A lipid nanoparticle complex, and methods of making and using the same

Abstract

The application discloses a kind of lipid nanoparticle complex and its preparation method and application, belong to molecular biology technical field.The method includes: after CdSe / ZnS quantum dot is treated by removing oleic acid ligand, with mercapto seven glycol monomethyl ether reaction, methoxyl polyethylene glycol modified water-soluble quantum dot is obtained;The water-soluble quantum dot is mixed with multi-thiol phosphonate single-stranded DNA, and DNA-quantum dot complex is formed;The DNA-quantum dot complex is mixed with complex lipid by microfluidic, and lipid nanoparticle complex is formed.The quantum dot of the application is encapsulated in the interior of lipid nanoparticle, avoids the interference of surface marker to the original property of nanoparticle, combines super-resolution imaging and single particle tracking technology, realizes real-time observation and analysis to single lipid nanoparticle transmembrane movement process under nanometer scale, and can be used for lipid nanoparticle formula screening, nucleic acid drug delivery efficiency evaluation and intracellular endocytosis mechanism research.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a lipid nanoparticle complex, its preparation method, and its application. Background Technology

[0002] Lipid nanoparticles (LNPs), as one of the breakthrough nanodelivery systems in recent years, have shown revolutionary application prospects in the biomedical field. A successful example is their role as a core delivery carrier for messenger RNA (mRNA) vaccines, playing a crucial role in addressing global public health crises. Furthermore, LNPs have also shown great potential in the targeted delivery of siRNA drugs, gene editing tools, and traditional chemotherapy drugs. However, the clinical translation and further optimization of LNPs still face significant challenges, one of the fundamental bottlenecks being insufficient understanding of the microscopic mechanisms of their intracellular transport behavior. The efficacy of LNPs highly depends on their completion of a complex journey from the extracellular to the intracellular target site, including key steps such as interaction with the cell membrane, endocytosis, escape from endocytic vesicles, and release of the payload in the cytoplasm. Among these, the selectivity of the endocytic pathway, the behavior within the vesicles after endocytosis, and the crucial endosome escape efficiency and kinetics are the core factors determining the success or failure of delivery. Unfortunately, due to the limitations of the spatiotemporal resolution of traditional research techniques, there is currently a lack of precise, quantitative, and real-time analytical capabilities for these microscopic, dynamic, and heterogeneous processes. As a result, the design of LNPs largely relies on empirical trial and error, making it difficult to rationally guide the optimization of their composition and structure.

[0003] The emergence of super-resolution fluorescence microscopy has provided a powerful tool for overcoming the aforementioned bottlenecks. It breaks the diffraction limit of traditional optical microscopes, elevating spatial resolution to the nanoscale, making it possible to directly visualize subcellular structures and nanoparticles within living cells. However, existing technologies still have the following drawbacks: Traditional optical microscopes are limited by the diffraction limit, with a spatial resolution of only about 200 nanometers, making it difficult to clearly observe LNPs, which are generally smaller than 100 nanometers, and the fine micro-domain structures on the cell membrane; although electron microscopes have nanoscale spatial resolution, they need to operate in a vacuum environment, making it impossible to perform real-time dynamic observation of living cells, and the complex sample pretreatment can easily introduce artifacts; although flow cytometry can achieve population statistical analysis, sample preparation may damage cells, and it is difficult to achieve dynamic tracking at the single-particle level; traditional fluorescent dyes are prone to photobleaching and fluorescence quenching, which is not conducive to long-term dynamic imaging, and existing fluorescent labels often modify the surface of nanoparticles, which may change the original physicochemical properties of LNP surfaces, thereby affecting their biological behavior. Therefore, how to observe and analyze the complete transmembrane motion process of a single lipid nanoparticle in real time with high spatiotemporal resolution over a long period of time at the nanoscale has become an urgent technical challenge. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for preparing lipid nanoparticle complexes. This invention encapsulates quantum dots inside lipid nanoparticles, avoiding the interference of traditional surface labeling on the chemical properties and biological functions of the outer layer, and more accurately reflects the surface properties and cell interaction mechanisms of LNPs themselves, reducing the biological behavior bias introduced by labeling.

[0005] Another objective of this invention is to provide a single-particle super-resolution tracking and analysis method for transmembrane dynamics of lipid nanoparticles. This method can spatially locate and dynamically track individual lipid nanoparticle complexes, distinguish different endocytic pathways, quantify motion trajectories and velocities, and detect key events such as endosome escape.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing lipid nanoparticle complexes, comprising the following steps: Oleic acid-modified CdSe / ZnS quantum dots were treated to remove oleic acid ligands, resulting in quantum dot precipitates. After dissolving the quantum dot precipitate, mercaptoheptaethylene glycol monomethyl ether was added to react and obtain water-soluble quantum dots modified with methoxy polyethylene glycol. The water-soluble quantum dots are mixed with polythiol phosphate single-stranded DNA to form a DNA-quantum dot complex; The DNA-quantum dot complex and the composite lipid were mixed by microfluidics and self-assembled to form a lipid nanoparticle complex.

[0007] Preferably, the oleic acid ligand removal treatment includes: adding anhydrous ethanol to oleic acid-modified CdSe / ZnS quantum dots, shaking, centrifuging and washing at high speed, repeating the washing process, and then drying.

