WO2018130000A1 - 可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂及其制备方法与应用 - Google Patents

可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂及其制备方法与应用 Download PDF

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WO2018130000A1
WO2018130000A1 PCT/CN2017/110896 CN2017110896W WO2018130000A1 WO 2018130000 A1 WO2018130000 A1 WO 2018130000A1 CN 2017110896 W CN2017110896 W CN 2017110896W WO 2018130000 A1 WO2018130000 A1 WO 2018130000A1
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self
penetrating
penetrating peptide
tandem
antibacterial agent
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PCT/CN2017/110896
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French (fr)
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何道航
何碧
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华南理工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to a tandem penetrating peptide, in particular to a self-assembled tandem penetrating peptide nanoparticle antibacterial agent capable of penetrating the blood brain barrier, and a preparation method and application thereof, the application of the nanoparticle antibacterial agent in the preparation of a brain infection and other Use in bacterial or fungal infections.
  • Brain infection has long been one of the most important causes of infectious death, and can be caused by bacteria such as Staphylococcus aureus, Escherichia coli and fungi such as Candida albicans.
  • bacteria such as Staphylococcus aureus, Escherichia coli and fungi such as Candida albicans.
  • high mortality may occur within a few hours, and survivors may suffer from permanent visual impairment, hearing loss, neurological dysfunction and mobility impairment.
  • patients with brain infections still have high morbidity and mortality due to the difficulty of penetrating the blood-brain barrier into the cerebrospinal fluid and brain tissue.
  • NP1 or NP2
  • NP1 is the first transmembrane peptide found in human NLBP protein by Sangho Lim et al in 2015. Very strong membrane and protein transport, and its sequence exists in many species.
  • Self-assembling peptide nanoparticles are expected to become new antibacterial drugs due to their broad-spectrum antibacterial activity and ability to inhibit drug-resistant microorganisms. Therefore, it is meaningful to develop new antibacterial drugs by improving transmembrane peptides.
  • the object of the present invention is to provide a self-assembled tandem penetrating peptide nanoparticle antibacterial agent which can penetrate the blood-brain barrier, and the invention is based on the fact that the transmembrane peptides NP1 (or NP2) and R 9 (or K 9 ) are connected in series and modified. Self-assembling tandem penetrating peptide nanoparticle antibacterial agent that can penetrate the blood-brain barrier, and which can overcome bacterial resistance and can be used for treating brain infections and other infectious diseases.
  • the structure of the antibacterial agent is C n -XY, wherein X and Y are both penetrating peptides, and X and Y are connected in series to form a hydrophilic part
  • C n represents a hydrophobic moiety fatty acid chain coupled to a penetrating peptide
  • the penetrating peptide is an L-form amino acid
  • the number of carbon atoms of the fatty acid chain is 12-20;
  • the X is a cationic penetrating peptide
  • the Y is NP1 or NP2; the NP1 sequence is KKDKKDERRRK; and the NP2 sequence is KIKKVKKKGRK.
  • the fatty acid is lauric acid, myristic acid, palmitic acid, stearic acid or arachidic acid.
  • the cationic penetrating peptide is nonameric arginine R 9 or nona-lysine K 9 .
  • the self-assembled tandem membrane-penetrating peptide nanoparticle antibacterial agent has an average diameter of from 100 to 500 nm.
  • the preparation method of the self-assembled tandem penetrating peptide nanoparticle antibacterial agent capable of penetrating the blood brain barrier comprises the following steps:
  • the Fmoc solid phase synthesis method was used to prepare the tandem penetrating peptide, the resin was firstly swelled, washed and deprotected, and the first Fmoc-amino acid and HoBt were condensed, deprotected and washed, and the deprotection was confirmed by sputum detection; The second amino acid is then condensed and the above steps are repeated from the C-terminus to the N-terminus until the polypeptide chain is synthesized;
  • the self-assembled tandem penetrating peptide nanoparticle antibacterial agent capable of penetrating the blood brain barrier is used for preparing anti-S. aureus, Escherichia coli, methicillin-resistant Staphylococcus aureus or Candida albicans drugs.
  • the self-assembled tandem penetrating peptide nanoparticle antibacterial agent capable of penetrating the blood brain barrier is used for preparing a medicament for treating a Staphylococcus aureus infection in the brain.
  • the self-assembling tandem penetrating peptide nanoparticle antibacterial agent that can penetrate the blood-brain barrier is prepared by coupling a fatty acid with the N-terminus of a cation penetrating peptide in a tandem penetrating peptide.
  • C n preferably palmitic acid
  • self-assembled nanoparticle series penetratin antibacterial structure C 16 -R 9 -NP1, C 16 -K 9 -NP1, C 16 -R 9 -NP2, C 16 -K 9 - NP2.
  • Any of the self-assembled tandem membrane-penetrating peptide nanoparticle antibacterial agents having an average diameter of from about 100 to about 500 nm.
  • the present invention has the following advantages and beneficial effects:
  • the present invention provides novel self-assembling nanopolypeptides that increase the type of self-assembling nanopolypeptides.
  • the N-terminus of the self-assembled tandem membrane-penetrating peptide nanoparticle antibacterial agent of the present invention is modified with a fatty acid to increase hydrophobicity, thereby facilitating the amphiphilicity of the peptide and facilitating self-assembly. It can self-assemble to form nanoparticles at a very low peptide concentration in pure water, without mechanical action such as vigorous stirring, sonication, and has good stability.
  • the self-assembled tandem-penetrating peptide nanoparticle antibacterial agent of the invention has good antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, fungi and drug-resistant bacteria, and the antibacterial effect thereof is far stronger than a single one. Transmembrane peptide. Therefore, it can be applied to the development of a novel nano antibacterial drug suitable for clinical use, which is beneficial to the treatment of microbial infectious diseases.
  • the self-assembled tandem membrane-penetrating peptide nanoparticle antibacterial agent comprises tandem penetrating peptides, and the self-assembled tandem penetrating peptides are distributed on the surface of the nanoparticles, which is beneficial to increase the permeability of the cell membrane and make self-assembly.
  • Transmembrane peptide nanoparticles can cross the blood-brain barrier.
