MRNA liposomes based on ionizable gold nanoparticles

Abstract

The invention relates to an mRNA (messenger Ribonucleic Acid) liposome based on ionizable gold nanoparticles. The ionizable gold nanoparticles are composed of gold nanoparticles and ionizable lipid, the ionizable lipid and gold atoms on the surfaces of the gold nanoparticles form a ligand structure, and therefore an ionizable lipid layer is formed on the surfaces of the gold nanoparticles. As the ionizable gold nanoparticles are combined with the negatively charged mRNA to form a compound, the energy of subsequent liposome micelle coating is reduced, so that liposome assembly is promoted, and the encapsulation efficiency is improved. The liposome provided by the invention has a better delivery effect on a living body level, and has correlation with the improvement of the encapsulation efficiency of the liposome.

Description

Technical Field

[0001] This patent relates to a novel mRNA liposome, particularly to an mRNA liposome based on ionizable gold nanoparticles and its preparation method. Background Art

[0002] To cope with the widespread global transmission of SARS-CoV-2, the US Food and Drug Administration (FDA) accelerated the clinical approval of the messenger ribonucleic acid vaccines (mRNA vaccines) of Moderna (mRNA-1273) and BioNtech and Pfizer (BNT162b2). Thanks to the great clinical success of mRNA vaccines, the liposome (lipid nanoparticles, LNPs) delivery system with a long history, low toxicity, and immunogenicity has returned to the public eye. Liposomes have the characteristics of adjustable surface physicochemical properties and size, making them the only mRNA delivery system approved by the US Food and Drug Administration so far. Liposomes are composed of four components, namely ionizable lipids, cholesterol, co-lipids, and polyethylene glycol lipids. The four components that make up liposomes each perform their own functions. Ionizable lipids are mainly responsible for the binding of mRNA and endosome-lysosome escape. Co-lipids and cholesterol are the key components for liposome formation. Polyethylene glycol lipids can extend the in vivo circulation time of liposomes, thereby improving the delivery efficiency. As is well known, the mRNA liposome complex is formed by increasing the solvent polarity. During the synthesis process, with the addition of the aqueous phase, the negatively charged mRNA in the aqueous phase binds to the positively charged ionizable lipids in the ethanol phase to form a hydrophobic and electron-dense liposome core. This structure places mRNA at the center of the liposome, enhancing its stability.

[0003] There are many ways to prepare liposomes, such as the traditional ethanol injection method and the thin film hydration method. With the progress of technology, Pieter R. Cullis et al. combined the ethanol injection method with microfluidic mixing technology to develop the latest generation of rapid mixing technology. Liposomes produced based on the rapid mixing technology have the properties of high mRNA encapsulation efficiency and stable and controllable size. The rapid mixing technology is a mixing technology based on chaotic flow with a high Reynolds number, and the chaotic flow with a high Reynolds number can improve the mixing efficiency of the organic phase and the aqueous phase. In a passive mixing chip such as a rapid mixing microfluidic chip, the structure in the microfluidic chip increases the Reynolds number of the fluid, improves the degree of chaos, and increases the contact area between the ethanol phase and the aqueous phase, thereby improving the mixing efficiency. However, the rapid mixing technology has many limitations. The high Reynolds number chaotic flow generated by the high liquid flow rate poses higher requirements for liquid pumps, system pressure control, and system design. The flow channel structure in the rapid mixing microfluidic chip is the key part of the chip. Fabricating the flow channel using the high-cost lithography method is full of challenges for both experimental exploration and industrial production. The inherent limitations of the rapid mixing technology have hindered the application and popularization of liposomes as excellent delivery systems. To reduce the preparation cost of liposomes, the rapid mixing method can be replaced by modifying the composition of liposomes. Summary of the Invention

