Use of terpenoids to improve their lymphatic targeting
Patent Information
- Application Number
- CN202210136410.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-02-15
- Publication Date
- 2026-11-03
- Estimated Expiration
- 2042-02-15
AI Technical Summary
Dendritic cell tumor vaccines have low lymph node migration and homing rates, and existing methods have poor safety, which affects efficacy.
Terpenoids such as myrcene and menthol are used in combination with dendritic cell tumor vaccines to reduce the expression of tight junction proteins in lymphatic endothelial cells, thereby promoting vaccine maturation, enhancing lymphatic migration and homing, and improving the clinical efficacy of tumor vaccines.
It significantly improved the lymph node homing rate of dendritic cell tumor vaccines, enhanced the proliferation of cytotoxic T cells in the body, improved the efficacy of tumor immunotherapy, and had high safety.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of a terpenoid compound in enhancing its lymphatic targeting effect. Background Technology
[0002] Terpenoids are compounds with the structure (C5H8). n Terpenes, along with their general formula and derivatives containing oxygen and varying degrees of saturation, can be considered a class of natural compounds formed by the linkage of isoprene or isopentane in various ways. Terpenes are widely found in nature, present in higher plants, fungi, microorganisms, insects, and marine organisms. They are an important class of compounds in traditional Chinese medicine, with many compounds identified as active ingredients. They are also important natural fragrances and indispensable raw materials for the cosmetics and pharmaceutical industries. They mainly include monoterpenes and sesquiterpenes.
[0003] Terpenoids, such as borneol, menthol, and citral, possess the effects of refreshing the mind, clearing heat and relieving pain, and promoting tissue regeneration and antiseptic properties. The *Shennong Bencao Jing* (Divine Farmer's Materia Medica) states, "Fragrance is the essence of qi; when the righteous qi is abundant, it dispels evil and filth." Most of these are plant-based medicines, hence they are also called "fragrant herbs" or "fragrant woods," possessing the characteristics of "aromatic, penetrating, mobile, and dispersing." In modern pharmacological research, aromatic medicinal materials or their main components are often developed into "penetration enhancers." Utilizing their "penetration-enhancing effect," they can guide other components through the physiological barrier into the lymphatic system, enhancing lymphatic drainage.
[0004] Dendritic cells (DCs) are currently known as the most potent antigen-presenting cells, and are classified into immature and mature dendritic cells based on their developmental differentiation. Immature dendritic cells express low levels of MHC II molecules, co-stimulatory molecules, and adhesion molecules. They take up antigens through phagocytosis and pinocytosis, which stimulates them to mature and develop into mature dendritic cells with a strong ability to activate and proliferate T cells. Immature dendritic cells have immunosuppressive effects, while mature dendritic cells are immunoreactive and play an important role in tumor immunotherapy. They present tumor antigens to cytotoxic T cells, stimulating their rapid and massive proliferation to exert tumor-killing effects.
[0005] Tumor vaccines prepared by dendritic cells phagocytizing tumor antigens need to migrate to lymphatic vessels and home to secondary immune organs such as lymph nodes to exert their immune effect. Therefore, efficient lymph node homing of dendritic cell tumor vaccines is crucial. However, clinical studies have shown that the lymph node homing rate of dendritic cell vaccines administered via various routes is only about 3-5%, resulting in unsatisfactory efficacy. Currently, a commonly used method to stimulate lymphatic migration is pre-stimulation with inflammatory factors, such as lipopolysaccharide, but its safety profile is poor, thus requiring further research. Summary of the Invention
[0006] To address the challenge of lymph node migration and homing in dendritic cell tumor vaccines, this invention provides the application of terpenoid compounds in enhancing their lymph node targeting effect. Furthermore, terpenoid compounds can enhance the lymph node migration effect of dendritic cell tumor vaccines, effectively increase the proliferation of cytotoxic T cells in the body, and thus significantly enhance the preventive and therapeutic efficacy of dendritic cell tumor vaccines.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0008] Application of a terpenoid compound in enhancing its lymphatic targeting effect.
