Synthesis method and application of < 31 > P magnetic resonance molecular probe based on lipid nanoparticles

Abstract

The invention relates to the field of nano biotechnology and medical detection, and particularly discloses a synthesis method and application of a lipid nanoparticle (LNP) mRNA molecular probe based on a 31P magnetic resonance spectrum (MRS). According to the probe, mRNA is entrapped by ionizable lipid, DSPC, cholesterol and DMG-PEG2000, the ratio and parameters are optimized by adopting a microfluidic technology, and LNP with uniform particle size and stable structure is prepared. According to the method, an mRNA phosphate skeleton is used as a 31P MRS signal source, Triton X-100 and R Nase A are used for processing and simulating release and splitting decomposition states of mRNA-LNP in vivo, and a 400-600MHz nuclear magnetic resonance spectrometer is used for detecting a phosphorus spectrum signal. Based on 31P signal intensity and chemical shift changes in different states, visual identification of the in-vivo metabolic state of mRNA-LNP is realized. The probe does not need additional marking, is simple to operate and good in repeatability, and has relatively high detection sensitivity and biological adaptability.

Description

Technical Field

[0001] This invention relates to the fields of nanobiotechnology and medical detection, specifically to a method for encapsulating mRNA in lipid nanoparticles. 31 A method for synthesizing the P MRS molecular probe. This probe simulates different metabolic states of LNP in vivo and utilizes the changes in phosphorus signal chemical shifts generated by LNP in the solution environment to reflect changes in phosphorus signals generated by the mRNA phosphate backbone, achieving highly sensitive dynamic detection of LNP behavior in vivo. It is suitable for the assessment and imaging analysis of metabolic processes in mRNA delivery systems. Background Technology

[0002] LNPs, as core vectors for mRNA delivery, have been widely used in vaccine development and gene therapy, demonstrating great potential in clinical translation. However, the lack of precise, dynamic, and non-invasive monitoring methods for the in vivo metabolic behavior of mRNA-LNPs, including mRNA loading stability, release efficiency, and degradation processes, has become a key technical challenge limiting their further application.

[0003] 31 PMRS technology, as a non-invasive technique for detecting phosphorus metabolites in vivo, possesses high specificity and spatial resolution, providing a new approach for in vivo tracking of phosphorus metabolites (LNPs). However, existing technologies still have significant shortcomings in the following aspects: 1. Traditional LNPs are mainly used as drug or gene delivery vectors and have not yet been developed into... 31 P MRS molecular probe; 2. Each nucleotide in the natural phosphodiester backbone of mRNA contains one phosphorus atom, and this endogenous phosphorus signal has not been fully utilized for MRS imaging. 3. Traditional LNP preparation processes (such as ethanol injection method) are prone to particle size inhomogeneity (PDI>0.2) and low loading rate (<90%), which makes it difficult to meet the requirements of MRS signal for particle stability and uniformity; 4. LNPs need to undergo stages such as complete encapsulation, membrane cleavage to release mRNA, and mRNA degradation into nucleotides in vivo. Existing technologies make it difficult to distinguish these states using a single detection method. 5. Conventional nucleic acid testing often relies on fluorescent labeling or invasive sampling, which can easily interfere with the natural behavior of LNPs and makes dynamic monitoring difficult.

[0004] Therefore, there is an urgent need to develop a detection strategy based on changes in phosphorus signaling caused by mRNA, which can dynamically distinguish the metabolic stages of LNP and does not depend on exogenous markers. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention optimizes the LNP preparation process to achieve high uniformity and high loading rate, and combines...31 P MRS technology is designed to simulate the detection process of LNP metabolic state. Based on the changes in characteristic P spectrum, it can realize dynamic, non-invasive and highly sensitive identification of LNP metabolic state in vivo, thereby breaking through the limitations of existing technologies in terms of label dependence, state resolution and signal stability.

