Patent Description:
Antimicrobial peptides, also known as host defense peptides (HDPs), are generally composed of <NUM> to <NUM> amino acid residues and are basic polypeptides, most of which are positively charged. Due to their broad-spectrum antibacterial effect, the antimicrobial peptides can effectively inhibit and kill pathogens such as fungi, viruses and parasites, and can selectively kill tumor cells. Moreover, the antimicrobial peptides are not easy to develop drug resistance, thus they constitute the first barrier for host defense against invasion of pathogenic microorganisms and are an important component of the body's immune system. The antimicrobial peptides have become a potential drug for the prevention and control of disease and have a broad development prospect as a substitute for antibiotics. Despite great application potential, only a few antimicrobial peptides are widely used clinically, mainly because of their stability and toxicity. Some researches suggest that the antimicrobial peptides can obtain better stability and reduce toxicity through nanocrystallization, which provides a new research direction for the application of antimicrobial peptides.

The immunomodulatory activities of antimicrobial peptides or HDPs have attracted more and more attention, including inducing the production of cytokines and chemokines by changing signal pathways, directly or indirectly recruiting effector cells including phagocytes, enhancing intracellular and extracellular bacterial killing, promoting dendritic cell maturation and macrophage differentiation, and mediating wound repair and apoptosis. Many antimicrobial peptides exhibit adjuvant activity due to their good immunomodulatory effects, which are achieved mainly by activating the natural immune response and mediating the acquired immune response.

The antimicrobial peptide DP7 is a type of antimicrobial peptide with higher bacterial recognition specificity and stronger antibacterial activity, which is obtained by replacing two amino acids of the template antimicrobial peptide HH2 (Patent No.: <CIT>) according to the computer aided design-based new antimicrobial peptide screening method. Researches show that the antimicrobial peptide DP7 has better antibacterial activity, lower hemolytic toxicity of red blood cells and stronger immunomodulatory activity than HH2. In the study of antibacterial activity in vitro, the antimicrobial peptide DP7 can obviously destruct bacterial cell wall and disrupt cell membrane, thus realizing antibacterial function. In the study of antibacterial activity in vivo, it is found from the abdominal cavity model of mice infected with pathogenic staphylococcus aureus that DP7 has a very good therapeutic effect by inducing immune cells to remove bacteria. However, the high concentration of DP7 will cause hemolysis of red blood cells and kill mice after administration by intravenous injection, indicating that the high concentration of DP7 is toxic to red blood cells and will destroy red blood cells. DP7 is a positively charged hydrophilic antimicrobial peptide and can be modified with a hydrophobic fragment to obtain an amphiphilic compound which can be self-assembled into nanoparticles, so as to greatly reduce the toxicity of intravenous administration, maintain its antibacterial and immunomodulatory effects in vivo, and act as a delivery system for small nucleic acid drugs. Thus, DP7 has a wide use in the drug field.

In <NPL> is disclosed as a positively charged, antimicrobial peptide (VQWRIRVAVIRK).

Examples of N-terminally modified, positively charged antimicrobial peptides of the prior art are disclosed in:.

The purpose of the invention is to provide a new modified antimicrobial peptide, a new and effective choice for anti-infection treatment, preparation of a new immunologic adjuvant, and preparation of siRNA carrier in the field.

To solve the above technical problems, the technical scheme adopted by the invention is to provide a hydrophobically modified antimicrobial peptide, and the hydrophobic modification is to couple a hydrophobic fragment at the nitrogen terminal of the antimicrobial peptide (or couple a hydrophobic compound at nitrogen terminal). The amino acid sequence of the antimicrobial peptide DP7 is VQWRIRVAVIRK (SEQ ID No.<NUM>). C-terminal of DP7 is usually modified by amidation to improve its stability, when the structure is VQWRIRVAVIRK-NH<NUM>.

For the hydrophobically modified antimicrobial peptide, the hydrophobic compound (hydrophobic fragment) is succinylated cholesterol.

For the hydrophobically modified antimicrobial peptide, the nitrogen terminal of the antimicrobial peptide is coupled to the hydrophobic segment (hydrophobic compound) by the amidation reaction of -CO-OH on the hydrophobic segment (hydrophobic compound) with -NH<NUM> on the antimicrobial peptide.

For the hydrophobically modified antimicrobial peptide, the structure is:
<CHM>
Wherein the R is a cholesterol compound, cholic acid or long-chain fatty acid.

For the hydrophobically modified antimicrobial peptide, the R is
<CHM>.

The invention further provides a micelle prepared from the hydrophobically modified antimicrobial peptide.

The micelle is self-assembled from the hydrophobically modified antimicrobial peptide in solution.

The micelle is subjected to freeze-drying treatment.

The micelle is further loaded with at least one of nucleic acid, small molecule drug, polypeptide or protein.

The invention further provides a method for prepare the hydrophobically modified antimicrobial peptide, comprising the following steps:.

The invention further provides a use of the hydrophobically modified antimicrobial peptide or the micelle in preparing an antimicrobial drug.

For the use in preparing the antimicrobial drug, the antimicrobial is antibacterial or antifungal.

For the use in preparing the antimicrobial drug, the bacteria are at least one of Staphylococcus aureus, Escherichia colibacillus, Acinetobacter baumanmii, Pseudomonas aeruginosa or Salmonella typhi.

For the use in preparing the antimicrobial drug, the fungi are at least one of Canidia Albicans or Candida parapsilosis.

The invention provides an antimicrobial drug prepared from the hydrophobically modified antimicrobial peptide or the micelle as the main active ingredient.

Further, the antimicrobial drug comprises other antimicrobial drugs.

Further, the other antimicrobial drugs are antibiotics.

Wherein, for the antimicrobial drug, the antibiotics are at least one of glycopeptide antibiotic, aminoglycoside antibiotic, macrolide antibiotic and β-lactam antibiotic.

Wherein, for the antimicrobial drug, the β-lactam antibiotic is at least one of penicillin antibiotic or cephalosporin antibiotic.

Further, the penicillin antibiotic is at least one of penicillin G, penicillin V, flucloxacillin, oxacillin, ampicillin, carboxybenzylpenicillin, pivampicillin, sulbenicillin, ticarcillin, piperacillin or amoxicillin. The cephalosporin antibiotic is at least one of cefadroxil, cefalexin, cefazolin, cefradine, cefprozil, cefuroxime, cefaclor, cefamandole, cefotaxime, ceftriaxone, cefixime, cefdinir, cefpirome, cefepime or cefuzonam.