[0009] The application of the terpenoid compounds in enhancing lymph node homing in dendritic cell tumor vaccines.
[0010] Furthermore, the terpenoid compounds are used to improve lymphatic homing of dendritic cell tumor vaccines and enhance the clinical efficacy of tumor vaccines.
[0011] The terpenoid compound is one or more of monoterpenoids, sesquiterpenoids, derivatives of the compound, or preparations of the compound and its derivatives.
[0012] The terpenoid compounds are one or more of the following: myrcene, ocimene, citronellol, geraniol, nerol, senna aldehyde, linalool, lavenderol, citral, citronellol, marigoldone, limonene, terpinene, phellandrene, menthol, menthone, terpineol, carvone, limonene, perillaldehyde, perillol, menthol, pinene, sennaol, myrtol, verbenaenool, camphor, borneol, borneol, thujone, thujone, paeoniflorin, longleaf aldehyde, farnesene, nerolidol, artemisinin, sennatoxin, sennaene, arbutin, caryophyllene, humulene, limonene, santalol, longleaf aldehyde, aristolochic acid, guaiacene, and eucalyptol.
[0013] The terpenoids are one or more of the following: myrcene, senna, citral, menthol, menthone, perillaldehyde, perillol, carvone, carvone, limonene, borneol, and borneol.
[0014] The formulation uses the compound and its derivatives as active substances, wherein the active substances account for 0.01%-10% of the formulation mass.
[0015] The formulation is a particulate formulation. The terpenoid compound formulation includes, but is not limited to, liposomes, nanoparticles, micelles, emulsions, etc.
[0016] The dendritic cell tumor vaccine is one or more of the following: renal cell carcinoma, brain tumor, malignant melanoma, prostate cancer, pancreatic cancer, malignant glioma, and gynecological tumors.
[0017] The terpenoid compound or its preparation may be administered simultaneously or in advance with the dendritic cell tumor vaccine, or may be used as a component of the dendritic cell tumor vaccine to co-prepare a tumor vaccine with the antigen.
[0018] The methods for preparing dendritic cell tumor vaccines include, but are not limited to, dendritic cell vaccines derived from peripheral blood and dendritic cell vaccines derived from bone marrow.
[0019] This invention has the following advantages:
[0020] (1) The combined use of terpenoids and dendritic cell vaccines can promote the migration of tumor cell vaccines to the lymph nodes and their homing to the lymph nodes, increase their exposure time to the lymph nodes, and enhance the effect of tumor immunotherapy.
[0021] (2) Terpenoids have a highly efficient permeation-promoting effect. They can promote the maturation of immature dendritic cells, enhance lymphatic migration by lymphatic and chemotactic orientations, and thus effectively guide the lymph nodes of dendritic cell vaccines to "homing" and improve their target organ bioavailability.
[0022] (3) Terpenoids have the effects of clearing the orifices, dispersing stagnant heat, removing pterygium and improving eyesight, reducing swelling and relieving pain. They have a synergistic effect with dendritic cell vaccines. This invention, when used in combination with microparticle formulations, fully leverages the effect of terpenoids in promoting the activation and proliferation of cytotoxic T cells by dendritic cell vaccines.
[0023] (4) Terpenoids have high safety. As pharmaceutical excipients or main drugs, they can reduce tight junction proteins in lymphatic vessels, thereby expanding lymphatic endothelial cells and reducing the permeability barrier of lymphatic endothelial cells. Terpenoids can upregulate the surface receptors of dendritic cell expression components, thereby improving the maturity of dendritic cell vaccines. Terpenoids can also play a key role in the expression of key cytokines in dendritic cell vaccines, and have a good promoting effect on lymphatic migration and homing of dendritic cell tumor vaccines. Therefore, when used in combination with dendritic cells, they have fewer toxic side effects, faster onset of action, and higher safety, thus enhancing the therapeutic and preventive efficacy of dendritic cell tumor vaccines. Attached Figure Description
[0024] Figure 1A The image shows a q-PCR result of menthol compounds reducing the expression of ZO-1 protein in lymphoendothelial cells, as provided in this embodiment of the invention.
