WO2024045275A1 - 一种超声响应型脂质体纳米颗粒及其制备方法和应用 - Google Patents

一种超声响应型脂质体纳米颗粒及其制备方法和应用 Download PDF

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WO2024045275A1
WO2024045275A1 PCT/CN2022/125025 CN2022125025W WO2024045275A1 WO 2024045275 A1 WO2024045275 A1 WO 2024045275A1 CN 2022125025 W CN2022125025 W CN 2022125025W WO 2024045275 A1 WO2024045275 A1 WO 2024045275A1
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liposome
ultrasound
responsive
biofilm
nanoparticle
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汪联辉
宇文力辉
修尉峻
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南京邮电大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/186—Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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

Definitions

  • the invention belongs to the field of biological preparations against bacterial biofilms, and specifically relates to an ultrasound-responsive liposome and its preparation method and application.
  • Biofilm is a highly organized and systematic multicellular community composed of bacteria and other microorganisms that secrete extracellular polymers (EPS) to wrap themselves during the growth process. Due to the coating of EPS, it is difficult for most drug molecules to penetrate into the biofilm and interact with internal bacteria, thus hindering the inhibitory effect of antibacterial agents on bacteria inside the biofilm.
  • EPS has certain obstacles to the diffusion of external oxygen, causing the oxygen content in the biofilm to gradually decrease from the outside to the inside.
  • Bacteria in the outer layer of the biofilm have high metabolic activity and are sensitive to antibacterial drugs; while bacteria in the inner layer of the biofilm have low metabolic activity and are highly resistant to antibacterial drugs. Therefore, the special fortress structure of the biofilm and the metabolic heterogeneity of the internal bacteria make it difficult for traditional drug treatments to completely eliminate the bacteria inside the biofilm and lead to stubborn chronic infections.
  • the present invention provides an ultrasound-responsive liposome, which contains a sonosensitizer and antibiotics to form liposome particles.
  • the liposome particles can produce a cavitation effect under the action of ultrasound and destroy bacteria.
  • Biofilm structure enhances drug penetration and achieves efficient anti-biofilm performance.
  • the invention is an ultrasound-responsive liposome nanoparticle, which contains dimyristoylphosphatidylcholine (DMPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), sonosensitizers, antibiotics, and perfluoropentane (PFP).
  • DMPC dimyristoylphosphatidylcholine
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DSPE-PEG distearoylphosphatidylethanolamine-polyethylene glycol
  • sonosensitizers antibiotics
  • PFP perfluoropentane
  • the invention also provides a method for preparing ultrasound-responsive liposome nanoparticles, which includes the following steps:
  • Step 1 Combine dimyristoylphosphatidylcholine (DMPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE) -PEG), sonosensitizer, and antibiotics are dissolved in chloroform, and rotary evaporated at 50°C for 5-10 minutes to form a liposome film;
  • DMPC dimyristoylphosphatidylcholine
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DSPE distearoylphosphatidylethanolamine-polyethylene glycol
  • sonosensitizer antibiotics
  • Step 2 After using ionized water to resuspend the liposome membrane, the hydrated liposomes are sonicated using a probe in an ice-water bath. During the treatment process, perfluoropentane (PFP) is slowly added dropwise, and the liposomes are ultrasonicated. During the hydration self-assembly process, perfluoropentane can be gradually wrapped to form a liposome nanoparticle dispersion;
  • PFP perfluoropentane
  • Step 3 Put the reacted liposome nanoparticle dispersion into a dialysis bag, and dialyze in a phosphate buffered saline solution to obtain drug-loaded liposome nanoparticles.
  • the dimyristoylphosphatidylcholine (DMPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), distearoylphosphatidylethanolamine-polyethylene described in step 1 The mass ratio of glycol (DSPE-PEG) was 5:1.5:1, and the final concentration of liposomes dissolved in chloroform was 10 mg/mL.
  • the sonosensitizer described in step 1 is chlorin, protoporphyrin, photoporphyrin, hematoporphyrin, hematoporphyrin methyl ether, chlorin, tetraethyl rhodamine, adriamycin cisplatin, cyclophosphamide, amphotericin b, 5-fluorouracil, cytarabine, mitomycin c, lomefloxacin, sparfloxacin, gatifloxacin, ciprofloxacin, levofloxacin, Methylene blue, toluidine blue, rose red, tetraiodotetrachlorofluorescein derivative, acridine orange, purpurin, phthalocyanine, naphthocyanine, cyanine, indocyanine green, curcumin, rhodophyllin, bamboo red Drugs with sonodynamic properties such as bacterio
  • the antibiotics described in step 1 are nitroimidazole antibiotics such as metronidazole, dimethylnidazole, isoprenidazole, secnidazole, ornidazole, tinidazole and lonidazole.