Wherein, for the antimicrobial drug, the aminoglycoside antibiotic is at least one of streptomycin, gentamicin, kanamycin, tobramycin, amikacin, neomycin, sisomicin, tobramycin, amikacin, netilmicin, ribozyme, micronomicin or azithromycin.

Wherein, for the antimicrobial drug, the polypeptide antibiotic is at least one of vancomycin, norvancomycin, polymyxin B or teicoplanin.

Wherein, for the antimicrobial drug, the macrolide antibiotic is at least one of erythromycin, albomycin, odorless erythromycin, erythromycin estolate, acetylspiramycin, midecamycin, j osamycin or azithromycin.

Further, the dosage form of the antimicrobial drug is injection.

The invention further provides a use of the hydrophobically modified antimicrobial peptide or the micelle in preparing an immunologic adjuvant.

The invention further provides an immunologic adjuvant prepared from the hydrophobically modified antimicrobial peptide or the micelle as the immunologic adjuvant and antigen.

The immunologic adjuvant further comprises a single-stranded oligodeoxyribonucleotide (CpG ODNs). Further, the ratio of the hydrophobically modified antimicrobial peptide to CpG ODNs is <NUM>:<NUM> to <NUM>:<NUM>.

The invention further provides a use of the hydrophobically modified antimicrobial peptide or the micelle in preparing a nucleic acid transporter.

For the use in preparing the nucleic acid transporter, the nucleic acid is RNA.

For the use in preparing the nucleic acid transporter, the nucleic acid is of message RNA, siRNA (small interfering RNA) for RNA interference, or SG RNA (small guide RNA) for genome editing.

The invention further provides a nucleic acid transporter obtained by the nucleic acid loaded in the hydrophobically modified antimicrobial peptide or the micelle.

For the nucleic acid transporter, the nucleic acid is RNA.

For the nucleic acid transporter, the nucleic acid is message RNA (mRNA), siRNA (small interfering RNA) for RNA interference, or sgRNA (small guide RNA) for genome editing.

For the nucleic acid transporter, the ratio by mass of the hydrophobically modified antimicrobial peptide to the nucleic acid is <NUM>: <NUM> to <NUM>:<NUM>.

The invention further provides a method for preparing the nucleic acid transporter, comprising the following steps:.

The coupled product of the antimicrobial peptide DP7 and the hydrophobic fragment can be stored in the form of lyophilized powder, and can be directly dissolved in sterile water or physiological saline in use.

The beneficial effects of the invention are as follows: due to small molecular weight, the antimicrobial peptide DP7 in the hydrophobically modified antimicrobial peptide DP7 can be conveniently synthesized by an Fmoc solid phase polypeptide synthesis method, and coupled to a hydrophobic segment by a chemical synthesis coupling method in a simple and easy way. The hydrophobically modified antimicrobial peptide DP7 of the invention can be self-assembled into micelles, develop better monodispersity and Zeta potential, and maintain stable after freeze-drying and redissolution. The micelles of the hydrophobically modified antimicrobial peptide DP7 can significantly reduce the toxicity of the antimicrobial peptide DP7 on red blood cell lysis and realize intravenous administration. Despite the extremely low antibacterial activity in vitro, the hydrophobically modified antimicrobial peptide DP7 has good antibacterial activity in zebrafish and mice. The antibacterial activity in vivo is achieved not by direct sterilization, but by recruiting macrophages, monocytes, neutrophils and other lymphocytes and regulating the expression of some immune cytokines, so as to provide the immune protection for the organism. In the meantime, the hydrophobically modified antimicrobial peptide DP7 can also be used as an immunologic adjuvant to induce a high immune response against the target antigen. In addition, the hydrophobically modified antimicrobial peptide DP7 cationic micelle of the invention can efficiently compound siRNA and introduce it into tumor cells such as colon cancer cells and melanoma cells, inhibit tumor tissue growth by intraperitoneal injection, intratumoral injection and caudal vein injection, and present high safety.

The English abbreviations involved in the invention are as follows:.

The strains involved in the invention are as follows:.

DP7 is a positively charged hydrophilic antimicrobial peptide and generally modified by C-terminal amidation. DP7 used in the embodiment of the invention is QWRIRVAVIRK-NH<NUM>. The hydrophobic fragments (or hydrophobic compounds) such as cholesterol, cholic acid and long-chain fatty acids may be coupled to the hydrophilic polypeptides, and may be self-assembled into nanostructures. In this study, the antimicrobial peptide DP7 was nanocrystallized by its coupling with the hydrophobic fragment, thus increasing its stability and reducing hemolytic toxicity, and realizing its intravenous administration. Because of its direct antibacterial and immunomodulatory effects, the nanoparticles formed by the hydrophobically modified DP7 have anti-infection effects.

The antimicrobial peptide DP7 has good immunomodulatory activity and can mediate the natural immune response, which makes it an immunologic adjuvant for vaccines. Co-immunization with antigens can enhance the host's cellular immunity and mediate the production of antigen-specific immunoglobulins. The research results show that the hydrophobic antimicrobial peptides exhibit good adjuvant properties. During in vitro tests, the hydrophobically modified DP7 can be self-assembled into nanoparticles, act as a new immunologic adjuvant by absorbing CpG through electrostatic interaction, enhance the antigen uptake by antigen presenting cells, and promote the maturation of antigen presenting cells and the expression of cytokines. In animal tumor model tests, the hydrophobically modified DP7/CpG complex prolonged the survival time of mice inoculated with tumor, significantly inhibited the growth of tumor and enhanced the level of antigen-specific antibody.

Meanwhile, as the DP7 is positively charged, the hydrophobically modified DP7 micelle formed by self-assembly is also positively charged in aqueous solution, showing its potential as a non-viral gene transmission vector, especially siRNA transmission vector. Combined with siRNA silencing against tumor targets, the hydrophobically modified DP7 will play an important role in the research and application of siRNA-based tumor therapy.

