WO2023159724A1 - 一种酸响应性抗癌肽及其制备方法 - Google Patents

一种酸响应性抗癌肽及其制备方法 Download PDF

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WO2023159724A1
WO2023159724A1 PCT/CN2022/087254 CN2022087254W WO2023159724A1 WO 2023159724 A1 WO2023159724 A1 WO 2023159724A1 CN 2022087254 W CN2022087254 W CN 2022087254W WO 2023159724 A1 WO2023159724 A1 WO 2023159724A1
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peptide
nanoparticle
add
acid
ppo
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PCT/CN2022/087254
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French (fr)
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鲍燕
姚燕丹
熊梦华
钟翠玉
李�杰
刘穗萍
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中山大学孙逸仙纪念医院
华南理工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention relates to the technical field of biopharmaceuticals, in particular to an artificially synthesized anticancer peptide and its acid-responsive nanoparticles.
  • TNBC Triple Negative Breast Cancer
  • HER2 human epidermal growth factor
  • endocrine and anti-HER2 Targeted monoclonal antibody therapy is ineffective for TNBC, and chemotherapy is still the standard treatment for TNBC.
  • TNBC has a high degree of tumor heterogeneity, and the expression of drug efflux pumps such as ATP-binding cassette transporters is significantly excessive, drug resistance is prone to occur, resulting in decreased chemotherapy sensitivity, leaving drug-resistant TNBC in a non-drug-resistant state. governance dilemma.
  • Anticancer peptides are cationic amphiphilic polypeptides with hydrophobic residues, which are adsorbed on the surface of tumor cell membranes through electrostatic interactions between positively charged regions and negatively charged lipids on the surface of tumor cell membranes, and then inserted into the cell membrane through hydrophobic domains Lipids that destroy the fluidity of the cell membrane can directly pass through the cell membrane or create holes, allowing the contents to leak out and kill tumor cells.
  • Anti-cancer peptides kill tumor cells by physically breaking the membrane, directly causing irreversible damage to the cell membrane, independent of cell metabolism and without entering the cell to play a role, so it is not easy to cause drug resistance.
  • Melittin has been proved to have good killing activity on liver cancer, lung cancer, breast cancer, gastric cancer and leukemia cell lines at the cellular level.
  • Anticancer peptides have entered clinical trials. Nevertheless, the anti-tumor therapy of anti-cancer peptides has not yet achieved a major breakthrough. Low selectivity, normal tissue cytotoxicity, hemolytic toxicity, instability in serum, low bioavailability, and the need for intratumoral administration are still the main bottlenecks that hinder the application of anticancer peptides in tumor therapy.
  • the present invention discloses a synthetic anti-cancer peptide and its prodrug acid-responsive anti-cancer peptide nanoparticles based on acid-responsive group modification; and further discloses the preparation method of the two.
  • the acid-responsive anticancer peptide nanoparticle of the present invention is formed by linking the anticancer peptide with amphiphilic monomethyl polyethylene glycol-polypropylene glycol polymer through specific acid-responsive chemical bonds.
  • the synthetic route diagram of its preparation method is shown in Figure 2.
  • the acid-responsive anticancer peptide nanoparticle molecular structure of the present invention is shown in the following formula:
  • the pH value of the acid response environment of the nanoparticles is 6.5-7.2, and the optimal pH value is 6.6-6.8.
  • the nanoparticles are amphiphilic particles with an average particle size of 60-70nm.
  • the present invention further discloses the application of the anti-cancer peptide and its acid-responsive anti-cancer peptide nanoparticles in the preparation of tumor drugs for treating breast cancer, colon cancer and pancreatic cancer, especially in the preparation and treatment of triple-negative breast cancer, including triple-negative Drug application in primary carcinoma of the mammary gland and metastases.
  • the anticancer peptide of the invention is a radial amphipathic anticancer peptide artificially synthesized for the first time using L-glutamic acid as a raw material. It has been proved by experiments that this anti-cancer peptide directly and irreversibly kills tumor cells by physically breaking the membrane, and has a significant killing effect on various breast cancer cell lines including triple-negative breast cancer, colon cancer and pancreatic cancer cell lines , has good broad-spectrum anticancer activity and anti-drug resistance advantages.
  • the anti-cancer peptide was passed through a specific Acid-responsive chemical bonds are bonded to amphiphilic monomethylpolyethylene glycol-polypropylene glycol polymers, which self-assemble into acid-responsive nanoparticle precursors.
  • the anticancer peptide is located inside the nanoparticle, which can effectively shield the cytotoxicity of the anticancer peptide, significantly reduce the hemolytic activity and increase the stability of the anticancer peptide.
  • the specific acid-responsive chemical bonds of this nanoparticle are selectively sensitive to the microenvironment of tumor microacidity (pH 6.5-7.2) and broken, which can completely release anti-cancer peptides and play an active role in targeting tumor cell lines.
  • the test results show that the nanoparticle has almost no membrane-breaking activity at pH 7.4, but the membrane-breaking activity at pH 6.8 in a slightly acidic environment is similar to that of free anticancer peptides, and has a good targeting effect.
  • the nanoparticles are amphiphilic, the average particle size is between 60-70nm, and the particle size distribution is good (PDI is lower than 0.3), so that the nanoparticles have better solubility. Studies have shown that nanoparticles in this particle size range have a longer half-life in vivo and are easy to accumulate in tumor sites, further improving the bioavailability of anticancer peptides.
  • the nanoparticle has no obvious damage to the liver and kidney function of mice through the detection of the maximum tolerated dose and liver and kidney toxicity in mice, and has the conditions for systemic administration without intratumoral administration.
  • the nanoparticles have significant therapeutic effects on triple-negative breast cancer in situ tumors and metastatic tumors, especially for drug-resistant triple-negative breast cancer, which has good clinical application potential.
  • the anticancer peptide and its acid-responsive nanoparticles of the present invention use L-glutamic acid as a raw material, the raw material is easy to obtain, the preparation method is simple, the cost is low, and it can be produced on a large scale.
  • Fig. 1 is the synthetic circuit diagram of anticancer peptide of the present invention
  • Fig. 2 is a synthesis circuit diagram of acid-responsive anticancer peptide nanoparticles of the present invention
  • Fig. 3 is the GPC characterization spectrum of intermediate product polymer C12-PButLG of the present invention.
  • Fig. 4 is the NMR spectrum of intermediate product polymer C12-PButLG of the present invention.
  • Fig. 5 is the NMR spectrum of the anticancer peptide C12-PButLG-CA of the present invention.
  • Fig. 6 is the NMR spectrum of the acid-responsive anticancer peptide nanoparticle intermediate product PEO-PPO-CDM of the present invention.
  • Fig. 7 is the NMR spectrum of acid-responsive anticancer peptide nanoparticles of the present invention.
  • Figure 8 is the nuclear magnetic hydrogen spectrum of (PEO-PPO-CDM) 2 -C12-PButLG-CA assembled in heavy water;
  • Figure 9 is the nuclear magnetic hydrogen spectrum of (PEO-PPO-CDM) 2 -C12-PButLG-CA nanoparticles after adding deuterated hydrochloric acid;
  • a in FIG. 11 is a graph showing the killing activity of anticancer peptides on different breast cancer cell lines, and B is a graph showing the killing activity of other tumor cell lines;
  • Fig. 12 is the hemolytic activity curve figure of NR Peptide, R Peptide and Peptide of different concentrations in the embodiment;
  • Figure 13 is a graph showing the inhibitory effect of NR Peptide, R Peptide and Peptide at different concentrations on tumor cells under pH 7.4 and pH 6.8 environments in the examples;
  • Fig. 14 is a graph showing the inhibitory effect of R Peptide on tumor cells at different pH values in the examples.
  • Figure 15 is the result picture of R Peptide acting on EMT6 cells under the scanning electron microscope in the embodiment under different pH environments;
  • a and B are the pictures of the untreated blank control group, and
  • C is the picture of the tumor cells treated with R Peptide in the pH 7.4 environment ,
  • D is a picture of tumor cells treated with R Peptide in a pH 6.8 environment,
  • ad is a partial enlarged view of the corresponding picture on the left, and the arrow indicates the position of the hole on the cell membrane surface;
  • Figure 16 is a graphical representation of the leakage of cell contents caused by R Peptide at pH 7.4 and pH 6.8 in the examples.
  • A is the leakage of intracellular LDH caused by NR Peptide and R Peptide;
  • B is the detection of R Peptide caused by silver staining. Leakage of intracellular proteins;
  • Figure 17 is the endocytosis inhibitor (2-deoxy-D-glucose, chlorpromazine, methyl- ⁇ -cyclodextrin and wortmannin) of different pathways in the embodiment after inhibiting tumor cell endocytosis, R Peptide at pH
  • Figure 18 is a schematic diagram of the administration scheme of the triple-negative breast tumor mouse model in the embodiment.
  • Figure 19 is a graphical representation of R Peptide inhibiting tumor growth in EMT6 tumor-bearing mice in vivo, A is the tumor growth curve, B is the tumor weight, and C is the tumor photo;
  • Figure 20 is a graphical representation of the inhibition of tumor growth in 4T1 tumor-bearing mice by R Peptide in the embodiment, A is the tumor growth curve, B is the tumor weight, and C is the tumor photo;
  • Figure 21 is a statistical histogram of the number of lung metastases of breast cancer cells in 4T1 tumor-bearing mice inhibited by Example R Peptide in vivo;
  • Figure 22 is a photo of HE stained sections of the lung and liver of the breast cancer of the 4T1 tumor-bearing mice inhibited by Example R Peptide in vivo, and the tumor tissue is inside the black dotted line;
  • Figure 23 is a histogram of the in vivo systemic toxicity test of R Peptide in mice.
  • Figure 24 is the nuclear magnetic hydrogen spectrum of NR Peptide in the embodiment.