  • nitroimidazole antibiotics such as metronidazole, dimethylnidazole, isoprenidazole, secnidazole, ornidazole, tinidazole and lonidazole.
  • the pH value of the solution described in step 1 is 7-7.4.
  • the concentration of the sonosensitizer in step 1 is 0.5-1 mg/mL, and the concentration of the antibiotic is 0.5-1 mg/mL.
  • the working conditions of the probe ultrasound in step 2 are: 5 seconds of operation, 2 seconds of intermission, 40% power, and 5 to 10 minutes of ultrasound time.
  • the volume proportion of the perfluoropentane added dropwise in the liposomes in step 2 is 1%-2%.
  • the molecular weight cutoff of the dialysis bag used in step 3 is 10 kDa, and the dialysis time is 24 to 48 hours.
  • the pH of the phosphate buffer saline solution used in step 3 is 7.4 and the concentration is 10mM.
  • the liposome nanoparticles prepared in the present invention have a size of 150 to 250 nm.
  • Ultrasound-responsive drug-loaded liposomes as described above will be used for the treatment of bacterial biofilm infections.
  • the present invention prepares ultrasound-responsive drug-loaded liposome nanoparticles, which can produce cavitation under the action of ultrasound, destroy biofilms, release sonosensitizers and antibiotics at the same time, and enhance their penetration inside biofilms. penetration.
  • Figure 1 is a schematic diagram of the preparation of liposome nanoparticles of the present invention.
  • Figure 2 is a transmission electron microscope image of liposome nanoparticles of the present invention.
  • Figure 3 is a particle size distribution diagram of liposome nanoparticles of the present invention.
  • Figure 4 is a Fourier transform infrared spectrum chart of liposome nanoparticles of the present invention.
  • Figure 5 is a crystal violet stained photo of the present invention to verify that the drug-loaded liposome nanoparticles destroy the biological membrane structure under the action of ultrasound.
  • Figure 6 shows the biomass of the present invention to verify that drug-loaded liposomes destroy biological membranes under the action of ultrasound.
  • Figure 7 shows how the present invention enhances the penetration of drug into biological membranes by drug-loaded liposomes under the action of ultrasound.
  • Figure 8 shows that the present invention uses drug-loaded liposomes to enhance the expression of nitroreductase-related genes in Pseudomonas aeruginosa biofilms under the action of ultrasound.
  • Figure 9 is the plate data statistics of the anti-biofilm performance of drug-loaded liposomes of the present invention.
  • Figure 10 is a confocal fluorescence microscope photo showing the anti-biofilm performance of drug-loaded liposomes of the present invention.
  • Figure 11 is a confocal fluorescence microscope photo showing that the drug-loaded liposome of the present invention inhibits the regrowth of biofilm after clearing the biofilm.
  • Figure 12 is the plate data statistics of the present invention's inhibition of biofilm regrowth after drug-loaded liposomes clear biofilms.
  • the invention is an ultrasound-responsive liposome nanoparticle.
  • the liposome nanoparticle includes lipid material, and the lipid material includes dimyristoyl phosphatidylcholine (DMPC), 1,2-dioleoyl -3-trimethylammonium-propane (DOTAP), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), the liposome nanoparticles also include sonosensitizers, antibiotics and perfluoropentane (PFP).
  • DMPC dimyristoyl phosphatidylcholine
  • DOTAP 1,2-dioleoyl -3-trimethylammonium-propane
  • DSPE-PEG distearoylphosphatidylethanolamine-polyethylene glycol
  • sonosensitizers antibiotics and perfluoropentane (PFP).
  • the preparation method of the present invention is:
  • DMPC dimyristoylphosphatidylcholine
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DSPE- PEG distearoylphosphatidylethanolamine-polyethylene glycol
  • the liposome film containing the drug is obtained by rotary evaporation at 50°C for 5 to 10 minutes.
  • the morphology of PLCM was observed using a transmission electron microscope.
  • the particle size distribution of PLCM in deionized water solution was observed using a dynamic light scattering particle size analyzer.
  • PLCM was mixed with potassium bromide powder and ground together.
