CN115227676A - Mineral coated particle material for slowly releasing mRNA (messenger ribonucleic acid) as well as preparation method and application thereof - Google Patents
Mineral coated particle material for slowly releasing mRNA (messenger ribonucleic acid) as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a mineral-coated particle material for slowly releasing mRNA, which comprises a mineral-coated particle MCM and mRNA coated in the MCM, and can be used for regulating macrophage to be transformed into tumor killing macrophage in real time. The preparation method comprises the following steps: (1) preparing liposome: preparing lipid-ethanol solution and mRNA-citric acid buffer solution, and mixing the two solutions by a microfluidic device to obtain mRNA liposome nanoparticles; (2) preparing MCM: firstly, preparing modified simulated body fluid mSBF; dispersing hydroxyapatite powder in modified simulated body fluid mSBF, and rotating and freeze-drying the obtained solution to obtain MCM; (3) And (3) adsorbing, namely dissolving the MCM and mRNA liposome nano-particles in a PBS buffer solution respectively, mixing and stirring the two solutions to obtain the mineral coated particle material for slowly releasing the mRNA. The invention not only provides a theoretical basis for developing high-efficiency and safe mRNA mineral-coated particle materials, but also provides a new strategy for tumor immunotherapy.
Description
Technical Field
The invention belongs to the technical field of biological medicine materials. More particularly, relates to a mineral coated particle material for slowly releasing mRNA for treating bone tumor, a preparation method and application thereof.
Background
Tumors remain one of the biggest threats and major causes of death to human health every year in recent decades, placing a heavy burden on the patient himself and the whole society. In fact, over 165,000 children diagnosed with cancer, the financial cost of cancer is estimated to be $ 1.16 trillion per year.
The macrophage has strong plasticity. In addition to the tumor growth promoting phenotype in the tumor microenvironment, macrophages can also be converted to tumor-killing macrophages (tumocidal macromacrophages) and have good therapeutic effects. Macrophages are able to kill tumor cells with excess or unknown tumor-specific antigens in a more direct manner than engineered T cells, making them likely to be successful in the case of inadequate T cell therapy. The conventional modification is active biological factor IFN γ stimulation, but drug delivery strategies for active biological factors are often limited by low biological activity in vivo, which results in the provision of supraphysiological doses of therapeutic proteins, which in turn often leads to negative side effects in clinical use.
Nucleic acid delivery is an attractive therapeutic approach, and compared to other nucleic acid delivery methods, non-viral delivery of mRNA has many attractive features, which may combine features of active expression and safety and stability. In particular, mRNA delivery does not require transport of nucleic acids to the nucleus, and thus mRNA can still be efficiently expressed in non-mitotic cell populations. In addition, mRNA is not expressed by homologous recombination or insertion of a mutagenized gene, and therefore, mRNA has safe stability.
However, mRNA has the disadvantage of being unstable. The invention uses Mineral-Coated Microparticles (MCM) to trigger a local delivery mechanism, stabilize mRNA, improve the transfection efficiency of the mRNA and simultaneously reduce the cytotoxicity related to the reagent. Furthermore, the nanostructured mineral coating can sequester and stabilize unstable growth factors to prevent denaturation, thereby allowing sustained protein release and prolonged stimulation of the desired biological response.
Disclosure of Invention
In order to overcome the defects that the titer of the existing recombinant protein is low, the existing mRNA product is unstable, the capability of regulating and controlling macrophage to be transformed into tumor killer macrophage in real time according to the experimental requirement is weak, and the like, the invention provides a novel bone tumor resistant material of 'mRNA-loaded mineral coated particle material'.
The technical scheme adopted by the invention is as follows:
a mineral-coated particulate material for sustained release of mRNA comprises a mineral-coated particulate MCM and mRNA coated in the MCM.
