CN114177283B - Magnetic drive nano motor Fe 3 O 4 /Ca@MnCO 3 Is prepared from (A) and its application in DC vaccine - Google Patents
Magnetic drive nano motor Fe 3 O 4 /Ca@MnCO 3 Is prepared from (A) and its application in DC vaccine Download PDFInfo
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- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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Abstract
The invention discloses a magnetic drive nano motor Fe 3 O 4 /Ca@MnCO 3 And their use in DC vaccines. In the invention, fe is 3 O 4 MnCO doped with nano particles as cores and Ca 3 Preparing the microsphere as a shell to obtain the Fe of the magnetic drive nano motor 3 O 4 /Ca@MnCO 3 . Fe prepared by the invention 3 O 4 /Ca@MnCO 3 The nanoparticle is not influenced by environment and energy supply, can autonomously move under a magnetic field, and can be monitored in vivo in real time by MRI; meanwhile, the nanoparticle can be used as an antigen delivery system to actively deliver antigens to cytoplasm of DCs so as to obtain magnetized DC vaccine, so that immune response can be remarkably enhanced, efficient homing of the DC vaccine to lymph nodes is expected to be realized, and a new strategy is provided for development of the DC vaccine.
Description
Technical Field
The invention belongs to the field of biomedical materials, and in particular relates to a magnetic drive nano-horseUp to Fe 3 O 4 /Ca@MnCO 3 And their use in DC vaccines.
Background
The tumor vaccine can induce tumor antigen specific immune response and kill tumor cells specifically by inoculating tumor antigen, and has important clinical application value. Among them, dendritic Cell (DCs) tumor vaccine is an important tumor vaccine with great potential for clinical application. DC vaccine is prepared by collecting patient DCs, culturing in vitro, and directly treating with tumor antigen. The DC vaccine re-vaccinates the patient, homing to secondary immune organs (primarily lymph nodes) and presents tumor antigens to T lymphocytes to further induce anti-tumor immune responses. The construction of the DC vaccine greatly saves the time for capturing antigen by DCs in vivo, starts immune response more quickly and effectively, and effectively induces cytotoxic T lymphocyte specificity to kill tumor cells. Among them, DC vaccine profnge for treating prostate cancer has been approved for clinical use by FDA in 2010. In recent years, more and more DC vaccines are studied successively and enter different clinical test stages, and have important clinical transformation and application prospects.
However, the clinical use of these DC vaccines remains a challenge. Among the most serious problems are DC vaccines that are vaccinated into patients, which often fail to effectively home to lymph nodes for antigen presentation to further induce anti-tumor immune responses. The intensity of the immune response induced by the DC vaccine is proportional to the number of DCs reaching the lymph nodes. Thus, for DC vaccines, the immune response can be enhanced by increasing the number of DC vaccines that home to the lymph nodes. Although a variety of DC vaccines have been tested in clinical trials today, in vitro culture has been found to reduce the survival and migration capacity of DCs, antigen-bearing DC vaccines are usually left at the injection site for a long period of time and cleared at the injection site within 48 hours, with only <5% of the DC vaccine reaching the lymph nodes, severely limiting the clinical therapeutic efficacy of the DC vaccine.
To address the homing problem of DC vaccines, researchers have taken a variety of approaches including: (1) inducing inflammation at the injection site. For example, pretreatment of the injection site with TNF- α or IL-1 promotes migration of DCs to lymph nodes. In addition, dudeck et al observed in real time by in vivo multiphoton microscopy, skin sensitization increased the rate of migration of DCs in vivo. (2) the injection site is subjected to laser irradiation and chemical stimulation. Laser irradiation and chemical stimulation can destroy collagen fibers and extracellular matrix, promoting migration of DCs to lymph nodes. (3) in vitro activation of DCs. Prins et al studies showed that imiquimod-activated DCs migrate to lymph nodes more significantly than vaccinated immature DCs. However, the above methods only indirectly promote the homing of DCs to lymph nodes, failing to achieve active, targeted homing of DCs vaccines to lymph nodes.