[0008] Preferably, the sequence of the polythiol phosphate single-stranded DNA includes a polythiol-modified adenine deoxynucleotide segment and a negatively charged single-stranded nucleotide segment.

[0009] More preferably, the sequence of the polythiol phosphate single-stranded DNA is shown in SEQ ID NO.1.

[0010] Preferably, the composite lipid comprises ionizable cationic lipids, distearate phosphatidylcholine, cholesterol, and polyethylene glycol-modified lipids.

[0011] Preferably, the ionizable cationic lipid is DLin-MC3-DMA, and the PEGylated lipid is dimyristic glycerol-polyethylene glycol 2000.

[0012] More preferably, in the microfluidic mixing, the volume ratio of the aqueous phase flow rate to the oil phase flow rate is 2:1 to 5:1.

[0013] The present invention also provides a lipid nanoparticle complex prepared by the aforementioned preparation method.

[0014] This invention also provides a single-particle super-resolution tracking and analysis method for transmembrane dynamics of lipid nanoparticles, comprising the following steps: The lipid nanoparticle complex was co-incubated with the cells to be tested, and the cells were imaged in real time using a structured light illumination microscope to acquire time-series images. The motion trajectory and coordinate information of a single lipid nanoparticle complex in the time series image are tracked. Based on the coordinate information, the mean square displacement and instantaneous velocity of each lipid nanoparticle complex are calculated; The transmembrane motion stage of the lipid nanoparticle complex is determined based on the mean square displacement curve, the instantaneous velocity, and the changing trend of the fluorescence intensity.

[0015] Preferably, the determination of the mean square displacement curve includes: when the mean square displacement curve shows an upward curving trend, it is determined to be directional motion; when the mean square displacement curve is approximately a straight line, it is determined to be random diffusion; when the mean square displacement curve shows a downward curving trend, it is determined to be restricted motion.

[0016] Compared with the prior art, the present invention has the following advantages: (1) This invention obtains a lipid nanoparticle complex (LNP@QD) by encapsulating quantum dots inside lipid nanoparticles. Transmission electron microscopy characterization confirms that the quantum dots are located inside the lipid nanoparticle complex rather than on the surface, successfully avoiding the alteration of the physicochemical properties of the nanoparticle surface by exogenous markers in traditional surface labeling methods, and truly restoring the cell interaction behavior of the lipid nanoparticles themselves. Dynamic light scattering analysis shows that the average particle size of the lipid nanoparticle complex is about 70 nm, and the polydispersity index (PDI) is less than 0.15, indicating that the obtained lipid nanoparticle complex has a highly uniform particle size distribution.

[0017] (2) This invention uses structured illumination microscopy (SIM) combined with the excellent photostability of quantum dots to achieve continuous imaging for 60 minutes at a acquisition rate of 1 frame / second. This overcomes the defects of traditional organic dyes, which are easy to photobleach and cannot be tracked for a long time. For the first time, it captures the complete transmembrane process of a single lipid nanoparticle from the cell membrane surface to the intracellular directional transport at the living cell level, including attachment, invading, internalization and directional transport.

[0018] (3) This invention quantitatively distinguishes different motion modes by analyzing the mean square displacement of the trajectory of a single particle: extracellular particles exhibit restricted motion, which transforms into directional motion after internalization. Statistical analysis shows that most particles exhibit directional motion after internalization, revealing the heterogeneity of the transmembrane transport process. Furthermore, by combining the instantaneous velocity-time relationship diagram and the fluorescence intensity-time relationship diagram, the transmembrane process can be divided into three stages: the endocytic site search period, the restricted motion period, and the cell internalization period. For the first time, a quantitative correlation between microscopic motion parameters and the process of endocytosis events has been established. Attached Figure Description

[0019] Figure 1 The results of agarose gel electrophoresis and transmission electron microscopy (TEM) imaging characterization of DNA-quantum dot complexes (DNA@QD).

[0020] Figure 2 The results are for the dynamic light scattering (DLS) and transmission electron microscopy (TEM) imaging characterization of LNP@QD.

[0021] Figure 3 The flowchart (A) for LNP@QD fabrication and the imaging operation diagram (B) are shown.

[0022] Figure 4 Screenshots and trajectory diagrams of LNP@QD movement along cellular microfilaments after internalization.

[0023] Figure 5 This describes the dynamics of LNP@QD particles and their trajectories.

[0024] Figure 6 The linear intensity profile of LNP@QD (t=111s).

[0025] Figure 7 This is a confocal microscopy image of the internalization time of LNP@QD, with the scale bar at 1 μm.

[0026] Figure 8 The figure shows the fluorescence intensity, diffusion rate distribution, and MSD analysis of LNP@QD. In the figure, A is the trajectory of LNP@QD, B is the fluorescence intensity and diffusion rate analysis of LNP@QD, C is the MSD analysis of stage I, and D is the MSD analysis of stages II and III.

[0027] Figure 9 The particle dynamics of LNP@QD on the cell membrane and its corresponding trajectory.

[0028] Figure 10 The plot shows the trajectory and velocity distribution of LNP@QD diffusion analyzed using MSD.

