CN115998714B - Lipid nanoparticle, delivery system and preparation method of delivery system - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a lipid nanoparticle, a delivery system and a preparation method of the delivery system, belongs to the field of biological medicine, and provides a lipid nanoparticle containing two cationic lipid molecules; the nanometer lipid particle can be used as a carrier to efficiently deliver mRNA to cells in a body, so that the mRNA molecules can be translated and expressed in the cells to express relevant vaccine antigens, and the acquired immune system can be activated under the background of starting a natural immune reaction system in the cells, and the encapsulation rate of the delivery system prepared by taking the lipid nanoparticle as the carrier to the mRNA molecules can reach more than 95%, and the effectiveness and the safety are equal to those of the existing LNP system.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to lipid nano particles, a delivery system and a preparation method of the delivery system.
Background
In the field of research and application of emerging mRNA vaccines and their gene drugs, one key technical link is the development of a mediating system capable of delivering mRNA molecules into cells, which is usually implemented in different forms of nanoparticle carriers. Among these different forms of nano-delivery vehicles, lipid nanoparticle (lipid nanoparticle, LNP) technology is an emerging field rapidly developed for the mRNA vaccine requirements of covd-19, which is based on the property of lipid molecules to fuse efficiently with cell membranes in specific constitutive forms to enable the delivery of mRNA molecules with translational capabilities into cells in vivo, thus enabling the translation of mRNA molecules into expression of relevant vaccine antigens within cells and the activation of the acquired immune system in the context of the initiation of the intracellular innate immune response system. The present LNP mainly comprises 4 basic lipid molecules, namely cationic lipid, phospholipid molecule, cholesterol and PEGylated lipid molecules. The specific forms of these molecules can vary, but this combination forms a more fixed pattern from the perspective of the LNP composition used for three mRNA vaccines that have been marketed (table below).
Currently, LNP, cholesterol, is one of the essential components that stabilize during the formation of lipid particles.
While the current art has provided the composition of a range of nanolipid particles, the nanolipid particles that provide new components remain of positive interest.
Disclosure of Invention
Cationic lipids have a key role in the formation of nanoparticle carriers, which together with ionizable lipids initiate the physicochemical processes of particle self-assembly, while having a key role in the encapsulation of mRNA molecules. The applicant of the invention prepares a nanoparticle carrier containing two cationic lipids through a large amount of experimental researches, cholesterol can be replaced through the proportion of the two cationic lipids, the encapsulation rate of the prepared delivery system to mRNA molecules reaches more than 95%, and the effectiveness and the safety are equal to those of the existing LNP system.
To this end, the present invention has a first object to provide a formulation of lipid nanoparticles, a second object to provide a delivery system using the lipid nanoparticles as a carrier, and a third object to provide a preparation method of the delivery system.
The first object of the invention is achieved by the following technical scheme:
the lipid nanoparticle comprises two cationic lipids, a neutral phospholipid molecule and a PEGylated lipid molecule. The two cationic lipids included were ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (hereinafter abbreviated to DHA) and (2, 3-dioleoyl-propyl) -trimethylammonium-chloride (DOTAP-Cl), respectively.
Further, the molar ratio of DHA to DOTAP-Cl is 48-58:12-20.
Further, the neutral phospholipid molecule is 1, 2-dioleoyl-SN-glycero-3-phosphorylcholine (DOPC); the PEGylated lipid molecule is 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (mPEG-DTA-2K).
Further, the mole ratio of DHA and DOTAP-Cl, DOPC, mPEG-DTA-2K is 48-58:12-20:23-28:1.8-2.5.
The second object of the invention is achieved by the following technical scheme:
the lipid nanoparticle is used as a carrier in the preparation of a bioactive substance delivery system.
Further, the bioactive substance delivery system is an mRNA vaccine or an mRNA vaccine of viroid structure.
Further, the vaccine is a vaccine for preventing viral infection.
Further, the virus is SARS-CoV-2.
