WO2022218284A1 - Nanoparticules de paroi de cellule de levure, leur procédé de préparation et leur application - Google Patents

Nanoparticules de paroi de cellule de levure, leur procédé de préparation et leur application Download PDF

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WO2022218284A1
WO2022218284A1 PCT/CN2022/086246 CN2022086246W WO2022218284A1 WO 2022218284 A1 WO2022218284 A1 WO 2022218284A1 CN 2022086246 W CN2022086246 W CN 2022086246W WO 2022218284 A1 WO2022218284 A1 WO 2022218284A1
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cell wall
yeast cell
yeast
tumor
nanoparticles
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汪超
徐嘉潞
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/063—Lysis of microorganisms of yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/066—Lysis of microorganisms by physical methods
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/13—Preparation or pretreatment of starting material involving cleaning, e.g. washing or peeling
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to a yeast cell wall nanoparticle and a preparation method and application thereof.
  • Cancer is a serious threat to human health. According to the latest statistics from the National Cancer Center, the death caused by malignant tumors accounts for 23.91% of all deaths. At present, the morbidity and mortality of malignant tumors are on the rise. Control the severe development trend. In recent years, tumor immunotherapy, a treatment method that uses the host immune system to achieve anti-tumor goals, has received extensive attention and achieved certain results. Despite this, the clinical response rate of tumor immunotherapy to solid tumors is low. More and more studies have shown that the inflammatory tumor microenvironment can make tumors sensitive to immune checkpoint inhibitors, and non-inflammatory tumors have immunosuppressive effects.
  • the tumor microenvironment is mainly characterized by inactivation of T cells embedded in the tumor stroma, abundant cells of myeloid origin, abnormal vascular distribution, and insensitivity to immune checkpoint inhibitors. Therefore, the development of immune stimulators with immune activating effects to transform the non-inflammatory tumor microenvironment into inflammatory ones and improve the sensitivity of tumors to immune checkpoint inhibitors is a current research hotspot.
  • microbe-based cancer immunotherapy is still in the early stages of development.
  • the present invention provides a yeast-derived nanoparticle system, which is prepared from the micron-scale cell wall of Saccharomyces cerevisiae by crushing and differential centrifugation.
  • the advantages of the present invention are as follows: First, because the cell wall of Saccharomyces cerevisiae has no reproductive ability, it will not cause the infection of microorganisms in the body, so it has good biological safety.
  • nano-scale Saccharomyces cerevisiae cell wall particles can be more easily enriched to tumors and lymph nodes than micro-scale particles, resulting in a strong anti-tumor immune response; It is inexpensive to produce and transport; finally, Saccharomyces cerevisiae is a probiotic that is acceptable to patients.
  • the first object of the present invention is to provide a yeast cell wall nanoparticle, the yeast cell wall nanoparticle is obtained by removing the contents of the yeast, and using ultrasonic crushing and differential centrifugation to obtain the collected yeast cell wall. Nanoparticles with a diameter of 10 to 1000 nm, and the potential of the yeast cell wall nanoparticles is -1mV to -50mV.
  • the particle size of the yeast cell wall nanoparticles is 10-100 nm, 100-500 nm or 500-1000 nm.
  • the second object of the present invention is to provide a preparation method of yeast cell wall nanoparticles, comprising the following steps:
  • yeast cells are subjected to wall breaking treatment, and cell wall components are collected;
  • step S2 washing the cell wall components collected in step S1, and drying to obtain cell wall powder;
  • step S4 centrifuge the supernatant of step S3 at 2000-3000g rotating speed, and collect the supernatant and precipitate respectively;
  • step S5 centrifuge the supernatant of step S4 at 8000-11000g rotating speed, and collect the supernatant and precipitate respectively;
  • step S6 centrifuge the supernatant of step S5 at 18000-22000g speed to collect the precipitate
  • the precipitate collected in step S4, S5 or S6 is the yeast cell wall nanoparticles
  • the conditions of ultrasonic fragmentation are as follows: under the ultrasonic power of 80-120W, ultrasonic treatment is performed 80-120 times according to the frequency of ultrasonic for 2-4 seconds and a gap of 6-8 seconds. .
  • step S1 the wall-breaking treatment is performed by suspending yeast cells in alkaline solution and heating at 70-90° C. for 0.5-2 hours.
  • the lye solution is 0.8 ⁇ 1.5M NaOH solution.
  • step S1 the cell wall fractions are collected by centrifugation at 1500-2500g centrifugal force for 10 minutes.
  • step S2 cleaning includes the following steps:
  • step S01 using dilute hydrochloric acid with a pH of 4-5 for the cell wall components collected in step S1, neutralizing the NaOH solution, heating at 50-60°C for 0.5-2 hours, and centrifuging at 1500-2500g for 10 minutes to collect the precipitate;
  • step S02 the precipitate collected in step S01 is washed successively with ultrapure water, isopropanol and acetone.
  • ultrapure water is used to remove water-soluble impurities first, and then isopropyl alcohol is used as a dehydrating agent to remove water in the precipitate, and finally acetone is used to clean, so as to facilitate drying.
  • the third object of the present invention is to provide the application of the yeast cell wall nanoparticles in the preparation of anti-tumor immune drugs.
  • anti-tumor immune drugs also include immune checkpoint inhibitors.
  • the present invention has at least the following advantages:
  • the nanoparticle system of the present invention is prepared from Saccharomyces cerevisiae by crushing and differential centrifugation, and has no reproductive ability in the injection body, so it has good safety.
  • the nano-sized yeast-derived particle system of the present invention has a strong ability to deliver to tumor-draining lymph nodes, can effectively regulate the microenvironment of tumor-draining lymph nodes, and induce immune response.
  • yeast-derived nanoparticles of the present invention are related to their nanometer size, which is the first time that this phenomenon has been found in microorganism-based tumor therapy.
  • the nano-formulation has good repeatability in preparation, can be produced and transported on a large scale, and has low cost.
  • 1 is a transmission electron microscope image of the yeast-derived microscale and nanoparticle system of the present invention
  • FIG. 2 is a particle size distribution diagram of the yeast-derived nanoparticle system of the present invention.
  • FIG. 3 is a graph showing the particle size change of the yeast-derived nanoparticle system of the present invention at room temperature and 4°C;
  • Fig. 4 is the surface protein content diagram of the yeast-derived nanoparticle system of the present invention.
  • FIG. 6 is a flow cytometry diagram of in vitro phagocytosis of the yeast-derived nanoparticle system of the present invention.
  • Figure 7 is a confocal image of the in vitro phagocytosis of the yeast-derived nanoparticle system of the present invention.
  • Figure 8 is a graph showing the maturation of bone marrow-derived dendritic cells induced by the yeast-derived nanoparticle system of the present invention.
