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EP-4737891-A1 - METHOD FOR PREDICTING TRANSFECTION EFFICIENCY OF FLUORINATED LIPID NANOPARTICLES USING NUCLEAR MAGNETIC RESONANCE

EP4737891A1EP 4737891 A1EP4737891 A1EP 4737891A1EP-4737891-A1

Abstract

The present invention relates to the field of analytical detection, and provides a method for predicting the transfection efficiency of fluorinated lipid nanoparticles (FLNPs) by utilizing nuclear magnetic resonance. The method includes the following steps repeated for multiple FLNPs containing different fluorine contents: (1) performing 19F magnetic resonance spectroscopy (19F MRS) on the FLNPs to obtain a fluorine spectrum of a control group; (2) transfecting the FLNPs into cells or living mice and performing 19F MRS to obtain a fluorine spectrum of an experimental group; (3) observing the transfection of the FLNPs using a confocal fluorescence microscope to quantify fluorescence intensity; (4) generating a calibration curve correlating post-transfection fluorescence intensity with the difference in the target peak area of the fluorinated lipid between the experimental and control groups; and (5) predicting FLNPs transfection using the calibration curve. This approach demonstrates excellent specificity, reproducibility, stability, and linearity, providing a robust technical foundation for large-scale screening of FLNPs-based nanomedicines.

Inventors

  • ZHOU, XIN
  • XIE, Kairu
  • CHEN, Daiqin
  • JIANG, Zhongxing
  • ZHU, LIJUN
  • CHEN, Shizhen

Assignees

  • Innovation Academy for Precision Measurement Science and Technology, CAS

Dates

Publication Date
20260506
Application Date
20251023

Claims (6)

  1. A method for predicting transfection efficiency of fluorinated lipid nanoparticles (FLNPs) by utilizing multi-nuclear magnetic resonance, comprising the following steps: (1) lysing untransfected cells to obtain a cell lysate, mixing the cell lysate with the FLNPs, and performing 19 F magnetic resonance spectroscopy (MRS) to obtain the fluorine spectrum of a control group; (2) transfecting the FLNPs into cells, lysing the transfected cells to obtain a lysate, and performing 19 F MRS to obtain the fluorine spectrum of an experimental group; (3) identifying the characteristic fluorine peak of the fluorinated lipid from both the control and experimental 19 F MRS obtained in steps (1) and (2), and calculating the difference in the target peak area between the two groups; (4) observing the transfection of the FLNPs following step (2) using a confocal fluorescence microscope, and quantifying the fluorescence intensity; (5) repeating steps (1)-(4) using FLNPs containing different fluorine contents; (6) plotting a calibration curve with the difference in the target peak area of the fluorinated lipid between the experimental and control groups on the x-axis, and the corresponding post-transfection fluorescence intensity on the y-axis, to establish a linear correlation between the two parameters; and (7) for any FLNPs to be evaluated, performing steps (1)-(3) and determining their transfection efficiency based on the calibration curve obtained in step (6); wherein the FLNPs are obtained by a conventional method, and their composition at least comprises fluorinated lipids and plasmids, and a structure of the comprised fluorinated lipids is as follows: wherein, Rf is selected from any one of perfluoro- tert -butoxy, trifluoroethoxy, difluoroethoxy, pentafluorothiobenzyloxy, p-fluorobenzyloxy, difluorobenzyloxy, bistrifluoromethylbenzyloxy, trifluoromethoxybenzyloxy, trifluoromethylthiobenzyloxy, and linear polyfluoroalkyl having a structure of -O(CH 2 ) 2 (CF 2 )nCF 3 , wherein n is a natural number of 3 to 20.
  2. The method according to claim 1, wherein the cells in the method are replaced with living experimental mice, and in this case, the aforementioned steps (1) and (2) are respectively: (1-1) performing 19 F MRS on the FLNPs to obtain the fluorine spectrum of the control group; and (2-1) injecting the FLNPs into mice, and after a defined incubation period, performing 19 F MRS at the injection site to obtain the fluorine spectrum of the experimental group.
  3. The method according to claim 1, wherein before the detection in the step (1) and the step (2), an external standard dissolved in a deuterated reagent is added to the corresponding cell lysate, wherein the deuterated reagent comprises any one of deuterated water, deuterated methanol, deuterated acetonitrile, and deuterated dimethyl sulfoxide; and 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, wherein a concentration of the external standard dissolved in the deuterated reagent is 0.5 to 5 mM; and in the step (1) and the step (2), a volume ratio of the corresponding cell lysate to the external standard is 1:(0.02 to 0.4).
  5. The method according to any one of claims 1-4, wherein a frequency of the nuclear magnetic resonance spectrometer used in the nuclear magnetic resonance fluorine spectrum test is 400 to 600 MHz.
  6. The method according to claim 5, wherein the nuclear magnetic resonance fluorine spectrum detection adopts a zgig pulse sequence or a rapid acquisition with relaxation enhancement sequence, and a test temperature is 297 ± 2 K; and/or a pulse width during the nuclear magnetic resonance fluorine spectrum detection is 11 to 12 µs; and/or a relaxation time during the nuclear magnetic resonance fluorine spectrum detection is 10 to 20 s; and/or a spectral width of the nuclear magnetic resonance fluorine spectrum detection is 7,000 to 8,000 Hz; and/or a radio frequency center frequency during the nuclear magnetic resonance fluorine spectrum detection is -45,000 to -35,000 Hz; and/or a number of acquisition points during the nuclear magnetic resonance fluorine spectrum detection is 125,000 to 135,000; and/or a sampling time during the nuclear magnetic resonance fluorine spectrum detection is 7 to 10 s; and/or the number of samplings during the nuclear magnetic resonance fluorine spectrum detection is 20 to 40; and/or the number of empty scans during the nuclear magnetic resonance fluorine spectrum detection is 3 to 6; and/or a gain during the nuclear magnetic resonance fluorine spectrum detection is 60 to 150.