  • the self-assembled tandem membrane-penetrating peptide nanoparticle antibacterial agent of the present invention can be used for preparing a medicament for treating a brain infection disease, and effectively inhibiting microorganisms in infected brain tissue.
  • Example 1 is a schematic view showing the three-dimensional molecular structure of the self-assembled tandem penetrating peptide obtained in Example 1.
  • Example 2 is a high performance liquid chromatogram of the self-assembled tandem penetrating peptide obtained in Example 1, and the result showed that its purity was 95.89%.
  • Figure 3 is a matrix-assisted laser desorption ionization time-of-flight mass spectrum of the self-assembled tandem penetrating peptide obtained in Example 1, which showed a molecular weight of 2984.6.
  • Example 4 is a molecular dynamics simulation diagram of self-assembly of self-assembled tandem penetrating peptides into nanoparticles in Example 3.
  • Figure 5 is a scanning electron micrograph of the self-assembled tandem membrane-penetrating peptide in Example 3 in an ultrapure aqueous solution, wherein the concentration of the peptide sample was 0.5 mg/ml.
  • Figure 6 is a diagram showing the microbial topography of the self-assembled tandem membrane-penetrating peptide nanoparticles before and after treatment in Example 4.
  • Figure 7 is a graph showing the hemolysis effect of self-assembled tandem membrane-penetrating peptide nanoparticles on human erythrocytes in Example 4.
  • Figure 8 is a diagram showing the matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of rat cerebrospinal fluid samples after self-assembly of tandem penetrating peptide nanoparticles by intravenous injection in Example 5.
  • Figure 9 is a graph showing the number of bacterial colonies in infected brain tissue after self-assembly of tandem penetrating peptide nanoparticles in Example 5.
  • the self-assembled tandem penetrating peptide nanoparticle antibacterial agent has the structural formula: C 16 -RRRRRRRRRKIKKVKKKGRK. The synthesis is as follows:
  • Palmitic acid Fmoc ⁇ Arg(pbf) ⁇ OH (N ⁇ methoxycarbonyl-2-2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonylarginine), Fmoc ⁇ Lys(Boc) ⁇ OH(N ⁇ methoxycarbonyl-N′ ⁇ tert-butoxycarbonyl-lysine), Fmoc ⁇ Ile ⁇ OH (N ⁇ methoxycarbonyl-isoleucine), Fmoc ⁇ Val ⁇ OH (N ⁇ methoxycarbonyl-proline), Fmoc ⁇ Gly ⁇ OH (N ⁇ methoxycarbonyl-glycine), Rink Amide-MBHA Resin, DBLK (hexahydropyridine + DMF), HBTU (O-benzotriazole-1-yl-N, N, N, N-tetramethyluron hexafluorophosphate) and HOBT (1-hydroxybenzotriazole); piperidine, acetic anhydride
  • the reaction was shaken for 30 min. After the reaction was completed, the resin was washed four times with 300 ml of DMF, and each washing time was 2 minutes, and a small amount of resin was taken for ninhydrin test, and the resin was negative.
  • Palmitic acid (51.23 g) was coupled with the arginine N-terminus of the cationic transmembrane peptide R 9 to obtain C 16 R 9 NP 2 as follows: Palmitic acid dissolved in 15 mL of DMF was slowly added to 5 mL of DMF containing 70 ⁇ L of triethylamine and tandem penetrating peptide at 0 °C with stirring. After 24 hours of reaction, DMF was removed from the mixture by purging dry nitrogen, and then the mixture was washed 3 times with diethyl ether to remove unreacted palmitic acid, i.e., N-terminal modification was completed.
  • a membrane having a molecular weight cutoff of 1000 Da was dialyzed against DMF for 6 days to further purify the crude product. DMF is then removed by vacuum drying to produce the final product to obtain the final product C 16 R 9 NP2. Successful synthesis of C 16 R 9 NP2 was confirmed by HPLC and MALDI-TOF analysis (see below).
  • the purity of the synthesized C 16 R 9 NP 2 was determined by reversed-phase high performance liquid chromatography HPLC.
  • a Cromasil-C-18 column (5 ⁇ m) was used as a stationary phase, and a mixture of acetonitrile and water was used as a mobile phase, and a peak shape of 0-30 min was recorded.
  • the detection results are shown in Fig. 2.
  • the corresponding peak at 14.032min is the target product peak, and the purity is determined to be 95.89% according to the percentage of peak area.
  • the other fatty acid-modified blood-brain barrier-permeable self-assembling tandem penetrating peptide nanoparticle antibacterial agent of the present invention can be replaced by Fmoc (fluorenylmethoxycarbonyl)-protected solid phase by replacing the amino acid and fatty acid in Example 1.
  • Fmoc fluorenylmethoxycarbonyl
  • the resin was swollen, and then washed with DMF, and the resin was washed by adding a piperidine/DBLK shaking reaction, and the amino acid raw material, HBTU, HOBT, and the like were added after the ninhydrin test was positive. NMM, DMF, the resin was washed with DMF after the shock reaction, and the ninhydrin test was negative. Then, according to the amino acid sequence of different tandem penetrating peptides, the different amino acid starting materials (from C-terminal to N-terminal) are sequentially added as described in the preparation method (2) of Example 1, and the shaking reaction, washing, and sputum are repeated. Ketone detection step.
  • the synthesis of the tandem penetrating peptide was completed by deprotection, DMF washing, lysate cleavage, diethyl ether precipitation, and purification.
  • the fatty acid was added to the DMF solution of triethylamine and tandem penetrating peptide in accordance with the preparation method (4) of Example 1, and the N-terminal modification was carried out by purging with nitrogen and washing with diethyl ether. Purification by dialysis membrane, drying to remove DMF to obtain the final product, that is, tandem transmembrane peptide modified with different fatty acids.
  • the product obtained by reversed-phase high performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry was further carried out according to the detection method in Example 1.
  • the fatty acid is preferably palmitic acid
  • the sequence is as follows: C 16 -R 9 -NP1, C 16 -K 9 -NP1 , C 16 -R 9 -NP 2 , C 16 -K 9 -NP 2 .