  • the anticancer peptide of this example is called Peptide for short, and the code name in the synthesis route is C12-PButLG-CA;
  • the acid-responsive anticancer peptide nanoparticles of this embodiment are referred to as R Peptide for short, and the code name in the synthesis route is: (PEO-PPO-CDM) 2 -C12-PButLG-CA;
  • the non-acid-responsive anticancer peptide nanoparticles of this embodiment are referred to as NR Peptide for short, and the code name in the synthesis route is: (PEO-PPO-SA) 2 -C12-PButLG-CA;
  • NR Peptide The molecular structural formula of NR Peptide is as follows:
  • the synthetic method comprises the following steps:
  • PEO-PPO polyethylene oxide-polypropylene oxide
  • SA maleic anhydride
  • DMAP 4-dimethylaminopyridine
  • 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 Biyuntian (China); MTT, hydrochloric acid Doxorubicin was purchased from Dalian Meilun (China); PBS phosphate buffer saline powder pack (2L/bag) was purchased from Wuhan Boster (China); fresh sheep blood was purchased from Guangzhou Future (China); Triton X-100 was purchased from From Sigma (USA); DMSO was purchased from Shanghai Sangong (China); Peptide, R Peptide and NR Peptide were synthesized by South China University of Technology.
  • All material concentrations in the test refer to the content of Peptide in the material.
  • 5mg/mL R Peptide Weigh 25mg R Peptide, add 400 ⁇ L DMSO to fully dissolve, that is, R Peptide mother solution containing 5mg/mL Peptide.
  • 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
  • 4T1 cells were cultured in 10% fetal bovine serum and 1% double antibody in DMEM medium
  • MCF-7/ADR cells were cultured in 1640 medium containing 10% fetal bovine serum, 1% double antibody and 1 ⁇ 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 experiments were not changed unless specifically required by the experiment. The cells were cultured in an incubator at 37°C, 5% CO2 and 95% humidity.
  • Tumor cells were planted in 96-well plates at a density of 1 ⁇ 104 cells per 100 ⁇ L per well, and cultured overnight in a cell incubator.
  • Nanoparticle preparation take a 10mL sample bottle, add 6mL PBS, place it on a magnetic stirrer, and adjust the rotation speed to 500rpm. Take 2 mL of 6 mg/mL R Peptide mother solution dropwise into the sample bottle, adjust the rotation speed to 400 rpm and stir for 15 minutes, then transfer the particles to a 14000 Da dialysis bag and put them in PBS for dialysis, change the PBS every 1 hour, and transfer the particles to the centrifuge after 4 hours of dialysis. tube and make up to 1.4mg/mL.
  • Group 1 64 ICR mice were randomly divided into 8 groups, 8 in each group.
  • Group 1 Group 2, Group 3, Group 4 and Group 5 were injected with 10mg/kg, 12.5mg/kg, 15mg/kg, 17.5mg/kg and 20mg/kg R Peptide nanoparticles by tail vein respectively.
  • Group 6, Group 7 and Group 8 were injected with 1.25mg/kg, 2.5mg/kg and 5mg/kg Peptide through tail vein respectively. After the administration, the mice were observed for three consecutive days.
  • CCK8 kit was purchased from GLPBIO (USA); cisplatin and propidium iodide (PI) were purchased from Dalian Meilun (China); Cellmask green and Protein marker were purchased from Thermo (USA); lactic acid dehydrogenation Enzyme Cytotoxicity Detection Kit, Rapid Silver Staining Kit, and 5 ⁇ protein loading buffer were purchased from Shanghai Biyuntian (China); Hepes buffer (100 ⁇ ) was purchased from Corning (USA); Sulfo-Cy5-NHS lipid was purchased from From AAT Bioquest (USA); Tris/glycine/SDS electrophoresis buffer (10 ⁇ ), PAGE gel rapid preparation kit were purchased from Shanghai Yazyme (China); Annexin V-FITC/PI apoptosis detection kit was purchased from Wuhan Elirut (China); 25% glutaraldehyde was purchased from Shanghai Fushen (China); absolute ethanol was purchased from Shanghai Sangong (China); 1,2-dioleoyl-sn-g
  • 50 ⁇ M wortmannin Take 14 mg wortmannin, add 654 ⁇ L DMSO to fully dissolve, and store at -20°C.
  • methyl- ⁇ -cyclodextrin Take 65mg of methyl- ⁇ -cyclodextrin, add 998 ⁇ L DMSO to dissolve, store at -20°C.
  • 1mg/mL Sulfo-Cy5-NHS Take 10mg Sulfo-Cy5-NHS, add 10mL DMSO to dissolve into a 1mg/mL Sulfo-Cy5-NHS stock solution, store at -20°C.
  • Buffer1 Take 710mg Na2HPO4 powder, add 450mL ultrapure water to dissolve, adjust the pH to 7.0, and dilute to 500mL with ultrapure water, that is, 10mM Na2HPO4.
  • Buffer2 Weigh 710mg Na2HPO4 powder and 5.26g NaCl powder, add 450mL ultrapure water to dissolve, adjust the pH to 7.4, and use ultrapure water to make up to 500mL, that is, 10mM Na2HPO4+90mM NaCl.
  • ANTS mother solution Weigh 15mg ANTS, add 2.81mL Buffer1 to fully dissolve, that is, 5.34mg/mL ANTS.
  • DPX mother solution Weigh 40mg DPX, add 2.11mL Buffer1 to fully dissolve, that is 18.99mg/mL DPX.
  • Tumor cells were planted in 96-well plates at a density of 1 ⁇ 105 cells per 100 ⁇ L per well, and cultured overnight in a cell culture incubator.
  • Panc02 cells were planted in 35mm porous glass-bottom dishes at a density of 5 ⁇ 105 per 500 ⁇ L per well, and cultured overnight. Remove the old medium, add 500 ⁇ L of 1640 medium, and add PI and Cellmask green at a ratio of 1:2000, and incubate for ten minutes. Add RPeptide-Cy5, which was pretreated for 1 h at pH 6.8 or pH 7.4, so that the final concentration was 10.5 ⁇ g/mL. Use a confocal microscope with a fixed field of view to collect pictures every 7s for 40 minutes to observe the dynamic changes of cells in real time. During the observation period, keep the temperature in the culture dish at 37°C. After the collection, the results were analyzed with ZEISS ZEN software.
  • EMT6 cells were planted in a 12-well plate (including sterile slides) at a density of 1 ⁇ 105 cells per 1 mL per well, and cultured overnight in a cell incubator.
  • Tumor cells were planted in 96-well plates at a density of 1 ⁇ 105 cells per 100 ⁇ L per well, and cultured overnight in a cell culture incubator.
  • LDH release rate (%) (absorbance of treated sample-absorbance of sample control well)/(absorbance of maximum cell enzyme activity-absorbance of sample control well) ⁇ 100%.
  • Tumor cells were planted in a 6-well plate at a density of 2 ⁇ 105 cells per 2 mL per well, and cultured overnight.
  • Lower gel lower gel solution 2.7mL; lower gel buffer 2.7mL, improved coagulant 60 ⁇ L.
  • Upper gel 0.75mL upper gel solution; 0.75mL upper gel buffer; 15 ⁇ L modified coagulant.
  • Electrophoresis Connect the electrophoresis tank and the electrophoresis instrument according to the order of the electrodes, select the constant voltage mode, set the voltage to 90V, electrophoresis for 15 minutes, then increase the voltage to 100V, and electrophoresis for about 60 minutes, until the bromophenol blue just runs out Stop electrophoresis.
  • the silver staining kit was used for staining, and the steps were as follows:
  • Fixative Mix 50mL of ethanol, 10mL of acetic acid and 40mL of Milli-Q pure water to obtain a fixative.
  • Silver dye sensitization solution (1 ⁇ ) Take 99mL Milli-Q grade pure water and add 1mL silver dye sensitizer solution (100 ⁇ ) and mix well to obtain silver dye sensitizer solution (1 ⁇ ) (2h after preparation) used within).
  • Silver solution (1 ⁇ ) Take 99ml of Milli-Q grade pure water and add 1mL of silver solution (100 ⁇ ) and mix well to obtain silver solution (1 ⁇ ). (Use within 2 hours after preparation).
  • Silver staining solution take 80mL Milli-Q grade pure water, add 20mL silver staining basic developing solution (5 ⁇ ) and 0.05mL silver staining developing solution (2000 ⁇ ), and mix well to obtain silver staining Chromogenic solution (use within 20 minutes after preparation).
  • Silver staining stop solution (1 ⁇ ) Take 95mL Milli-Q grade pure water and add 5mL silver staining stop solution (20 ⁇ ) and mix well to obtain the silver stain stop solution (1 ⁇ ) (use on the same day after preparation).
  • Fixation After electrophoresis, remove the glass plate and put the gel into 100mL fixative solution, place it on a shaker, and shake it at room temperature at 60-70rpm for 20min.
  • Washing with water Discard the silver solution, add 100mL Milli-Q grade pure water, and shake on a shaker at room temperature at 60-70rpm for 1min.
  • Termination discard the silver staining solution, add 100mL silver staining termination solution (1 ⁇ ), and shake on a shaker at room temperature at 60-70rpm for 10min.
  • step 3 100 ⁇ L of the dilution in step 3 was added to each well. And set up the control group of pH 6.8 medium and pH 7.4 medium respectively.
  • CCK8 assay was used to detect the killing effect of different concentrations of R Peptide on tumor cells under acidic and neutral conditions.
  • the results showed that the killing effect of R Peptide on triple-negative breast cancer cells at pH 6.8 was significantly stronger than that at pH 7.4, and had no significant difference from the killing effect of free Peptide. There was almost no killing effect on tumor cells (as shown in Figure 13).
  • R Peptide also showed the same effect in breast cancer phyllodes tumors.
  • the results in Figure 14 suggest that the killing effect of R Peptide on tumor cells gradually increases as the pH decreases, and when the pH reaches 6.8, lowering the pH of the environment has little effect on the killing effect of the material.
  • R Peptide can regulate the interaction between anticancer peptides and tumor cell membranes
  • the inventors bonded Cy5 fluorescent dye to anticancer peptides to form fluorescently labeled R Peptide-Cy5, and labeled tumor cell membranes with green membrane dyes.
  • the results showed that R Peptide-Cy5 could release anti-cancer peptides after pH 6.8 pretreatment, and the red fluorescent Peptide-Cy5 quickly aggregated on the tumor cell membrane and showed the phenomenon of bubbling, which led to the entry of PI into the tumor cell nucleus.