[0029] Figure 11Statistical analysis of LNP@QD internalization and intracytoplasmic movement; in the figure, A is the cell outline and the movement trajectory of LNP@QD in the cytoplasm, B is the frequency distribution histogram of diffusion index α obtained by mean square displacement (MSD) analysis of all LNP@QD trajectories, C is the frequency distribution histogram of the number of capture frames of all LNP@QD, and D is the scatter plot of the relationship between the number of capture frames of all LNP@QD and displacement. Detailed Implementation

[0030] This invention provides a method for preparing lipid nanoparticle complexes, comprising the following steps: Oleic acid-modified CdSe / ZnS quantum dots were treated to remove oleic acid ligands, resulting in quantum dot precipitates. After dissolving the quantum dot precipitate, mercaptoheptaethylene glycol monomethyl ether was added to react and obtain water-soluble quantum dots modified with methoxy polyethylene glycol. The water-soluble quantum dots are mixed with polythiol phosphate single-stranded DNA to form a DNA-quantum dot complex; The DNA-quantum dot complex and the composite lipid were mixed by microfluidics and self-assembled to form a lipid nanoparticle complex.

[0031] In this invention, to achieve efficient removal of oleic acid ligands from the surface of quantum dots for subsequent hydrophilic modification, the oleic acid-modified CdSe / ZnS quantum dots are treated as follows: A 3 mg / mL solution of oleic acid-modified CdSe / ZnS quantum dots (core-shell structure, emission wavelength approximately 605 nm) is taken, anhydrous ethanol is added, and the mixture is thoroughly shaken to ensure homogeneity. Ethanol, acting as an antisolvent, effectively reduces the solubility of quantum dots in the organic phase, promoting precipitation. The mixture is then centrifuged at high speed for 10-15 min, and the supernatant containing free oleic acid and organic solvent is discarded, retaining the precipitate. The washing steps of "adding anhydrous ethanol, shaking, and centrifuging" are repeated three times to ensure thorough removal of the oleic acid ligands and residual organic solvent. Finally, the precipitate is placed in a vacuum drying oven and dried overnight at room temperature to completely remove residual solvent, resulting in a dry, ligand-free quantum dot precipitate. The key to this step lies in separating the quantum dots from the original solution using an ethanol antisolvent precipitation method, and minimizing the interference of surface ligands on subsequent modification reactions through multiple washing and vacuum drying, thereby providing a clean and highly reactive quantum dot surface for subsequent methoxy polyethylene glycol (mPEG) modification. The oleic acid-modified CdSe / ZnS quantum dots used in this invention were purchased from Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd., catalog numbers Q1525 (emission wavelength 525nm±5nm) and Q1605 (emission wavelength 605nm±5nm). These quantum dots have a core-shell structure, a particle size of 7-12nm, a quantum dot yield greater than 80%, a concentration of 3mg / mL, and are produced using an organic solvent. It should be understood that the quantum dots of this invention are not limited to the specific sources and models mentioned above. Other core-shell structured oil-soluble quantum dots, such as InP / ZnS quantum dots and CdZnSe / ZnS quantum dots, can be used in this invention as long as surface ligands can be removed and subsequently modified to achieve water solubility. The emission wavelength of quantum dots can be selected according to experimental needs. For example, when used for multicolor imaging, quantum dots with different emission wavelengths can be selected to distinguish different labeled objects.

[0032] In this invention, to achieve methoxy polyethylene glycol (mPEG) modification on the surface of quantum dots and impart water solubility, the quantum dot precipitate after oleic acid ligand removal is preferably subjected to the following operation: the dried quantum dot precipitate is dissolved in chloroform, allowing the quantum dots to redisperse in the organic phase, resulting in a clear and transparent quantum dot chloroform solution; a 0.2 mg / mL tetrabutylammonium bromide (TBAB) chloroform solution is added to this solution as a phase transfer catalyst, and mercaptoheptaethylene glycol monomethyl ether is added, wherein the mercapto (-SH) end forms a stable coordination bond with the zinc atoms on the quantum dot surface, anchoring the mPEG molecules to the quantum dot surface, while the methoxy (-OCH3) end provides hydrophilicity. The quantum dots were capped with inert end-capsulation solution and stirred at room temperature to allow for complete ligand exchange. After the reaction, NaOH aqueous solution was added and the mixture was vortexed thoroughly. At this point, the mPEG ligands on the surface of the quantum dots made them hydrophilic, and with the assistance of the phase transfer catalyst TBAB, the quantum dots transferred from the chloroform phase to the aqueous phase. The upper aqueous phase was then separated by low-speed centrifugation, and the lower organic phase was discarded. The resulting aqueous phase was quickly transferred to a dialysis bag and dialyzed in ultrapure water. The ultrapure water was replaced every 12 hours to completely remove residual organic solvents, unreacted mPEG-SH, and salt impurities, ultimately yielding a purified aqueous solution of methoxy polyethylene glycol-modified water-soluble quantum dots (QD-mPEG).