  • the mass ratio of sample to potassium bromide powder was 1:100.
  • the ground mixture was pressed into tablets and placed in an infrared spectrometer for detection to prove the successful loading of the drug. .
  • FIG 1 is a schematic diagram of the synthesis process of PLCM.
  • PLCM is a spherical nanoparticle with a size distribution of 150 to 250nm.
  • the following specifically takes Pseudomonas aeruginosa biofilm as an example to verify that the PLCM of the present invention can destroy the biofilm through the ultrasonic cavitation effect under the action of ultrasound, enhance the penetration of sonosensitizers and antibiotics in the biofilm, and achieve high-efficiency antibacterial biofilm performance.
  • PAO1 wild-type Pseudomonas aeruginosa
  • LB Luria-Bertani
  • the PAO1 biofilm was grown inside a 6-hole Transwell chamber with a pore diameter of 2 ⁇ m, and PLCM dispersion was added to the chamber.
  • the ultrasound treatment group used 1MHz ultrasound at a power of 1W/ cm2 for 10 minutes. After incubation for 1h, 3h, 6h, and 12h, the solution in the lower chamber of the Transwell was collected, and the content of Ce6 and MNZ was analyzed by UV-visible absorption spectroscopy. The results are shown in Figure 7
  • the PAO1 biofilm was grown in a 6-well plate, and phosphate buffer and PLCM were added respectively.
  • the ultrasound treatment group used 1MHz ultrasound at 1W/ cm2 power for 10 minutes; after treatment, it was incubated for 24h, and PAO1 was extracted using a bacterial RNA extraction kit.
  • the RNA of the bacteria inside the biofilm was extracted, and further reverse transcribed using a reverse transcription kit to synthesize DNA complementary to the bacterial RNA; finally, the expression level of the bacterial nitroreductase-related expression gene (NsfB) was measured using a real-time fluorescence quantitative PCR instrument. Proc-based Because of the internal reference gene, the experimental results are shown in Figure 8.
  • PAO1 biofilms were grown in confocal dishes and 96-well plates, and PFP-loaded liposomes (PL), PFP- and Ce6-loaded liposomes (PCL), and PFP-, Ce6-, and MNZ-loaded liposomes ( PLCM).
  • PL PFP-loaded liposomes
  • PCL PFP- and Ce6-loaded liposomes
  • PLCM PFP-, Ce6-, and MNZ-loaded liposomes
  • nitroreductase of Pseudomonas aeruginosa under anaerobic conditions can activate nitroimidazole drugs and produce imidazole fragments to kill bacteria.
  • Ce6 produces sonodynamic properties under the action of ultrasound, converting oxygen into active oxygen, killing active bacteria on the surface of the biofilm, aggravating the anoxic state deep in the biofilm, enhancing the expression of bacterial nitroreductase, and further activating metronidazole, Kill low-active bacteria deep in the biofilm.
  • the Pseudomonas aeruginosa nitroreductase-related expression gene NsfB was significantly higher than that in the PLCM treatment group and the control group, proving that oxygen consumption caused by sonodynamic treatment of PLCM can significantly enhance patina. Expression of Pseudomonas nitroreductase.
  • the amount of bacteria inside the Pseudomonas aeruginosa biofilm in the PLCM treatment group and the PL combined with ultrasound treatment group has no significant change compared with the control group (Control), while the number of bacteria in the PLC combined with ultrasound treatment group Compared with the control group, it decreased by 4.1 orders of magnitude, proving that the sonodynamic performance of PLC has a significant antibacterial effect.
  • the bacteria in the PLCM combined with ultrasound treatment group decreased by 5.9 orders of magnitude compared with the control group, proving that sonodynamically activated MNZ can further inhibit bacteria inside the biofilm.
  • PAO1 biofilms were grown in confocal culture dishes and 96-well plates, and the antibiotic piperacillin (Pip) dispersed in TSB, a mixture of Pip and MNZ, PCL, and PLCM were added respectively.
  • TSB antibiotic piperacillin
  • PCL PCL
  • PLCM PLCM
  • 1MHz ultrasound was used at 1W/cm 2 power for 10 minutes, and cultured in a 37°C incubator.
  • the biofilm in the confocal dish after incubation for 1d, 2d, and 3d was stained with Calcien-AM for half an hour, and viable bacteria in the biofilm were observed through confocal fluorescence microscopy imaging.
  • the experimental results are shown in Figure 11.