  • Table 1 The different synthetic experimental parameters and test results in this process are shown in Table 1 below:
  • C 16 R 9 NP2 The self-assembly morphology of C 16 R 9 NP2 in aqueous solution was observed by cold field emission scanning electron microscope (JSM-6330F).
  • C 16 R 9 NP2 was formulated into a 0.5 mg/mL peptide solution with ultrapure water, and 20 ⁇ L of the peptide solution was placed on a mica plate and allowed to air dry at room temperature. The mica sheet was fixed on an aluminum column and then gold plated for observation.
  • Figure 5 is a scanning electron micrograph of an aqueous solution of C 16 R 9 NP2 at an acceleration voltage of 15.0 keV at 14000 times. The results show that the palmitic acid modified tandem penetrating peptide C 16 R 9 NP2 self-assembles in water to form particles smaller than 150 nm in diameter. .
  • the N-terminus of the self-assembled tandem membrane-penetrating peptide nanoparticle antibacterial agent is modified with a fatty acid to increase hydrophobicity, thereby facilitating the amphiphilicity of the peptide and facilitating self-assembly. It can self-assemble to form nanoparticles at a very low peptide concentration in pure water, without mechanical action such as vigorous stirring, sonication, and has good stability.
  • the tested strains were all from the Guangdong microbial germplasm resource bank.
  • Staphylococcus aureus ATCC 29213, Escherichia coli CMCC 44825, methicillin-resistant Staphylococcus aureus ATCC 43300 was cultured in a nutrient agar medium at 37 °C.
  • Candida albicans ATCC 10231 was cultured in potato glucose medium at 37 °C.
  • the minimum inhibitory concentration MIC of the peptide nanoparticles was determined using a microdilution method.
  • the minimum inhibitory concentration of Staphylococcus aureus was 8.4 ⁇ M, and the minimum inhibitory concentration against Candida albicans was 50.3 ⁇ M.
  • the single transmembrane peptide R 9 that is not self-assembled has extremely low inhibition ability for testing bacteria and fungi, and exhibits an extremely high minimum inhibitory concentration.
  • the formation of the nanoparticles significantly enhances the antibacterial ability of the peptide and reduces the minimum inhibitory concentration. Therefore, the self-assembled tandem penetrating peptide nanoparticles of the present invention can be developed into an effective antibacterial agent for inhibiting the proliferation of microorganisms.
  • the cultured bacterial solution and the bacterial solution treated with tandem penetrating peptide nanoparticles were centrifuged at 6000 rpm for 10 min, and the cells were washed three times with phosphate buffered saline (PBS), and then fixed overnight in PBS containing 5% formaldehyde. Dehydration was carried out with a series of concentration gradients of ethanol, then lyophilized and fixed on an aluminum column. The samples were coated with gold prior to SEM analysis.
  • PBS phosphate buffered saline
  • a is a blank control of Staphylococcus aureus, the cell structure is full and complete, the cell surface is smooth and round;
  • b is Staphylococcus aureus treated by tandem penetrating peptide nanoparticles (minimum inhibitory concentration), Significant changes in morphology, obvious cell rupture and cell debris were observed;
  • c was a blank control of Candida albicans with a smooth cell surface;
  • d was treated with tandem penetrating peptide nanoparticles (minimum inhibitory concentration) Candida albicans, its morphology changed significantly, showing cell membrane dissolving, cell lysis and death.
  • Microorganism morphology changes observed with a scanning electron microscope to verify the present invention which is series penetratin peptide nanoparticle antibacterial C 16 R 9 NP2, indicating its main mechanism for the destruction of microbial cell structure.
  • Fresh human erythrocytes were washed with PBS and diluted to 4% by volume. 100 ⁇ L of red blood cell suspension was placed in each well of a 96-well plate, and then 100 ⁇ L of peptide nanoparticles or amphotericin was added to each well. Solution B (water soluble), incubated at 37 ° C for 1 hour. Thereafter, the cell suspension was taken out and centrifuged at 1000 g to obtain an aliquot of the supernatant (100 ⁇ L), transferred to a 96-well plate, and hemoglobin release was monitored at 540 nm using a microplate reader.
  • Hemolysis (%) [(nano-particles in solution OD OD 540nm 540nm -PBS in) / (in 0.1% TritonX-100 OD OD 540nm -PBS of 540nm)] x100
  • the results are shown in Figure 7.
  • the nanoparticles have a low hemolysis effect.
  • the minimum inhibitory concentration against Escherichia coli and methicillin-resistant Staphylococcus aureus has a hemolysis effect of less than 4%; at 50 mg/L, gold
  • the tandem penetrating peptide nanoparticles had a lower hemolytic effect than the positive control drug amphotericin (water-soluble). It is clinically considered that the medical material with less than 5% hemolysis is safe. Therefore, the low hemolysis of the self-assembled tandem membrane-penetrating peptide nanoparticle C 16 R 9 NP2 shows its safety as an effective antibacterial agent and can be used for infection. Treatment of the disease.
  • Self-assembled tandem penetrating peptide nanoparticle antibacterial agent C 16 R 9 NP2 penetrates blood-brain barrier detection and its application in the treatment of brain infection
  • the experimental procedure is referred to by Yi-Yan Yang et al. (Self-assembled etching peptide nanoparticles as an efficient antimicrobial agent, Nature nanotechnology, 2009).
  • 50 ⁇ L of Staphylococcus aureus solution was injected into the medullary sac of SD rats, and C 16 R 9 NP2 nanoparticle solution was injected every 12 hours through the tail vein.
  • the blank control group was injected with physiological saline, and the positive control group was injected with vancomycin solution.
  • the rat brain tissue was weighed and homogenized, and 100 ⁇ L of the homogenate was applied to the nutrient agar medium to calculate the colony number, that is, the colony forming unit, expressed as lg colony forming unit/gram brain tissue.
  • the results are shown in Fig. 9.
  • the number of colonies in rat brain tissue was significantly lower than that in the blank control group (ie, untreated group), which was similar to that in the amphotericin-treated group, indicating that it was in the treated rats.
