Application of sunflower nano pollen particles in preparation of anti-tumor immune response oral drug
By nano-nancing and binding to the immune checkpoint inhibitor PD1 antibody, the problem of low bioavailability of micron-level pollen and unclear mechanism of action of anti-tumor immune molecules is solved, and the effect of efficient activation of immune cells and significantly inhibiting tumor growth is achieved.
Patent Information
- Application Number
- CN202510037364.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-04-25
AI Technical Summary
In the prior art, the bioavailability of micron-scale pollen is limited, and the mechanism of action of anti-tumor immune molecules is unclear, making it difficult to effectively activate immune cells in the body to achieve anti-tumor effects.
Sunflower pollen was nano-sized by mechanical grinding method, with particle size controlled between 200 and 400 nm, and combined with the immune checkpoint inhibitor PD1 antibody, oral drugs for anti-tumor immune response were prepared.
Nanopollen can significantly improve bioavailability, enhance the activation effect on immune cells, significantly inhibit tumor growth, and increase sensitivity to immune checkpoint inhibitors.
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Figure CN119868531A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biomaterials, and in particular relates to the application of sunflower nano-pollen particles in the preparation of oral anti-tumor immune response drugs. Background Art
[0002] Malignant tumors are major diseases that seriously threaten human health and life safety, among which solid tumors account for more than 90% of all tumors. At present, tumor treatment is still mainly based on surgery, supplemented by comprehensive strategies such as targeted immunotherapy. Although certain effects have been achieved, the prognosis of many solid tumors is still poor. Therefore, exploring new tumor treatment methods to improve patient survival and reduce tumor recurrence and mortality has become an urgent clinical need.
[0003] As a traditional Chinese medicine, sunflower pollen has attracted much attention because of its rich resources and immunomodulatory effects. Structurally, most pollens have a double wall. The inner layer is composed of carbohydrates such as cellulose / hemicellulose and pectin, which can protect male gametes, other biomolecules and nutrients. The outer layer is composed of a biopolymer of pollenin that can resist acids, alkalis and organic solvents. In the pollen structure, proteins are present in large quantities in the core, and some of these proteins can cause allergies. Pollen contains various chemical and biologically active compounds and has a variety of therapeutic effects. The effects of some pollens have been used in traditional Chinese medicine, and the therapeutic effects of pollen have also been evaluated in animal and clinical studies. Among them, the chemical and biologically active compounds include carbohydrates (fructose, glucose, sucrose, fiber), amino acids, nucleic acids, polyphenols, proteins and lipids, as well as minerals and trace elements and various vitamins, enzymes and coenzymes. Because pollen grains contain these compounds, they have many therapeutic properties, such as antibacterial, antifungal, antioxidant, immunomodulatory, anti-osteoporosis, anti-inflammatory, chemoprevention, anti-tumor, anti-aging, radiation protection and liver protection.
[0004] With the renewed awareness of natural therapies and alternative medicine, the anti-tumor properties of pollen have begun to attract widespread attention from scientific researchers. A double-blind study evaluated the effects of non-estrogenic pollen extract PCC-100 on 300 women aged 50 to 65 who experienced vasomotor symptoms during adjuvant hormone therapy for breast cancer. PCC-100 has been shown to be useful for treating vasomotor symptoms in postmenopausal women receiving adjuvant hormone therapy and menopausal women who have never had breast cancer. In addition, pollen itself can act as an exogenous antigen to activate antigen presenting cells and initiate Th2 cell-dependent anti-tumor immune responses, activate B cells to produce IgE / IgG antibodies and have pro-inflammatory effects. For example, grass pollen immunotherapy can improve the quality of life of patients with seasonal allergic rhinitis and reduce the symptoms of seasonal asthma and pulmonary hyperresponsiveness. However, although pollen can promote local inflammatory responses and activate the body's immune response, the bioavailability of micron-sized pollen is still limited and the mechanism of action of anti-tumor immune molecules is still unclear. Summary of the invention
[0005] Due to the in vivo metabolic pathways and biodistribution of oral nanopollen, the activation of immune cells in the mesenteric lymph nodes or spleen, the activation of immune-related signals, and its anti-tumor immune response mechanism are still unclear.