Currently, many studies have been carried out on the formation of nanoparticles by coupling the hydrophobic fragments with water-soluble peptides, and no report has been made on the coupling of the hydrophobic fragments with antimicrobial peptides. The antibacterial and immunomodulatory effects and mechanisms of coupling the hydrophobic fragments with antimicrobial peptides are to be studied. In the study, the cationic micelles formed by DP7-C conjugates successfully realized efficient siRNA introduction to tumor cells, and effectively inhibited the growth of various tumor models to obtain a new siRNA transfer vector. At present, the gene transfer vector based on antimicrobial peptide conjugates has not been reported.

The invention will be described in detail in combination with drawings and embodiment. It should be understood that the examples are only considered to be illustrative for the technical scheme of the invention instead of limitation thereto.

The antimicrobial peptide DP7 and the hydrophobic fragment conjugate were synthesized by the synthetic route as shown in <FIG>, wherein the hydrophobic fragment comprises cholesterol, cholic acid, palmitic acid, stearic acid and lauric acid.

<NUM>-chlorotrityl chloride Resin; Fmoc-Rink Amide MBHA Resin, <NUM>-(<NUM>', <NUM>'-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxyl-acetamido-methylbe nzhydrylamine resin; Fmoc, fluorenylmethoxycarbonyl; pbf, tbu, Otbu, Trt and Boc are all protecting groups named <NUM>, <NUM>, <NUM>, <NUM>, <NUM>-pentamethyldihydrobenzofuran-<NUM>-sulfonyl, tertiary butyl, tert-butoxy, triphenylmethyl, t-butyloxycarboryl respectively.

The specific synthesis method is as follows:.

Swelling: weigh <NUM> of Rink MBHA (<NUM>-(<NUM>',<NUM>'-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxyl-acetamido-met hylbenzhydrylamine resin) resin (substitution value: <NUM>. 36mmol/g) and put into a reaction vessel of a polypeptide synthesizer where the swelling activation is performed by DCM/DMF (<NUM>:<NUM>); shake the reaction vessel up and down to fully swell the resin, and remove the solvent after about <NUM> of the swelling process.

Deprotection: remove the Fmoc protecting group from the resin with <NUM> of <NUM>% Piperdine/DMF (N,N-dimethylformamide) solution; after <NUM> of reaction, wash the resin with DCM/DMF (dichloromethane /N, N-dimethylformamide) alternately for three times; wash a small amount of resin with methanol/DCM/DMF in turn, and then add to an EP tube filled with anhydrous ethanol solution containing <NUM>% ninhydrin; bath the tube in boiling water for three minutes until the resin turns into blue, i.e. positive reaction; then wash twice and continue the next reaction, otherwise continue the deprotection step.

Weigh Fmoc-Lys (Boc)-OH (<NUM>. 44mmol, <NUM>, 4eq), put into a reaction vessel after dissolved in DMF (about <NUM>), add <NUM> of HBTu (benzotriazole-N,N,N',N'-tetramethyluronium hexafluophosphate ) (<NUM>. 44mmol, <NUM>, 4eq) and <NUM> of DIEA (diisopropylethylamine) (<NUM> mmol, <NUM>, 8eq) into the DMF solution, and finally supplement <NUM> of DMF to start reaction. Shake the reaction vessel up and down for <NUM> and remove the reaction fluid. Wash a small amount of resin with CH<NUM>OH/DCM/DMF in turn, and add to an EP tube filled with absolute ethanol solution containing <NUM>% of ninhydrin. Bath the tube in boiling water for three minutes until the resin turns into yellow or light blue, i.e. negative reaction, which indicates that the reaction is completed. Then, wash the resin in the reaction vessel with DCM/DMF alternately for three times, remove the solvent and carry out the subsequent reaction. If the resin turns into dark blue or reddish brown, the reaction is not complete and the condensation reaction should be repeated. But the condensation reaction is generally complete for it is easy to perform. The raw material used in this step is Fmoc-Rink Amide MBHA Resin comprising Rink Amine Linker modified by MBHA Resin linked to Fmoc. In the invention, the substitution degree is <NUM>. 36mmol/g, but other substitution degrees can achieve the same or similar technical effect and are also within the scope of the invention. The substitution degree of <NUM>. 36mmol/g used in the invention is the best value obtained by balancing various factors such as yield, purity, resin utilization rate and the like of synthetic fragments.

After Fmoc is removed from Fmoc-Lys (Boc) -MBHA Resin obtained in step <NUM>, add N,N-dimethylformamide, Fmoc-Arg(pbf)-OH, <NUM>-hydroxybenzotriazole, benzotriazole-N,N,N',N '-tramethyluronium hexafluorophosphate and N,N'-diisopropylethylamine, and allow them to react under nitrogen protection to obtain Fmoc-Arg(pbf)-Lys (Boc) -MBHA Resin. Then connect 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 and Fmoc-Val-OH sequentially according to the same method.

The above synthesis yields a fully protected DP7 sequence peptide.

In case that the condensation reaction is not ideal, the following steps are generally required so as not to affect the subsequent reaction due to the missing peptide: perform the acetylation blocking on the free amino groups, add the prepared blocking solution (acetic anhydride: pyridine: DMF: <NUM>: <NUM>: <NUM>) to the reaction vessel filled with resin, shake the reaction vessel up and down for about <NUM>, and test with ninhydrin. If the resin turns into yellow, the reaction is complete and the subsequent reaction could be carried out. In case of incomplete blocking, the blocking time should be prolonged or the ratio of blocking solution should be adjusted to make the reaction as complete as possible.

Weigh <NUM> (<NUM>. 44mmol, <NUM> eq) of succinylated cholesterol, dissolve in DCM (about <NUM>) and add to the reaction vessel. Then, add <NUM> of HBTu (<NUM>. 44mmol, <NUM>, 4eq) and DIEA (<NUM> mmol, <NUM>, 8eq) into the DMF solution respectively. Shake the reaction vessel up and down for <NUM> and remove the reaction fluid. Wash a small amount of resin with CH<NUM>OH/DCM/DMF in turn, and then add to an EP tube filled with absolute ethanol solution containing <NUM>% of ninhydrin. Bath the tube in boiling water for three minutes until the resin turns into yellow or light blue, i.e. positive reaction, indicating that the reaction is complete. Then, wash the resin in the reaction vessel with DCM/DMF alternately for three times, and remove the solvent. Add the obtained resin into a reaction vessel filled with lysate, seal the reaction vessel, and fix to a cradle for reaction. After about <NUM> of the reaction process, remove the resin by filtration, wash the resin with DCM for several times, collect the filtrate, remove the TFA and solvent by rotary evaporation, add anhydrous ether of ice to the remaining liquid to produce a large amount of white flocculent precipitate, and centrifuge the white precipitate at high speed (4000r/min) to obtain the crude product. The crude product is further purified and refined by HPLC to obtain the target product, i.e. cholesterol-containing DP7 (DP7-C). Its mass spectrum is shown in <FIG>, and the measured molecular weight is as expected.