  • the biofilm in the 96-well plate was dispersed and transferred to a 1.5mL centrifuge tube. Use physiological saline to adjust the volume to 1mL, and then perform gradient dilutions of 10, 10 2 , 10 3 , 10 4 , and 10 5 respectively. , 10 6 times, and then add 100 ⁇ L of the original solution and the bacterial solution diluted 10 4 , 10 5 , and 10 6 times onto the petri dish containing the solid LB medium for plate quantification.
  • the experimental results are shown in Figure 12.
  • the sonodynamic force of Ce6 can efficiently kill metabolically active bacteria in the biofilm, aggravate the hypoxic microenvironment in the membrane, enhance bacterial nitroreductase gene expression, activate the antibiotic MNZ, and kill low metabolically active bacteria in the biofilm, achieving It has high-efficiency Pseudomonas aeruginosa biofilm inhibition function and inhibits the re-growth of Pseudomonas aeruginosa biofilm, and the therapeutic effect is significantly better than the traditional antibiotic Pip used to treat Pseudomonas aeruginosa infection.

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Abstract

一种超声响应型脂质体纳米颗粒及其制备方法和应用，该脂质体纳米颗粒包括二肉豆蔻酰磷脂酰胆碱、1,2-二油酰基-3-三甲基铵-丙烷、二硬脂酰基磷脂酰乙醇胺-聚乙二醇、声敏剂、抗生素和全氟戊烷。该脂质体纳米颗粒用于细菌生物膜感染的治疗，该制备方法为制备脂质体膜，脂质体膜水化负载全氟戊烷、声敏剂和抗生素，在超声作用下，该脂质体产生超声空化效应，破坏细菌生物膜结构，释放声敏剂与抗生素并增强其在生物膜内的渗透，通过声敏剂的声动力性能杀灭部分细菌，并提高细菌硝基还原酶表达以激活硝基咪唑类药物，进一步杀灭残余顽固菌，实现高效的抗细菌生物膜性能。

Description

[根据细则26改正 09.11.2023]一种超声响应型脂质体纳米颗粒及其制备方法和应用 技术领域
本发明属于生物制剂抗细菌生物膜领域，具体的说是涉及一种超声响应型脂质体及其制备方法和应用。
背景技术
生物膜是细菌等微生物在生长过程中分泌胞外聚合物(EPS)将自身包裹构成的高度组织化、系统化的多细胞群落。由于EPS的包覆，大部分的药物分子难以渗透进入生物膜与内部细菌相互作用，从而阻碍了抗菌剂对生物膜内部细菌的抑制效果。此外，EPS对外部氧气的扩散具有一定的阻碍，使得生物膜内氧气含量由外而内逐渐降低。生物膜外层的细菌代谢活性高，对抗菌药物敏感；而生物膜内部的细菌代谢活性低，对抗菌药物具有较高的耐受性。因此，生物膜特殊的堡垒结构以及内部细菌的代谢异质性使得传统药物治疗难以彻底清除生物膜内部细菌，并导致顽固的慢性感染。
