Method for predicting transfection efficiency of fluorinated lipid nanoparticles using multi-nuclear magnetic resonance imaging

Abstract

The present invention relates to the field of analytical detection technology, and specifically provides a method for predicting the transfection efficiency of fluorinated lipid nanoparticles using multi-nuclear magnetic resonance. The method comprises the following steps: (1) performing nuclear magnetic resonance fluorine spectrum detection on the fluorinated nanoparticles to obtain a nuclear magnetic fluorine spectrum of the control group; (2) transfecting the fluorinated nanoparticles into cells or mice in vivo, performing nuclear magnetic resonance fluorine spectrum detection on the cells or mice in vivo, and obtaining a nuclear magnetic fluorine spectrum of the experimental group; (3) calculating the difference in target peak area of ​​the fluorinated lipids in the experimental group and the control group to predict the transfection efficiency of the fluorinated lipid nanoparticles. The method has good performance in terms of specificity, repeatability, stability, linearity, etc., and can provide effective technical support for large-scale screening of nanopharmaceuticals using fluorinated lipid nanoparticles.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for predicting the transfection efficiency of fluorinated lipid nanoparticles by using multi-nuclear magnetic resonance. Background Art

[0002] Nucleic acid therapy has great potential in treating and preventing diseases. Messenger RNA-based technologies have achieved impressive clinical results in vaccines, protein supplementation, and gene editing (Han X, Xu J, Xu Y, et al. Nat Commun. 2024; 15(1): 1762). However, nucleic acids are negatively charged and unstable macromolecules that require carriers for effective intracellular delivery.

[0003] Lipid nanoparticles (LNPs) are currently the most advanced non-viral nucleic acid delivery platform in clinical practice. Studies have shown that only a small amount of nucleic acid carried by LNPs can escape lysosomes and enter the cytoplasm (Gilleron J, Querbes W, Zeigerer A, et al. Nat Biotechnol. 2013; 31(7): 638-646.). In order to further improve the nucleic acid delivery performance of LNPs, fluorinated lipids are used to improve the transport efficiency of nucleic acids in cells. This is mainly due to the fact that the fluorinated lipids added to fluorinated LNPs can significantly promote the cellular uptake and lysosomal escape of LNPs (Zhang H, Meng C, Yi X, et al. ACS Nano. 2024; 18(11): 7825-7836.). After the fluorinated LNPs successfully escape the lysosomes, they release the carried nucleic acid and fluorinated lipids, which helps in subsequent nucleic acid transfection and protein expression. Therefore, the release of fluorinated lipids is correlated with subsequent protein expression.

[0004] Currently, the most intuitive method for measuring LNP transfection efficiency in vitro is to observe the expression of fluorescent proteins using a confocal microscope. However, this method cannot avoid the differences in LNP delivery in vitro and in vivo. The most common method for measuring LNP transfection efficiency in vivo is to inject LNPs into mice for a certain period of time and then obtain tissue from the injection site for immunofluorescence staining to analyze protein expression. However, this method requires the sacrifice of a large number of mice, so usually only a small number of LNPs can be screened in vivo. Summary of the Invention

[0005] Based on the above, the present invention aims to provide a method for predicting the transfection efficiency of fluorinated lipid nanoparticles by using nuclear magnetic resonance (NMR) to detect the fluorine spectra of fluorinated nanoparticles before and after transfection into cells or mice. This method overcomes the inaccurate and cumbersome procedures associated with prior art transfection efficiency measurements of fluorinated nanoparticles. It enables simpler, faster, and more accurate prediction of the transfection efficiency of fluorinated lipid nanoparticles, enabling large-scale screening of fluorinated lipid nanoparticles in non-human primate models.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for predicting the transfection efficiency of fluorinated lipid nanoparticles using multi-nuclear magnetic resonance imaging, comprising the following steps:

[0008] (1) Lysing untransfected cells to obtain cell lysate, mixing the cell lysate with fluorinated lipid nanoparticles, and performing nuclear magnetic resonance fluorine spectrum detection to obtain a nuclear magnetic resonance fluorine spectrum of the control group;