Weigh <NUM> (<NUM> mmol, <NUM> eq) of cholic acid, dissolve in DCM (about <NUM>), put into a reaction vessel, then add <NUM> of HBTU (<NUM> mmol, <NUM>, <NUM> eq) and DIEA (<NUM> mmol, <NUM>, <NUM> eq) into the DMF solution respectively. Shake the reaction vessel up and down for <NUM> and remove the reaction fluid. Wash a small amount of resin with CH<NUM>OH/DCM/DMF in turn, and then add to an EP tube filled with absolute ethanol solution containing <NUM>% of ninhydrin. Bath the tube in boiling water for three minutes until the resin turns into yellow or light blue, i.e. negative reaction, indicating that the reaction is complete. Then, wash the resin in the reaction vessel with DCM/DMF alternately for three times, and remove the solvent. Add the obtained resin into a reaction vessel filled with lysate, seal the reaction vessel, and fix to a cradle for reaction. After about <NUM> of the reaction process, remove the resin by filtration, wash the resin with DCM for several times, collect the filtrate, remove the TFA and solvent by rotary evaporation, add anhydrous ether of ice to the remaining liquid to produce a large amount of white flocculent precipitate, and centrifuge the white precipitate at high speed (<NUM> r/min) to obtain the crude product. The crude product is further purified and refined by HPLC to obtain the target product, i.e. cholesterol DP7 (DP7-CA).

Weigh <NUM> (<NUM> mmol, <NUM> eq) of stearic acid, dissolve in DCM (about <NUM>), put into a reaction vessel, then add <NUM> of HBTU (<NUM> mmol, <NUM>, <NUM> eq) and DIEA (<NUM> mmol, <NUM>, <NUM> eq) into the DMF solution respectively. Shake the reaction vessel up and down for <NUM> and remove the reaction fluid. Wash a small amount of resin with CH<NUM>OH/DCM/DMF in turn, and then add to an EP tube filled with absolute ethanol solution containing <NUM>% of ninhydrin. Bath the tube in boiling water for three minutes until the resin turns into yellow or light blue, i.e. negative reaction, indicating that the reaction is complete. Then, wash the resin in the reaction vessel with DCM/DMF alternately for three times, and remove the solvent. Add the obtained resin into a reaction vessel filled with lysate, seal the reaction vessel, and fix to a cradle for reaction. After about <NUM> of the reaction process, remove the resin by filtration, wash the resin with DCM for several times, collect the filtrate, remove the TFA and solvent by rotary evaporation, add anhydrous ether of ice to the remaining liquid to produce a large amount of white flocculent precipitate, and centrifuge the white precipitate at high speed (4000r/min) to obtain the crude product. The crude product is further purified and refined by HPLC to obtain the target product, i.e. stearic acid DP7 (DP7-SA). Its mass spectrum is shown in <FIG>, and the measured molecular weight is as expected.

Weigh <NUM> (<NUM> mmol, <NUM> eq) of palmitic acid, dissolve in DCM (about <NUM>), put into a reaction vessel, then add <NUM> of HBTU (<NUM> mmol, <NUM>, <NUM> eq) and DIEA (<NUM> mmol, <NUM>, <NUM> eq) into the DMF solution respectively. Shake the reaction vessel up and down for <NUM> and remove the reaction fluid. Wash a small amount of resin with CH<NUM>OH/DCM/DMF in turn, and then add to an EP tube filled with absolute ethanol solution containing <NUM>% of ninhydrin. Bath the tube in boiling water for three minutes until the resin turns into yellow or light blue, i.e. negative reaction, indicating that the reaction is complete. Then, wash the resin in the reaction vessel with DCM/DMF alternately for three times, and remove the solvent. Add the obtained resin into a reaction vessel filled with lysate, seal the reaction vessel, and fix to a cradle for reaction. After about <NUM> of the reaction process, remove the resin by filtration, wash the resin with DCM for several times, collect the filtrate, remove the TFA and solvent by rotary evaporation, add anhydrous ether of ice to the remaining liquid to produce a large amount of white flocculent precipitate, and centrifuge the white precipitate at high speed (4000r/min) to obtain the crude product. The crude product is further purified and refined by HPLC to obtain the target product, i.e. palmitic acid DP7 (DP7-PA).

Weigh <NUM> (<NUM> mmol, <NUM> eq) of lauric acid, dissolve in DCM (about <NUM>), put into a reaction vessel, then add <NUM> of HBTU (<NUM> mmol, <NUM>, <NUM> eq) and DIEA (<NUM> mmol, <NUM>, <NUM> eq) into the DMF solution respectively. Shake the reaction vessel up and down for <NUM> and remove the reaction fluid. Wash a small amount of resin with CH<NUM>OH/DCM/DMF in turn, and then add to an EP tube filled with absolute ethanol solution containing <NUM>% of ninhydrin. Bath the tube in boiling water for three minutes until the resin turns into yellow or light blue, i.e. negative reaction, indicating that the reaction is complete. Then, wash the resin in the reaction vessel with DCM/DMF alternately for three times, and remove the solvent. Add the obtained resin into a reaction vessel filled with lysate, seal the reaction vessel, and fix to a cradle for reaction. After about <NUM> of the reaction process, remove the resin by filtration, wash the resin with DCM for several times, collect the filtrate, remove the TFA and solvent by rotary evaporation, add anhydrous ether of ice to the remaining liquid to produce a large amount of white flocculent precipitate, and centrifuge the white precipitate at high speed (<NUM> r/min) to obtain the crude product. The crude product is further purified and refined by HPLC to obtain the target product, i.e. lauric acid DP7 (DP7-DA).