A preparation method of a mineral-coated microparticle material for slowly releasing mRNA comprises the following steps:
(1) Preparing liposome: preparing lipid-ethanol solution and mRNA-citric acid buffer solution, and mixing the two solutions by a microfluidic device to obtain mRNA liposome nanoparticles; the lipid-ethanol solution is an ethanol solution of ionizable cationic lipid DLin-MC3-DMA, phosphatidyl ethanolamine HSPE-MPEG2000, distearoyl phosphatidyl choline DSPC and cholesterol;
(2) Preparing MCM: mixing NaCl, KCl and MgSO 4 ·7H 2 O、MgCl 2 ·6H 2 O、NaHCO 3 、Hepes、CaCl 2 ·2H 2 O、KH 2 PO 4 And NaF are dissolved in ultrapure water to obtain modified simulated body fluid mSBF; mixing the hydroxyapatiteDispersing stone powder in the modified simulated body fluid mSBF, rotating and freeze-drying the obtained suspension to obtain MCM;
(3) And (3) adsorbing, namely dissolving the MCM and mRNA liposome nanoparticles in a PBS buffer solution respectively, mixing and stirring the two solutions to obtain the mineral-coated particle material for slowly releasing the mRNA.
Further, in the step (1), the molar ratio of DLin-MC3-DMA, HSPE-MPEG2000, DSPC and cholesterol in the lipid-ethanol solution is 50.
Further, in the step (1), the preparation method of the mRNA-citric acid buffer solution comprises the following steps: preparing a mixed solution of citric acid and sodium citrate by using ultrapure water, adding DEPC, standing for 30 minutes, then carrying out high-pressure sterilization to remove DEPC, and carrying out constant volume by using DEPC water after sterilization to obtain a citric acid buffer solution with pH = 4; diluting mRNA to a required concentration by using a citric acid buffer solution to obtain an mRNA-citric acid buffer solution; in the mixed solution, the molar ratio of citric acid to sodium citrate is 33.
Further, the microfluidic device mixing of step (1) is specifically: filtering the lipid-ethanol solution and the mRNA-citric acid buffer solution through 0.22 micron filter membranes respectively; respectively sucking the lipid-ethanol solution and the mRNA-citric acid buffer solution into the injector, and exhausting air in the injector; connecting the outlet of the injector with the sample introducing pipe, and fixing the injector on the injection pump; adjusting the flow rate and mixing volume of the injection pump according to specific needs; operating, and collecting the liquid flowing out by a collecting pipe after observing the stable flow rate of the outflow pipe; wherein the volume ratio of the mixed lipid-ethanol solution to the mRNA-citric acid buffer solution is 1:3.
furthermore, after collecting the effluent liquid by a collecting pipe, the nano-particles are obtained, and the nano-particles can be not filtered. If filtering is needed, the following method is adopted: immediately after sample collection, the ethanol concentration was diluted to below 1% with 30 volumes of PBS and ultrafiltered using a Millipore 30KD ultrafiltration tube, centrifuged at 3000g for 20 minutes.
Further, in the step (2), naCl, KCl, mgSO 4 ·7H 2 O、MgCl 2 ·6H 2 O、NaHCO 3 、Hepes、CaCl 2 ·2H 2 O、KH 2 PO 4 And NaF at a molar ratio of 141.5; hydroxyapatite powder was suspended in a concentration of 1mg/ml in modified simulated body fluid mSBF.
Further, in the step (2), the operation of rotating and freeze-drying is as follows: the suspension was spun at 37 ℃ for 5 days, and after 5 days, washed 3 times with deionized water, filtered through a 40 μm pore cell filter, suspended in 15ml of distilled water, and lyophilized.
Further, in the step (3), the mass ratio of the mRNA liposome nanoparticles to the MCM in the mixed solution is 1:5, stirring for 30min-1h at 4 ℃.
The invention also provides application of the mineral coated particle material for slowly releasing the mRNA, which can be used for preparing a medicament for inducing cell differentiation, wherein the cell differentiation is as follows: converting macrophage to tumor killing macrophage.
The beneficial effects of the invention are:
1. the invention utilizes a non-viral delivery mode of mRNA to regulate the phenotype of macrophage TAM, and has the characteristics of active expression and safety and stability, in particular, mRNA delivery does not need to transport nucleic acid to nucleus, so that mRNA can still be effectively expressed in a non-mitotic cell population. In addition, mRNA is not expressed by homologous recombination or insertion of a mutagenized gene, and therefore, mRNA has safe stability.