纳米技术的发展给细菌生物膜感染的治疗带了的新的曙光。尽管在实验室研究阶段，许多纳米试剂被开发用于破坏生物膜结构，增强抗生素在生物膜内部的渗透以实现更高效的杀菌效果。然而，目前的基于纳米试剂增强抗生物渗透的治疗模式虽能杀灭生物膜外层代谢高活性菌，对生物膜深处代谢低活性菌的治疗效果有限，且易造成生物膜的复发。因此，在增强药物渗透的同时，能够高效杀灭生物膜内部异质代谢的细菌是根除生物膜，解决生物膜感染易反复发作问题的关键。
发明内容
针对上述技术问题，本发明提供了一种超声响应型脂质体，并包载声敏剂和抗生素制成脂质体颗粒，该脂质体颗粒可在超声作用下产生空化效应，破坏细菌生物膜结构，增强药物渗透，并实现高效的抗生物膜性能。
为了达到上述目的，本发明是通过以下技术方案实现的：
本发明是一种超声响应型脂质体纳米颗粒，所述脂质体纳米颗粒包含二肉豆蔻酰磷脂酰胆碱(DMPC)、1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)、声敏剂、抗生素和全氟戊烷(PFP)。
本发明还提供了一种超声响应型脂质体纳米颗粒的制备方法，包括如下步骤：
步骤1：将二肉豆蔻酰磷脂酰胆碱(DMPC)、1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)、声敏剂、抗生素溶解于氯仿，在50℃下旋蒸5-10min，形成一层脂质体膜；
步骤2：利用离子水重悬脂质体膜后，将水化后的脂质体在冰水浴中利用探头超声处理，处理过程中缓慢滴加全氟戊烷(PFP)，脂质体在超声水化自组装的过程中能逐渐包裹全氟戊烷，形成写脂质体纳米颗粒分散液；
步骤3：将反应后的脂质体纳米颗粒分散液至于透析袋，在磷酸缓冲盐溶液中透析，得到载药脂质体纳米颗粒。
优选的，步骤1中所述二肉豆蔻酰磷脂酰胆碱(DMPC)、1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)的质量比为5：1.5：1，溶解于氯仿的最终脂质体的浓度为10mg/mL。
优选的，步骤1中所述声敏剂为二氢卟吩、原卟啉、光卟啉、血卟啉、血卟啉甲醚、苯并二氢卟酚、四乙基罗丹明、阿霉素、顺铂、环磷酰胺、两性霉素b、5-氟尿嘧啶、阿糖胞苷、丝裂霉素c、洛美沙星、司帕沙星、加替沙星、环丙沙星、左氧氟沙星、亚甲基蓝、甲苯胺蓝、玫瑰红、四碘四氯荧光素衍生物、吖啶橙、红紫素、酞菁、萘菁、花菁、吲哚菁绿、姜黄素、竹红菌素、竹红菌乙素、竹黄菌素、叶绿素衍生物、金丝桃素或黄连素等具有声动力性能的药物。
优选的，步骤1中所述抗生素为甲硝唑、二甲硝咪唑、异丙硝唑、塞可硝唑、奥硝唑、替硝唑和洛硝哒唑等硝基咪唑类抗生素。
优选的，步骤1中所述的溶液的pH值为7-7.4。
优选的，步骤1中声敏剂的浓度为0.5-1mg/mL、抗生素的浓度为0.5-1mg/mL。
优选的，步骤2中探头超声的工作条件为：工作5s，间歇2s，功率40％，超声时间5～10min。
优选的，步骤2中滴加的全氟戊烷在脂质体中的体积占比为1％-2％。
优选的，步骤3中所用的透析袋截留分子量为10kDa，透析时间为24～48h。
优选的，步骤3中所用的磷酸缓冲盐溶液pH为7.4，浓度为10mM。
优选的，本发明制备的脂质体纳米颗粒尺寸为150～250nm。
如以上所述的超声响应载药脂质体将用于细菌生物膜感染的治疗。
本发明的有益效果是：
(1)本发明制备了一种超声响应载药脂质体纳米颗粒，其可在超声作用下产生空化作用，破坏生物膜，同时释放声敏剂和抗生素，并增强其在生物膜内部的渗透。
(2)通过声敏剂的声动力性能杀灭部分细菌，并提高细菌硝基还原酶表达以激活硝基咪唑类药物，进一步杀灭残余顽固菌，实现高效的抗细菌生物膜性能。
附图说明
图1是本发明脂质体纳米颗粒的制备示意图。
图2是本发明脂质体纳米颗粒的透射电镜图。
图3是本发明脂质体纳米颗粒的粒径分布图。
图4是本发明脂质体纳米颗粒的傅里叶红外光谱图。
图5是本发明为验证载药脂质体纳米颗粒在超声作用破坏生物膜结构的结晶紫染色照片。
图6是本发明为验证载药脂质体在超声作用破坏生物膜的生物量。
