CN116333989A - Application of sodium magnesium lithium silicate in preparation of dendritic cell function promoter - Google Patents
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to application of sodium magnesium lithium silicate in preparation of dendritic cell function promoters. The research of the invention discovers that the sodium magnesium lithium silicate can promote cytoskeletal rearrangement after being ingested by dendritic cells, further can improve the maturity and migration capacity of the dendritic cells before adoptive infusion, has no obvious toxic or side effect, and further improves the in vitro movement capacity of the dendritic cells and the homing capacity of in vivo lymphoid tissues; meanwhile, in the invention, sodium magnesium lithium silicate has good biocompatibility and is easy to be enriched by dendritic cells, and the adoptive dendritic cell vaccine which is locally injected or intravenous infusion can be used as an accelerator to greatly improve the migration efficiency of the adoptive dendritic cell vaccine to a T cell enrichment area, thereby providing a new choice and path for clinical transformation of the dendritic cell vaccine.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of sodium magnesium lithium silicate in preparation of dendritic cell function promoters.
Background
Dendritic Cells (DCs) are considered to be the most potent, most specialized Antigen Presenting Cells (APCs). It shuttles between lymphoid and non-lymphoid tissues, and is effective in uptake and presentation of antigen, causing specific T cell responsesThe bridge should be established and established between innate immunity and adaptive immunity. Under normal physiological conditions, DCs act as immune defenses, phagocytose and treat invasive pathogens, and sense pathogenic components such as LPS and CpG through cell surface Toll-like receptors, and convert from resting immature DCs (imDCs) to activated mature DCs (mDCs). mDCs can activate a variety of immune cells, such as CD4, either directly or indirectly + Helper T cell, cytotoxic CD8 + T cells, natural killer cells, and natural killer T cells, thereby removing foreign substances from the cells.
Along with the establishment of the in-vitro amplification culture technology of DCs, people can obtain a large amount of imDCs from peripheral blood mononuclear cells or bone marrow stem cells, thereby laying a solid foundation for DCs immunotherapy. Recent studies have begun to expand the understanding of the role of DCs in anti-tumor immune responses by those skilled in the art and have proposed new methods for the production of DCs vaccines. Personalized DCs vaccines loaded with various antigenic forms have successfully initiated and remodel specific immune responses and mobilize endogenous effector T cells for infectious diseases and cancers. Although some clinical trials have demonstrated the safety and immunogenicity of DCs vaccines, traditional vaccine formulations still face serious cellular functional deficiencies, such as insufficient antigen expression, migration capacity and cytokine release, which make it impossible to overcome the immunosuppressive environment that limits DCs and immune effector cell function. Therefore, how to induce imDCs more efficiently as mDCs is a problem that many immunologists are working to solve, and will also directly determine how efficient DCs activate the immune system in vivo.
The most commonly used vaccine stimulator for DCs in clinical use today is the cytokine cocktail (cytokine cocktail), which consists of IL-6, IL-1 beta, TNF-alpha and PGE 2. The cocktail can increase the expression of CD40/CD80/CD86 on the surface of DCs, promote the secretion of TH1 inflammatory factors, and further promote the conversion of imDCs into mDCs. However, it was soon found that the immune activation capacity of mature mDCs stimulated by cytokine cocktail was far lower than expected.
Disclosure of Invention
The invention aims to provide application of sodium magnesium lithium silicate in preparation of dendritic cell function promoters, wherein the sodium magnesium lithium silicate can improve the maturity, migration capacity and T cell activation capacity of dendritic cells, and has no obvious toxic or side effect.
The invention provides application of sodium magnesium lithium silicate in preparation of dendritic cell function promoters.
Preferably, the sodium magnesium lithium silicate comprises sodium magnesium lithium silicate tablets.
Preferably, the sodium magnesium lithium silicate sheet comprises sodium magnesium lithium silicate nano sheet and/or sodium magnesium lithium silicate micro sheet.
Preferably, the sheet diameter of the sodium magnesium lithium silicate nano sheet is 25-30 nm.