2. mRNA has the disadvantage of being unstable. The Mineral-Coated Microparticles (MCM) provided by the invention can trigger a local delivery mechanism, stabilize mRNA, improve the transfection efficiency of the mRNA and reduce the cytotoxicity related to a reagent. Furthermore, the nanostructured mineral coating can sequester and stabilize unstable growth factors to prevent denaturation, thereby allowing sustained protein release and prolonged stimulation of the desired biological response.
3. The mineral coated particle material for slowly releasing mRNA accurately and efficiently conveys mRNA into tumor-related macrophages, and TAM is differentiated into M1 type macrophages with cancer inhibition effect by regulating and controlling a series of gene expression, so that anti-tumor immune response is initiated.
4. The invention not only provides a theoretical basis for developing high-efficiency and safe mRNA mineral-coated particle materials, but also provides a new strategy for tumor immunotherapy.
Drawings
FIG. 1 is a schematic diagram of a mineral coated particulate material for sustained release of mRNA;
FIG. 2 is a transmission electron micrograph of mRNA liposome nanoparticles;
FIG. 3 is a scanning electron micrograph of an MCM;
FIG. 4 is GFP mRNA LNP @ MCM transfection efficiency;
FIG. 5 is a drawing of the resorption of mineral coated microparticles;
FIG. 6 is a mineral coated particulate material that sustains mRNA release to promote the activation of TAMs;
FIG. 7 is the mineral coated particulate material with sustained release of mRNA to induce activation of TAMs.
Detailed Description
The present invention is described in detail with reference to the drawings, but the scope of the present invention is not limited to the following embodiments, and all simple equivalent changes and modifications made by the claims and the contents of the specification of the present invention are still within the scope of the present invention.
Example 1: preparation of mRNA-containing liposome nanoparticles
Preparing a lipid-ethanol solution: DLin-MC3-DMA (mw 321.1 mg/mmmol), HSPE-MPEG2000 (mw 37.6 mg/mmmol), DSPC (mw 79.0 mg/mmmol), cholesterol (mw 148.9 mg/mmmol), four molar ratios 50.
Preparing an mRNA-citric acid buffer solution: 50ml of each of 100mM citric acid (molecular weight: 210.14, 1.05g by weight) and sodium citrate (molecular weight: 294.10, 1.47g by weight) solutions was prepared using ultrapure water. And (3) mixing 33.0ml of citric acid solution and 17.0ml of sodium citrate solution, adding DEPC, standing for 30 minutes, then carrying out autoclaving to remove DEPC, and carrying out sterilization, and then using DEPC water to fix the volume to 100ml to obtain 50mM citric acid buffer solution with the pH value of = 4. The mRNA was measured for concentration and diluted to the desired concentration using citrate buffer according to lipid concentration.
Operating by means of a microfluidic device: 1. the lipid-ethanol solution and the mRNA-citric acid buffer were filtered through 0.22 micron filters, respectively. 2. The lipid-ethanol solution was aspirated into a 1ml syringe (at least 0.5ml if necessary), the mRNA-citric acid buffer was aspirated into a 3ml syringe (at least 1.5 ml), and the air in the syringe was evacuated. 3. The outlet of the syringe was connected to the sample introduction tube and fixed to the syringe pump. 4. The flow rate and mixing volume of the syringe pump are adjusted according to the specific needs. 5. In operation, the collection tube was used to collect the effluent after the flow rate through the effluent tube was observed to stabilize. Finally, the cmRNA liposome nanoparticle is obtained, and the transmission electron microscope image of the nanoparticle is shown in FIG. 2.
Example 2: preparation of MCM and characterization thereof
mSBF (modified mock body fluid) was prepared from 141mM NaCl, 4mM KCl, 0.5mM MgSO 4 ·7H 2 O、1mM MgCl 2 ·6H 2 O、4.2mM NaHCO 3 、20mM Hepes、5mM CaCl 2 ·2H 2 O、2mM KH 2 PO 4 And 1mM NaF in ultrapure water.