[0006] To this end, the present invention intends to analyze the activation of immune-related signaling pathways of antigen-presenting cells activated by nano-pollen through in vitro transcriptome sequencing, including its role in stimulating cytokine release and enhancing cell-to-cell interactions; molecular biological analysis methods will be used to study how nano-pollen affects the activity and anti-tumor mechanism of immune cells by regulating signaling pathways such as TLR2, NF-κB, PI3K / Akt, etc.; single-cell transcriptome sequencing technology will be used to analyze changes in the tumor immune microenvironment after treatment to reveal changes in different immune cell subpopulations and their functional states, providing a molecular basis for the immunomodulatory properties of sunflower pollen. The present invention not only has important scientific value, but also has broad social application potential.
[0007] The invention discloses an application of sunflower nano pollen particles in the preparation of an oral anti-tumor immune response drug, wherein the particle size of the sunflower nano pollen particles is 200 to 400 nm, and the Zeta potential of the sunflower nano pollen is 0 to 0.025 mV.
[0008] The oral medicament further comprises a pharmaceutically acceptable carrier or excipient: a preservative, an antioxidant, a flavoring agent, an aromatic, a solubilizing agent, an emulsifier, a pH buffering substance, a binder, a filler or a lubricant.
[0009] The sunflower nano-pollen particles are prepared by the following steps: (1) grinding sunflower pollen, dispersing crude sunflower pollen in a PBS solution, grinding the pollen using a cryo-grinder, and then subjecting the grinding to ultrasonic treatment;
[0010] (2) Centrifugation and particle size analysis: The micron-sized pollen with a larger particle size that is easy to precipitate is separated and removed by centrifugation, and then the supernatant is taken and freeze-dried to obtain the sunflower nano-pollen particles.
[0011] The grinding parameters in step (1) are 60 Hz, 5 minutes, 3 cycles, 1 minute intervals; after grinding, ultrasonic treatment is performed at 70% power, and each ultrasonic treatment is continued for 2 minutes in a cycle of 2 seconds for each ultrasonic treatment and 3 seconds for rest, for a total of three times.
[0012] The centrifugation parameter in step (2) is set to 3000 rpm / min for a total of 5 min.
[0013] The sunflower nano-pollen particles are combined with immune checkpoint inhibitors to prepare drugs for treating tumors.
[0014] The PD1 inhibitor includes an anti-PD-1 antibody (BioLegend, Inc (USA)) or an antigen-binding fragment thereof.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] 1. Use mechanical grinding to physically grind sunflower pollen to destroy its outer wall structure, and obtain nano-pollen of about 200 to 400 nm by centrifugation. Nano-pollen can not only improve the bioavailability of drugs, but also enhance their therapeutic effects in the body. By co-culturing nano-pollen with primary dendritic cells, it was found that compared with micron-sized pollen, nano-pollen can be efficiently taken up by them and effectively activate the maturation of dendritic cells.
[0017] 2. After oral administration to a mouse subcutaneous tumor model, it was found that nano pollen can significantly inhibit tumor growth, verifying its effectiveness in treating tumors.
[0018] 3. Nano pollen can activate the TLR2 signaling pathway to exert an immunomodulatory effect, and at the same time, nano pollen can increase its sensitivity to immune checkpoint inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The plant image of sunflower nanopollen, the scanning electron microscope (SEM) image of a single pollen, and the pollen processing process are shown;
[0020] Figure 2 This is a transmission electron microscope (TEM) image of sunflower nanopollen;
[0021] Figure 3 This is the particle size distribution diagram of sunflower nano pollen before and after grinding;
[0022] Figure 4 Zeta potential diagram of sunflower nanopollen and original pollen;
[0023] Figure 5 Confocal fluorescence images of nanopollen and original pollen being taken up by DCs2.4 immune cells;
[0024] Figure 6 The effects of Nanopollen and Pollen on the co-stimulatory factors CD80 and CD86 on the surface of BMDCs.