The invention also provides a dendritic cell function promoter, and the effective components of the dendritic cell function promoter comprise sodium magnesium lithium silicate.
Preferably, the sodium magnesium lithium silicate is the only active ingredient in the dendritic cell function promoting agent.
Preferably, the dendritic cell function promoting agent includes a sodium magnesium lithium silicate dispersant.
Preferably, the concentration of the sodium magnesium lithium silicate in the dendritic cell function promoter is 25-200 mug/mL.
The invention also provides a preparation method of the dendritic cell function promoter by the technical method, which comprises the following steps: dispersing sodium magnesium lithium silicate in a solvent through ultrasonic treatment to obtain the dendritic cell function promoter.
The invention also provides the application of the dendritic cell function promoter or the dendritic cell function promoter prepared by the preparation method in preparation of dendritic cell vaccines.
The beneficial effects are that:
the invention provides application of sodium magnesium lithium silicate in preparation of dendritic cell function promoters, and researches show that the sodium magnesium lithium silicate can promote increase of lipid peroxidation in cells after being ingested by the dendritic cells, promote cytoskeletal rearrangement, further can improve maturity, migration capacity and T cell activation capacity of the dendritic cells before the dendritic cells are infused, has no obvious toxic or side effect, and further improves the in vitro exercise capacity and homing capacity of in vivo lymphoid tissues of the dendritic cells;
in addition, in the invention, sodium magnesium lithium silicate has good biocompatibility and is easy to be enriched by dendritic cells, and the application of the sodium magnesium lithium silicate as an accelerator can greatly improve the migration efficiency of the adoptive DCs vaccine through local injection or intravenous infusion to a T cell enrichment region, thereby providing a new choice and path for clinical transformation of the dendritic cell vaccine.
In addition, compared with cytokine cocktails, sodium magnesium lithium silicate has the characteristics of low cost, stable structure, easy acquisition and relatively single composition, is beneficial to clinical transformation in the preparation of dendritic cell promoters and dendritic cell vaccines, and more importantly, has significant advantages in promoting the maturation and migration of DCs.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a graph showing the characterization of sodium magnesium lithium silicate nanoplatelets in example 1;
FIG. 2 shows phagocytosis of Dendritic Cells (DCs) after their action on Lap in example 2;
FIG. 3 is a graph showing the evaluation of the biocompatibility of Lap with DCs cells in example 3;
FIG. 4 is a graph showing the expression of surface markers of DCs after different concentrations of Lap in example 4 have been reacted with DCs;
FIG. 5 shows cytoskeletal rearrangement of DCs after the effect of Lap with DCs at different incubation concentrations in example 5;
FIG. 6 is an in vitro motility assay of DCs following the effect of different concentrations of Lap with DCs in example 6;
FIG. 7 shows the ability of DCs to home lymphoid tissues in vivo after different concentrations of Lap have been reacted with DCs in example 7.
Detailed Description
The invention provides application of sodium magnesium lithium silicate in preparing dendritic cell function promoters, and more preferably dendritic cell adoptive therapy promoters.
In the present invention, the sodium magnesium lithium silicate includes sodium magnesium lithium silicate sheets, more preferably sodium magnesium lithium silicate nano sheets and/or sodium magnesium lithium silicate micro sheets. The specification, the grade and the source of the sodium magnesium lithium silicate tablet are not particularly limited, and any sodium magnesium lithium silicate tablet capable of preparing the dendritic cell function promoter belongs to the protection scope of the invention, such as Laponite RDS & XLS, laponite S, laponite JS or the sodium magnesium lithium silicate tablet layer; more preferably, the size of the diameter of the sodium magnesium lithium silicate nano sheet is 25-30 nm, and more preferably 28-30 nm; the thickness is about 1nm, more preferably 0.988nm.
The dendritic cell function promoting agent of the present invention preferably has a function of increasing one or more of the maturation degree, the migration ability and the T cell activation ability of dendritic cells, more preferably has a function of increasing the maturation degree, the migration ability and the T cell activation ability of dendritic cells. The dendritic cells of the present invention preferably include bone marrow or peripheral blood mononuclear cell-derived dendritic cells.