[0004] To address the deficiencies of the prior art, based on the composition of existing mRNA liposomes, the inventors introduced an ionizable hydrophobic nanoparticle into the aqueous phase. Before mixing the aqueous phase and the ethanol phase, the ionizable positively charged hydrophobic nanoparticle was co-incubated with negatively charged mRNA to form a complex. Then, the aqueous phase containing the complex was mixed with the ethanol phase to prepare an mRNA vaccine or drug. Since in the preparation process of existing mRNA liposomes, mRNA exists in a free and extended state in the aqueous phase with a low encapsulation efficiency. In contrast, in the present invention, the mRNA attached to the ionizable hydrophobic nanoparticle has a smaller size due to the aggregation of mRNA, thus reducing the energy required for subsequent liposome micelle coating and improving the liposome assembly efficiency. When the aqueous phase (containing the ionizable hydrophobic nanoparticle - mRNA complex) is mixed with the ethanol phase (containing the four components of liposomes), the four components of liposomes are wrapped around the ionizable hydrophobic nanoparticle - mRNA complex through electronic force and hydrophobic force to form a core-shell structure with mRNA delivery ability.

[0005] The present invention is achieved through the following technical solutions: First, ionizable gold nanoparticles (IL-AuNPs) are successfully developed through ligand exchange technology. IL-AuNPs are composed of gold nanoparticles and ionizable lipids. The ionizable lipids form ligand structures with gold atoms on the surface of the gold nanoparticles, thereby forming an ionizable lipid layer on the surface of the gold particles. The gold nanoparticles have a particle size of less than 100 nm, preferably 10-30 nm. Typically, the ionizable lipids have amine groups, an acid dissociation constant (pKa ~ 6.5), and are positively charged in solutions with a pH of 4. Therefore, IL-AuNPs are positively charged in an aqueous mRNA solution used for liposome synthesis. Because the mRNA in the solution is negatively charged, the free mRNA is captured on the surface of the IL-AuNPs due to the interaction between the two due to Coulombic forces, forming an IL-AuNPs-mRNA complex. When the IL-AuNPs-mRNA complex is mixed into an ethanol solution containing the four components of liposomes, the liposomes self-assemble on the surface of the IL-AuNPs-mRNA complex due to the combined action of Coulomb force and hydrophobic force.

[0006] The ionizable lipid comprises at least three parts: (a) a head with an ionizable amine group, (b) a linker group containing a -COC- group between the head and the tail, and (c) one or more tails consisting of hydrophobic groups. In an acidic solution, the amine group of the head can react with free H + The -COC- group in the linker is the key group for the interaction between the ionizable lipid and the gold particle to form the ligand structure. The linker containing the -COC- group is preferably -(CH2) 3-6 -C(=O)-OC- or -(CH2) 3-6 -OC(=O)-C-. The tail contains a hydrophobic group with a carbon skeleton, such as a straight-chain alkyl, olefin, or diene group, and the number of carbon atoms is usually between 6 and 18. The hydrophobic tail is the key to the solubility and dispersion of ionizable lipids in chloroform. There are one or two linking groups in the ionizable lipid molecule, and each linking group has one or two hydrophobic tails. For example, the ionizable lipid molecule R-(CH2) 3-6 -C(=O)-O-CH-R 1 (R 2 ) or R-[(CH2) 3-6 -OC(=O)-CH-R 1 (R 2 )]2, where R 1 、R 2Each represents the same or different hydrophobic groups (i.e., tails), R represents the head with an ionizable amine group. The former has a linking group and two tails, and the latter has two linking groups, each with two hydrophobic tails.

[0007] Preferred ionizable lipids have an acid dissociation constant (pKa ∼ 6.5) and are positively charged in a solution at pH = 4. Examples include, but are not limited to, 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester (Dlin-MC3-DMA), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid 1-octylnonyl ester, heptadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((decoxy)hexyl)amino)octanoate) (SM102), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315). In the molecular structure of Dlin-MC3-DMA, there is a linking group (-(CH2)3-C(=O)-O-CH-) and two hydrophobic tails; while in the molecular structure of ALC-0315, there are two linking groups (-(CH2)6-O-C(=O)-CH-), each with two hydrophobic tails.