[0025] Figure 1B WB diagram showing the reduction of ZO-1 protein expression in lymphoendothelial cells by menthol compounds provided in this embodiment of the invention.
[0026] Figure 2AThe image shows a q-PCR diagram of how borneol compounds reduce the expression of Occludin protein in lymphoendothelial cells, as provided in this embodiment of the invention.
[0027] Figure 2B WB diagram showing the reduction of Occludin protein expression in lymphoendothelial cells by borneol compounds provided in this embodiment of the invention.
[0028] Figure 3 The diagram shows the expansion of intercellular spacing in lymphatic endothelial cells using citral, menthol, and borneol compounds provided in this embodiment of the invention.
[0029] Figure 4 The diagram shows the level of interstitial fluid drainage into lymphatic vessels provided by citral liposomes, menthol nanoparticles, and borneol micelles in the embodiments of the present invention.
[0030] Figure 5 Transwell experiment diagram showing how citral, menthol, and borneol compounds promote dendritic cell migration, as provided in embodiments of the present invention.
[0031] Figure 6 The diagram shows a cell scratch assay illustrating how citral, menthol, and borneol compounds promote dendritic cell migration, as provided in an embodiment of the present invention.
[0032] Figure 7 shows the q-PCR (A) and flow cytometry (B) of the citral compound upregulating the secretion of CCR7 by dendritic cells provided in the embodiments of the present invention.
[0033] Figure 8 shows the q-PCR (A) and ELISA (B) results of the borneol compound upregulating the expression of CCL21 in lymphatic endothelial cells provided in the embodiments of the present invention.
[0034] Figure 9 Flow cytometry plot showing the upregulation of maturation markers CD80 and CD86 in dendritic cells by menthol compounds provided in this embodiment of the invention.
[0035] Figure 10 Optical and electron microscope images of the use of citral, menthol, and borneol compounds to enhance the maturity of dendritic cell B16 tumor vaccines, as provided in embodiments of the present invention.
[0036] Figure 11 The image shows in vivo animal imaging results of the lymph node homing effect of citral liposomes, menthol nanoparticles, and borneol micelles provided in the embodiments of the present invention.
[0037] Figure 12 The citral nanoparticles, menthol liposomes, and borneol emulsion provided in this embodiment of the invention improve the tumor volume in a mouse model of dendritic cell vaccine against melanoma.
[0038] Figure 13The citral nanoparticles, menthol liposomes, and borneol emulsion provided in this embodiment of the invention improve the body weight change in a dendritic cell vaccine anti-melanoma model mouse.
[0039] Figure 14 The citral nanoparticles, menthol liposomes, and borneol emulsion provided in this embodiment of the invention increase tumor weight in mice using a dendritic cell vaccine against melanoma.
[0040] Figure 15 The citral nanoparticles, menthol liposomes, and borneol emulsion provided in this embodiment of the invention enhance tumor volume in a dendritic cell vaccine-induced melanoma model mouse.
[0041] Figure 16 The image shows the HE staining results of tumors in a mouse model of dendritic cell vaccine against melanoma, provided in the embodiments of the present invention, using citral nanoparticles, menthol liposomes, and borneol emulsion. Detailed Implementation
[0042] The present invention will be further described below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.
[0043] This invention utilizes the application of terpenoid compounds in enhancing lymph node homing of dendritic cell tumor (DCMT) vaccines. Terpenoid-related formulations can be administered simultaneously with or prior to DCMT vaccines, or as components of DCMT vaccines. Terpenoid compounds can reduce the expression of tight junction proteins in lymphatic endothelial cells, promote DCMT vaccine maturation, enhance lymphatic exposure through lymphatic circulation, increase lymph uptake and retention, and improve the proliferation of cytotoxic T lymphocytes, thereby enhancing the therapeutic effect of DCMT vaccines.