The sodium magnesium lithium silicate (Lap) of the invention can be phagocytosed and enriched by DCs, and can promote the up-regulation of the expression of the surface co-stimulatory molecules CD40, CD80, CD86 and MHC class II molecules and the significant up-regulation of the expression of migration related markers CCR7 and CXCR 4; lap can promote cytoskeletal rearrangement including microfilaments and microtubules; enhancing the in vitro motor capacity and migration rate of DCs. In addition, compared with other nano vaccines which adopt nano materials as an accelerant or an immune function regulator for direct immunization, the invention has better biological safety because only a small amount of Lap which is directly adhered or phagocytized into DCs enters the receptor, and increases the possibility of using sodium magnesium lithium silicate as a dendritic cell accelerant.
Based on the advantages, the invention also provides a dendritic cell function promoter, and the effective components of the dendritic cell function promoter comprise sodium magnesium lithium silicate. The sodium magnesium lithium silicate is preferably the only effective component of the dendritic cell function promoting agent. The sodium magnesium lithium silicate comprises sodium magnesium lithium silicate sheets, more preferably sodium magnesium lithium silicate nano sheets and/or sodium magnesium lithium silicate micro sheets. The specification, the grade and the source of the sodium magnesium lithium silicate tablet are not particularly limited, and any sodium magnesium lithium silicate tablet capable of preparing the dendritic cell function promoter belongs to the protection scope of the invention, such as Laponite RDS & XLS, laponite S, laponite JS or the sodium magnesium lithium silicate tablet; more preferably, the size of the diameter of the sodium magnesium lithium silicate nano sheet is 25-30 nm, and more preferably 28-30 nm; the thickness is 1-2 nm. The purity of the sodium magnesium lithium silicate according to the present invention is preferably not less than 99%. The dendritic cell function promoting agent of the present invention preferably comprises a sodium magnesium lithium silicate dispersing agent; the solvent in the dispersing agent preferably comprises PBS buffer or cell complete medium; the pH of the PBS buffer is preferably 7.4. The concentration of the sodium magnesium lithium silicate in the dendritic cell function promoter is 25-200 mu g/mL, and more preferably 5-100 mu g/mL.
The invention also provides a preparation method of the dendritic cell function promoter by the technical method, which comprises the following steps: dispersing sodium magnesium lithium silicate in a solvent through ultrasonic treatment to obtain the dendritic cell function promoter. The power of the ultrasonic treatment is preferably 100W; the temperature is preferably 25 ℃; the time is preferably 15 minutes.
The invention also provides the application of the dendritic cell function promoter or the dendritic cell function promoter prepared by the preparation method in preparation of dendritic cell vaccines. The invention uses the good biocompatibility of sodium magnesium lithium silicate and the characteristic of easy enrichment by dendritic cells, can greatly improve the migration efficiency of adoptive DCs vaccine through local injection or intravenous infusion to a T cell enrichment region by using the sodium magnesium lithium silicate as an accelerator, and provides a new choice and path for clinical transformation of the dendritic cell vaccine.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
The sources of biological and nanomaterials used in the examples described below are broad, and any biological and nanomaterials that are available without violating law and ethics can be used instead as suggested in the examples. Any person skilled in the relevant art can implement the described application of the invention according to the principles of the invention using sodium magnesium lithium silicate sheets (Laponite, lap) of other specifications and grades, such as Laponite RDS & XLS, laponite S, laponite JS or mixtures of the above, and Lap-based materials modified with other reagents or molecules. In addition, the dendritic cells described in the present invention are not limited to dendritic cells derived from mouse bone marrow, and dendritic cells derived from other sources (human or other animals) are also included without departing from the basic concept described in the present invention. Accordingly, such modifications and different applications are intended to be within the scope of this invention.
Example 1
Characterization of sodium magnesium lithium silicate nanoplatelets (Laponite, lap)
Sodium magnesium lithium silicate nanoplates for use in the present invention are purchased from pick chemistry (BYK, USA).
