A method for the determination of the encapsulation efficiency of fluorine-containing nanoparticles by multinuclear magnetic resonance

Abstract

The present application relates to the technical field of analytical detection, and specifically provides a method for measuring the encapsulation efficiency of fluorine-containing nanoparticles by multi-nuclear magnetic resonance, comprising the following steps: S1, dissolving fluorine-containing lipids in a fluorine-free solvent as a control, adding quantifiable fluorine-containing substances as an internal standard, then performing nuclear magnetic resonance fluorine spectrum detection to determine the peak shape and peak position of the fluorine-containing lipids; S2, combining nucleic acids with the fluorine-containing lipids to prepare fluorine-containing lipid nanoparticles, dissolving the nanoparticles in a fluorine-free solvent, adding quantifiable fluorine-containing substances as an internal standard, then performing nuclear magnetic resonance fluorine spectrum detection; S3, quantitatively calculating the amount of free fluorine-containing lipids by integrating the peak areas of the internal standard and the fluorine-containing lipids in the nuclear magnetic fluorine spectrum; S4, calculating the nucleic acid encapsulation efficiency of the fluorine-containing nanoparticles. The method can realize efficient and non-destructive detection of samples, and can provide effective technical support for detecting the combination of carriers and nucleic acids in fluorine-containing drugs and nucleic acid drugs.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and specifically to a method for determining the encapsulation efficiency of fluorine-containing nanoparticles using multinuclear magnetic resonance imaging. Background Technology

[0002] In the field of biomedicine and drug delivery, lipid nanoparticles, as a broad category of lipid-based nanocarriers, encompass various types including liposomes, solid lipid nanoparticles (SLNs), dendritic macromolecular vesicle nanoparticles (DNPs), nanostructured lipid carriers (NLCs), lipid-polymer hybrid nanoparticles (LPHNPs), and ionizable lipid nanoparticles. With their excellent biocompatibility, structural tunability, and efficient drug loading capacity, they have become a core delivery platform for therapeutic molecules such as small molecule drugs, mRNA, and siRNA. The encapsulation efficiency of these carriers directly determines the stability, in vivo delivery efficiency, and clinical safety of the drug—insufficient encapsulation efficiency can lead to drug exposure to in vivo degrading enzymes or trigger immune side effects. Therefore, accurate encapsulation efficiency detection is a crucial prerequisite for carrier formulation optimization and clinical translation.

[0003] Current methods for detecting the encapsulation efficiency of lipid nanoparticles mainly rely on traditional separation-detection methods and fluorescent probe methods, but their technical limitations are becoming increasingly apparent. Offline separation methods (such as column chromatography, dialysis, and ultracentrifugation) require the separation of free drug and carrier first. During this process, mechanical forces and changes in osmotic pressure can easily damage the lipid nanoparticle structure, leading to leakage of the encapsulated drug. This is particularly unsuitable for structurally fragile carrier types such as LPHNPs and NLCs, resulting in distorted detection results. While the Ribogreen fluorescence method, as a mainstream approach, is relatively simple to operate, it cannot distinguish between the target drug and impurity nucleic acid fragments, and it is difficult to adapt to the detection of multi-component drug-loaded systems, limiting its application in the development of complex carriers. More importantly, existing methods are mostly endpoint detection methods, unable to dynamically capture real-time changes in encapsulation efficiency during preparation, nor can they be linked to in vivo circulation and release behaviors within the carrier for systematic analysis. This makes it difficult to establish a direct correlation between "encapsulation efficiency and delivery efficiency," hindering the rapid iterative optimization of lipid nanoparticles.