  • Significant effects have been achieved in S. aureus infection, which inhibits bacterial growth. Therefore, the self-assembled tandem penetrating peptide nanoparticles of the present invention have a good application prospect in the preparation of an antibacterial agent for treating brain infection diseases, and can effectively inhibit the growth of infected brain bacteria.

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Abstract

本发明公开了可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂及其制备方法与应用。该抗菌剂的结构通式为Cn-X-Y，其中X和Y均为穿膜肽，且X和Y串联形成亲水部分；Cn表示与穿膜肽偶联的疏水部分脂肪酸链；穿膜肽为L型氨基酸；所述脂肪酸链的碳原子个数为12-20。本发明所述自组装串联穿膜肽纳米颗粒具有较好的抗细菌(包括耐药细菌)和抗真菌作用，其能透过血脑屏障，且溶血毒性较低，可用于研制治疗脑部感染及其他感染疾病的新型抗菌药物。

Description

可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂及其制备方法与应用 技术领域
本发明涉及串联穿膜肽，特别是涉及可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂及其制备方法与应用，该应用涉及纳米颗粒抗菌剂在制备治疗脑部感染及其他细菌或真菌感染疾病药物中的应用。
背景技术
脑部感染长期作为最重要的感染性致死原因之一，可由细菌如金黄色葡萄球菌、大肠杆菌和真菌如白色念珠菌引起。在急性脑膜炎患者中，即使细菌是敏感菌，并且很早给予抗生素治疗，高死亡率仍可能发生在几小时内，且幸存者可能遭受永久性视觉损害、听力丧失、神经功能障碍及行动障碍。尽管抗生素治疗的重大进展，由于药物很难穿透血脑屏障进入脑脊液和脑组织，脑部感染患者仍然存在很高的致病率和死亡率。临床上只有少数抗生素可用于治疗脑部感染，但其对中枢神经系统及肝肾等器官的较高的毒性限制了其使用。影响治疗效果的另一个因素是近年来不断加强的细菌耐药性及增多的耐药菌株。
近年来，穿膜肽因其超强的穿膜能力及携带分子进入细胞的载体功能，引起了广泛关注。聚精氨酸R9(或聚赖氨酸K9)是有名的穿膜肽之一，而NP1(或NP2)是2015年Sangho Lim等从人NLBP蛋白中首次发现的穿膜肽，其有极强的穿膜及蛋白运输作用，且其序列存在于许多物种体内。
自组装肽纳米颗粒因其广谱抗菌活性和抑制耐药微生物的能力，有望成为新型的抗菌药物。因此，通过改进穿膜肽开发新型抗菌药物十分有意义。
发明内容
本发明的目的在于提供可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂，发明基于将穿膜肽NP1(或NP2)及R9(或K9)串联并进行修饰，设计了可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂，且其能克服细菌耐药性，可用于治疗脑部感染及其他感染疾病。
本发明的目的通过以下技术方案实现：
可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂：该抗菌剂的结构通 式为Cn-X-Y，其中X和Y均为穿膜肽，且X和Y串联形成亲水部分；Cn表示与穿膜肽偶联的疏水部分脂肪酸链；穿膜肽为L型氨基酸；所述脂肪酸链的碳原子个数为12-20；
所述X为阳离子穿膜肽；
所述Y为NP1或NP2；所述NP1序列为KKDKKDERRRK；所述NP2序列为KIKKVKKKGRK。
为进一步实现本发明目的，优选地，所述脂肪酸为月桂酸、肉豆蔻酸、棕榈酸、硬脂酸或花生酸。
优选地，所述阳离子穿膜肽为九聚精氨酸R9或九聚赖氨酸K9。
优选地，所述自组装串联穿膜肽纳米颗粒抗菌剂具有100-500nm的平均直径。
所述可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂的制备方法，包括如下步骤：
1)采用Fmoc固相合成法制备串联穿膜肽，先溶涨树脂，洗涤并脱保护后，将第一个Fmoc-氨基酸和HoBt进行缩合，脱保护并洗涤后，茚检确认脱保护完全；然后缩合第二个氨基酸，重复以上步骤，由C-端向N-端，直至多肽链合成完成；
2)将合成完成的多肽链用裂解液裂解，减压过滤后用冷乙醚沉淀获得粗肽，并通过液相色谱进一步纯化；
3)脂肪酸与串联穿膜肽中阳离子穿膜肽的N-端偶联，将棕榈酸加至含有三乙胺和串联穿膜肽的DMF中搅拌反应后，通氮气去除DMF，用二乙醚清洗，并使用透析膜进一步纯化粗产物。
所述可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂在制备抗金黄色葡萄球菌、大肠杆菌、耐甲氧西林金葡菌或白色念珠菌药物中的应用。
所述可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂在制备治疗脑部金黄色葡萄球菌感染药物中的应用。
所述可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂的制备方法是将脂肪酸与串联穿膜肽中阳离子穿膜肽的N-端偶联。Cn优选棕榈酸时，自组装串联穿膜肽纳米颗粒抗菌剂结构为：C16-R9-NP1、C16-K9-NP1、C16-R9-NP2、 C16-K9-NP2。任意一项自组装串联穿膜肽纳米颗粒抗菌剂，具有约100到约500nm的平均直径。