  • R Peptide-Cy5 pretreated at pH 7.4 did not show the phenomenon of red fluorescence gathering on the tumor cell membrane because it could not release the anticancer peptide.
  • Tumor cells were treated with R Peptide at pH 7.4 and pH 6.8, and then the cell culture fluid was collected to detect the release of LDH and protein in tumor cells by LDH release experiment, SDS-PAGE gel electrophoresis and silver staining.
  • the results showed that R Peptide was in the The release of LDH and protein in tumor cells can be caused under the condition of pH 6.8, while the release of intracellular LDH and protein is hardly caused under the condition of pH 7.4 (the results are shown in Figure 16).
  • the mode of tumor cell death caused by anticancer peptide was studied by Annexin V/PI staining and flow cytometry detection. The results showed that the traditional chemotherapy drug cisplatin can induce tumor cell apoptosis, while anticancer peptide directly leads to tumor cell necrosis.
  • mice 5-week-old female Balb/c mice were purchased from Slack Jingda Experimental Animal Co., Ltd. The rest are the same as above experimental animals and cell lines.
  • Tumor model establishment EMT6 cells were subcultured and cultured one day in advance, and the tumor cells in good culture state were starved for 4 h with serum-free medium the next day, and then the cells were digested with 0.25% trypsin, and the cells were collected and resuspended in sterile PBS and Count, dilute the cell suspension to 4 ⁇ 106 cells/mL, and place on ice. Then inject 50 ⁇ L of cell suspension into the second left fat pad of each BALB/c mouse with an insulin syringe, taking care to avoid leakage.
  • Treatment and detection when the average size of the tumor reached 40mm3, the mice were randomly divided into 3 groups, the treatment group was injected with 6mg/kg R Peptide through the tail vein, the control group was injected with 6mg/kg NR Peptide through the tail vein, and the blank control group was injected with the tail vein Equal volume of PBS was administered on the 6th day after tumor implantation, and the dosage regimen was as shown in 18-1 of Figure 18.
  • the body weight of the mice was regularly measured with an electronic balance and the length and width of the tumor was measured with a vernier caliper.
  • Tumor model establishment 4T1 cells were subcultured and cultured one day in advance, and the tumor cells in good culture state were starved with serum-free medium for 4 hours the next day, and then the cells were digested with 0.25% trypsin, and the cells were collected, resuspended with sterile PBS and Count, dilute the cell suspension to 4 ⁇ 106 cells/mL, and place on ice. Then inject 50 ⁇ L of cell suspension into the second left fat pad of each BALB/c mouse with an insulin syringe, taking care to avoid leakage. After the treatment, the mice were killed by cervical dislocation, and the tumor tissues were photographed and weighed.
  • Treatment and detection when the average size of the tumor reached 40mm3, the mice were randomly divided into two groups.
  • the treatment group was injected with 6mg/kg R Peptide through the tail vein, and the blank control group was injected with the same volume of PBS through the tail vein.
  • the administration plan is shown in Figure 18. 18-2.
  • the body weight of the mice was regularly measured with an electronic balance and the length and width of the tumor was measured with a vernier caliper.
  • Tumor model establishment 4T1 cells were subcultured and cultured one day in advance, starved 4T1 cells in good culture state with serum-free medium for 4 hours the next day, then digested with 0.25% trypsin, collected and resuspended with sterile PBS Count, dilute the cell suspension to 1 ⁇ 105 cells/mL, and place on ice. Subsequently, 100 ⁇ L of the cell suspension was injected into the tail vein of each BALB/c mouse with an insulin syringe, taking care to avoid leakage.
  • mice On the third day after the injection of tumor cells, the mice were randomly divided into two groups. The treatment group was injected with 6 mg/kg R Peptide through the tail vein (all dosages in this article refer to the content of Peptide in the material), and the blank control group was injected with 6 mg/kg R Peptide through the tail vein. Intravenous injection of equal volume of PBS, the dosing scheme is shown in Figure 18-18-3, and the weight of the mice was measured regularly with an electronic balance.
  • mice were killed by cervical dislocation, the lungs and trachea of the mice were exposed, and 15% Indian ink was injected into the trachea with a 20mL syringe, and the injection was stopped when the ink reflux appeared in the nasal cavity, and the lung tissue was taken out and placed in 4% polymer Formaldehyde fixed.
