WO2018001383A1 - 抗菌肽衍生物及其用途 - Google Patents

抗菌肽衍生物及其用途 Download PDF

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WO2018001383A1
WO2018001383A1 PCT/CN2017/091390 CN2017091390W WO2018001383A1 WO 2018001383 A1 WO2018001383 A1 WO 2018001383A1 CN 2017091390 W CN2017091390 W CN 2017091390W WO 2018001383 A1 WO2018001383 A1 WO 2018001383A1
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Prior art keywords
antimicrobial peptide
micelle
nucleic acid
modified antimicrobial
hydrophobically modified
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PCT/CN2017/091390
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English (en)
French (fr)
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杨莉
门可
何谷
魏于全
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SI CHUAN UNIV
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SI CHUAN UNIV
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Priority to JP2018567676A priority Critical patent/JP2019527044A/ja
Priority to EP17819379.3A priority patent/EP3480208B1/en
Priority to US16/313,228 priority patent/US20190216939A1/en
Publication of WO2018001383A1 publication Critical patent/WO2018001383A1/zh
Anticipated expiration legal-status Critical
Priority to US19/029,005 priority patent/US20250249110A1/en
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    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Definitions

  • the invention belongs to the field of biomedicine, and mainly relates to antibacterial peptide derivatives and uses thereof, in particular to hydrophobically modified antimicrobial peptide DP7 derivatives and uses thereof.
  • Antibacterial peptides also known as Host Defense Peptides (HDPs), generally consist of 12 to 100 amino acid residues, are a class of basic polypeptides, and most of the antimicrobial peptides are positively charged.
  • Antibacterial peptide has a broad-spectrum antibacterial effect, can effectively inhibit pathogens such as fungi, viruses, parasites, and can selectively kill tumor cells, is not easy to produce drug resistance, and constitutes the first barrier for host to prevent invasion of pathogenic microorganisms. It is an important component of the body's immune system. Antibacterial peptides have become potential drugs to replace antibiotics in preventing and controlling diseases, and have broad development prospects.
  • antimicrobial peptides Despite the enormous potential of antimicrobial peptides, only a very small number of antimicrobial peptides are widely used clinically, mainly due to its stability and toxicity. Some studies have shown that antimicrobial peptide nanocrystallization can achieve better stability and reduce toxicity, which provides a new research direction for the application of antimicrobial peptides.
  • the immunomodulatory activities of antimicrobial peptides or HDPs are receiving increasing attention, including the induction of cytokine and chemokine production by altering signaling pathways, direct or indirect recruitment of effector cells including phagocytic cells, enhancement of intracellular and extracellular bacterial killing, Promotes dendritic cell maturation and macrophage differentiation as well as mediating wound repair and apoptosis.
  • a variety of antimicrobial peptides exhibit adjuvant activity due to their good immunomodulatory effects, primarily by activating the innate immune response and mediating the acquired immune response.
  • the antibacterial peptide DP7 is a screening method for computer-assisted design of novel antibacterial peptides.
  • the antibacterial peptide with higher bacterial recognition specificity and stronger antibacterial activity obtained by replacing two amino acids with the template antibacterial peptide HH2 (HH2 patent number) :WO08022444).
  • HH2 patent number HH2 patent number
  • HH2 patent number HH2 patent number
  • HH2 patent number HH2 patent number
  • DP7 In the in vivo antibacterial activity study, the abdominal model of mice infected with pathogenic Staphylococcus aureus found that DP7 showed a very good therapeutic effect by inducing immune cells to clear bacteria. However, high concentration of DP7 after intravenous administration may cause hemolysis of red blood cells and death of mice, indicating that high concentrations of DP7 are toxic to red blood cells and destroy red blood cells.
  • DP7 is a positively charged hydrophilic antibacterial peptide. We can modify it with hydrophobic fragments to obtain amphiphilic compounds with self-assembly into nanoparticles, which can greatly reduce the toxicity of intravenous administration, maintain its antibacterial effect and immune regulation in vivo. Function, and can be used as a delivery system for small nucleic acid drugs The domain has a wide range of uses.
  • the object of the present invention is to provide a novel modified antimicrobial peptide for the purpose of providing a new and effective choice for anti-infective treatment, preparation of novel immunoadjuvants and preparation of siRNA delivery vehicles in the field.
  • the technical solution adopted by the present invention is to provide a hydrophobically modified antimicrobial peptide which is coupled to a hydrophobic moiety (or a nitrogen-terminated coupling hydrophobic compound) at the nitrogen end of the antimicrobial peptide.
  • the amino acid sequence of the antimicrobial peptide DP7 is VQWRIRVAVIRK (SEQ ID No. 1).
  • the carbon end of DP7 is generally amidated, and its structure is VQWRIRVAVIRK-NH 2 .
  • the hydrophobic compound is a sterol compound or a saturated linear fatty acid.
  • the sterol compound is a cholesterol compound or a cholic acid compound.
  • the sterol compound is succinylated cholesterol, cholic acid or deoxycholic acid.
  • the long-chain fatty acid is a C6 to C20 fatty acid.
  • the saturated linear fatty acid is at least one of C8 to C18.
  • the long-chain fatty acid is at least one of stearic acid (C18), palmitic acid (C16), lauric acid (C12) or n-octanoic acid (C8).
  • the nitrogen terminal of the antimicrobial peptide is coupled to the hydrophobic segment (hydrophobic compound) by -CO-OH on the hydrophobic segment (hydrophobic compound) and -NH 2 on the antimicrobial peptide The amidation reaction is formed.
  • hydrophobically modified antimicrobial peptide In the above hydrophobically modified antimicrobial peptide, the structure of the hydrophobically modified antimicrobial peptide is:
  • R is a cholesterol compound, a bile acid, or a long chain fatty acid.
  • the present invention also provides a micelle made of the above hydrophobically modified antimicrobial peptide.
  • the micelle is a hydrophobically modified antimicrobial peptide self-assembled in a solution.
  • the micelles are subjected to freeze-drying treatment.
  • the micelle is further loaded with at least one of a nucleic acid, a small molecule drug, a polypeptide or a protein.
  • the present invention also provides the above method for hydrophobically modifying an antimicrobial peptide, comprising the steps of:
  • the present invention also provides the use of the above hydrophobically modified antimicrobial peptide or the above-mentioned micelle in the preparation of an antibacterial agent.
  • the antibacterial is antibacterial or antifungal.
  • the bacterium is Staphylococcus aureus, Escherichia colibacillus, Acinetobacter baumanmii, Pseudomonas aeruginosa or typhoid fever. At least one of the genus (Salmonella typhi).
  • the fungus is at least one of Canidia Albicans or Candida parapsilosis.
  • the present invention also provides an antibacterial agent prepared by using the above hydrophobically modified antimicrobial peptide or the above micelle as a main active ingredient.
  • antibacterial drugs also contain other antibacterial drugs.
  • the other antibacterial agent described in the above antibacterial agent is an antibiotic.
  • the antibiotic according to the above antibacterial agent is at least one of a glycopeptide antibiotic, an aminoglycoside antibiotic, a macrolide antibiotic, and a ⁇ -lactam antibiotic.
  • the ⁇ -lactam antibiotic described in the above antibacterial agent is at least one of a penicillin antibiotic or a cephalosporin antibiotic.
  • the penicillin antibiotic is penicillin G, penicillin V, flucloxacillin, oxacillin, At least one of ampicillin, carbenicillin, piracetin, sulfacillin, ticarcillin, piperacillin or amoxicillin.
  • the cephalosporin antibiotics are: cefadroxil, cephalexin, cefazolin, cefradine, cefprozil, cefuroxime, cefaclor, cefmendo, cefotaxime, ceftriaxone, cephalosporin At least one of gram, cefdinir, ceftapi, cefepime or ceftizox.
  • aminoglycoside antibiotics mentioned in the above antibacterial drugs are streptomycin, gentamicin, kanamycin, tobramycin, amikacin, neomycin, sisomicin, and At least one of brothmycin, amikacin, netilmicin, ribomycin, saponin or azin.
  • polypeptide antibiotic described in the antibacterial agent is at least one of vancomycin, norvancomycin, polymyxin B or teicoplanin.
  • the macrolide antibiotic described in the above antibacterial agent is erythromycin, leucomycin, odorless erythromycin, erythromycin, acetylspiramycin, medimycin, josamycin or azithromycin. At least one of them.
  • the above antibacterial agent is in the form of an injection.
  • the present invention also provides the use of the above hydrophobically modified antimicrobial peptide or the above-mentioned micelles in the preparation of an immunoadjuvant.
  • the present invention also provides an immunological adjuvant prepared by the above hydrophobized modified antimicrobial peptide or the above micelle as an immunological adjuvant and an antigen.
  • the above immunoadjuvant further contains single-stranded deoxyoligonucleotides (CpG ODNs). Further, the ratio of the hydrophobically modified antimicrobial peptide to the CpG ODNs is 1:0.5 to 1:5.
  • the present invention also provides the use of the above hydrophobically modified antimicrobial peptide or the above-mentioned micelles in the preparation of a nucleic acid delivery carrier.
  • the nucleic acid is RNA.
  • the nucleic acid is messenger RNA (mRNA), siRNA (Small interfering RNA) for RNA interference or sgRNA (small guide RNA) for genome editing.
  • mRNA messenger RNA
  • siRNA Small interfering RNA
  • sgRNA small guide RNA
  • the present invention also provides a nucleic acid delivery vector obtained by loading the nucleic acid by the above-described hydrophobized modified antimicrobial peptide or the above-mentioned micelle.
  • the nucleic acid is RNA.
  • the nucleic acid described above is messenger RNA (mRNA), siRNA (Small interfering RNA) for RNA interference, or sgRNA (small guide RNA) for genome editing.
  • mRNA messenger RNA
  • siRNA Small interfering RNA
  • sgRNA small guide RNA
  • the mass ratio of the hydrophobically modified antimicrobial peptide to the nucleic acid is 1:1 to 20:1.
  • the invention also provides methods of making the nucleic acid delivery vectors described above.
  • the method includes the following steps:
  • the above-mentioned antibacterial peptide DP7 and the hydrophobic fragment coupling product can be stored in the form of a lyophilized powder, and can be directly dissolved in sterile water or physiological saline when used.
  • the antimicrobial peptide DP7 in the hydrophobically modified antimicrobial peptide DP7 of the present invention has a small molecular weight, can be conveniently synthesized by the Fmoc solid phase polypeptide synthesis method, and is simple and easy to be coupled with the hydrophobic segment by chemical synthesis.
  • the hydrophobized DP7 of the invention can self-assemble into micelles, has good monodispersity and zeta potential, and is stable by lyophilization and re-dissolution; hydrophobized DP7 micelle can significantly reduce the toxicity of antibacterial peptide DP7 on erythrocyte lysis.
  • hydrophobicated DP7 has low antibacterial activity in vitro, it has excellent antibacterial activity in zebrafish and mice, and antibacterial activity in vivo is not through direct sterilization, but through recruitment of macrophages. Lymphocytes such as monocytes and neutrophils, as well as regulating the expression of some immune cytokines, thereby achieving immune protection against the body.
  • hydrophobized DP7 can also act as an immunological adjuvant, inducing the body to produce a higher immune response against the antigen of interest.
  • hydrophobized DP7 cationic micelle of the present invention can efficiently compound siRNA and introduce into tumor cells such as colon cancer and melanoma, and inhibit tumor tissue growth by intraperitoneal injection, intratumoral injection and tail vein injection. Role and show a high level of security.
  • Staphylococcus aureus strains ATCC 33591, ATCC 25923
  • Figure 1 Scheme for the synthesis of antimicrobial peptide DP7 and hydrophobic fragment conjugates.
  • FIG. 1 Mass spectrum after modification of the DP7 hydrophobic fragment.
  • A is a mass spectrum of DP7-C after cholesterol modification;
  • B is a mass spectrum of DP7-SA after stearic acid modification.
  • FIG. 3 Self-assembly of DP7-C micelles and determination of critical micelle concentration: A is a schematic diagram of DP7-C micelle self-assembly; B is the determination of critical micelle concentration of DP7-C micelles.
  • Figure 4 Physical characteristics of DP7-C micelles: A is the particle size; B is the Zeta potential; C is the atomic force microscope; D is the appearance.
  • Figure 5 Comparison of in vitro hemolytic activity of DP7 and DP7-C micelles: A is a hemoglobin measurement curve; B is an external view of a hemolysis experiment.
  • Figure 6 In vivo antibacterial activity of DP7-C: A is a gross and fluorescent map of the pyelocyte infection of Pseudomonas aeruginosa; B is a schematic diagram of fluorescence gradation integration; C is a schematic diagram of the number of colonies in the model of intraperitoneal infection of S. aureus mice.
  • Figure 7 In vivo antibacterial mechanism of DP7-C: A is the flow pattern of monocyte and neutrophil detection in the peritoneal cavity after DP7-C stimulation; B is the detection of macrophages in the peritoneal cavity after DP7-C stimulation Flow cytometry; C is a schematic diagram comparing the number of total cells, macrophages, neutrophils and monocytes in the peritoneal cavity after DP7-C stimulation; D is the detection of immune-related cytokines by Q-PCR after stimulation of PBMC by DP7-C The expression was compared. E is the comparison of the expression of immune-related cytokines by Q-PCR after DP7-C combined with LPS stimulation of PBMC.
  • Figure 8 Detection of immune effect of DP7-C/CpG complex: A is a schematic diagram of anti-OVA antibody titer in different immunization groups at week 5; B is a tumor growth curve of each group in a prophylactic model; C is prevention In the sexual model, the survival of each group of mice changed; D is the growth curve of the tumors of each group in the therapeutic model.
  • Figure 10 Test of DP7-C promoting DC uptake of OVA antigen.
  • Figure 11 Test of CpG/DP7-C complex to stimulate innate immune response.
  • Figure 12 Test of CpG/DP7-C complex to stimulate cellular immune response.
  • Figure 13 CpG/DP7-C adjuvant challenged a highly potent anti-tumor immunity test.
  • FIG. 14 Electron microscopic morphology of DP7-C micelle/siRNA complex: A is DP7-C micelle; B is DP7-C/siRNA complex.
  • Figure 15 In vitro transfection efficiency of DP7-C carrying siRNA: A is a comparison of DP7-C and PEI25K and lipofectamine2000 carrying siRNA transfected tumor cells; B is a schematic diagram of transfection efficiency of B16 cells by flow detection; Flow cytometry to detect the transfection efficiency of C26 cells.
  • Figure 16 Cytotoxicity assay of DP7-C (CCK-8 method).