In our earlier studies, ca@MnCO was found as a tumor antigen carrier 3 Antigens can be delivered significantly to DCs while activating DCs to induce an anti-tumor immune response, but fail to direct driving DCs homing to lymph nodes.
Disclosure of Invention
The primary purpose of the invention is to overcome the defects and shortcomings of the prior art and provide a magnetic drive nano motor Fe 3 O 4 /Ca@MnCO 3 Is prepared by the preparation method of (1).
Another object of the present invention is to provide the magnetic driving nanomotor Fe prepared by the method 3 O 4 /Ca@MnCO 3 。
It is still another object of the present invention to provide the magnetic-driven nanomotor Fe 3 O 4 /Ca@MnCO 3 The application in preparing tumor vaccine immunoadjuvant.
It is still another object of the present invention to provide the magnetic-driven nanomotor Fe 3 O 4 /Ca@MnCO 3 The application of the vaccine as an immune adjuvant of tumor vaccine in preparing DC tumor vaccine.
The aim of the invention is achieved by the following technical scheme:
magnetic drive nano motor Fe 3 O 4 /Ca@MnCO 3 The preparation method is that Fe is used 3 O 4 MnCO doped with nano particles as cores and Ca 3 Microsphere (Ca@MnCO) 3 ) Is prepared from shell; the method specifically comprises the following steps:
(1) Will beCaCl 2 And MnCl 2 Adding into glycerol water solution, mixing uniformly to obtain CaCl 2 And MnCl 2 Is a mixed solution of (a) and (b); then Fe is added 3 O 4 Stirring and mixing the nano particles uniformly to obtain the Fe-containing material 3 O 4 、CaCl 2 And MnCl 2 Is a mixed solution of (a) and (b);
(2) NH is added to 4 HCO 3 Dissolving in glycerol water solution to obtain NH 4 HCO 3 A solution; then NH is added 4 HCO 3 Adding the solution to the Fe-containing solution obtained in the step (1) 3 O 4 、CaCl 2 And MnCl 2 Stirring and reacting, centrifuging after the reaction is finished, taking precipitate, washing, and freeze-drying to obtain the magnetic drive nano motor Fe 3 O 4 /Ca@MnCO 3 。
The glycerol aqueous solution in the steps (1) and (2) is a solution obtained by mixing glycerol and water according to a volume ratio of 1:1.
CaCl as described in step (1) 2 And MnCl 2 The concentration of the mixed solution of (C) is preferably 0.016mol/L.
CaCl as described in step (1) 2 And MnCl 2 Ca in the mixed solution of (2) 2+ And Mn of 2+ The molar ratio of (2) is 1:1.
Fe as described in step (1) 3 O 4 The amount of nanoparticles added is per milliliter (mL) of CaCl 2 And MnCl 2 Is mixed with 0.5mg Fe 3 O 4 Nanoparticle calculation.
The stirring conditions in the step (1) are as follows: stirring at 250-350 rpm for 25-35 min; preferably, it is: stirring at 300rpm for 30min.
NH described in step (2) 4 HCO 3 The concentration of the solution is preferably 0.16mol/L.
CaCl as described in step (2) 2 、MnCl 2 And NH 4 HCO 3 The molar ratio of (2) is 1:1:10.
The stirring conditions in the step (2) are as follows: stirring for 0.5-1.5 h at 50+ -5 ℃ and 550-650 rpm; preferably, it is: stirring at 50℃and 600rpm for 1h.
The centrifugation conditions in step (2) are: centrifuging for 4-6 min at 3000-5000; preferably, it is: centrifuge at 4000rpm for 5min.
The washing in the step (2) is alternately washing with absolute ethyl alcohol and deionized water; preferably, the washing is performed with dry ethanol and deionized water alternately three or more times.
The freeze drying time in the step (2) is 22-26 hours; preferably 24h.
Magnetic drive nano motor Fe 3 O 4 /Ca@MnCO 3 Prepared by the method of any one of the above.
The magnetic driving nano motor Fe 3 O 4 /Ca@MnCO 3 The application in preparing tumor vaccine immunoadjuvant.