[0044] Example 1: Preparation of liposomes containing different amounts of various terpenoid compounds:
[0045] Citral, menthol, and borneol were ground separately and passed through a 150-mesh sieve. Different amounts (0.2 mg, 2 mg, 10 mg, 20 mg) of citral, menthol, or borneol powder were accurately weighed and then mixed with 100 mg of egg yolk lecithin, 25 mg of cholesterol, and 5 mg of PEG, respectively. 5 mL of anhydrous ethanol was added, and the mixture was rotary evaporated for 30 min to form a film. After adjusting the volume with 2 mL of purified water, the film was sonicated for 5 min and then passed through a membrane to prepare liposomes with concentrations of 0.1 mg / mL (0.01%), 1 mg / mL (0.1%), 5 mg / mL (0.5%), and 10 mg / mL (1%).
[0046] Example 2: Preparation of nanoparticles containing different amounts of various terpenoid compounds:
[0047] Accurately weigh 10 mg of egg yolk lecithin and mix it with 2 mg of PEG-DSPE. Dissolve the mixture in 5 mL of anhydrous ethanol, then slowly add it dropwise to 10 mL of ultrapure water until the volume is brought to a final volume. Stir continuously for 5 min to obtain a lipid aqueous solution. Separately, mix 15 mg of PLGA with different amounts (10 mg, 100 mg, 500 mg, 1000 mg) of citral, menthol, or borneol powder, dissolve them in acetonitrile, sonicate the mixture, and then slowly add it dropwise to the above lipid aqueous solution. Sonicate the mixture for 10 min, stir for 30 min, and then remove the residual organic solvent using a rotary evaporator. Finally, filter the mixture through a membrane to remove unpackaged drugs, and then filter it through a membrane at 0.22 μm to obtain different amounts of citral, menthol, and borneol polymer nanoparticles at concentrations of 1 mg / mL (0.1%), 10 mg / mL (1%), 50 mg / mL (5%), and 100 mg / mL (10%).
[0048] Example 3: Preparation of micelles containing different amounts of various terpenoid compounds:
[0049] Citral, menthol, and borneol were ground and passed through a 150-mesh sieve. Different amounts (0.5 mg, 5 mg, 25 mg, 50 mg) of citral, menthol, or borneol powder were accurately weighed and dissolved in 1 mL of propylene glycol. 0.2 mL of PEG and 0.1 mL of Tween-80 were added, mixed thoroughly, and then diluted to volume with 5 mL of purified water to obtain micelle solutions of citral, menthol, and borneol at concentrations of 0.1 mg / mL (0.01%), 1 mg / mL (0.1%), 5 mg / mL (0.5%), and 10 mg / mL (1%).
[0050] Example 4: Preparation of emulsions containing different amounts of various terpenoid compounds:
[0051] Accurately weigh 0.9 g of Span 85 and 0.6 g of Tween 80, and mix them with different amounts (2 mg, 20 mg, 200 mg, 2000 mg) of citral, menthol, or borneol powder to form the oil phase. Add 0.5 mL of purified water to form the inner aqueous phase. Dissolve 0.3 g of Tween 80 in 20 mL of PBS solution to form the outer aqueous phase. Add the inner aqueous phase dropwise to the mixed oil phase and stir to obtain the primary emulsion solution. Pour the primary emulsion into the outer aqueous phase containing Tween 80 under high-speed shearing at 10000 rpm and continue shearing for 2 minutes to obtain the secondary emulsion. Homogenize at 900 bar for 2 minutes to obtain different amounts of citral, menthol, and borneol nanoemulsions of 0.1 mg / mL (0.01%), 1 mg / mL (0.1%), 10 mg / mL (1%), and 100 mg / mL (10%).
[0052] Example 5: Terpenoid compounds reduce the expression of ZO-1 protein in lymphoendothelial cells:
[0053] DMSO solutions were prepared to a concentration of 100 mg / mL for each terpene compound, and then diluted to 10, 50, and 100 μg / mL using DMEM medium (containing 5% fetal bovine serum). These solutions were then reacted with 2 × 10⁻⁶ ppm of DMSO solution. 5 Mouse lymphatic endothelial cells (cells / mL) were co-incubated in DMEM medium (containing 5% fetal bovine serum) for 6 hours, with 100 μg / mL DMSO solution as a control. Real-time quantitative PCR and Western blotting were used to investigate the effects of various terpenoid compounds on the expression of tight junction protein ZO-1 in mouse lymphatic endothelial cells at both the gene and protein levels.