[0025] Figure 7 The effects of Nanopollen and Pollen on BMDCs cell morphology;
[0026] Figure 8 TEM images of cells treated with PBS (control group), LPS and the same concentration of nano-pollen and original pollen; sunflower nano-pollen is taken up by antigen-presenting cells and activates primary bone marrow-derived antigen-presenting cells;
[0027] Fig. 9 The graphs show the expression levels of specific proteins in nano-pollen, original pollen treated with PBS, and BMDCs treated with LPS;
[0028] Fig.10 The results of intragastric administration of sunflower nano-pollen showed that it inhibited the growth of subcutaneous Hepal-6 tumors in C57BL6 mice;
[0029] Fig.11 The results show that oral administration of sunflower nanopollen combined with PD1 inhibitor inhibited the growth of subcutaneous Hepal-6 tumors in C57BL6 mice. DETAILED DESCRIPTION
[0030] The present invention will now be described in further detail with reference to the accompanying drawings.
[0031] Example 1. Method for preparing nano-pollen and its biosafety evaluation
[0032] 1. Preparation method of nano pollen:
[0033] (1) Sample pretreatment: sunflower pollen is centrifuged using a mixture of water and ethanol to separate and wash the pollen to obtain a crude pollen product.
[0034] (1) Grinding of sunflower pollen: 10 mg of crude sunflower pollen was dispersed in PBS (phosphate buffered saline) solution and placed in an Eppendorf tube. The pollen was ground using a freezing grinder (JXFSTPRP-CL.) from Shanghai Jingxin Industrial Development Co., Ltd. The grinding parameters were 60 Hz for 5 minutes, 3 cycles, and 1 minute intervals. After grinding, the pollen was treated with an ultrasonic cell crusher model SCIENTZ-IID, with the maximum output power set to 70%, a single ultrasonic time of 2 minutes, and a working mode of 2.0 seconds working / 3.0 seconds intermittent cycle mode for each ultrasonication, and a total of three ultrasonications.
[0035] (2) Centrifugation and particle size analysis: The micron-sized pollen with larger particle sizes that are easy to precipitate was separated and removed by centrifugation (3000 rpm / min, 5 min). The supernatant was then taken and freeze-dried to obtain nano-pollen particles with a particle size range of approximately 200 to 400 nm. The particles were weighed to obtain their mass and then dissolved in deionized water. The particle size distribution of the nano-pollen particles was analyzed using dynamic light scattering (DLS) technology.
[0036] Obtaining nano-pollen particles: Through the above steps, sunflower nano-pollen particles with a particle size of about 200 nm are finally obtained. The obtained nano-pollen particles are dispersed in anhydrous ethanol for storage and subsequent use.
[0037] like Figure 1 Shown is an image of sunflower pollen taken using a scanning electron microscope (SEM). Figure 1 Middle b shows the overall morphology of the pollen and the details of the spikes on the pollen surface. Figure 1 Middle c is the pollen processing process. Figure 2 The microscopic morphology of sunflower nanopollen is shown, which is much smaller than the original pollen. The size of these nanopollen is between 200 and 400nm.
[0038] Figure 3 The particle size distribution diagram compares the particle size distribution of nanopollen and original pollen. The particle size distribution of nanopollen (red) is mainly concentrated around 200-400nm, while the particle size of original pollen (blue) is significantly larger, distributed around 10μm. This shows that the preparation process of sunflower nanopollen effectively reduces the size of pollen particles. Figure 4 is the Zeta potential of nanopollen, which reflects the charge characteristics of the particle surface; Figure 4 As shown in the figure, the Zeta potential of nanopollen is -30.2mV.
[0039] Example 2: Activation of antigen presenting cells and T cell immune response by sunflower nanopollen
[0040] 1. Inoculate DC2.4 cells (purchased from Fenghui Bio) into a 24-well plate 24 h in advance, and then co-culture with sunflower original pollen, sunflower nanopollen, and LPS (lipopolysaccharide, an immunostimulant) for 48 hours, respectively, and collect the cell culture supernatant and cells, respectively.
[0041] Figure 5 This is a cell staining image, which shows the fluorescent staining images of mouse dendritic cells DC 2.4 under different treatment conditions. The cell membrane stain Cy-5 is shown in red, the cell nucleus stain DAPI is shown in blue, the merged image shows the superposition effect of the three stains, and the bright field image shows the morphology of the cells.