The solid-phase peptide synthesis technology is almost mature, and the conjugate of the antimicrobial peptide DP7 and the hydrophobic fragment can also be synthesized by relevant companies. For example, the DP7-C modified by succinylated cholesterol (Chol-suc-VQWRIRVAVIRK-NH<NUM>) is synthesized by Shanghai Ketai Biotechnology Co. based on the solid-phase peptide synthesis method. The synthesized DP7-C is purified by HPLC, with the purity more than <NUM>%; and the molecular weight of DP7-C is determined by MS. The synthesized polypeptide is stored at - <NUM> and prepared into <NUM>/ml of mother liquor by MillQ water for use. For the DP7-C synthesized by the solid-phase peptide synthesis method (Chol-suc-VQWRIRVAVIRK-NH<NUM>), N-terminal is coupled to the hydrophobic cholesterol through ester bonds on the basis of DP7, and C-terminal is connected to a molecule -NH<NUM> for protection. The mass spectrogram of DP7-C is shown in <FIG>. Its molecular weight is <NUM>, and the main peak on MS is <NUM>, indicating that the correct DP7-C is synthesized.

Since the experiments show that the hydrophobically modified DP7 prepared above has similar properties, the following examples are described in detail in combination with cholesterol DP7 (DP7-C).

DP7-C can be self-assembled into micelles in aqueous solution. We detected the critical micelle concentration (CMC) of DP7-C by the pyrene fluorescence probe spectroscopy.

Pyrene is insoluble in water, and its solubility in water is about <NUM>×<NUM>-<NUM>mol/L, but it is easily soluble in ethanol and diethyl ether. The fluorescence emission spectrum of pyrene in aqueous solution has five fluorescence peaks, and the ratio of the first emission spectrum light intensity I1 to the third emission spectrum light intensity I3 in aqueous solution (the ratio of fluorescence intensity at <NUM> to that at <NUM>) is about <NUM>. According to the literature, the surfactant may solubilize nonpolar organic compounds, and the surfactant at different concentrations may solubilize pyrene to varying degrees. Thus, the solubilizing ability of the solution will have an obvious mutation point after the concentration of surfactant exceeds the critical micelle concentration (CMC). If a curve is drawn according to the change of I1/I3 with the surfactant concentration, the midpoint of the curve mutation is the CMC of the substrate to be tested. Therefore, the CMC of pyrene in the solution can be determined by measuring the fluorescence spectra of pyrene in DP7-C solutions with different concentrations. Detection method of CMC: weigh DP7-C and dissolve with Milliq water in <NUM> water bath to obtain DP7-C mother liquor with a concentration of <NUM>/mL. Prepare the pyrene solution of <NUM>×<NUM>-<NUM> mol/L by taking methanol as solvent. After methanol is volatilized and dried in a dark and ventilated place, add <NUM> of <NUM>/mL DP7-C solution, sonicate at <NUM> for <NUM> and shake on a cradle for <NUM>. Then, detect I1 and I3 by a fluorescence spectrophotometer. Add different volumes of sample mother liquor to BD tubes containing trace pyrene to reach the concentrations of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>/ml respectively, and determine the fluorescence spectra of each solution. The critical micelle concentration can be calculated according to the fluorescence spectrum, wherein the excitation wavelength of fluorescence scanning is <NUM>, the emission wavelength is <NUM> and <NUM>, the excitation slit is set to <NUM>, the emission slit is set to <NUM>, and the scanning speed is <NUM>/min.

<FIG> is a schematic diagram of the DP7-C self-assembled into micelles in an aqueous solution, of which the CMC measurement value is <NUM>. 47µg/mL (see <FIG> for the results).

We detected some physical characteristics such as particle size and Zeta potential of DP7-C micelles by the atomic force microscope and Malvin particle size meter.

The results are given in <FIG>. Specifically, the DP7-C micelle is spherical or ellipsoidal (<FIG>), the particle size of DP7-C micelle is about <NUM> ± <NUM> (<FIG>), and Zeta potential is about <NUM> ± <NUM> mV (<FIG>). DP7-C lyophilized powder is white fluffy, soluble in water, and colorless and transparent.

In the hemolysis test, DP7 and DP7-C at the same concentration were detected for the hemolysis of red blood cells.

The calculation formula of the percentage of hemolysis is: <MAT>.

The hemolysis test results are given in <FIG>. When the drug concentration is more than <NUM>/mL, the degree of lysis of DP7-C micelles to red blood cells is much less than DP7, indicating that the antimicrobial peptide DP7 could be coupled to cholesterol for greatly reducing the toxicity to red blood cells. <FIG> visually shows the hemolysis of red blood cells under different conditions. Specifically, in the negative control group (PBS) (group a), no hemolysis is observed and red blood cells are settled at the bottom of the vessel. In the positive control group (<NUM>% Tween-<NUM>) (group b) and DP7 solution group (group c), red blood cells are lysed. In the DP7-C micelles (group d), red blood cells are not lysed but uniformly suspended in the solution due to their micelle characteristics.

The previous experiments show that the cholesterol-containing DP7-C micelles have low antibacterial activity in vitro (MIC><NUM>/L for multiple strains). Further, we detected the antibacterial activity of the intra-abdominally infected mouse and zebrafish model in vivo.

The anti-infection activity of DP7-C is determined by the intra-abdominally infected models. The experimental groups are as follows:.

The therapeutic outcome of DP7-C in treating the abdominally infected mouse model is given in <FIG>. The results show that the average colony forming unit (CFU) of Staphylococcus aureus in the DP7-C group (<NUM>/kg) and the positive drug group (<NUM>/kg VAN) after intravenous administration are significantly lower than those in NS group (p<<NUM>). In addition, the antibacterial effect of DP7-C group is basically the same as that of the positive drug group, with no statistical difference. It shows that DP7-C has a good antibacterial activity in the systemically infected mouse model.

Next, we established an abdominally infected zebrafish model through Pseudomonas aeruginosa with fluorescence (PAO1-GFP) to further verify the anti-infection effect of DP7-C.

The male and female AB wild zebrafish were mated according to the mating and breeding procedures. After spawning, the roe was collected, placed in an incubator at <NUM> and incubated in the seawater containing PTU. The seawater containing PTU was changed once a day. When zebrafish was incubated for <NUM>, Pseudomonas aeruginosa with fluorescence (PAO1-GFP) diluted with DP7-C and normal saline was intraperitoneally injected, and photographs were taken at <NUM>, <NUM> and <NUM> under a fluorescence microscope to observe the growth of PAO1-GFP in the abdomen of zebrafish.