  • One mouse was randomly selected without lung ink staining, and the lungs were directly removed 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.
  • Tissues such as tumors or organs removed from animals are directly fixed in 4% paraformaldehyde, and the fixative is at least 10 times the volume of the tissue to avoid extrusion.
  • Wax immersion and embedding soak the transparent tissue block in melted paraffin, and the wax immersion process needs to be carried out in a wax melting box. After wax immersion, place the tissue block into an embedding box filled with paraffin, and cool it down rapidly after it solidifies.
  • R Peptide had a good therapeutic effect on EMT6 mouse triple-negative breast cancer in situ tumor model.
  • the average volume of tumors in the R Peptide nanoparticle group was 137.3mm3, and the average weight was 0.1802g, while PBS and NR Peptide nanoparticles
  • the average tumor volumes of mice in the treatment groups were 610.3 mm3 and 587.6 mm3, and the average weights were 0.7793 g and 0.6573 g, respectively (see FIG. 19 ).
  • R Peptide nanoparticles also had a significant therapeutic effect in the 4T1 mouse triple-negative breast cancer in situ tumor model.
  • the average volume of tumors in the R Peptide group was 256.1mm3, and the average weight was 0.2881 g, while the average volume of mice in the PBS group was 575.6mm3, and the average weight was 0.4630g (see Figure 20).
  • the body weight of the mice in the R Peptide nanoparticle group did not decrease significantly in the two triple-negative breast cancer mouse models during the experiment.
  • R Peptide also has a good therapeutic effect on triple-negative breast cancer metastases.
  • the results showed that the number of lung metastases in the mice treated with R Peptide was significantly less than that in the PBS control group.
  • the statistics of the number of metastases are shown in Figure 21.
  • HE staining was performed on the lungs and livers of the mice in the treatment group and the PBS control group, and it was found that the mice in the PBS group had more cancer metastases, while the mice in the treatment group had fewer metastatic tumors.
  • the results are shown in Figure 22.
  • the body weight of the mice in the R Peptide group did not decrease significantly during the treatment.
  • R Peptide and NR Peptide were administered to ICR mice through the tail vein respectively, and the mouse serum was taken 24 hours after the last administration to detect ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALB (total protein), CREA (blood Creatinine) and UREA (blood urea), the results showed that both R Peptide and NR Peptide had no significant damage to the liver and kidney function of mice, the results are shown in Figure 23.

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Abstract

一种人工合成抗癌肽及其酸响应性纳米粒前体。本纳米粒由抗癌肽与两亲性的单甲基聚乙二醇-聚丙二醇聚合物通过酸响应的化学键相连而成。经试验证明，本抗癌肽通过破膜活性杀伤肿瘤细胞，具有优良的广谱抗癌活性及抗耐药优势。本纳米粒选择性地对肿瘤微酸性环境敏感，可完全释放抗癌肽，发挥靶向肿瘤细胞系的活性作用。且具有抗溶血、血浆稳定性高，体内系统毒性低，可系统给药等优点。本纳米粒对包括三阴性乳腺癌在内的多种肿瘤细胞具有显著的抑制作用，特别是对耐药三阴性乳腺癌具有较好的临床应用潜力。

Description

一种酸响应性抗癌肽及其制备方法 技术领域
本发明涉及生物制药技术领域，具体涉及一种人工合成抗癌肽及其酸响应性纳米粒。
背景技术
易耐药和选择性差一直是化疗抗癌药物亟待解决的问题。以乳腺癌亚型三阴性乳腺癌(Triple Negative Breast Cancer，TNBC)为例，由于雌激素受体、孕激素受体和人表皮生长因子(HER2)受体低表达或缺失，导致内分泌和抗HER2靶向单克隆抗体治疗都对TNBC无效，化疗仍是TNBC的标准治疗方案。然而，由于TNBC具有高度的肿瘤异质性，且ATP结合盒转运蛋白等药物外排泵的表达明显过多，很容易出现耐药性，导致化疗敏感性降低，使耐药TNBC处于无药可治的困境。
研究表明，一些抗菌肽可发挥破膜活性而具有广谱的抗癌作用，因此又称为“抗癌肽”。抗癌肽是一类阳离子并带有疏水残基的两亲性多肽，通过正电荷区域与肿瘤细胞膜表面带负电荷的脂类发生静电作用而吸附于肿瘤细胞膜表面，进而通过疏水结构域插入细胞膜的脂质，破坏细胞膜流动性，可直接穿过细胞膜或产生孔洞，使内容物外泄而杀死肿瘤细胞。抗癌肽通过物理破膜的方式杀伤肿瘤细胞，直接造成细胞膜的不可逆损伤，不依赖于细胞代谢状态且无需进入细胞内发挥作用，因而不容易引起耐药问题。鉴于抗癌肽低耐药的独特优势，使其成了业界热点，例如：蜂毒肽Melittin被证明在细胞水平对肝癌、肺癌、乳腺癌、胃癌和白血病细胞系都具有良好的杀伤活性，少数抗癌肽已经进入临床试验阶段。尽管如此，抗癌肽的抗肿瘤治疗仍未取得较大突破。选择性低，正常组织细胞毒性，溶血毒性，在血清中不稳定，生物利用率低和需瘤内给药等缺点，仍是阻碍抗癌肽应用于肿瘤治疗的主要瓶颈。
发明内容
为克服以上技术缺陷，本发明公开了一种合成抗癌肽及其基于酸响应基团修饰而获得的前体药物酸响应性抗癌肽纳米粒；并进一步公开两者的制备方法。
本发明抗癌肽的分子结构如下式所示：
Figure PCTCN2022087254-appb-000001
其制备方法为：以L-谷氨酸为原料经聚合形成聚合肽后再作阳离子修饰而得到，合成线 路图如图1所示。
本发明的酸响应性抗癌肽纳米粒是由本抗癌肽与两亲性的单甲基聚乙二醇-聚丙二醇聚合物通过特定的酸响应化学键相连而成。其制备方法的合成线路图如图2所示。
本发明的酸响应性抗癌肽纳米粒分子结构如下式所示：
Figure PCTCN2022087254-appb-000002
本纳米粒的酸响应环境的pH值为6.5～7.2，最优pH值为6.6～6.8。
本纳米粒是平均粒径为60-70nm的两亲性颗粒。
本发明进一步公开所述抗癌肽及其酸响应性抗癌肽纳米粒在制备治疗乳腺癌、结肠癌和胰腺癌等肿瘤药物中的应用，尤其是在制备治疗三阴性乳腺癌，包括三阴性乳腺原体癌和转移癌药物中的应用。
本发明抗癌肽是以L-谷氨酸为原料首次人工合成的放射状两亲性抗癌肽。经试验证明，本抗癌肽以物理破膜的方式直接不可逆地杀伤肿瘤细胞，对包括三阴性乳腺癌在内的多种乳腺癌细胞系、结肠癌和胰腺癌细胞系都有显著的杀伤作用，具有很好的广谱抗癌活性和抗耐药优势。
为降低本抗癌肽对正常细胞的毒性并提高其在血浆中的稳定性，利用肿瘤微环境pH值低于正常组织呈微酸性(pH 6.5～7.2)的特点，将抗癌肽通过特定的酸响应化学键与两亲性的单甲基聚乙二醇-聚丙二醇聚合物键合，自行组装成酸响应性纳米粒前体。
本发明的酸响应性抗癌肽纳米粒经试验证明具有以下特性及优点：
1.抗癌肽位于纳米颗粒内部，可有效屏蔽抗癌肽的细胞毒性，显著减少溶血活性并增加抗癌肽的稳定性。