[0004] With the functionalization and diversification of lipid nanoparticles, especially the application of novel modification technologies such as fluorination, traditional detection methods are no longer sufficient to meet the demands for precise, end-to-end monitoring. Developing an encapsulation efficiency detection technology that is non-destructive, highly specific, provides real-time dynamic monitoring, and has broad compatibility has become a core requirement for overcoming current technological bottlenecks. Summary of the Invention

[0005] Based on the above, the present invention is based on 19Nuclear magnetic resonance fluorine spectroscopy (NMR) detection technology, leveraging the unique physical properties of the F-core, offers a novel approach to solving this challenge due to its advantages of zero background interference and precise carrier signal capture. It holds promise for quantitative prediction of the encapsulation efficiency of different types of lipid nanoparticles, while simultaneously linking carrier structure and delivery performance analysis, providing crucial technical support for the efficient development and clinical translation of lipid nanoparticle carriers. The specific objective is to... 19 1F NMR spectroscopy was used to quantitatively calculate the amount of free fluorinated lipids in fluorinated lipid nanoparticles, and then the nucleic acid encapsulation efficiency was calculated based on the amount of fluorinated lipids in the nanoparticles. This provides effective technical support for real-time, non-destructive, and high-throughput determination of nucleic acid encapsulation efficiency in fluorinated lipid nanoparticles.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] This invention provides a method for determining the encapsulation efficiency of fluorine-containing nanoparticles using multinuclear magnetic resonance imaging (MMRI), the method comprising the following steps:

[0008] S1. Reference standard detection: Dissolve the fluorinated lipid in a fluorine-free solvent, add a quantifiable fluorinated substance as the first internal standard, and perform nuclear magnetic resonance fluorine spectroscopy to determine the characteristic peaks of the fluorinated lipid.

[0009] S2, Sample Detection: Nucleic acid is combined with the fluorinated lipid to be detected to prepare fluorinated lipid nanoparticles. After purification, they are dissolved in a fluorine-free solvent. A quantifiable fluorinated substance is added as a second internal standard. Nuclear magnetic resonance fluorine spectrum detection is performed. The free fluorinated lipid corresponds to the characteristic peak of the fluorinated lipid in S1.

[0010] S3. Quantitative Calculation: By integrating the peak areas of the internal standard and fluorinated lipids in the NMR fluorine spectrum of S2, the amount of free fluorinated lipids not bound to nucleic acids is calculated. The amount of free fluorinated lipids is calculated as follows: ;

[0011] S4. Encapsulation efficiency calculation: The nucleic acid encapsulation efficiency is calculated according to the following formula: ;

[0012] Furthermore, the fluorine-free solvent is any one of pure water, ethanol, PBS, and physiological saline, preferably pure water.

[0013] Furthermore, the nucleic acid is any one of DNA, mRNA, siRNA, and miRNA, preferably Fluc mRNA.

[0014] Furthermore, the structural formula of the fluorinated lipid is shown below:

[0015] ,

[0016] Rf is selected from perfluorotert-butyl Trifluoroethyl Trifluoromethyl difluoroethyl Pentafluorothiobenzyl Monofluorobenzyl difluorobenzyl bis(trifluoromethyl)benzyl Trifluoromethoxybenzyl Trifluoromethylbenzyl And having O(CH2)2(CF2) m The CF3 structure contains any one of the straight-chain fluorinated alkoxy groups, Hy is selected from any one of the hydrophilic functional groups such as hydroxyl, carboxyl, amino, sulfonic acid, phosphoric acid, mercapto, amide, quaternary ammonium, cyano, aldehyde, and nitro, m is a natural number from 1 to 10, n is a natural number from 3 to 20, k is a natural number from 3 to 20, and y is a natural number from 0 to 20.

[0017] Furthermore, the fluorinated lipid is 3-(8,8,8-trifluorooctoxy)-2,2-bis((8,8,8-trifluorooctoxy)methyl)propyl-4-(4-(2-hydroxyethyl)piperazin-1-yl)butyrate.

[0018] Furthermore, the amount of substance can be quantified by mass, number of fluorine atoms, or amount of fluorine atoms.

[0019] Furthermore, whether the first internal standard and the second internal standard are the same or different, they both meet the following requirements: they do not affect the peak of the fluorinated lipid, the number of F atoms is known, and the spin of the F atoms is unrestricted, so that a complete F signal can be generated.