  • Figure 17 DP7-C transmission of VEGF siRNA in a C26 mouse colon cancer peritoneal metastasis model.
  • Figure 18 DP7-C transmission of VEGF siRNA in a C26 mouse colon cancer subcutaneous xenograft model.
  • Figure 19 DP7-C transmission of VEGF siRNA in a B16 mouse melanoma lung metastasis model.
  • DP7 is a positively charged hydrophilic antimicrobial peptide, and is usually in the form of amidation modification of its carbon terminal, and DP7 used in the embodiment of the present invention is QWRIRVAVIRK-NH 2 .
  • Hydrophobic fragments or hydrophobic compounds
  • cholesterol, bile acids, long-chain fatty acids, etc. coupled to hydrophilic polypeptides, may have the ability to self-assemble to form nanostructures.
  • the antimicrobial peptide was nano-sized by the hydrophobic fragment-conjugated antimicrobial peptide DP7, thereby increasing its stability and reducing hemolytic toxicity, and its intravenous administration was achieved. Due to its direct antibacterial and immunomodulatory effects, the nanoparticles formed by hydrophobization DP7 have anti-infective effects.
  • the antibacterial peptide DP7 has good immunomodulatory activity and can mediate the natural immune response. This property makes it a vaccine immunoadjuvant. Co-immunization with the antigen can enhance the host's cellular immunity and mediate antigen-specific immunoglobulin production. Studies have shown that hydrophobicized antimicrobial peptides exhibit good adjuvant properties. In vitro, hydrophobicized DP7 can self-assemble into nanoparticles, and CpG can be used as a novel immunoadjuvant by electrostatic adsorption, which can enhance the antigen uptake by antigen presenting cells, promote the maturation of antigen presenting cells and the expression of cytokines. In the animal tumor model test, the hydrophobized Dp7/CpG complex prolonged the survival of tumor-inoculated mice, significantly inhibited tumor growth, and enhanced the level of antigen-specific antibodies.
  • hydrophobized DP7 micelles formed by self-assembly are positively charged in aqueous solution, demonstrating the potential as a non-viral gene delivery, especially siRNA delivery vector.
  • hydrophobicated DP7 will play an important role in siRNA-based tumor therapy research and applications.
  • hydrophobic fragments include: cholesterol, cholic acid, palmitic acid, stearic acid, lauric acid and the like.
  • 2-chlorotrityl chloride Resin the name is 2-chlorotrityl chloride resin;
  • Fmoc-Rink Amide MBHA Resin the name is 4-(2',4'-dimethoxyphenyl-fluorenylmethoxycarbonyl- Aminomethyl)-phenoxyacetamido-methylbenzhydrylamine resin;
  • Fmoc: fluorenylmethoxycarbonyl; pbf, tbu, Otbu, Trt, Boc are all protecting groups, the names are 2, 2, 4, 6 respectively , 7-pentamethyldihydrobenzofuran-5-sulfonyl, tert-butyl, tert-butoxy, trityl, tert-butoxycarbonyl.
  • Deprotection Remove 15% of the Fmoc protecting group on the resin with 20% Pipedine/DMF (N,N-dimethylformamide) solution, react for 15 min, use DCM/DMF (dichloromethane/N, N-dimethylmethyl)
  • the amide was washed alternately three times with a small amount of resin, washed successively with methanol/DCM/DMF, and then added to an EP tube containing 5% ninhydrin in absolute ethanol. The bath was bathed for three minutes and the resin appeared blue, ie positive. After the reaction, the reaction is continued twice, and the next step is continued, otherwise the deprotection process is repeated.
  • the starting material used in this step was Fmoc-Rink Amide MBHA Resin. It was composed of Rink Amide Linker modified by MBHA Resin-linked Fmoc protection.
  • the degree of substitution in the present invention is 0.36 mmol/g, but other substitution degrees can also achieve the same or similar technical effects, and are also within the scope of the present invention.
  • the degree of substitution used in the present invention of 0.36 mmol/g is the optimum value from the balance of the yield, purity, and utilization ratio of the synthetic fragment.
  • step 2 Fmoc-Lys(Boc)-MBHA Resin is de-Fmoc, and then N,N-dimethylformamide, Fmoc-Arg(pbf)-OH, 1-hydroxybenzotriazole, benzotriazole are added.
  • Fmoc-Arg(pbf)-Lys(Boc) -MBHA Resin Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Arg(pbf)-OH, Fmoc-Ile-OH, Fmoc are sequentially connected in this way.
  • the free amino group is generally acetylated and blocked, and the prepared blocking solution (acetic anhydride: pyridine: DMF 3:3:4) is added to the resin.
  • the reaction bottle the reaction bottle is shaken up and down for about 20 minutes, and the ninhydrin is detected. If the resin is yellow, the reaction is complete, and the subsequent reaction can be carried out. If it is not completely closed, it is necessary to extend the sealing time or adjust the ratio of the blocking solution to Make the reaction as complete as possible.
  • the boiling water bath for three minutes the resin is yellow or light blue, that is, the negative reaction, the reaction is completed, and the resin in the reaction flask is alternately washed three times with DCM/DMF, and the solvent is removed.
  • the resin obtained above was added to a reaction flask containing a lysate, the reaction bottle was sealed, and fixed on a shaker to carry out a reaction. After about 2 hours, the resin was removed by filtration, and the resin was washed several times with DCM, and the filtrate was collected and rotated.
  • the TFA and the solvent were removed by evaporation, and iced anhydrous diethyl ether was added to the remaining liquid, and a large amount of white flocculent precipitate appeared, and a white precipitate of high-speed centrifugation (4000 r/min) was a crude product.
  • the crude product is further purified by preparative high performance liquid chromatography to obtain the target product cholic acid DP7 (DP7-CA).
  • the resin showed yellow or light blue, that is, the negative reaction, the reaction was completed, and the resin in the reaction flask was alternately washed three times with DCM/DMF, and the solvent was removed.
  • the resin obtained above was added to a reaction flask containing a lysate, the reaction bottle was sealed, and fixed on a shaker to carry out a reaction. After about 2 hours, the resin was removed by filtration, and the resin was washed several times with DCM, and the filtrate was collected and rotated.
  • the TFA and the solvent were removed by evaporation, and iced anhydrous diethyl ether was added to the remaining liquid, and a large amount of white flocculent precipitate appeared, and a white precipitate of high-speed centrifugation (4000 r/min) was a crude product.
  • the crude product was further purified by preparative high performance liquid chromatography to obtain the target product stearic acid DP7 (DP7-SA).
  • the mass spectrum is shown in Fig. 2B, and the molecular weight is determined to be in agreement with the expected value.
  • the resin showed yellow or light blue, that is, the negative reaction, the reaction was completed, and the resin in the reaction flask was alternately washed three times with DCM/DMF, and the solvent was removed.
  • the resin obtained above was added to a reaction flask containing a lysate, the reaction bottle was sealed, and fixed on a shaker to carry out a reaction. After about 2 hours, the resin was removed by filtration, and the resin was washed several times with DCM, and the filtrate was collected and rotated.
  • the TFA and the solvent were removed by evaporation, and iced anhydrous diethyl ether was added to the remaining liquid, and a large amount of white flocculent precipitate appeared, and a white precipitate of high-speed centrifugation (4000 r/min) was a crude product.
  • the crude product was further purified by preparative high performance liquid chromatography to obtain the target product palmitized DP7 (DP7-PA).
  • the boiling water bath for three minutes the resin is yellow or light blue, that is, the negative reaction, the reaction is completed, and the resin in the reaction flask is alternately washed three times with DCM/DMF, and the solvent is removed.
  • the resin obtained above was added to a reaction flask containing a lysate, the reaction bottle was sealed, and fixed on a shaker to carry out a reaction. After about 2 hours, the resin was removed by filtration, and the resin was washed several times with DCM, and the filtrate was collected and rotated.
  • the TFA and the solvent were removed by evaporation, and iced anhydrous diethyl ether was added to the remaining liquid, and a large amount of white flocculent precipitate appeared, and a white precipitate of high-speed centrifugation (4000 r/min) was a crude product.
  • the crude product is further purified by preparative high performance liquid chromatography to obtain the target product stearified DP7 (DP7-DA).
  • the conjugate of the antimicrobial peptide DP7 and the hydrophobic segment can also be synthesized by a synthetic company.
  • the succinylated cholesterol-modified DP7-C ((Chol-suc-VQWRIRVAVIRK-NH 2 ) was synthesized by Shanghai Kep Biotechnology Co., Ltd., and the peptide was synthesized by solid phase synthesis.
  • the synthesized DP7-C was purified by HPLC. , purity >95% or more, the molecular weight of DP7-C was determined by MS.
  • the synthesized polypeptide was stored at -20 ° C, and was mixed with Millq water to prepare 5 mg/ml mother liquor before use.
  • DP7-C synthesized by solid phase synthesis (Chol -suc-VQWRIRVAVIRK-NH 2 ), which is based on DP7 and is coupled to hydrophobic cholesterol via an ester bond at the N-terminus, and protected by a molecule of -NH 2 at the C-terminus.
  • the mass spectrum of DP7-C is shown in Figure 2. The molecular weight is 1991.3408, and the main peak on the MS map is 996.1748, suggesting that the correct DP7-C is synthesized.
  • DP7-C can be assembled into micelles in aqueous solution.
  • Pyrene is insoluble in water, and its solubility in water is about 6 ⁇ 10 -7 mol/L, but it is easily soluble in ethanol and ether.
  • the fluorescence emission spectrum of yttrium has five fluorescent peaks, and the ratio of the first emission spectral light intensity I1 to the third emission spectral light intensity I3 in aqueous solution (the fluorescence intensity at 373 nm and the ratio at 384 nm) is about 1.8.
  • the solubilizing ability of different concentrations of surfactants on bismuth is different. After exceeding the critical micelle concentration (CMC), the solution is solubilized.
  • the ability has a significant point of mutation; the curve is plotted as a function of surfactant concentration as a function of I1/I3, and the midpoint of the curve mutation is the CMC of the substrate to be detected. Therefore, the CMC of the solution can be determined by measuring the fluorescence spectrum of ruthenium in different concentrations of DP7-C solution.
  • CMC detection method DP7-C was weighed and dissolved in MilliQ water in a 37 ° C water bath to obtain a DP7-C mother liquor having a concentration of 1.5 mg/mL mL. Prepare a 6 ⁇ 10 -5 mol/L hydrazine solution with methanol as solvent.
  • the excitation wavelength of the fluorescence scanning was 334 nm
  • the emission wavelength was 373 nm and 384 nm
  • the excitation slit was set to 8.0 nm
  • the emission slit was set to 2.5 nm
  • the scanning speed was 1200 nm. According to the fluorescence spectrum, the critical micelle concentration was calculated.
  • Fig. 3A is a schematic view showing the self-assembly of DP7-C to form micelles in an aqueous solution, and the micelles of the micelles have a CMC measurement value of 3.47 ⁇ g/mL (see Fig. 3B for the results).
  • Atomic force microscopy Configure different concentrations of DP7-C solution, add to the mica plate, dry naturally, and place the DP7-C coated mica plate on an atomic force microscope.
  • Particle size and zeta potential detection DP7-C was dissolved in MilliQ water, and the particle size and zeta potential were measured by a Malvern particle size analyzer. Each sample was tested 4 times and averaged.
  • DP7-C appearance and solution state Observe the appearance of MilliQ water, DP7-C aqueous solution and lyophilized powder, and take a picture with a camera.
  • a MilliQ water
  • b DP7-C dissolved in an aqueous solution
  • c is a state after reconstitution of DP7-C lyophilized powder
  • d is a form and color of DP7-C lyophilized powder.
  • DP7-C micelles The shape of DP7-C micelles is spherical or ellipsoidal (Fig. 4C), the particle size of DP7-C micelles is about 24.3 ⁇ 1.5 nm (Fig. 4A), and the zeta potential is about 28.8 ⁇ 0.27 mV. 4B).
  • DP7-C lyophilized powder is white fluffy, soluble in water, and the solution is colorless and transparent.
  • the red blood cells were slowly mixed and washed with PBS, centrifuged at 400 ⁇ g for 10 minutes, the supernatant was removed, and the cell pellet was washed with PBS and repeated 3 times.
  • Figure 5B visually shows the hemolysis of red blood cells under different conditions, in which the negative control group (PBS) (group a) does not undergo hemolysis, the red blood cells settle in the bottom of the bottle, the positive control group (2% Tween-20) (group b) and The DP7 solution (group c) lysed red blood cells to present a clear red solution, while the DP7-C micelles (group d) did not lyse red blood cells, but due to its micelle characteristics, red blood cells were uniformly suspended in the solution.
  • PBS negative control group
  • group a the positive control group
  • group 2% Tween-20 group 2% Tween-20
  • the anti-infective activity of DP7-C was determined by intraperitoneal infection model.
  • the experimental groups are as follows:
  • NS group 100 ⁇ L saline/mouse
  • DP7-C group 0.3 mg/kg
  • Vancomycin (VANC) positive control group 10 mg/kg.
  • mice (2) Establish a model of abdominal infection in mice. Each mouse was intraperitoneally injected with 1.5 ⁇ 10 8 cfu/0.mL 5 mL.
  • mice were intraperitoneally injected with 5 mL of normal saline, gently abdomen, and sacrificed. Disinfect with 75% alcohol, cut the abdominal epithelium after 5 minutes, open a small mouth in the abdominal cavity, and draw as much ascites from the 1 mL syringe. Then transfer to a sterile EP tube and mix.
  • the results of the DP7-C treatment of mouse peritoneal infection model are shown in Figure 6C.
  • the results show that the average colony of Staphylococcus aureus in the DP7-C group (1 mg/kg) and the positive drug group (20 mg/kg vancomycin) after intravenous administration.
  • the formation units (CFU) were significantly lower than the NS group (p ⁇ 0.01).
  • the antibacterial effects of the DP7-C group and the positive drug group were basically the same, and there was no statistical difference. It is indicated that DP7-C has a good antibacterial activity in a systemic infection model in mice.
  • the male and female AB wild-type zebrafish were paired according to the paired breeding program. After the eggs were laid, the eggs were collected, and then the eggs were cultured in seawater containing PTU, and cultured in a thermostat at 28 ° C, and the PTU was changed once a day.
  • seawater zebrafish were intraperitoneally injected with DP7-C and saline diluted fluorescent Pseudomonas aeruginosa (PAO1-GFP) 48 hours after incubation. The cells were observed under a fluorescence microscope at 3, 8 and 18 h to observe PAO1-GFP in zebrafish. Abdominal growth.
  • Fig. 6 The results are shown in Fig. 6 (A, B).