[0008]

[0009] The IL-AuNPs described in the present invention can be synthesized by the following method: (1) Gold particle synthesis: Gold particles of the required size are prepared by a known method such as sodium citrate reduction method. The present invention requires that the particle size of the gold nanoparticles is controlled within 100 nm, preferably the particle size is 10 - 30 nm, such as 13 - 25 nm, and the 13-nm particles can better achieve the encapsulation efficiency of ionizable gold nanoparticles for mRNA; (2) Ligand modification: The prepared gold particles are modified with at least one polymer that can undergo ligand binding with them. For example, thiol polyethylene glycol (SH-PEG) is added with stirring in a solution with pH = 8, and the thiol groups will adsorb on the surface of the gold particles, enabling the gold particles to have the ability to cross the water-chloroform interface; (3) Ligand exchange: That is, the modified ligand and the ionizable lipid are exchanged to form a ligand structure between the ionizable lipid and the gold atoms on the surface of the gold particles, thereby forming an ionizable lipid layer on the surface of the gold particles, and thus preparing IL-AuNPs. The gold particles modified with thiol polyethylene glycol dispersed in an aqueous solution are added to a chloroform solution containing ionizable lipids, and the solution will be divided into two layers, the upper layer is the aqueous phase and the lower layer is the hydrophobic phase. The gold particles modified with thiol polyethylene glycol have the ability to shuttle between the hydrophobic phase and the aqueous phase, and enter the hydrophobic phase due to gravity, and the thiol groups are replaced by the surrounding ionizable lipids to form IL-AuNPs.

[0010] The mRNA liposome based on ionizable gold nanoparticles described in the present invention has a core-shell structure, and from the inside to the outside includes IL-AuNPs, target mRNA and a lipid layer, wherein the target mRNA and IL-AuNPs are encapsulated by the lipid layer in the form of a complex. The mRNA liposome can be prepared into an mRNA vaccine or a drug. The specific preparation method includes the following steps: (1) Co-incubating the mRNA aqueous solution and ionizable gold nanoparticles to obtain an aqueous phase; (2) Mixing the aqueous phase and the ethanol phase to prepare the mRNA liposome. In step (1), it is better to control the mass ratio of mRNA to gold nanoparticles in the range of 0.2 to 0.6. Among them, the preferred mass ratio of mRNA to ionizable gold nanoparticles is 0.5, that is, mRNA: ionizable gold nanoparticles = 1:2. In step (2), the ethanol phase contains four components of liposome, which is common knowledge in the art and will not be elaborated here; those skilled in the art can optimize its specific components and proportions according to actual needs. The four components of the liposome form the outermost lipid layer in the liposome core-shell structure, and encapsulate the target mRNA and ionizable gold nanoparticles in the form of a complex.

[0011] The structure of the mRNA liposome based on ionizable gold nanoparticles includes three parts, and the morphology is like Figure 4As shown, from the inside to the outside are the IL-AuNPs core, the target mRNA to be delivered, and the lipid layer. The ligand of the IL-AuNPs core is an ionizable lipid with a pKa of approximately 6.5, which is designed to bind to the target mRNA under acidic conditions and release the mRNA in a neutral environment. The main difficulty in mRNA delivery lies in the endosome-lysosome escape part. The nano-delivery carrier enters the cell through endocytosis and is encapsulated by a membrane structure. After the nano-delivery carrier experiences endosome-lysosome escape, it is released into the cytoplasm. During endocytosis, the internal environment of the membrane becomes acidic and rich in various enzymes. At this time, the nano-delivery carrier is most likely to be damaged, making it unable to protect the internal mRNA, resulting in delivery failure due to mRNA degradation. When the delivery carrier has a pKa, the acidic internal environment of the membrane will cause the co-protonation of the internal IL-AuNPs and the outer liposome layer, tightly encapsulating the internal target mRNA. At the same time, the mRNA encapsulated by the outer liposome layer is not degraded until endosome-lysosome escape is completed. Finally, the mRNA is released into the cytoplasm and further binds to ribosomes to initiate the translation process. Description of the Drawings