图7是本发明为载药脂质体在超声作用下增强药物在生物膜内的渗透量。
图8是本发明为载药脂质体在超声作用下增强铜绿假单胞菌生物膜内硝基还原酶相关基因表达。
图9是本发明为载药脂质体抗生物膜性能的涂板数据统计。
图10是本发明为载药脂质体抗生物膜性能的共聚焦荧光显微镜照片。
图11是本发明为载药脂质体清除生物膜后抑制生物膜再生长的共聚焦荧光显微镜照片。
图12是本发明为载药脂质体清除生物膜后抑制生物膜再生长的涂板数据统计。
具体实施方式
以下将以图式揭露本发明的实施方式，为明确说明起见，许多实务上的细节将在以下叙述中一并说明。然而，应了解到，这些实务上的细节不应用以限制本发明。也就是说，在本发明的部分实施方式中，这些实务上的细节是非必要的。
本发明是一种超声响应型脂质体纳米颗粒，该脂质体纳米颗粒包括脂质材料，所述脂质材料包括二肉豆蔻酰磷脂酰胆碱(DMPC)、1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)，所述脂质体纳米颗粒还包括声敏剂、抗生素和全氟戊烷(PFP)。
下面以二氢卟吩为声敏剂，甲硝唑为抗生素来解释本发明的制备方法。
本发明的制备方法为：
S1、将二肉豆蔻酰磷脂酰胆碱(DMPC)，1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)，二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)以质量比为5：1.5：1溶解于氯仿中，使得终溶液中脂质体浓度为10mg/mL。
S2、进一步在脂质体溶液中加入声敏剂二氢卟吩(Ce6)和抗生素甲硝唑(MNZ)， 二氢卟吩和甲硝唑在脂质体溶液中的浓度为0.5-1mg/mL。最后在50℃下旋蒸5～10min，得到含有药物的脂质体薄膜。
S3、加入去离子水重悬脂质体膜后，在冰水浴下使用超声细胞破碎仪超声5-10min，工作5s，间歇2s，功率40％，超声过程中逐滴加入全氟戊烷(PFP)使其在终溶液中的体积比为1.5％，最终得到脂质体纳米颗粒分散液；
S4、将反应后的脂质体纳米颗粒分散液用截留分子量为10kDa的透析袋在pH为7.4，浓度为10mM的磷酸盐缓冲液中透析24-48h，最终得到包裹PFP，负载Ce6和MNZ的脂质体纳米颗粒PLCM。
利用透射电子显微镜观察PLCM的形貌。利用动态光散射粒度分析仪观察PLCM在去离子水溶液中的粒径分布。将PLCM冻干后与溴化钾粉末混合后共同研磨，样品与溴化钾粉末质量比为1:100，并将研磨后的混合物压片并置于红外光谱仪中进行检测，证明药物的成功负载。
图1为PLCM的合成过程示意图。从图2和图3可以看出，PLCM为球状的纳米颗粒，尺寸分布在150～250nm。图4中可以观察到PLCM的红外光谱图含有Ce6在1590cm -1处N-H键的弯曲振动峰以及MNZ在1485cm -1处N＝O键的伸缩振动峰，证明Ce6和MNZ在脂质体上的成功负载。
下面具体以铜绿假单胞菌生物膜为例来验证本发明PLCM可以在超声作用下课通过超声空化效果破坏生物膜，增强声敏剂和抗生素在生物膜内的渗透，实现高效抗细菌生物膜的性能。
1、载药脂质体的超声空化效应破坏生物膜结构并增强药物渗透
(1)铜绿假单胞菌生物膜的培养
划取单菌落野生型铜绿假单胞菌(PAO1)于5mL的Luria-Bertani(LB)培养基中，在37℃恒温摇床下孵育生长10～12h，转速：220rpm，得到对数生长期的PAO1悬液。将PAO1悬液用生理盐水(0.85％NaCl溶液)清洗三遍，离心条件：12000rpm，3min，并用酶标仪定量，OD 600＝0.1时，菌液浓度为1×10 8CFU/mL。将洗过的菌液用胰酪大豆胨液体培养基(TSB)稀释至1×10 6CFU/mL，分别加入96孔板，置于37℃恒温箱中孵育24h，得到PAO1生物膜。
(2)评估超声响应载药脂质体对生物膜的破坏效果