2.本纳米粒特定的酸响应化学键选择性地对肿瘤微酸性(pH 6.5～7.2)微环境敏感而断裂，可完全释放抗癌肽，发挥靶向肿瘤细胞系的活性作用。试验结果显示，pH 7.4条件下本纳米粒几乎没有破膜活性，但在微酸环境pH 6.8的破膜活性与游离抗癌肽相近，具有良好的靶向作用。
3.本纳米粒具有两亲性，平均粒径在60-70nm之间，粒径分布较好(PDI均低于0.3)，使纳米粒具有较好的溶解度。研究证明该粒径范围的纳米粒在体内具有较长的半衰期且容易 在肿瘤部位积累，进一步提高了抗癌肽的生物利用度。
4.经小鼠最大耐受剂量及肝肾毒性检测发现本纳米粒对小鼠肝肾功能无明显损伤，具备系统给药，无需瘤内给药的条件。
5.经动物体内试验验证，本纳米粒对三阴性乳腺癌原位瘤和转移瘤均具有显著的治疗效果，特别是对于耐药的三阴性乳腺癌具有较好的临床应用潜力。
6.本发明抗癌肽及其酸响应纳米粒以L-谷氨酸为原料，原料易得，制备方法简单，成本低，可规模化生产。
附图说明
图1是本发明抗癌肽的合成线路图；
图2是本发明酸响应性抗癌肽纳米粒的合成线路图；
图3是本发明中间产物聚合物C12-PButLG的GPC表征图谱；
图4是本发明中间产物聚合物C12-PButLG的核磁氢图谱；
图5是本发明抗癌肽C12-PButLG-CA的核磁氢图谱；
图6是本发明酸响应性抗癌肽纳米粒中间产物PEO-PPO-CDM的核磁氢图谱；
图7是本发明酸响应性抗癌肽纳米粒的核磁氢图谱；
图8是(PEO-PPO-CDM) 2-C12-PButLG-CA在重水中组装的核磁氢图谱；
图9是(PEO-PPO-CDM) 2-C12-PButLG-CA纳米颗粒在加入氘代盐酸后的核磁氢图谱；
图10是(PEO-PPO-CDM) 2-C12-PButLG-CA在pH＝6.8下的降解图谱；
图11中的A是抗癌肽对不同乳腺癌细胞系的杀伤活性曲线图，B是其他肿瘤细胞系的杀伤活性曲线图；
图12是实施例中不同浓度的 NRPeptide、 RPeptide和Peptide的溶血活性曲线图；
图13是实施例中在pH 7.4和pH 6.8环境下，不同浓度的 NRPeptide、 RPeptide和Peptide对肿瘤细胞抑制效应曲线图；
图14是实施例中不同pH值下 RPeptide对肿瘤细胞抑制效应曲线图；
图15是实施例中扫描电镜下，不同pH环境下 RPeptide作用于EMT6细胞的结果图片；A和B为未处理的空白对照组图片，C为pH 7.4环境下 RPeptide处理后的肿瘤细胞图片，D为pH 6.8环境下 RPeptide处理后的肿瘤细胞图片，a-d为左侧相应图片的局部放大图，箭头指示细胞膜表面孔洞位置；
图16是实施例中在pH 7.4和pH 6.8环境下， RPeptide引起细胞内容物泄漏的情况图示， A为 NRPeptide和 RPeptide引起细胞内LDH泄漏的情况；B为银染检测 RPeptide引起细胞内蛋白泄漏的情况；
图17是实施例中不同途径的内吞抑制剂(2-脱氧-D-葡萄糖、氯丙嗪、甲基-β-环糊精和渥曼青霉素)抑制肿瘤细胞内吞后， RPeptide在pH 7.4和pH 6.8环境下对肿瘤细胞活力抑制作用的柱状图，数据展示为均值±标准差，n.s.指无显著性差异；
图18是实施例中三阴性乳腺肿瘤小鼠模型的给药方案示图；
图19是实施例中 RPeptide在体内抑制EMT6荷瘤小鼠肿瘤生长情况的图示，A为肿瘤生长曲线，B为肿瘤重量，C为肿瘤照片；
图20是实施例中 RPeptide体内抑制4T1荷瘤小鼠肿瘤生长情况的图示，A为肿瘤生长曲线，B为肿瘤重量，C为肿瘤照片；
图21是实施例 RPeptide体内抑制4T1荷瘤小鼠乳腺癌细胞肺脏转移瘤个数统计柱状图；
图22是实施例 RPeptide体内抑制4T1荷瘤小鼠乳腺癌肺脏及肝脏HE染色切片照片，黑色虚线内为肿瘤组织；
图23是实施例中 RPeptide小鼠体内系统毒性试验的柱状图；
图24是实施例中 NRPeptide的核磁氢图谱。
具体实施方式
以下结合实施例对本发明作进一步说明，但不作为对本发明保护范围的限制。
名称及对应缩写或代号说明：
本实施例的抗癌肽简称为Peptide，合成路线中的代号为C12-PButLG-CA；
本实施例的酸响应性抗癌肽纳米粒简称为 RPeptide，合成路线中的代号为:(PEO-PPO-CDM) 2-C12-PButLG-CA；
本实施例的非酸响应性抗癌肽纳米粒简称为 NRPeptide，合成路线中代号为:(PEO-PPO-SA) 2-C12-PButLG-CA；
1.Peptide、 RPeptide和 NRPeptide的制备
1.1 Peptide的合成及表征
Peptide的合成线路图如图1所示，具体合成方法步骤为：
(1)谷氨酸衍生物ButLG的合成
称取10.0g的L-谷氨酸于圆底烧瓶中，加15mL的3-丁烯-1-醇，置于冰浴中充分搅拌，缓慢滴加4.0mL浓硫酸，室温反应24h，抽掉未反应的3-丁烯-1-醇，加饱和碳酸氢钠中和 到pH＝7，抽滤，水洗，得到固体，加到4.0mL异丙醇和4.0mL去离子水中，80℃溶解，降温重结晶，抽滤，乙醚洗涤，得到固体为谷氨酸衍生物ButLG。
(2)活性单体ButLG-NCA的合成
称取5.0g的谷氨酸衍生物ButLG于圆底烧瓶中，加入约150mL的无水四氢呋喃(THF)，置于冰浴搅拌，加入7.2g的三光气，50℃反应2.5h，抽干溶剂，转入手套箱过柱纯化，得到活性单体ButLG-NCA。
(3)聚合物C12-PButLG的合成
称取2.5g的ButLG-NCA单体于圆底烧瓶中，加入5.0mL的无水二甲基甲酰胺(DMF)，和200mg的十二烷基胺，搅拌反应24h，抽掉二甲基甲酰胺(DMF)，用二氯甲烷溶解，滴加到体积比为1:1的乙醚和正己烷混合溶液中沉淀，去掉上清，抽干，得聚合物C12-PButLG，测GPC和核磁氢谱，所得图谱见图3和图4。
通过图3的GPC表征可以看出，所制备的C12-PButLG具有单分散性。
通过核磁氢谱图4可以看出，聚肽C12-PButLG成功合成，5.75ppm和5.1ppm处为典型的双键特征峰，通过计算，聚合度为10。
(4)阳离子聚肽C12-PButLG-CA的合成
取1.0g的聚合物C12-PButLG溶解于4mL的二甲基甲酰胺(DMF)，并加入1.2g的巯基乙胺盐酸盐，通入氮气15min，排尽空气，加入15mg的催化剂2,2-二甲氧基-2-苯基苯乙酮，避光，继续通入氮气15min，用365nm激光光照反应1h，在去离子水中透析24h，冻干，得阳离子聚肽C12-PButLG-CA，测核磁氢谱(图谱见图5)，确定为目标产品抗癌肽。
所Peptide得核磁氢谱和聚合物C12-PButLG比较，双键特征峰消失，说明点击完全。
1.2  RPeptide的合成及表征
RPeptide的合成线路图如图2所示，具体合成方法步骤：
(1)PEO-PPO-CDM的合成
称取30mg的2,5-二羟基-4-甲基-2,5-二氧代-3-呋喃丙酸(CDM)溶于2mL的二氯甲烷，置于冰浴上搅拌，加入100μL的草酰氯，并加入10μL的二甲基甲酰胺(DMF)催化反应30min，室温反应2h，抽掉二甲基甲酰胺(DMF)和过量的草酰氯，得到2,5-二羟基-4-甲基-2,5-二氧代-3-呋喃丙酰氯(CDM-Cl)；称取1.0g的聚环氧乙烷-聚环氧丙烷(PEO-PPO)溶于二氯甲烷，置于冰浴上搅拌，并将2,5-二羟基-4-甲基-2,5-二氧代-3-呋喃丙酰氯(CDM-Cl)溶于二氯甲烷，加入到聚环氧乙烷-聚环氧丙烷(PEO-PPO)二氯甲烷溶液中，加入20μL的吡啶，反应30 min后，室温反应过夜，浓缩后沉淀在乙醚中，低温离心，得到PEO-PPO-CDM；测核磁氢谱表征图谱如图6所示。
(2)(PEO-PPO-CDM) 2-C12-PButLG-CA的合成
将1.0g的PEO-PPO-CDM溶于6mL二氯甲烷，将80mg的C12-PButLG-CA溶于2mL甲醇加到PEO-PPO-CDM中，并加入20μL的三乙胺，反应24h，浓缩后沉淀到无水乙醚中，低温离心，去上清液，抽干，得到(PEO-PPO-CDM)2-C12-PButLG-CA，测核磁氢谱，如图7所示，确定为所述目标产品 RPeptide。
1.3  NRPeptide(PEO-PPO-SA)2-C12-PButLG-CA)的合成及表征
NRPeptide的分子结构式如下：
Figure PCTCN2022087254-appb-000003
合成方法包括以下步骤：
(1)PEO-PPO-SA的合成
称取1.0g的聚环氧乙烷-聚环氧丙烷(PEO-PPO)溶于二氯甲烷，称取20mg的顺丁烯二酸酐(SA)溶于2mL的二氯甲烷，加到反应中，搅拌反应，称取10mg的4-二甲氨基吡啶(DMAP)加入到反应中，反应12h后，浓缩后沉淀在乙醚中，低温离心，得到PEO-PPO-SA，测核磁氢谱，证明顺丁烯二酸酐(SA)成功键合到聚环氧乙烷-聚环氧丙烷(PEO-PPO)上。
(2)(PEO-PPO-SA)2-C12-PButLG-CA的合成
将1.0g的PEO-PPO-SA溶于6mL二氯甲烷，加入100μL的草酰氯，并加入10μL的二甲基甲酰胺(DMF)催化反应30min，室温反应2h，抽掉二甲基甲酰胺(DMF)和过量的草酰氯，得到PEO-PPO-SA-Cl，将80mg的C12-PButLG-CA溶于2mL甲醇加到PEO-PPO-SA-Cl中，并加入20μL的三乙胺，反应24h，浓缩后沉淀到无水乙醚中，低温离心，去掉上清，抽干，得到PEO-PPO修饰的非响应聚肽(PEO-PPO-SA) 2-C12-PButLG-CA，核磁氢谱如图24所示，证明 NRPeptide制备成功。
1.4响应性验证
1.4.1核磁证明C12-PButLG-CA在酸环境下逃逸
将20mg的(PEO-PPO-CDM) 2-C12-PButLG-CA溶于200μL的二甲基亚砜(DMSO)，滴加到2mL的重水中，搅拌并组装成纳米颗粒，用重水超滤洗涤8次，并浓缩，得到约600μL的重水溶液，测核磁，结果如图8所示，聚肽的信号被屏蔽，只能看到聚环氧乙烷-聚环氧丙烷(PEO-PPO)的部分信号。在样品中加10μL的DCl，一个小时后继续测核磁氢谱，结果如图9所示。发现核磁中有聚肽的信号，说明聚肽已经可以和重水相互作用，可能到达颗粒表面，甚至逃离到重水中。
1.4.2 HPLC证明聚肽在酸环境下的降解
将(PEO-PPO-CDM) 2-C12-PButLG-CA溶于甲醇中(0.1mg/mL)，取甲醇溶液和pH＝6.8的PB按照1:1混合，HPLC测试0、30、60min的降解情况，如图10所示。提示键合聚肽基本上在pH＝6.8条件下1h可以降解得到游离的聚肽C12-PButLG-CA。
2.Peptide和 RPeptide的理化性质检测及药理活性试验
2.1实验材料
2.1.1实验动物及细胞系
5周龄雌性ICR小鼠，购自斯莱克景达实验动物有限公司。乳腺癌细胞系4T1、EMT6、MDA-MB-231及MCF-7、结肠癌细胞系CT26和胰腺癌细胞系Panc02购自美国ATCC，MCF-7阿霉素耐药(MCF-7/ADR)细胞由华南理工大学提供。乳腺癌叶状肿瘤(PTB)细胞由中山大学孙逸仙纪念医院从临床标本中分离培养提供。