The results are given in <FIG> (A, B). The growth rate of PAO1-GFP in zebrafish abdominal cavity is positively correlated with the total intensity of green fluorescence, and the growth rate of PAO1-GFP of the DP7-C group is much lower than that of NS group, indicating that the DP7-C micelles with low concentration (<NUM>/mL) have good antibacterial activity in zebrafish.

During the detection of DP7-C antibacterial activity, it was found that DP7-C had low in vitro antibacterial activity but high in vivo antibacterial activity. The possible reason lies in that DP7-C has achieved the antibacterial effect by regulating the organism immune system. We detected the effects of DP7-C on the immune function from the cellular level and cytokine level.

Grouping: The mice are randomly divided into two groups (NS group and DP7-C group) with <NUM> mice in each group. The detected immunocytes and their surface markers are shown in the table below:.

Each mouse in the DP7-C group is given 200µL of <NUM>/mL DP7-C, while each mouse in the NS group is injected with 200µL of normal saline. The mice of both groups are killed <NUM> later. Each mouse is intraperitoneally injected with <NUM> of normal saline. By gently massaging the abdomen, the ascites in the abdominal cavity is aspirated to calculate the cell concentration and total cell count there. The lymphocyte typing of the ascites is detected by a flow cytometry, the percentage of macrophages, neutrophils and inflammatory monocytes in total cells is analyzed, and the number of macrophages, neutrophils and inflammatory monocytes is calculated according to the results of flow cytometry and the total number of cells in ascites.

The results are given in <FIG>. After the mice are intraperitoneally injected with DP7-C, the total number of cells in the abdominal cavity is increased significantly and the number of macrophages, neutrophils and monocytes is significantly different. Especially, the percentage of monocytes is increased from <NUM>% to <NUM>% (<FIG>) and the percentage of macrophages is increased from <NUM>% to <NUM>% (<FIG>).

In order to determine the changes of cytokines in mouse PBMC after DP7-C stimulation, the isolated mouse PBMC is diluted to <NUM>×<NUM><NUM> cells/mL and added to a <NUM>-well plate at <NUM>/well, in which the stimulation concentration of DP7-C is 200µg/ml and the stimulation time is <NUM>. After stimulation, the cells are collected and stored in a - <NUM> refrigerator. Meanwhile, in order to verify whether DP7-C can reverse LPS-induced inflammatory reaction, an experiment is set up to stimulate PBMC together with LPS: PBMC is diluted to <NUM>×<NUM><NUM> cells/ml and added to a <NUM>-well plate at <NUM>/well in which the stimulation concentration of DP7-C is 200µg/mL; after <NUM>, stimulated by LPS for <NUM>, and cells are collected and stored in - <NUM> refrigerator. After all samples are collected, the following steps are performed, including centrifugation, washing, total RNA extraction, reverse transcription and real-time quantitative PCR.

The expression of cytokines related to PBMC stimulated by DP7-C is shown in <FIG>. The expression of IL-<NUM>, IL-<NUM>, MCP-<NUM>, M-CSF, TNF-α and other cytokines related to immune activation is greatly increased; meanwhile, when DP7-C and LPS stimulate PBMC together, it is found that the gene expression of major cytokines related to cytokine storm, such as IL-<NUM>, MCP-<NUM> and TNF-α is significantly reduced. This indicates that DP7-C could reduce the degree of injury caused by sepsis and other related infectious diseases.

In previous test, it is found that DP7-C could significantly up-regulate the expression of immune-related cytokines in mouse PBMC. Thus, it is predicted that DP7-C combined with CpG ODNs could be used as a new immunologic adjuvant, and DP7-C could spontaneously form micelles, which also inspires the inventors to use it as a possible substitute for aluminum adjuvant. In this study, the inventors studies the immunomodulatory effect of DP7-C/CpG complex adjuvant, and the antitumor effect in the preventive and therapeutic tumor model of the mouse by taking the OVA as the model antigen.

Therapeutic immune tumor model: subcutaneously inoculate <NUM>×<NUM><NUM> of EG7-OVA tumor cells at the back of each mouse on day <NUM>, and immunize on day <NUM> (once a week, for <NUM> consecutive times). Measure at intervals of <NUM> days and observe the survival time after the tumor grows out.

The results are given in <FIG>. In the preventive model (A and B), the OVA-specific antibodies produced in the DP7-C/CpG group are significantly higher than those produced in other groups on week <NUM>, showing statistical differences. Tumor growth in DP7-C/CpG group is significantly inhibited on day <NUM> after tumor inoculation. In the therapeutic model (C and D), the survival time of mice inoculated with tumor is significantly prolonged in the DP7-C/CpG group and the growth of tumor is greatly inhibited. The results show that DP7-C/CpG is a new immunologic adjuvant with good immunostimulatory activity.

The mechanism of adjuvant-activated immune response probably lies in its promotion to the maturation of DC cells to improve their antigen uptake and processing. We studied the effect of DP7-C micelles on DC activity in vitro.

The maturation of dendritic cells (DC) largely determines the immune response or tolerance of the body. Their surface antigens CD80, CD86 and MHC-II are obviously up-regulated with the maturation of DC. Therefore, we detected the effect of DP7-C adjuvant on dendritic cell maturation by flow cytometry.

The results are given in <FIG>. After DP7-C adjuvant is added to DCS cells, the expression of cell maturation molecule MHC II and co-expressed CD80 and CD86 is increased, and their difference from the NS group is statistically significant.

DC cells are the most powerful antigen presenting cells in the body, which can absorb foreign substances, process and present them to T cells to stimulate immune response. DC cells play an important role in T cell immune response and production of T cell dependent antibody. We examined the effect of DP7-C on DC presenting antigen by flow cytometry.

The results are given in <FIG>. The uptake rate of OVA antigen by DC cells alone is low, and the uptake of OVA by DC cells is slightly enhanced through 40µg/ml of DP7-C to a certain extent. However, <NUM>. 5µg/ml of DP7-C could significantly increase the uptake of antigen by DC cells, and the uptake effect is increased with DP7-C concentration.

Innate immunity plays an important role in anti-tumor immunity. We detected the killing activity of mouse NK cells after immunization with DP7-C adjuvant.

After <NUM> of primary immunization, kill the mice by the cervical dislocation, and isolate splenic lymphocyte from the lymphocyte separation medium as effector cells.

Resuscitate and incubate YAC-<NUM> cells, transfer one day before the experiment, and keep <NUM>×<NUM><NUM>/<NUM>. Stain with trypan blue. Available when activity is more than <NUM>%.