  • the growth rate of PAO1-GFP in the peritoneal cavity of zebrafish was positively correlated with the total intensity of green fluorescent fluorescence.
  • the growth rate of PAO1-GFP in DP7-C group was much smaller than that of NS, indicating that the concentration of DP7 was low.
  • -C micelles (1 mg/mL) have excellent antibacterial activity in zebrafish.
  • DP7-C In the detection of DP7-C antibacterial activity, DP7-C was found to have low in vitro activity, but it has high in vivo antibacterial activity, which may be due to DP7-C regulating the body's immune system to achieve antibacterial effect.
  • mice were randomly divided into two groups (NS group and DP7-C group), 5 in each group.
  • Detected immune cells and The surface markers are as follows:
  • DP7-C group 200 ⁇ l of 1 mg/mL DP7-C was administered to each mouse, and 200 ⁇ l of normal saline was injected into each mouse of the NS group. After 24 hours, the mice were sacrificed, and each mouse was intraperitoneally injected with 5 mL of physiological saline, and the abdominal cavity was gently. Then, the ascites in the abdominal cavity was aspirated, and the cell concentration and the total number of cells in the ascites were calculated. Flow cytometry was used to detect the typing of lymphocytes in ascites, and the proportion of macrophages, neutrophils and inflammatory monocytes in total cells was analyzed. The results of flow cytometry and total number of cells in ascites were calculated. The number of macrophages, neutrophils, and inflammatory monocytes.
  • Fig. 7C The results are shown in Fig. 7C.
  • the total number of cells in the peritoneal cavity increased significantly, including the number of macrophages, neutrophils and monocytes, especially mononuclear cells.
  • the proportion increased from 2.7% to 32.8% (Fig. 7A) and the proportion of macrophages increased from 38.8% to 50.8% (Fig. 7B).
  • mice PBMC Changes in cytokines after stimulation with DP7-C were measured using mouse PBMC.
  • the isolated mouse PBMC was diluted to 1 ⁇ 10 6 cells/mL, and added to a 6-well plate at 2 mL/well.
  • the stimulation concentration of DP7-C was 200 ⁇ g/mL, and the stimulation time was 4 h. After the stimulation was over, the cells were collected at - Save in 80 refrigerators.
  • PBMC was diluted to 1 ⁇ 10 6 cells/mL and added to a 6-well plate at 2 mL/well, DP7-C The stimulation concentration was 200 ⁇ g/mL, and after 1 h, it was further stimulated with LPS for 4 h, and the collected cells were stored in a -80 refrigerator. After all samples have been collected, centrifuge, wash, extract total RNA, reverse transcription, and perform real-time quantitative PCR.
  • PBMC-associated cytokines stimulated by DP7-C is shown in Figure 7D.
  • the expression of IL-1 ⁇ , IL-6, MCP-1, M-CSF and TNF- ⁇ and immune activation-related cytokines are greatly improved, and DP7-
  • C and LPS co-stimulate PBMC, it was found that the expression of major cytokines related to cytokine storm, such as IL-1 ⁇ , MCP-1 and TNF- ⁇ , was significantly decreased, indicating that DP7-C can reduce the related infectious diseases such as sepsis. degree of damage.
  • DP7-C was found to significantly up-regulate the expression of mouse PBMC immune-associated cytokines in previous experiments, we predicted that DP7-C combined with CpG ODNs could be used as a novel immunoadjuvant, and DP7-C could form spontaneously.
  • Micelles have inspired the inventors to replace the use of aluminum adjuvants.
  • OVA is a model antigen study for the immunomodulatory effects of DP7-C/CpG complex adjuvants, as well as anti-tumor effects in mouse prophylactic and therapeutic tumor models.
  • mice C57BL/6J female mice were randomly divided into 4 groups, 10 in each group, as follows:
  • NS group 100 ⁇ L saline
  • CpG group 10 ⁇ g OVA+20 ⁇ g CpG
  • DP7-C group 10 ⁇ g OVA+40 ⁇ g DP7-C
  • CpG/DP7-C group 10 ⁇ g OVA+40 ⁇ g DP7-C+20 ⁇ g CpG
  • Prophylactic immune tumor model multiple subcutaneous immunizations were performed at 0, 2, and 4 weeks, and total antibody titers before tumor inoculation at week 5 were examined. At the 5th week, each mouse was subcutaneously inoculated with tumor cells EG7-OVA: 2 ⁇ 10 6 , and the tumor was measured after the tumor grew. Every 3 days, the tumor volume calculation formula was 0.52 ⁇ length ⁇ width 2 .
  • Therapeutic immune tumor model On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 6 EG7-OVA tumor cells on the back, and immunization was started on the 5th day, once a week, and serially immunized 3 times, every 3 days after tumor growth. The tumor was measured once a day while observing the survival period.
  • the mechanism by which an adjuvant activates an immune response may be to promote maturation of DC cells and increase their uptake and processing of antigens.
  • DCs dendritic cells
  • C57/BL mouse primary bone marrow cells were isolated and cultured on day 5 with an induction medium containing 10 ng/ml GM-CSF and 10 ng/ml IL-4.
  • DC cells are the most powerful antigen-presenting cells in the body. They can take up foreign substances, process them, and present them to T cells to stimulate immune response. DC cells play an important role in T cell immune responses and T cell-dependent antibody production. We used flow cytometry to examine the effect of DP7-C on DC-presenting antigens.
  • C57/BL mouse primary bone marrow cells were isolated and cultured on day 5 with an induction medium containing 10 ng/ml GM-CSF and 10 ng/ml IL-4.
  • OVA protein-labeled FITC, OVA or DP7-C/OVA was added to DC cell culture and mixed for 3 h.
  • DC cells have a lower uptake rate of OVA antigen alone.
  • 40 ⁇ g/ml DP7 slightly enhances OVA uptake by DC cells to a certain extent, while 2.5 ⁇ g/ml DP7-C can significantly increase DC cells to antigen.
  • Example 9 DP7-C stimulates innate immune response
  • Innate immunity plays an important role in anti-tumor immunity.
  • mice were sacrificed by cervical dislocation after 48 h of initial immunization, and the mouse spleen lymphocytes were isolated as effector cells by mouse lymphocyte separation.
  • YAC-1 cells were cultured in the resuscitation, and passaged 1 day before the experiment, maintaining 2 ⁇ 10 6 /20 ml. Dyeing with trypan blue. Available when the activity is >95%.
  • the supernatant was centrifuged at 2250 g to perform LDH release detection.
  • the results are shown in Fig. 11.
  • the NS group had almost no NK cell activity, CpG and DP7-C showed slightly stronger NK cell killing activity, and the CpG/DP7-C complex could effectively enhance the activity of NK cells, and the difference was significant.
  • Example 10 DP7-C stimulates cellular immune response
  • DP7-C and CpG can stimulate the specific immune response of the antigen, and can effectively inhibit tumor growth and metastasis, and prolong the survival of the mouse. Moreover, DP7-C and CpG can significantly up-regulate the specific antibody titer of OVA antigen and enhance the humoral immune response, so we further detect the cellular immune response activated by DP7-C.
  • mouse spleen lymphocytes were isolated, counted and diluted to 5 x 10 6 /ml.
  • a 6-well plate was plated at 25 ⁇ 10 6 /well, and 10 ⁇ g/ml OVA whole protein was added to each well for stimulation for 1 h at 37 ° C.
  • the negative control was treated with DMSO and the positive control was treated with 5 ⁇ g/ml conA.
  • Example 11 CpG/DP7-C adjuvant stimulates highly potent anti-tumor immunity
  • mice in each group were divided into 5 groups: NS, CpG, DP7-C and CpG/DP7-C groups, 10 mice in each group, and the doses of each mouse were as follows:
  • 1NS group 100 ⁇ l PBS
  • 2CpG group 5 ⁇ g NY-ESO-1 protein + 20 ⁇ g CpG;
  • 3DP7-C group 5 ⁇ g NY-ESO-1 protein + 40 ⁇ g DP7-C;
  • 4CpG/DP7-C group 5 ⁇ g NY-ESO-1 protein + 20 ⁇ g CpG + 40 ⁇ g DP7-C;
  • the total dose volume per mouse was 100 ⁇ l, and less than 100 ⁇ l was added to 100 ⁇ l of sterile PBS.
  • Prophylactic immune tumor model multiple subcutaneous immunizations were performed at 0, 2, and 4 weeks, and total antibody titers before tumor inoculation at week 5 were detected.
  • each mouse was subcutaneously inoculated with tumor cells NY-ESO-1 + B16: 2 ⁇ 10 5 , and the tumor was measured after the tumor grew. Every 3 days, the tumor volume was calculated to be 0.52 ⁇ length ⁇ width. 2 ;
  • Therapeutic immune tumor model On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 5 NY-ESO-1 + B16 tumor cells on the back, and immunization was started on the 5th day, once a week, 3 times consecutively, to be tumor Tumors were measured every 3 days after growth, and survival was observed.
  • the DP7-C micelles formed by the self-assembly are positively charged in the aqueous solution, showing As a non-viral gene transmission, especially the potential of siRNA delivery vectors.
  • the morphology of the micelles formed by DP7-C by self-assembly and the DP7-C/siRNA complex were observed by transmission electron microscopy (H-6009IV, Hitachi, Japan). Before the observation, 1 mg ⁇ mL of the micelle solution was diluted with distilled water and coated on a copper grid, covered with nitrocellulose, then negatively stained with tungstophosphoric acid, dried at room temperature, and observed under an electron microscope.
  • the DP7-C micelles were spherical and uniform in distribution. Under the electron microscope, the particle size of DP7-C micelle is about 60 nm, and the particle size after compounding with siRNA is about 100 nm (see Figure 14 for the results).
  • C26 mouse colon cancer cells or B16 mouse melanoma cells were plated in a 6-well plate at a density of 5 ⁇ 10 4 cells per well, and 2 mL of DMEM medium supplemented with 20% FBS was added to each well.
  • FAM-modified nonsense siRNA FAM-modified nonsense siRNA (FAM-siRNA) was used as a reporter gene for detecting transfection efficiency, and PEI25K and Lipofectamin2000 were used as positive controls.
  • FAM-siRNA FAM-modified nonsense siRNA
  • PEI25K and Lipofectamin2000 were used as positive controls.
  • the medium was first replaced with 1 mL of serum-free DMEM medium. Subsequently, gene transfection complexes mixed in different ratios were added to each well, and each complex contained 1 ⁇ g of FAM-siRNA.
  • the ratios of siRNA/DP7-C, siRNA/PEI25K, and siRNA/Lipofectamin2000 were 1:5, 1:2, and 1:2, respectively.
  • the medium was replaced with DMEM medium supplemented with 20% FBS.
  • the transfection was observed using a microscope and photographed. All cells including the floating and adherent wells were collected, washed twice with pre-cooled PBS, and the total fluorescence intensity of each group was counted using a flow cytometer (Epics Elite ESP, USA).
  • the transfection results are shown in Figure 15.
  • the results of the assay showed that DP7-C was able to transfect FAM-modified fluorescent siRNA into Ct26 (Fig. 15c) and B16 (Fig. 15b) cells with transfection efficiencies of 42.4 ⁇ 3.5, respectively. % and 53.3 ⁇ 4.0%; and the corresponding transfection efficiencies of PEI complex were 19.0 ⁇ 1.8% and 72.0 ⁇ 4.1%, respectively; the corresponding transfection efficiencies of Lipofectamin2000 complex were 29.5 ⁇ 3.2% and 25.6 ⁇ 4.2%, respectively.
  • Our results show that DP7-C micelles have higher siRNA transfection efficiency relative to PEI25K and Lipofectamin2000.
  • cytotoxicity of cations is one of the important factors that restrict the application of siRNA transfection.
  • 293T cells were plated in a 96-well plate at a density of 3 ⁇ 10 3 cells per well, and 100 ⁇ L of DMEM/20% FBS medium was added to each well, and culture was carried out for 24 hours.
  • Three groups of DP7-C, PEI25K and Lipofectamin2000 were set up in the experiment, and each group was set to correspond to 10 concentration gradients: 0, 12.5, 18.75, 25, 37, 50, 75, 100, 150, 200 ⁇ g/mL.
  • the survival of the cells was measured by the CCK-8 method, and 10 ⁇ L of CCK-8 solution was added to each well 24 hours after the administration, and the cells were incubated at 37 ° C for 2 hours.
  • the absorbance per well was measured at 630 nm and 450 nm using a microplate reader, a standard curve was plotted and the IC 50 was calculated. The results were averaged over 6 sets of parallel experiments.
  • Example 15 DP7-C transmission of VEGF siRNA for C26 mouse colon cancer peritoneal metastasis model
  • mice were randomly divided into 4 groups, 8 in each group and labeled.
  • mice were intraperitoneally injected with 10 doses of normal saline (blank control group), blank DP7-C micelles (17.5 ⁇ g), and DP7-C/sense control siRNA (Scramble siRNA) complex (17.5 ⁇ g). /3.5 ⁇ g), or DP7-C/VEGF siRNA complex (17.5 ⁇ g/3.5 ⁇ g).
  • mice in the control group were already very weak, all the mice were sacrificed by the cervical dislocation method, and the intra-abdominal tumor tissues and the heart, liver, spleen, lung and kidney tissues were immediately collected, weighed and analysis. The ascites volume of each group of mice was also collected and measured.
  • CD31 immunohistochemical staining analysis was performed on the tumor tissues of each experimental group; HE staining analysis was performed on the collected heart, liver, spleen, lung and kidney tissues.
  • Figure 17 shows the effect of intraperitoneal injection of the DP7-C/VEGF siRNA complex for the intraperitoneal metastasis C26 tumor model.
  • Figure 17a is a photograph of the abdominal cavity of a representative mouse in each group of animals. Among them, the average tumor weight of the DP7-C/VEGF siRNA complex treatment group was 1.07 ⁇ 0.5g, the weight of the saline control group was 8.82 ⁇ 0.63g, and the weight of the empty DP7-C group was 7.94 ⁇ 0.53g, DP7-C/ The nonsense control siRNA complex was 5.3 ⁇ 0.72 g (Fig. 17c). The number of abdominal metastatic tumor nodules in mice treated with the DP7-C/VEGF siRNA complex was significantly less than in the other treatment groups. Therefore, tumor growth in mice treated with the DP7-C/VEGF siRNA complex was greatly inhibited.
  • CD31 immunohistochemical staining showed that the number of neovascularizations in the tumor tissue of the DP7-C/VEGF siRNA complex treatment group was lower than that of the other three experimental groups, confirming that DP7-C transmits siRNA against VEGF. Tumor tissue blood vessel formation was more effectively inhibited (Fig. 17d).