[0006] This invention aims to provide a method that can be used 31 The mRNA-LNP molecular probes detected by P MRS can accurately distinguish different metabolic states of LNP in vivo, solving the problems of difficulty in monitoring LNP behavior in vivo, weak signal, and the need for additional labeling in existing technologies.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A lipid nanoparticle-based 31 The synthesis method of p-magnetic resonance molecular probes is as follows: (1) Stock solutions of ionized lipids, auxiliary phospholipids, cholesterol and PEG lipids were prepared by anhydrous ethanol and mixed to form an ethanol phase; (2) Dissolve the mRNA in citrate buffer and mix to form an aqueous phase; (3) Using microfluidic technology, the above ethanol and aqueous phases were mixed, and the LNP sample was collected and then purified to obtain lipid nanoparticle-based products. 31 P-magnetic resonance molecular probe.

[0008] Furthermore, the ionizable lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate)SM102; and / or The auxiliary phospholipid is distearate phosphatidylcholine (DSPC); and / or The PEG lipid is 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol 2000 DMG-PEG2000.

[0009] Furthermore, the molar ratio of SM102, DSPC, cholesterol, and DMG-PEG2000 is 50:10:38.5:1.5.

[0010] Furthermore, the mRNA includes, but is not limited to, encoding luciferase Fluc mRNA, red fluorescent protein mCherry mRNA, and enhanced green fluorescent protein mEGFP mRNA.

[0011] Furthermore, the control parameters of the microfluidic technology are as follows: SHM chip is selected, the volume ratio of lipid ethanol solution to mRNA buffer is 1:3, the flow rate is 0.1-12 mL / min, wherein the volume of the initial waste liquid is 0.1-0.2 mL and the volume of the subsequent waste liquid is 0.1-0.2 mL.

[0012] The nanoparticles obtained by the above synthesis method have a particle size of 130-160 nm, a polydispersity index (PDI) of less than 0.2, and an mRNA loading rate of not less than 90%. Characteristic phosphorus spectra can be obtained by magnetic resonance detection.

[0013] The present invention also provides the application of the molecular probe obtained by the synthesis method in the detection of LNP metabolic state, wherein the application is as follows: a characteristic phosphorus spectrum is obtained by nuclear magnetic resonance phosphorus spectroscopy, and the metabolic state is determined by the change in the peak position in the spectrum.

[0014] Furthermore, during the phosphorus spectroscopy detection using nuclear magnetic resonance, a 400-600 MHz nuclear magnetic resonance spectrometer (preferably 400 MHz) is employed, with the following specific parameters: zgig pulse sequence, test temperature 299 K, pulse width 30 μs, D1=1s, spectral width 81521.7 Hz, radio frequency center frequency 161.9 Hz, LB=100 Hz, number of acquisition points 65536-32768, number of sampling accumulations 8192-32768, number of empty scans 0-3, and gain 2050.

[0015] In this invention, the mRNA phosphate backbone is used as 31 The P MRS signal source was used to simulate the release and cleavage states of mRNA-LNP in vivo by processing with Triton X-100 and RNase A. Phosphorus spectrum signals were then detected using NMR spectra, and the results were analyzed based on the different states. 31 The changes in P signal intensity and chemical shift (such as the characteristic peak of LNP in the intact encapsulated state at -1.33 ppm; the characteristic peak of LNP cleavage and mRNA free state at -1.38 ppm and a secondary peak at -3.88 ppm; and the characteristic peak of completely degraded mRNA at -1.41 ppm and multiple secondary peaks at 0.59 ppm, -3.56 ppm, and -3.77 ppm) enable the visual identification of the metabolic state of mRNA-LNP in vivo.