[0009] (2) After transfecting cells (preferably 293T cells) with fluorinated lipid nanoparticles, the cells are lysed to obtain a transfected cell lysate, and the cell lysate is subjected to nuclear magnetic resonance fluorine spectrum detection to obtain a nuclear magnetic resonance fluorine spectrum of the experimental group;

[0010] (3) obtaining the target peak of the fluorinated lipid from the nuclear magnetic resonance fluorine spectra of the experimental group and the control group obtained in step (1) and step (2), and calculating the difference in the target peak area of ​​the fluorinated lipid between the experimental group and the control group;

[0011] (4) using a confocal fluorescence microscope to observe the transfection status of the fluorinated lipid nanoparticles after cell transfection in step (2), and quantitatively analyzing the fluorescence intensity;

[0012] (5) selecting fluorinated lipid nanoparticles with different fluorinated lipid contents to carry out the above steps (1)-(4);

[0013] (6) Plotting a curve with the difference in target peak area of ​​fluorinated lipids between the experimental group and the control group as the horizontal axis and the fluorescence intensity after cell transfection as the vertical axis to obtain the linear curves of the two;

[0014] (7) The fluorinated lipid nanoparticles to be tested are subjected to the above steps (1) to (3), and the corresponding transfection efficiency is obtained using the curve of step (6).

[0015] In the above method of the present invention, the cells can also be replaced by living experimental mice. In this case, the above steps (1) and (2) are respectively:

[0016] (1-1) performing nuclear magnetic resonance fluorine spectrum detection on the fluorinated lipid nanoparticles to obtain a nuclear magnetic resonance fluorine spectrum of the control group;

[0017] (2-1) Fluorinated lipid nanoparticles were injected into mice, and after a period of incubation, nuclear magnetic resonance fluorine spectrum of the injection site was detected to obtain the nuclear magnetic resonance fluorine spectrum of the experimental group.

[0018] The fluorinated lipid nanoparticles are obtained by conventional methods, and their composition at least includes fluorinated lipids and plasmids. The structure of the fluorinated lipids is as follows:

[0019] wherein Rf is selected from perfluoro-tert-butyloxy, trifluoroethoxy, difluoroethoxy, pentafluorothiobenzyloxy, p-fluorobenzyloxy, difluorobenzyloxy and other fluorobenzyloxy, bis-trifluoromethylbenzyloxy, trifluoromethoxybenzyloxy, trifluoromethylthiobenzyloxy and -O(CH2)2(CF2) n Any linear polyfluoroalkyl group of CF3 structure, wherein n is a natural number of 3 to 20, preferably di(12-(perfluoro-tert-butoxy)dodecyl)amine.

[0020] Preferably, the fluorinated lipid nanoparticles are prepared by the following method: an ethanol solution containing di(12-(perfluoro-tert-butoxy)dodecyl)amine, heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanoxy)hexyl)amino)octanoate), distearoylphosphatidylcholine, cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 is mixed with a pEGFP plasmid solution containing an EGFP plasmid at a certain volume ratio (preferably 1:3), The molar ratio of the di(12-(perfluorotert-butoxy)dodecyl)amine, heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanoxy)hexyl)amino)octanoate), distearoylphosphatidylcholine, cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 is: (12.5-37.5): (12.5-37.5): 10: 38.5: 1.5; the mass ratio of the plasmid to cholesterol is 150 μg: 0.6 mg.

[0021] In order to perform a more accurate quantitative analysis of the fluorine content in step (1) and step (2), before the detection in step (1) and step (2), an external standard dissolved in a deuterated reagent is added to the corresponding cell lysate, wherein the deuterated reagent includes any one of deuterated water, deuterated methanol, deuterated acetonitrile, and deuterated dimethyl sulfoxide, preferably deuterated water;

[0022] Described external standard comprises any one in trifluoroacetic acid, 2-chloro-3-fluoropyridine, sodium trifluoromethanesulfonate, 4-fluorobenzoic acid, is preferably sodium trifluoromethanesulfonate, and the chemical shift of its target peak is-75ppm~-85ppm, is preferably-78ppm~-80ppm.As the preferred technical scheme of this invention, can guarantee the stability and accuracy of characteristic peak target integration result using sodium trifluoromethanesulfonate as external standard.And sodium trifluoromethanesulfonate and two (12-(perfluorinated tert-butoxy) dodecyl) amine all only produce a corresponding fluorine signal, and distance is closer and separates completely, does not have interference phenomenon, further improves the accuracy of characteristic target peak integral area result.