  • the results of HE staining showed (Fig. 17e) that the DP7-C micelles administered through the abdominal cavity did not produce significant pathological changes in the main organ tissues, confirming that the side effects of DP7-C were small.
  • Example 16 DP7-C transmission of VEGF siRNA for treatment of C26 mouse colon cancer subcutaneous xenograft model
  • mice were randomly divided into 4 groups, 8 rats in each group, and labeled.
  • mice were injected intratumorally with 10 doses of normal saline (blank control group), blank DP7-C micelles (25 ⁇ g), DP7-C/negless control siRNA complex (25 ⁇ g/5 ⁇ g), or DP7- C/VEGF siRNA complex (25 ⁇ g/5 ⁇ g).
  • mice in the control group were already very weak, all the mice were sacrificed by the cervical dislocation method, and the intra-abdominal tumor tissues and the heart, liver, spleen, lung and kidney tissues were immediately collected, weighed and analysis. The ascites volume of each group of mice was also collected and measured.
  • CD31 immunohistochemical staining analysis was performed on the tumor tissues of each experimental group; HE staining analysis was performed on the collected heart, liver, spleen, lung and kidney tissues.
  • Figure 18 shows the effect of intratumoral injection of the DP7-C/VEGF siRNA complex on the C26 subcutaneously transplanted tumor model.
  • Figure 18a shows photographs of subcutaneous tumors in mice in each group of animals. Among them, the average tumor volume of the DP7-C/VEGF siRNA complex treatment group was 261.4 ⁇ 115.51 mm 3 , the weight of the saline control group was 577.21 ⁇ 107.46 mm 3 , and the weight of the empty DP7-C group was 357.64 ⁇ 30.56 mm 3 , DP7. The -C/sense control siRNA complex was 474.43 ⁇ 120.67 mm 3 . The subcutaneous tumor volume of mice treated with the DP7-C/VEGF siRNA complex was significantly smaller relative to the other treatment groups (Fig. 18b). Therefore, tumor growth in mice treated with the DP7-C/VEGF siRNA complex was greatly inhibited.
  • Example 17 DP7-C transmission of VEGF siRNA for treatment of B16 mouse melanoma lung metastasis model
  • mice were randomly divided into 4 groups, 8 in each group and labeled.
  • mice were injected with 7 doses of normal saline (blank control group), blank DP7-C micelles (60 ⁇ g), DP7-C/sense control siRNA complex (60 ⁇ g/12 ⁇ g), or DP7-, respectively.
  • C/VEGF siRNA complex 60 ⁇ g/12 ⁇ g).
  • mice in the control group were already very weak, all the mice were sacrificed by the cervical dislocation method, and the lung tumor tissues and heart, liver, spleen, lung and kidney tissues were immediately collected, weighed and Analysis; HE staining analysis of collected heart, liver, spleen, lung and kidney tissues.
  • Figure 19 shows the effect of intravenous injection of the DP7-C/VEGF siRNA complex on a B16 lung metastasis tumor model.
  • Figure 19a shows photographs of mouse lung tumors in each group of animals. Among them, the average tumor nodules in the DP7-C/VEGF siRNA complex treatment group were 30 ⁇ 12, the physiological saline control group was 172 ⁇ 16, and the empty DP7-C group was 132 ⁇ 22, DP7-C/ The nonsense control siRNA complex was 106 ⁇ 15.
  • the average lung tissue weight of the DP7-C/VEGF siRNA complex treatment group was 0.22 ⁇ 0.02 g
  • the weight of the saline control group was 0.52 ⁇ 0.18 g
  • the weight of the empty DP7-C group was 0.41 ⁇ 0.1 g, DP7-C.
  • the nonsense control siRNA complex was 0.42 ⁇ 0.17 g.
  • the number of lung metastatic tumor nodules in mice treated with the DP7-C/VEGF siRNA complex was significantly less than in the other treatment groups (Fig. 19a), and the mean lung weight of the mice was significantly lighter than in the other treatment groups (Fig. 19). 19b). Therefore, tumor growth in mice treated with the DP7-C/VEGF siRNA complex was greatly inhibited.

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Abstract

一种疏水化修饰的抗菌肽及其用途。所述的疏水化修饰为在抗菌肽的氮末端偶联疏水片段。还提供了上述疏水化修饰的抗菌肽制备得到的胶束、上述疏水化修饰的抗菌肽和上述胶束在制备抗菌药物、制备核酸转运载体、制备免疫佐剂等方面的用途。所述的抗菌肽分子量小，可以方便的使用Fmoc固相多肽合成法合成，与疏水片段经化学合成偶联方法简单易行。

Description

抗菌肽衍生物及其用途 技术领域
本发明属于生物医药领域，主要涉及抗菌肽衍生物及其用途，尤其是疏水化修饰的抗菌肽DP7衍生物及其用途。
背景技术
抗菌肽，又称为宿主防御肽(Host Defense Peptides,HDPs)，一般由l2到100个氨基酸残基组成，是一类碱性多肽，绝大部分的抗菌肽带正电荷。抗菌肽具有广谱的抗菌作用，能有效地抑制杀伤真菌、病毒、寄生虫等病原体，并能选择性的杀伤肿瘤细胞，不易产生耐药性，构成宿主防御病原微生物入侵的第一道屏障，是机体免疫系统的重要成分，抗菌肽已成为替代抗生素预防和控制疾病的潜在药物，且具有广阔的开发前景。尽管抗菌肽有巨大的应用潜力，但只有极少的抗菌肽在临床上广泛使用，主要原因是它的稳定性和毒性。一些研究表明，抗菌肽纳米化可以使其获得较好稳定性及减少毒性，这为抗菌肽的应用提供一个新研究方向。
抗菌肽或HDPs的免疫调节活性受到越来越多的关注，包括通过改变信号通路诱导细胞因子和趋化因子的产生、直接或间接招募效应细胞包括吞噬细胞、增强细胞内和细胞外细菌杀伤、促进树突状细胞成熟和巨噬细胞分化以及介导伤口修复、凋亡。多种抗菌肽因其良好的免疫调节作用而展现出了佐剂活性，主要方式为激活天然免疫反应和介导获得性免疫反应。
抗菌肽DP7是用计算机辅助设计新型抗菌肽的筛选方法，通过对模板抗菌肽HH2替换了2个氨基酸得到的具有更高的细菌识别特异性与更强的抗菌活性的抗菌肽(HH2的专利号：WO08022444)。经过研究表明，相较于HH2，抗菌肽DP7具有更好的抗菌活性、更低的红细胞溶血毒性及更强的免疫调节活性。在体外抗菌活性研究中，抗菌肽DP7具有很明显的细菌细胞壁的破坏作用和细胞膜的扰乱作用，从而实现抗菌的功能。在体内抗菌活性研究中，从致病性金黄色葡萄球菌感染小鼠腹腔模型研究发现，通过诱导免疫细胞清除细菌的作用，DP7表现出了非常好的治疗效果。但是高浓度DP7经静脉注射方法给药后，会导致红细胞发生溶血现象和小鼠死亡的现象出现，说明高浓度的DP7对红细胞有毒性，会破坏红细胞。DP7是带正电荷的亲水性抗菌肽，我们将其用疏水片段进行修饰可得到具有自组装成纳米粒子的两亲化合物，可大大降低静脉给药的毒性，保持其体内抗菌作用、免疫调节作用，并能作为小核酸药物的传递系统，在药物领 域具有较广的用途。
发明内容
本发明的目的是提供一种新型的修饰的抗菌肽，以期为本领域的抗感染治疗、新型免疫佐剂的制备及siRNA运载载体制备提供一种新的有效选择。
为了解决上述技术问题，本发明采取的技术方案是提供一种疏水化修饰的抗菌肽，所述的疏水化修饰为在抗菌肽的氮末端偶联疏水片段(或叫氮末端偶联疏水化合物)。所述的抗菌肽DP7的氨基酸序列为VQWRIRVAVIRK(SEQ ID No.1)。为提高稳定，DP7的碳末端一般会进行酰胺化修饰，此时其结构为VQWRIRVAVIRK-NH2。
上述疏水化修饰的抗菌肽中，所述的疏水化合物(疏水片段)为甾醇类化合物或饱和直链脂肪酸。优选的，所述甾醇类化合物为胆固醇类化合物或胆酸类化合物。
上述疏水化修饰的抗菌肽中，所述的甾醇类化合物为丁二酰化胆固醇、胆酸或去氧胆酸。
上述疏水化修饰的抗菌肽中，所述的长链脂肪酸为C6到C20的脂肪酸。优选的，所述饱和直链脂肪酸为C8～C18中的至少一种。
上述疏水化修饰的抗菌肽中，所述的长链脂肪酸为硬脂酸(C18)、软脂酸(C16)、月桂酸(C12)或正辛酸(C8)中的至少一种。
上述疏水化修饰的抗菌肽中，所述的抗菌肽的氮端与疏水片段(疏水化合物)偶联的方式为通过疏水片段(疏水化合物)上的-CO-OH与抗菌肽上的-NH2酰胺化反应生成。
上述疏水化修饰的抗菌肽中，所述疏水化修饰的抗菌肽的结构为：
Figure PCTCN2017091390-appb-000001
其中，所述的R为胆固醇类化合物、胆酸、长链脂肪酸。
上述疏水化修饰的抗菌肽中，所述的R为
Figure PCTCN2017091390-appb-000002
Figure PCTCN2017091390-appb-000003
本发明还提供了由上述疏水化修饰的抗菌肽制成的胶束。
上述胶束中，所述的胶束为的疏水化修饰的抗菌肽在溶液中自组装而成。
上述胶束中，所述的胶束经过了冷冻干燥处理。
上述胶束中，所述的胶束里还装载有核酸、小分子药物、多肽或者蛋白中的至少一种。
本发明还提供了上述疏水化修饰的抗菌肽的方法，包括以下步骤：
A、准备多肽VQWRIRVAVIRK-NH2；