[0012] Figure 1 As shown, after adding the modified gold particles to the chloroform solution containing the ionizable lipid, the solution is divided into two layers, with the upper layer being the aqueous phase and the lower layer being the hydrophobic phase;

[0013] Figure 2 As shown is the synthesis schematic diagram of IL-Au-mRNA@LNPs;

[0014] Figure 3 As shown is the transmission electron microscope image of IL-AuNPs;

[0015] Figure 4 As shown is the transmission electron microscope image of IL-AuNPs-mRNA@LNPs;

[0016] Figure 5 As shown is the chemiluminescence intensity graph of the firefly luciferin substrate in 293 cells;

[0017] Figure 6 As shown is the in vivo imaging of mice with chemiluminescence of the firefly luciferin substrate. Detailed Embodiments

[0018] Example 1: Preparation of IL-AuNPs

[0019]

Synthesis of Gold Particles

[0020] The gold particle synthesis was carried out by the sodium citrate reduction method, which included two steps: solution preparation and particle synthesis. Solution preparation: A commercial chloroauric acid reagent was prepared into a chloroauric acid stock solution with a concentration of 200 mg / mL, and a commercial sodium citrate reagent was prepared into a sodium citrate working solution with a concentration of 0.01 g / mL. Particle synthesis: 15 μL of chloroauric acid with a stock solution concentration of 200 mg / mL was added to 30 mL of deionized water, stirred and heated to boiling, and then 900 μL of sodium citrate working solution with a concentration of 0.1 g / mL was added for reduction, and the reaction was carried out for 10 min. The obtained gold particles could obtain gold nanoparticles with relatively uniform particle sizes without further purification. Due to the evaporation of water during the reaction, the concentration of the reaction solution needed to be redetermined, and all the reaction solution was poured into a graduated cylinder to measure the volume, and then deionized water was added to make up the volume to 30 mL. After detection by transmission electron microscopy, the measured particle size of the gold particles synthesized by this method was about 13 nm.

[0021]

SH-PEG Modification

[0022] The initial ligand modification of gold particles was completed by modifying gold particles with gold-sulfur bonds. The relative molecular mass of SH-PEG was 750. 500 μL of an ethanol solution of SH-PEG with a concentration of 10 mg / mL was added to the above reaction solution, and 30 μL of a NaOH solution with a concentration of 2 M was added to adjust the pH value of the solution to 8. After 24 h, centrifugation was carried out, and after washing three times, it was resuspended with deionized water, centrifuged at 12000 g for 20 - 30 min, and the supernatant was discarded to obtain SH-PEG modified gold particles, which were reconstituted to 3 mL, and 100 μL was taken for standby.

[0023]

DLin-MC3-DMA Ligand Exchange

[0024] The Dlin-MC3-DMA ligand modification of gold particles was completed by ligand exchange. The Dlin-MC3-DMA modification of gold particles was carried out in three steps: 1. Solution preparation: 5 μL of the stock solution of 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl ester (DLin-MC3-DMA) was added to 200 μL of chloroform to form a ligand exchange solution for gold particles. At the same time, after centrifuging the gold particles, they were weighed and reconstituted with deionized water to form a SH-PEG modified gold particle solution with a concentration of 1 mg / mL. 2. Ligand exchange process: 200 μL of the ligand solution was added to a 1.5 mL centrifuge tube, and then 100 μL of the SH-PEG modified gold particle solution was added to the solution. After the solution was added, a vortex mixer was used to shake until the gold particles in water were completely exchanged into the chloroform phase. 3. Purification: The IL-AuNPs particles dispersed in chloroform were washed with ethanol and then resuspended with a 50 mM acetic acid solution with pH = 4, and centrifuged at 12000 g for 20 - 30 min. AsFigure 1 As shown, IL-AuNPs were prepared. The transmission electron microscope image of IL-AuNPs is as Figure 2 shown.