[0033] In this invention, to achieve effective recombination of water-soluble quantum dots and polythiol phosphate single-stranded DNA to form a DNA-quantum dot complex suitable for subsequent lipid nanoparticle encapsulation, the following operation is preferably performed: 0.5 mL of a 1 μM polythiol phosphate single-stranded DNA solution is gradually added dropwise to the prepared QD-mPEG aqueous solution under stirring at room temperature. To achieve efficient recombination of quantum dots and lipid nanoparticles, this invention designs and synthesizes a polythiol phosphate single-stranded DNA with a bifunctional segment structure, the sequence of which includes a polythiol-modified adenine deoxynucleotide segment and a negatively charged single-stranded nucleotide segment. Specifically, the 5' end of the DNA sequence consists of multiple consecutive polythiol-modified adenine deoxynucleotides, where each A is chemically modified to introduce a thiol (-SH) group at the base or phosphate backbone position, forming a polyvalent thiol-anchored segment; the 3' end of the DNA sequence is a negatively charged single-stranded nucleotide segment of 15-20 nucleotides in length, preferably with the sequence GGCGAAGACAGCGGGTTGGG. This segment does not form a complex secondary structure, remains in a single-stranded state, and its phosphodiester backbone carries a dense negative charge in solution. In a specific embodiment of the present invention, the DNA sequence is preferably 5'-A. A A A A A A A A A A A A A A A A A A A GGCGAAGACAGCGGGTTGGG-3' (SEQ ID NO.1), where A The reaction mixture consists of thiol-modified adenine deoxynucleotides, each A carrying a thiol group, which can form stable coordination bonds with zinc atoms on the quantum dot surface. After addition, the mixture is stirred vigorously at 900 rpm overnight at room temperature to allow DNA molecules to fully bind to the quantum dot surface via multi-thiol anchoring. Simultaneously, the phosphate groups on the DNA backbone provide a large amount of negative charge, making the complex negatively charged overall. After the reaction, agarose gel electrophoresis is used to verify the binding effect of DNA and quantum dots. The reaction solution is loaded onto a 1% agarose gel, and under UV light, the quantum dot fluorescent bands and DNA bands are completely superimposed, with no free DNA band residue, indicating that DNA has been successfully bound to the quantum dot surface. Further characterization of the complex morphology using transmission electron microscopy reveals that the particle size is approximately 8 nm, the particles are uniformly distributed, and there is no obvious aggregation, confirming the successful preparation of the DNA-quantum dot complex.

[0034] In this invention, to achieve efficient encapsulation of DNA-quantum dot complexes within lipid nanoparticles, the DNA-quantum dot complex prepared above is mixed and assembled with composite lipids using microfluidic technology. The preferred specific operation is as follows: the DNA-quantum dot complex solution prepared above is quantified by fluorescence intensity and then diluted with citrate-sodium citrate buffer to prepare an aqueous phase; simultaneously, ionizable cationic lipids DLin-MC3-DMA, distearate phosphatidylcholine (DSPC), and cholesterol (Cholesterol) are added... A composite lipid-ethanol solution was prepared by dissolving a mixture of sterol and dimyristic glycerol-polyethylene glycol 2000 (DMG-PEG2000) in anhydrous ethanol, which served as the oil phase. A microfluidic preparation device (such as MPE-L2), whose core component is a microfluidic chip, was used to inject the aqueous phase and oil phase into the two inlets of the microfluidic chip, respectively, and control the flow rates of the aqueous phase and oil phase. The volume ratio of the aqueous phase flow rate to the oil phase flow rate was preferably 2:1 to 5:1, more preferably 3:1, so that the two phase solutions could be rapidly mixed in the microchannels of the chip. In this process, an acidic buffer (pH=4.0) protonates the ionizable cationic lipids, making them positively charged. These lipids then interact electrostatically with the negatively charged DNA-quantum dot complex, forming a complex core. Simultaneously, the lipid molecules self-assemble to form a lipid bilayer structure, encapsulating the DNA-quantum dot complex within. The mixed sample is collected in a centrifuge tube and immediately placed at 4°C. It is then dialyzed overnight with 1×PBS buffer to remove ethanol and adjust the pH to the physiological range (pH=7.4), thereby obtaining a stable lipid nanoparticle complex (LNP@QD). The resulting product is characterized, and dynamic light scattering (DLS) analysis shows an average particle size of approximately 70 nm.

[0035] This invention also provides a single-particle super-resolution tracking and analysis method for transmembrane dynamics of lipid nanoparticles, comprising the following steps: The lipid nanoparticle complex was co-incubated with the cells to be tested, and the cells were imaged in real time using a structured light illumination microscope to acquire time-series images. The motion trajectory and coordinate information of a single lipid nanoparticle complex in the time series image are tracked. Based on the coordinate information, the mean square displacement and instantaneous velocity of each lipid nanoparticle complex are calculated; The transmembrane motion stage of the lipid nanoparticle complex is determined based on the mean square displacement curve, the instantaneous velocity, and the changing trend of the fluorescence intensity.