B、在多肽VQWRIRVAVIRK-NH2的氮端偶联疏水片段。
本发明还提供了上述疏水化修饰的抗菌肽或者上述胶束在制备抗菌药物中的用途。
上述在制备抗菌药物中的用途中，所述的抗菌为抗细菌或抗真菌。
上述在制备抗菌药物中的用途中，所述的细菌为金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia colibacillus)、鲍曼不动杆菌(Acinetobacter baumanmii)、绿脓杆菌(Pseudomonas aeruginosa)或伤寒杆菌(Salmonella typhi)中的至少一种。
上述在制备抗菌药物中的用途中，所述的真菌为白色念珠菌(Canidia Albicans)或近平滑念珠菌(Candida parapsilosis)中的至少一种。
本发明还提供了一种抗菌药物，是由上述疏水化修饰的抗菌肽或者上述胶束作为主要活性成分制备而成。
进一步的，上述抗菌药物中还含有其他抗菌药物。
更进一步的，上述抗菌药物中所述的其他抗菌药物为抗生素。
其中，上述抗菌药物中所述的抗生素为糖肽类抗生素、氨基苷类抗生素、大环内脂类抗生素、β-内酰胺类抗生素中的至少一种。
其中，上述抗菌药物中所述的β-内酰胺类抗生素为青霉素类抗生素或头孢菌素类抗生素中的至少一种。
进一步的，所述的青霉素类抗生素为青霉素G、青霉素V、氟氯西林、苯唑青霉素、 氨苄西林、羧苄西林、匹氨西林、磺苄西林、替卡西林、哌拉西林或阿莫西林中的至少一种。所述的头孢菌素类抗生素为：头孢羟氨苄、头孢氨苄、头孢唑啉、头孢拉啶、头孢丙烯，头孢呋辛脂、头孢克洛、头孢孟多、头孢噻肟、头孢曲松、头孢克肟、头孢地尼、头孢皮罗、头孢吡肟或头孢唑南中的至少一种。
其中，上述抗菌药物中所述的氨基苷类抗生素为链霉素、庆大霉素、卡那霉素、妥布霉素、丁胺卡那霉素、新霉素、西索米星、妥布霉素、阿米卡星、奈替米星、核糖霉素、小诺霉素或阿斯霉素中的至少一种。
其中，上述抗菌药物中所述的多肽类抗生素为万古霉素、去甲万古霉素、多粘菌素B或替考拉宁中的至少一种
其中，上述抗菌药物中所述的大环内脂类抗生素为红霉素、白霉素、无味红霉素、依托红霉素、乙酰螺旋霉素、麦迪霉素、交沙霉素或阿奇霉素中的至少一种。
进一步的，上述抗菌药物的其剂型为注射剂。
本发明还提供了上述疏水化修饰的抗菌肽或者上述胶束在制备免疫佐剂中的用途。
本发明还提供了一种免疫佐剂，是由上述疏水化修饰的抗菌肽或者上述胶束作为免疫佐剂与抗原制备而成。
上述免疫佐剂中，还含有单链脱氧寡核苷酸(CpG ODNs)。进一步的，所述的疏水化修饰的抗菌肽与CpG ODNs之间的配比为：1:0.5～1:5。
本发明还提供了上述疏水化修饰的抗菌肽或者上述胶束在制备核酸转运载体中的用途。
上述在制备核酸转运载体中的用途中，所述的核酸为RNA。
上述在制备核酸转运载体中的用途中，所述的核酸为信使RNA(message RNA,mRNA)、用于RNA干扰的siRNA(Small interfering RNA)或者为用于基因组编辑的sgRNA(small guide RNA)。
本发明还提供了核酸转运载体，由上述的疏水化修饰的抗菌肽或者上述述的胶束装载核酸而得。
上述核酸转运载体中，所述核酸为RNA。
上述核酸转运载体中，中所述的核酸为信使RNA(message RNA,mRNA)、用于RNA干扰的siRNA(Small interfering RNA)或者为用于基因组编辑的sgRNA(small guide RNA)。
上述核酸转运载体中，所述的疏水化修饰的抗菌肽与核酸的质量比为1:1～20:1。
本发明也提供了制备上述核酸转运载体的方法。该方法包括以下步骤：
a、称取适量权利要求1～9任一项所述的疏水化修饰的抗菌肽，加入溶液溶解，自发形成胶束；或取权利要求9～12任一项所述的胶束形成溶液；
b、将核酸加入胶束溶液，室温孵育，即得。
上述的抗菌肽DP7与疏水片段偶联产物可以以冻干粉形式保存，使用时直接用无菌水或者生理盐水溶解即可。
本发明的有益效果在于：本发明涉及的疏水化修饰的抗菌肽DP7中的抗菌肽DP7分子量小，可以方便的使用Fmoc固相多肽合成法合成，与疏水片段经化学合成偶联方法简单易行。本发明的疏水化DP7能自组装成胶束，有较好的单分散性和Zeta电位，冻干再溶解也很稳定；疏水化DP7的胶束能显著降低抗菌肽DP7对红细胞的裂解等毒性，实现了静脉系统给药；虽然疏水化DP7在体外抗菌活性很低，但在在斑马鱼及小鼠体内具有很好的抗菌活性，在体内抗菌不是通过直接杀菌，而是通过募集巨噬细胞、单核细胞、中性粒细胞等淋巴细胞以及调控一些免疫细胞因子的表达，从而实现对机体免疫保护作用。同时，疏水化DP7也可作为免疫佐剂，诱导机体产生针对目的抗原的较高的免疫应答。此外，本发明的疏水化DP7阳离子胶束，能够高效地复合siRNA并导入到结肠癌和黑色素瘤等肿瘤细胞中，通过腹腔注射、瘤内注射和尾静脉注射的方式实现对肿瘤组织生长的抑制作用，并展示出较高的安全性。
本发明涉及到的英文缩写如下：
(1)万古霉素：VAN
本发明所涉及到的菌株：
(1)金黄色葡萄球菌菌株：ATCC 33591、ATCC 25923
(2)大肠杆菌菌株：ATCC 25922
(3)绿脓杆菌菌株:ATCC 10145、ATCC 10145GFP
附图说明
图1：抗菌肽DP7与疏水片段偶联物的合成路线图。
图2：DP7疏水片段修饰后的质谱图。A为胆固醇修饰后DP7-C的质谱图；B为硬脂酸修饰后DP7-SA的质谱图。
图3：DP7-C胶束的自组装及临界胶束浓度测定：A为DP7-C胶束自组装示意图；B为DP7-C胶束的临界胶束浓度的测定。
图4：DP7-C胶束的物理特征：A为粒径大小；B为Zeta电位；C为原子力显微镜图谱；D为外观形态。
图5：DP7和DP7-C胶束体外溶血活性比较：A为血红蛋白测定曲线图；B为溶血实验外观图。
图6：DP7-C的体内抑菌活性：A为绿脓杆菌斑马鱼腹腔感染模型大体及荧光图；B为荧光灰度积分示意图；C为金黄色葡萄球菌小鼠腹腔感染模型菌落数示意图。
图7：DP7-C的体内抑菌的机制研究：A为DP7-C刺激后腹腔中单核细胞、中性粒细胞检测的流式图谱；B为DP7-C刺激后腹腔中巨噬细胞检测的流式图谱；C为DP7-C刺激后腹腔中总细胞、巨噬细胞、中性粒细胞及单核细胞数量比较示意图；D为DP7-C刺激PBMC后Q-PCR检测免疫相关细胞因子的表达情况比较；E为DP7-C联合LPS刺激PBMC后Q-PCR检测免疫相关细胞因子的表达情况比较。
图8：DP7-C/CpG复合物的免疫效果检测：A为第5周不同免疫组的抗OVA抗体滴度示意图；B为预防性模型中，各组小鼠肿瘤的生长曲线；C为预防性模型中，各组小鼠的生存期变化；D为治疗性模型中，各组小鼠肿瘤的生长曲线。
图9：DP7-C促进树突状细胞的成熟的试验。
图10：DP7-C促进DC对OVA抗原的摄取的试验。
图11：CpG/DP7-C复合物激发固有免疫反应的试验。
图12：CpG/DP7-C复合物激发细胞免疫反应的试验。
图13：CpG/DP7-C佐剂激发高效的抗肿瘤免疫的试验。
图14：DP7-C胶束/siRNA复合物的电镜形态图：A为DP7-C胶束；B为DP7-C/siRNA复合物。
图15：DP7-C携带siRNA的体外转染效率检测：A为DP7-C与PEI25K和lipofectamine2000携带siRNA转染肿瘤细胞的效果比较图；B为流式检测B16细胞的转染效率示意图；C为流式检测C26细胞的转染效率示意图。
图16：DP7-C的细胞毒性测定(CCK-8法)。
图17：DP7-C传输VEGF siRNA治疗C26小鼠结肠癌腹腔转移瘤模型。
图18：DP7-C传输VEGF siRNA治疗C26小鼠结肠癌皮下移植瘤模型。
图19：DP7-C传输VEGF siRNA治疗B16小鼠黑色素瘤肺转移瘤模型。
具体实施方式
DP7是一个带正电荷的亲水性抗菌肽，常用的是其碳末端进行酰胺化修饰的形式，在本发明实施例使用的DP7是QWRIRVAVIRK-NH2。疏水片段(或叫疏水化合物)如胆固醇、胆酸、长链脂肪酸等与亲水性多肽偶联，可能具有自组装形成纳米结构的能力。 本研究通过疏水片段偶联抗菌肽DP7将抗菌肽纳米化，从而增加其稳定性和减轻溶血毒性，可实现其静脉给药。由于其直接抗菌及免疫调节作用，疏水化DP7形成的纳米粒子具备抗感染作用。
抗菌肽DP7具有良好的免疫调节活性，能够介导天然免疫反应，此特性使其成为疫苗免疫佐剂，与抗原共同免疫能够增强宿主的细胞免疫并介导抗原特异性的免疫球蛋白的产生。经研究表明，疏水化的抗菌肽展现了良好的佐剂特性。在体外试验中，疏水化DP7可自组装成纳米粒子，通过静电作用吸附CpG可作为新型的免疫佐剂，可增强抗原呈递细胞对抗原的摄取，促进抗原呈递细胞的成熟以及细胞因子的表达。在动物肿瘤模型试验中，疏水化Dp7/CpG复合物延长了接种肿瘤小鼠的生存期，明显抑制了肿瘤的生长，增强了抗原特异性抗体的水平。
同时，由于DP7带阳电荷，使得所自组装形成的疏水化DP7胶束在水溶液中呈正电，展示出作为一种非病毒基因传输，特别是siRNA传输载体的潜力。结合针对肿瘤靶点的siRNA沉默作用，疏水化DP7将在基于siRNA的肿瘤治疗研究和应用中发挥重要作用。
目前，疏水片段偶联水溶性多肽形成纳米的研究很多，与抗菌肽偶联还未见报道，疏水片段偶联抗菌肽的抗菌及免疫调节作用及机制也有待于研究。同时，本研究还利用DP7-C偶联物所形成的阳离子胶束，成功实现对肿瘤细胞的高效siRNA导入，并有效抑制多种肿瘤模型的生长，得到一种新型的siRNA传输载体。而目前基于抗菌肽偶联物的基因传输载体也尚未见报道。
以下将结合附图和实施例详细说明本发明，应当说明的是，实施例是对本发明的技术方案的进一步说明，并不表明本发明仅限于这些实施例。
实施例1抗菌肽DP7与胆固醇偶联物(DP7-C)的合成
抗菌肽DP7与疏水片段偶联物是由图1所示合成路线进行合成的。其中，疏水片段包括：胆固醇、胆酸、软脂酸、硬脂酸、月桂酸等。
2-chlorotrityl chloride Resin，其名称为2-氯三苯甲基氯树脂；Fmoc-Rink Amide MBHA Resin，其名称是4-(2’，4’-二甲氧基苯基-芴甲氧羰基-氨甲基)-苯氧基乙酰氨基-甲基二苯甲胺树脂；Fmoc：芴甲氧羰基；pbf、tbu、Otbu、Trt、Boc都是保护基，名称分别是2，2，4，6，7-五甲基二氢苯并呋喃-5-磺酰基、叔丁基、叔丁氧基、三苯甲基、叔丁氧羰基。
具体的合成方法如下所述：
1.树脂的溶胀活化及脱保护：
溶胀：称取1.0g Rink MBHA(4-(2′,4′-二甲氧基苯基-芴甲氧羰基-氨甲基)-苯氧基乙酰氨基-甲基二苯甲胺)树脂(取代值0.36mmol/g)放入多肽合成仪反应瓶中，用DCM/DMF(1：1)进行溶胀活化,上下摇动反应瓶，使树脂充分溶胀，溶胀大约20min,去除溶剂。
脱保护：用20％Piperdine/DMF(N，N-二甲基甲酰胺)溶液15mL脱除树脂上Fmoc保护基,反应15min,用DCM/DMF(二氯甲烷/N，N-二甲基甲酰胺)交替洗涤树脂三次，取少量树脂用甲醇/DCM/DMF依次洗涤后加入到装有5％茚三酮的无水乙醇溶液的EP管中，沸水浴三分钟，树脂呈现蓝色，即阳性反应，则洗涤两次后继续进行下步反应，否则继续重复脱保护过程。
2.第一个氨基酸(Lys)的偶联
称取Fmoc-Lys(Boc)-OH(1.44mmol，0.93g,4eq)，用DMF(约5mL)溶解后加入反应瓶，随后再加入5mL HBTu(苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸酯)(1.44mmol,0.54g,4eq)、5mL DIEA(二异丙基乙胺)(2.88mmol,0.474mL,8eq)DMF溶液,最后再补加5mLDMF，开始反应，上下来回震荡反应瓶50min,而后排出反应液，取出少量树脂用CH3OH/DCM/DMF依次洗涤后加入到装有5％茚三酮的无水乙醇溶液的EP管中，沸水浴三分钟，树脂呈现黄色或浅蓝色，即阴性反应，则反应完成，将反应瓶中树脂用DCM/DMF交替洗涤三次后，抽去溶剂，进行后续反应，若树脂呈现深蓝色或红褐色则反应未完全，需要重新缩合。此步反应较易进行，一般缩合较彻底。该步骤中使用的原料是Fmoc-Rink Amide MBHA Resin.其是由MBHA Resin连接Fmoc保护修修饰的Rink Amide Linker构成。本发明使用的是取代度为0.36mmol/g，但其他取代度的也可以达到相同或类似的技术效果，也在本发明的保护范围内。本发明使用的取代度0.36mmol/g是从合成片段的产率、纯度、树脂的利用率等因素平衡的最佳取值。
3.氨基酸链的延长
将步骤2得到Fmoc-Lys(Boc)-MBHA Resin脱Fmoc后，加入N，N-二甲基甲酰胺、Fmoc-Arg(pbf)-OH、1-羟基苯丙三唑、苯并三氮唑-N，N，N’，N’-四甲基脲六氟磷酸酯、N，N’-二异丙基乙胺，在氮气保护下进行反应得到Fmoc-Arg(pbf)-Lys(Boc)-MBHA Resin；按照该方法再依次连接Fmoc-Ile-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Arg(pbf)-OH、Fmoc-Ile-OH、Fmoc-Arg(pbf)-OH、Fmoc-Trp(Boc)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Val-OH，
上述合成得到即为全保护的DP7序列肽。
4.乙酰化封闭
当缩合反应不理想时，为避免缺失肽对后续反应影响，一般要对游离氨基进行乙酰化封闭，将配好的封闭液(醋酐：吡啶：DMF 3:3:4)加入到装有树脂的反应瓶中，上下震荡反应瓶约20min,茚三酮检测，若树脂呈现黄色即反应完全，可进行后续反应，若未封闭完全，则需延长封闭时间，或调整封闭液的配比，以使反应尽量完全。
5.DP7的疏水化修饰
1)用丁二酰化胆固醇对DP7进行疏水化修饰