[0020] Furthermore, the concentration of the internal standard does not interfere with the fluorine spectrum signal of fluorinated lipids and fluorinated lipid nanoparticles.

[0021] Furthermore, the internal standard is sodium trifluoromethanesulfonate, preferably a deuterated aqueous solution of sodium trifluoromethanesulfonate at a concentration of 0.12 mg / mL.

[0022] Furthermore, the nuclear magnetic resonance fluorine spectroscopy test was performed using a nuclear magnetic resonance spectrometer with a frequency of 400-850 MHz.

[0023] Furthermore, the NMR fluorine spectrum detection employs a ZGFLQN pulse sequence or a fast spin echo sequence, with a test temperature of 297±2 K; and / or the pulse width during NMR fluorine spectrum detection is 11-12 μs; and / or the relaxation time during NMR fluorine spectrum detection is 10-20 s; and / or the spectral width during NMR fluorine spectrum detection is 7000-8000 Hz; and / or the radio frequency center frequency during NMR fluorine spectrum detection is -45000 to -35000 Hz. Hz; and / or the number of sampling points during the nuclear magnetic resonance fluorine spectrum detection is 125,000-135,000; and / or the sampling time during the nuclear magnetic resonance fluorine spectrum detection is 7-10s; and / or the number of samplings during the nuclear magnetic resonance fluorine spectrum detection is 20-40; and / or the number of blank scans during the nuclear magnetic resonance fluorine spectrum detection is 3-6; and / or the gain during the nuclear magnetic resonance fluorine spectrum detection is 1-150.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] This invention addresses the industry pain points of traditional lipid nanoparticle encapsulation efficiency detection methods, such as sample destruction, result distortion, insufficient specificity, and inability to dynamically monitor. It pioneers a method based on… 19 A quantitative detection method for the encapsulation efficiency of fluorinated lipid nanoparticles using NMR fluorine spectroscopy. This method breaks through the traditional technical logic of direct nucleic acid detection, achieving precise quantification through the intrinsic characteristics of zero background interference and high specificity of fluorine nuclei, fundamentally avoiding detection interference from nucleic acid degradation fragments and excipient impurities. The method requires no destructive sample pretreatment, achieving completely non-destructive detection and avoiding result distortion caused by nucleic acid leakage. The detection accuracy is highly consistent with the industry gold standard RiboGreen fluorescent probe method. Furthermore, the method is simple to operate, with standardized detection parameters, compatible with mainstream NMR equipment, enabling high-throughput rapid detection of batch samples. It can also dynamically track real-time changes in the binding state of lipids and nucleic acids under different conditions, filling the technical gap in dynamic monitoring in this field and providing reliable detection basis for subsequent performance evaluation of nanoparticles. Attached Figure Description

[0026] Figure 1 The nuclear magnetic resonance fluorine spectrum of the fluorinated lipid in pure water in Example 2;

[0027] Figure 2 The NMR fluorine spectrum of mRNA encapsulated by fluorinated lipid nanoparticles in Example 3 in pure water;

[0028] Figure 3 The fluorine NMR spectrum of mRNA encapsulated in fluorinated lipid nanoparticles in Example 4 after sonication in a pure water:methanol = 1:1 solvent for 0.5 h.

[0029] Figure 4 The fluorine NMR spectrum of mRNA encapsulated in fluorinated lipid nanoparticles in Example 5 after sonication in pure water:methanol = 1:1 solvent for 1 h.

[0030] Figure 5 This is an integrated NMR fluorine spectrum from Examples 2-5, showing the peak positions and peak area trends of fluorinated lipids, fluorinated nanoparticles, and the internal standard sodium trifluoromethanesulfonate. Detailed Implementation

[0031] The technical solution of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0032] The main reagents and materials used in the following examples are described below: Fluc mRNA was purchased from Shanghai Unimicron Biotechnology Co., Ltd.; the 5 mm NMR tube used for NMR fluorine spectroscopy was purchased from Wilmad, USA. The NMR spectrometer used for the NMR fluorine spectroscopy was 500 MHz, purchased from Bruker, Switzerland, with the following parameters: zgflqn pulse sequence, test temperature 297±2 K, pulse width 12 μs, relaxation time 10-20 s, spectral width 8000 Hz, RF center frequency -45000 Hz, number of acquisition points 125000, sampling time 8 s, number of samplings 20-40, number of blank scans 4, and gain 120.