The magnetic driving nano motor Fe 3 O 4 /Ca@MnCO 3 The application of the vaccine adjuvant as tumor vaccine immunoadjuvant in preparing vaccine preparation.
The vaccine is a tumor vaccine; preferably a DC tumor vaccine, i.e. Fe 3 O 4 /Ca@MnCO 3 The DC cells can be activated for constructing DC tumor vaccine.
The vaccine also includes an antigen.
The antigen is preferably Ovalbumin (OVA).
A vaccine preparation is prepared by mixing the above magnetic drive nanomotor Fe 3 O 4 /Ca@MnCO 3 Adding into an antigen Ovalbumin (OVA) solution, stirring at room temperature, and incubating for 2-4 h to obtain Fe 3 O 4 /Ca@MnCO 3 OVA vaccine formulation.
The magnetic driving nano motor Fe 3 O 4 /Ca@MnCO 3 The mass ratio of the egg white protein to the Ovalbumin (OVA) is 50:3.
The Ovalbumin (OVA) solution is preferably an ovalbumin solution prepared by normal saline.
The stirring speed is preferably 100rpm.
A DC vaccine is prepared by mixing the above vaccine preparation (i.e. Fe 3 O 4 /Ca@MnCO 3 OVA vaccine formulation) was added to DC cells using magnetismThe field promotes DC cells to ingest vaccine preparations, and magnetized DC vaccines are obtained, can move along the direction of a magnetic field, and are hopeful to realize that the DC vaccines home lymph nodes and further induce immune response.
The DC cell is a DC2.4 cell.
The magnetic field is generated by a neodymium-iron-boron permanent magnet disk.
Compared with the prior art, the invention has the following advantages and effects:
1、Fe 3 O 4 has magnetism, but has high magnetization intensity, easy aggregation and low antigen loading capacity, and aims to overcome Fe 3 O 4 The invention uses Ca@MnCO to solve the problems of low aggregation and loading capacity and the like 3 The advantages of good stability, dispersibility, high loading capacity and the like, and the Fe is constructed by a one-pot method 3 O 4 The nano particles are cores and Ca@MnCO 3 Biodegradable magnetically driven nanomotor Fe for a shell 3 O 4 /Ca@MnCO 3 To improve Fe 3 O 4 The magnetic driving nano motor is hopeful to actively deliver the antigen to cytoplasm of DCs so as to obtain magnetized DC vaccine, and simultaneously ensures that the composite material has magnetism, and can further efficiently and directionally drive DC vaccine to home to lymph node for antigen presentation, thereby triggering further immune response.
2. Fe in the present invention 3 O 4 /Ca@MnCO 3 Simple synthesis, low cost, economy, environmental protection and degradation of released Mn 2+ Has vaccine adjuvant effect, can obviously enhance immune response and realize in vivo MRI imaging.
3. Magnetic Fe in the present invention 3 O 4 /Ca@MnCO 3 The nanoparticles can perform autonomous motion in the presence of a magnetic field without environmental restrictions and without the need for energy.
4. The invention adopts magnetic Fe for the first time 3 O 4 /Ca@MnCO 3 The nano particles are used as antigen delivery systems, promote the uptake of the antigen by DC2.4 cells, drive the antigen into DCs more rapidly after increasing a magnetic field, and construct magnetized DC vaccine, which is beneficial to the cross presentation of the antigen and DC finenessActivation of cells, and therefore has important application value in the field of vaccine delivery.
5. The magnetized DC vaccine can directionally move by adjusting the direction of the magnetic field, is hopeful to realize efficient homing of the DC vaccine to the lymph nodes, enhances the vaccine effect and provides a new strategy for developing a novel DC vaccine.