[0023] Preferably, the concentration of the external standard dissolved in the deuterated reagent is 0.5-5 mM, preferably 1 mM; in the steps (1) and (2), the volume ratio of the corresponding cell transfection solution to the external standard is 1: (0.02-0.4); preferably 10:1.

[0024] Preferably, the frequency of the nuclear magnetic resonance spectrometer used in the nuclear magnetic resonance fluorine spectrum test is 400-600 MHz.

[0025] Preferably, the nuclear magnetic resonance fluorine spectrum detection adopts a zgig pulse sequence or a fast spin echo sequence, and the test temperature is 297±2K; and / or

[0026] The pulse width during nuclear magnetic resonance fluorine spectrum detection is 11-12 μs, preferably 11.1-11.5 μs; and / or

[0027] The relaxation time during nuclear magnetic resonance fluorine spectrum detection is 10-20s, preferably 14-16s; and / or

[0028] The spectrum width of the nuclear magnetic resonance fluorine spectrum detection is 7000-8000 Hz, preferably 7300-7500 Hz; and / or

[0029] The radio frequency center frequency during the nuclear magnetic resonance fluorine spectrum detection is -45000 to -35000 Hz, preferably -41000 to -40000 Hz; and / or

[0030] The number of collection points during the nuclear magnetic resonance fluorine spectrum detection is 125,000-135,000, preferably 130,000-132,000; and / or

[0031] The sampling time for the nuclear magnetic resonance fluorine spectrum detection is 7-10s, preferably 8-9s; and / or

[0032] The sampling times during the nuclear magnetic resonance fluorine spectrum detection is 20-40 times, preferably 30-35 times; and / or

[0033] The number of empty scans during the nuclear magnetic resonance fluorine spectrum detection is 3-6 times, preferably 3-5 times; and / or

[0034] The gain during the nuclear magnetic resonance fluorine spectrum detection is 60-150, preferably 120-135.

[0035] In the method for determining the content of fluorinated lipids described in the present invention, considering that the response signal range of the fluorine spectrum is relatively wide, in order to reduce the error of phase adjustment and ensure the accuracy of integration, the radio frequency center frequency is set at the middle position between the external standard peak and the sample peak to ensure the accuracy of the target peak integration.

[0036] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0037] The present invention utilizes nuclear magnetic resonance fluorine spectrum detection of fluorinated lipid nanoparticles and the fluorine spectrum after the fluorinated lipid nanoparticles are transfected into cells or mice in vivo to predict the transfection efficiency of the fluorinated lipid nanoparticles. Specifically, it relates to nuclear magnetic resonance fluorine spectrum detection of fluorinated lipid content. By optimizing the experimental parameters to give the detection method the characteristics of simple operation, good repeatability, high stability, high accuracy and routine use, the accuracy of the characteristic target peak integration result is guaranteed by selecting a suitable external standard sodium trifluoromethanesulfonate, while ensuring the accuracy of the fluorinated lipid content determination. This method has good performance in terms of specificity, precision, repeatability, stability, linear relationship, etc., and not only meets the prediction of the transfection efficiency of fluorinated LNPs at the cell and in vivo levels, but also can provide technical support for the large-scale screening of fluorinated lipid nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : The NMR fluorine spectra of the cells before and after the fluorinated LNP transfection in Example 2;

[0039] Figure 2 Representative confocal images of cells transfected with fluorinated LNPs in Example 3;

[0040] Figure 3 This is a statistical graph of fluorescence intensity after cells were transfected with fluorinated LNPs;

[0041] Figure 4 The linear relationship between the peak area difference of the target fluorine spectrum before and after fluorinated LNP transfection and the fluorescence intensity;

[0042] Figure 5 This is the fluorine magnetic resonance spectrum before and after the injection of fluorinated LNP into the gastrocnemius muscle of mice;

[0043] Figure 6 Representative images of immunofluorescence-stained sections of mouse gastrocnemius muscle 48 h after injection of fluorinated LNPs. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is described in detail below with reference to specific embodiments and drawings.