[0033] 8-Bromo-1,1,1-Trifluorooctane was purchased from Shanghai Haohong Biomedical Technology Co., Ltd.; the PD-10 desalting chromatography column (catalog number 17085101) was purchased from GE; the Quant-iT RiboGreen RNA kit (catalog number R11490) was purchased from Thermo Fisher Scientific; and the Triton X-100 (catalog number T8200) was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0034] Methanol was purchased from Sinopharm Chemical Reagent Co., Ltd. 0.5 M citrate buffer was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., pH=3. Preparation of 50 mM citrate buffer: 3 mL of pH=3 0.5 M citrate buffer was added to 27 mL of RNase-free ddH2O, mixed thoroughly, and diluted to obtain 50 mM citrate buffer.

[0035] The internal standard was prepared as follows: 50 μL of a deuterated aqueous solution of 0.12 mg / mL sodium trifluoromethanesulfonate was prepared as one internal standard. The amount of sodium trifluoromethanesulfonate in one internal standard was 3.5 × 10⁻⁶. -7 mol, 0.06 mg.

[0036] Synthesis of fluorinated lipids:

[0037] The fluorinated lipid 3-(8,8,8-trifluorooctoxy)-2,2-bis((8,8,8-trifluorooctoxy)methyl)propyl-4-(4-(2-hydroxyethyl)piperazin-1-yl)butyrate was prepared by the following method:

[0038] Step a: Dissolve 7.5 g of sodium hydroxide in 7.5 mL of water, add 0.545 g of pentaerythritol, heat to 80 °C, and stir to react for 1 h.

[0039] Step b: After step a is completed, 4 g of 8-bromo-1,1,1-trifluorooctane and 0.516 g of tetrabutylammonium bromide (TBAB) are added to the reaction solution from step a, and the mixture is stirred at 80 °C for 5 h. After the reaction is complete, the reaction solution is extracted three times with an equal volume of water and a DCM mixture (volume ratio 1:1, the same below). The combined organic phases are dried over anhydrous sodium sulfate, filtered, and the filtrate is purified by column chromatography after being evaporated to dryness to obtain the product fluoroalkyl alcohol 2, which is a brownish-yellow oily liquid (0.8 g, 32% yield). 1 H NMR(500 MHz, Chloroform-d) δ 3.69 (s, 2H), 3.42 (s, 6H), 3.37 (s, 6H), 2.04 (s,6H), 1.53 (s, 12H), 1.32 (s, 18H). 13 C NMR (176 MHz, Chloroform-d) δ 128.02,71.57, 71.50, 44.71, 33.57, 29.43, 28.95, 28.61, 25.88, 21.77, 21.76. 19 F NMR (471 MHz, Chloroform-d) δ -66.49.

[0040] Step c: 3.15 mmol of fluorinated alkyl alcohol 2 was added to a reaction flask, dissolved in dichloromethane (DCM, 15 mL), followed by the addition of triethylamine (Et3N, 637.5 mg, 6.3 mmol) and 4-dimethylaminopyridine (DMAP, 154 mg, 1.26 mmol). After stirring at 0 °C for 10 min, 15 mL of a DCM solution of 4-bromobutyryl chloride (3.8 mmol) was added dropwise, and the reaction was continued for 2 h. After the reaction was complete, the reaction solution was extracted three times with an equal volume of water and DCM. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was purified by column chromatography after evaporation to obtain activated fluorinated alkyl ester 3, which was a brownish-yellow oily liquid (1.85 g, 75% yield). 1HNMR (500 MHz, Chloroform-d) δ 4.11 (s, 2H), 3.44 (s, 2H), 3.35 (s, 12H), 2.47(s, 2H), 2.14 (s, 2H), 2.04 (s, 6H), 1.50 (s, 12H), 1.31 (s, 18H) 13 C NMR (126MHz, Chloroform-d) δ 172.32, 128.49, 71.52, 69.62, 64.38, 44.55, 33.89,32.69, 32.57, 29.58, 29.09, 28.75, 27.96, 26.03, 21.89. 19 F NMR (471 MHz, Chloroform-d) δ -66.55.