相对于现有技术，本发明具有如下优点和有益效果：
(1)本发明提供了新型的自组装纳米多肽，增加了自组装纳米多肽的类型。
(2)本发明自组装串联穿膜肽纳米颗粒抗菌剂N端以脂肪酸修饰以增加疏水性，便于提高肽的两亲性，更易于自组装的进行。其在纯水中即可在很低的肽浓度下自组装形成纳米颗粒，无需剧烈搅拌、超声处理等机械作用，且具有较好的稳定性。
(3)本发明自组装串联穿膜肽纳米颗粒抗菌剂对革兰氏阳性细菌、革兰氏阴性菌，真菌及耐药细菌均有较好的抑菌作用，且其抗菌效果远强于单一的穿膜肽。因此，可应用于研制适于临床上使用的新型纳米抗菌药物，有利于微生物感染疾病的治疗。
(4)本发明自组装串联穿膜肽纳米颗粒抗菌剂中包含了串联的穿膜肽，且自组装后串联穿膜肽分布在纳米颗粒表面，有利于增加细胞膜的通透性，使得自组装穿膜肽纳米颗粒能穿过血脑屏障。
(5)本发明自组装串联穿膜肽纳米颗粒抗菌剂可用于制备治疗脑部感染疾病药物，有效抑制受感染的脑组织内的微生物。
附图说明
图1是实施例1所得自组装串联穿膜肽的三维分子结构示意图。
图2是实施例1所得自组装串联穿膜肽的高效液相色谱图，结果显示它的纯度是95.89％。
图3是实施例1所得自组装串联穿膜肽的基质辅助激光解吸电离飞行时间质谱图，结果显示它的分子量为2984.6。
图4是实施例3中自组装串联穿膜肽自组装成纳米颗粒的分子动力学模拟图。
图5是实施例3中自组装串联穿膜肽在超纯水溶液中的扫描电镜纳米形貌图，其中肽样品的浓度是0.5mg/ml。
图6是实施例4中自组装串联穿膜肽纳米颗粒处理前后的微生物形貌图。
图7是实施例4中自组装串联穿膜肽纳米颗粒对人红细胞的溶血作用图。
图8是实施例5中，通过静脉注射自组装串联穿膜肽纳米颗粒后，大鼠脑脊液样品的基质辅助激光解吸电离飞行时间质谱分析图。
图9是实施例5中，通过自组装串联穿膜肽纳米颗粒治疗后，受感染的脑组织中的细菌菌落数。
具体实施方式
为了更好的理解本发明，下面结合附图和实施例对本发明作进一步说明，但本发明要求保护的范围并不局限于实施例表示的范围。
实施例1
当X为九聚精氨酸R9，Y为NP2，Cn为棕榈酸长链时，自组装串联穿膜肽纳米颗粒抗菌剂的结构通式为：C16-R-R-R-R-R-R-R-R-R-K-I-K-K-V-K-K-K-G-R-K。其合成方式如下：
1、材料
棕榈酸、Fmoc‐Arg(pbf)‐OH(N‐芴甲氧羰酰基‐2，2，4，6，7‐五甲基二氢苯并呋喃‐5‐磺酰精氨酸)、Fmoc‐Lys(Boc)‐OH(N‐芴甲氧羰酰基‐N'‐叔丁氧羰酰基‐赖氨酸)、Fmoc‐Ile‐OH(N‐芴甲氧羰酰基‐异亮氨酸)、Fmoc‐Val‐OH(N‐芴甲氧羰酰基‐缬氨酸)、Fmoc‐Gly‐OH(N‐芴甲氧羰酰基‐甘氨酸)、Rink Amide一MBHA Resin、DBLK(六氢吡啶+DMF)、HBTU(O‐苯并三唑‐1‐基‐N、N、N、N‐四甲基尿六氟磷酸脂)和HOBT(1‐羟基苯并三氮唑)；哌啶、醋酸酐、DMF(N、N‐二甲基甲酰胺)、TFA(三氟乙酸)、NMM(N‐甲基吗啉)、乙醚、甲醇、DCM(二氯甲烷)。
2、制备方法
采用Fmoc(芴甲氧羰酰基)保护的固相合成法，其工艺步骤如下：
(1)称取20g 0.5mmol/g的Rink Amide-MBHA Resin(树脂)于肽合成器皿中，取200ml DCM溶涨树脂30min，然后抽滤，再取用300ml DMF洗涤树脂，分三次进行，每次的洗涤时间为2min，抽滤干洗涤液后向肽合成器中加入100ml哌啶/DBLK(体积比20％)震荡反应30min，反应结束后，再抽滤出反应液，再用400mlDMF分四次洗涤树脂，洗毕后取少量树脂做茚三酮检测试验，树脂呈阳性，向肽合成器皿中加入以下原料：
Figure PCTCN2017110896-appb-000001
上述原料加完后，震荡反应30min，反应结束后，用300ml DMF分四次洗涤树脂，每次洗涤时间2分钟，取少许树脂做茚三酮试验检测，树脂呈阴性。
(2)向肽合成器皿中加入5ml哌啶/DBLK(体积比20％)震荡反应30min，反应结束后抽滤出反应液，再用40ml DMF分四次洗涤树脂，洗毕取少许树脂做茚三酮试验检测，结果树脂呈阳性，向反应器皿中加入以下原料：
Figure PCTCN2017110896-appb-000002
上述原料加完后，震荡反应40min，反应结束后，用40ml DMF分四次洗涤树脂，每次洗涤时间为2min，取少许树脂做茚三酮检测，树脂呈阴性。(3)变换步骤(2)中的(a)原料，保持(b)(c)(d)(e)原料及加入量不变，重复步骤(2)的操作，由C-端向N-端依次缩合氨基酸。即(a)原料依次替换为Fmoc-Gly-OH(9.37g)、Fmoc-Lys(Boc)-OH(18.74g)、Fmoc-Lys(Boc)-OH(18.74g)、Fmoc-Lys(Boc)-OH(18.74g)、Fmoc-Val-OH(13.58g)、Fmoc-Lys(Boc)-OH(18.74g)、Fmoc-Lys(Boc)-OH(18.74g)、Fmo-Ile-OH(14.14g)、Fmoc-Lys(Boc)-OH(18.74g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)，(b)HBTU(15.16g)、(c)HOBT(5.40g)、(d)NMM(4.39ml)、(e)DMF(160ml)加入量不变。
(4)最后一个Arg合成结束后，脱出Fmoc-保护基，再加入5ml哌啶/ DBLK(体积比1:5)反应30min，洗净树脂，加入160ml醋酸酐/DMF(体积比1:2)反应30min，用40ml DMF洗净树脂，再用甲醇洗涤树脂8次，除去DMF。氮气吹干后，用TFA(三氟乙酸)/DCM强酸裂解液(体积比1:1)，裂解3小时，将合成得到的多肽从树脂上裂解下来，同时脱去所有保护基团，收集溶有合成肽的裂解液，然后减压过滤，收集滤液，用冷乙醚沉淀溶解在滤液中的多肽，再抽滤得到白色固体，即得串联穿膜肽的肽粗品。肽粗品经高效液相色谱纯化，收集主峰，经过冷冻干燥后，将棕榈酸(51.23g)与阳离子穿膜肽R9的精氨酸N-端偶联获得C16R9NP2，步骤如下：在0℃搅拌下将溶于15mL DMF的棕榈酸缓慢添加至5mL含有70μL三乙胺和串联穿膜肽的DMF中。在反应24小时后，通过吹扫干燥氮气从混合物去除DMF，然后用二乙醚清洗混合物3次以去除未反应的棕榈酸，即完成N端修饰。使用分子量截断值为1000Da的膜用DMF透析6天以进一步纯化粗产物。然后通过真空干燥去除DMF以产生终产物获得终产物C16R9NP2。通过HPLC和MALDI-TOF分析证明了C16R9NP2的成功合成(如下)。