  • Fig. 9 is a graph showing the cytokine secretion of bone marrow-derived dendritic cells induced by the yeast-derived nanoparticle system of the present invention.
  • Figure 10 is a graph showing tumor growth after intratumoral injection of the anti-tumor immune drug of the present invention.
  • Figure 11 is the tumor H&E slice images of the untreated group and the small-diameter yeast cell wall treatment group
  • Figure 12 is a flow cytometry analysis of the tumor microenvironment in the untreated group and the small-diameter yeast cell wall treatment group;
  • Figure 13 is an in vitro imaging image of the anti-tumor immune drug of the present invention delivered to tumor draining lymph nodes;
  • Figure 14 is a mathematical modeling diagram of the ability of the anti-tumor immune drug of the present invention to migrate to tumor-draining lymph nodes and size effect;
  • Figure 15 is a diagram showing the distribution of anti-tumor immune drugs of the present invention in tumor-draining lymph nodes
  • Figure 16 is a flow cytometry diagram of T cells and B cells in tumor-draining lymph nodes activated by anti-tumor immune drugs of the present invention
  • Figure 17 is a flow cytometry analysis of dendritic cells in tumor-draining lymph nodes activated by anti-tumor immune drugs of the present invention.
  • Figure 18 is a graph showing the tumor growth curve of the anti-tumor immune drug of the present invention in the treatment of melanoma;
  • Figure 19 is a graph showing the survival curve of the anti-tumor immune drug of the present invention in the treatment of melanoma;
  • Figure 20 is the H&E stained section diagram and the mouse body weight diagram of the main organ of the anti-tumor immune drug of the present invention for treating melanoma;
  • Figure 21 is a tumor growth curve diagram of the anti-tumor immune drug of the present invention in the treatment of melanoma metastases;
  • Fig. 22 is a small animal fluorescence imaging image of the anti-tumor immunodrug of the present invention for treating melanoma metastases.
  • C57BL/6 and BALB/c female mice aged 6-8 weeks were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All mouse experiments were performed in accordance with the animal experimental protocol approved by the Laboratory Animal Center of Soochow University.
  • BMDCs Bone marrow-derived dendritic cells
  • Immune checkpoint inhibitor PD-L1 antibody (anti-PD-L1) was purchased from Biox cell company, (the antibody number is 10F.9G2).
  • Example 1 Preparation of a yeast-derived nanoparticle system
  • Example 2 Characterization of a yeast-derived nanoparticle system
  • TEM Transmission electron microscopy
  • TEM Transmission electron microscopy
  • DLS Dynamic light scattering
  • the surface proteins were analyzed by BCA protein quantification kit and SDS-PAGE gel electrophoresis. The results are shown in Figure 4 and Figure 5.
  • the three nano-sized yeast cell walls contain the same protein composition and content, which indicates that in addition to There are differences in size, otherwise consistent.
  • Example 3 Immunological effects of yeast-derived nanoparticle systems on dendritic cells
  • the three nano-sized yeast cell walls were stained with Cy5.5, and after co-incubating with DC2.4 for 24 hours, the cells were collected by centrifugation at 300g for 3 minutes, and the effect of dendritic cells phagocytosing the three nanoparticles was analyzed by flow cytometry.
  • the three nanometer-sized yeast cell walls stained with Cy5.5 were co-incubated with dendritic cells for 24 hours, and after fixation with 4% paraformaldehyde, the nuclei were stained with DAPI to locate the cells, and confocal microscopy images were taken for analysis. .
  • the results are shown in Fig. 6 and Fig. 7, the three nano-sized yeast cell walls can be effectively phagocytosed by dendritic cells, and as the particle size decreases, the phagocytosis effect is better.
  • mice bone marrow-derived dendritic cells were extracted according to the existing method, and when their maturity reached about 8%, they were incubated with LPS and three nano-sized yeast cell walls for 24 hours, and the supernatant was collected and stored at -80°C
  • BMDCs were collected by centrifugation at 300g for 3 minutes to analyze the expression of costimulatory factors (CD80, CD86).
  • CD80, CD86 costimulatory factors
  • Example 4 In vivo immunological effects of yeast-derived nanoparticle systems
  • Yeast-derived nanoparticle system inhibits tumor growth by remodeling the tumor microenvironment
  • the yeast-derived nanoparticle system can inhibit the growth of mouse melanoma, and as the particle size decreases, the effect of suppressing the tumor is better.
  • the H&E sections of the tumors in the control and treatment groups reflected the same results.
  • T cell infiltration, bone marrow-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and dendritic cells in the tumor microenvironment were analyzed. The results are shown in Figure 12.
  • the small-diameter yeast cell wall treatment group could significantly increase the proportion of CD8 + T cells and CD4 + T cells in the tumor, and the treatment group also significantly improved the tumor immunosuppressive microenvironment.
  • the proportion of MDSCs, Tregs, and TAMs in the tumor decreased significantly, and at the same time, the maturity of DCs reached about 35%, which further demonstrated that the yeast-derived nanoparticle system can be used as an anti-tumor immune drug.
  • Cy5.5 was detected in major immune cells such as dendritic cells, macrophages, T cells, B cells, etc. , and its content is negatively correlated with the size of the nanoparticle system.
  • the activation of immune cells plays a key role in the antitumor immune response, and next, we evaluated the activation of major immune cells.
  • Dendritic cells as professional antigen-presenting cells, play a crucial role in the antitumor process.
  • the expression of costimulatory molecules (CD80, CD86, CD40, MHCII) on dendritic cells in the tumor-draining lymph nodes of the treatment group were all increased compared with the control group. Small-sized yeast cell walls had stronger stimulatory effects on major immune cells in mice, which may be related to their higher enrichment in tumor-draining lymph nodes.
  • mice treated with the yeast-derived nanoparticle system combined with the immune checkpoint inhibitor were well tolerated.
  • the results are shown in Figure 20. . These results intuitively demonstrate that the combination of small particle size yeast cell walls and PD-L1 blockade produces a significant synergistic antitumor immune response.
  • Small-sized yeast cell walls combined with anti-PD-L1 can destroy tumors, resulting in tumor cell lysates, which subsequently co-migrate to tumor-draining lymph nodes and promote dendritic cell maturation and activation of T and B cells. From the growth curve in Figure 21 and the fluorescence imaging of small animals in Figure 22, the combination treatment not only inhibited the growth of the in situ tumor, but also significantly inhibited the contralateral tumor, indicating that the yeast-derived nanoparticle system and immune checkpoint inhibition As an anti-tumor composition, the combination of the drug can induce a systemic anti-tumor immune response, thereby reducing tumor metastasis.
  • yeast-derived nanoparticle system of the present invention as an anti-tumor immune drug, induces an anti-tumor immune response by remodeling tumor-draining lymph nodes and tumor microenvironment, inhibits tumor growth and reduces tumor metastasis.