Preparation of MCM hydroxyapatite powder (Plasma Biotal Limited) was used as a microparticle core, suspended at a concentration of 1mg/ml in mSBF, and rotated at 37 ℃ for 5 days. After 5 days, the MCM was washed 3 times with 50ml Deionized (DI) water, filtered through a 40 μm pore cell filter, suspended in 15ml distilled water, and lyophilized to give MCM. The lyophilized MCMs were then analyzed for nanotopography and calcium release. Scanning Electron Microscope (SEM) images were obtained using a JSM-6510LV electron microscope (Japanese JEOL) as shown in FIG. 3.
Example 3: transfection efficiency of GFP mRNA LNP @ MCM
Stirring liposome nanoparticles containing GFP mRNA (GFP mRNA LNP) with MCM in a PBS buffer environment of enucleated enzyme at 4 ℃ for 30 minutes at the ratio of 1; 1; 1;1, 10, GFP mRNA LNP @ MCM was obtained. RAW264.7 cells were incubated in complete medium with the above GFP mRNA LNP @ MCM for 24 hours and the fluorescence intensity was counted by inverted fluorescence microscopy. As shown in fig. 4, GFP mRNA LNP was expressed in RAW264.7, but the expression was slightly weak, while with the increase of MCM content, the expression of GFP gradually increased, reflecting that MCM could slowly release and stabilize mRNA LNP, thereby promoting mRNA expression.
Example 4: re-adsorption experiments for MCM
GFP and MCM were added together to a complete medium for RAW264.7 culture, and after 48 hours of co-incubation, the fluorescence intensity was counted by an inverted fluorescence microscope. As shown in fig. 1, 5, the significantly increased fluorescence intensity of the MCM particles, greater than the surrounding fluid, represents the MCM particles re-adsorbing the GFP protein to isolate and stabilize the unstable free protein from denaturation, thereby allowing sustained protein release and prolonged stimulation of the desired biological response.
Example 5: mineral coated particulate material for sustained release of IFN gamma mRNA to promote activation of TAMs
The liposome nanoparticle containing IFN γ mRNA (IFN γ mRNA LNP) was stirred with MCM at 4 ℃ for 30 minutes under an enucleation environment at a ratio of 1. RAW264.7 in complete medium, add the above IFN gamma mRNA LNP @ MCM, after 48 hours incubation by flow cytometry detection. As shown in fig. 6, an increase in the number of RAW264.7 cells expressing iNOS and CD86 in the IFN γ mRNA LNP @ mcm group compared to the IFN γ mRNA LNP group represents an increase in macrophages with tumor killing. It is demonstrated that IFN γ mRNA LNP @ MCM prevents denaturation by re-adsorbing and stabilizing labile IFN γ, allowing sustained IFN γ release and prolonged stimulation of the desired biological response, thus achieving potential efficacy against tumors with high efficacy.
Example 6: mineral coated particulate material for sustained release of IFN gamma mRNA to promote activation of TAMs
Liposomal nanoparticles containing IFN γ mRNA (IFN γ mRNA LNP) were stirred with MCM for 30min at 4 ℃ in an enucleated enzyme environment at a ratio of 1. RAW264.7 in complete medium, adding the IFN gamma mRNA LNP @ MCM, after 48 hours incubation by flow cytometry detection. As shown in FIG. 7, the increased number of RAW264.7 cells expressing iNOS and CD86 in IFN γ mRNA LNP @ MCM group compared with recombinant protein (recombiant) IFN γ group represents the increase of macrophages with tumor killing property. The IFN gamma mRNA LNP @ MCM is shown to prolong the stimulation of expected biological reaction through autocrine IFN gamma and slow-release IFN gamma after reabsorption, thereby achieving the potential effect of high-efficiency anti-tumor.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A mineral-coated particulate material having mRNA sustained release comprising a mineral-coated particulate MCM and mRNA coated in the MCM.