A process for preparing an mRNA delivery system using lipid nanoparticles as a carrier, comprising the steps of:
s1, dissolving DHA, DOTAP-CL, DOPC and mPEG-DTA-2K in absolute ethyl alcohol, and fully dissolving to obtain an organic phase; mRNA was diluted with NaAc 10mM, trehalose 0.0001% buffer to give an aqueous phase.
S2, preparing an organic phase and a water phase of the step S1 through micro-flow control to obtain mRNA-ADT (the system is named as letter T in A, DOPC of DHA and letter A in DOTAP in nano lipid particles, and is named as ADT), diluting 15-20 times by using a buffer solution containing NaAc 10mM and trehalose of 0.0001%, and carrying out ultrafiltration concentration by using a 100Kda ultrafiltration centrifuge tube to obtain suspension of ADT lipid particles loaded with mRNA, wherein the molar ratio of lipid particles to nitrogen and phosphorus in the mRNA is 4-6:1.
The invention has the beneficial effects that:
1. the lipid nanoparticle formula of the invention can still achieve good carrier delivery effect without using cholesterol as a stabilizer, and a delivery system constructed by using the lipid nanoparticle as a carrier does not need to add cholesterol as a stabilizer, so that the encapsulation rate of mRNA molecules reaches more than 95%, and the effectiveness and the safety are equal to those of the existing LNP.
2. The diameter of ADT formed by the delivery system prepared by the lipid nanoparticle is 50-60nm before encapsulation, the diameter of the ADT is 70-80nm when the ADT is encapsulated by mRNA molecules (the mRNA molecules prepared by transcription of S1 sequences of SARS-CoV-2, which have the sequence length of 2471 and nt when the mRNA molecules contain 5'-UTR and 3' -UTR and polyA), and the encapsulated particle has a uniform spherical capsule structure, so that the encapsulation rate is more than 95%.
3. The delivery system prepared by taking the lipid nanoparticle provided by the invention as a carrier can enable the mRNA molecules with the same dosage to generate translation expression efficiency similar to that of a Moderna company formula in cells. The prepared delivery system can efficiently deliver mRNA to cells in a body, so that the mRNA molecules can be translated and expressed in the cells to form relevant vaccine antigens, and the acquired immune system can be activated under the background of starting a natural immune reaction system in the cells.
Drawings
FIG. 1 is an electron microscopy morphology comparison of the delivery system of the present invention for pre-and post-mRNA encapsulation;
FIG. 2 is a comparison of electrophoretic detection of mRNA by a delivery system of the present invention before and after encapsulation;
FIG. 3 is a comparison of the expression of EGFP fluorescent protein encapsulated by a delivery system of the present invention;
FIG. 4 is a comparison of transfection efficiencies of delivery systems of the present invention;
FIG. 5 is a representation of S1 expression of transfected cells with the delivery system of the present invention;
FIG. 6 is a comparison of serum antibody titers after immunization of mice with S1mRNA from different delivery systems;
FIG. 7 is a comparison of virus neutralizing antibody titers in serum after immunization of mice with different delivery systems S1 mRNA;
FIG. 8 is a comparison of cellular immune response assays after immunization of mice with S1mRNA from different delivery systems;
FIG. 9 is an illustration of the efficient expression of antigenic proteins following entry of mRNA molecules into cells;
FIG. 10 immunohistochemical pathology results.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the technical solutions of the present invention will be described in detail below, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, those skilled in the art may obtain other embodiments without making any creative effort, which fall within the protection scope of the present invention.
The invention is realized by the following technical scheme:
example 1
Nanometer lipid particle and application
The lipid nanoparticle of the present invention is cholesterol free and comprises two cationic lipids, a neutral phospholipid molecule and a pegylated lipid molecule.
The first cationic lipid was:
((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (abbreviated as DHA)
English name: (4-Hydroxybutyl) azanediyl) bis (2-hexydecanoate)
The second cationic lipid was:
(2, 3-dioleoyl-propyl) -trimethylammonium-chloride salt (DOTAP-Cl)
English name: (2, 3-Dioleyloxy-propyl) -trimethylcam-chloride
Preferably, the neutral phospholipid molecule is:
(1, 2-dioleoyl-SN-glycerol-3-phosphorylcholine) (DOPC);
english name: (1, 2-diolyl-sn-glycero-3-phosphaline).