2.1.2实验试剂
实验试剂：胎牛血清、0.25％胰蛋白酶、1640培养基、DMEM培养基及DMEM/F12培养基购自Gibco(美国)；抗青-链霉素购自上海碧云天(中国)；MTT、盐酸多柔比星购自大连美仑(中国)；PBS磷酸盐缓冲液粉包(2L/袋)购自武汉博士德(中国)；新鲜羊血购自广州未来(中国)；Triton X-100购自Sigma(美国)；DMSO购自上海生工(中国)；Peptide、 RPeptide和 NRPeptide由华南理工大学合成提供。
试验中所有材料浓度均指材料中Peptide的含量。例如：5mg/mL  RPeptide：称取25mg  RPeptide，加入400μL DMSO充分溶解，即含5mg/mL Peptide的 RPeptide母液。
2.2实验方法
2.2.1细胞培养
EMT6细胞、MDA-MB-231细胞、MCF-7细胞、Panc02细胞及CT26细胞都培养在含10％胎牛血清和1％双抗的1640培养基中，4T1细胞培养在含10％胎牛血清和1％双抗的DMEM培养基中。MCF-7/ADR细胞培养在1640培养基中，内含10％胎牛血清、1％双抗及1μM阿霉素，以维持细胞的阿霉素耐药性。PTB细胞培养在含15％胎牛血清和1％双抗的DMEM/F12培养基中。除非实验有特殊需要，否则实验中所用的所有培养基不会更换。细胞置于37℃、5％CO2及95％湿度的培养箱中培养。
2.2.2细胞活力实验
(1)肿瘤细胞分别以1×104个细胞每100μL每孔的密度种植在96孔板中，并置于细胞培养箱培中培养过夜。
(2)将10mg/mL Peptide母液用1640平培养基分别稀释成40μg/mL、20μg/mL、10μg/mL、5μg/mL。
(3)吸除96孔板中的旧培养基，并在每孔加入100μL含不同浓度Peptide的培养液，置于细胞培养箱中孵育1h。
(4)将MTT储液(5mg/mL)用1640培养基稀释成1mg/mL，1h后吸除含材料的培养基，每孔加入100μL含MTT培养基，不含细胞的孔作为空白对照。将96孔板置于培养箱中继续孵育3h。随后弃去上清，每孔加入100μL DMSO并置于摇床上震荡10min充分溶解甲瓒，通过多功能酶标仪检测在490nm处各孔的吸光值。
2.2.3抗癌肽纳米颗粒制备及表征
取2个10mL样品瓶，分别加3mL PBS，置于磁力搅拌器上，转速为500rpm。取5mg/mL的 RPeptide及 NRPeptide母液各500μL分别滴加至样品瓶，调节转速400rpm搅拌15min。后将制备的颗粒转移到14000Da透析袋至PBS中透析，每1h更换一次PBS，透析4h，后将颗粒转移至离心管并定容至625μg/mL。
分别取50μL制备好的颗粒，用PBS稀释10倍后测粒径。分别取10μL浓度为5mg/mL的 RPeptide及 NRPeptide母液，检测Zeta电位。纳米颗粒的粒径及zata电位均使用动态光散射粒度仪(Malvern Zetasizer Nano ZS90)检测。
2.2.4纳米颗粒粒径稳定性测试
分别将625μg/mL的 RPeptide及 NRPeptide颗粒用1640培养基和终浓度为10％的胎牛血清稀释10倍，稀释后分别于即刻及孵育0.5h、1h、2h、3h、4h、5h、6h、8h、10h、12h使用动态光散射仪测粒径。
2.2.5溶血实验
(1)取适量新鲜羊血于离心管中，在4℃，3000rpm下离心3min，将上清弃去。再加入10倍体积的PBS把红细胞沉淀重悬，随后在4℃，3000rpm下离心3min，弃上清。用PBS重复洗涤三遍，将洗涤好的羊红细胞加入PBS重悬稀释成4％(v/v)。
(2)分别取适量浓度为5mg/mL的 RPeptide和 NRPeptide母液和10mg/mL Peptide溶液用pH 7.4的PBS稀释成400μg/mL，接着再用PBS分别释成以下浓度梯度：400mg/mL、200mg/mL、100mg/mL、50mg/mL、25mg/mL、12.5mg/mL。
(3)取200μL稀释好的红细胞悬液于离心管分别加入200μL上述不同浓度稀释液，阴性对照加入200μL PBS，阳性对照加入200μL 0.2％Triton X-100。37℃孵育1h后3000rpm离心3min。取100μL上清液至96孔板，并于576nm处测各孔吸收值。溶血率计算公式如下：溶血率(％)＝(样品吸光度-阴性对照吸光度)/(阳性对照吸光度-阴性对照吸光度)×100％。
2.2.6最大耐受剂量检测
(1)纳米颗粒制备：取1个10mL样品瓶，加入6mL PBS，置于磁力搅拌器上，调节转速为500rpm。取6mg/mL的 RPeptide母液2mL滴加至样品瓶，调节转速至400rpm搅拌15min，后将颗粒转移至14000Da透析袋放至PBS中透析，每1h更换一次PBS，透析4h后将颗粒转移至离心管并定容至1.4mg/mL。
(2)取10mg/mL Peptide母液用PBS稀释成1.4mg/mL。
(3)取64只ICR小鼠，随机分8组，每组8只。第1组、第2组、第3组、第4组和第5组分别尾静脉注射10mg/kg、12.5mg/kg、15mg/kg、17.5mg/kg和20mg/kg  RPeptide纳米颗粒。第6组、第7组和第8组分别尾静脉注射1.25mg/kg、2.5mg/kg和5mg/kg Peptide。给药后连续三天观察观察小鼠情况。
2.3试验结果(数据均采用Graph prism 6.0软件进行分析。)
2.3.1 Peptide对肿瘤细胞的杀伤作用
通过MTT试验检测Peptide对肿瘤细胞的杀伤作用，结果如图11所示，显示Peptide对包括三阴性乳腺癌在内的多种乳腺癌细胞系及结肠癌和胰腺癌细胞系都有明显的杀伤作用。
2.3.2  RPeptide和 NRPeptide的粒径和电势检测
通过透析法制备 RPeptide和 NRPeptide，对其粒径和电势进行检测，结果显示 RPeptide和 NRPeptide的平均粒径均位于60-70nm，粒径分布较好(PDI均低于0.3)，在pH 7.4和pH  6.8环境下纳米颗粒粒径无明显改变，且两者电势均接近于0mV。
2.3.3  RPeptide和 NRPeptide在血清中的稳定性
检测 RPeptide和 NRPeptide在含10％FBS的培养基中不同时间点的粒径，结果显示两种颗粒在12h内粒径随时间的推移未发生明显变化，表明纳米颗粒在血清中可稳定存在无明显的聚集或崩解现象。
2.3.4  RPeptide和 NRPeptide对Peptide细胞毒性的屏蔽作用
用溶血实验检测 RPeptide和 NRPeptide及游离Peptide对正常细胞的毒性。结果如图12所示， RPeptide和 NRPeptide的溶血活性都显著低于游离Peptide。
向小鼠体内注射 RPeptide及游离Peptide检测小鼠的最大耐受剂量。结果显示，小鼠对 RPeptide纳米颗粒的最大耐受剂量为12.5mg/kg，明显高于对Peptide的最大耐受剂量2.5mg/mL。
3. RPeptide微酸环境选择性破损肿瘤细胞膜试验
3.1实验材料
3.1.1实验细胞系：同上实验动物和细胞系。
3.1.2实验试剂
实验试剂：CCK8试剂盒购自GLPBIO(美国)；顺铂、碘化丙啶(Propidium Iodide,PI)购自大连美仑(中国)；Cellmask green、Protein marker购自Thermo(美国)；乳酸脱氢酶细胞毒性检测试剂盒、快速银染试剂盒、5×蛋白上样缓冲液购自上海碧云天(中国)；Hepes缓冲液(100×)购自Corning(美国)；Sulfo-Cy5-NHS脂购自AAT Bioquest(美国)；Tris/甘氨酸/SDS电泳缓冲液(10×)、PAGE凝胶快速制备试剂盒购自上海雅酶(中国)；Annexin V-FITC/PI凋亡检测试剂盒购自武汉伊莱瑞特(中国)；25％戊二醛购自上海复申(中国)；无水乙醇购自上海生工(中国)；1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、Sephadex G-50购自Sigma(美国)；1-棕榈酰基-2-油酰基磷脂酰甘油(POPG)购自Cordenpharma(瑞士)；氯丙嗪、渥曼青霉素、2-脱氧-D-葡萄糖和甲基-β-环糊精购自上海麦克林(中国)；8-氨基-1,3,6-萘三磺酸二钠盐水合物(ANTS)、1,1'-[1,4-亚苯基双(亚甲基)]双(1-吡啶鎓)二溴化物(DPX)购自TCI(日本)；Na2HPO4购自阿拉丁(中国)。其他试剂同上。
3.1.3实验所需溶液
10mg/mL氯丙嗪：称取10mg氯丙嗪，加入1mL DMSO充分溶解，-20℃保存。
50μM渥曼青霉素：取14mg渥曼青霉素，加入654μL DMSO充分溶解，-20℃保存。
0.9M2-脱氧-D-葡萄糖：取74mg 2-脱氧-D-葡萄糖，加501μL纯水溶解，-20℃保存。
50mM甲基-β-环糊精：取65mg甲基-β-环糊精，加998μL DMSO溶解，-20℃保存。
1mg/mL Sulfo-Cy5-NHS：取10mg Sulfo-Cy5-NHS，加10mL DMSO溶解成1mg/mL的Sulfo-Cy5-NHS贮液，-20℃保存。
Buffer1：取710mg Na2HPO4粉末，加入450mL超纯水溶解，调节pH至7.0，用超纯水定容至500mL，即10mM Na2HPO4。
Buffer2：称取710mg Na2HPO4粉末及5.26g NaCl粉末，加450mL超纯水溶解，调节pH至7.4，用超纯水定容至500mL，即10mM Na2HPO4+90mM NaCl。
ANTS母液：称取15mg ANTS，加入2.81mL Buffer1充分溶解，即5.34mg/mL ANTS。
DPX母液：称取40mg DPX，加入2.11mL Buffer1充分溶解，即18.99mg/mL DPX。
20mg/mL DOPE：称取20mg POPE，加入1mL氯仿充分溶解。
20mg/mL POPG：称取20mg POPG，加入1mL氯仿充分溶解。
3.2实验方法
3.2.1 CCK8法检测酸响应抗癌肽纳米颗粒对肿瘤细胞的杀伤效应
(1)肿瘤细胞分别以1×105个细胞每100μL每孔的密度种植在96孔板中，并置于细胞培养箱培养过夜。
(2)将5mg/mL的 RPeptide和 NRPeptide母液和10mg/mL Peptide母液分别用pH 6.8和pH 7.4含血清1640培养基稀释成40μg/mL、20μg/mL、10μg/mL、5μg/mL(DMSO已配平)。
(3)取出96孔板，弃去旧培养基，每孔加入100μL上述稀释液，各三个副孔。并分别设置pH 6.8培养基和pH 7.4培养基的空白对照。