[0016] Compared with the prior art, the technical solution of this application has the following advantages and beneficial effects: This invention utilizes phosphorus NMR spectroscopy to achieve dynamic visualization and differentiation of different metabolic stages of nucleic acid LNPs, based on the NMR spectra. 31 The different elution positions of P reflect the nucleic acid metabolism process in LNP. By optimizing experimental parameters, this detection method is characterized by its simplicity, excellent repeatability, and high accuracy. It also has the advantage of being non-destructive, as it does not rely on exogenous labels. This effectively avoids the interference that exogenous labels may cause and the potential impact on the inherent properties of the sample, providing reliable technical support for the real-time monitoring of the release behavior of nucleic acid drugs in vivo. Attached Figure Description

[0017] Figure 1 shows the particle size distribution of the LNP sample prepared in Example 1, with a main peak at 153 nm and PDI = 0.13. Figure 2 is a cryo-transmission electron microscope image of the LNP sample prepared in Example 1 (scale bar: 100 nm), showing that the prepared LNP sample has a spherical structure and uniform particle size. Figure 3 shows the different LNP treatment groups (LNP, LNP + Triton, and LNP + Triton + RNaseA) in Example 3. 31 P nuclear magnetic resonance spectrum.

[0018] Figure 4 For example 4, LNP was incubated in cells for different times (2 h, 24 h). 31 P nuclear magnetic resonance spectrum. Detailed Implementation

[0019] The technical solution of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. The present invention is not limited to the specific forms listed in the following embodiments, unless otherwise stated.

[0020] The main reagents and materials used in the examples are described below: Heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate)SM102 was purchased from Xiamen Sinobond. Distearate phosphatidylcholine DPSC and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 DMG-PEG2000 were purchased from Ruixi Biotechnology. Cholesterol was purchased from Sigma. PD-10 desalting chromatography column (catalog number 17085101) was purchased from GE. Fluc mRNA was purchased from Unimicron Biotechnology Co., Ltd. (catalog number abs60178, concentration used 1 μg / μL). The RediPlate™ 96 RiboGreen® RNA Quantitation Kit (R-32700) was purchased from Invitrogen. 4T1 cells were purchased from Shanghai Cell Bank.

[0021] Phosphorus NMR spectroscopy was performed using a 400 MHz NMR spectrometer (Bruker, Switzerland) and a 10 mm NMR tube (purchased from Wilmad, USA).

[0022] Preparation of 50 mM citrate buffer: Add 300 μL of pH = 3, 0.5 M citrate buffer to 2.7 mL of RNase-free ddH2O and mix well to obtain 50 mM citrate buffer. The pH = 3, 0.5 M citrate buffer was purchased from Shanghai Maclean Biotechnology Co., Ltd. The RNase-free ddH2O was purchased from Sangon Biotech Co., Ltd.

[0023] Example 1: Preparation of LNP-mRNA molecular probes (1) Preparation of lipid stock solution: Weigh 10 mg each of SM102, DSPC, cholesterol, and DMG-PEG2000, dissolve them in anhydrous ethanol, and incubate at 60°C until dissolved to prepare a 10 mg / mL stock solution. Separately, dissolve 100 μL of DMG-PEG2000 (10 mg / mL) in 900 μL of anhydrous ethanol to prepare a 1 mg / mL stock solution.

[0024] (2) Lipid component mixture (ethanol phase) Take 150 μL of SM102 stock solution (10 mg / mL), 33.4 μL of DSPC stock solution (10 mg / mL), 62.9 μL of cholesterol stock solution (10 mg / mL), and 159 μL of DMG-PEG2000 stock solution (1 mg / mL) and mix them to form the ethanol phase.

[0025] (3) mRNA solution (aqueous phase) Dissolve 150 μL of Fluc mRNA (150 μg) in 1065.8 μL of 50 mM citrate buffer to form an aqueous phase.

[0026] (4) Microfluidic assembly LNP synthesis was performed online using the above aqueous and ethanol phases: the ethanol and aqueous phases were mixed using a NanoAssemblr microfluidic instrument. The specific microfluidic parameters were: using an SHM chip, an ethanol-to-aqueous phase volume ratio of 1:3, a flow rate of 4 mL / min, and setting the initial waste liquid at 0.1 mL and the subsequent waste liquid at 0.1 mL.

