tail of each BALB/c mouse with an insulin syringe, taking care to avoid leakage.

Treatment and detection: mice were randomly divided into 2 groups on the third day after tumor cell injection, and 6 mg/kg was injected into tail vein of treatment group^RPeptide (all doses referred to herein as the Peptide content of the material), the blank control group was injected with an equal volume of PBS into the tail vein, and the dosing schedule was as shown in 18-3 of fig. 18, while the body weight of the mice was measured periodically on an electronic balance.

On day 20, the mice were sacrificed by cervical dislocation to expose the lungs and trachea, 15% india ink was injected into the trachea with a 20 mL syringe until the ink flowed back into the nasal cavity, and the lung tissue was removed and fixed in 4% paraformaldehyde. One mouse was randomly selected and the lung was directly taken out without lung ink staining and fixed in 4% paraformaldehyde for subsequent HE staining. The liver was removed and fixed in 4% paraformaldehyde. The number of lung metastases was counted and photographed.

Pathological section

(1) Material taking and fixing: the tumor or organ taken from animal is directly fixed in 4% paraformaldehyde, and the volume of the fixing liquid is at least 10 times of that of the tissue, so as to avoid extrusion.

(2) Trimming, dehydrating and transparentizing: after fixation, the tissue was placed in an embedding cassette and washed with running water for 30 minutes (to remove the fixative). The tissue is sequentially placed in ethanol with different concentrations, and the ethanol from low concentration to high concentration is used as a dehydrating agent to gradually remove the water in the tissue. The tissue was then placed in xylene to be transparent.

(3) Wax dipping and embedding: and (3) soaking the transparent tissue block in molten paraffin, wherein the paraffin soaking process needs to be carried out in a paraffin dissolving box. After the paraffin immersion was completed, the tissue blocks were placed in an embedding cassette filled with paraffin and cooled rapidly after the surface showed solidification.

(4) Slicing, spreading and baking: cutting paraffin block into 4-6 μm slices, flattening, sticking on glass slide, and oven drying at 45 deg.C.

HE staining

(1) Slice dewaxing and to water: placing the tissue slices into a 60 deg.C oven, oven drying for 1.5 h, sequentially placing the slices into xylene I, xylene II, and xylene III, each for 10min, sequentially placing anhydrous ethanol, 95% ethanol, and 70% ethanol, each for 5 min. And finally washing with distilled water for 1-2 min.

(2) Hematoxylin staining: the slices are placed into Harris hematoxylin liquid for dip dyeing for 5-8 min (scum on the slices is removed before dyeing), washed by tap water, differentiated for 1-2 s by 8% hydrochloric acid alcohol, washed by tap water, rewetted by warm water for 1-5 min, and washed by 95% ethanol for 1 min.

(3) Eosin staining: the sections were dip-stained in eosin for 1-2 s.

(4) Dewatering and sealing: placing the tissue slices in 95% alcohol I, 95% alcohol II, anhydrous alcohol I, anhydrous alcohol II, xylene I, and xylene II in sequence, each for 5min, air drying, and sealing with neutral gum.

4.3 data analysis and results

The results show that the method has the advantages of high yield,^Rthe Peptide has a good treatment effect on an EMT6 mouse triple negative breast cancer orthotopic tumor model. At the start of treatment, there was little difference in tumor volume in the three groups of mice, and on day 11 of treatment,^Rthe average volume of the tumor in the mice of the Peptide nanoparticle group was 137.3 mm3, the average weight was 0.1802 g, and PBS and^NRthe average volumes of tumors in mice in the Peptide nanoparticle-treated group were 610.3 mm3 and 587.6 mm3, respectively, and the average weights were 0.7793 g and 0.6573 g, respectively (see fig. 19). Similarly, in the 4T1 mouse triple negative breast cancer orthotopic tumor model^RThe Peptide nanoparticles also had significant therapeutic effects, with the drug on day 9 of treatment,^Rthe mean volume of the tumors was 256.1 mm3 and the mean weight was 0.2881 g in the Peptide group mice, while the mean volume was 575.6 mm3 and the mean weight was 0.4630 g in the PBS group mice (see FIG. 20). In addition, two triple negative breast cancer miceModel in the course of experiment^RNo significant weight loss occurred in mice with the Peptide nanoparticle group.

^RThe Peptide also has a better treatment effect on triple negative breast cancer metastasis. Results display^RThe number of lung metastases was significantly less in mice of the Peptide-treated group than in the PBS control group. Statistics of the number of metastases are shown in FIG. 21. HE staining of the lungs and liver of the treated and PBS control mice revealed more metastases, and fewer metastases in the PBS mice, as shown in figure 22. In addition, during the course of treatment^RNo significant weight loss occurred in mice in the Peptide group.

5. ^RIn vivo systemic toxicity assay for Peptide

ICR mice were given separately via tail vein^RPeptide and^NRpeptide, serum of mice is taken 24 hours after the last administration to detect ALT (glutamic pyruvic transaminase), AST (glutamic oxaloacetic transaminase), ALB (total protein), CREA (serum creatinine) and UREA (serum UREA) in serum, and the result shows that^RPeptide and^NRthe Peptide has no obvious damage to the liver and kidney functions of the mice, and the result is shown in figure 23.

Claims (7)

1. An acid-responsive anticancer peptide nanoparticle, which is characterized in that: the molecular structure of the nanoparticle is shown as the following formula:

。

2. a nanoparticle according to claim 1, wherein: the pH value of the acid response environment of the nanoparticles is 6.5-7.2.

3. A nanoparticle according to claim 2, wherein: the pH value of the acid response environment of the nanoparticles is 6.6-6.8.

4. A nanoparticle according to claim 1, wherein: the average grain diameter of the nanoparticles is 60-70 nm.

5. The method for synthesizing nanoparticles according to claim 1, comprising the following steps:

(1) synthesis of PEO-PPO-CDM

Dissolving 30 mg of 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furylpropionic acid in 2mL of dichloromethane, stirring in ice bath, adding 100 mu L of oxalyl chloride and 10 mu L of dimethylformamide to perform catalytic reaction for 30min, reacting at room temperature for 2h, and pumping off the dimethylformamide and excessive oxalyl chloride to obtain 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furylpropionyl chloride; dissolving 1.0 g of polyethylene oxide-polypropylene oxide in dichloromethane, stirring in an ice bath, dissolving 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionyl chloride in dichloromethane, adding into the polyethylene oxide-polypropylene oxide, adding 20 mu L of pyridine, reacting for 30min, reacting overnight at room temperature, concentrating, precipitating in diethyl ether, and centrifuging at low temperature to obtain PEO-PPO-CDM;

（2）(PEO-PPO-CDM)₂synthesis of-C12-PButLG-CA

Dissolving 1.0 g PEO-PPO-CDM in 6 mL dichloromethane, dissolving 80 mg C12-PButLG-CA in 2mL methanol, adding to PEO-PPO-CDM, adding 20 μ L triethylamine, reacting for 24 h, concentrating, precipitating in anhydrous ether, and centrifuging at low temperature to obtain (PEO-PPO-CDM)₂-C12-PButLG-CA, i.e. said acid-responsive anticancer peptide nanoparticles.

6. The use of nanoparticles according to claim 1 for the preparation of a medicament for the treatment of breast tumors.

7. The use of claim 6, wherein said breast tumor comprises a triple negative breast tumor.

Images