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

Abstract

The invention relates to the technical field of analysis and detection, and particularly provides a method for predicting the transfection efficiency of fluorinated lipid nanoparticles by using multi-nuclear magnetic resonance, and the method comprises the following steps: (1) carrying out nuclear magnetic resonance fluorine spectrum detection on fluorinated nanoparticles to obtain a nuclear magnetic fluorine spectrum of a control group; (2) transfecting the fluorinated nanoparticles into a cell or a mouse living body, and performing nuclear magnetic resonance fluorine spectrum detection on the cell or the living body to obtain a nuclear magnetic fluorine spectrum of an experimental group; and (3) calculating the difference value of the target peak areas of the fluorinated lipid in the experimental group and the control group to predict the transfection efficiency of the fluorinated lipid nanoparticles. The method has good performance in the aspects of specificity, repeatability, stability, linear relation and the like, and can provide effective technical support for large-scale screening of nano medicaments of fluorinated lipid nanoparticles.

Description

Technical Field

[0001] The 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 the treatment and prevention of diseases. Messenger RNA-based technologies have achieved considerable clinical results in vaccines, protein supplementation therapy, and gene editing therapy (Han X, Xu J, Xu Y, et al. Nat Commun. 2024; 15(1): 1762.). However, nucleic acid is a negatively charged and unstable macromolecule that requires a carrier 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 LNP 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 LNP, fluorinated lipids are used to improve the transport efficiency of nucleic acids in cells, which 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 LNP successfully escapes the lysosome, it will release the carried nucleic acid and fluorinated lipids, which is helpful for subsequent nucleic acid transfection and protein expression, so the release of fluorinated lipids is correlated with subsequent protein expression.

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

[0005] Based on the above situation, the purpose of the present invention is to provide a method for predicting the transfection efficiency of fluorinated lipid nanoparticles by detecting the fluorine spectrum of fluorinated nanoparticles before and after transfection into cells or mice in vivo by nuclear magnetic resonance. This method can overcome the problems of inaccurate detection of transfection efficiency of fluorinated nanoparticles and cumbersome operation in the prior art. It can more simply, quickly and accurately predict the transfection efficiency of fluorinated lipid nanoparticles for 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, comprising the following steps:

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

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

[0010] (3) obtaining the target peak of the fluorinated lipid from the NMR 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 the cell transfection in step (2), and quantitatively analyzing the fluorescence intensity;

[0012] (5) selecting fluorinated lipid nanoparticles having different fluorinated lipid contents to carry out the above steps (1) to (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 coordinate and the fluorescence intensity after cell transfection as the vertical coordinate to obtain linear curves for both;

[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 may 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 a control group;

[0017] (2-1) After the fluorinated lipid nanoparticles are injected into mice, the injection site is cultured for a period of time and then the nuclear magnetic resonance fluorine spectrum is 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 of the linear polyfluoroalkyl groups of CF3 structure, wherein n is a natural number of 3 to 20, and 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-((undecanyloxy)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 encoding 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-((undecanyloxy)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] The external standard comprises any one of trifluoroacetic acid, 2-chloro-3-fluoropyridine, sodium trifluoromethanesulfonate, and 4-fluorobenzoic acid, preferably sodium trifluoromethanesulfonate, and the chemical shift of its target peak is -75ppm to -85ppm, preferably -78ppm to -80ppm. As the preferred technical solution of the present invention, the stability and accuracy of the characteristic peak target integral result can be guaranteed by using sodium trifluoromethanesulfonate as the external standard. Moreover, sodium trifluoromethanesulfonate and di(12-(perfluorotert-butoxy)dodecyl)amine only produce one corresponding fluorine signal, which is close and completely separated, and there is no interference phenomenon, which further improves the accuracy of the 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 instrument 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 125000-135000, preferably 130000-132000; and / or

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

[0032] The sampling times of 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 the 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 the detection of fluorinated lipid content by nuclear magnetic resonance fluorine spectrum. 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 ensured by selecting a suitable external standard sodium trifluoromethanesulfonate, and the accuracy of the fluorinated lipid content determination is ensured at the same time. The method has good performance in terms of specificity, precision, repeatability, stability, linear relationship, etc., which not only meets the prediction of the transfection efficiency of fluorinated LNP at the cell and in vivo level, 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 fluorinated LNPs before and after transfection of cells in Example 2;

