WO2022218284A1

WO2022218284A1 - Yeast cell wall nanoparticles and preparation method therefor and application thereof

Info

Publication number: WO2022218284A1
Application number: PCT/CN2022/086246
Authority: WO
Inventors: 汪超; 徐嘉潞
Original assignee: 苏州大学
Priority date: 2021-04-16
Filing date: 2022-04-12
Publication date: 2022-10-20
Also published as: CN113143977B; CN113143977A

Abstract

Provided are yeast cell wall nanoparticles and a preparation method therefor and an application thereof. The yeast cell wall nanoparticles are nanoparticles having a particle size of 10-1000 nm obtained by removing an inclusion of yeast and treating collected yeast cell walls in a differential centrifugation mode, and the potential of the yeast cell wall nanoparticles is -1 mV to -50 mV. The nano-sized yeast-derived particle system has a strong ability to deliver to tumor draining lymph nodes, and can effectively regulate a microenvironment of the tumor draining lymph nodes, thereby causing an immune response.

Description

Yeast cell wall nanoparticle and its preparation method and application

technical field

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.

Background technique

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.

Recent studies have shown that bacteria, viruses and/or fungi are ubiquitous in cancer, and they are key elements in cancer immunotherapy and can be used to treat tumor metastasis. In recent years, microbe-based cancer immunotherapies, including bacteria, viruses, and fungi, have been used to activate innate immunity and boost adaptive immunity, thereby enhancing antitumor immune responses. Engineering of exogenous bacterial and viral agents has made significant progress for cancer therapy, especially as a powerful immunotherapy option or neoadjuvant. There are two drugs approved by the U.S. Food and Drug Administration (FDA): T-VEC (an oncolytic virus modified from herpes simplex virus type I (HSV-1)) and bacteria to treat advanced melanoma of Mycobacterium (BCG).

Although numerous studies have shown that microbial-based therapeutic strategies offer new directions for cancer immunotherapy, limitations still exist. First, the live microbiota can cause systemic infections and severe systemic toxicity in patients, causing the immune system to attack healthy cells. Second, the tumor regression rate caused by bacterial therapy is low, and the efficiency of anti-tumor immunity still needs to be improved; third, the above-mentioned large-scale production, quality control, and stability based on live bacteria and viruses are difficult to guarantee; Acceptability is also an issue worth considering. Therefore, microbe-based cancer immunotherapy is still in the early stages of development.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, 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. Second, 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.

Further, 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:

S1. The yeast cells are subjected to wall breaking treatment, and cell wall components are collected;

S2, washing the cell wall components collected in step S1, and drying to obtain cell wall powder;

S3, resuspend the cell wall powder in the step S2 in the buffer, carry out ultrasonic fragmentation, and centrifuge at 600-1200g speed after fragmentation, and collect the supernatant;

S4, centrifuge the supernatant of step S3 at 2000-3000g rotating speed, and collect the supernatant and precipitate respectively;

S5, centrifuge the supernatant of step S4 at 8000-11000g rotating speed, and collect the supernatant and precipitate respectively;

S6, centrifuge the supernatant of step S5 at 18000-22000g speed to collect the precipitate;

Wherein, 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. .

Further, in 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.

Further, the lye solution is 0.8～1.5M NaOH solution.

Further, in step S1, the cell wall fractions are collected by centrifugation at 1500-2500g centrifugal force for 10 minutes.

Further, in step S2, cleaning includes the following steps:

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;

S02, the precipitate collected in step S01 is washed successively with ultrapure water, isopropanol and acetone.

In the present invention, 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.

Further, the anti-tumor immune drugs also include immune checkpoint inhibitors.

By means of the above scheme, the present invention has at least the following advantages:

1. 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.

2. 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.

3. The ability of the yeast-derived nanoparticles of the present invention to be delivered to tumor-draining lymph nodes and their anti-tumor efficacy are related to their nanometer size, which is the first time that this phenomenon has been found in microorganism-based tumor therapy.

4. Compared with live microbial therapy, the nano-formulation has good repeatability in preparation, can be produced and transported on a large scale, and has low cost.

The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and implement it according to the content of the description, the preferred embodiments of the present invention are described in detail below.