Characterization of Lap by Atomic Force Microscope (AFM) and Transmission Electron Microscope (TEM) is shown in FIG. 1, wherein a is a structural schematic diagram of Lap; b is an atomic force microscope representative image of Lap; c is a transmission electron microscope representative image of Lap in ultrapure water and cell complete medium, respectively.
The AFM results are shown in a of FIG. 1, from which it can be seen that Lap is uniformly dispersed in an aqueous solution and is a nearly circular lamellar material; the analysis of the results shows that the diameter of Lap is about 28.086nm and the thickness is about 0.988nm, as shown in the graph b of FIG. 1, which is consistent with the literature report;
TEM results are shown in FIG. 1 c, with Lap showing a monolayer disk-like morphology consistent with AFM results. Taken together, the Lap of the present invention meets its classical characteristics.
Example 2
In vitro phagocytosis of Lap by mouse bone marrow-derived DCs
Co-incubating Lap with DCs in RPMI-1640 complete medium for 48h, namely the experimental group, and marking as Lap treatment; PBS buffer was co-incubated with DCs in RPMI-1640 complete medium for 48h, a negative control, designated PBS control. DCs in the experimental group and the negative control group are respectively collected and fixed for 12 hours by using 2% glutaraldehyde, and are observed after the samples are prepared according to the requirements of conventional transmission electron microscope preparation, and the results are shown in figure 2, wherein a is a transmission electron microscope representative image of internalization of the DCs into the Lap (gray arrow shows that the Lap is internalized by the DCs through cup-shaped invagination) after PBS control and Lap treatment; b is a fluorescent confocal microscope (CLSM) representative image of the DCs after treatment with Lap versus internalization with Lap.
As shown in fig. 2 a, there were fewer phagocytic vesicles in PBS group DCs (0 μg/mL group) in a, and cleaner in vesicles, with no apparent aggregates, whereas Lap-treated groups visible Lap was phagocytized by DCs and Lap aggregation was seen in cytoplasmic phagocytic vesicles, grey arrows indicate details of the process by which DCs phagocytize Lap and phagocytic vesicles formed; b and c observe the localization of the fluorescent-labeled Lap in the DCs by the CLSM, and quantitatively analyze the fluorescence value to confirm the internalization of the Lap by the DCs.
Example 3
Evaluation of biosafety of Lap to DCs in Co-incubation System
Co-incubating Lap and DCs in RPMI-1640 complete medium for 48h, wherein ultrasonic treatment is carried out during the co-incubation, and the ultrasonic treatment conditions are as follows: ultrasound at 25 ℃ is carried out at 100W, so that the final incubation concentration of the Lap is 25 mu g/mL, 50 mu g/mL and 100 mu g/mL are respectively carried out, and the MTT cell proliferation and cytotoxicity detection kit (C0009S, biyun) is used for detecting the cell viability (cell viability) of DCs in the co-incubation system, and the result is shown in figure 3, wherein a is the influence of different concentrations of Lap on the cell viability of DCs; b is apoptosis of DCs after Lap treatment at different incubation concentrations (NS: P > 0.05 compared to PBS; P <0.05 compared to PBS).
As can be seen from a, compared with the DCs (0 mug/mL group) of the PBS group, the Lap does not influence the cell viability of the DCs in the concentration range of 25-100 mug/mL; b, the auxiliary evidence Lap detected by apoptosis has good biocompatibility, and can be safely used within the range of 25-100 mu g/mL.
