A method for enhancing multi-nuclear magnetic resonance signals based on lipid nanoparticle loaded with hole-phanes and application thereof

Abstract

The application belongs to the technical field of biological medicine and nanomaterials, and particularly relates to a method for enhancing multi-nuclear magnetic resonance signals based on lipid nanoparticle loaded with hole banana. 129 Xe molecular imaging and related biomedical applications provide a new technical path.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedicine and nanomaterials, specifically relating to a highly efficient delivery system based on lipid nanoparticles (LNPs) loaded with monocarboxylic acid benzo[a]-A and its application in hyperpolarization. 129 A method for signal enhancement in Xe nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI). Through the synergistic effect of the hydrophilic outer layer and hydrophobic core of the ligand (LNP), the dispersion stability of LNP in aqueous phase and its binding efficiency with xenon are significantly improved, laying the foundation for ultrasensitive molecular imaging applications. Background Technology

[0002] Cryptophanes are supramolecular compounds with a rigid, cage-like structure. Their internal cavities can selectively trap small gas molecules, particularly xenon (Xe). Hyperpolarization... 129 After Xe is introduced into the cavity, the complex contains 129 Xe produces unique chemical shift peaks under magnetic resonance conditions, thus possessing extremely high sensitivity and specificity, making it a preferred probe in the field of molecular imaging and widely used in early tumor diagnosis, inflammation detection, and research on nervous system diseases.

[0003] Despite the cave dwellings and 129 While Xe exhibits excellent binding performance in in vitro solutions, its application in in vivo imaging faces multiple technical bottlenecks:

[0004] 1. Extremely low solubility: The saturated solubility of vacuole molecules in aqueous media is usually less than 1 μM, making it difficult to achieve effective delivery.

[0005] 2. Aggregation and clearance in blood: Free adenosine monophosphate readily aggregates and becomes inactive in plasma, and is rapidly cleared by the reticuloendothelial system (RES), severely limiting its bioavailability;

[0006] 3. Limitations of the delivery system: Existing methods mostly improve water solubility and delivery efficiency through chemical modification (such as introducing sulfonic acid groups or PEG chains) or polymer nanocarriers (such as PLGA, dendritic polymers), but all of them have significant drawbacks:

[0007] 4. Chemical modification steps are complex, often disrupting the cage-like structure and weakening its interaction with... 129 Xe's binding ability;

[0008] 5. Due to the interfacial barrier between the hydrophobic core and the hydrophilic shell inside the polymer carrier, only the surface cavitation layer can participate. 129Xe exchange means that many molecules deep within the core cannot contribute NMR signals. Summary of the Invention

[0009] To address the bottlenecks in the prior art, such as low delivery efficiency, aggregation and inactivation, and rapid clearance of crypto-A in aqueous phases, this invention proposes a highly efficient delivery system (Crypto-LNPs) based on lipid nanoparticles loaded with monocarboxylated crypto-A, significantly improving its solubility and dispersion stability in aqueous phases while maintaining its compatibility with... 129 Xe binding ability is suitable for high sensitivity 129 Xe NMR molecular imaging.

[0010] A multinuclear magnetic resonance signal enhancement method based on lipid nanoparticles loaded with silanium, wherein the method utilizes lipid nanoparticles loaded with silanium, and the specific preparation steps of the lipid nanoparticles loaded with silanium are as follows:

[0011] 1. Preparation of Pre-solution of Adenophora-A

[0012] A pre-solution of cryptanalyte-A was prepared by dissolving monocarboxylated cryptanalyte-A in dimethyl sulfoxide.

[0013] 2. Preparation of lipid organic and aqueous phases

[0014] (1) Stock solutions of ionizable lipids, auxiliary phospholipids and PEG lipids were prepared by anhydrous ethanol and mixed as needed to form a lipid organic phase;

[0015] (2) Use citrate buffer as the aqueous phase;

[0016] 3. Crypto-LNPs Preparation

[0017] (1) Mix the pre-solution of succinate-A (preferably with a concentration of 10–50 mg / mL) with the lipid organic phase to form an organic phase containing succinate;

[0018] (2) The organic phase containing cavitary elements is rapidly mixed with the aqueous phase using microfluidic technology to form a preliminary nanoparticle suspension;

[0019] (3) Remove organic solvents and free components from the initial nanoparticle suspension (preferably by PD-10 desalting chromatography column, ultrafiltration, centrifugation, etc.) to obtain stable lipid nanoparticles loaded with cytoplasm.

[0020] The lipid nanoparticles loaded with kaempferol can interact with hyperpolarization. 129 Xe fully exchanges, enhancing 129 Xe-interaction sites are used to enhance multinuclear magnetic resonance signals.