[0036] In this invention, to achieve real-time dynamic observation of the interaction between lipid nanoparticle complexes and living cells, the following cell incubation and super-resolution imaging operations are preferably performed: The LNP@QD solution prepared above is diluted 20-100 times with preheated cell culture medium. The diluted LNP@QD solution is gently added to a glass-bottomed culture dish pre-cultured with HeLa cells. After addition, the culture dish is gently shaken to distribute the LNP@QD evenly. The culture dish is then placed on the stage of a structured light illumination microscope, and imaging is performed using a 60x oil immersion objective. The excitation light source is preferably a solid-state laser with a wavelength of 488nm, and the emitted light passes through a 525nm±10nm bandpass filter. The imaging parameters were set as follows: imaging field of view size of 1024×1024 pixels, exposure time of 100ms per frame, and temporal resolution of 1 frame / second. Time-series images were continuously acquired for 60 minutes, resulting in a total of 3600 frames. During the imaging process, the microscope's autofocus system maintained focal plane stability in real time, compensating for focal plane drift caused by cell movement or temperature fluctuations, ensuring that LNP@QD particles were always in a clear imaging state. The acquired raw images were stored in 16-bit TIFF format. Each frame contained fluorescence signals of multiple individual LNP@QD particles and cell background signals, providing high signal-to-noise ratio and high spatiotemporal resolution image data for subsequent single-particle tracking.

[0037] In this invention, to extract motion information of individual lipid nanoparticle complexes from acquired time-series images, single-particle tracking technology is used to process and analyze the image data. The preferred specific operation is as follows: The acquired time-series images are imported into ImageJ image analysis software, and after noise removal, the automatic tracking plugin of Plugins-Tracking-Trackmate is used to capture and connect individual particles. Simultaneously, an image with superimposed motion trajectories is generated through a visualization module, using different colors to distinguish the trajectories of different particles, and displaying the relative position of the trajectory to the cell membrane. For possible focal plane drift or tracking interruptions caused by particle movement along the Z-axis, the continuity of the trajectory is manually checked. Trajectories that clearly belong to the same particle but are broken due to temporary loss are manually connected to ensure the acquisition of complete motion trajectory information.

[0038] In this invention, to quantitatively analyze the transmembrane dynamics of a single lipid nanoparticle complex from its trajectory, the mean square displacement (MSD) is calculated based on the coordinate information obtained from tracking, and the motion mode is determined according to the shape characteristics of the MSD curve. The preferred specific operation is as follows: The coordinate data (x(t), y(t)) of the single LNP@QD trajectory obtained above are imported into a MATLAB or Python data analysis platform. First, each trajectory is preprocessed, and short trajectories with a length of less than 20 consecutive frames are removed to ensure the reliability of subsequent statistical analysis. For each valid trajectory, its mean square displacement is calculated over a time interval Δt, using the following formula: Where Δt is the time interval, which takes values ​​of 1 frame, 2 frames, ..., and does not exceed 1 / 4 of the total trajectory length. This represents averaging over all initial times t; the calculated MSD versus Δt is plotted in a log-log coordinate system to obtain the MSD curve, which is then further analyzed using a power-law function. The curve is fitted nonlinearly, where A is the diffusion coefficient correlation constant and α is the diffusion index; the motion mode is determined based on the fitted α value and the macroscopic shape of the MSD curve.

[0039] In this invention, the calculation of instantaneous velocity is based on the bit removal of particles between two adjacent frames by a time interval, i.e. The instantaneous velocity magnitude of each frame is calculated, and the directional change of velocity is recorded based on the direction of the displacement vector. For each trajectory, a curve showing the change of instantaneous velocity over time can be plotted, and the average velocity, maximum velocity, and the number of frames with consecutively consistent velocity directions can be counted. Through the above calculations, the mean square displacement fitting parameter α of each LNP@QD particle, as well as the magnitude and directional characteristics of the instantaneous velocity, can be obtained, providing a quantitative basis for subsequent comprehensive determination of the transmembrane motion stage in conjunction with changes in fluorescence intensity.