The PEGylated lipid molecules are:
2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (mPEG-DTA-2K);
the English name is: 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide.
Wherein, the mol ratio of DHA and DOTAP-Cl, DOPC, mPEG-DTA-2K is 48-58:12-20:23-28:1.8-2.5.
The use of the nanolipid particles of this example as a carrier in the preparation of a bioactive substance delivery system.
Example 2
A method of preparing an mRNA-ADT delivery system with the lipid nanoparticle of example 1 as a carrier, comprising the steps of:
s1: DHA, DOTAP-CL, DOPC, mPEG-DTA-2K are respectively used for preparing the polypeptide according to the molecular concentration: 20%, 10%, 20%, 10% in GR grade absolute ethanol, and fully dissolved. The dissolution temperature is preferably 37-42 ℃.
S2, according to DHA: DOTAP-CL: DOPC: mPEG-DTA-2k=54.7: 16.5:26.7:2.1 the absolute ethanol solution of step S1 was mixed to prepare an organic phase.
S3: the aqueous phase was prepared by diluting the mRNA with NaAc 10mM buffer containing 0.0001% trehalose (the mRNA content may be determined according to the required dosage, but the molar ratio of nitrogen to phosphorus (N/P value) is required to be 4-6:1).
S4: through NanoAssemblr Ignite TM And the micro-fluidic device prepares mRNA-ADT according to the flow rate ratio of the organic phase to the aqueous phase of 1:3 (ml/min) and the total flow rate of 15-20 ml/min. Diluting mRNA-ADT with buffer solution containing NaAc 10mM and trehalose 0.0001%, diluting with 15-20 times, and ultrafiltering with 100Kda ultrafiltration centrifuge tube for concentration. Centrifugation conditions: 15℃at 2500 Xg for 20 minutes until the volume of the dilution is near before dilution. Its final nitrogen to phosphorus ratio N/p=4-6.
Physicochemical characterization of the prepared ADT system:
physicochemical property analysis based on the different types of LNPs present shows that LNP particles capable of efficiently delivering mRNA into cells are typically 60-120nm in diameter and exhibit corresponding surface potentials (Zeta potentials) and polymer dispersion index f (PDI).
The corresponding characterization of the ADT lipid nanoparticle formed in this example, the comparison of the encapsulated mRNA molecule and the free mRNA molecule, etc., shows that the ADT lipid nanoparticle formed in this example has a diameter of 50-60nm prior to encapsulation, and a diameter of 70-80nm in the case of encapsulated mRNA molecules (mRNA molecules prepared by transcription of the S1 sequence of SARS-CoV-2, which have a sequence length of 2471 nt in the case of 5'-UTR and 3' -UTR and poly a tail, and PDI and Zeta potentials of 0.1-0.2 and 30-40 mv, respectively). The specific analytical data are shown in Table 2.
TABLE 2 characterization of mRNA-LNP delivery System encapsulation mRNA before and after encapsulation
Morphological electron microscopy observation before and after encapsulation of mRNA molecules showed that the encapsulated particles were in a uniform spherical capsule structure (FIG. 1).
Meanwhile, the encapsulation efficiency of the ADT coated mRNA is detected (detected by using a Quant-iT ™ riboGreen ™ RNA Reagent and Kit Catalog Numbers R11490 kit), and the result shows that the encapsulation efficiency is more than 95%.
Table-3 encapsulation efficiency of mRNA by ADT delivery system
The gel electrophoresis results also demonstrate this result (FIG. 2).
Transfection effects of the mRNA-ADT lipid nanoparticle formed in this example on in vivo and in vitro cells
Research shows that the main index of ADT lipid particle performance analysis with mRNA molecule intracellular delivery capability is the transfection effect on in vivo and in vitro cells.