[0042] 2. BMDCs (bone marrow-derived dendritic cells) were inoculated into 24-well plates 24 hours in advance, and sunflower original pollen, sunflower nano pollen (0.5μg / mL, 1μg / mL, 5μg / mL, 10μg / mL, 20μg / mL), and LPS (lipopolysaccharide, an immunostimulant) were co-cultured with BMDCs (bone marrow-derived dendritic cells) for 48 hours; the effects of sunflower pollen, sunflower nano pollen, and LPS on the activation (CD40, CD11c, CD80, CD86) and MHC (MHC-I and MHC-II) phenotypes of BMDCs were analyzed by flow cytometry; the cytokines and inflammatory cytokine levels (TNF-α, IFN-γ, IL-6, IL-12, IL-1β) released by BMDCs after stimulation with sunflower pollen, sunflower nano pollen, and LPS were analyzed by ELISA kits, and the effects of different treatments on the activation of BMDCs were compared; Figure 6 This figure shows the effect of different concentrations of nanopollen and LPS on the expression of surface markers CD80 and CD86 of bone marrow-derived dendritic cells (BMDCs) by flow cytometry analysis. Bone marrow-derived dendritic cells (BMDCs) have high expression of CD11c, Figure 6 A in the figure shows a scatter plot of cells under different treatment conditions, where the colors represent the cell density, and the data in the figure provide the percentage of cells expressing CD80 and CD86 at different concentrations. Figure 6 The bar graph in middle b shows the expression levels of CD80 and CD86 on the cell surface after treatment with different concentrations of nano-pollen and LPS. The results show that nano-pollen can significantly increase the expression of CD80 and CD86 at low concentrations, while high concentrations of LPS have a similar effect.
[0043] Figure 7The results are for the percentage of dendritic cells expressing CD80 and CD86. This figure shows the effects of different concentrations of nano-pollen and original pollen on dendritic cells. The results show that nano-pollen can significantly increase the expression percentage of CD80 and CD86 at low concentrations, while original pollen requires higher concentrations.
[0044] Figure 8 Transmission electron microscopy (TEM) images show TEM images of BMDCs cells treated with PBS (control group), LPS, and the same concentration of nano-pollen and original pollen. The red circles in the image show the effects of nano-pollen and original pollen on the morphology of BMDCs.
[0045] Among them, nano-pollen can effectively promote the morphological differentiation of BMDCs into multi-antenna-shaped mature BMDCs cells with a higher proportion, while the original pollen and PBS control groups cannot effectively promote the occurrence of this morphology and the proportion is low.
[0046] Conclusion: Nanopollen can significantly increase the expression of immune cell surface markers at low concentrations, showing a strong immunostimulatory effect. Compared with original pollen, nanopollen has a stronger activation effect on immune cells at the same concentration. These results suggest that nanopollen may have the potential to be an immunomodulator, especially in the field of vaccine adjuvants or immunotherapy.
[0047] 3. Collect RNA from the original sunflower pollen (80ug), sunflower nano-pollen (80ug), and BMDCs treated with LPS in step 1 above for Western blot experiments, and analyze and study whether nano-pollen can efficiently activate specific immune-related molecular signaling pathways (such as TLR2) in BMDCs;
[0048] Here are the steps:
[0049] Protein sample preparation: First, total protein was extracted from original sunflower pollen, sunflower nanopollen, and LPS-treated BMDCs. Lysis buffer was used to lyse cells and extract proteins.
[0050] Protein quantification: Use a BCA protein quantification kit or other quantitative methods to determine the protein concentration in each sample.
[0051] SDS-PAGE electrophoresis: Mix equal amounts of protein samples with SDS-PAGE sample buffer and then separate them by electrophoresis on polyacrylamide gel. The concentration of the gel should be selected according to the molecular weight of the target protein.
[0052] Membrane transfer: Transfer the separated proteins from the gel to a nitrocellulose or PVDF membrane.
[0053] Blocking: After transfer, use blocking solution (such as 5% skim milk powder or PBS-T) to block non-specific binding sites on the membrane to reduce background noise.
[0054] Primary antibody incubation: Incubate the membrane with primary antibody against the target protein (e.g., antibody against TLR2) overnight at 4°C.