[0045] The main reagents and materials used in the following examples are described as follows: 293T cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. RIPA lysis buffer was purchased from Boster (Cat. No. AR0102). Sodium trifluoromethanesulfonate was purchased from Beijing Bailingwei. 5 mm NMR tubes were purchased from Wilmad, USA. 500 MHz and 400 MHz AVANCE NEO NMR spectrometers were purchased from Bruker, Switzerland. 1 mm capillaries were purchased from the Instrument Factory of West China University of Medical Sciences. Heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate) was purchased from Xiamen Sinobond. Distearoylphosphatidylcholine and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were purchased from Ruixi Biotechnology. Cholesterol was purchased from Sigma.

[0046] Prepare a pEGFP plasmid solution encoding EGFP protein: Use a high-purity plasmid extraction kit to extract the EGFP-expressing plasmid from Escherichia coli, and then dilute it to 1 mg / mL with 50 mM citrate buffer, pH 3, for subsequent experiments. The high-purity plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. DP116.

[0047] Example 1 Preparation of fluorinated lipid nanoparticles, the specific steps are as follows:

[0048] (1) An ethanol solution containing di(12-(perfluorotert-butyloxy)dodecyl)amine (abbreviated as F12C), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate (abbreviated as SM102), distearoylphosphatidylcholine (abbreviated as DSPC), cholesterol (abbreviated as Chol) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (abbreviated as (DMG-PEG2000)) was mixed with 1.2158 mL of the p-hydroxybenzoate prepared above. The EGFP plasmid solution (containing 150 μg of plasmid) was mixed in a volume ratio of 1:3 to prepare four types of LNPs, namely LNP-SM102, LNP-25% F12C, LNP-50% F12C, and LNP-75% F12C. They were then diluted with 1× PBS (0.0067 M, the same below) for subsequent cell experiments. The molar ratios of F12C, SM102, DSPC, Cholesterol, and DMG-PEG2000 are shown in Table 1 (Example: The formula of the LNP-SM102 group is: 1.5 mg SM102, 0.33 mg DSPC, 0.6 mg Chol, and 0.16 mg DMG-PEG2000 dissolved in 405.3 μL of anhydrous ethanol. The amounts of Chol and DMG-PEG2000 in the other groups remained unchanged, and the amounts of other substances were adjusted):

[0049] Table 1

[0050]

[0051] (2) Characterization data of four LNPs are shown in Table 2:

[0052] Table 2

[0053] LNP Name Hydrated particle size (nm) PDI LNP-SM102 106.5±12.7 0.132 LNP-25%F12C 96.7±12.7 0.176 LNP-50%F12C 116.8±7.9 0.188 LNP-75%F12C 91.9±11.0 0.212

[0054] Example 2 Calculate the difference in target peak area of ​​the nuclear magnetic resonance fluorine spectrum before and after the fluorinated LNP is transfected into the cells. The specific steps are as follows:

[0055] (1) 293T cells were transfected with LNP-25% F12C, LNP-50% F12C, and LNP-75% F12C prepared in Example 1 (pEGFP in each LNP group was 98 μg). After 48 h, approximately 5E+7 cells were collected from each group and lysed with 1 mL of RIPA lysis buffer. After centrifugation at 12000 g, the supernatant was collected and placed in a new EP tube for later use, thereby obtaining cell lysates of the LNP-25% F12C experimental group, cell lysates of the LNP-50% F12C experimental group, and cell lysates of the LNP-75% F12C experimental group.

[0056] (2) To reduce the influence of variables, 5E+7 untreated 293T cells were lysed with 900 μL RIPA lysis buffer, centrifuged at 12000 g, and the supernatant was placed in a new EP tube for later use, thus obtaining the control group cell lysate;

[0057] (3) Preparation of capillary external standard: Dissolve 0.172 mg of sodium trifluoromethanesulfonate in 1 mL of deuterated water to prepare a 1 mM sodium trifluoromethanesulfonate solution. Use a microsyringe to take 50 μL of the solution and load it into a capillary tube with a diameter of 1 mm. Seal both ends of the capillary tube to obtain the sodium trifluoromethanesulfonate external standard.