In vitro cultured 293 cells were transfected at different doses using the preparation of the coated EGFP mRNA molecules prepared in example 2, while fluorescent protein expression was observed within 24 hours after transfection using the Moderna LNP formulation as a reference (FIG. 3), and the results indicate: the delivery system of the present invention has good transfection capacity, and its transfection efficiency is as high as 80% (fig. 4), without significant difference from the LNP formulation of the Moderna company.
293 cells were transfected with the S1 gene mRNA molecule-coated SARS-CoV-2 preparation prepared in example 2, cell cultures were collected 24h after transfection, recombinant proteins were separated by 12% SDS-PAGE, and proteins were transferred onto polyvinylidene fluoride (PVDF) membranes by electrotransformation. 5% skim milk was prepared using TBST, blocked for 1H at room temperature, washed 3 times, incubated overnight at 4℃with the addition of a specific primary antibody against protein S, washed 3 times, then incubated with a secondary antibody labeled with peroxidase (HRP) (anti-IgG (H+L) antibody) for 1H at room temperature, and washed 3 times. Finally, the PVDF film treated as described above was subjected to exposure and color development by ECL ultrasensitive chemiluminescence method in a Bio-Rad gel imager. The transfection efficiency of the ADT system was thus determined. Meanwhile, we used the formulation composition of Moderna company as a reference. The results indicate that the ADT system formulations of the present invention allow the same dose of mRNA molecules to produce a translational expression efficiency in cells similar to that of Moderna (FIG. 5).
Immune effect analysis of delivery systems for mRNA vaccines
In the research field of different LNP systems for mRNA vaccines, the formation of animal-specific immune responses and the effectiveness of clinical immune protection thereof are the most important indicators for judging the application value of the LNP system.
The S1 gene of SARS-CoV-2 spike protein is used, the S1 gene mainly comprises RBD part of virus combined with cell ACE2 receptor, the coded transcribed mRNA molecule, 5'-UTR and 3' -UTR carried by the mRNA molecule, wherein the 5'-UTR is the 5' -UTR of YFV 17D strain gene, 3'-UTR is 3' -UTR of ribosome in human mitochondrial gene, the ADT system of example 2 is used for preparing experimental vaccine, the experimental vaccine of mRNA with total lipid mass of 1.95mg/dose is formed according to the corresponding mRNA dosage range, balb/c mice are immunized according to 0, 21 days immune mode, the immune route is intramuscular injection, the antibody titer, cell immune response and other indexes are detected 14 days and 28 days after the second immune mode, the result shows that: SARS-CoV-2 S1 gene mRNA molecule mediated by ADT lipid particle system can induce obvious antibody reaction in mouse body, its binding antibody titer is up to 10 5-6 Above (fig. 6). Its euvirus neutralizing antibody titer is also 10 2-3 Above (fig. 7). The corresponding IFN-. Gamma.and IL-4 specific Elispot assays also suggested that immunized mice developed a systemic immune response against the specific S antigen (FIG. 8).
Safety analysis:
in previous studies of multiple mRNA vaccines, a large number of relevant LNP preparation lipid molecular pharmacology data have been accumulated, and in the compositional formulation of the ADT lipid particle system of the present invention, all lipid raw materials used have been observed for relevant pathology and confirmed for their safety. The safety of ADT formed by these lipid raw materials in the formulation composition of this example was emphasized, mainly from the general pathological observations of the tissue distribution formed by the immune body and the tissues of the organs after application after their formation of mRNA vaccine formulations. The observations indicate that the ADT system, after immunization with S1mRNA molecules, can first efficiently express antigen proteins in muscle tissue after the mRNA molecules enter cells (FIG. 9), with a certain dynamic change in expression over time. Expressed to peak in 24-48 hours (fig. 9). Meanwhile, the presence of the mRNA molecule was not detected in each tissue organ (Table 4).
TABLE 4 expression of mRNA in different tissues of mice
Note that: "-" indicates that no mRNA antigen molecule is detected; "+" indicates that mRNA antigen molecules are detected.