[0021] The structural formula of the monocarboxylated cryptanalyte-A is as follows: Me stands for methyl group.

[0022] Furthermore, the ionizable lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate)SM102; and / or

[0023] The auxiliary phospholipid is distearate phosphatidylcholine (DSPC); and / or

[0024] The PEG lipid is 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol 2000 DMG-PEG2000.

[0025] Furthermore, the molar ratio of SM102, DSPC, and DMG-PEG2000 is 50:10:1.5.

[0026] Furthermore, the control parameters of the microfluidic technology are as follows: SHM chip is selected, the volume ratio of organic phase containing silage to aqueous phase is 1:3, the flow rate is 0.1-12 mL / min (preferably 4 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.

[0027] In this invention, nucleic acids (DNA, mRNA, siRNA, or miRNA) are dissolved in citrate buffer as the aqueous phase and the above operations are performed. The nucleic acid encapsulation rate is 91.5%, proving that nucleic acid drugs can be encapsulated and delivered.

[0028] This invention also provides the above method in high sensitivity 129 Applications of Xe NMR molecular imaging.

[0029] Compared with the prior art, this application has the following advantages and beneficial effects:

[0030] (1) Significantly improves accessibility to aqueous phase: The flexible lipid bilayer gently encapsulates the cryptanalyte, avoiding damage to the molecular structure caused by chemical modification; by controlling the lipid ratio and PEG density, multiple inter- and inter-regional micro-regions are formed on the core surface, so that the cryptanalyte is both uniformly dispersed and close to the aqueous phase interface; the hydrophilic outer layer and hydrophobic core of LNPs work together to achieve efficient encapsulation and stable dispersion of cryptanalyte-A in the aqueous phase.

[0031] (2) Enhance 129 Xe NMR signal: hyperpolarization 129 Xe can rapidly penetrate the lipid membrane to the micro-region boundary, where it fully exchanges with pituitary cells distributed near the surface, significantly enhancing observable microenvironments. 129Xe–caecin interaction sites. In vitro experiments showed that caecin-A in Crypto-LNPs exhibited a significant characteristic peak (in-cage xenon signal) at 75.9 ppm, which was completely separated from the dissolved Xe signal (195 ppm).

[0032] (3) Excellent biocompatibility and in vivo stability: PEG-modified lipid chains endow particles with long-circulation properties, reduce RES clearance, and prolong the imaging window period;

[0033] (4) Functional expansion potential: The PEG end can be attached with active functional groups (such as maleimide, azide, etc.), which facilitates subsequent coupling with targeted ligands and has the potential to be transformed into targeted imaging and drug delivery. It can also encapsulate nucleic acids, providing a new approach for the integration of imaging and gene therapy.

[0034] In summary, this invention successfully constructs a highly efficient and scalable supramolecular compound delivery platform by encapsulating a single carboxyl cryptanalyte-A within LNPs, providing a new platform for the delivery of supramolecular compounds. 129 Xe molecular imaging and related biomedical applications offer a new technological pathway. Attached Figure Description

[0035] Figure 1 This is a particle size distribution diagram of CLNPs loaded with different contents in Example 1.

[0036] Figure 2 The image shows cryo-electron microscopy (cryo-TEM) images of CLNPs loaded with different amounts in Example 1.

[0037] Figure 3 The Xe NMR spectrum of the unloaded cryptabolic lipid nanoparticles (blank LNPs) in Example 1 is shown.

[0038] Figure 4 The image shows the nuclear magnetic resonance Xe spectrum of CLNPs (the concentration of cytosine in the cytosine pre-solution was 50 mg / mL) in Example 1.

[0039] Figure 5 The image shows the Xe NMR spectrum of mRNA-CLNPs (the concentration of clava in the clava pre-solution was 50 mg / mL) in Example 1. Detailed Implementation

[0040] To facilitate understanding and implementation of the present invention by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific embodiments. However, it should be understood that these embodiments are only for illustrating the present invention and are not intended to limit the scope of protection of the present invention.