[0058] (4) 500 μL of the cell lysate obtained in step (1) was taken and mixed with the sodium trifluoromethanesulfonate external standard obtained in step (3) and transferred to a 5 mm diameter nuclear magnetic resonance tube to obtain the LNP-25% F12C experimental group test solution, the LNP-50% F12C experimental group test solution and the LNP-75% F12C experimental group test solution. The nuclear magnetic resonance spectra of the LNP-25% F12C experimental group, the LNP-50% F12C experimental group and the LNP-75% F12C experimental group were obtained by nuclear magnetic resonance detection using a 500 MHz VANCE NEO nuclear magnetic resonance instrument ( Figure 1 );

[0059] (5) The control group cell lysate obtained in step (2) (about 900 μL) was mixed evenly with the LNP-25% F12C, LNP-50% F12C and LNP-75% F12C obtained and diluted in step (1) (pEGFP in each LNP was 98 μg, and the volume was about 100 μL), and 500 μL of the mixed solution and the sodium trifluoromethanesulfonate external standard obtained in step (3) were taken and transferred to a nuclear magnetic resonance tube with a diameter of 5 mm to obtain LNP-25% F12C control group test solution, LNP-50% F12C control group test solution and LNP-75% F12C control group test solution. The nuclear magnetic resonance spectra of the LNP-25% F12C control group, LNP-50% F12C control group and LNP-75% F12C control group were obtained by nuclear magnetic resonance detection using a 500MHz VANCE NEO nuclear magnetic resonance instrument ( Figure 1 );

[0060] The sampling parameters of the nuclear magnetic resonance fluorine spectrum in this embodiment are as follows: a zgig pulse sequence is used, the test temperature is 297K, the pulse width is 11.1μs, the relaxation time is 15s, the spectrum width is 7462.8Hz, the RF center frequency is -40664.92Hz, the number of acquisition points is 131000, the sampling time is 8.78s, the number of sampling times is 32 times, the number of empty scans is 4 times, and the gain is 128.

[0061] The NMR fluorine spectra obtained in step (4) and step (5) are as follows: Figure 1 As shown, the characteristic peak with a chemical shift of -79.6 ppm is the external standard sodium trifluoromethanesulfonate, and the characteristic peak with a chemical shift of -71.3 ppm is F12C (fluorinated lipid);

[0062] (6) The NMR fluorine spectrum data were processed using MestReNova v14.2.3 software. After calibrating the baseline and adjusting the phase, the target peaks of F12C and the external standard were integrated. The target peak area of ​​the external standard was set to 1, and the target peak area of ​​the fluorinated lipid in the experimental group was calculated as S a The target peak area of ​​the fluorinated lipid in the control group was calculated as S b The difference between the target peak area of ​​the fluorinated lipids in the experimental group and the target peak area of ​​the fluorinated lipids in the control group was calculated as S Δ , the calculation formula is: S Δ =S a -S b The difference between the target peak area of ​​the fluorinated lipids in the experimental group and the target peak area of ​​the fluorinated lipids in the control group is shown in Table 3:

[0063] Table 3

[0064] LNP Name <![CDATA[S Δ ]]> LNP-25%F12C 0.35 LNP-50%F12C 4.64 LNP-75%F12C 6.71

[0065] Example 3: Confocal fluorescence microscopy was used to observe the cell transfection efficiency of different fluorinated lipid nanoparticles. The specific steps are as follows:

[0066] (1) 293T cells were transfected using the lipid nanoparticles prepared in Example 1. Specifically: an appropriate amount of 293T cells were planted in a glass-bottomed confocal dish with a diameter of 2 cm and waited for them to adhere to the wall (equally divided into LNP-SM102 group, LNP-25% F12C group, LNP-50% F12C group and LNP-75% F12C group). After 12 hours, the corresponding diluted LNP was added to the confocal dish of each group. The LNP volume was 19.6 μL, and the pEGFP content was 0.39 μg. After the cells were incubated at 37°C and 5% CO2 for 48 hours, the culture medium was removed, and the cells were fixed with paraformaldehyde and the cell nuclei were stained with DAPI stain. Fluorescence visualization was captured using a fluorescence confocal microscope ( Figure 2 , scale bar = 100 μm).