[0041] Step d: Add an anhydrous acetonitrile (CH3CN, 50 mL) solution of potassium carbonate (K2CO3, 0.25 mmol, 34.55 mg), 1-(2-hydroxyethyl)piperazine (1.3 mmol, 157 mg), and fluorinated alkyl ester 3 (1.08 mmol, 850 mg) to a round-bottom flask. The reaction solution was then refluxed in a 95 °C oil bath for 3 h. After the reaction was completed, the solution was cooled to room temperature (25 °C, the same below), acetonitrile was evaporated, and the solution was extracted three times with an equal volume of water and DCM. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was evaporated and purified by column chromatography to obtain fluorinated lipid 4, namely 3-(8,8,8-trifluorooctoxy)-2,2-bis((8,8,8-trifluorooctoxy)methyl)propyl-4-(4-(2-hydroxyethyl)piperazin-1-yl)butyrate, which was a colorless oily liquid (599.8 mg, 66% yield). 1 HNMR (500 MHz, Chloroform-d) δ 4.07 (s, 2H), 3.58 (s, 2H), 3.33 (s, 12H), 2.51(s, 10H), 2.31 (s, 2H), 2.02 (s, 6H), 1.77 (s, 2H), 1.48 (s, 12H), 1.29 (s, 18H). 13C NMR (126 MHz, Chloroform-d) δ 173.30, 128.45, 71.49, 69.59, 64.08,59.42, 57.81, 57.67, 53.17, 52.94, 44.54, 33.87, 29.56, 29.06, 28.72, 25.99,22.25, 21.88. 19 F NMR (471 MHz, Chloroform-d) δ -66.48. HRMS m / z:[M+H] + calcdfor C 39 H 69 F9N2O6; 833.97, Found 833.50.

[0042] Example 1: Preparation of fluorinated lipid nanoparticles, the specific steps are as follows:

[0043] (1) Take 2.4 × 10 -6 2 mg of 3-(8,8,8-trifluorooctoxy)-2,2-bis((8,8,8-trifluorooctoxy)methyl)propyl-4-(4-(2-hydroxyethyl)piperazine-1-yl)butyrate was dissolved in 200 μL of anhydrous ethanol and mixed thoroughly to obtain a fluorinated ethanol phase lipid solution.

[0044] (2) Dissolve 50 μg of firefly luciferase mRNA (Fluc mRNA) in 2 mL of 50 mM citrate buffer to obtain an aqueous nucleic acid solution.

[0045] (3) The ethanol phase and the aqueous phase nucleic acid solution were mixed uniformly at a volume ratio of 1:10 to obtain fluorinated lipid nanoparticles (F-DNP).

[0046] (4) Using pure water as the developing solvent, F-DNP was purified by using a PD-10 desalting chromatography column to remove citric acid, and purified fluorinated lipid nanoparticles F-DNP were obtained.

[0047] Example 2: Detection of fluorine content in fluorinated lipids by nuclear magnetic resonance fluorine spectroscopy:

[0048] (1) Dissolve 0.2 mg of 3-(8,8,8-trifluorooctoxy)-2,2-bis((8,8,8-trifluorooctoxy)methyl)propyl-4-(4-(2-hydroxyethyl)piperazine-1-yl)butyrate in 500 μL of pure water, mix well, and pack into a 5 mm NMR tube.