称取丁二酰化胆固醇0.67g(1.44mmol,4.0eq)，用DCM(约10mL)溶解后加入反应瓶，随后再加入随后再加入HBTu(1.44mmol,0.54g,4eq)、DIEA(2.88mmol,0.474mL,8eq)DMF溶液各5mL,上下来回震荡反应瓶50min,而后排出反应液，取出少量树脂用CH3OH/DCM/DMF依次洗涤后加入到装有5％茚三酮的无水乙醇溶液的EP管中，沸水浴三分钟，树脂呈现黄色或浅蓝色，即阴性反应，则反应完成，将反应瓶中树脂用DCM/DMF交替洗涤三次后，抽去溶剂。将上述得到的树脂加入到装有裂解液的反应瓶中，将反应瓶密封好，固定到摇床上进行反应，反应约2h后，过滤除去树脂，用DCM洗涤树脂几次后，收集滤液，旋转蒸发除去TFA和溶剂，将冰的无水乙醚加入到剩余液体中，有大量白色絮状沉淀出现，高速离心(4000r/min)的白色沉淀即为粗品。通过制备高效液相色谱法进一步对粗品进行纯化精制后可得到目标产物胆固醇化的DP7(DP7-C)，其质谱图见图2A，测定分子量与预期一致。
2)用胆酸对DP7进行疏水化修饰
称取胆酸0.59g(1.44mmol,4.0eq)，用DCM(约10mL)溶解后加入反应瓶，随后再加入随后再加入HBTu(1.44mmol,0.54g,4eq)、DIEA(2.88mmol,0.474mL,8eq)DMF溶液各5mL,上下来回震荡反应瓶50min,而后排出反应液，取出少量树脂用CH3OH/DCM/DMF依次洗涤后加入到装有5％茚三酮的无水乙醇溶液的EP管中，沸水浴三分钟，树脂呈现黄色或浅蓝色，即阴性反应，则反应完成，将反应瓶中树脂用DCM/DMF交替洗涤三次后，抽去溶剂。将上述得到的树脂加入到装有裂解液的反应瓶中，将反应瓶密封好，固定到摇床上进行反应，反应约2h后，过滤除去树脂，用DCM洗涤树脂几次后，收集滤液，旋转蒸发除去TFA和溶剂，将冰的无水乙醚加入到剩余液体中，有大量白色絮状沉淀出现，高速离心(4000r/min)的白色沉淀即为粗品。通过制备高效液相色谱法进一步对粗品进行纯化精制后可得到目标产物胆酸化的DP7 (DP7-CA)。
3)用硬脂酸对DP7进行疏水化修饰
称取硬脂酸0.41g(1.44mmol,4.0eq)，用DCM(约10mL)溶解后加入反应瓶，随后再加入随后再加入HBTu(1.44mmol,0.54g,4eq)、DIEA(2.88mmol,0.474mL,8eq)DMF溶液各5mL,上下来回震荡反应瓶50min,而后排出反应液，取出少量树脂用CH3OH/DCM/DMF依次洗涤后加入到装有5％茚三酮的无水乙醇溶液的EP管中，沸水浴三分钟，树脂呈现黄色或浅蓝色，即阴性反应，则反应完成，将反应瓶中树脂用DCM/DMF交替洗涤三次后，抽去溶剂。将上述得到的树脂加入到装有裂解液的反应瓶中，将反应瓶密封好，固定到摇床上进行反应，反应约2h后，过滤除去树脂，用DCM洗涤树脂几次后，收集滤液，旋转蒸发除去TFA和溶剂，将冰的无水乙醚加入到剩余液体中，有大量白色絮状沉淀出现，高速离心(4000r/min)的白色沉淀即为粗品。通过制备高效液相色谱法进一步对粗品进行纯化精制后可得到目标产物硬脂酸化的DP7(DP7-SA)，其质谱图见图2B，测定分子量与预期值一致。
4)用软脂酸对DP7进行疏水化修饰
称取软脂酸0.37g(1.44mmol,4.0eq)，用DCM(约10mL)溶解后加入反应瓶，随后再加入随后再加入HBTu(1.44mmol,0.54g,4eq)、DIEA(2.88mmol,0.474mL,8eq)DMF溶液各5mL,上下来回震荡反应瓶50min,而后排出反应液，取出少量树脂用CH3OH/DCM/DMF依次洗涤后加入到装有5％茚三酮的无水乙醇溶液的EP管中，沸水浴三分钟，树脂呈现黄色或浅蓝色，即阴性反应，则反应完成，将反应瓶中树脂用DCM/DMF交替洗涤三次后，抽去溶剂。将上述得到的树脂加入到装有裂解液的反应瓶中，将反应瓶密封好，固定到摇床上进行反应，反应约2h后，过滤除去树脂，用DCM洗涤树脂几次后，收集滤液，旋转蒸发除去TFA和溶剂，将冰的无水乙醚加入到剩余液体中，有大量白色絮状沉淀出现，高速离心(4000r/min)的白色沉淀即为粗品。通过制备高效液相色谱法进一步对粗品进行纯化精制后可得到目标产物软脂酸化的DP7(DP7-PA)。
5)用软月桂酸对DP7进行疏水化修饰
称取月桂酸0.29g(1.44mmol,4.0eq)，用DCM(约10mL)溶解后加入反应瓶，随后再加入随后再加入HBTu(1.44mmol,0.54g,4eq)、DIEA(2.88mmol,0.474mL,8eq)DMF溶液各5mL,上下来回震荡反应瓶50min,而后排出反应液，取出少量树脂用CH3OH/DCM/DMF依次洗涤后加入到装有5％茚三酮的无水乙醇溶液的EP管中，沸水 浴三分钟，树脂呈现黄色或浅蓝色，即阴性反应，则反应完成，将反应瓶中树脂用DCM/DMF交替洗涤三次后，抽去溶剂。将上述得到的树脂加入到装有裂解液的反应瓶中，将反应瓶密封好，固定到摇床上进行反应，反应约2h后，过滤除去树脂，用DCM洗涤树脂几次后，收集滤液，旋转蒸发除去TFA和溶剂，将冰的无水乙醚加入到剩余液体中，有大量白色絮状沉淀出现，高速离心(4000r/min)的白色沉淀即为粗品。通过制备高效液相色谱法进一步对粗品进行纯化精制后可得到目标产物硬脂酸化的DP7(DP7-DA)。
由于固相合成多肽技术已经较为成熟，抗菌肽DP7与疏水片段的偶联物还可交由合成公司合成。比如将丁二酰化胆固醇修饰的DP7-C((Chol-suc-VQWRIRVAVIRK-NH2)交由上海科肽生物科技有限公司合成，采用固相合成法合成多肽。合成的DP7-C用HPLC纯化，纯度>95％以上，经MS确定DP7-C分子量。合成的多肽于-20℃保存，使用前用MillQ水配成5mg/ml的母液备用。采用固相合成法合成的DP7-C(Chol-suc-VQWRIRVAVIRK-NH2)，是在DP7的基础上N末端通过酯键与疏水性的胆固醇偶联，其C末端接一分子-NH2进行保护。DP7-C的质谱图如图2，分子量为1991.3408，MS图上主峰是996.1748，提示合成了正确的DP7-C。
由于实验表明上述制得的疏水化修饰的DP7具有相似的性质，故以下的实施例均以胆固醇化的DP7(DP7-C)进行具体的描述。
实施例2 DP7-C的临界胶束浓度
DP7-C在水溶液中可发组装成胶束，我们采用芘荧光探针光谱法检测DP7-C的临界胶束浓度(CMC)。
芘(Pyrene)不溶于水，芘在水中的溶解度约为6×10-7mol/L，但是易溶于乙醇、乙醚。芘的水溶液荧光发射光谱有5个荧光峰，芘在水溶液中第1个发射光谱光强度I1与第3个发射光谱光强度I3值比值(在373nm处荧光强度与在384nm处的比值)约为1.8。根据文献报道，由于表面活性剂对非极性有机化合物有着增溶的作用，不同浓度的表面活性剂对芘的增溶能力不一样，在超过临界胶束浓度(CMC)后，溶液的增溶能力会有一个明显的突变点；根据I1/I3随着表面活性剂浓度的变化绘制曲线，曲线突变的中点就是所要检测底物的CMC。因此通过测定不同浓度DP7-C溶液中芘的荧光光谱，可以确定其在溶液的CMC。CMC的检测方法：称取DP7-C，用MilliQ水在37℃水浴溶解得到浓度为1.5mg/mLmL的DP7-C母液。以甲醇为溶剂配制6×10-5mol/L的芘溶液，待甲醇在黑暗通风处挥发干燥后，加入4mL的1.5mg/mLmL DP7-C溶液，37℃下超声4h 并在摇床上震荡8h，用荧光分光光度计检测I1和I3。在含微量芘的BD管中分别加入不同体积的样品母液，使其浓度为00.0001、0.001、0.01、0.025、0.05、0.10、0.125、0.25、0.5、1.0、1.5mg/mLmL，分别测定各溶液的荧光光谱值。荧光扫描的激发波长为334nm，发射波长为373nm和384nm，激发狭缝设置为8.0nm，发射狭缝设置为2.5nm,扫描速度为1200nm，根据荧光光谱，可计算出临界胶束浓度。
图3A为DP7-C在水溶液中自组装形成胶束的示意图，其胶束的CMC测定值为3.47μg/mL(结果见如图3B)。
实施例3 DP7-C胶束的物理特征
我们采用原子力显微镜和马尔文粒径仪检测DP7-C胶束的粒径、Zeta电位等一些物理特征。
1.原子力显微镜拍照：配置不同浓度DP7-C溶液，滴加到云母片，自然晾干，将涂有DP7-C的云母片置于原子力显微镜上拍照。
2.粒径及Zeta电位检测：用MilliQ水溶解DP7-C，采用马尔文粒径仪测定粒径和Zeta电位，每个样品检测4次，取平均值。
3.DP7-C外观形态及溶液状态：观察MilliQ水、DP7-C水溶液及冻干粉的外观，并用相机拍照。a为MilliQ水，b为溶于水溶液中的DP7-C，c为DP7-C冻干粉复溶后的状态，d为DP7-C冻干粉的形态和颜色。
结果见图4，DP7-C胶束形态呈球形或椭球形(图4C)，DP7-C胶束的粒径约为24.3±1.5nm(图4A)，Zeta电位约为28.8±0.27mV(图4B)。DP7-C冻干粉呈白色蓬松状，易溶于水，溶液呈无色透明状。
实施例4 DP7和DP7-C胶束体外溶血活性
在溶血试验中，用同一浓度下的DP7与DP7-C对红细胞的裂解溶血情况进行检测。
1.取健康自愿者的血液于抗凝管中，轻轻混匀。
2.取出抗凝血并向其加入等体积的生理盐水，缓慢混匀，400×g离心10分钟，去上清。
3.用PBS将红细胞缓慢混匀洗涤，400×g离心10分钟，去上清，细胞沉淀用PBS洗涤，重复3次。
4.用PBS稀释，得到20％(v/v)红细胞溶液，可立即使用或于4摄氏度保存2周左右(未发生溶血)。
5.用PBS将药物稀释成一系列浓度梯度，取100μLL药物对应加入到96孔板中， 阳性对照用100μLL 2％的吐温20，阴性对照加入100μL PBS，每个浓度药物设置3个复孔。
6.取20％(v/v)的红细胞溶液，用PBS按照体积比1:20稀释，混匀后加入96孔板中，药物浓度分别为100、200、400、600、800、1000、1200、1600μg/mL，37℃孵育1h。
7.药物和红细胞混合物孵育后，900×g离心10分钟，分别取上清160μLL加入新的96孔板中，在OD 450nm处读取吸光值。
8.根据A450的吸光值，计算出各个浓度药物所对应的溶血百分比。溶血百分比的计算公式为：
Figure PCTCN2017091390-appb-000004
溶血实验结果见图5A，当药物浓度大于0.8mg/mLmL时，DP7-C胶束对红细胞的裂解程度远小于DP7，说明了抗菌肽DP7偶联胆固醇后能够极大地降低对红细胞毒性的作用。图5B直观展示了红细胞在不同条件下的溶血情况，其中阴性对照组(PBS)(a组)不发生溶血，红细胞沉降于瓶底，阳性对照组(2％Tween-20)(b组)和DP7溶液(c组)红细胞裂解，呈现澄清的红色溶液，而DP7-C胶束(d组)则不裂解红细胞，但由于其胶束特性，红细胞均匀悬浮于溶液中。
实施例5 DP7-C的体内抑菌活性
我们之前的试验表明胆固醇化的DP7-C胶束在体外抗菌活性较低(对多个菌株的MIC>1024mɡ/L)，我们分别采用小鼠及斑马鱼的腹腔感染模型进一步检测其体内的抗菌活性。
(一)、DP7-C在小鼠体内的抗菌活性检测(革兰氏阳性菌——金黄色葡萄球菌)
采用腹腔感染模型测定DP7-C的抗感染活性，实验分组如下：
NS组：100μL生理盐水/只小鼠；
DP7-C组：0.3mg/kg；
万古霉素(VANC)阳性对照组：10mg/kg。
实验步骤如下：
(1)实验前一天用MHB活化金黄色葡萄球菌33591，实验当天取活化后的菌株，用生理盐水洗涤两次，测定菌液浓度后备用。
(2)建立小鼠的腹腔感染模型，每只小鼠腹腔注射菌液1.5×108cfu/0.mL5mL。
(3)感染1小时后，按分组分别静脉注射药物，0.mL1mL/只。
(4)24小时后向小鼠腹腔注射5mL的生理盐水，轻柔腹部，并处死，用75％酒精消毒，5min后剪开腹腔上皮，在腹腔开个小口，用1mL注射器从中吸取尽量多的腹水，然后转移至无菌的EP管中混匀。
(5)用生理盐水进行10倍的梯度稀释，如稀释10倍，100倍，1000倍，10000倍等，并取三个合适的浓度的菌液20μL，涂MHA平板，细菌孵箱培养过夜。
(6)挑取一个长有20～200个菌落的平板数菌落数，换算成计算5mL腹水中每毫升的菌量。
DP7-C治疗小鼠腹腔感染模型的结果见图6C，结果表明静脉给药后的DP7-C组(1mg/kg)和阳性药物组(20mg/kg万古霉素)的金黄色葡萄球菌平均集落形成单位(CFU)均显著低于NS组(p<0.01)。此外，DP7-C组与阳性药物组的抑菌效果基本一致，无统计学差异。说明在小鼠的全身感染模型中，DP7-C具有很好的抗菌活性。
(二)、DP7-C在斑马鱼体内的抗菌活性(革兰氏阴性菌——绿脓杆菌)
其次，我们采用带有荧光的绿脓杆菌(PAO1-GFP)建立斑马鱼的腹腔感染模型进一步验证DP7-C的抗感染作用。
将雌雄AB野生型斑马鱼按照配对繁殖程序进行配对，待其产卵后，收集鱼卵，然后将鱼卵培养于含PTU的海水中，于28℃温箱中培养，每天换一次含PTU的海水，斑马鱼孵化后48h开始腹腔注射采用DP7-C和生理盐水稀释的带荧光的绿脓杆菌(PAO1-GFP)，在3、8、18h荧光显微镜下观察拍照，观察PAO1-GFP在斑马鱼腹部生长情况。
结果见图6(A、B)，斑马鱼腹腔中PAO1-GFP的生长速率与绿色荧光荧光总强度成正相关，DP7-C组的PAO1-GFP生长速率远远小于NS，结果表明低浓度的DP7-C胶束(1mg/mL)在斑马鱼具有很好的抗菌活性。
实施例6 DP7-C的体内抑菌的机制研究
对DP7-C抗菌活性检测中，发现DP7-C具有较低的体外活性，然而却具有很高的体内抗菌活性，这可能是由于DP7-C调节机体免疫系统来实现抗菌作用。我们从细胞水平及细胞因子水平检测DP7-C对机体免疫功能的影响。
(一)、激活免疫相关细胞的检测
分组：将小鼠随机分成两组(NS组和DP7-C组)，每组5只。检测的免疫细胞及 其表面marker如下表：
  细胞标记