[0040] This invention comprehensively determines the transmembrane motion stage of lipid nanoparticle complexes based on the calculated mean square displacement curve, instantaneous velocity, and fluorescence intensity trends. As a preferred embodiment, for each tracked LNP@QD particle, the macroscopic shape of its mean square displacement curve is first observed, and the diffusion index α value obtained from power-law fitting is read; simultaneously, the instantaneous velocity change curve over time is analyzed, and the fluorescence intensity change trajectory over time is examined concurrently. The transmembrane movement stage is determined by the following rules: When the mean square displacement curve shows a slightly downward trend (α < 1), the instantaneous velocity shows a decreasing trend, and the fluorescence intensity is relatively stable (fluctuations less than 20% of the initial value), it is determined to be the restricted movement stage on the cell membrane surface. This stage corresponds to the process in which particles continuously decelerate and contact the cell membrane in a "Kiss-and-Run" pattern to find suitable endocytic sites. When the mean square displacement curve shows a significant downward trend (α < 1 or even close to 0), the instantaneous velocity approaches zero, and the fluorescence intensity continuously weakens to less than 50% of the initial value, it is determined to be the internalization stage. At this time, the particles leave the microscope focal plane along the Z-axis and are confined in the limited space of the clathrin-coated pits or other endocytic structures formed at the endocytic site, gradually entering the cell interior. When the fluorescence intensity... When a particle experiences a momentary drop (usually to 50%-80% of its initial value) followed by a rapid recovery to near its original level, and the trajectory at the corresponding time point shows significant particle displacement, it is considered an internalization attempt failure event. This indicates that the particle attempted internalization at the clathrin-coated pits but failed and continues to search for a suitable endocytic site. When the mean square displacement curve shows an upward curving trend (α>1), the instantaneous velocity gradually increases, the trajectory direction remains consistent across multiple frames, and the fluorescence intensity is relatively stable, it is considered the intracytoplasmic directed movement stage, indicating that the particle has successfully internalized and is actively transported along the cytoskeleton. When the mean square displacement curve shows an approximately linear trend (α≈1), the instantaneous velocity direction changes randomly, and the fluorescence intensity is relatively stable, it is considered the random diffusion stage, which is often seen in the free movement of particles in free solution or giant vesicles (such as lysosomes). By combining the shape of the mean square displacement curve, instantaneous velocity characteristics, and fluorescence intensity change trend, the above-mentioned comprehensive judgment method can accurately distinguish the different stages of LNP@QD in the transmembrane process, such as attachment, confinement, internalization, internalization failure, intracellular directional transport, and random diffusion, providing a reliable basis for determining the complete transmembrane dynamics of a single lipid nanoparticle.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0043] Example 1: Preparation of lipid nanoparticle complex (LNP@QD) A method for preparing a lipid nanoparticle complex, comprising the following steps: (1) Removal of surface oleic acid ligands from CdSe / ZnS quantum dots: Take 1 mL of oleic acid-modified CdSe / ZnS quantum dots (emission wavelength 605 nm) with a concentration of 3 mg / mL, add anhydrous ethanol to a total volume of 5 mL, shake thoroughly to mix, centrifuge at 10000 rpm for 15 min, discard the supernatant, repeat the above washing steps three times to remove organic solvent, place the precipitate in a vacuum drying oven and dry overnight to obtain dried quantum dot precipitate without ligand modification.

[0044] (2) CdSe / ZnS quantum dot-modified mPEG ligand: The above quantum dot precipitate was dissolved in 1 mL of chloroform, and 1 mL of 0.2 mg / mL tetrabutylammonium bromide (TBAB) chloroform solution and 250 μL of mercaptoheptaethylene glycol monomethyl ether (mPEG-SH, molecular weight approximately 350 Da) were added. The mixture was stirred at 600 rpm at room temperature for 30 min. After the reaction was completed, 2 volumes of 0.2 M NaOH aqueous solution were added, and the mixture was vortexed thoroughly to transfer QD-mPEG to the aqueous phase. The aqueous phase was separated by centrifugation at 3000 rpm for 1 min and quickly dialyzed in ultrapure water for 3 days, changing the water every 12 hours to obtain the QD-mPEG aqueous solution.

[0045] (3) DNA encapsulation with QD-mPEG: Take 1 mL of the above QD-mPEG solution and gradually add 0.5 mL of 1 μM polythiol phosphate single-stranded DNA (sequence of SEQ ID NO.1: 5'-A) A A A A A A A A A A A A A A A A A A A GGCGAAGACAGCGGGTTGGG-3', where A The DNA@QD complex was formed by vigorous stirring at 900 rpm overnight at room temperature with thiol-modified adenine deoxynucleotides. The binding of DNA to quantum dots was verified by agarose gel electrophoresis (1% agarose, 100V, 30min), showing that the DNA band completely migrated to the fluorescent region of the quantum dots. Transmission electron microscopy (TEM) characterization showed that the DNA@QD particle size was approximately 8 nm (see...). Figure 1 ).

[0046] (4) LNP-encapsulated DNA@QD: After quantifying the above DNA@QD solution by fluorescence intensity, dilute it to 66.7 nM with 20 mM citrate-sodium citrate buffer (pH=4.0), with a volume of approximately 1.5 mL. Prepare a 10 mg / mL composite lipid-ethanol solution with ionizable cationic lipids DLin-MC3-DMA, distearate phosphatidylcholine (DSPC), cholesterol (Cholesterol), and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000) at a molar ratio of 50:10:38.5:1.5, with a volume of approximately 0.5 mL. Mixing was performed using microfluidic technology: Using a microfluidic preparation instrument (MPE-L2), 0.5 mL of a 10 mg / mL composite lipid-ethanol solution and 1.5 mL of a 20 mM citrate-sodium citrate buffer containing 66.7 nM DNA@QD were added to two syringes, respectively. The flow rate ratio of the aqueous phase (DNA@QD buffer) to the oil phase was 3:1, the total flow rate was 20 mL / min, and the total product volume was 2 mL. After rapid mixing, the sample was collected. Dialysis was performed with 1×PBS at 4°C for 2 h, followed by dialyzing with 1×PBS at 4°C for 12 h. 50 nM LNP@QD was obtained, and the particle size was approximately 70 nm as determined by DLS and TEM characterization.

[0047] (5) Characterization of LNP@QD: The particle size of LNP@QD was approximately 70 nm using dynamic light scattering (DLS) and transmission electron microscopy (TEM) (see Figure 2 A schematic diagram of the preparation process of the lipid nanoparticle complex of the present invention is shown below. Figure 3 .