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Abstract

L'invention concerne des nanoparticules de paroi de cellule de levure, leur procédé de préparation et leur application. Les nanoparticules de paroi de cellule de levure sont des nanoparticules ayant une taille de particule de 10-1 000 nm obtenues par élimination d'une inclusion de levure et par traitement de parois de cellule de levure collectées dans un mode de centrifugation différentielle, et le potentiel des nanoparticules de paroi de cellule de levure est de -1 mV à -50 mV. Le système de particules nanométriques issues de levure a une forte capacité de production de ganglions lymphatiques drainant les tumeurs, et peut réguler efficacement un microenvironnement des ganglions lymphatiques drainant les tumeurs, ce qui provoque une réponse immunitaire.
PCT/CN2022/086246 2021-04-16 2022-04-12 Nanoparticules de paroi de cellule de levure, leur procédé de préparation et leur application WO2022218284A1 (fr)

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CN113143977B (zh) * 2021-04-16 2022-04-05 苏州大学 酵母细胞壁纳米颗粒及其制备方法与应用
CN114425088B (zh) * 2021-12-08 2023-06-27 深圳先进技术研究院 一种酵母仿生免疫微纳生物机器人及其制备方法和应用
JP2023164363A (ja) * 2022-04-28 2023-11-10 学校法人帝京大学 免疫機能活性化剤、ワクチンアジュバントおよび免疫誘導方法

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