中性粒细胞 Gr1+(PE)、F4/80-(APC)
单核细胞 Gr1+(PE)、F4/80+(APC)
巨噬细胞 F4/80+(APC)、CD11b+(PE)
DP7-C组每只小鼠给药200μl的1mg/mL DP7-C，NS组每只小鼠注射200μl生理盐水，24小时后处死小鼠，每只小鼠腹腔注射5mL生理盐水，轻柔腹腔，然后吸取腹腔中的腹水，计算腹水中的细胞浓度及总细胞数。采用流式细胞仪检测腹水中淋巴细胞的分型，分析巨噬细胞、中性粒细胞、炎性单核细胞在总细胞中所占比例；根据流式细胞仪结果和腹水中总细胞数量计算出巨噬细胞、中性粒细胞、炎性单核细胞的数量。
结果如图7C所示，小鼠腹腔注射DP7-C后，腹腔内细胞总数显著上升，包括巨噬细胞、中性粒细胞及单核细胞的数量都有显著差异，特别是单核细胞所占比例由2.7％提高到32.8％(图7A)和巨噬细胞所占比例由38.8％提高到50.8％(图7B)。
(二)、与免疫相关细胞因子的测定
采用小鼠PBMC测定经DP7-C刺激后的细胞因子的变化情况。将分离得到的小鼠PBMC稀释至1×106cells/mL，加至六孔板2mL/孔，DP7-C的刺激浓度为200μg/mL，刺激时间为4h，刺激结束后，收集细胞于-80冰箱中保存。同时，为了验证DP7-C是否可逆转LPS诱导的炎症反应，设置与LPS同时刺激作用的实验：将PBMC稀释至1×106cells/mL，加至六孔板2mL/孔，DP7-C的刺激浓度为200μg/mL，1h后，用LPS再刺激4h，收集细胞于-80冰箱中保存。待所有样品收集完毕后，离心，洗涤，提取总RNA，反转录，做实时定量PCR。
DP7-C刺激PBMC相关细胞因子的表达结果见图7D，IL-1β、IL-6、MCP-1、M-CSF和TNF-α等与免疫激活相关细胞因子的表达均大幅提高，同时DP7-C与LPS共同刺激PBMC时，发现与细胞因子风暴相关的主要细胞因子，如IL-1β、MCP-1和TNF-α的基因表达明显降低，说明DP7-C会降低败血症等相关感染性疾病的损伤程度。
实施例7 DP7-C作为免疫佐剂的研究
由于在之前的试验中，发现DP7-C能显著上调小鼠PBMC免疫相关细胞因子的表达，我们预测DP7-C与CpG ODNs联合可作为一种新型的免疫佐剂，且DP7-C可以自发形成胶束，更是启发了发明人是否有可能替代铝佐剂的使用。本研究中，发明人以 OVA为模式抗原研究DP7-C/CpG复合佐剂的免疫调节作用，以及在小鼠预防性和治疗性肿瘤模型中的抗肿瘤效果。
(1)动物分组：
C57BL/6J雌性小鼠随机分为4组，每组10只，具体如下：
NS组：100μL生理盐水
CpG组:10μg OVA+20μg CpG
DP7-C组:10μg OVA+40μg DP7-C
CpG/DP7-C组:10μg OVA+40μg DP7-C+20μg CpG
(2)预防性免疫肿瘤模型：0、2、4周多点皮下免疫，检测第5周接种肿瘤前总抗体滴度。第5周每只小鼠背部皮下接种肿瘤细胞EG7-OVA:2×106个，待肿瘤长出后测量肿瘤，每隔3天一次，瘤体积计算公式为0.52×长×宽2。
治疗性免疫肿瘤模型：第0天每只小鼠背部皮下接种2×106个EG7-OVA肿瘤细胞，第5天开始免疫，每周一次，连续免疫3次，待肿瘤长出后每隔3天一次测量肿瘤，同时观察生存期。
结果见图8，在预防性模型中(A和B)，对第5周的OVA特异性抗体进行检测发现，DP7-C/CpG组产生的抗OVA特异性抗体显著升高，与其他组相比均具统计学差异。在接种肿瘤后第26天，DP7-C/CpG组的肿瘤生长受到明显抑制。在治疗性模型中(C和D)，DP7-C/CpG组显著地延长了接种肿瘤小鼠的生存期，并很大程度上抑制了肿瘤的生长。结果表明，DP7-C/CpG是一种具有良好的免疫刺激活性的新型免疫佐剂。
实施例8 DP7-C体外促抗原摄取作用的研究
佐剂激活免疫反应的机理可能在于促进DC细胞的成熟，提高其对抗原的摄取和加工。我们研究了DP7-C胶束在体外对DC活性的影响。
(一)促进树突状细胞的成熟
树突状细胞(dendritic cell,DC)的成熟状况在很大程度上决定机体产生免疫应答或免疫耐受。随着DC的成熟，其表面抗原CD80、CD86、MHC-II明显上调。因此我们用流式细胞术检测了DP7-C佐剂对树突状细胞成熟的影响。
实验步骤如下：
(1)分离C57/BL小鼠原代骨髓细胞，用含10ng/ml GM-CSF和10ng/ml IL-4的诱导培养基培养至第5天。
(2)将DP7-C佐剂加入DC细胞培养物中混匀，孵育16h。
(3)收集刺激后的DC细胞，经PBS洗2次后，用100μl PBS重悬细胞，加入1μl anti-mouse CD16/CD32 4℃封闭30min。
(4)用PBS洗3次，再加入1μg FITC-anti-CD80，APC-anti-CD86，PE-anti-MHC-II，室温孵育30min后，PBS洗3次后用200μl PBS重悬，进行流式检测。
结果见图9，DP7-C佐剂作用于DCs细胞后，细胞成熟分子MHCII以及共表达分子CD80和CD86的表达增强，并且与NS相比，差异具有统计学意义。
(二)增强树突状细胞的抗原摄取
DC细胞是机体内功能最强的抗原呈递细胞，可摄取外源物质，对其进行加工处理，并将其呈递给T细胞，激发免疫反应。DC细胞在T细胞免疫应答及T细胞依赖的抗体生成中具有重要的作用。我们采用流式细胞数检测了DP7-C对DC呈递抗原的作用。
实验步骤如下：
(1)分离C57/BL小鼠原代骨髓细胞，用含10ng/ml GM-CSF和10ng/ml IL-4的诱导培养基培养至第5天。
(2)将OVA蛋白标记FITC，OVA或DP7-C/OVA加入DC细胞培养物中混匀，孵育3h。
(3)收集刺激后的DC细胞，经PBS洗2次后，用300μl PBS重悬细胞，进行流式检测
结果见图10，DC细胞对单独的OVA抗原摄取率较低，40μg/ml DP7在一定程度上稍微增强DC细胞对OVA的摄取，而2.5μg/ml DP7-C能够显著增加DC细胞对抗原的摄取，并且随着DP7-C的浓度增加，摄取作用增强。
实施例9 DP7-C激发固有免疫反应
固有免疫在抗肿瘤免疫中起着重要作用。我们检测了小鼠免疫DP7-C佐剂后，对小鼠NK细胞的杀伤活性。
实验步骤如下：
(1)效应细胞制备
初次免疫48h后颈椎脱臼法处死小鼠，用小鼠淋巴细胞分离液分离小鼠的脾淋巴细胞作为效应细胞。
(2)靶细胞制备
复苏培养YAC-1细胞，实验前1天传代，保持2×106/20ml。用苔盼蓝染色。当活性＞95％时可用。
(3)效应细胞与靶细胞共孵育
①按靶细胞10000个/孔，靶效比＝1:25，1:50，1:100，1:200四个梯度将靶细胞和效应细胞加入96孔板中，37℃共孵育4h。
②250g离心取上清，进行LDH释放检测。
结果见图11，NS组基本无NK细胞活性，CpG和DP7-C展示了稍强的NK细胞杀伤活性，而CpG/DP7-C复合物能够有效的增强NK细胞的活性，差异具有显著性。
实施例10 DP7-C激发细胞免疫反应
上述结果中，我们得知DP7-C与CpG联用可以刺激抗原的特异性免疫反应，并能有效抑制肿瘤生长及转移，延长小鼠的生存期。并且，DP7-C与CpG能显著上调OVA抗原的特异性抗体滴度，增强体液免疫反应，因而我们进一步检测DP7-C激活的细胞免疫反应。
具体步骤如下：
(1)脾淋巴细胞体外刺激。
①3次免疫7天后，分离小鼠脾脏淋巴细胞，计数并将其稀释为5×106/ml。
②5×106/孔铺6孔板，每孔加入10μg/ml OVA全蛋白37℃刺激1h，阴性对照用DMSO，阳性对照用5μg/ml conA。
③每孔加入1μl Golgiplug混匀后再孵育6-12h。
(2)荧光抗体标记
①收集细胞，加入2ml 1×BD Pharmlyse涡旋，避光室温孵育10min，500g离心5min弃上清。
②经1×instaining buffer清洗后，用100μl1×instaining buffer重悬，加入1μl anti-mouse CD16/CD32 4℃孵育15min封闭。
③经1×instaining buffer清洗后，用100μl staining buffer重悬细胞，分别加入1μl PE-anti-mouse CD8、perCP-cy5-anti-mouse CD4 4℃避光孵育30min。
④经staining buffer清洗2次后，加入250μl fixation/permeabilization涡旋充分悬起，室温避光孵育20min固定透化细胞。
(7)加入2ml BD Perm/wash buffer，500g离心5min弃上清。
(8)加入100μl BD perm/wash buffer重悬细胞，加入胞内抗原抗体(FITC-IFN-γ或PE-IL-17A)室温避光孵育30min，然后加入2ml BD Perm/wash buffer，500g离心5min弃上清，加入500μl含2％多聚甲醛的PBS重悬，涡旋充分悬起，即可进行流式分析。
结果见图12，NS组中CD4+/IFN-γ+、CD8+/IFN-γ+和CD4+/IL-17A+细胞所占比例较低，而小鼠免疫CpG和DP7-C佐剂后，细胞所占比例有所增加，而免疫CpG/DP7-C复合物的小鼠脾脏中，这三种细胞占有较高比例，与NS组相比，差异具有统计学意义。
实施例11 CpG/DP7-C佐剂激发高效的抗肿瘤免疫
上述结果中，我们得知在EG7-OVA肿瘤模型中，免疫CpG/DP7-C复合物的小鼠肿瘤生长受到抑制。我们进一步以NY-ESO-1为抗原，与CpG/DP7-C佐剂形成疫苗，检测此疫苗是否能够抑制高表达该抗原的黑色素瘤的生长。
具体步骤如下：
(1)疫苗制备
20μg CpG和40μg DP7-C在37℃孵育15min；最后加入5μg NY-ESO-1蛋白，并用无菌PBS补足至体积100μl。
(2)分组及给药方案
在本研究中动物实验共分为5组：NS、CpG、DP7-C和CpG/DP7-C组，每组10只小鼠，每只小鼠给药剂量如下：
①NS组:100μl PBS；
②CpG组:5μg NY-ESO-1蛋白+20μg CpG；
③DP7-C组:5μg NY-ESO-1蛋白+40μg DP7-C；
④CpG/DP7-C组:5μg NY-ESO-1蛋白+20μg CpG+40μg DP7-C；
每只小鼠总给药量体积为100μl，不足100μl的加入无菌PBS补至100μl。
(3)预防性免疫肿瘤模型：0、2、4周多点皮下免疫，检测第5周接种肿瘤前总抗体滴度。第5周每只小鼠背部皮下接种肿瘤细胞NY-ESO-1+B16:2×105个，待肿瘤长出后测量肿瘤，每隔3天一次，瘤体积计算公式为0.52×长×宽2；治疗性免疫肿瘤模型：第0天每只小鼠背部皮下接种2×105个NY-ESO-1+B16肿瘤细胞，第5天开始免疫，每周一次，连续免疫3次，待肿瘤长出后每隔3天一次测量肿瘤，同时观察生存期。
结果见图13，无论在预防性(图13A)还是治疗性模型(图13B)中，NS组小鼠的肿瘤生长迅速。而免疫单独的CpG或者DP7-C佐剂在一定程度上能够抑制肿瘤的生长。而免疫CpG/DP7-C复合物佐剂能够有效地抑制肿瘤的生长，与NS组相比，差异具有显著性差异。
实施例12 电镜观察DP7-C胶束/siRNA复合物形态
由于DP7带阳电荷，使得所自主装形成的DP7-C胶束在水溶液中呈正电，展示出 作为一种非病毒基因传输，特别是siRNA传输载体的潜力。DP7-C通过自主装所形成的胶束及DP7-C/siRNA复合物的形态通过透射电镜(H-6009IV，日立公司，日本)观察。观察前，1mg\mL的胶束溶液用蒸馏水稀释并且涂布于一张铜载网上，并用硝酸纤维素覆盖，随后用磷酸钨酸负染，室温干燥后，于电镜下观察。结果可见DP7-C胶束为球状，分布均一。电镜下DP7-C胶束粒径约为60nm，与siRNA复合后粒径约为100nm(结果见图14)。
实施例13 DP7-C/siRNA复合物体外转染效率检测
转染前24小时，C26小鼠结肠癌细胞或B16小鼠黑色素瘤细胞以5×104个细胞每孔的密度铺于6孔板中，每孔加入添加20％FBS的DMEM培养基2mL。FAM修饰的无义siRNA(FAM-siRNA)用作报告基因用于检测转染效率,以PEI25K、Lipofectamin2000作为阳性对照。转染时，先将培养基置换为1mL无血清DMEM培养基。随后，将不同比例混合的基因转染复合物加入每孔中，每种复合物包含1μg FAM-siRNA。其中，siRNA/DP7-C、siRNA/PEI25K以及siRNA/Lipofectamin2000的比例分别为1:5、1:2和1：2。37度孵育4小时后，培养基替换为添加20％FBS的DMEM培养基继续培养。24小时后，使用显微镜观察转染情况并拍照。收集包括漂浮的和贴壁的孔内所有细胞，用预冷的PBS洗2遍，使用流式细胞计数仪(Epics Elite ESP，美国)计数各组的总荧光强度。
转染结果如图15所示，检测观察均显示：DP7-C能够将FAM修饰的荧光siRNA转染到Ct26(图15c)和B16(图15b)细胞中，其转染效率分别为42.4±3.5％和53.3±4.0％；而PEI复合物的对应转染效率分别为19.0±1.8％和72.0±4.1％；Lipofectamin2000复合物的对应转染效率分别为29.5±3.2％和25.6±4.2％，这也能够从图15a中直观体现出来。我们的结果显示，DP7-C胶束相对于PEI25K和Lipofectamin2000具有更高的siRNA转染效率。
实施例14 DP7-C细胞毒性检测
阳离子的细胞毒性是制约siRNA转染应用的重要因素之一。我们通过细胞存活实验检测DP7-C胶束对正常细胞——293T人胚肾细胞株的毒性。293T细胞以每孔3×103个细胞的密度铺于96孔板中，每孔加入添加DMEM/20％FBS培养基100μL，培养24小时。实验设置DP7-C、PEI25K以及Lipofectamin2000 3个组，每个组设置对应10个浓度梯度，分别为：0、12.5、18.75、25、37、50、75、100、150、200μg/mL。细胞的生存情况通过CCK-8法检测，加药后24小时，每孔加入CCK-8溶液10μL，37℃孵育2 小时。使用酶标仪在630nm和450nm波长处检测每孔吸收值，绘制标准曲线并计算IC50，结果取6组平行实验的平均值。