[0041] Unless otherwise stated, all raw materials, solvents, and reagents used in this invention are commercially available analytical grade or pharmaceutical grade reagents. The main reagents and materials used in the following examples are described below: The sample solution was placed in a 10 mm NMR tube (Wilmad, USA) and subjected to NMR using a 400 MHz NMR spectrometer (Bruker, Germany). 129 Xe spectrum detection. Monocarboxylated bismuth-A was purchased from Zhengzhou Hongshang Technology Co., Ltd.; heptadecano-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate)SM102 was purchased from Xiamen Sinobond Biotechnology Co., Ltd.; distearate phosphatidylcholine DSPC and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol DMG-PEG2000 were purchased from Ruixi Biotechnology; cholesterol (Chol) was purchased from Sigma; and the PD-10 desalting chromatography column (catalog number 17085101) was purchased from GE. 0.5 M citrate buffer (pH = 3) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and RNase-free ddH2O was purchased from Sangon Biotech (Shanghai) Co., Ltd. Fluc mRNA was purchased from Unimicron Biotechnology Co., Ltd. (catalog number abs60178, concentration used: 1 μg / μL), and the RediPlate™ 96 RiboGreen® RNA Quantitation Kit (R-32700) was purchased from Invitrogen.

[0042] Preparation of 50 mM citrate buffer: Add 300 μL of 0.5 M citrate buffer (pH = 3) to 2.7 mL of RNase-free ddH2O, mix well to obtain 50 mM citrate buffer.

[0043] The structural formula of the monocarboxylated cryptanalyte-A used is as follows: Me stands for methyl group.

[0044] Example 1: Preparation of lipid nanoparticles

[0045] The specific steps for preparing blank LNPs (control group) without cysteine ​​loading, lipid nanoparticles CLNPs (experimental group) loaded with cysteine, and lipid nanoparticles mRNA-CLNPs loaded with cysteine ​​and mRNA (encapsulation efficiency detection group) are as follows:

[0046] (1) Prepare ethanol solutions of SM-102, DSPC and Chol at 10 mg / mL and ethanol solutions of DMG-PEG2000 at 1 mg / mL respectively.

[0047] (2) Take 150 μL of SM-102, 33.4 μL of DSPC, 62.9 μL of Chol and 159 μL of DMG-PEG2000 in ethanol solution respectively and mix them to complete the preparation of lipid organic phase solution.

[0048] (3) Take 1215 μL of citrate buffer (50 mM) as the aqueous phase solution.

[0049] (4) Blank LNPs of the control group: The lipid organic phase solution (150 μL SM-102, 33.4 μL DSPC, 62.9 μL Chol and 159 μL DMG-PEG2000 in ethanol solution) of (2) and the aqueous phase solution of (3) were mixed evenly by NanoAssemblr microfluidic instrument, separated by a PD-10 gel filter column that was pre-buffered and balanced to remove free small molecule impurities, and then the lipid nanoparticles were obtained. They were then placed in a 100 kDa ultrafiltration tube, centrifuged at 4000 rpm and concentrated to 2 mL to obtain a blank LNPs dispersion without cytoplasm loading.

[0050] (5) CLNPs of the experimental group: Monocarboxylated cryptanalyte-A was dissolved in DMSO to prepare a 63 μL cryptanalyte pre-solution (50 mg / mL). 150 μL of SM-102, 33.4 μL of DSPC, 62.9 μL of cryptanalyte pre-solution and 159 μL of DMG-PEG2000 were mixed to form an organic phase solution. The organic phase solution was then mixed with the aqueous phase solution of (3) by a microfluidic mixer to form a nanoparticle suspension. The nanoparticles were separated by a PD-10 gel filter column that was pre-equilibrated with buffer to remove free cryptanalyte and small molecule impurities, and then the dispersed and stable lipid nanoparticles loaded with cryptanalyte were obtained. The nanoparticles were then centrifuged in a 100 kDa ultrafiltration tube at 4000 rpm and concentrated to 2 mL to obtain a cryptanalyte-loaded CLNPs dispersion.

[0051] (6) Encapsulation efficiency detection group mRNA-CLNPs: Replace the Fluc mRNA solution in (5) with the aqueous solution (150 μF Fluc mRNA was dissolved in 1065 μL 50 mM citrate buffer), and the rest of the steps are the same to obtain mRNA-CLNPs loaded with cytosine and mRNA.

[0052] (7) Prepare pre-solutions of 10 mg / mL, 25 mg / mL and 50 mg / mL respectively to obtain CLNPs dispersions loaded with different contents of cyanidins. (5)

[0053] In steps (4)-(5), the specific microfluidic parameters are as follows: using an SHM chip, the flow rate is 4 mL / min, the initial waste liquid is set to 0.1 mL, and the subsequent waste liquid is set to 0.1 mL.

[0054] Example 2 Characterization of lipid nanoparticles

[0055] DLS detection: Take 100 μL of CLNPs dispersions with different pore contents, and perform dynamic light scattering (DLS) detection for particle size analysis at 25℃. Figure 1 The PDI is around 0.2;

[0056] Cryo-TEM observation: 1 μL of CLNP dispersions with different cavitation contents was dropped onto a carbon film copper grid, rapidly frozen in liquid nitrogen, and analyzed by TEM at -196℃. See [image / description]. Figure 2 Spherical particles are visible;

[0057] Encapsulation efficiency assay: 5 μL of mRNA-CLNPs sample solution was taken and measured using the RediPlate™ 96 RiboGreen® RNA Quantitation Kit. The mRNA encapsulation efficiency was 91.5%.