[0067] (2) The fluorescence images were analyzed using Image J software to analyze the fluorescence expression intensity ( Figure 3 ).

[0068] Example 4 The linear relationship between the peak area difference of the target fluorine spectrum before and after fluorinated LNP transfection and the fluorescence intensity is determined by the following steps:

[0069] The difference in target peak area of ​​the nuclear magnetic resonance fluorine spectrum before and after the fluorinated LNP obtained in Example 2 was used as the x-axis, and the fluorescence expression intensity after the fluorinated LNP obtained in Example 3 was used as the y-axis (LNP-SM102 does not contain fluorinated lipids, so the x-axis is set to 0 and the fluorescence intensity is used as the y-axis). The fitting coefficient (R 2 )( Figure 4 ).

[0070] Analysis of results: The higher the fluorescence intensity after fluorinated LNP transfection, the greater the difference in peak area of ​​the target fluorine spectrum before and after transfection. The model fitting coefficient of linear regression analysis was 0.9629, indicating that the two had a good correlation, that is, the difference in peak area of ​​the target fluorine spectrum before and after transfection can be used to predict the transfection efficiency of fluorinated lipid nanoparticles.

[0071] Example 5: Detecting the nuclear magnetic resonance fluorine spectra of fluorinated LNPs before and after injection into mice and the corresponding protein expression. The specific steps are as follows:

[0072] (1) collecting a nuclear magnetic resonance fluorine spectrum of the LNP-75% F12C obtained in Example 1 to obtain a nuclear magnetic resonance fluorine spectrum of the fluorinated LNP before injection; ( Figure 5 , LNP-75% F12C)

[0073] (2) The LNP-75% F12C obtained in Example 1 was injected into the gastrocnemius muscle of Balb / c nude mice, with 50 μL injected into the gastrocnemius muscle on one side. Mice that received an intramuscular injection of 50 μL PBS served as a control group. 48 hours later, the mice were anesthetized by intraperitoneal injection of 150 μL 1% sodium pentobarbital solution. The NMR fluorine spectrum of the mice's legs was collected using a 400 MHz AVANCE NEO NMR spectrometer, and the NMR fluorine spectrum after injection was obtained ( Figure 5 , in vivo);

[0074] The sampling sequence was a rapid spin echo (RARE) sequence with the following sampling parameters: repetition time: 500 ms; average number: 18,000; repetition number: 1.

[0075] (3) The mice were dissected and their gastrocnemius muscle tissues were fixed in 4% paraformaldehyde solution for 24 h. The gastrocnemius muscle tissues were then embedded in paraffin and sliced, and immunofluorescence staining was performed. Subsequently, the EGFP protein expressed in the gastrocnemius muscle tissues was observed using a confocal fluorescence microscope ( Figure 6 , where the control group is the control group).

[0076] Analysis of results: The peak area difference of the target fluorine spectrum of the fluorinated LNP before and after injection into mice was large, with signal-to-noise ratios (SNRs) of 48.58 and 5.54, respectively, demonstrating that the release of nucleic acids and fluorinated lipids was complete, consistent with the results of the cell experiments. Secondly, immunofluorescence staining of mouse gastrocnemius muscle sections showed a large amount of EGFP protein expression in the muscle tissue, demonstrating the superiority of this vector in delivering nucleic acids and expressing proteins. These results demonstrate that magnetic resonance fluorine spectroscopy can be used to predict the transfection efficiency of fluorinated lipid nanoparticles in vivo.