检测结果如图16所示，PEI25K和Lipofectamin2000均展示出极强的毒性，其IC50小于20μg/mL；而DP7-C胶束的毒性则小很多，其IC50超过了200μg/mL，具有很好的安全性
实施例15 DP7-C传输VEGF siRNA治疗C26小鼠结肠癌腹腔转移瘤模型
为了进一步表征DP7-C胶束在肿瘤治疗中的应用潜力，我们建立了C26小鼠结肠癌腹腔转移模型，并使用DP7-C传输抗VEGF的siRNA治疗基因用于治疗研究。
(1)建立腹腔转移模型：第0天时，6-8周龄雌性BALB/c小鼠腹腔注射0.1mL细胞悬液(约含1×105个C26细胞)。
(2)第3天时，小鼠被随机分为4组，每组8只并标记。
(3)4组小鼠分别腹腔注射10次剂量的生理盐水(空白对照组)、空白DP7-C胶束(17.5μg)、DP7-C/无义对照siRNA(Scramble siRNA)复合物(17.5μg/3.5μg)、或DP7-C/VEGF siRNA复合物(17.5μg/3.5μg)。
(4)第20天时，当对照组的小鼠已经非常虚弱时，断颈法处死所有的小鼠，其腹腔内肿瘤组织以及心、肝、脾、肺和肾脏组织被立即采集、称重和分析。每组小鼠的腹水体积也同样被收集和测量，对每个实验组的肿瘤组织进行CD31免疫组化染色分析；对所收集的心、肝、脾、肺、肾组织进行HE染色分析。
图17显示了使用DP7-C/VEGF siRNA复合物经过腹腔注射治疗腹腔转移C26肿瘤模型的效果。图17a为每组动物中具有代表性的小鼠的腹腔照片。其中，DP7-C/VEGF siRNA复合物治疗组的平均肿瘤重量为1.07±0.5g，生理盐水对照组重量为8.82±0.63g，空载DP7-C组重量为7.94±0.53g，DP7-C/无义对照siRNA复合物为5.3±0.72g(图17c)。用DP7-C/VEGF siRNA复合物治疗的小鼠的腹腔转移肿瘤结节数量相对于其他治疗组明显更少。因此，用DP7-C/VEGF siRNA复合物治疗的小鼠的肿瘤生长得到了极大的抑制。
同时，如同图17b显示，各组的小鼠所产生的腹水体积差别具有显著性差异。DP7-C/VEGF siRNA复合物治疗组的平均肿瘤重量为0.2±0.1mL，生理盐水对照组重量为1.17±0.4mL，空载DP7-C组重量为1.17±0.51mL，DP7-C/无义对照siRNA复合物为0.58±0.11mL。没有使用DP7-C/VEGF siRNA复合物治疗的各组小鼠的腹水呈明显的血样红色，证实这些组的小鼠存在严重的肠系膜损伤。相对于其他实验组，DP7-C/VEGF  siRNA复合物更有效地抑制了C26腹腔转移肿瘤模型的生长，以及所伴随的严重肿瘤浸润和炎症的产生。
此外，CD31免疫组化染色结果显示，相对于其他三个实验组，DP7-C/VEGF siRNA复合物治疗组的肿瘤组织中，新生血管的数量更少，证实DP7-C通过传输抗VEGF的siRNA更有效地抑制了肿瘤组织血管的生成(图17d)。此外，HE染色结果显示(图17e)，DP7-C胶束通过腹腔关注给药并未在主要脏器组织中产生明显的病理变化，证实DP7-C的副作用较小。
实施例16 DP7-C传输VEGF siRNA治疗C26小鼠结肠癌皮下移植瘤模型
为了进一步表征DP7-C胶束在肿瘤治疗中的应用潜力，我们建立了C26小鼠结肠癌皮下移植瘤模型，并使用DP7-C传输抗VEGF的siRNA治疗基因用于治疗研究。
(1)第0天时，6-8周龄雌性BALB/c小鼠皮下接种0.1mL细胞悬液(约含1.5×106个C26细胞)。
(2)第8天时，待扪及肿瘤，小鼠被随机分为4组，每组8只并标记。
(3)小鼠分别瘤内注射10次剂量的生理盐水(空白对照组)、空白DP7-C胶束(25μg)、DP7-C/无义对照siRNA复合物(25μg/5μg)、或DP7-C/VEGF siRNA复合物(25μg/5μg)。
(4)第20天时，当对照组的小鼠已经非常虚弱时，断颈法处死所有的小鼠，其腹腔内肿瘤组织以及心、肝、脾、肺和肾脏组织被立即采集、称重和分析。每组小鼠的腹水体积也同样被收集和测量，对每个实验组的肿瘤组织进行CD31免疫组化染色分析；对所收集的心、肝、脾、肺、肾组织进行HE染色分析。
图18显示了使用DP7-C/VEGF siRNA复合物经过瘤内注射治疗C26皮下移植肿瘤模型的效果。图18a展示的是每组动物中小鼠皮下肿瘤的照片。其中，DP7-C/VEGF siRNA复合物治疗组的平均肿瘤体积为261.4±115.51mm3，生理盐水对照组重量为577.21±107.46mm3，空载DP7-C组重量为357.64±30.56mm3，DP7-C/无义对照siRNA复合物为474.43±120.67mm3。用DP7-C/VEGF siRNA复合物治疗的小鼠的皮下肿瘤体积相对于其他治疗组明显更小(图18b)。因此，用DP7-C/VEGF siRNA复合物治疗的小鼠的肿瘤生长得到了极大的抑制。
此外，CD31免疫组化染色结果显示(图18c)，相对于其他三个实验组，DP7-C/VEGF siRNA复合物治疗组的肿瘤组织中，新生血管的数量更少，证实DP7-C通过传输抗VEGF的siRNA更有效地抑制了肿瘤组织血管的生成。此外，HE染色结果显示(图18d)， DP7-C胶束通过腹腔关注给药并未在主要脏器组织中产生明显的病理变化，证实DP7-C的副作用较小。
实施例17 DP7-C传输VEGF siRNA治疗B16小鼠黑色素瘤肺转移瘤模型
为了进一步表征DP7-C胶束在肿瘤治疗中的应用潜力，我们建立了B16小鼠黑色素瘤肺转移瘤模型，并使用DP7-C传输抗VEGF的siRNA治疗基因用于治疗研究。
(1)第0天时，6-8周龄雌性C57小鼠皮下接种0.1mL细胞悬液(约含2.5×105个B16细胞)。
(2)第3天时，小鼠被随机分为4组，每组8只并标记。
(3)小鼠分别尾静脉注射7次剂量的生理盐水(空白对照组)、空白DP7-C胶束(60μg)、DP7-C/无义对照siRNA复合物(60μg/12μg)、或DP7-C/VEGF siRNA复合物(60μg/12μg)。
(4)第20天时，当对照组的小鼠已经非常虚弱时，断颈法处死所有的小鼠，其肺部肿瘤组织以及心、肝、脾、肺和肾脏组织被立即采集、称重和分析；对所收集的心、肝、脾、肺、肾组织进行HE染色分析。
图19显示了使用DP7-C/VEGF siRNA复合物经过静脉注射治疗B16肺转移肿瘤模型的效果。图19a展示的是每组动物中小鼠肺部肿瘤的照片。其中，DP7-C/VEGF siRNA复合物治疗组的平均肿瘤结节数为30±12个，生理盐水对照组为172±16个，空载DP7-C组为132±22个，DP7-C/无义对照siRNA复合物为106±15个。此外，DP7-C/VEGF siRNA复合物治疗组的平均肺组织重量为0.22±0.02克，生理盐水对照组重量为0.52±0.18克，空载DP7-C组重量为0.41±0.1克，DP7-C/无义对照siRNA复合物为0.42±0.17克。用DP7-C/VEGF siRNA复合物治疗的小鼠的肺转移肿瘤结节个数相对于其他治疗组明显更少(图19a)，小鼠肺部平均重量相对于其他治疗组明显更轻(图19b)。因此，用DP7-C/VEGF siRNA复合物治疗的小鼠的肿瘤生长得到了极大的抑制。
此外，HE染色结果显示(图19c)，DP7-C胶束通过腹腔关注给药并未在主要脏器组织中产生明显的病理变化，证实DP7-C的副作用较小。

Claims (38)

  1. 疏水化修饰的抗菌肽，其特征在于所述的疏水化修饰为在抗菌肽的氮末端偶联疏水片段；所述的抗菌肽的氨基酸序列为VQWRIRVAVIRK。
  2. 根据权利要求1所述的疏水化修饰的抗菌肽，其特征在于抗菌肽VQWRIRVAVIRK碳端酰胺化修饰为VQWRIRVAVIRK-NH2。
  3. 根据权利要求1或2所述的疏水化修饰的抗菌肽，其特征在于所述疏水片段为甾醇类化合物或饱和直链脂肪酸。
  4. 根据权利要求1～3任一项所述的疏水化修饰的抗菌肽，其特征在于所述的甾醇类化合物为胆固醇类化合物或胆酸类化合物。
  5. 根据权利要求所述的疏水化修饰的抗菌肽，其特征在于所述的甾醇类化合物为丁二酰化胆固醇、胆酸或去氧胆酸中的至少一种。
  6. 根据权利要求3所述的疏水化修饰的抗菌肽，其特征在于所述饱和直链脂肪酸为C6～C20中的至少一种。
  7. 根据权利要求6所述的疏水化修饰的抗菌肽，其特征在于所述饱和直链脂肪酸为C8～C18中的至少一种。
  8. 根据权利要求7所述的疏水化修饰的抗菌肽，其特征在于所述的长链脂肪酸为硬脂酸、软脂酸、月桂酸或正辛酸中的至少一种。
  9. 根据权利要求1～8任一项所述的疏水化修饰的抗菌肽，其特征在于所述的抗菌肽的氮端与疏水片段偶联的方式为通过疏水片段上的-CO-OH与抗菌肽上的-NH2酰胺化反应生成。
  10. 根据权利要求1～9任一项所述的疏水化修饰的抗菌肽，其特征在于所述疏水化修饰的抗菌肽的结构为：
    Figure PCTCN2017091390-appb-100001
    其中，所述的R为甾醇类化合物或饱和直链脂肪酸。
  11. 根据权利要求10的疏水化修饰的抗菌肽，其特征在于所述的R为：
    Figure PCTCN2017091390-appb-100002
  12. 权利要求1～11任一项所述的疏水化修饰的抗菌肽制成的胶束。
  13. 根据权利要求12所述的胶束，其特征在于：所述的胶束为疏水化修饰的抗菌肽在溶液中自组装而成。
  14. 根据权利要求12或13所述的胶束，其特征在于：所述的胶束里还装载有核酸、小分子药物或者蛋白中的至少一种。
  15. 权利要求1～11任一项所述的疏水化修饰的抗菌肽或者权利要求12～14任一项所述胶束在制备抗菌药物中的用途。
  16. 根据权利要求15所述的用途，其特征在于：所述的抗菌为抗细菌或抗真菌。
  17. 根据权利要求16所述的用途，其特征在于：所述的细菌为金黄色葡萄球菌、大肠杆菌、鲍曼不动杆菌、绿脓杆菌或伤寒杆菌中的至少一种。
  18. 根据权利要求15所述的用途，其特征在于：所述的真菌为白色念珠菌或近平滑念珠菌中的至少一种。
  19. 抗菌药物，其特征在于是由权利要求1～11任一项所述的疏水化修饰的抗菌肽或者权利要求12～14任一项所述的胶束作为主要活性成分制备而成。
  20. 根据权利要求19所述的抗菌药物，其特征在于还含有其他抗菌药物。
  21. 根据权利要求20所述的抗菌药物，其特征在于其他抗菌药物为抗生素。
  22. 根据权利要求21所述的抗菌药物，其特征在于所述的抗生素为糖肽类抗生素、氨基苷类抗生素、大环内脂类抗生素、β-内酰胺类抗生素中的至少一种。
  23. 根据权利要求22所述的抗菌药物，其特征在于所述的青霉素类抗生素为青霉素G、青霉素V、氟氯西林、苯唑青霉素、氨苄西林、羧苄西林、匹氨 西林、磺苄西林、替卡西林、哌拉西林或阿莫西林中的至少一种；所述的头孢菌素类抗生素为：头孢羟氨苄、头孢氨苄、头孢唑啉、头孢拉啶、头孢丙烯，头孢呋辛脂、头孢克洛、头孢孟多、头孢噻肟、头孢曲松、头孢克肟、头孢地尼、头孢皮罗、头孢吡肟或头孢唑南中的至少一种。
  24. 根据权利要求22所述的抗菌药物，其特征在于所述的氨基苷类抗生素为链霉素、庆大霉素、卡那霉素、妥布霉素、丁胺卡那霉素、新霉素、西索米星、妥布霉素、阿米卡星、奈替米星、核糖霉素、小诺霉素或阿斯霉素中的至少一种。
  25. 根据权利要求22所述的抗菌药物，其特征在于所述的多肽类抗生素为万古霉素、去甲万古霉素、多粘菌素B或替考拉宁中的至少一种。
  26. 根据权利要求22所述的抗菌药物，其特征在于所述的大环内脂类抗生素为红霉素、白霉素、无味红霉素、依托红霉素、乙酰螺旋霉素、麦迪霉素、交沙霉素或阿奇霉素中的至少一种。
  27. 权利要求1～11任一项所述的疏水化修饰的抗菌肽或者权利要求12～14任一项所述的胶束在制备免疫佐剂中的用途。
  28. 免疫佐剂，其特征在于：是由权利要求1～11任一项所述的疏水化修饰的抗菌肽或者权利要求12～14任一项所述的胶束作主要成分制成。
  29. 根据权利要求28所述的免疫佐剂，其特征在于：还含有单链脱氧寡核苷酸CpG ODNs。
  30. 根据权利要求28或29所述的免疫佐剂，其特征在于：所述的疏水化修饰的抗菌肽与CpG ODNs之间的配比为：1:0.5～1:5。
  31. 权利要求1～11任一项所述的疏水化修饰的抗菌肽或者权利要求12～14任一项所述的胶束在制备核酸转运载体中的用途。
  32. 根据权利要求31所述的用途，其特征在于：所述的核酸为RNA。
  33. 根据权利要求32所述的用途，其特征在于：所述的核酸为信使RNA、用于RNA干扰的siRNA或者为用于基因组编辑的sgRNA中的至少一种。
  34. 核酸转运载体，其特征在于：由权利要求1～11任一项所述的疏水化修饰的抗菌肽或者权利要求12～14任一项所述的胶束装载核酸而得。
  35. 根据权利要求34所述的核酸转运载体，其特征在于：所述核酸为RNA。
  36. 根据权利要求34所述的核酸转运载体，其特征在于：所述的核酸为信使RNA、siRNA或者为sgRNA。
  37. 根据权利要求34～36任一项所述的核酸转运载体，其特征在于：所述的疏水化修饰的抗菌肽与核酸的质量比为1:1～20:1。
  38. 制备权利要求34～36任一项所述的核酸转运载体的方法，其特征在于包括以下步骤：
    a、称取适量权利要求1～11任一项所述的疏水化修饰的抗菌肽，加入溶液溶解，自发形成胶束；或取权利要求12～14任一项所述的胶束形成溶液；
    b、将核酸加入胶束溶液，室温孵育，即得。
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