At the same time, no abnormal pathological changes were detected in the histopathological and gross pathological observations of the animals at different times after immunization, and only a certain degree of inflammatory cell aggregation and mild tissue damage was observed locally by injection. (Table 5 and FIG. 10)
TABLE 5 detection of histopathology at different time points after local immunization of mRNA
Note that: "-" indicates that no histopathological changes were detected; "++" indicates that lymphocyte infiltration is detected; "+++". Representation of detection of lymph cell infiltration and tissue mild injury.
The work shows that the ADT lipid particle delivery system designed and constructed by the invention realizes the function of effectively delivering vaccine mRNA molecules on the basis of autonomous formulation, and still has corresponding safety characteristics. Can be used for further application research of mRNA vaccine, especially SARS-CoV-2 mRNA vaccine.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (10)
1. A lipid nanoparticle consisting of two cationic lipid molecules, a neutral phospholipid molecule and a pegylated lipid molecule;
the two cationic lipid molecules are ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate) and (2, 3-dioleoyl-propyl) -trimethylammonium-chloride, respectively;
the neutral phospholipid molecule is 1, 2-dioleoyl-SN-glycero-3-phosphorylcholine; the PEGylated lipid molecule is 2- [ (polyethylene glycol) -2000] -N, N-tetracosanamide;
wherein the molar ratio of ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) to (2, 3-dioleoyl-propyl) -trimethylammonium-chloride salt is 48-58:12-20.
2. The lipid nanoparticle of claim 1, wherein the molar ratio of ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), (2, 3-dioleoyl-propyl) -trimethylammonium-chloride, 1, 2-dioleoyl-SN-glycero-3-phosphorylcholine, 2- [ (polyethylene glycol) -2000] -N, N-tetracosanamide, is 48-58:12-20:23-28:1.8-2.5.
3. The lipid nanoparticle of claim 2, wherein the molar ratio of ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), (2, 3-dioleoyl-propyl) -trimethylammonium-chloride, 1, 2-dioleoyl-SN-glycero-3-phosphorylcholine, 2- [ (polyethylene glycol) -2000] -N, N-tetracosanamide, is 54.7:16.5:26.7:2.1.
4. use of a lipid nanoparticle according to any one of claims 1-3 as a carrier for the preparation of a bioactive substance delivery system.
5. The use according to claim 4, wherein the biologically active substance delivery system is an mRNA vaccine.
6. The use according to claim 4, wherein the biologically active substance delivery system is an mRNA vaccine of viroid structure.
7. The use according to claim 5 or 6, wherein the vaccine is a vaccine for the prevention of viral infections.
8. The use according to claim 7, wherein the virus is SARS-CoV-2.
9. A gene delivery system comprising the lipid nanoparticle vector of any one of claims 1-3 and a genetic material.
10. A process for preparing an mRNA delivery system using lipid nanoparticles as a carrier, wherein the lipid nanoparticles comprise two cationic lipid molecules ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) and (2, 3-dioleoyl-propyl) -trimethylammonium-chloride, the preparation method comprising the steps of:
s1, dissolving ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate), (2, 3-dioleoyl-propyl) -trimethylammonium-chloride salt, 1, 2-dioleoyl-SN-glycero-3-phosphorylcholine and 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide in absolute ethyl alcohol, and fully dissolving to obtain an organic phase; diluting mRNA with NaAc 10mM buffer solution with trehalose of 0.0001% to obtain water phase;
s2, preparing the organic phase and the water phase of the step S1 through micro-flow control to obtain mRNA-ADT, diluting 15-20 times by using a buffer solution containing NaAc 10mM and trehalose of 0.0001%, and carrying out ultrafiltration concentration by using a 100Kda ultrafiltration centrifuge tube to obtain suspension of ADT lipid particles loaded with mRNA, wherein the molar ratio of the lipid particles to nitrogen and phosphorus in the mRNA is 4-6:1.
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