(4)将CCK8原液用培养基稀释10倍，弃去旧培养基，每孔加入含CCK8培养基100μL，不含细胞的孔作为空白对照。将96孔板置于培养箱中继续孵育3h。通过多功能酶标仪检测在490nm处各孔的吸光值。
3.2.2 Cy5标记的抗癌肽纳米颗粒制备
取5mg/mL  RPeptide母液500μL，265μL 1mg/mL Sulfo-Cy5-NHS(Peptide与Cy5的摩尔比为：10:4)，混匀室温孵育8h。取1个样品瓶，加1mL PBS，置于磁力搅拌器上，转速500rpm，将上述混合液缓慢滴入PBS内，转速400rpm避光搅拌1h。再将溶液转移至超滤管内，1000rpm，离心20min，离心至底部的液体呈浅蓝色，加入500μL PBS，1000rpm 离心20min，离心至底部的液体仍呈浅蓝色，重复加入PBS及离心直至底部液体为无色。离心完毕后收集颗粒并定容至4mL即625μg/mL。
3.2.3动态观察抗癌肽纳米颗粒与肿瘤细胞膜的相互作用
Panc02细胞以5×105个每500μL每孔的密度种植在35mm多孔玻底皿中，培养过夜。去除旧培养基，加入500μL1640培养基，并以1：2000的比例分别加入PI和Cellmask green，孵育十分钟。加入提前在pH 6.8或pH 7.4环境下预处理1h的RPeptide-Cy5，使终浓度为10.5μg/mL。用共聚焦显微镜，固定视野，每隔7s采集一次图片，连续采集40min，实时观察细胞的动态变化，观察期间保持培养皿内温度为37℃。采集结束后用ZEISS ZEN软件进行结果分析。
3.2.4扫描电镜观察抗癌肽纳米颗粒对细胞膜的破坏情况
(1)EMT6细胞以1×105个细胞每1mL每孔的密度种植在12孔板(含无菌爬片)中，置于细胞培养箱培养过夜。
(2)将5mg/mL  RPeptide母液分别用pH 6.8和pH 7.4含血清1640培养基稀释成20μg/mL(DMSO已配平)。
(3)取出12孔板，弃去旧培养基，每孔加入500μL上述稀释液，并分别设置pH 6.8培养基和pH 7.4培养基的空白对照，置于培养箱孵育30min。
(4)30min后弃去培养基加入PBS洗涤3次，加2.5％戊二醛室温固定1min。取样合适大小的离心管，灌满2.5％戊二醛，并将细胞爬片转移至离心管内。
(5)取细胞爬片，先后在30％、50％、70％、90％、100％的乙醇中进行梯度脱水。随后将细胞爬片放入超临界干燥机中进行临界点干燥，取出样品喷金后，扫描电镜观察。
3.2.5 LDH释放实验
(1)肿瘤细胞分别以1×105个细胞每100μL每孔的密度种植在96孔板中，并置于细胞培养箱培养过夜。
(2)将5mg/mL RPeptide和 NRPeptide母液分别用pH 6.8和pH 7.4的不含血清1640培养基稀释成40μg/mL(DMSO已配平)。
(3)96孔板每孔加入200μL上述稀释液，各3个副孔。同时在对照孔加入200μL培养基，“样品最大酶活性对照孔”中加入200μL培养基及20μL LDH释放试剂并混匀，置于培养箱中孵育1h。
(4)到达预定时间后，将96孔板置于离心机400g离心5min。分别取各孔的上清液120 μL，加入到一新的96孔板相应孔中。
(5)按试剂盒方法配置工作液，各孔分别加入60μL LDH检测工作液，混匀，室温避光孵育30min(用铝箔包裹后置于水平摇床上缓慢摇动)。孵育结束后在490nm处测定各孔吸光度。LDH释放的计算公式为：LDH释放率(％)＝(处理样品吸光度－样品对照孔吸光度)/(细胞最大酶活性的吸光度－样品对照孔吸光度)×100％。
3.2.6银染检测胞内蛋白释放情况
样品制备
(1)肿瘤细胞以2×105个细胞每2mL每孔的密度种植在6孔板中，培养过夜。
(2)将5mg/mL RPeptide母液分别用pH 6.8和pH 7.4无血清1640培养基稀释成30μg/mL和15μg/mL(DMSO已配平)。
(3)取出6孔板，弃去旧培养基，用PBS洗涤三遍。分别在每个孔加入500μL上述稀释液，空白对照分别加入pH 7.4和pH 6.8的不含血清1640培养基500μL(DMSO已配平)。1h后每皿取培养液300μL，5000rpm离心5min，取上清，置于冰上。
(4)分别取第3步的上清40μL，及含0.05％FBS的1640培养基40μL，各加入10μL 5×Loading Buffer，混匀后95℃金属浴10min，冷却至室温后8000rpm离心1.5min。SDS-PAGE电泳
(1)检查电泳所需仪器，清洗玻璃板，晾干后将玻璃板对齐，垂直卡在架子上。
(2)配胶：用雅酶公司的PAGE凝胶快速制备试剂盒10％PAGE凝胶，用移液枪加下层胶至梳子下约1cm，观察有无漏胶，下层胶凝固后用移液枪加上层胶至低板玻璃线，插梳子。
下层胶：下层胶溶液2.7mL；下层胶缓冲液2.7mL，改良型促凝剂60μL。
上层胶：上层胶溶液0.75mL；上层胶缓冲液0.75mL；改良型促凝剂15μL。
(3)将胶同玻璃板一起安装到电泳槽上，加入电泳缓冲液。每孔上样20μL，Marker 4μL。继续加缓冲液至700mL，直至内槽加满，其余加至外槽。
(4)电泳：按电极顺序连接好电泳槽及电泳仪，选择恒压模式，电压设为90V，电泳15min，随后将电压增大至100V，电泳60min左右，至溴酚兰刚跑出即可终止电泳。
采用银染试剂盒进行染色，步骤如下：
试剂配制：
(1)固定液：取50mL乙醇、10mL乙酸和40mL Milli-Q级纯水混匀，得固定液。
(2)30％乙醇：取30mL无水乙醇加入70mL Milli-Q级纯水混匀。
(3)银染增敏液(1×)：取99mL Milli-Q级纯水加入1mL银染增敏液(100×)混匀，即为银染增敏液(1×)(配制后2h内使用)。
(4)银溶液(1×)：取99ml Milli-Q级纯水加入1mL银溶液(100×)混匀，即为银溶液(1×)。(配制后2h内使用)。
(5)银染显色液：取80mL Milli-Q级纯水加入20mL银染基本显色液(5×)及0.05mL银染显色加速液(2000×)，混匀后即为银染显色液(配制后20min内使用)。
(6)银染终止液(1×)：取95mL Milli-Q级纯水加入5mL银染终止液(20×)混匀，即为银染终止液(1×)(配置后当天使用)。
实验步骤：
(1)固定：电泳结束后，取下玻璃板并将凝胶取出放入100mL固定液中，置于摇床，60-70rpm室温摇动20min。
(2)30％乙醇洗涤：弃固定液，加100mL 30％乙醇，60-70rpm室温摇床摇动10min。
(3)水洗涤：弃30％乙醇，加200mL Milli-Q级纯水，60-70rpm室温摇床摇动10min。
(4)增敏：洗涤完毕，加100mL银染增敏液(1×)，60-70rpm室温摇床摇动2min。
(5)水洗涤(共2次)：将银染增敏液弃去，加入200mL Milli-Q级纯水，置于摇床，60-70rpm室温摇动1min。重复洗涤一次。
(6)银染：洗涤完毕后，加入100mL银溶液(1×)，60-70rpm室温摇床摇动10min。
(7)水洗涤：弃银溶液，加100mL Milli-Q级纯水，60-70rpm室温摇床摇动1min。
(8)显色：洗涤完毕后，加100mL银染显色液，置于摇床，60-70rpm室温摇动2-5min，直到出现蛋白条带。
(9)终止：弃银染显色液，加100mL银染终止液(1×)，60-70rpm室温摇床摇动10min。
(10)水洗涤：弃终止液，加100mL Milli-Q级纯水，60-70rpm，室温摇床摇动3min。
(11)保存：将凝胶置于Milli-Q级纯水中保存。
3.2.7内吞抑制实验
(1)4T1细胞以1×105个细胞每100μL每孔的密度种植在96孔板中，培养过夜。
(2)取10mg/mL氯丙嗪母液，用培养基稀释成10μg/mL，实验组每孔加入100μL；取50μM渥曼青霉素母液，用培养基稀释成50nM，实验组每孔加入100μL；取0.9M 2-脱 氧-D-葡萄糖母液，用培养基稀释成50mM，实验组每孔加入100μL；取50mM甲基-β-环糊精母液，用培养基稀释成50μM，实验组每孔加入100μL。置于培养中箱孵育30min。
(3)将5mg/mL RPeptide母液分别用pH 6.8和pH 7.4的1640培养基稀释成40μg/mL(DMSO已配平)，同时再次加入各抑制剂(浓度与预孵育时一致)。
(4)处理30min后取出96孔板，将含抑制剂的培养基弃去，每孔加入100μL第3步的稀释液。并分别设置pH 6.8培养基和pH 7.4培养基的对照组。
(5)将MTT储液(5mg/mL)用1640培养基稀释成1mg/mL，1h后吸除含材料的培养基，每孔加入100μL含MTT培养基，不含细胞的孔作为空白对照。将96孔板置于培养箱中继续孵育3h。随后弃去上清，每孔加入100μL二甲亚砜并置于摇床上震荡10min充分溶解甲瓒，通过多功能酶标仪检测在490nm处各孔的吸光值。
3.2.8流式检测细胞死亡方式
(1)4T1细胞分别以5×105个细胞每100μL每孔的密度种植在6孔板中，培养过夜。
(2)取5mM顺铂母液，用1640培养基分别稀释成20μM和10μM。取10mg/mL Peptide母液，用1640培养基分别稀释成10μg/mL和5μg/mL。
(3)取出6孔板，弃去旧培养基，每孔加入2mL第2步述稀释液，空白对照孔加入2mL培养基，置于培养箱孵育24h。
(4)取出6孔板，用不含EDTA胰酶消化细胞，并用旧培养基重悬细胞，1000rpm，离心3min，弃上清，用PBS重悬细胞，400目尼龙网过滤1次，1000rpm离心3min，收集细胞于1.5mL EP管。
(5)每管加入600μL Binding Buffer重悬细胞，然后取三个EP管，分别标记NA，FITC单染，PI单染，从6管细胞悬液各取50μL到这三个EP管。处理组和空白对照组细胞悬液每管取300μL至相应的新EP管。
(6)处理组和空白对照每管先加入3μL Annexin v-FITC混匀，再加入3μL PI混匀。FITC单染管加入3μL Annexin v-FITC混匀，PI单染管加入3μL PI混匀。室温下避光反应15min。
(7)用流式细胞仪检测细胞荧光，坏死细胞为FITC+/PI+，凋亡细胞为FITC+/PI-。
3.3实验结果
数据分析：数据均采用Graph prism 6.0及SPSS25.0软件进行分析，P＜0.05表示差异具有统计学意义。
CCK8实验检测酸性和中性条件下不同浓度 RPeptide对肿瘤细胞的杀伤效应。结果显示 RPeptide在pH 6.8条件下对三阴性乳腺癌细胞的杀伤力明显强于pH 7.4的条件环境，与游离Peptide的杀伤效果无明显差别，而 NRPeptide纳米颗粒在pH 7.4和6.8条件下对肿瘤细胞都几乎没有杀伤效果(如图13所示)。 RPeptide在乳腺癌叶状肿瘤中也显示了同样的效果。图14结果提示， RPeptide对肿瘤细胞的杀伤作用随着pH的降低逐渐增强，当pH到达6.8后再降低环境的pH对材料的杀伤作用影响不大。在不同温度下进行CCK8试验，观察在低温环境下细胞内各种代谢酶作用降低时 RPeptide的作用是否受到影响。结果显示无论在pH 7.4条件下还是pH 6.8条件下， RPeptide在4℃和37℃环境下的杀伤效果都没有区别。