[0054] Specifically, taking menthol compounds as an example, see [link to relevant documentation]. Figure 1A As observed by q-PCR, 50 μg / mL and 100 μg / mL of menthol significantly reduced the mRNA level of the tight junction protein ZO-1, while the mRNA level in the control group remained unchanged. Western blot analysis showed that the gray values of the bands corresponding to 50 μg / mL and 100 μg / mL menthol compounds after incubation with endothelial cells were significantly lower than those in the control group, indicating that menthol reduces ZO-1 protein expression in endothelial cells. This reduction in ZO-1 protein significantly promotes lymph node homing in dendritic cell tumor vaccines.
[0055] Example 6: Terpenoid compounds reduce the expression of Occludin protein in lymphoendothelial cells:
[0056] DMSO solutions were prepared to a concentration of 100 mg / mL for each terpene compound, and then diluted to 10, 50, and 100 μg / mL using DMEM medium (containing 5% fetal bovine serum). These solutions were then reacted with 2 × 10⁻⁶ ppm of DMSO solution. 5 Mouse lymphatic endothelial cells (cells / mL) were co-incubated in DMEM medium (containing 5% fetal bovine serum) for 6 hours, with 100 μg / mL DMSO solution as a control. Real-time quantitative PCR and Western blotting were used to investigate the effects of various terpenoid compounds on the expression of the tight junction protein Occludin in mouse lymphatic endothelial cells at both the gene and protein levels.
[0057] Specifically, taking borneol compounds as an example, see [link to relevant documentation]. Figure 2A and 2Bq-PCR showed that 50 μg / mL and 100 μg / mL of borneol compound significantly reduced the mRNA level of the tight junction protein Occludin, while the mRNA level in the control group remained unchanged. Western blotting showed that the gray values of the bands corresponding to 50 μg / mL and 100 μg / mL borneol compound incubation with endothelial cells were significantly lower than those in the control group, indicating that borneol reduces the expression of Occludin protein in endothelial cells. The reduction in Occludin protein significantly promoted lymph node homing in dendritic cell tumor vaccines.
[0058] Example 7: Terpenoid compounds expand the intercellular spacing of lymphatic endothelial cells:
[0059] DMSO solution was prepared to a concentration of 100 mg / mL for each terpene compound, then diluted to 50 μg / mL using DMEM medium (containing 5% fetal bovine serum). The solutions were then reacted with 2 × 10⁻⁶ ppm of DMSO solution. 5 Mouse lymphatic endothelial cells (cells / mL) were co-incubated in DMEM medium (containing 5% fetal bovine serum) for 24 hours, with 50 μg / mL PBS buffer as a control. The effects of various terpenoid compounds on the intercellular spacing of the lymphatic endothelial cells were observed using an optical microscope.
[0060] Specifically, taking citral, menthol, and borneol compounds as examples, from Figure 3 It is evident that, compared with the control group, the intercellular distance of endothelial cells was significantly increased when 50 μg / mL of citral, menthol, and borneol were co-incubated with them. The increase in intercellular distance indirectly indicates that the permeability of the lymphatic endothelial barrier is increased, thereby improving the lymph node homing effect of the dendritic cell tumor vaccine.
[0061] Example 8: Citral liposomes, menthol nanoparticles, and borneol micelles increase interstitial fluid drainage into lymphatic vessels:
[0062] In the experiment, SD rats were subcutaneously injected with 50 μL of 1 mg / mL (0.1%) citral liposomes, menthol nanoparticles, and borneol micelles, respectively. Lymphatic vessels were then stained with FITC-labeled LYVE antibody for visualization, and lymphatic vessel dilation was observed under a microscope. The lymphatic vessel diameter was used to characterize the drug's effect on lymphatic vessel dilation level (see [link to experiment]). Figure 4 The control group rats were injected subcutaneously with 50 μL of PBS buffer.