3、产物检测
合成得到的C16R9NP2二维分子结构通过ChemBioDraw软件绘制，其三维分子模型通过ChemBio 3D软件绘制。如图1所示，通过该图可知其氨基酸及脂肪酸链的空间分布。图中可见，串联穿膜肽中阳离子穿膜肽N-端与棕榈酸偶联，NP2为末端结构。
通过反相高效液相色谱HPLC检测合成得到的C16R9NP2纯度。以Cromasil-C-18柱(5μm)作为固定相，乙腈和水的混合物作为流动相，记录0-30min的峰形。检测结果见图2，图中14.032min处对应的峰即为目标产物峰，根据峰面积百分比结果确定其纯度达到95.89％。
将等体积的肽溶液(1.0mg/mL)与α-氰基-4-羟基肉桂酸(HCCA)溶液(饱和于体积比为50％的乙腈水溶液)混合，通过基质辅助激光解吸电离飞行时间质谱MALDI-TOF检测合成得到的C16R9NP2。检测结果见图3，图中最大强度峰，即对应数值为2584.6的峰为产物的分子离子峰，表示其分子量为2584.6，与由结构式计算的理论分子量一致，结果证明了目标产物C16R9NP2的合成。
实施例2
本发明所述其他脂肪酸修饰的可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂可以通过替换实施例1中的氨基酸及脂肪酸，采用Fmoc(芴甲氧羰酰基)保护的固相合成法，按照实施例1所述制备方法合成纯化得到。
即按实施例1的制备方法(1)中所述，溶胀树脂，然后用DMF洗涤，加入哌啶/DBLK震荡反应后洗涤树脂，经茚三酮检测呈阳性后加入氨基酸原料、HBTU、HOBT、NMM、DMF，震荡反应结束后用DMF洗涤树脂，茚三酮检验呈阴性。然后按照实施例1的制备方法(2)中所述，根据不同串联穿膜肽的氨基酸序列，依次加入不同氨基酸原料(由C-端向N-端顺序)，重复震荡反应、洗涤、茚三酮检测步骤。再按实施例1的制备方法(3)中所述，经脱保护、DMF洗涤、裂解液裂解、乙醚沉淀、纯化，完成串联穿膜肽的合成。最后按照实施例1的制备方法(4)中所述，将脂肪酸加至三乙胺和串联穿膜肽的DMF溶液中反应，经氮气吹扫、二乙醚清洗完成N端修饰。通过透析膜纯化，干燥去除DMF后得到终产物即不同脂肪酸修饰的串联穿膜肽。
再按实施例1中检测方法进行反相高效液相色谱检测和基质辅助激光解吸电离飞行时间质谱检测得到的产物。脂肪酸优选棕榈酸时，得到序列如下：C16-R9-NP1、C16-K9-NP1、C16-R9-NP2、C16-K9-NP2。在此过程中不同的合成实验参数及检测结果如下表1所示：
表1本发明所述部分自组装串联穿膜肽纳米颗粒抗菌剂的不同合成实验参数及检测结果
Figure PCTCN2017110896-appb-000003
实施例3
自组装串联穿膜肽纳米颗粒抗菌剂C16R9NP2的自组装分子动力学模拟及扫描电镜形貌观察
1、分子动力学模拟
对实施例1中自组装串联穿膜肽纳米颗粒抗菌剂C16R9NP2进行分子动力学模拟。所用软件为Material Studio，通过粗粒化模型，计算其与水作用的Flory-Huggins参数，进一步建立力场，进行分子动力学模拟。模拟结果如图4所示，串联穿膜肽在水溶液中自组装形成了纳米颗粒，其中内部为疏水性棕榈酸，外部为串联穿膜肽。
2、扫描电镜形貌观察
利用冷场发射扫描电镜(JSM-6330F)观察C16R9NP2在水溶液中的自组装形态。用超纯水将C16R9NP2配制成0.5mg/mL肽溶液，取20μL肽溶液置于云母片上，并在室温下自然风干。将云母片固定于铝柱上，然后镀金用于观察。图5为14000倍下加速电压为15.0keV时的C16R9NP2水溶液的扫描电镜图，结果表明棕榈酸修饰的串联穿膜肽C16R9NP2在水中自组装形成了直径小于150nm的颗粒。
该实施例自组装串联穿膜肽纳米颗粒抗菌剂N端以脂肪酸修饰以增加疏水性，便于提高肽的两亲性，更易于自组装的进行。其在纯水中即可在很低的肽浓度下自组装形成纳米颗粒，无需剧烈搅拌、超声处理等机械作用，且具有较好的稳定性。
实施例4
自组装串联穿膜肽纳米颗粒抗菌剂C16R9NP2在制备新型纳米抗菌药物中的应用
1、最小抑菌浓度
受试菌株均来自广东微生物种质资源库。金黄色葡萄球菌ATCC 29213，大肠杆菌CMCC 44825，耐甲氧西林-金黄色葡萄球菌ATCC 43300在37℃的营养琼脂培养基中进行培养。白色念珠菌ATCC 10231在37℃的马铃薯葡萄糖培养基中进行培养。利用微量稀释法来测定肽纳米颗粒的最小抑菌浓度MIC。简单来说，将50μl浓度为4.2到100.4μM的自组装串联穿膜肽纳米颗粒溶液和对照品R9肽溶液置于96孔板的每个孔中，再将相同体积的受试菌液添加到每个孔中以得到600nm处0.1到0.2的光密度读数。放置于37℃的振荡培养箱中温育10～16h，每隔2小时在酶标仪测量600nm处的光密度值，以观 察不到细菌生长的最低药物浓度即为该样品的最低抑菌浓度。试验重复三次。
实验结果，自组装串联穿膜肽C16R9NP2纳米颗粒以及阳性对照穿膜肽R9的最低抑菌浓度测定结果如表2所示。
表2最低抑菌浓度测定结果
Figure PCTCN2017110896-appb-000004
结果表明，本发明所述的自组装串联穿膜肽纳米颗粒对测试微生物均具有较好的抑菌效果，其对金黄色葡萄球菌的最小抑菌浓度为13.4μM，对大肠杆菌、耐甲氧西林金葡菌最小抑菌浓度为8.4μM，对白色念珠菌的最小抑菌浓度为50.3μM。而未自组装的单一穿膜肽R9对测试细菌和真菌的抑制能力极低，表现为极高的最小抑菌浓度。纳米颗粒的形成明显增强了肽的抗菌能力，降低了最小抑菌浓度，因此本发明所述自组装串联穿膜肽纳米颗粒可发展成为有效的抑制微生物增殖的抗菌药物。