6. The magnetic drive nanomotor is used for constructing DC vaccine and has multiple advantages: first, since the magnetic field has the characteristic of wireless propagation, the magnetic Fe 3 O 4 The nano particles are not influenced by environment and energy supply, can move autonomously under a magnetic field, can be monitored in vivo in real time by MRI, and overcomes the defect of pure Fe 3 O 4 The magnetic nano particles have the defects of low loading capacity, easy aggregation and the like due to high magnetization intensity; secondly, adopting degradable Ca@MnCO 3 Coating to Fe 3 O 4 The nano particles are subjected to surface modification, so that the aggregation problem is hopeful to be solved, and the antigen loading capacity is improved; third, mn generated by degradation of the motor 2+ Has been shown to have vaccine adjuvant effects, to significantly enhance immune responses, and to achieve in vivo tracking by MRI.
Drawings
FIG. 1 is Fe 3 O 4 And Fe (Fe) 3 O 4 /Ca@MnCO 3 TEM image of nanoparticles.
FIG. 2 is a graph of IR and ICP analysis results; wherein A is Fe 3 O 4 And Fe (Fe) 3 O 4 /Ca@MnCO 3 FT-IR spectrogram of the nanoparticle; b is Fe 3 O 4 /Ca@MnCO 3 Element content map in nanoparticle.
FIG. 3 is a Cy5.5-OVA supported Fe 3 O 4 /Ca@MnCO 3 Is a confocal microscopy image of (2).
FIG. 4 is Fe 3 O 4 And Fe (Fe) 3 O 4 /Ca@MnCO 3 Antigen loading ratio of nanoparticles.
FIG. 5 is a graph of the mean fluorescence intensity of Cy5.5-OVA in DC2.4 cells.
Fig. 6 is a diagram of a moving track of the magnetization DC under a magnetic field.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
Ovalbumin (OVA) referred to in the examples of the present invention was purchased from Sigma.
Fe involved in the embodiment of the invention 3 O 4 Nanoparticles were purchased from Macklin corporation.
The DC2.4 cells involved in the examples of the present invention were purchased from Shanghai Huiyi Biotechnology Co.
EXAMPLE 1 preparation of Fe 3 O 4 /Ca@MnCO 3 Nanoparticles
Fe 3 O 4 /Ca@MnCO 3 The nanoparticle is prepared by the following steps:
(1) Preparation of CaCl from glycerol/Water solution (1/1, v/v) 2 And MnCl 2 Mixed solution (Ca) 2+ And Mn of 2+ The molar ratio of (2) was 1:1, and the concentration of the mixed solution was 0.016M).
(2) Fe is added to 3 O 4 Nanoparticle (5 mg) was added to CaCl as described above 2 And MnCl 2 The mixture (10 mL) was stirred at 300rpm for 30min at room temperature.
(3) NH to be formulated with glycerol/Water solution (1/1, v/v) 4 HCO 3 The solution (10 mL, 0.16M) was then added to the mixture obtained in step (2) in its entirety, and stirred at 50℃and 600rpm for 1 hour to obtain a precipitate.
(4) Centrifuging (4000 rpm,5 min) the precipitate, alternately centrifuging with deionized water and absolute ethanol for 3 times, and lyophilizing for 24 hr to obtain Fe 3 O 4 /Ca@MnCO 3 The nano particles are stored in a sealed way at room temperature.
Using Transmission Electron Microscopy (TEM)) Observation of Fe 3 O 4 And Fe (Fe) 3 O 4 /Ca@MnCO 3 The morphology of the nanoparticle is shown in fig. 1: fe (Fe) 3 O 4 The size of the nano particles is about 200nm, ca@MnCO is adopted 3 Modification of Fe 3 O 4 After the nano particles, the prepared Fe 3 O 4 /Ca@MnCO 3 The nanoparticles are flower-shaped and have a size of about 900nm.