[0049] (2) Take one internal standard and place it in the above 5 mm NMR tube for NMR fluorine spectroscopy characterization. Figure 1 ), Figure 1 In the figure, the peak positions of -67.78 ppm and -79.6 ppm correspond to free fluorinated lipids and the internal standard, respectively. Direct comparison of the free fluorinated lipids and the internal standard... 19 The amount of free fluorolipids can be obtained by integrating the peak area of ​​the F-magnetic resonance signal. The method for calculating the amount of free fluorolipids is as follows:

[0050] ;

[0051] .

[0052] Example 3: Detection of F-DNP fluoride in pure water by nuclear magnetic resonance spectroscopy:

[0053] (1) Centrifuge and concentrate the purified F-DNP obtained in step (4) of Example 1 to 2500 μL, then take 250 μL into a 1.5 mL enzyme-free sterile centrifuge tube, add 250 μL of pure water, vortex for 5 seconds, mix evenly, and put into a 5 mm NMR tube.

[0054] (2) Take one internal standard and place it in the 5 mm NMR tube containing the sample to be tested, and perform NMR fluorine spectrum characterization. Figure 2 ).

[0055] (3) Comparison Figure 1 The peak position, according to Figure 2 The peak area ratio of free fluorinated lipids to the internal standard sodium trifluoromethanesulfonate was used to quantitatively calculate the amount of free fluorinated lipids. Since fluorinated lipids are released from the lysis of fluorinated nanoparticles, the nucleic acid encapsulation efficiency can be calculated as: total fluorinated lipids - released fluorinated lipids / total fluorinated lipids. 100%.

[0056] Specifically, in this embodiment... 100% = 92.22%.

[0057] Example 4: Nuclear magnetic resonance fluorine spectrum detection of F-DNP after ultrasonic treatment in pure water:methanol = 1:1 (volume ratio) solvent for 0.5 hours:

[0058] (1) The purified F-DNP obtained in step (4) of Example 1 was concentrated to 2500 μL by centrifugation. 250 μL was taken into a 1.5 mL enzyme-free sterile centrifuge tube, and 250 μL of methanol was added (the nanoparticles were dissolved in pure water during preparation, so the volume ratio of pure water to methanol was 1:1). The mixture was sonicated for 0.5 h (40 kHz, the same below) and then placed into a 5 mm NMR tube.

[0059] (2) Take one internal standard and put it into the 5 mm NMR tube containing the sample to be tested.

[0060] (3) Perform nuclear magnetic resonance fluorine spectroscopy characterization ( Figure 3 ), calculate the encapsulation ratio: 100% = 65.49%.

[0061] Example 5: Nuclear magnetic resonance fluorine spectrum detection of F-DNP after ultrasonic treatment in pure water:methanol = 1:1 (volume ratio) solvent for 1 hour:

[0062] (1) The purified F-DNP obtained in step (4) of Example 1 was concentrated to 2500 μL by centrifugation. 250 μL was taken into a 1.5 mL enzyme-free sterile centrifuge tube, and 250 μL of methanol was added. The mixture was sonicated for 1 h and then placed into a 5 mm NMR tube.

[0063] (2) Take one internal standard and put it into the 5 mm NMR tube containing the sample to be tested.

[0064] (3) Perform nuclear magnetic resonance fluorine spectroscopy characterization ( Figure 4 ), calculate the encapsulation ratio: 100% = 19.31%.

[0065] Figure 5 The integrated NMR fluorine spectrum diagrams of Examples 1-4 show the positional relationship of the peaks of fluorinated lipids, fluorinated nanoparticles, and the internal standard sodium trifluoromethanesulfonate on the fluorine spectrum. Furthermore, it can be seen that as the degree of fragmentation increases, the peak area of ​​fluorinated lipids increases and the peak height increases, while the peak area of ​​fluorinated nanoparticles decreases and the peak height decreases.