[0039] Figure 2 This is a representative confocal image of cells transfected with fluorinated LNP in Example 3;

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

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

[0042] Figure 5 This is the fluorine NMR spectrum before and after the fluorinated LNP was injected into the gastrocnemius muscle of mice;

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

[0044] The technical solution of the present invention is described in detail below in conjunction with 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 (Article No. AR0102). Sodium trifluoromethanesulfonate was purchased from Beijing Bailingwei. 5mm NMR tubes were purchased from Wilmad, USA. 500MHz and 400MHz AVANCE NEO NMR instruments were purchased from Bruker, Switzerland. 1mm 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, distearoyl phosphatidylcholine and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were purchased from Ruixi Biotechnology, and cholesterol was purchased from Sigma.

[0046] Prepare pEGFP plasmid solution containing EGFP protein: Use a high-purity plasmid extraction kit to extract the EGFP expression plasmid from Escherichia coli, and then dilute it to 1 mg / mL with pH=3, 50 mM citrate buffer 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-(perfluoro-tert-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-PEG 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. Then it was diluted with 1×PBS (0.0067M, the same below) for subsequent cell experiments. The molar ratios of F12C, SM102, DSPC, Cholesterol and DMG-PEG2000 are shown in Table 1 (for 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, and the other groups kept the amount of Chol and DMG-PEG2000 unchanged, and adjusted the amount of other substances):

[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 cell. The specific steps are as follows:

[0055] (1) Using the LNP-25% F12C, LNP-50% F12C and LNP-75% F12C prepared in Example 1, 293T cells were transfected respectively (pEGFP in each group of LNPs was 98 μg), and about 5E+7 cells were collected from each group after 48 h, and the cells were lysed using 1 mL RIPA lysis buffer, respectively. After centrifugation at 12000 g, the supernatant was taken and loaded into a new EP tube for standby use, thereby obtaining the cell lysate of the LNP-25% F12C experimental group, the cell lysate of the LNP-50% F12C experimental group and the cell lysate 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 cell lysate of the control group;

[0057] (3) Preparation of capillary external standard: 0.172 mg of sodium trifluoromethanesulfonate was dissolved in 1 mL of deuterated water to prepare a sodium trifluoromethanesulfonate solution with a concentration of 1 mM. 50 μL of the solution was taken using a microsyringe and loaded into a capillary with a diameter of 1 mm. Both ends of the capillary were sealed to obtain the sodium trifluoromethanesulfonate external standard.

[0058] (4) Take 500 μL of the experimental group cell lysate obtained in step (1) and mix it with the sodium trifluoromethanesulfonate external standard obtained in step (3) and transfer it 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. Use a 500 MHz VANCE NEO nuclear magnetic resonance instrument to perform nuclear magnetic resonance detection to obtain the nuclear magnetic fluorine spectra of the LNP-25% F12C experimental group, the LNP-50% F12C experimental group and the LNP-75% F12C experimental group ( 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 5 mm diameter nuclear magnetic resonance tube to obtain LNP-25% F12C control group test solution, LNP-50% F12C control group test solution and LNP-75% F12C control group test solution. A 500 MHz VANCE NEO nuclear magnetic resonance instrument was used to perform nuclear magnetic resonance detection to obtain the nuclear magnetic fluorine spectra of the LNP-25% F12C control group, the LNP-50% F12C control group and the LNP-75% F12C control group ( Figure 1 );

[0060] In this embodiment, the sampling parameters of the nuclear magnetic resonance fluorine spectrum 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 collection 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 target peaks of 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 is 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 The cell transfection efficiency of different fluorinated lipid nanoparticles was observed using a confocal fluorescence microscope. The specific steps are as follows:

[0066] (1) The lipid nanoparticles prepared in Example 1 were used to transfect 293T cells. 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 incubating the cells at 37°C and 5% CO2 for 48 hours, the culture medium was removed, the cells were fixed with paraformaldehyde, and the cell nuclei were stained with DAPI stain. Fluorescence visualization was captured using a fluorescent confocal microscope ( Figure 2 , scale bar = 100 μm).