[0063] Depend on Figure 4 It is evident that 1 mg / mL (0.1%) of citral liposomes, menthol nanoparticles, and borneol micelles can significantly increase lymphatic vessel dilation, thus indicating that citral, menthol, and borneol can increase the drainage of interstitial fluid into lymphatic vessels, which is crucial for the migration of dendritic cell tumor vaccines into lymphatic vessels.
[0064] Example 9: The effect of various terpenoid compounds on promoting dendritic cell migration:
[0065] The effects of various terpenoid compounds on the migration of dendritic cells (DC2.4 cells) were investigated using transwell and cell scratch techniques.
[0066] In the Transwell experiment, the upper chamber was 2 × 10⁻⁶. 5 Dendritic cells, lower chamber 2 × 10 5 We used 10 lymphatic endothelial cells and 50 μg / mL of various terpenoid compounds to observe, using a regular light microscope, whether the terpenoid compounds promoted the migration of dendritic cells to the lower chamber. A 50 μg / mL PBS buffer was used as a control group.
[0067] The scratch test was 2×10 5 Dendritic cells per mL were treated with scratch therapy after being added to 1640 medium (containing 5% fetal bovine serum) with 50 μg / mL of various terpenoid compounds. After 24 hours of culture, the degree of scratch healing was observed, and the extent to which the terpenoid compounds promoted the migration of dendritic cells to the scratch site was examined using a regular optical microscope. PBS was used as a control group.
[0068] Taking citral, menthol, and borneol compounds as examples, from Figure 5 As can be seen, compared with the control group, the addition of citral, menthol, and borneol compounds significantly increased the migration of dendritic cells in the lower chamber, indicating that citral, menthol, and borneol can significantly promote the longitudinal migration of dendritic cells. As shown in Figure 6, compared with the control group, the scratch healing in the culture medium supplemented with citral, menthol, and borneol compounds was significantly improved, indicating that citral, menthol, and borneol can significantly promote the lateral migration of dendritic cells.
[0069] Example 10: Terpenoids promote the secretion of cytokine CCR7 by dendritic cells
[0070] DMSO solutions were prepared to a concentration of 100 mg / mL for each terpene compound, and then diluted to 10, 50, and 100 μg / mL using 1640 medium (containing 5% fetal bovine serum). These solutions were then reacted with 2 × 10⁻⁶ mol / mL solutions of each terpene compound. 5 Mouse dendritic cells (CCR7) at concentrations of 100 μg / mL were co-incubated in 1640 medium (containing 5% fetal bovine serum) for 6 hours. Real-time quantitative PCR and flow cytometry were used to investigate the effects of different concentrations of various terpenoid compounds on CCR7 expression in mouse bone marrow-derived dendritic cells at both the gene and protein levels. A control group was prepared using 100 μg / mL DMSO buffer.
[0071] For example, see citral compounds. Figure 7Aq-PCR showed that citral compounds at concentrations of 50 μg / mL and 100 μg / mL significantly increased the mRNA level of factor CCR7, while the mRNA level in the control group remained unchanged. (See also...) Figure 7B Flow cytometry results showed that the fluorescence intensities of citral compounds at concentrations of 10 μg / mL, 50 μg / mL, and 100 μg / mL were significantly higher than those in the control group, indicating that different concentrations of citral compounds increased the secretion of CCR7 by dendritic cells. Upregulation of the CCR7 cytokine plays a crucial role in promoting lymph node homing of dendritic cell tumor vaccines.