Subsequently, characterization was performed using a Fourier transform infrared spectrometer (FT-IR) and an inductively coupled plasma spectrometer (ICP). FT-IR spectrum (FIG. 2A) shows that it is equivalent to Fe 3 O 4 Compared with nano particles, fe 3 O 4 /Ca@MnCO 3 Nanoparticles with CO 3 2- Characteristic peaks of functional groups of (2), ICP elemental analysis results (FIG. 2B) indicate synthesized Fe 3 O 4 /Ca@MnCO 3 The nanoparticles contain iron, manganese and calcium elements. These can all account for Fe 3 O 4 Ca@MnCO is successfully modified on the surface of the nanoparticle 3 。
Example 2 magnetic drive nanomotor actively facilitates antigen entry into DC to obtain magnetized DC
1. Fe (Fe) 3 O 4 /Ca@MnCO 3 Antigen loading of nanoparticles
(1) 5mg of Fe prepared in example 1 3 O 4 /Ca@MnCO 3 The nanoparticle was added to 1mL of a solution of Cy5.5-labeled OVA (300. Mu.g, prepared with physiological saline) (Cy5.5-fluorescent-labeled OVA was obtained from Siam Aziyue Biotechnology Co., ltd.; abbreviated as Cy5.5-OVA), stirred at 100rpm for 4 hours at room temperature, and the resulting Fe was obtained 3 O 4 /Ca@MnCO 3 The OVA nanoparticles were stored at 4 ℃. The results are shown in FIG. 3, fe 3 O 4 /Ca@MnCO 3 After being loaded with Cy5.5-OVA, the material shows red fluorescence, which indicates Fe 3 O 4 /Ca@MnCO 3 Nanoparticles can successfully support OVA.
(2) Subsequently, the same dosage of Fe was detected and compared 3 O 4 And Fe (Fe) 3 O 4 /Ca@MnCO 3 Amount of nanoparticle adsorbed OVA at different time points (2 h and 4 h) (preparation method same as above in step (1)), centrifuging the suspension, collecting supernatant, and using BCThe a kit detects OVA concentration in the supernatant. The BCA kit detection results are shown in FIG. 4, fe 3 O 4 /Ca@MnCO 3 The level of the nanoparticle-loaded OVA is higher, and 300 mug of OVA can be completely loaded in 4 hours.
2. Preparation of vaccine formulations
1mL of an OVA (300. Mu.g-containing) solution was prepared using ovalbumin OVA as a model antigen and physiological saline as a solvent. The mixed solution was added to 5mg of Fe 3 O 4 /Ca@MnCO 3 Stirring the nano particles for 4h (100 rpm) at room temperature to prepare Fe 3 O 4 /Ca@MnCO 3 OVA vaccine formulation. At the same time set Fe 3 O 4 /Ca@MnCO 3 group/OVA (M), i.e. simultaneous addition of Fe 3 O 4 /Ca@MnCO 3 Vaccine formulation of/OVA and magnetic field (M) (magnetic field is generated by NdFeB permanent magnet disk purchased from Cheng Yu machine), vaccine formulation prepared by mixing aluminium adjuvant with OVA was used as positive control (without Fe) 3 O 4 /Ca@MnCO 3 ) Saline was used as a blank (Saline) and 300. Mu.g/mL of OVA solution (prepared with Saline) was used as a negative control (OVA). The vaccine formulation was stored at 4 ℃. Vaccines of different formulations were formulated as in table 1.
Table 1 vaccine formulations for subcutaneous injection
3. Magnetically driven nanomotors actively promote antigen entry into DCs
DC2.4 cells were seeded on 24-well plates (1X 10) 5 cells/well) were incubated in an incubator at 37℃for 24h. Washing twice with PBS buffer, and adding physiological Saline (Saline), cy5.5-OVA, and Fe respectively 3 O 4 /Ca@MnCO 3 Cy5.5-OVA and Fe 3 O 4 /Ca@MnCO 3 Cy5.5-OVA (M) (according to the above-mentioned ratio of Table 1, usedCy5.5 fluorescent-labeled OVA (Cy5.5-OVA) the test solutions (25. Mu.g of Cy5.5-OVA was added to each well) were each set in three replicates and incubated at 37℃for a further 30min. Finally, the culture broth was aspirated, the free particles were removed by washing with PBS buffer, the cells were resuspended, and detection was performed using a flow cytometer to obtain the average fluorescence intensity of DC2.4 cells.