Claims

1. A method for predicting the transfection efficiency of fluorinated lipid nanoparticles using multi-nuclear magnetic resonance imaging, characterized in that: The method comprises the following steps: (1) Lysing untransfected cells to obtain cell lysate, mixing the cell lysate with fluorinated lipid nanoparticles, and performing nuclear magnetic resonance fluorine spectrum detection to obtain a nuclear magnetic resonance fluorine spectrum of the control group; (2) After transfecting cells with fluorinated lipid nanoparticles, the cells are lysed to obtain transfected cell lysate, and nuclear magnetic resonance fluorine spectrum detection is performed to obtain the nuclear magnetic resonance fluorine spectrum of the experimental group; (3) obtaining the target peak of the fluorinated lipid from the nuclear magnetic resonance fluorine spectra of the experimental group and the control group obtained in step (1) and step (2), and calculating the difference in the target peak area of ​​the fluorinated lipid in the experimental group and the control group; (4) using a confocal fluorescence microscope to observe the transfection status of the fluorinated lipid nanoparticles after cell transfection in step (2), and quantitatively analyzing the fluorescence intensity; (5) selecting fluorinated lipid nanoparticles with different fluorinated lipid contents to perform the above steps (1)-(4); (6) Plotting a curve with the difference in target peak area of ​​fluorinated lipids between the experimental group and the control group as the horizontal axis and the fluorescence intensity after cell transfection as the vertical axis to obtain the linear curves of the two; (7) subjecting the fluorinated lipid nanoparticles to be tested to the above steps (1)-(3), and obtaining the corresponding transfection efficiency using the curve of step (6); The fluorinated lipid nanoparticles are obtained by conventional methods, and their composition at least includes fluorinated lipids and plasmids. The structure of the fluorinated lipids is as follows: wherein Rf is selected from perfluoro-tert-butyloxy, trifluoroethoxy, difluoroethoxy, pentafluorothiobenzyloxy, p-fluorobenzyloxy, difluorobenzyloxy, bis-trifluoromethylbenzyloxy, trifluoromethoxybenzyloxy, trifluoromethylthiobenzyloxy and -O(CH2)2(CF2) n Any linear polyfluoroalkyl group of CF3 structure, wherein n is a natural number of 3-20.

2. The method according to claim 1, characterized in that The cells in the method are replaced with living experimental mice. In this case, the above steps (1) and (2) are respectively: (1-1) performing nuclear magnetic resonance fluorine spectrum detection on the fluorinated lipid nanoparticles to obtain a nuclear magnetic resonance fluorine spectrum of the control group; (2-1) Fluorinated lipid nanoparticles were injected into mice, and then the injection site was cultured for a period of time to obtain the nuclear magnetic resonance fluorine spectrum of the experimental group.

3. The method according to claim 1, characterized in that Before performing the detection in step (1) and step (2), an external standard dissolved in a deuterated reagent is added to the corresponding cell lysate, wherein the deuterated reagent includes any one of deuterated water, deuterated methanol, deuterated acetonitrile, and deuterated dimethyl sulfoxide; The external standard is any one of trifluoroacetic acid, 2-chloro-3-fluoropyridine, sodium trifluoromethanesulfonate, and 4-fluorobenzoic acid.

4. The method according to claim 3, characterized in that The concentration of the external standard dissolved in the deuterated reagent is 0.5-5 mM; in the steps (1) and (2), the volume ratio of the corresponding cell transfection solution to the external standard is 1: (0.02-0.4).

5. The method according to any one of claims 1 to 4, characterized in that The frequency of the nuclear magnetic resonance instrument used in the nuclear magnetic resonance fluorine spectrum test is 400-600MHz.

6. The method according to claim 5, characterized in that The nuclear magnetic resonance fluorine spectrum detection adopts a zgig pulse sequence or a fast spin echo sequence, and the test temperature is 297±2K; and / or The pulse width during nuclear magnetic resonance fluorine spectrum detection is 11-12 μs; and / or The relaxation time during nuclear magnetic resonance fluorine spectrum detection is 10-20s; and / or The spectrum width of the nuclear magnetic resonance fluorine spectrum detection is 7000-8000 Hz; and / or The radio frequency center frequency during the nuclear magnetic resonance fluorine spectrum detection is -45000 to -35000 Hz; and / or The number of collection 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 sampling times during the nuclear magnetic resonance fluorine spectrum detection is 20-40 times; and / or The number of empty scans during the nuclear magnetic resonance fluorine spectrum detection is 3-6 times; and / or The gain during the nuclear magnetic resonance fluorine spectrum detection is 60-150.

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