[0072] Example 11: Terpenoids promote the secretion of cytokine CCL21 by lymphatic endothelial cells
[0073] DMSO solutions were prepared to a concentration of 100 mg / mL for each terpene compound, and then diluted to 10, 50, and 100 μg / mL using DMEM medium (containing 5% fetal bovine serum). These solutions were then reacted with 2 × 10⁻⁶ ppm of DMSO solution. 5 Mouse lymphatic endothelial cells (CCL21) were incubated in DMEM (containing 5% fetal bovine serum) medium for 6 hours. The effects of various terpenoid compounds on CCL21 expression in lymphatic endothelial cells were investigated at both the gene and protein levels using real-time quantitative PCR and ELISA. A control group of 100 μg / mL DMSO buffer was also included.
[0074] For example, see borneol compounds. Figure 8A q-PCR showed that 50 μg / mL and 100 μg / mL of borneol significantly increased the mRNA level of factor CCL21, while the mRNA level in the control group remained unchanged. (See also...) Figure 8B ELISA results showed that 50 μg / mL and 100 μg / mL of borneol significantly increased the secretion of factor CCL21. Increased secretion of CCL21 by lymphatic endothelial cells enhances the attraction of dendritic cell vaccines, thereby promoting the migration of dendritic cell tumor vaccines to lymphatic vessels and homing to lymph nodes.
[0075] Example 12: Various terpenoid compounds upregulate the expression of maturation marker receptors in dendritic cells:
[0076] DMSO solutions were prepared to a concentration of 100 mg / mL for each terpene compound, and then diluted to 10, 50, and 100 μg / mL using 1640 medium (containing 5% fetal bovine serum). These solutions were then reacted with 2 × 10⁻⁶ mol / mL solutions of each terpene compound. 5Mouse dendritic cells per mL were cultured in 1640 medium (containing 5% fetal bovine serum) for 24 hours. Dendritic cells were stained and labeled with FITC-CD80 and PE-CD86 fluorescent antibodies. Flow cytometry was used to assess the effects of various terpenoid compounds on the maturation markers CD80 and CD86 on the surface of mouse bone marrow-derived dendritic cells.
[0077] For example, see menthol compounds. Figure 9 Flow cytometry results showed that 10, 50, and 100 μg / mL menthol significantly increased the expression of CD80 and CD86 on the surface of dendritic cells. These surface receptors are characteristic molecules of dendritic cell maturity, indicating that menthol can increase dendritic cell maturation, which is crucial for its lymphatic migration and lymph node homing.
[0078] Example 13: Citral, menthol, and borneol compounds promote the maturation of dendritic cell tumor vaccines:
[0079] 50 μg / mL of citral, menthol, and borneol compounds were reacted with 5 × 10 6 After co-incubating with B16 tumor dendritic cell vaccine at a concentration of cells / mL for 24 hours, the morphology of the dendritic cell vaccine, including changes such as pseudopodia, was observed using optical microscopy and scanning electron microscopy (see [link to article]). Figure 10 The preparation process of the B16 tumor dendritic cell vaccine is as follows: 2×10 6 After lysing mouse melanin B16 tumor cells / mL (protein concentration 0.35 mg / mL), 5 × 10⁻⁶ cells were added. 6 The product is obtained by culturing mouse bone marrow-derived dendritic cells at a density of 1 / mL for 24 hours.
[0080] See Figure 10 It was found that citral, menthol, and borneol compounds can significantly improve the maturity of dendritic cell tumor vaccines. The improvement in maturity has a significant promoting effect on the lymph node homing of the vaccine, that is, citral, menthol, and borneol compounds can increase the lymph node homing of dendritic cell vaccines.
[0081] Example 14: Citral liposomes, menthol nanoparticles, and borneol micelles enhance lymph node homing of B16 tumor dendritic cell vaccine:
[0082] In vitro, B16 tumor dendritic cell vaccine was labeled with a DIR fluorescent probe. C57BL / 6 mice were first subcutaneously injected with 10 μL of citral liposomes, menthol nanoparticles, and borneol micelles at a drug concentration of 5 mg / mL (0.5%). Half an hour later, 2 × 10⁻⁶ C16- ... 6Cells / mL of mouse melanin B16 tumor dendritic cell vaccine. Timing began at this point, and small animal in vivo imaging techniques were used to characterize the DIR fluorescence intensity near the popliteal lymph nodes in mice at different time points to indicate the amount of dendritic cell vaccine homing to lymph nodes (see [link to documentation]). Figure 11 ).