The results are shown in FIG. 5: in all Fe-containing ratios to Cy5.5-OVA alone 3 O 4 /Ca@MnCO 3 In the group (a), the OVA internalization ratio is significantly higher; wherein Fe under magnetic field treatment 3 O 4 /Ca@MnCO 3 The internalization ratio of antigen OVA was highest at 30min for the nanoparticle. This indicates that Fe 3 O 4 /Ca@MnCO 3 The nanoparticle may accelerate the active presentation of antigen to DC under a magnetic field.
4. Preparation of magnetized DC and migration under the action of magnetic field
DC2.4 cells were seeded into 10cm cell culture dishes (about 5X 10) 5 Individual cells/dish) and incubated for 24h. Washing twice with PBS buffer, adding Fe 3 O 4 /Ca@MnCO 3 The nanoparticles were subjected to a magnetic field treatment (magnetic field generated by neodymium-iron-boron permanent magnet disk) for 30min, and then the cells were collected (magnetized DC), and the movement track of the cells under the magnetic field was observed with an inverted microscope, and then the particles were subjected to a magnetic field treatment with Fe 3 O 4 /Ca@MnCO 3 And (5) performing comparison. Three replicates were set up for each group.
The results are shown in FIG. 6: fe (Fe) 3 O 4 /Ca@MnCO 3 Both the nanoparticle and the magnetization DC move in the direction of the magnetic field. These results indicate that the successful preparation of magnetized DCs allows accurate arrival at the destination, which is expected to achieve homing of the DC vaccine to the lymph nodes, further inducing immune responses.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (3)
1. Magnetic drive nanomotor Fe for antigen delivery 3 O 4 /Ca@MnCO 3 The preparation method of (2) is characterized by comprising the following steps:
(1) CaCl is added with 2 And MnCl 2 Adding into glycerol water solution, mixing uniformly to obtain CaCl 2 And MnCl 2 Is a mixed solution of (a) and (b); then Fe is added 3 O 4 Stirring and mixing the nano particles uniformly to obtain the Fe-containing material 3 O 4 、CaCl 2 And MnCl 2 Is a mixed solution of (a) and (b);
(2) NH is added to 4 HCO 3 Dissolving in glycerol water solution to obtain NH 4 HCO 3 A solution; then NH is added 4 HCO 3 Adding the solution to the Fe-containing solution obtained in the step (1) 3 O 4 、CaCl 2 And MnCl 2 Stirring and reacting, centrifuging after the reaction is finished, taking precipitate, washing, and freeze-drying to obtain the magnetic drive nano motor Fe 3 O 4 /Ca@MnCO 3 ；
The glycerol aqueous solution in the steps (1) and (2) is a solution obtained by mixing glycerol and water according to a volume ratio of 1:1;
CaCl as described in step (1) 2 And MnCl 2 Ca in the mixed solution of (2) 2+ And Mn of 2+ The molar ratio of (2) is 1:1;
fe as described in step (1) 3 O 4 The addition amount of the nano particles is per milliliter CaCl 2 And MnCl 2 Is mixed with 0.5mg Fe 3 O 4 Calculating nano particles;
CaCl as described in step (2) 2 、MnCl 2 And NH 4 HCO 3 The molar ratio of (2) is 1:1:10;
the stirring conditions in the step (1) are as follows: stirring at 250-350 rpm for 25-35 min;
the stirring conditions in the step (2) are as follows: stirring at 50+ -5 deg.C and 550-650 rpm for 0.5-1.5-h.
2. For use according to claim 1Antigen delivery magnetically driven nanomotor Fe 3 O 4 /Ca@MnCO 3 The preparation method of (2) is characterized in that:
the centrifugation conditions in step (2) are: centrifuging for 4-6 min at 3000-5000;
the washing in the step (2) is alternately washing with absolute ethyl alcohol and deionized water;
the freeze-drying time in the step (2) is 22 to 26 and h.
3. Magnetic drive nanomotor Fe for antigen delivery 3 O 4 /Ca@MnCO 3 The method is characterized in that: prepared by the method of any one of claims 1-2.
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