为了进一步验证 RPeptide可调控抗癌肽与肿瘤细胞膜的相互作用，发明人将Cy5荧光染料键合到抗癌肽上，形成荧光标记 RPeptide-Cy5，并通过绿色膜染料标记肿瘤细胞膜，在共聚焦显微镜下动态观察pH 7.4和pH 6.8预处理后 RPeptide-Cy5与肿瘤细胞膜的相互作用。结果显示，pH 6.8预处理后 RPeptide-Cy5可释放抗癌肽，带红色荧光的Peptide-Cy5迅速聚集在肿瘤细胞膜上并呈现吐泡泡现象，从而导致PI进入肿瘤细胞核。而pH 7.4预处理的 RPeptide-Cy5由于无法释放抗癌肽，未出现红色荧光聚集在肿瘤细胞膜的现象。
在pH 7.4和pH 6.8条件下，通过扫描电镜观察用 RPeptide处理后的肿瘤细胞膜形态。结果如图15所示，pH 7.4条件下 RPeptide处理的EMT6细胞形态与未处理的对照组正常细胞相似，细胞表面有较多的微绒毛，呈梭形或多角形，无孔洞；pH 6.8条件下 RPeptide处理的肿瘤细胞表面微绒毛减少，有大小不一的孔洞形成，且细胞胀大。
在pH 7.4和pH 6.8条件下用 RPeptide处理肿瘤细胞，随后收集细胞培养液通过LDH释放实验、SDS-PAGE凝胶电泳及银染检测肿瘤细胞内LDH和蛋白质的释放情况，结果显示 RPeptide在pH 6.8条件下可引起肿瘤细胞内LDH和蛋白质的释放，而在pH 7.4条件下几乎不引起细胞内LDH和蛋白质的释放(结果如图16所示)。
为了进一步证明 RPeptide无需进入细胞内发挥作用，用不同内吞抑制剂处理细胞后观察 RPeptide对肿瘤细胞的杀伤作用。结果表明，在巨胞饮抑制剂渥曼青霉素、能量依赖性内吞抑制剂2-脱氧-D-葡萄糖、网格蛋白依赖性内吞抑制剂氯丙嗪、小窝蛋白依赖性内吞抑制剂甲基-β-环糊精等不同机制内吞抑制剂抑制内吞的情况下，抗癌肽纳米颗粒在微酸环境杀伤肿瘤细胞的作用几乎不受影响，结果如图17所示。
通过Annexin V/PI染色及流式细胞仪检测研究抗癌肽引起肿瘤细胞死亡方式，结果显示，传统化疗药物顺铂可诱导肿瘤细胞凋亡，而抗癌肽直接导致肿瘤细胞坏死。
4. RPeptide对三阴性乳腺癌细胞的抑制作用
4.1实验材料
4.1.1实验动物及细胞系
5周龄的雌性Balb/c小鼠，购自斯莱克景达实验动物有限公司。其余同以上实验动物及细胞系。
4.1.2实验试剂
4％多聚甲醛购自广州永津(中国)，二甲苯购自阿拉丁(中国)，苏木素染液、伊红染液购自武汉塞维尔(中国)，中性树胶购自福州迈新(中国)，印度墨汁购自大连美仑(中国)。其余同上。
4.2实验方法
EMT6原位肿瘤模型建立及治疗效果评估
肿瘤模型建立：提前一天将EMT6细胞传代扩增培养，次日将培养状态良好的肿瘤细胞用无血清培养基饥饿4h，随后用0.25％胰酶消化细胞，收集细胞后用无菌PBS重悬并计数，将细胞悬液稀释至4×106个/mL，置于冰上。随后用胰岛素注射器在每只BALB/c小鼠左侧第二个脂肪垫中注射50μL细胞悬液，注意避免漏液。
治疗及检测：当肿瘤的平均大小达40mm3时，将小鼠随机分为3组，治疗组尾静脉注射6mg/kg  RPeptide，对照组尾静脉注射6mg/kg NRPeptide，空白对照组尾静脉注射等体积PBS，在植瘤后的第6天开始给药，给药方案如图18之18-1。同时定期用电子天平测量小鼠体重及用游标卡尺测量肿瘤长宽，肿瘤体积的计算公式为：体积＝0.5×长×宽2。第18天用颈椎脱臼法处死小鼠，取肿瘤组织拍照并称重。
4T1原位肿瘤模型建立及治疗效果评估
肿瘤模型建立：提前一天将4T1细胞传代扩增培养，次日将培养状态良好的肿瘤细胞用无血清培养基饥饿4h，随后用0.25％胰酶消化细胞，收集细胞后用无菌PBS重悬并计数，将细胞悬液稀释至4×106个/mL，置于冰上。随后用胰岛素注射器在每只BALB/c小鼠左侧第二个脂肪垫中注射50μL细胞悬液，注意避免漏液。治疗结束后，用颈椎脱臼法处死小鼠，取肿瘤组织拍照并称重。
治疗及检测：当肿瘤的平均大小达40mm3时，将小鼠随机分为2组，治疗组尾静脉注射6mg/kg  RPeptide，空白对照组尾静脉注射等体积PBS，给药方案如图18之18-2。同时定期用电子天平测量小鼠体重及用游标卡尺测量肿瘤长宽，肿瘤体积的计算公式为：体积＝0.5×长 ×宽2。第13天用颈椎脱臼法处死小鼠，取肿瘤组织拍照并称重。
4T1转移瘤模型建立及治疗效果评估
肿瘤模型建立：提前一天将4T1细胞传代扩增培养，次日将培养状态良好的4T1细胞用无血清培养基饥饿4h，随后用0.25％胰酶消化细胞，收集细胞后用无菌PBS重悬并计数，将细胞悬液稀释至1×105个/mL，置于冰上。随后用胰岛素注射器在每只BALB/c小鼠尾静脉注射100μL细胞悬液，注意避免漏液。
治疗及检测：注射肿瘤细胞后的第三天将小鼠随机分为2组，治疗组尾静脉注射6mg/kg  RPeptide(本文中所有给药量均指材料中Peptide含量)，空白对照组尾静脉注射等体积PBS，给药方案如图18之18-3，同时定期用电子天平测量小鼠体重。
第20天用颈椎脱臼法处死小鼠，暴露小鼠的肺脏和气管，用20mL注射器向气管注入15％印度墨汁，至鼻腔出现墨汁反流后停止注射，取出肺脏组织，置于4％多聚甲醛固定。随机选取一只小鼠不进行肺脏墨汁染色，直接取出肺脏于4％多聚甲醛固定，用于后续HE染色。取出肝脏，置于4％多聚甲醛固定。计算肺脏转移瘤数量并拍照。
病理切片
(1)取材、固定：从动物中取下的肿瘤或脏器等组织直接置于4％多聚甲醛中固定，固定液至少为组织的10倍体积，避免挤压。
(2)修剪、脱水、透明：固定结束后将组织放入包埋盒，流水冲洗30分钟(除去固定液)。将组织依次置于不同浓度的乙醇中，从低浓度到高浓度乙醇作为脱水剂，逐渐将组织内水份脱净。随后将组织置于二甲苯中透明。
(3)浸蜡、包埋：将透明后的组织块置于溶化的石蜡中浸泡，浸蜡过程需在溶蜡箱中进行。浸蜡完成后将组织块置入装满石蜡的包埋盒中，待表明凝固后迅速冷却。
(4)切片、展片、烤片：将石蜡块切成4-6μm薄片，展平后贴至载玻片上，置于45℃恒温箱中烘干。
HE染色
(1)切片脱蜡及至水：将组织切片放入60℃烘箱中烘1.5h，随后将切片依次放入二甲苯Ⅰ、二甲苯Ⅱ、二甲苯Ⅲ，各10min，随后依次放入无水乙醇、95％乙醇、70％乙醇，各5min。最后蒸馏水洗1-2min。
(2)苏木素染色：将切片放入Harris苏木素液中浸染5-8min(染色前去上面的浮渣)，自来水冲洗，8％盐酸酒精分化1-2s，自来水冲洗，温水返蓝1-5min，95％乙醇清洗1min。
(3)伊红染色：将切片放入伊红中浸染1-2s。
(4)脱水及封片：将组织切片依次放入95％酒精I、95％酒精II、无水乙醇Ⅰ、无水乙醇Ⅱ、二甲苯Ⅰ、二甲苯Ⅱ，各5min，切片晾干后用中性树胶封片。
4.3数据分析及结果
结果表明， RPeptide对EMT6小鼠三阴性乳腺癌原位瘤模型有较好的治疗效果。在开始治疗时，三组小鼠肿瘤体积几乎没有差别，在治疗的第11天， RPeptide纳米颗粒组小鼠肿瘤的平均体积为137.3mm3，平均重量为0.1802g，而PBS和 NRPeptide纳米颗粒处理组小鼠肿瘤的平均体积分别为610.3mm3、587.6mm3，平均重量分别为0.7793g、0.6573g(见图19)。类似地，在4T1小鼠三阴性乳腺癌原位瘤模型中 RPeptide纳米颗粒也有明显的治疗效果，在治疗的第9天， RPeptide组小鼠肿瘤的平均体积为256.1mm3，平均重量为0.2881g，而PBS组小鼠的平均体积为575.6mm3，平均重量为0.4630g(见图20)。此外，两种三阴性乳腺癌小鼠模型在实验过程中 RPeptide纳米颗粒组小鼠体重未出现明显下降。
RPeptide对三阴性乳腺癌转移瘤也有较好治疗效果。结果显示 RPeptide治疗组小鼠肺转移瘤数量明显少于PBS对照组。转移瘤数量统计见图21所示。对治疗组和PBS对照组小鼠的肺脏和肝脏进行HE染色，发现PBS组小鼠出现较多的癌转移，而治疗组小鼠较少出现转移瘤，结果见图22所示。此外，在治疗过程中 RPeptide组小鼠体重未出现明显下降。
5. RPeptide的体内系统毒性试验
分别通过尾静脉给予ICR小鼠 RPeptide及 NRPeptide，最后一次给药24h后取小鼠血清检测血清中的ALT(谷丙转氨酶)、AST(谷草转氨酶)、ALB(总蛋白)、CREA(血肌酐)和UREA(血尿素)，结果表明 RPeptide及 NRPeptide都对小鼠肝肾功能无明显损伤，结果见图23。

Claims (7)

  1. 一种酸响应性抗癌肽纳米粒，其特征在于：所述纳米粒的分子结构如下式所示：
    Figure PCTCN2022087254-appb-100001
  2. 如权利要求1所述的纳米粒，其特征在于：所述纳米粒的酸响应环境的pH值为6.5～7.2。
  3. 如权利要求2所述的纳米粒，其特征在于：所述纳米粒的酸响应环境的pH值为6.6～6.8。
  4. 如权利要求1所述的纳米粒，其特征在于：所述纳米粒平均粒径为60-70nm。
  5. 如权利要求1所述的纳米粒的合成方法，其特征在于，具体包括以下步骤：
    (1)PEO-PPO-CDM的合成
    取30mg的2,5-二羟基-4-甲基-2,5-二氧代-3-呋喃丙酸溶于2mL的二氯甲烷，冰浴搅拌，加入100μL的草酰氯和10μL的二甲基甲酰胺催化反应30min，室温反应2h，抽掉二甲基甲酰胺和过量的草酰氯，得2,5-二羟基-4-甲基-2,5-二氧代-3-呋喃丙酰氯；取1.0g的聚环氧乙烷-聚环氧丙烷溶于二氯甲烷，冰浴搅拌，并将2,5-二羟基-4-甲基-2,5-二氧代-3-呋喃丙酰氯溶于二氯甲烷，加入到聚环氧乙烷-聚环氧丙烷中，加入20μL的吡啶，反应30min后，室温反应过夜，浓缩后沉淀在乙醚中，低温离心，得到PEO-PPO-CDM；
    (2)(PEO-PPO-CDM) 2-C12-PButLG-CA的合成
    将1.0g的PEO-PPO-CDM溶于6mL二氯甲烷，将80mg的C12-PButLG-CA溶于2mL甲醇加到PEO-PPO-CDM中，并加入20μL的三乙胺，反应24h，浓缩后沉淀到无水乙醚中，低温离心，得到(PEO-PPO-CDM) 2-C12-PButLG-CA，即所述的酸响应性抗癌肽纳米粒。
  6. 如权利要求1所述的纳米粒在制备治疗乳腺肿瘤药物中的应用。
  7. 如权利要求6所述的应用，其特征在于，所述的乳腺肿瘤包括三阴性乳腺肿瘤。
PCT/CN2022/087254 2022-02-23 2022-04-17 一种酸响应性抗癌肽及其制备方法 WO2023159724A1 (zh)

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