[0066] Example 6: Determination of F-DNP encapsulation efficiency using a fluorescent probe method:

[0067] mRNA standards:

[0068] (1) Take a 50 mL conical tube, add 2.5 mL of 20X TE Buffer (pH 7.4), then add 47.5 mL of RNase-free water (DEPC water), vortex mix evenly to obtain 1X TE Buffer, 10 mM Tris + 1 mM EDTA.

[0069] (2) Dilute the Quant-iT Ribogreen reagent 200 times with 1X TE Buffer (e.g., when preparing 2 mL, take 10 μL of Ribogreen reagent and mix with 1990 μL of 1X TE Buffer, vortex and shake well, and use immediately).

[0070] (3) Add 0.5 mL of Triton-X 100 to a 50 mL conical tube, then add 49.5 mL of 1X TEBuffer, and stir gently for 30 minutes to avoid foaming, to obtain Triton buffer.

[0071] (4) Use 100 μg / mL λRNA from the Quant-iT RiboGreen RNA kit as the mRNA standard, and dilute it to 4 μg / mL stock solution (25-fold dilution). That is, take 20 μL of 100 μg / mL mRNA standard, add 480 μL of 1X TE Buffer, mix well, and then use 1X TE Buffer to prepare mRNA standards of 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL.

[0072] (5) Add 100 μL of mRNA standards at concentrations of 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL to each well of a 96-well plate, respectively. Add 100 μL of 1X TE Buffer (or Triton Buffer) and 100 μL of Quant-iT Ribogreen reagent diluted 200 times to each well of the plate to adapt to the two scenarios of "unbroken membrane (1×TE Buffer, only free nucleic acid luminescence)" and "broken membrane (Triton Buffer, total nucleic acid luminescence)". At the same time, set up a blank control group, add only 100 μL of 1X TE Buffer (or Triton Buffer) and 100 μL of Quant-iT Ribogreen reagent diluted 200 times. Cover the plate and incubate gently at 100 r / min for 10 minutes.

[0073] Sample groups of Examples 3-5:

[0074] (6) Add 5 μL of the sample treated in step (1) of Examples 3-5 and 95 μL of 1X TE Buffer (or Triton buffer) to each well of the 96-well plate, then add 100 μL of Quant-iT Ribogreen reagent diluted 200 times, cover the plate, and gently shake at 100 r / min for 10 minutes.

[0075] (7) Place the 96-well plate that has been incubated above into the SpectraMax instrument, set the excitation wavelength to 480 nm and the emission wavelength to 520 nm, and detect the fluorescence intensity value of each well according to the above layout order.

[0076] (8) Plot the concentration on the x-axis and the fluorescence intensity on the y-axis to fit two independent standard curves for different concentrations of mRNA standards (i.e., nucleic acids) in TE Buffer and Triton buffer. Substitute the fluorescence intensity values ​​obtained in steps (6) and (7) into the corresponding standard curves to obtain the corresponding nucleic acid concentrations. Calculate the nucleic acid encapsulation efficiency in F-DNP using the following formula:

[0077] 100%.

[0078] Table 1. Encapsulation efficiency of F-DNP nucleic acid determined by fluorescent probe method and nuclear magnetic resonance fluorine spectroscopy:

[0079]

[0080] Table 1 summarizes and statistically analyzes the F-DNP nucleic acid encapsulation rates measured by fluorescence probe method and nuclear magnetic resonance fluorine spectroscopy in Examples 3, 4, and 5 above. It can be found that the encapsulation rates measured by the traditional fluorescence probe method and the encapsulation rates measured by nuclear magnetic resonance fluorine spectroscopy in this patent are basically consistent and can correspond to each other.