Example 4
Surface marker expression profile of DCs after co-incubation with Lap
Co-incubating Lap and DCs in a culture medium for 48h, wherein ultrasonic treatment is carried out during the co-incubation, and the ultrasonic treatment conditions are as follows: sonicating at 25 ℃ at 100w to give final Lap concentrations of 25, 50 and 100 μg/mL, respectively, detecting expression of CD40, CD80, CD86 and MHC class II molecules in the co-incubated system by flow cytometry, and migration of chemokine receptor 7 (C-C motif chemokine receptor 7, CCR 7) and chemokine receptor 4 (C-X-C motif chemokine receptor 4, CXCR 4) on the relevant markers, as shown in FIG. 4, wherein a is a statistical plot of co-stimulatory molecule CD40, CD80, CD86 and MHC class II expression on the flow cytometry detected DCs; b is a statistical graph of CCR7 and CXCR4 expression in flow cytometry detected DCs (< 0.05 compared to PBS group).
As can be seen from fig. 4 a, when the incubation concentration of Lap is 50 μg/mL and 100 μg/mL, the expression rates of co-stimulatory molecules CD40, CD80, CD86 and MHC ii on the surface of the stimulated DCs are all significantly higher than those of the PBS group, and the difference is statistically significant (P < 0.05), indicating that Lap can stimulate DCs to mature.
Expression of CCR7 and CXCR4 is critical for chemotactic homing of DCs to lymphoid tissues and recruitment to sites of inflammation, as can be seen in fig. 4 b, laps promote significant upregulation of CCR7 and CXCR4 expression at incubation concentrations of 25-100 μg/mL. The data indicate that Lap can fully up-regulate the expression level of key chemokines of DCs, suggesting that Lap can promote in vitro movement and homing ability of DCs.
Example 5
Lap significantly promotes cytoskeletal rearrangement of DCs
To further demonstrate the enhancement of the migration motility of Lap to DCs, the cytoskeleton of Lap-treated DCs (including microfilaments and Tubulin) was stained with phalloidin and anti-mouse β -Tubulin antibodies, then photographed by a fluorescent light focusing microscope, and the average fluorescence intensities of both were statistically analyzed to indicate the extent of cytoskeletal rearrangement, as shown in fig. 5, where a is a CLSM representative image of the cytoskeleton of DCs treated at different concentrations of Lap; b is a statistical plot of Actin (action) and Tubulin (β -Tubulin) expression levels (< 0.05 compared to PBS group).
As shown in fig. 5 a, with increasing Lap incubation concentration, DCs microfilaments and microtubules were stained more strongly, the level of dead polarization was increased, and the formation of adhesive spots was increased; b is a fluorescent semi-quantitative statistical result of microfilament and microtubule expression quantity, which shows that Lap greatly promotes the cytoskeletal rearrangement of DCs.
Example 6
Lap significantly improves the in vitro movement migration capacity of DCs
In order to explore the influence of Lap on the migration capacity of DCs, CLSM is used for dynamically imaging DCs in different treatments, and the motion trail of the DCs is recorded and analyzed, and the result is shown in FIG. 6, wherein a is an in-vitro dynamic migration path analysis chart recorded by the CLSM of the DCs in different treatments; b is a DCs in-vitro dynamic migration rate statistical chart under unit length; and c is an in-vitro dynamic migration length statistical chart of DCs in unit time (I: PBS group, II: 25 μg/mL Lap treatment group, III: 50 μg/mL Lap treatment group, IV: 100 μg/mL Lap treatment group; P <0.05 compared with PBS group).
As can be seen from fig. 6 a, the movement ability of DCs is significantly enhanced with increasing Lap incubation concentration, the migration distance is longer, the movement speed is faster in the same time, and the directional migration tendency is stronger with increasing Lap dose;
b is a statistical graph of migration rate and length, and it can be seen that 100. Mu.g/mL of Lap-treated group DCs have the longest movement distance of 1.8+ -0.2 times the movement distance of PBS-group DCs, and also have a movement speed faster than 25. Mu.g/mL and 50. Mu.g/mL of group DCs, which is 1.6+ -0.1 times the movement speed of PBS-group DCs. The data show that Lap can significantly improve the in vitro motility of DCs.
Example 7
Lap significantly improves homing ability of DCs to local lymphoid tissues
The promotion of the homing and migration modes of the DCs in vivo by the Lap treatment is verified by in vivo animal experiments, namely, the co-incubation process of the DCs derived from bone marrow of C57BL/6J transgenic mice which are used for expressing firefly luciferase (firefly luciferase, fluc) and the Lap is adopted.