[0027] (5) Purification via PD-10 column The LNP samples collected by microfluidics were purified using a PD-10 column to remove unreacted lipids and citric acid.

[0028] (6) LNP concentration and purification The sample obtained after column purification was placed in a 100 kDa ultrafiltration tube, centrifuged at 4000 rpm, and concentrated to a final volume of 2 mL to obtain the LNP sample solution.

[0029] Example 2: Characterization of LNP-mRNA probes DLS detection: Take 100 μL of LNP sample solution and detect at 25℃. See [the table below]. Figure 1 Particle size 153.8 nm, PDI = 0.13; Cryo-TEM observation: 1 μL of LNP sample solution was dropped onto a carbon film copper grid, rapidly frozen in liquid nitrogen, and its TEM was detected at -196℃. See [link to Cryo-TEM observation]. Figure 2 The visible particles are spherical, about 150 nm in diameter, with amorphous mRNA distribution inside.

[0030] Encapsulation efficiency assay: 5 μL of LNP sample solution was taken and measured using the RediPlate™ 96 RiboGreen® RNA Quantitation Kit. The encapsulation efficiency was 95.39%.

[0031] Example 3: 31 P MRS detection Instrument parameters A Bruker 400 MHz NMR spectrometer was used, employing a zgig pulse sequence. The test temperature was 299 K, the pulse width was 30 μs, D1 was 1 s, the spectral width was 81521.7 Hz, the RF center frequency was 161.9 Hz, LB=100 Hz, the number of sampling points was 65536, the number of sampling accumulations was 8192, the number of empty scans was 3, and the gain was 2050.

[0032] Testing process (1) Take 2 mL of LNP sample solution to simulate the state of fully loaded mRNA and detect it. 31 P-spectrum (e.g.) Figure 3 mFluc@LNP), with the main peak located at -1.33 ppm; (2) Add 100 μL of 5% Triton X-100 to the above sample, shake well to simulate membrane rupture and release, and detect the reaction. 31 P-spectrum (e.g.) Figure 3 mFluc@LNP+Trition), with the main peak at -1.38 ppm and a secondary peak at -3.88 ppm; (3) Add 5 μL of 100 mg / mL RNase A, shake well to simulate complete degradation to nucleotide state, and then detect. 31 P-spectrum (e.g.) Figure 3 mFluc@LNP+Trition+R NaseA), with the main peak at -1.41 ppm and secondary peaks at 0.59 ppm, -3.56 ppm and -3.77 ppm.

[0033] Under identical detection conditions, the phosphorus spectrum of the LNP group showed only a single peak at -1.33 ppm. After adding Triton X-100 to cleave LNPs, the main peak shifted to -1.38 ppm, and a secondary peak at -3.88 ppm was detected. Since Triton X-100 does not contain phosphorus, this demonstrates that the chemical shift change originated from the single variable, LNP cleavage. Similarly, adding R NaseA to cleave long mRNA chains caused corresponding chemical shift changes. Changes in chemical shifts in the phosphorus spectrum can dynamically reflect nucleic acid metabolism processes.

[0034] Example 4: Cell incubation and 31 P MRS detection 100 μL of LNP sample solution (cell concentration approximately 10*10) was added to each of the two groups of 4T1 cells. 6 Cells were incubated at 37°C and 5% CO2 for 2 h and 24 h, respectively. After centrifugation at 1500 rpm for 5 min, the cells were resuspended in 1x PBS to a final volume of 2 mL, bringing the cell concentration to approximately 1×10⁻⁶ cells / ml. 7 / ml, to obtain a cell suspension loaded with LNP for use 31 P MRS detection; Instrument parameters A Bruker 400 MHz NMR spectrometer was used, employing a zgig pulse sequence. The test temperature was 299 K, the pulse width was 30 μs, D1 was 1 s, the spectral width was 81521.7 Hz, the RF center frequency was 161.9 Hz, LB=100 Hz, the number of sampling points was 65536, the number of sampling accumulations was 8192, the number of empty scans was 3, and the gain was 2050.