[0083] See Figure 11 The results showed that the DIR fluorescence intensity in the lymphatic vessels and lymph nodes of animals given 10 μL of 5 mg / mL citral liposomes, 5 mg / mL menthol nanoparticles, and 5 mg / mL borneol micelles was significantly higher than that in the control group. In other words, the various terpenoid compound preparations increased the lymphatic migration and lymph node homing of the dendritic cell vaccine.
[0084] Example 15: Citral nanoparticles, menthol liposomes, and borneol emulsion enhance the efficacy of dendritic cell vaccine against mouse melanoma:
[0085] Forty-eight C57BL / 6 mice were acclimatized to normal feeding for one week, and then subcutaneously injected with 2×10⁻⁶ ppm of the solution near the thigh and groin. 6 Mouse melanoma B16 tumor cells were injected at a rate of 1 cell / mL. One week later, tumors with a diameter of approximately 1 mm formed. Mice were randomly divided into 8 groups and subcutaneously injected sequentially with 20 μL of PBS, 5 mg / mL of menthol liposomes, and 4 × 10⁶ cells / mL of PBS. 6 Dendritic cells / mL, 5 mg / mL menthol liposomes plus 4 × 10 6 Dendritic cells / mL, 4×10 6 Melanin B16 dendritic cell vaccine (number / mL), citral nanoparticles plus 4×10 6 Melanin B16 dendritic cell vaccine (number / mL), menthol liposomes plus 4×10 6 Melanin B16 dendritic cell vaccine (number / mL), borneol emulsion plus 4×10 6 The melanin B16 dendritic cell vaccine was administered at a concentration of 100 cells / mL. One week later, each group of mice underwent the same administration once more, and were then fed normally for 18 days (day 0). Tumor volume and weight changes were measured every two days. At the end of the experiment, mice were dissected to examine changes in tumor volume and weight, and HE staining was used to observe apoptosis within the tumor cells. The preparation process of the B16 tumor dendritic cell vaccine was as follows: 2 × 10⁻⁶ cells / mL of melanin B16 dendritic cell vaccine was administered. 6 After lysing mouse melanin B16 tumor cells / mL (protein concentration 0.35 mg / mL), 5 × 10⁻⁶ cells were added. 6 The mouse bone marrow-derived dendritic cells were cultured for 24 hours at a density of 1 cell / mL.
[0086] Depend on Figure 12As can be seen, autopsy of the model mice at the end of the experiment revealed that the tumor volume in the groups treated with different terpenoid compounds was significantly smaller than that in the other experimental groups. Figure 13 As can be seen, there was no significant difference in body weight changes among the groups of mice, indicating that the terpenoid compounds had no obvious toxic effects. Figure 14 It is evident that the tumor weight in the mouse groups treated with each terpene compound was significantly lower than that in the other groups. Figure 15 It is evident that administration of various terpenoid compounds significantly inhibited tumor growth in mice. Figure 16 It is evident that the administration of various terpenoid compounds significantly induced apoptosis in mouse tumor tissues. These experimental results indicate that the various terpenoid compounds significantly enhanced the therapeutic effect of the B16 melanoma dendritic cell vaccine.
[0087] In summary, the present invention utilizes terpenoid compounds or their preparations to enhance lymph node homing of dendritic cell tumor vaccines, reduce the expression of tight junction proteins in lymphatic endothelial cells, promote the maturation of dendritic cell tumor vaccines, enhance lymphatic exposure through lymphatic circulation, increase lymph uptake and retention, and improve the proliferation of cytotoxic T lymphocytes in the body, thereby enhancing the therapeutic effect of dendritic cell tumor vaccines.
[0088] The above embodiments are merely preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. The application of a terpenoid compound and a B16 melanoma dendritic cell vaccine in the preparation of a drug for treating melanoma, characterized in that: The terpenoid compound is borneol.