[0025] Example 2: Preparation of mRNA liposomes based on ionizable gold nanoparticles

[0026] As Figure 2 shown, the synthesis of liposomes was obtained by mixing an aqueous phase and an ethanol phase.

[0027] Aqueous phase: mRNA and the ionizable gold particles prepared in Example 1 were mixed and incubated for 20 min in an acetic acid buffer solution with a pH of 4 and a concentration of 50 mM at a mass ratio of 1:2. The mRNA carrying firefly luciferase was used as the target nucleic acid.

[0028] Ethanol phase: The ionizable lipid, co-lipid, cholesterol, and polyethylene glycol lipid were blended in the molar ratio shown in the following table to obtain the ethanol phase.

[0029]

[0030] Mixing the aqueous phase and the ethanol phase can prepare the mRNA liposome preparation IL-AuNPs@LNP based on ionizable gold nanoparticles. IL-AuNPs@LNP has three parts, and the morphology is as Figure 4 shown. From the inside to the outside, they are the IL-AuNPs core, the delivered target mRNA, and the lipid layer.

[0031] Example 3: Encapsulation efficiency and biological activity of the mRNA preparation

[0032] To verify the encapsulation efficiency and effectiveness of the IL-AuNPs prepared in Example 2, a comparative experiment was conducted on liposomes with or without IL-AuNPs. Utilizing the characteristic of the RNA dye Ribogreen to sensitively recognize RNA, the specific concentration of RNA in the solution can be distinguished. Then, by combining the property that liposomes are easily disrupted in a 1% Triton solution, the concentrations of RNA inside and outside the liposomes can be measured.

[0033] The liposomes prepared in Example 2 were diluted 100-fold in enzyme-free sterile water and 1% Triton solution respectively. The Ribogreen dye was diluted to the working concentration according to the product instructions. After adding 1 μL of the sample to be tested to 99 μL of enzyme-free sterile water and 1% Triton solution respectively, 100 μL of the Ribogreen dye at the working concentration was added, and the mixture was tested on a microplate reader. The excitation wavelength was 485 nm and the emission wavelength was 525 nm. The fluorescence intensity of the RNA dye in the enzyme-free sterile water was denoted as I1, and the fluorescence intensity of the RNA dye in the Triton solution was denoted as I2. The encapsulation efficiency was calculated using the formula: Encapsulation efficiency = (I2 - I1) / I2. The results showed that the encapsulation efficiency of the liposomes without IL-AuNPs in the aqueous phase was approximately 63.16%, while the encapsulation efficiency of the liposomes with IL-AuNPs mixed in the aqueous phase was as high as 91.58%.

[0034] The luciferase assay was performed using a firefly luciferase assay kit. The specific steps were as follows: 100 μL of cell lysis buffer was added to every 100,000 293 cells. After lysing for 10 min, the mixture was centrifuged at 4°C and 11,000 rpm for 5 min, and then 10 μL of the supernatant was taken and added to 50 μL of the firefly luciferin substrate. In the experiment, the target mRNA was the mRNA encoding firefly luciferase (Firefly-luciferase mRNA; FLuc mRNA). Firefly luciferase would react with the firefly luciferin substrate, promoting the oxidation of the firefly luciferin substrate to generate chemiluminescence, which significantly increased the fluorescence intensity of the substrate. At the same time, the amount of enzyme added was positively correlated with the fluorescence intensity of the substrate, that is, when the same amount of substrate emitted light, within a certain range within a specified time, the more enzyme there was, the higher the fluorescence intensity. In the experiment to verify the effectiveness of IL-AuNPs, the total amount of mRNA and lipids input during the synthesis of liposomes containing IL-AuNPs and liposomes without IL-AuNPs was the same to determine the role of IL-AuNPs during the synthesis process.