The results are given in <FIG>. Specifically, the NS group does not show NK cell activity basically; the CpG group and the DP7-C group show slightly strong NK cell killing activity; and CpG/DP7-C complex could effectively enhance NK cell activity. The differences among them are significant.

From the above results, we know that DP7-C could be combined with CpG to stimulate the specific immune response of antigen, effectively inhibit tumor growth and metastasis, and prolong the survival time of mice. Moreover, DP7-C and CpG could significantly up-regulate specific antibody titer of OVA antigen and enhance humoral immune response. So we further detected DP7-C activated cellular immune response.

The results are given in <FIG>. The percentage of CD4+/IFN-γ+, CD8+/IFN-γ+ and CD4+/IL-17A+ cells is low in the NS group; while the percentage of such cells in mice immunized with CpG and DP7-C adjuvant is increased. However, the percentage of these three cells in mice spleen immunized with CpG/DP7-C complex is high, and the difference is statistically significant compared with the NS group.

From the above results, we know that the tumor growth of mice immunized with CpG/DP7-C complex is inhibited in the EG7-OVA tumor model. We further used NY-ESO-<NUM> as antigen to form a vaccine with CpG/DP7-C adjuvant, aim at testing whether the vaccine can inhibit the growth of melanoma with high expression of the antigen.

Incubate 20µg of CpG and 40µg of DP7-C at <NUM> for <NUM>, add 5µg of NY-ESO-<NUM> protein, and replenish to a volume of 100µl with sterile PBS.

In this study, animal experiments are divided into <NUM> groups: NS, CpG, DP7-C and CpG/DP7-C, <NUM> mice for each group, and the dosage of each mouse is as follows:.

The total dosage of each mouse is 100µl; if the dosage is less than 100µl, sterile PBS may be added to reach 100µl.

(<NUM>) Preventive immunity tumor model: perform subcutaneous immunotherapy at multiple sites on weeks <NUM>, <NUM> and <NUM>, and detect total antibody titers before inoculate tumor on week <NUM>. Subcutaneously inoculate the tumor cells NY-ESO-<NUM>+B16: <NUM>×<NUM><NUM> at the back of each mouse. After the tumor grows out, measure it at intervals of <NUM> days. Calculation formula of tumor volume: <NUM> × length ×width<NUM>. Therapeutic immune tumor model: subcutaneously inoculate <NUM>×<NUM><NUM> of NY-ESO-<NUM>+B16 tumor cells at the back of each mouse on day <NUM>, and immunize on day <NUM> (once a week, for <NUM> consecutive times). Measure at intervals of <NUM> days and observe the survival time after the tumor grows out.

The results are given in <FIG>. In both preventive model (<FIG>) and therapeutic model (<FIG>), the tumors of NS group mice grow rapidly; the CpG alone or DP7-C adjuvant could inhibit tumor growth to some extent; and the immune CpG/DP7-C complex adjuvant could effectively inhibit the growth of tumor, which is significantly different from NS group.

As the DP7 is positively charged, the DP7-C micelle formed by self-assembly is also positively charged in aqueous solution, showing its potential as a non-viral gene transmission vector, especially siRNA transmission vector. The morphology of DP7-C micelles formed by self-assembly and DP7-C/siRNA complexes is observed under a transmission electron microscopy (H-6009IV, Hitachi, Japan). First, <NUM>\mL of micellar solution is diluted with distilled water and coated on a copper mesh, covered with nitrocellulose, then negatively stained with phosphotungstic acid, dried at room temperature and observed under an electron microscope. The results show that DP7-C micelles are spherical and distributed uniformly. Under the electron microscope, the particle size of DP7-C micelles is about <NUM>, and the particle size of DP7-C micelle/siRNA complex is about <NUM> (see <FIG> for the results).

In <NUM> before transfection, the C26 mouse colon cancer cells or B16 mouse melanoma cells are laid in a <NUM>-well plate with a density of <NUM>× <NUM><NUM> cells per well, and <NUM> of DMEM medium containing <NUM>% FBS is added to each well. FAM-modified no-sense siRNA (FAM-siRNA) is used as a reporter gene to detect transfection efficiency, while PEI25K and Lipofectamin2000 are used as positive controls. During transfection, the medium is firstly replaced with <NUM> of serum-free DMEM medium. Subsequently, the gene transfection complexes mixed at different proportions are added to each well, and each complex contains 1µg of FAM-siRNA. Among them, the ratio of siRNA/DP7-C, siRNA/PEI25K and siRNA/Lipofectamin2000 is <NUM>:<NUM>, <NUM>:<NUM> and <NUM>:<NUM> respectively. After the medium is incubated at <NUM> for <NUM>, it is replaced by DMEM medium supplemented with <NUM>% FBS and sequentially incubated. After <NUM>, the transfection is observed under a microscope and photographed. All cells in the wells including floating and adherent cells are collected, washed twice with precooled PBS, and the total fluorescence intensity of each group is counted by flow cytometer (EPICS Elite ESP, USA).

The transfection results are given in <FIG>. The detection and observation results show that DP7-C could transfect FAM-modified fluorescent siRNA into Ct26 (<FIG>) and B16 (<FIG>) cells with transfection efficiencies of <NUM>±<NUM>% and <NUM>±<NUM>% respectively; the corresponding transfection efficiency of PEI complex is <NUM>±<NUM>% and <NUM>±<NUM>% respectively; and the corresponding transfection efficiency of Lipofectamin2000 complex is <NUM>±<NUM>% and <NUM>±<NUM>% respectively, which could be directly reflected in <FIG>. The results show that DP7-C micelles have higher siRNA transfection efficiency than PEI25K and Lipofectamin2000.

Cytotoxicity of cations is one of the important factors that restrict the application of siRNA transfection. The toxicity of DP7-C micelles to 293T human embryonic kidney cell line, a normal cell, is detected through cell survival experiments. 293T cells are laid in a <NUM>-well plate at a density of <NUM>× <NUM><NUM> cells per well, and 100µL of DMEM/<NUM>% FBS medium is added to each well and incubated for <NUM>. In the experiment, three groups are set up, i.e. DP7-C, PEI25K and Lipofectamin2000; and each group had <NUM> concentration gradients of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>µg/mL respectively. The survival of the cells was detected by the CCK-<NUM> method. After <NUM> of dosing, 10µL of CCK-<NUM> solution is added to each well and incubated at <NUM> for <NUM>. The absorption value of each well is detected at the wavelength of <NUM> and <NUM> by a microplate reader. The standard curve is drawn and IC<NUM> is calculated, and the average value of <NUM> groups of parallel experiments is taken as the final result.