[0067] (2) Use Image J software to analyze the fluorescence expression intensity of the fluorescent images obtained. Figure 3 ).

[0068] Example 4 The linear relationship between the peak area difference of the target fluorine spectrum before and after the fluorinated LNP transfection of cells and the fluorescence intensity is as follows:

[0069] The difference in target peak area of ​​the nuclear magnetic resonance fluorine spectrum before and after the fluorinated LNP transfection cells obtained in Example 2 was used as the x-axis, and the fluorescence expression intensity after the fluorinated LNP transfection cells obtained in Example 3 was used as the y-axis (LNP-SM102 does not contain fluorinated lipids, that is, the x-axis is counted as 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 peak area difference 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 peak area difference 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 LNP 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, and 50 μL was injected into the gastrocnemius muscle of the unilateral side. Mice injected with 50 μL PBS intramuscularly were used as the control group. After 48 hours, the mice were anesthetized by intraperitoneal injection of 150 μL 1% sodium pentobarbital solution, and the nuclear magnetic resonance fluorine spectrum of the mouse legs was collected using a 400MHz AVANCE NEO nuclear magnetic resonance instrument to obtain the nuclear magnetic resonance fluorine spectrum after injection ( Figure 5 , in vivo);

[0074] The sampling sequence was a rapid spin echo sequence (RARE) with the following sampling parameters: repetition time: 500 ms; average times: 18,000; repetition times: 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] Results analysis: The peak area difference of the target fluorine spectrum of fluorinated LNP before and after injection into mice was large, and the signal-to-noise ratio (SNR) was 48.58 and 5.54, respectively, proving that it completed the release of nucleic acids and fluorinated lipids, which was consistent with the results of cell experiments. Secondly, the results of immunofluorescence staining of mouse gastrocnemius sections showed that a large amount of EGFP protein expression appeared in the muscle tissue, proving the superiority of the vector in delivering nucleic acids and expressing proteins. The above results prove 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 transfection efficiency of fluorinated lipid nanoparticles using multi-nuclear magnetic resonance, characterized in that: The method comprises the following steps: (1) Lysing the untransfected cells to obtain a cell lysate, mixing the cell lysate with the fluorinated lipid nanoparticles, and performing nuclear magnetic resonance fluorine spectrum detection to obtain a nuclear magnetic resonance fluorine spectrum of a control group; (2) After the cells are transfected with fluorinated lipid nanoparticles, the cells are lysed to obtain a transfected cell lysate, and a nuclear magnetic resonance fluorine spectrum is detected to obtain a nuclear magnetic resonance fluorine spectrum of the experimental group; (3) obtaining the target peak of the fluorinated lipid from the NMR 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; (4) using a confocal fluorescence microscope to observe the transfection status of the fluorinated lipid nanoparticles after the cell transfection in step (2), and quantitatively analyzing the fluorescence intensity; (5) selecting fluorinated lipid nanoparticles having different fluorinated lipid contents to carry out the above steps (1) to (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 coordinate and the fluorescence intensity after cell transfection as the vertical coordinate to obtain linear curves for both; (7) Subjecting the fluorinated lipid nanoparticles to be tested to the above steps (1)-(3), and using the curve of step (6) to obtain the corresponding transfection efficiency.

2. The method according to claim 1, characterized in that The cells in the method are replaced with living experimental mice. At this time, 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 a control group; (2-1) After the fluorinated lipid nanoparticles are injected into mice, the injection site is cultured for a period of time and then the nuclear magnetic resonance fluorine spectrum is detected to obtain the nuclear magnetic resonance fluorine spectrum of the experimental group.

3. The method according to claim 1 or 2, characterized in that: 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 and other fluorobenzyloxy, bis-trifluoromethylbenzyloxy, trifluoromethoxybenzyloxy, trifluoromethylthiobenzyloxy and -O(CH2)2(CF2) n Any of the linear polyfluoroalkyl groups of CF3 structure, wherein n is a natural number of 3-20.

4. The method according to claim 1, characterized in that: 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; The external standard is any one of trifluoroacetic acid, 2-chloro-3-fluoropyridine, sodium trifluoromethanesulfonate, and 4-fluorobenzoic acid.

5. The method according to claim 4, 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).

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

7. The method according to claim 6, 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 125000-135000; 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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