Description of drawings

1 is a transmission electron microscope image of the yeast-derived microscale and nanoparticle system of the present invention;

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;

5 is an SDS-PAGE gel electrophoresis image 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.

Detailed ways

The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, and the protection scope of the present invention can be more clearly defined.

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.

Mouse melanoma B16-luc tumor cells, colon cancer cells CT26, macrophage RAW264.7, and dendritic cells DC2.4 were purchased from the Cell Bank of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Bone marrow-derived dendritic cells (BMDCs) were extracted from the bone marrow cavity of 7-8 week old C57BL/6 mice according to established methods. Cells subcultured at the third or fourth passage were used in the various examples of the present invention.

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

100 g of Saccharomyces cerevisiae powder was weighed and suspended in 1M NaOH solution, and heated at 80°C for 1 hour. After cooling, centrifuge at 2000g for 10 minutes to collect insoluble material containing yeast cell walls. The insoluble material was suspended in 1 L of ultrapure water adjusted to pH 4-5 with HCl and incubated at 55°C for 1 hour. After cooling to room temperature, insoluble substances were collected by centrifugation at 2000 g for 10 minutes, and washed once with 1 L of ultrapure water, 4 times with 200 mL of isopropanol, and twice with 200 mL of acetone. The obtained insoluble material is placed in a container, and dried at room temperature to obtain a fine white powder, which is a micron-scale yeast cell wall. Subsequently, yeast cell walls of different nanometer sizes were prepared by gradient centrifugation. Specifically, 25 mg of white powder was weighed and dissolved in 8 mL of PBS (pH=7.4), and a 100W cell disruptor was used to perform ultrasonic disruption according to ultrasonic time: 3 seconds; gap time: 7 seconds; working times: 99 times , after crushing, centrifuged at 1000g for 5 minutes to remove large particles of insoluble substances, the obtained supernatant was centrifuged at 2500g for 10 minutes, and the obtained precipitate was resuspended in PBS to be large-diameter yeast cell wall particles; The medium-sized yeast cell wall particles were obtained by resuspending in PBS; finally, the precipitate obtained after the supernatant was centrifuged at 20,000 g for 10 minutes was resuspended in PBS to obtain small-sized yeast cell wall particles.

Example 2: Characterization of a yeast-derived nanoparticle system

Transmission electron microscopy (TEM) was used to characterize the internal structure of micron-scale yeast cell walls and three nano-sized yeast cell walls. shape and uniform distribution. Dynamic light scattering (DLS) analysis of particle size and particle size changes of three nano-sized yeast cell walls with time under different storage conditions, the results are shown in Figure 2 and Figure 3, both at room temperature and 4 ℃ storage conditions Stable storage for at least 2 weeks. 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

(1) In vitro cell uptake analysis

First, 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. At the same time, 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.

(2) Immunological effect analysis of bone marrow-derived dendritic cells in vitro

The mouse 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 For subsequent detection of cytokines, BMDCs were collected by centrifugation at 300g for 3 minutes to analyze the expression of costimulatory factors (CD80, CD86). The results are shown in Figure 8 and Figure 9, the three nano-sized yeast cell walls stimulated the maturation of BMDCs well, and the effect was comparable to the LPS group (positive control). , BMDC effectively secreted cytokines such as TNF-α, IL-1β, IL-12p70, which provided the basis for the subsequent induction of strong anti-tumor immune responses in vivo.

Example 4: In vivo immunological effects of yeast-derived nanoparticle systems

(1) Yeast-derived nanoparticle system inhibits tumor growth by remodeling the tumor microenvironment

Three kinds of nano-sized yeast cell walls were injected intratumorally, and their tumor growth inhibition effects were judged by monitoring their tumor growth. The results are shown in Figure 10. 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. At the same time, as shown in Figure 11, 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. Compared with the untreated group, 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.