[0055] Secondary antibody incubation: Incubate with HRP-conjugated secondary antibody corresponding to the primary antibody for 1 hour at room temperature.
[0056] Color development: Use a chemiluminescent substrate (such as ECL) to detect the location of secondary antibody binding, thereby determining the presence and expression level of the target protein.
[0057] Internal reference protein detection: In order to correct for differences in sample loading, the expression level of the internal reference protein (such as β-Actin) was also detected.
[0058] Analysis of results: By comparing the band intensities of the target protein and the reference protein in different samples, the activation effect of nanopollen on specific immune-related molecular signaling pathways in BMDCs was evaluated.
[0059] like Fig. 9 The Western blot images on the right were used to analyze the expression levels of specific proteins in nanopollen, raw pollen treated with PBS (phosphate buffered saline), and BMDCs treated with LPS (lipopolysaccharide, a known immunostimulant). These proteins include:
[0060] TLR-2 (89kDa) Activation of TLR2 can lead to the production of pro-inflammatory cytokines, which in turn regulate the activation and recruitment of immune cells and are crucial for inflammatory responses and immune defense;
[0061] MyD88 (33 kDa) can connect proteins that receive extracellular signals with proteins that transmit signals within cells, and is involved in signal transduction in a variety of tissue cells;
[0062] p-JNK (phosphorylated JNK, 54 kDa) refers to the phosphorylated c-Jun amino-terminal kinase. When JNK is activated, it undergoes phosphorylation, thereby acquiring or enhancing its kinase activity;
[0063] JNK (46kDa) is involved in a variety of biological processes including cell proliferation and differentiation, morphology maintenance, skeleton construction, apoptosis and malignant transformation;
[0064] β-Actin (42 kDa, as an internal reference protein) is often used as an internal reference protein in Western Blot experiments to correct for differences in sample loading amounts and ensure the accuracy and comparability of experimental results.
[0065] By comparison, it can be seen that the TLR-2, MyD88, and p-JNK band intensities of the nanopollen treatment group were higher than those of the PBS treatment group and the original sunflower pollen treatment group, which may indicate that nanopollen can activate the TLR-2, MyD88, and JNK signaling pathways.
[0066] Example 3: Anti-tumor immune efficacy of nano-pollen
[0067] 1. Construct a mouse Hepa1-6 subcutaneous tumor model.
[0068] ① Digest and obtain mouse liver cancer cell Hepa1-6 cells in the logarithmic growth phase, count and resuspend in PBS to 1.5×10 7 / ml cell suspension, select 30 C57BL / 6 female mice aged 4-5 weeks with the same growth condition and good condition, remove the hair under the armpits of C57BL / 6 mice, use a 1ml syringe to extract Hepa1-6 cell suspension, and inject 200ul subcutaneously under the armpit of each mouse. After pulling out the needle tip, quickly press the puncture point with a cotton swab for 2 minutes. The number of days starts to be counted after the injection of mouse liver cancer cell Hepa1-6 cells.
[0069] ② The above 30 mice were randomly divided into 3 groups, and 10 mice in each group started treatment (such as Fig.10 After inoculation, the subcutaneous tumor is about 60 mm. 3 The mice were gavaged once every 2 days (on the 6th, 8th, 10th, 12th and 14th days) with 40 μg / mouse and 80 μg / mouse nano-pollen, and PBS was used as the control group. A total of 5 gavages were performed.
[0070] During this period, the tumor size was measured with a vernier caliper, and the tumor size was measured with a vernier caliper every 2 days. The tumor volume (V) was calculated as follows: V = AB 2 / 2. A is the long diameter of the tumor, and B is the short diameter of the tumor.
[0071] Tumor growth curve and anatomical results Fig.10 As shown in b, it shows that sunflower nano pollen has the ability to inhibit the progress of tumor growth. Fig.10 Panel c shows the size of tumors removed from different mice in each group after sacrifice on the 26th day.