[0058] Example 3 129 Xe NMR signal detection

[0059] Nuclear magnetic resonance of lipid nanoparticle dispersion 129 Xe spectrum detection, thereby obtaining its nuclear magnetic resonance Xe spectrum ( Figure 5 ).

[0060] Nuclear magnetic resonance of lipid nanoparticle dispersion 129 The Xe spectrum acquisition parameters are as follows: ZG pulse sequence was used; the test temperature was 299 K; the pulse width was 31.8 μs; the D1 was 0.1 s; the spectral width was 403.3 ppm; the RF center frequency was 110.7 Hz; the LB was 20 Hz; the number of acquisition points was 65536; the number of sampling accumulations was 32; the number of blank scans was 0; and the gain was 15.56. The NMR spectrometer used was at a frequency of 400 MHz to evaluate the signal enhancement factor.

[0061] Results Analysis

[0062] Nuclear magnetic resonance Xe spectroscopy results showed that, excluding the dissolved state 129 Xe NMR signal (195 ppm), blank LNPs (without styrax loading) in the control group showed no other specificity. 129The experimental group's CLNPs dispersion showed a significant characteristic peak at 75.9 ppm, completely separated from the dissolved Xe signal (195 ppm). Similarly, the mRNA-CLNPs dispersion containing mRNA also showed a significant characteristic peak at 75.9 ppm, completely separated from the dissolved Xe signal (195 ppm). These results indicate that LNPs themselves do not possess the ability to adsorb Xe and cannot generate in-cage Xe. 129 Xe NMR signal; using LNPs to load cavitary molecules into a hydrophobic core, the cavitary molecules can be effectively dispersed in the aqueous phase. Furthermore, the cavitary molecules loaded with LNPs retain the integrity of their cage-like structure. After binding with xenon, they alter the electron cloud distribution outside the xenon nucleus through supramolecular interactions (including van der Waals forces and size-matching effects), inducing a chemical shift. 129 The Xe NMR spectrum shows distinguishable characteristic signals, and the inclusion of nucleic acid does not affect these distinguishable characteristic signals.

Claims

1. A method for preparing lipid nanoparticles loaded with silage, comprising the following steps: (1) Preparation of pre-solution of cypermethrin-A A pre-solution of cryptanalyte-A was prepared by dissolving monocarboxylated cryptanalyte-A in dimethyl sulfoxide. (2) Preparation of lipid organic phase and aqueous phase (2-1) Stock solutions of ionizable lipids, auxiliary phospholipids and PEG lipids were prepared by anhydrous ethanol and mixed to form a lipid organic phase; (2-2) Use citrate buffer as the aqueous phase; (3) Preparation of Crypto-LNPs (3-1) Mix the pre-solution of cytosine-A with the lipid organic phase to form an organic phase containing cytosine; (3-2) The organic phase containing cryptanalytes is rapidly mixed with the aqueous phase using microfluidic technology to form a preliminary nanoparticle suspension; the control parameters of the microfluidic technology are: SHM chip is selected, the volume ratio of the organic phase containing cryptanalytes and lipids to the aqueous phase containing citrate buffer is 1:3, and the flow rate is 0.1-12 mL / min. (3-3) Remove organic solvents and free components from the initial nanoparticle suspension to obtain stable lipid nanoparticles loaded with cytoplasm; The lipid nanoparticles loaded with kaempferol can interact with hyperpolarization. 129 Xe fully exchanges, enhancing 129 Xe-interaction sites are used to enhance multinuclear magnetic resonance signals. The structural formula of the monocarboxylated cryptanalyte-A is as follows: Me stands for methyl group; The ionizable lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate) (SM102); the cofactor phospholipid is distearate phosphatidylcholine (DSPC); the PEG lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000); the molar ratio of the ionizable lipid, cofactor phospholipid, and PEG lipid is 50:10:1.

5.

2. The method for preparing lipid nanoparticles loaded with cytoplasm according to claim 1, characterized in that, The concentration of the cypermethrin-A pre-solution is 10-50 mg / mL.

3. The lipid nanoparticles loaded with cryptanalyte prepared by the method of any one of claims 1-2 are used to prepare highly sensitive cryptanalyte nanoparticles. 129 Applications of probes in Xe NMR molecular imaging.

Images