[0035] Testing process (1) Take 2 mL of the 2 h LNP-incubated cell suspension test solution and test. 31 The P spectrum shows the main peak at -2.612 ppm. (See...) Figure 4 Mid-red spectrum; (2) Take 2 mL of the 24 h LNP-incubated cell suspension test solution and test. 31 The P spectrum shows the main peak at -0.971 ppm. Figure 4 Mid-blue spectrum; Results analysis: Using the mRNA phosphate backbone as... 31A P MRS signal source was used to simulate the release and cleavage states of mRNA-LNP in vivo by treatment with Triton X-100 and R Nase A, and the phosphorus spectrum signal was detected using a 400MHz NMR spectrometer. The characteristic peak of the intact LNP sample was -1.33 ppm; after Triton X-100 cleavage, the characteristic peak was -1.38 ppm with a secondary peak of -3.88 ppm; after R Nase A degradation, the characteristic peak was -1.41 ppm with multiple secondary peaks of 0.59 ppm, -3.56 ppm, and -3.77 ppm, indicating that the chemical environment of phosphorus atoms in the phospholipids of mRNA-LNP, the phosphate groups in the nucleic acid, and the phosphorus atoms in the degraded phosphate group are not the same. Furthermore, the phosphorus spectrum signal was detected using a 400MHz NMR spectrometer after incubation of mRNA-LNP in cells for different times. The characteristic peak of mRNA-LNP was -2.612 ppm after incubation in cells for 2 h; the characteristic peak was -0.971 ppm after incubation in cells for 24 h. This study investigated the effects of different conditions on LNP solution and incubation times in cells. 31 Changes in P signal intensity and chemical shift enable the visual identification of the metabolic state of mRNA-LNP in vivo.

Claims

1. A lipid nanoparticle-based 31 The synthesis method of p-magnetic resonance molecular probes is as follows: (1) Stock solutions of ionized lipids, auxiliary phospholipids, cholesterol and PEG lipids were prepared by anhydrous ethanol and mixed to form an ethanol phase; (2) Dissolve the mRNA in citrate buffer and mix to form an aqueous phase; (3) Using microfluidic technology, the above ethanol and aqueous phases were mixed, and the LNP sample was collected and then purified to obtain lipid nanoparticle-based products. 31 P-magnetic resonance molecular probe.

2. The synthesis method according to claim 1, characterized in that, The ionizable lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate)SM102; and / or The auxiliary phospholipid is distearate phosphatidylcholine (DSPC); and / or The PEG lipid is 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol 2000 DMG-PEG2000.

3. The synthesis method according to claim 2, characterized in that, The molar ratio of SM102, DSPC, cholesterol, and DMG-PEG2000 is 50:10:38.5:1.

5.

4. The synthesis method according to claim 1, characterized in that, The mRNA is any one of the following: luciferase Fluc mRNA, red fluorescent protein mCherry mRNA, and enhanced green fluorescent protein mEGFP mRNA.

5. The synthesis method according to claim 1, characterized in that, The control parameters of the microfluidic technology are as follows: SHM chip is selected, the volume ratio of ethanol phase to water phase is 1:3, and the flow rate is 0.1-12 mL / min.

6. The application of the molecular probe obtained by the synthesis method according to any one of claims 1-5 in the detection of LNP metabolic state, wherein the application is: obtaining a characteristic phosphorus spectrum by nuclear magnetic resonance phosphorus spectroscopy, and judging its metabolic state by the change in the peak position in the spectrum; Its phosphate diester skeleton as 31 The signal source for P MRS; The nanoparticles have a particle size of 130-160 nm, a polydispersity index (PDI) of less than 0.2, and an mRNA loading rate of not less than 90%. Characteristic phosphorus spectra can be obtained by magnetic resonance detection.

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