[0072] Example 4: Analysis of the anti-tumor immune efficacy and immune regulation of nano-pollen
[0073] 1. Nano-pollen can increase the sensitivity of tumors to immune checkpoint inhibitors
[0074] 1) Digested mouse liver cancer cell Hepa1-6 cells in the logarithmic growth phase, counted them, and resuspended them in PBS to 1.5×10 7 / ml cell suspension, select 50 C57BL / 6 female mice aged 4-5 weeks with the same growth condition and good condition, remove the hair under the armpits of C57BL / 6 mice, use a 1ml syringe to extract Hepa1-6 cell suspension, and inject 200uL subcutaneously under the armpit of each mouse. After pulling out the needle tip, quickly press the puncture point with a cotton swab for 2 minutes. The number of days starts to be counted after the injection of mouse liver cancer cell Hepa1-6 cells.
[0075] 2) The above 20 mice were randomly divided into 4 groups, with 5 mice in each group to start treatment. After tumor inoculation, subcutaneous tumors of about 60 mm were observed. 3 Starting from the 6th day, 80 μg / head of nano-pollen was given by gavage, once every 2 days (on the 10th, 12th, 14th, 16th, and 18th days), for a total of 5 times. Or 5 mg / kg of anti-PD1 antibody was injected intraperitoneally, once every 2 days (on the 11th, 13th, 15th, and 17th days), for a total of 4 times), or 80 μg / head of nano-pollen and 50 μg / head of anti-PD1 antibody were injected intraperitoneally (the injection time points were the same as above), and PBS was used as the control group.
[0076] PD1 antibody was purchased from BioLegend, Inc.
[0077] During this period, the tumor size was measured using a vernier caliper and the weight and survival of the mice were recorded. Fig.11 As shown;
[0078] PBS group: As a control group, the tumor volume of mice treated with phosphate buffered saline (PBS) increased rapidly, showing no effect in inhibiting tumor growth.
[0079] Nano Pollen Group: The tumor volume of mice treated with nano pollen grew slower than that of the PBS group, indicating that nano pollen has a certain effect in inhibiting tumor growth.
[0080] PD-1 group: The tumor volume growth of mice treated with anti-PD1 antibodies was also slower than that of the PBS group, indicating that anti-PD1 antibodies can inhibit tumor growth.
[0081] Nano Pollen+PD-1 group: The tumor volume of mice treated with both nano pollen and anti-PD1 antibodies grew the slowest, and even decreased during the experiment, indicating that the combined use of nano pollen and anti-PD1 antibodies has a synergistic effect and can more effectively inhibit tumor growth.
[0082] This indicates that nano pollen can enhance the inhibitory effect of anti-PD1 antibodies on subcutaneous tumors in C57BL / 6 mice.
Claims
1. An application of sunflower nano-pollen particles in the preparation of oral anti-tumor immune response drugs, characterized in that: The particle size of the sunflower nano pollen particles is 200-400 nm, and the Zeta potential of the sunflower nano pollen is 0-0.025 mV.
2. The use according to claim 1, characterized in that: The oral medicament further comprises a pharmaceutically acceptable carrier or excipient: a preservative, an antioxidant, a flavoring agent, an aromatic, a solubilizing agent, an emulsifier, a pH buffering substance, a binder, a filler or a lubricant.
3. The use according to claim 1, characterized in that: The sunflower nano-pollen particles are prepared by the following steps: (1) grinding sunflower pollen, dispersing crude sunflower pollen in a PBS solution, grinding the pollen using a cryo-grinder, and then subjecting the grinding to ultrasonic treatment; (2) Centrifugation and particle size analysis: The micron-sized pollen with a larger particle size that is easy to precipitate is separated and removed by centrifugation, and then the supernatant is taken and freeze-dried to obtain the sunflower nano-pollen particles.
4. The use according to claim 3, characterized in that: The grinding parameters in step (1) are 60 Hz for 5 minutes, 3 cycles, and 1 minute intervals; After grinding, the product was treated with ultrasound. Each treatment was continued for 2 minutes in a cycle of 2 seconds on and 3 seconds off, for a total of three times. The centrifugation parameters in step (2) are set to 3000 rpm / min for a total of 5 min.
5. The use according to claim 1, characterized in that: The sunflower nano-pollen particles are combined with immune checkpoint inhibitors to prepare drugs for treating tumors.
6. The use according to claim 1, characterized in that: The PD1 inhibitor includes an anti-PD-1 antibody or an antibody-binding fragment thereof.