[0048] Example 2: Cellular uptake and single-particle super-resolution tracking and analysis of LNP@QD A single-particle super-resolution tracking and analysis method for transmembrane dynamics of lipid nanoparticles includes the following steps: (1) Cell culture: HeLa cells were seeded in 35mm glass bottom culture dishes and cultured at 37℃ and 5% CO2 until the density reached about 70% for later use.

[0049] (2) Co-incubation of LNP@QD with cells: Take the LNP@QD solution prepared in Example 1, dilute it 20 times with cell culture medium, take 100 μL and add it to the cell surface, and place it on the stage of a structured light illumination microscope (SIM) for observation.

[0050] (3) Super-resolution imaging and single-particle tracking: A 60× oil immersion objective (numerical aperture 1.49), an excitation wavelength of 488 nm, and a frame rate of 1 frame / second were used for continuous imaging for 60 minutes to obtain time-series images. The time-series images were imported into ImageJ software, and after noise reduction, the automatic tracking plugin Plugins-Tracking-Trackmate was used to capture and connect single particles, obtaining the transmembrane motion trajectory map of LNP@QD. For example... Figure 4 This image shows screenshots and trajectory diagrams of LNP@QD movement along cellular microfilaments after internalization. This invention verifies the co-localization relationship between LNP@QD and the cytoskeleton using fluorescence localization mapping and extracts and analyzes the movement trajectory of internalized LNP@QD. Multiple trajectories in the image show that LNP@QD's movement within the cell is primarily directional, migrating along cellular microfilaments. Unlike random displacement caused by free diffusion, this directional movement pattern relies on the cytoskeleton network, particularly the active transport process mediated by microfilaments (actin filaments). In living cells, microfilaments typically work synergistically with myosin motor proteins to provide transport tracks for vesicles or endosomes from the cell periphery to the interior (retrograde transport) or from the interior to the periphery (anterograde transport). LNP@QD exhibits a typical "run-stop-run" jumping motion characteristic within the cell, and its trajectory is often linearly distributed along the cell's long axis or stress fiber direction, further supporting the cytoskeleton-dependent nature of its transport process.

[0051] (5) Instantaneous velocity and fluorescence intensity analysis: After obtaining the coordinate position of LNP@QD, the instantaneous velocity-time relationship diagram, fluorescence intensity-time relationship diagram, and mean square displacement (MSD) analysis diagram of the trajectory can be calculated and plotted to analyze the transmembrane motion process and heterogeneity of LNP@QD one by one. Figure 5 The data shows that LNP@QDs undergo strictly restricted movement with enhanced restraint on the outer surface of the cell membrane. Colocalization analysis clearly demonstrates significant colocalization between LNP@QDs and the cell membrane, indicating dynamic movement on the cell membrane surface. Trajectory analysis further reveals that after a brief migration, LNP@QDs enter a near-static, highly restricted movement state. Figure 6Specifically, in the first 5 seconds, LNP@QD diffuses to a limited extent on the membrane surface to find suitable endocytosis sites; in the following 350 seconds, its movement is extremely limited, exhibiting a clear restricted characteristic; and in the last second, the particle disappears rapidly, indicating that it has successfully achieved cell internalization. Figure 7 It is worth noting that the internalization process of LNP@QD is not limited to two-dimensional movement within the cell membrane plane, but may involve a three-dimensional endocytic pathway along the Z-axis.

[0052] To further elucidate its internalization mechanism, this invention divides the movement trajectory of LNP@QD into three stages: Stage I is the endocytic site search phase, Stage II is the restricted movement phase, and Stage III is the cell internalization phase. Fluorescence intensity analysis shows ( Figure 8 A and Figure 8 In stage I, LNP@QD interacted transiently with the cell membrane multiple times and attempted endocytosis twice at existing clathrin-coated pits (CCPs), but both attempts failed (manifested as a transient decrease in fluorescence intensity). Subsequently, LNP@QD successfully localized to a third endocytic site on the membrane surface, entering a highly restricted movement stage (stage II). During this process, the fluorescence intensity gradually decreased, suggesting that it underwent clathrin-mediated endocytosis along the Z-axis, ultimately achieving cell internalization (stage III). Furthermore, mean square displacement (MSD) analysis further supports the above observations. Figure 8 C and Figure 8 (D) shows that in stages II and III, the degree of restriction on particle movement gradually increases, which is highly correlated with the endocytosis process.

[0053] (6) Mean Square Displacement (MSD) Analysis and Motion Pattern Determination: To further analyze the overall fate of LNP@QD from intracellular to extracellular, its transmembrane movement process was divided into two macroscopic stages ( Figure 9 The first stage is the restricted internalization stage on the cell membrane surface. In this stage, LNP@QD particles first diffuse in a limited manner on the membrane surface to find endocytic sites, and then are confined within clathrin-coated pits to perform highly restricted movement. Finally, they are internalized into the cytoplasm along the Z-axis. The fluorescence intensity gradually decreases, and the MSD curve shows a downward bending trend (α < 1), indicating restricted movement. Figure 10 The second stage is the directional migration stage within the cytoplasm after internalization. After entering the cytoplasm, LNP@QD particles undergo long-range directional migration along cellular microfilaments, exhibiting a clear directional trajectory, distinct from random diffusion or simply restricted movement. Mean square displacement (MSD) analysis of this stage shows that the MSD curve curves upwards, and the fitted diffusion coefficient α > 1, indicating directional migration. Figure 10 ).