2、扫描电子显微镜观察微生物形貌变化
将培养好的菌溶液及经串联穿膜肽纳米颗粒处理的菌溶液，在6000rpm离心10min后，用磷酸缓冲盐溶液(PBS)洗涤细胞三次，然后在含有5％甲醛的PBS中隔夜固定。用一系列浓度梯度乙醇进行脱水，然后冷冻干燥，固定于铝柱上。在SEM分析前用金包被样品。图6中a为空白对照的金黄色葡萄球菌，细胞结构饱满完整，细胞表面呈光滑的圆形；b为经串联穿膜肽纳米颗粒(最小抑菌浓度)处理后的金黄色葡萄球菌，其形貌发生明显变化，可观察到明显的细胞破裂和细胞碎片；c为空白对照的白色念珠菌，其细胞表面完整光滑；d为经串联穿膜肽纳米颗粒(最小抑菌浓度)处理后的的白色念珠菌，其形貌发生明显变化，可见细胞膜消溶，细胞裂解死亡。扫描电子显微镜观察到的微生物形貌变化，验证了本发明串联穿膜肽纳米颗粒C16R9NP2的抗菌能力，表明其作用机制主要为破坏微生物细胞结构。
3、溶血作用检测
用PBS洗涤新鲜的人红细胞，并稀释成4％(按体积计)悬液，取100μL红细胞悬液置于96孔板的每个孔中，然后向每孔添加100μL肽纳米颗粒或两性霉素B溶液(水溶性)，在37℃温育1小时。之后取出细胞悬液于1000g离心取等份上清(100μL)转移到96孔板，利用酶标仪在540nm监测血红蛋白释放。
使用下列公式计算溶血作用百分比:
溶血作用(％)＝[(纳米颗粒溶液中的O.D.540nm-PBS中的O.D.540nm)/(0.1％TritonX-100中的O.D.540nm-PBS中的O.D.540nm)]x100
结果如图7所示，纳米颗粒具有低溶血作用，在25mg/L，对大肠杆菌和耐甲氧西林金葡菌的最小抑菌浓度，其溶血作用小于4％；在50mg/L，对金黄色葡萄球菌的最小抑菌浓度，其溶血作用小于5％。在同等浓度下，串联穿膜肽纳米颗粒比阳性对照药物两性霉素(水溶性)的溶血作用低。临床上认为小于5％溶血作用的医用材料是安全的，因此，自组装串联穿膜肽纳米颗粒C16R9NP2的低溶血作用显示出其作为有效的抗菌药物的安全性，能用于感染疾病的治疗。
实施例5
自组装串联穿膜肽纳米颗粒抗菌剂C16R9NP2穿透血脑屏障的检测，以及在治疗脑部感染中的应用
所有涉及动物的程序均得到中国医学科学院实验动物研究所实验动物使用与管理委员会(IACUC)的批准，并且按照National Institute of Health Guide for the Care and Use of Laboratory Anima1s(NIH Publications NO.85-23，1996年修订)中阐述的指南进行。
1、自组装串联穿膜肽纳米颗粒的穿血脑屏障作用
实验过程参考任长虹等(大鼠脑脊液抽取的新方法，实验动物科学，2012)，通过尾静脉给成年SD大鼠(重量约为250g)注射C16R9NP2纯纳米颗粒溶液。取脑脊液，1000g离心取上清，通过基质辅助激光解吸电离飞行时间质谱检测。脑脊液样品检测结果如图8所示，分子离子峰对应的数值为2984.254，与C16R9NP2标品2984.56一致，故为肽纳米颗粒的特征峰，结果表明肽纳米颗粒能够穿过大鼠血脑屏障，进入脑脊液，进而能进一步进入脑组织。
2、自组装串联穿膜肽纳米颗粒在治疗脑部细菌感染中的作用
实验过程参考Yi-Yan Yang等(Self-assembled cationic peptide nanoparticles as an efficient antimicrobial agent，Nature nanotechnology，2009)。向SD大鼠延髓池注入50μL的金黄色葡萄球菌液，通过尾静脉每12h注射C16R9NP2纳米颗粒溶液，空白对照组注射生理盐水，阳性对照组注射万古霉素溶液。48h后取大鼠脑组织称重并匀浆，取100μL匀浆液在营养琼脂培养基上涂布，计算其菌落数，即菌落形成单位，以lg菌落形成单位/克脑组织表示。结果如图9所示，经肽纳米颗粒治疗后，大鼠脑组织菌落数相较空白对照组(即未治疗组)明显减少，与经两性霉素治疗组结果相近，表明其在治疗大鼠脑部金葡菌感染中取得了明显效果，抑制了细菌增长。因此，本发明所述自组装串联穿膜肽纳米颗粒在制备治疗脑部感染疾病的抗菌药物中具有良好的应用前景，可有效抑制受感染的脑部细菌的生长。
本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (7)

  1. 可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂，其特征在于，该抗菌剂的结构通式为Cn-X-Y，其中X和Y均为穿膜肽，且X和Y串联形成亲水部分；Cn表示与穿膜肽偶联的疏水部分脂肪酸链；穿膜肽为L型氨基酸；所述脂肪酸链的碳原子个数为12-20；
    所述X为阳离子穿膜肽；
    所述Y为NP1或NP2；所述NP1序列为KKDKKDERRRK；所述NP2序列为KIKKVKKKGRK。
  2. 权利要求1所述的自组装串联穿膜肽纳米颗粒抗菌剂，其特征在于，所述脂肪酸为月桂酸、肉豆蔻酸、棕榈酸、硬脂酸或花生酸。
  3. 权利要求1所述的自组装串联穿膜肽纳米颗粒抗菌剂，其特征在于，所述阳离子穿膜肽为九聚精氨酸R9或九聚赖氨酸K9。
  4. 权利要求1所述的自组装串联穿膜肽纳米颗粒抗菌剂，其特征在于，所述自组装串联穿膜肽纳米颗粒抗菌剂具有100-500nm的平均直径。
  5. 权利要求1所述可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂的制备方法，其特征在于包括如下步骤：
    1)采用Fmoc固相合成法制备串联穿膜肽，先溶涨树脂，洗涤并脱保护后，将第一个Fmoc-氨基酸和HoBt进行缩合，脱保护并洗涤后，茚检确认脱保护完全；然后缩合第二个氨基酸，重复以上步骤，由C-端向N-端，直至多肽链合成完成；