2. The method for preparing the mRNA sustained-release mineral-coated particulate material of claim 1, comprising the steps of:
(1) Preparing liposome: preparing lipid-ethanol solution and mRNA-citric acid buffer solution, and mixing the two solutions by a microfluidic device to obtain mRNA liposome nanoparticles; the lipid-ethanol solution is an ethanol solution of ionizable cationic lipid DLin-MC3-DMA, phosphatidylethanolamine HSPE-MPEG2000, distearoyl phosphatidylcholine DSPC and cholesterol;
(2) Preparing MCM: mixing NaCl, KCl and MgSO 4 ·7H 2 O、MgCl 2 ·6H 2 O、NaHCO 3 、Hepes、CaCl 2 ·2H 2 O、KH 2 PO 4 And NaF are dissolved in ultrapure water to obtain modified simulated body fluid mSBF; dispersing hydroxyapatite powder in the modified simulated body fluid mSBF, and rotating and freeze-drying the obtained suspension to obtain MCM;
(3) And (3) adsorbing, namely dissolving the MCM and mRNA liposome nanoparticles in a PBS buffer solution respectively, mixing and stirring the two solutions to obtain the mineral-coated particle material for slowly releasing the mRNA.
3. The method for preparing a mineral-coated particulate material with sustained release of mRNA according to claim 2, wherein in the lipid-ethanol solution of step (1), the molar ratio of DLin-MC3-DMA, HSPE-MPEG2000, DSPC and cholesterol is 50.
4. The method for preparing the mRNA slow-release mineral-coated particle material according to claim 2, wherein in the step (1), the mRNA-citric acid buffer solution is prepared by the following steps: preparing a mixed solution of citric acid and sodium citrate by using ultrapure water, adding DEPC, standing for 30 minutes, then carrying out high-pressure sterilization to remove DEPC, and carrying out constant volume by using DEPC water after sterilization to obtain a citric acid buffer solution with pH = 4; diluting mRNA to a required concentration by using the citric acid buffer solution to obtain an mRNA-citric acid buffer solution; in the mixed solution, the molar ratio of citric acid to sodium citrate is 33.
5. The method for preparing the mRNA sustained-release mineral-coated particulate material of claim 2, wherein the microfluidic device mixing of the step (1) is specifically: filtering the lipid-ethanol solution and the mRNA-citric acid buffer solution through a 0.22 micron filter membrane respectively; respectively sucking the lipid-ethanol solution and the mRNA-citric acid buffer solution into the injector, and exhausting air in the injector; connecting an outlet of the injector with a sample introducing pipe, and fixing the injector on an injection pump; adjusting the flow rate and mixing volume of the injection pump according to specific needs; operating, and collecting the liquid flowing out by a collecting pipe after observing the stable flow rate of the outflow pipe; wherein the volume ratio of the mixed lipid-ethanol solution to the mRNA-citric acid buffer solution is 1:3.
6. the method for preparing the mRNA slow-release mineral-coated particulate material of claim 2, wherein in the step (2), naCl, KCl, mgSO 4 ·7H 2 O、MgCl 2 ·6H 2 O、NaHCO 3 、Hepes、CaCl 2 ·2H 2 O、KH 2 PO 4 And NaF at a molar ratio of 141.5; hydroxyapatite powder was suspended in modified simulated body fluid mSBF at a concentration of 1 mg/ml.
7. The method for preparing the mRNA sustained-release mineral-coated particulate material according to claim 2, wherein in the step (2), the operation of rotating and lyophilizing is: the suspension was spun at 37 ℃ for 5 days, and after 5 days, it was washed 3 times with deionized water, filtered through a 40 μm pore cell filter, suspended in 15ml of distilled water, and lyophilized.
8. The method for preparing the mRNA sustained-release mineral-coated particulate material of claim 2, wherein in the step (3), the mass ratio of the mRNA liposome nanoparticles to the MCM in the mixed solution is 1:5, stirring for 30min-1h at 4 ℃.
9. Use of a mineral-coated particulate material that releases mRNA slowly according to claim 1, for the preparation of a medicament for inducing cell differentiation into: to convert macrophage into tumor killing macrophage.
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