Claims

1. A method for determining the encapsulation efficiency of fluorine-containing nanoparticles using multinuclear magnetic resonance imaging, characterized in that, The method includes the following steps: S1. Reference standard detection: Dissolve the fluorinated lipid in a fluorine-free solvent, add a quantifiable fluorinated substance as the first internal standard, and perform nuclear magnetic resonance fluorine spectroscopy to determine the characteristic peaks of the fluorinated lipid. S2, Sample Detection: Nucleic acid is combined with the fluorinated lipid to be detected to prepare fluorinated lipid nanoparticles. After purification, they are dissolved in a fluorine-free solvent. A quantifiable fluorinated substance is added as a second internal standard. Nuclear magnetic resonance fluorine spectrum detection is performed. The free fluorinated lipid corresponds to the characteristic peak of the fluorinated lipid in S1. S3. Quantitative Calculation: By integrating the peak areas of the internal standard and fluorinated lipids in the NMR fluorine spectrum of S2, the amount of free fluorinated lipids not bound to nucleic acids is calculated. The amount of free fluorinated lipids is calculated as follows: ; S4. Encapsulation efficiency calculation: The nucleic acid encapsulation efficiency is calculated according to the following formula: .

2. The method according to claim 1, characterized in that, The fluorine-free solvent is any one of pure water, ethanol, PBS, or physiological saline.

3. The method according to claim 1, characterized in that, The nucleic acid can be any one of DNA, mRNA, siRNA, or miRNA.

4. The method according to claim 1, characterized in that, The structural formula of the fluorinated lipid is shown below: ， Rf is selected from perfluorotert-butyl Trifluoroethyl difluoroethyl Trifluoromethyl Pentafluorothiobenzyl Monofluorobenzyl difluorobenzyl bis(trifluoromethyl)benzyl Trifluoromethoxybenzyl Trifluoromethylbenzyl And having O(CH2)2(CF2) m The CF3 structure is any one of the straight-chain fluorinated alkoxy groups, Hy is selected from any one of hydroxyl, carboxyl, amino, sulfonic acid, phosphoric acid, mercapto, amide, quaternary ammonium, cyano, aldehyde, and nitro groups, m is a natural number from 1 to 10, n is a natural number from 3 to 20, k is a natural number from 3 to 20, and y is a natural number from 0 to 20.

5. The method according to claim 4, characterized in that, The fluorinated lipid is 3-(8,8,8-trifluorooctoxy)-2,2-bis((8,8,8-trifluorooctoxy)methyl)propyl-4-(4-(2-hydroxyethyl)piperazin-1-yl)butyrate.

6. The method according to claim 1, characterized in that, Whether the first internal standard and the second internal standard are the same or different, they both meet the following requirements: they do not affect the peaks of fluorinated lipids, the number of F atoms is known, and the spin of F atoms is unrestricted, so that a complete F signal can be generated; the concentration does not interfere with the fluorine spectrum signal of fluorinated lipids and fluorinated lipid nanoparticles.

7. The method according to claim 6, characterized in that, The internal standard is sodium trifluoromethanesulfonate.

8. The method according to claim 1, characterized in that, The nuclear magnetic resonance (NMR) spectrometer used for fluorine spectroscopy testing has a frequency of 400-850 MHz.

9. The method according to claim 8, characterized in that, The nuclear magnetic resonance fluorine spectroscopy detection employs a zgflqn pulse sequence or a fast spin echo sequence, with a test temperature of 297±2 K; and / or The pulse width during the nuclear magnetic resonance fluorine spectroscopy detection is 11-12 μs; and / or The relaxation time during the nuclear magnetic resonance fluorine spectroscopy detection is 10-20 s; and / or The spectral width for the nuclear magnetic resonance fluorine spectrum detection is 7000-8000 Hz.

10. The method according to claim 9, characterized in that, The radio frequency center frequency for the nuclear magnetic resonance fluorine spectrum detection is -45000 to -35000 Hz; and / or The number of sampling points during the nuclear magnetic resonance fluorine spectroscopy detection is 125,000-135,000; and / or The sampling time for the nuclear magnetic resonance fluorine spectroscopy detection is 7-10 seconds; and / or The number of samples taken during the nuclear magnetic resonance fluorine spectroscopy detection is 20-40; and / or The number of empty scans during the nuclear magnetic resonance fluorine spectroscopy detection is 3-6; and / or The gain during nuclear magnetic resonance fluorine spectroscopy detection is 1-150.

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