Co-incubating Lap with DCs in a culture medium for 48h respectively, so that the final concentration of the Lap is 25, 50 and 100 mug/mL respectively, centrifugally collecting the DCs, injecting the DCs into a mouse foot pad, monitoring homing of the DCs to popliteal lymph nodes and inguinal lymph nodes in real time by in-vivo fluorescence imaging, and setting untreated DCs as a control group (PBS group), wherein a is a dynamic migration representative image of the DCs after injecting the foot pad; b is a statistical plot of the percentage of DCs migrating to the popliteal lymph nodes and inguinal lymph nodes (note:. P <0.05 compared to PBS group).
Experiments were performed to monitor the lymphatic homing of DCs at 0, 12, 24 and 48 hours after infusion, respectively, where a is a representative image of DCs along the lymphatic homing, and b is analysis of the luminous intensity data of each lymphoid tissue using in vivo imaging software, and further calculate the resulting migration rate of DCs.
The results show that the signal intensity of the Lap at the incubation concentration of 25-100 mug/mL, which enables the DCs to migrate to the popliteal lymph node area, is significantly higher than that of the PBS group (P < 0.05) at 4, 24 and 48 hours. This example demonstrates that Lap enhances the local migration of subcutaneously infused DCs and homing to lymphoid tissues. The results of migration in vivo were consistent with the results of chemokines, i.e., the Lap treated group was superior to the control group.
From the above examples, it can be seen that the sodium magnesium lithium silicate nanosheets of the present invention are taken into DCs, causing a severe rearrangement of the cytoskeleton of DCs, thereby significantly promoting the migration ability of DCs and homing ability of lymphatic tissues in vivo.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The application of sodium magnesium lithium silicate in preparing dendritic cell function promoter.
2. The use according to claim 1, wherein the sodium magnesium lithium silicate comprises sodium magnesium lithium silicate tablets.
3. Use according to claim 2, characterized in that the sodium magnesium lithium silicate tablets comprise sodium magnesium lithium silicate nano-tablets and/or sodium magnesium lithium silicate micro-tablets.
4. The use according to claim 3, wherein the sodium magnesium lithium silicate nanoplatelets have a sheet diameter size of 25 to 30nm.
5. A dendritic cell function promoter, characterized in that the effective component of the dendritic cell function promoter comprises sodium magnesium lithium silicate.
6. The dendritic cell function promoter according to claim 5, wherein the sodium magnesium lithium silicate is the only active ingredient in the dendritic cell function promoter.
7. The dendritic cell function promoting agent according to claim 6, wherein the dendritic cell function promoting agent comprises a sodium magnesium lithium silicate dispersant.
8. The dendritic cell function promoter of claim 7, wherein the concentration of sodium magnesium lithium silicate in the dendritic cell function promoter is 25-200 μg/mL.
9. The method for producing a dendritic cell function promoter according to any one of claims 5 to 8, comprising: dispersing sodium magnesium lithium silicate in a solvent through ultrasonic treatment to obtain the dendritic cell function promoter.
10. The dendritic cell function promoter according to any one of claims 5 to 8 or the dendritic cell function promoter prepared by the preparation method according to claim 9, and the use thereof in the preparation of a dendritic cell vaccine.
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| WO2016159877A1 (en) * | 2015-03-27 | 2016-10-06 | Nanyang Polytechnic | A drug delivery composition |
| CN108836980A (en) * | 2018-07-25 | 2018-11-20 | 中国人民解放军军事科学院军事医学研究院 | Graphene oxide is preparing the application in dendritic cell function promotor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016159877A1 (en) * | 2015-03-27 | 2016-10-06 | Nanyang Polytechnic | A drug delivery composition |
| CN108836980A (en) * | 2018-07-25 | 2018-11-20 | 中国人民解放军军事科学院军事医学研究院 | Graphene oxide is preparing the application in dendritic cell function promotor |
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