除去96孔板生长好PAO1生物膜中上的清液，每孔分别加入200μL磷酸盐缓冲液和PLCM分散液，置于37℃恒温箱，超声治疗组使用1MHz的超声在1W/cm 2功率下超声 10min，孵育6h后，将生物膜上清液取出，使用福尔马林固定10min，吸去福尔马林，每孔加入100μL 0.2％结晶紫染色液，染色30min后，每孔用生理盐水洗涤三次，然后在倒置显微镜观察并拍照，成像结果如图5所示。观察后，每孔加入200μL乙醇溶解结晶紫，脱色3h后通过酶标仪测每孔在590nm处的吸光度，计算生物膜生物量，结果如图6所示。
(3)评估超声响应载药脂质体促进药物在生物膜内的渗透
将PAO1生物膜生长于孔径为2μm的6孔Transwell小室内部，并在小室中加入PLCM分散液，超声治疗组使用1MHz的超声在1W/cm 2功率下超声10min。孵育1h，3h，6h，12h后，收集Transwell下室溶液，并通过紫外-可见吸收光谱分析Ce6和MNZ的含量，结果如图7所示
从图5和图6结果可以看出，PLCM在超声作用下产生的空化效应在PAO1生物膜内部产生了明显的孔洞，并显著降低了PAO1生物膜的生物量。此外，图7结果也表明经PLCM和超声处理后，Ce6和MNZ渗透穿过生物膜的量也明显提高，证明PLCM结合超声可以破坏生物膜结果，并增强药物在生物膜内的渗透。
2、超声响应载药催化微泡清除生物膜效果
(1)硝基还原酶相关基因表达测定
将PAO1生物膜生长于6孔板中，分别加入磷酸盐缓冲液和PLCM，超声治疗组使用1MHz的超声在1W/cm 2功率下超声10min；治疗后孵育24h，使用细菌RNA提取试剂盒将PAO1生物膜内部细菌的RNA提取，并通过逆转录试剂盒进一步逆转录合成与细菌RNA互补的DNA；最后通过实时荧光定量PCR仪测定细菌硝基还原酶相关表达基因(NsfB)的表达量，Proc基因为内参基因，实验结果如图8所示。
(2)评估超声响应载药脂质体对生物膜的抑制效果
在共聚焦培养皿和96孔板中生长PAO1生物膜，分别加入负载PFP的脂质体(PL)、负载PFP和Ce6的脂质体(PCL)和负载PFP，Ce6与MNZ的脂质体(PLCM)。对于超声治疗组，使用1MHz的超声在1W/cm 2功率下超声10min，于37℃恒温箱下培养12h。96孔板中的生物膜分散后转移至1.5mL离心管中，使用生理盐水定容至1mL，再分别进行梯度稀释10、10 2、10 3、10 4、10 5、10 6倍，然后将100μL原液及稀释10 4、10 5、10 6倍的菌液加到含有固体LB培养基的培养皿上进行涂板定量，实验结果如图9所示。共聚焦皿中的生物膜经Calcien-AM染色半小时后，通过共聚焦荧光显微镜成像观察生物膜内活菌，实验结果如图10所示。
铜绿假单胞菌在乏氧状态下高表达的硝基还原酶可以激活硝基咪唑类药物，产生咪唑 片段杀灭细菌。Ce6在超声作用下产生声动力性能将氧气转化为活性氧，杀灭生物膜表层活性菌，并加剧生物膜深处的乏氧状态、增强细菌硝基还原酶的表达，进一步激活甲硝唑，杀灭生物膜深层低活性菌。如图8所示，经PLCM结合超声治疗后，铜绿假单胞菌硝基还原酶相关表达基因NsfB显著高于PLCM治疗组和对照组，证明PLCM的声动力治疗导致的氧气消耗可显著增强铜绿假单胞菌硝基还原酶的表达。如图9-图10所示，PLCM治疗组和PL结合超声治疗组中铜绿假单胞菌生物膜内部细菌量相较对照组(Control)没有明显的变化，而PLC结合超声治疗组中的细菌相较于对照组下降了4.1个数量级，证明PLC的声动力性能具有显著的抗菌作用。PLCM结合超声治疗组中的细菌相较于对照组下降了5.9个数量级，证明声动力激活的MNZ可进一步抑制生物膜内部的细菌。
3、超声响应载药催化微泡抑制生物膜再生长
在共聚焦培养皿和96孔板中生长PAO1生物膜，分别加入分散于TSB的抗生素哌拉西林(Pip)、Pip与MNZ的混合液、PCL、PLCM。对于超声治疗组，使用1MHz的超声在1W/cm 2功率下超声10min，于37℃恒温箱下培养。分别将孵育1d、2d、3d后共聚焦皿中的生物膜用Calcien-AM染色半小时，通过共聚焦荧光显微镜成像观察生物膜内活菌，实验结果如图11所示。孵育1d、2d、3d后96孔板中的生物膜分散后转移至1.5mL离心管中，使用生理盐水定容至1mL，再分别进行梯度稀释10、10 2、10 3、10 4、10 5、10 6倍，然后将100μL原液及稀释10 4、10 5、10 6倍的菌液加到含有固体LB培养基的培养皿上进行涂板定量，实验结果如图12所示。