Description

TECHNICAL FIELD The present invention relates to the technical field of analytical detection, and in particular to a method for predicting transfection efficiency of fluorinated lipid nanoparticles by utilizing multi-nuclear magnetic resonance. BACKGROUND Nucleic acid therapies have great potential in the treatment and prevention of diseases. Messenger ribonucleic acid-based technologies have achieved considerable clinical effects in vaccines, protein supplementation therapies, and gene editing therapies (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 efficient intracellular delivery. Lipid nanoparticles (LNPs) are the most advanced non-viral nucleic acid delivery platform in clinic currently. Studies have shown that only a small amount of nucleic acids carried by the 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 the LNPs, fluorinated lipids are used for improving the efficiency of nucleic acid delivery in cells, which is mainly attributable to the fact that the fluorinated lipids added into fluorinated LNPs can significantly promote the cellular uptake and lysosomal escape of the 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 will release the carried nucleic acids and fluorinated lipids, which facilitates the subsequent nucleic acid transfection and protein expression. Therefore, the release of the fluorinated lipids is correlated with the subsequent protein expression. Currently, the most intuitive method for detecting LNP transfection efficiency at an in vitro level 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 commonly used method for detecting LNP transfection efficiency at an in vivo level is to inject LNPs into mice for a certain period of time, and then acquire tissues from the injection sites 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 number of LNPs can be screened in vivo. SUMMARY Based on the aforementioned situation, an objective of the present invention is to provide a method for predicting transfection efficiency of fluorinated lipid nanoparticles (FLNPs) by detecting fluorine spectra before and after transfection into cells or living mice using magnetic resonance spectroscopy (MRS). This method addresses limitations of conventional techniques, which often involve inaccurate measurements and complex procedures. The proposed approach enables simple, rapid, and accurate prediction of FLNPs transfection efficiency, facilitating large-scale screening of FLNPs in non-human primate models. To achieve the objective of the present invention, the following technical solution is proposed: The present invention provides a method for predicting transfection efficiency of FLNPs using multi-nuclear magnetic resonance analysis. The method includes the following steps: (1) lysing untransfected cells to obtain a cell lysate, mixing the cell lysate with the FLNPs, and performing 19F MRS to obtain the fluorine spectrum of a control group;(2) transfecting the FLNPs into cells (preferably 293T cells), lysing the transfected cells to obtain a lysate, and performing 19F MRS to obtain the fluorine spectrum of an experimental group;(3) identifying the characteristic fluorine peak of the fluorinated lipid from both the control and experimental 19F MRS obtained in steps (1) and (2), and calculating the difference in the target peak area between the two groups;(4) observing the transfection of the FLNPs following step (2) using a confocal fluorescence microscope, and quantifying the fluorescence intensity;(5) repeating steps (1)-(4) using FLNPs containing different fluorine contents;(6) plotting a calibration curve with the difference in the target peak area of the fluorinated lipid between the experimental and control groups on the x-axis, and the corresponding post-transfection fluorescence intensity on the y-axis, to establish a linear correlation between the two parameters; and(7) for any FLNPs to be evaluated, performing steps (1)-(3) and determining their transfection efficiency based on the calibration curve obtained in step (6). In the aforementioned method of the present invention, the cells can also be replaced with living experimental mice, and in this case, the aforementioned steps (1) and (2) are respectively: (1-1) performing 19F MRS on the FLNPs to obtain the fluorine spectrum of the control group; and(2-1) injecting the FLNPs into mice, and after a defined incubation period, performing 19F MRS at