[0035] Figure 5 It is a chemiluminescence intensity graph of the firefly luciferin substrate. 100,000 293 cells were transfected with 250 ng of Fluc mRNA. After lysing the cells and adding an equal amount of firefly luciferin substrate, it was found that the fluorescence intensity of the firefly luciferase catalyzing the substrate produced by the cells transfected with the IL-AuNPs liposomes was higher, indicating that the cells transfected with the IL-AuNPs liposomes produced more firefly luciferase. Compared with the liposomes without IL-AuNPs, the liposomes containing IL-AuNPs had a better delivery effect.

[0036] To further verify whether the liposomes containing IL-AuNPs have biological activity in vivo, we selected 7-week-old female BALB / c as the experimental subjects for the experiment. The experimental results are as Figure 6As shown, the chemiluminescence intensity of luciferin in the legs of BALB / c mice containing IL-AuNPs liposomes was higher. This indicates that the liposomes containing IL-AuNPs also have good delivery effects at the in vivo level, and there is a positive correlation with the improvement of its encapsulation efficiency.

Claims

1. An ionizable gold nanoparticle, composed of a gold nanoparticle and an ionizable lipid, wherein the ionizable lipid forms a ligand structure with the gold atoms on the surface of the gold nanoparticle, thereby forming an ionizable lipid layer on the surface of the gold particle; Among them, The particle size of the gold nanoparticles is within 100 nm, preferably 10 - 30 nm; The ionizable lipid comprises at least three parts, namely: (a) a head with an ionizable amino group, (b) a linking group containing a -C-O-C- group between the head and the tail, and (c) one or more tails composed of hydrophobic groups.

2. The ionizable gold nanoparticles according to claim 1, wherein the linking group containing a -C-O-C- group is -(CH2) 3-6 -C(=O)-O-C- or -(CH2) 3-6 -O-C(=O)-C-.

3. The ionizable gold nanoparticle according to claim 1, wherein the tail contains a hydrophobic group with a carbon skeleton such as a straight-chain alkyl group, an olefin group or a diene group, and the number of carbon atoms is generally between 6 and 18.

4. The ionizable gold nanoparticles according to claim 1, wherein the ionizable lipid molecular structure is R-(CH2) 3-6 -C(=O)-O-CH-R 1 (R 2 ) or R-[(CH2) 3-6 -O-C(=O)-CH-R 1 (R 2 )]2, wherein R 1 , R 2 respectively represent the same or different hydrophobic groups (i.e., tails), and R represents a head with an ionizable amine group.

5. The ionizable gold nanoparticle according to claim 4, wherein the ionizable lipid has an acid dissociation constant (pKa ~ 6.5) and is positively charged in a solution with pH = 4.

6. The ionizable gold nanoparticle according to claim 4, wherein the ionizable lipid is selected from 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester (Dlin-MC3-DMA), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid 1-octylnonyl ester, heptadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((decoxy)hexyl)amino)octanoate) (SM102), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) or ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

7. An mRNA liposome, having a core-shell structure, comprising, from the inside to the outside, the ionizable gold nanoparticle (IL-AuNPs) according to claim 1, the target mRNA, and the outermost lipid layer, wherein the target mRNA and the IL-AuNPs are encapsulated by the lipid layer in the form of a complex.

8. A method for preparing the mRNA liposome according to claim 7, comprising the following steps: (1) co-incubating an aqueous solution of the target mRNA and the ionizable gold nanoparticle to obtain an aqueous phase; (2) mixing the aqueous phase and an ethanol phase to prepare the mRNA liposome.

9. The method for preparing the mRNA liposome according to claim 8, wherein in step (1), the mass ratio of the mRNA to the ionizable gold nanoparticle is controlled within the range of 0.2 to 0.6.

Images