(2) Analysis of the ability of anti-tumor immune drugs to migrate to tumor-draining lymph nodes

First, we injected Cy5.5-labeled three nano-sized yeast cell walls into B16 tumors, euthanized the mice 48 hours later, and collected their tumor-draining lymph nodes for in vivo fluorescence imaging. As shown in Figure 13, the small-sized yeast cell wall is favorable for entering and targeting the tumor-draining lymph nodes, followed by the medium-sized yeast cell wall, and the large-sized yeast cell wall has the worst targeting ability to the tumor-draining lymph node. In order to better explain that the ability of the yeast-derived nanoparticle system to target tumor-draining lymph nodes is related to the size, through mathematical modeling, the data fitting results show that, as shown in Figure 14, the migration ability of the nanosystem is related to the particle size. The square root is inversely proportional, ie E=D- ^1/2 . Next, we evaluated the distribution of the yeast-derived nanoparticle system in tumor-draining lymph nodes, and as shown in Figure 15, 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. As shown in Figure 16, the expression of CD69 on both T (CD4 ⁺ and CD8 ⁺ ) cells and B cells (CD19 ⁺ ) was up-regulated 48 hours after intratumoral injection, indicating that both T cells and B cells were effectively treated after treatment activation. Dendritic cells, as professional antigen-presenting cells, play a crucial role in the antitumor process. As shown in Figure 17, 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.

(3) Efficacy and safety evaluation of yeast-derived nanoparticle system combined with immune checkpoint blockade therapy

After immunological evaluation analysis of tumors, the results showed that the expressions of PD-1 and PD-L1 on T cells were significantly up-regulated after treatment with small-sized yeast cell walls. Therefore, we combined small-sized yeast cell walls with anti-PD- L1 combination to treat mouse melanoma. The results are shown in Figure 18. Compared with the single-administration treatment group, the immunotherapy based on the yeast-derived nanoparticle system combined with anti-PD-L1 showed a great synergistic effect in controlling tumor growth, and the combined treatment The tumors in the mice were completely regressed, and Figure 19 shows that the survival of the mice was significantly prolonged. At the same time, by monitoring the body weight of the mice during the treatment period and observing the H&E slices of the main organs, the 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.

In conclusion, the 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.

The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the technical principles of the present invention. , these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

A yeast cell wall nanoparticle, characterized in that, the yeast cell wall nanoparticle is obtained by removing the contents of the yeast, and applying ultrasonic crushing and differential centrifugation to the collected yeast cell wall to obtain a particle size of 10-1000 nm. The nanoparticle of the yeast cell wall has a potential of -1mV to -50mV.
The yeast cell wall nanoparticles according to claim 1, wherein the yeast cell wall nanoparticles have a particle size of 10-100 nm, 100-500 nm or 500-1000 nm.
A method for preparing yeast cell wall nanoparticles according to any one of claims 1 to 2, characterized in that, comprising the steps of:

S1. The yeast cells are subjected to wall breaking treatment, and cell wall components are collected;

S2, washing the cell wall components collected in step S1, and drying to obtain cell wall powder;

S3, resuspend the cell wall powder in the step S2 in the buffer, carry out ultrasonic fragmentation, and centrifuge at 600-1200g speed after fragmentation, and collect the supernatant;

S4, centrifuge the supernatant of step S3 at 2000-3000g rotating speed, and collect the supernatant and precipitate respectively;

S5, centrifuge the supernatant of step S4 at 8000-11000g rotating speed, and collect the supernatant and precipitate respectively;

S6, centrifuge the supernatant of step S5 at 18000-22000g speed to collect the precipitate;

Wherein, 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.
The method according to claim 3, wherein in 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.
method according to claim 4, is characterized in that, described lye solution is 0.8～1.5M NaOH solution.
The method according to claim 3, wherein, in step S1, collecting the cell wall fraction is centrifuged at 2000 g for 10 minutes.
The method according to claim 3, wherein in step S2, cleaning comprises the following steps:

S01, adopting dilute hydrochloric acid with a pH of 4-5 for the cell wall fraction collected in step S1, heating at 50-60° C. for 0.5-2 hours, and centrifuging at 2000-3000 g centrifugal force to collect the precipitate;

S02, the precipitate collected in step S01 is washed successively with ultrapure water, isopropanol and acetone.
Application of the yeast cell wall nanoparticles according to any one of claims 1 to 2 in the preparation of anti-tumor immune drugs.
The application according to claim 8, wherein the anti-tumor immune drug induces a systemic anti-tumor immune response by remodeling the tumor microenvironment and the tumor-draining lymph node microenvironment.
The application according to claim 8, wherein the anti-tumor immune drug comprises an immune checkpoint inhibitor.