[0054] In this embodiment, the mean square displacement is calculated according to the formula. Fitting is performed. When the mean square displacement (MSD) curve shows an upward bending trend (α > 1), it is determined to be directional motion; when the MSD curve is approximately a straight line (α ≈ 1), it is determined to be random diffusion; when the MSD curve shows a downward bending trend (α < 1), it is determined to be restricted motion.

[0055] Example 3: Statistical analysis and heterogeneity analysis of the transmembrane motion process of LNP@QD This embodiment performed statistical analysis on multiple LNP@QDs, obtaining statistically significant data on LNP@QDs. The results showed that after internalization, most LNP@QDs exhibited long-range directional movement, with only a small portion exhibiting restricted movement, which is distinctly different from the movement state outside the cell membrane. To further investigate the movement behavior of LNP@QDs under different environments, this embodiment first analyzed their particle heterogeneity. The results are as follows... Figure 11 As shown in Figure A, LNP@QD exhibits typical Brownian motion (i.e., free diffusion) in free solution; when it approaches or binds to the cell membrane, its motion pattern changes to restricted motion; and only after successful internalization into the cell does it exhibit directional motion driven by external force. Based on this, this embodiment statistically analyzed the motion trajectories of multiple internalized LNP@QDs and fitted their diffusion index α. The frequency distribution histogram of α values ​​clearly shows that the vast majority of particles have α > 1, indicating that internalized LNP@QDs mainly exhibit directional motion, with only a few particles still exhibiting restricted motion (…). Figure 11 B). Furthermore, regarding acquisition time, the observable duration of directional motion events is relatively short. This may be attributed to the fact that their motion is not strictly two-dimensional planar motion; displacement along the Z-axis easily causes particles to deviate from the confocal focal plane, thus shortening the effective tracking time. Figure 11 C); however, in terms of displacement, the displacement of directional motion is significantly greater than that of restricted motion (C). Figure 11 (D) This observation is consistent with the results of the MSD analysis, further verifying the motion characteristics of internalized particles driven by active transport mechanisms.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a lipid nanoparticle complex, characterized in that, Includes the following steps: Oleic acid-modified CdSe / ZnS quantum dots were treated to remove oleic acid ligands, resulting in quantum dot precipitates. After dissolving the quantum dot precipitate, mercaptoheptaethylene glycol monomethyl ether was added to react and obtain water-soluble quantum dots modified with methoxy polyethylene glycol. The water-soluble quantum dots are mixed with polythiol phosphate single-stranded DNA to form a DNA-quantum dot complex; The DNA-quantum dot complex and the composite lipid were mixed by microfluidics and self-assembled to form a lipid nanoparticle complex.

2. The preparation method according to claim 1, characterized in that, The oleic acid ligand removal process includes: adding anhydrous ethanol to oleic acid-modified CdSe / ZnS quantum dots, shaking, centrifuging and washing at high speed, repeating the washing process, and then drying.

3. The preparation method according to claim 1, characterized in that, The sequence of the polythiol phosphate single-stranded DNA includes a polythiol-modified adenine deoxynucleotide segment and a negatively charged single-stranded nucleotide segment.

4. The preparation method according to claim 3, characterized in that, The sequence of the polythiol phosphate single-stranded DNA is shown in SEQ ID NO.

1.

5. The preparation method according to claim 1, characterized in that, The complex lipids comprise ionizable cationic lipids, distearate phosphatidylcholine, cholesterol, and polyethylene glycol-modified lipids.

6. The preparation method according to claim 5, characterized in that, The ionizable cationic lipid is DLin-MC3-DMA, and the PEGylated lipid is dimyristic glycerol-polyethylene glycol 2000.

7. The preparation method according to claim 1, characterized in that, In the microfluidic mixing, the volume ratio of the aqueous phase flow rate to the oil phase flow rate is 2:1 to 5:

1.

8. A lipid nanoparticle complex, prepared by the preparation method according to any one of claims 1-7.

9. A single-particle super-resolution tracking and analysis method for transmembrane dynamics of lipid nanoparticles, characterized in that, Includes the following steps: The lipid nanoparticle complex of claim 8 was co-incubated with the cells to be tested, and the cells were imaged in real time using a structured light illumination microscope to acquire time-series images. The motion trajectory and coordinate information of a single lipid nanoparticle complex in the time series image are tracked. Based on the coordinate information, the mean square displacement and instantaneous velocity of each lipid nanoparticle complex are calculated; The transmembrane motion stage of the lipid nanoparticle complex is determined based on the mean square displacement curve, the instantaneous velocity, and the changing trend of the fluorescence intensity.

10. The method according to claim 9, characterized in that, The determination of the mean square displacement curve includes: when the mean square displacement curve shows an upward curving trend, it is determined to be directional motion; when the mean square displacement curve is approximately a straight line, it is determined to be random diffusion; when the mean square displacement curve shows a downward curving trend, it is determined to be restricted motion.

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