The detection results are given in <FIG>. PEI25K and Lipofectamin2000 are highly toxic with IC<NUM> less than <NUM>µg/mL; while DP7-C micelle has small toxicity with IC<NUM> exceeding <NUM>µg/mL and thus it is safe.

In order to further characterize the application potential of DP7-C micelles in tumor therapy, we established a C26 mouse colon cancer peritoneal metastasis model and used DP7-C to transmit anti-VEGF siRNA therapeutic genes for therapeutic research.

<FIG> shows the therapeutic effect of DP7-C/VEGF siRNA complex which is intraperitoneally injected to treat abdominal metastasis C26 tumor model. <FIG> is a photograph of abdominal cavity of a representative mouse in each group of animals. Among them, the average tumor weight is <NUM>±<NUM> for the DP7-C/VEGF siRNA complex treatment group, <NUM>±<NUM> for the normal saline control group, <NUM>±<NUM> for the no-load DP7-C group, and <NUM>±<NUM> for the DP7-C/no-sense control siRNA complex group (<FIG>). The number of metastatic tumor nodule in abdominal cavity is significantly smaller in the mice treated with DP7-C/VEGF siRNA complex, compared with other treatment groups. Therefore, tumor growth in mice treated with DP7-C/VEGF siRNA complex is greatly inhibited.

Meanwhile, as shown in <FIG>, the volume of ascites produced by mice in each group is significantly different. The average tumor weight is <NUM>±<NUM> for the DP7-C/VEGF siRNA complex treatment group, <NUM>±<NUM> for the normal saline control group, <NUM>±<NUM> for the blank DP7-C micelle group, and <NUM>±<NUM> for the DP7-C/no-sense control siRNA complex group. For mice in each group that are not treated with DP7-C/VEGF siRNA complex, the ascites shows obvious blood red, confirming that mice in these groups have serious mesenteric injury. Compared with other experimental groups, DP7-C/VEGF siRNA complex effectively inhibits the growth of C26 peritoneal metastasis tumor model and the production of accompanying serious tumor infiltration and inflammation.

In addition, CD31 immunohistochemical staining results show that DP7-C/VEGF siRNA complex treatment group has fewer new blood vessels in tumor tissue than other three experimental groups, confirming that DP7-C could effectively inhibit tumor tissue angiogenesis by transmitting anti-VEGF siRNA (<FIG>). Meanwhile, HE staining results show (<FIG>) that no significant pathological changes are observed in major organ tissues after DP7-C micelles have been intraperitoneally injected, confirming that DP7-C has few side effects.

In order to further characterize the application potential of DP7-C micelles in tumor therapy, we established a subcutaneously implanted tumor model of C26 mouse colon cancer and used DP7-C to transmit anti-VEGF siRNA therapeutic genes for therapeutic research.

<FIG> shows the therapeutic effect of DP7-C/VEGF siRNA complex which is intratumorally injected to treat C26 subcutaneously implanted tumor model. <FIG> shows a photograph of mouse subcutaneous tumors in each group of animals. Among them, the average tumor weight is <NUM>±<NUM><NUM> for the DP7-C/VEGF siRNA complex treatment group, <NUM>±<NUM><NUM> for the normal saline control group, <NUM>±<NUM><NUM> for the blank DP7-C micelle group, and <NUM>±<NUM><NUM> for the DP7-C/no-sense control siRNA complex group. The volume of subcutaneous tumor of mice treated with DP7-C/VEGF siRNA complex is significantly smaller than that of other treatment groups (<FIG>). Therefore, tumor growth in mice treated with DP7-C/VEGF siRNA complex is greatly inhibited.

In addition, CD31 immunohistochemical staining results (<FIG>) show that DP7-C/VEGF siRNA complex treatment group has fewer new blood vessels in tumor tissue than other three experimental groups, confirming that DP7-C could effectively inhibit tumor tissue angiogenesis by transmitting anti-VEGF siRNA. Meanwhile, HE staining results (<FIG>) show that no significant pathological changes are observed in major organ tissues after DP7-C micelles have been intraperitoneally injected, confirming that DP7-C has few side effects.

In order to further characterize the application potential of DP7-C micelles in tumor therapy, we established a B16 mouse model of metastatic melanoma in the lung and used DP7-C to transmit anti-VEGF siRNA therapeutic genes for therapeutic research.

<FIG> shows the therapeutic effect of DP7-C/VEGF siRNA complex which is intravenously injected to treat B16 mouse model of metastatic melanoma in the lung. <FIG> shows a photograph of lung tumors in mouse in each group of animals. Among them, the average number of tumor nodes is <NUM>±<NUM> for the DP7-C/VEGF siRNA complex treatment group, <NUM>±<NUM> for the normal saline control group, <NUM>±<NUM> for the blank DP7-C micelle group, and <NUM>±<NUM> for the DP7-C/no-sense control siRNA complex group. In addition, the average weight of lung tissue is <NUM>±<NUM> for the DP7-C/VEGF siRNA complex treatment group, <NUM>±<NUM> for the normal saline control group, <NUM>±<NUM> for the blank DP7-C micelle group, and <NUM>±<NUM> for the DP7-C/no-sense control siRNA complex group. The number of metastatic tumor lung nodules in the lung of mice treated with DP7-C/VEGF siRNA complex is significantly less than that in other treatment groups (<FIG>), and the average lung weight of mice is significantly lighter than that in other treatment groups (Fig. 19b). Therefore, tumor growth in mice treated with DP7-C/VEGF siRNA complex is greatly inhibited.

Claim 1:
A hydrophobically modified antimicrobial peptide, characterized in that the hydrophobic modification is to couple a hydrophobic fragment at the nitrogen terminal of the antimicrobial peptide, and the amino acid sequence of the antimicrobial peptide is VQWRIRVAVIRK,
characterized in that,
the hydrophobic fragment is succinylated cholesterol, and
the nitrogen terminal of the antimicrobial peptide is coupled to the hydrophobic segment by the amidation reaction of -CO-OH on the hydrophobic segment with -NH<NUM> on the antimicrobial peptide.