    2)将合成完成的多肽链用裂解液裂解，减压过滤后用冷乙醚沉淀获得粗肽，并通过液相色谱进一步纯化；
    3)脂肪酸与串联穿膜肽中阳离子穿膜肽的N-端偶联，将棕榈酸加至含有三乙胺和串联穿膜肽的DMF中搅拌反应后，通氮气去除DMF，用二乙醚清洗，并使用透析膜进一步纯化粗产物。
  6. 权利要求1所述可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂在制备抗金黄色葡萄球菌、大肠杆菌、耐甲氧西林金葡菌或白色念珠菌药物中 的应用。
  7. 权利要求1所述可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂在制备治疗脑部金黄色葡萄球菌感染药物中的应用。
PCT/CN2017/110896 2017-01-13 2017-11-14 可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂及其制备方法与应用 WO2018130000A1 (zh)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113135984A (zh) * 2021-05-06 2021-07-20 中国医学科学院放射医学研究所 一种病理微环境响应的原位自组装多肽衍生物及其应用
CN114699387A (zh) * 2022-03-17 2022-07-05 常州大学 一种核壳结构的载药纳米颗粒及其制备方法和应用
CN116808231A (zh) * 2023-07-07 2023-09-29 中国药科大学 一种细胞穿膜肽偶联舒必利前药体系以及制备方法和应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106916228B (zh) * 2017-01-13 2019-06-18 华南理工大学 可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂及其制备方法与应用
CN110123658B (zh) * 2019-05-22 2022-07-15 上海璞萃生物科技有限公司 一种具有自组装聚集体结构的超分子多肽及其制备方法
WO2023277628A1 (ko) * 2021-07-02 2023-01-05 주식회사 아임뉴런 신규한 세포 투과성 펩타이드 및 이의 용도
CN114788872A (zh) * 2022-05-06 2022-07-26 太阳雨林(北京)生物医药有限公司 预防、阻止或治疗微生物感染的复合物及制备和用途
CN115607677A (zh) * 2022-05-06 2023-01-17 太阳雨林(北京)生物医药有限公司 预防、阻止或治疗微生物感染的复合物及其制备和应用
CN115536730B (zh) * 2022-09-20 2023-08-15 南开大学 一种穿越血脑屏障的多肽及其制备方法、纳米结构及其制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103467579A (zh) * 2013-08-26 2013-12-25 华南理工大学 一类阳离子两亲性自组装纳米抗菌肽及其应用
KR20140046996A (ko) * 2012-10-09 2014-04-21 한양대학교 산학협력단 인간 ｎｌｂｐ 유래의 ｎｐ12 폴리펩티드 또는 ｎｐ21 폴리펩티드를 포함하는 세포 투과 펩티드 및 이를 이용한 카고 전달 시스템
CN106916228A (zh) * 2017-01-13 2017-07-04 华南理工大学 可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂及其制备方法与应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140046996A (ko) * 2012-10-09 2014-04-21 한양대학교 산학협력단 인간 ｎｌｂｐ 유래의 ｎｐ12 폴리펩티드 또는 ｎｐ21 폴리펩티드를 포함하는 세포 투과 펩티드 및 이를 이용한 카고 전달 시스템
CN103467579A (zh) * 2013-08-26 2013-12-25 华南理工大学 一类阳离子两亲性自组装纳米抗菌肽及其应用
CN106916228A (zh) * 2017-01-13 2017-07-04 华南理工大学 可穿透血脑屏障的自组装串联穿膜肽纳米颗粒抗菌剂及其制备方法与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XU, XIAODING: "Self-assembly of Small Peptides and Their Applications in Biomedical Field", ENGINEERING SCIENCE AND TECHNOLOGY I, 15 April 2015 (2015-04-15), pages 11; 16 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113135984A (zh) * 2021-05-06 2021-07-20 中国医学科学院放射医学研究所 一种病理微环境响应的原位自组装多肽衍生物及其应用
CN114699387A (zh) * 2022-03-17 2022-07-05 常州大学 一种核壳结构的载药纳米颗粒及其制备方法和应用
CN116808231A (zh) * 2023-07-07 2023-09-29 中国药科大学 一种细胞穿膜肽偶联舒必利前药体系以及制备方法和应用
CN116808231B (zh) * 2023-07-07 2024-03-26 中国药科大学 一种细胞穿膜肽偶联舒必利前药体系以及制备方法和应用

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