如图11-12所示，PLCM结合超声治疗组生物膜中的细菌数量逐渐降低，且没有再生长，其他治疗组的生物膜均有明显的细菌数量上升以及再生长现象，证明超声响应载药脂质体(PLCM)可高效杀灭生物膜内部细菌的同时抑制生物膜再生长。
以上研究结果证明PLCM在超声作用下可通过超声空化效用破坏铜绿假单胞菌生物膜结构、增强声敏剂Ce6和抗生素MNZ在生物膜内的渗透。Ce6的声动力可以高效杀灭生物膜中的代谢活性菌，并加剧膜内乏氧微环境，增强细菌硝基还原酶基因表达，激活抗生素MNZ，杀灭生物膜内的低代谢活性菌，实现高效的铜绿假单胞菌生物膜抑制功能，并抑制铜绿假单胞菌生物膜的再生长，且治疗效果显著优于传统用于治疗铜绿假单胞菌感染的抗生素Pip。
以上所述仅为本发明的实施方式而已，并不用于限制本发明。对于本领域技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原理的内所作的任何修改、等同替换、改进等，均应包括在本发明的权利要求范围之内。

Claims (13)

  1. 一种超声响应型脂质体纳米颗粒，所述脂质体纳米颗粒包含脂质材料、声敏剂，其特征在于：所述脂质材料包括二肉豆蔻酰磷脂酰胆碱、1,2-二油酰基-3-三甲基铵-丙烷、二硬脂酰基磷脂酰乙醇胺-聚乙二醇，所述脂质体纳米颗粒还包括抗生素和全氟戊烷。
  2. 根据权利要求1所述的一种超声响应型脂质体纳米颗粒，其特征在于：所述全氟戊烷在脂质体中的体积占比为1％-2％。
  3. 根据权利要求1所述的一种超声响应型脂质体纳米颗粒，其特征在于：所述声敏剂在脂质体中的浓度为0.5-1mg/mL。
  4. 根据权利要求3所述的一种超声响应型脂质体纳米颗粒，其特征在于：所述声敏剂为二氢卟吩。
  5. 根据权利要求1所述的一种超声响应型脂质体纳米颗粒，其特征在于：所述抗生素在脂质体中的浓度为0.5-1mg/mL。
  6. 根据权利要求5所述的一种超声响应型脂质体纳米颗粒，其特征在于：所述抗生素为甲硝唑。
  7. 根据权利要求1所述的一种超声响应型脂质体纳米颗粒，其特征在于：二肉豆蔻酰磷脂酰胆碱、1,2-二油酰基-3-三甲基铵-丙烷、二硬脂酰基磷脂酰乙醇胺-聚乙二醇的质量比为5：1.5：1。
  8. 一种如权利要求1-7任一项所述的一种超声响应型脂质体纳米颗粒的制备方法，其特征在于：该脂质体纳米颗粒的制备方法包括如下步骤：
    步骤1：将二肉豆蔻酰磷脂酰胆碱、1,2-二油酰基-3-三甲基铵-丙烷、二硬脂酰基磷脂酰乙醇胺-聚乙二醇、声敏剂、抗生素溶解于氯仿，在50℃下旋蒸5-10min，形成一层脂质体膜；
    步骤2：利用离子水重悬脂质体膜后，将水化后的脂质体在冰水浴中利用探头超声处理，处理过程中缓慢滴加全氟戊烷，脂质体在超声水化自组装的过程中能逐渐包裹全氟戊烷，形成脂质体纳米颗粒分散液；
    步骤3：将反应后的脂质体纳米颗粒分散液至于透析袋，在磷酸缓冲盐溶液中透析，得到载药脂质体纳米颗粒。
  9. 根据权利要求8所述的一种超声响应型脂质体纳米颗粒的制备方法，其特征在于：步骤2中探头超声的工作条件为：工作5s，间歇2s，功率40％，超声时间5-10min。
  10. 根据权利要求8所述的一种超声响应型脂质体纳米颗粒的制备方法，其特征在于：步骤3中所用的透析袋截留分子量为10kDa，透析时间为24-48h。
  11. 根据权利要求8所述的一种超声响应型脂质体纳米颗粒的制备方法，其特征在于：步骤3中透析所用的磷酸缓冲盐溶液pH为7.4，浓度为10mM。
  12. 根据权利要求8所述的一种超声响应型脂质体的制备方法，其特征在于：步骤3中超声响应型脂质体为载药脂质体纳米颗粒，脂质体纳米颗粒尺寸为150-250nm。
  13. 一种如权利要求1所述的一种超声响应型脂质体纳米颗粒在抗细菌生物膜感染治疗中的应用。
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