A method for quantifying traceable lipid nanoparticle fluorescent dyes

Abstract

This invention provides a quantitative method for traceable lipid nanoparticles containing fluorescent dyes, comprising the following steps: S1, serially diluting DiR fluorescent dye to obtain working solutions of various concentrations; S2, diluting the lipid nanoparticle solution containing DiR fluorescent dye, breaking the membrane, centrifuging, removing the lower layer and dispersing it to obtain the test solution; S3, detecting the absorbance of the working solutions and the test solution of various concentrations; plotting a standard curve based on the concentration of the working solutions and their detected absorbance; substituting the absorbance of the test solution into the working curve to obtain the concentration of the test solution. This invention provides a simple quantitative method for traceable lipid nanoparticles, enabling rapid and batch quantification of the DIR content in traceable lipid nanoparticles, ensuring consistency in fluorescence signal intensity between batches of liposome samples, and providing a consistent basis for subsequent fluorescence intensity-based experiments.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials, and more specifically to a quantitative method for tracing fluorescent dyes in lipid nanoparticles. Background Technology

[0002] Lipid nanoparticles, as a new generation of nanomedicine delivery carriers, have attracted much attention in the field of biomedical materials in recent years. They are composed of lipid-like substances with a uniform lipid core, typically containing four key components: ionizable lipids, polyethylene glycol-modified lipids, phospholipids, and cholesterol (Theranostics. 2022, 12, 7509). Compared with traditional lipid particles, lipid nanoparticles have smaller particle sizes and narrower distributions, giving them unique advantages in drug delivery. Since its discovery by Bangham in the 1960s, liposomes have demonstrated broad application prospects in various fields such as drug delivery, gene therapy, and vaccine development due to their excellent biocompatibility, biodegradability, and non-immunogenicity. The application of lipid nanoparticles can be traced back to 2018, when the first siRNA drug based on this technology, Onpattro®, received FDA approval, marking a successful leap from the laboratory to the clinical application of lipid nanoparticle technology. In recent years, the great success of mRNA vaccines based on lipid nanoparticle technology has further confirmed its enormous potential in the field of nucleic acid drug delivery. These successes not only demonstrate the safety and efficacy of lipid nanoparticles but also promote the development and application of this technology in a wider range of biomedical fields. With the continuous advancement of lipid nanoparticle technology, accurate quantitative characterization of it has become a core element in ensuring product quality, controlling batch-to-batch consistency, and achieving clinical translation. Traditional quantitative methods, such as UV-Vis spectrophotometry, fluorescence spectroscopy, atomic absorption / emission spectroscopy, high-performance liquid chromatography, mass spectrometry, nuclear magnetic resonance, and nanoparticle tracking analysis, while each with its own advantages, all have significant limitations. Ultraviolet-visible spectrophotometry is not only cumbersome and difficult to completely separate liposomes from free drugs, but it is also plagued by matrix interference. The absorption peaks of lipid components in the ultraviolet region often overlap significantly with those of drugs, resulting in insufficient sensitivity. Atomic absorption / emission spectroscopy is limited to the analysis of drugs containing specific metal elements. High-performance liquid chromatography (HPLC) is difficult to handle large batches of samples due to its time-consuming analysis and low efficiency. Complex pretreatment steps are prone to introducing errors, and different demulsifiers and extraction solvents have a significant impact on drug recovery. In addition, the chromatographic columns are expensive, resulting in high overall costs. Mass spectrometry, although powerful, is expensive and complex to operate, and ion suppression effects further weaken its sensitivity. Nuclear magnetic resonance (NMR) has high operating costs, long analysis cycles, and limited sensitivity, making it unsuitable for routine liposome quantification. Nanoparticle tracking analysis is highly dependent on conditions such as concentration, temperature, and light, is time-consuming, and has weak processing capabilities for high-concentration and small-particle samples. Traditional fluorescence spectroscopy is limited by fluorescence quenching, making it difficult to construct reliable working curves. In contrast, fluorescent labeling technology stands out due to its advantages such as ease of operation, high sensitivity, and real-time monitoring, making it an ideal tool for studying the dynamic behavior of lipid nanoparticles. Among many fluorescent dyes, DiR, with its unique near-infrared emission characteristics and strong binding ability to lipids, shows broad application prospects in the characterization of lipid nanoparticles (J Biomed Opt. 2011,16, 096013).

[0003] DiR (1,1'-dioctyl-3,3,3',3'-tetramethylindole iodide), an important hydrophobic long-chain dialkyl carbocyanine dye, has attracted much attention due to its unique molecular configuration and optical properties. Its molecule consists of two octadecyl chains and a tricarboncyanine dye core. This structure not only endows DiR with excellent lipophilicity but also gives it efficient membrane binding ability. DiR is almost insoluble in water but exhibits strong fluorescence in organic solvents and lipid environments, with its fluorescence emission peak located in the near-infrared region (775-780 nm). This characteristic brings significant advantages to its application in bioimaging (CurrDrug Deliv. 2016, 13, 40). The binding of DiR to lipid nanoparticles mainly relies on hydrophobic interactions. Thanks to the presence of the two long alkyl chains in the molecule, DiR can rapidly embed into the lipid bilayer, forming a stable membrane-bound structure and is not prone to significant dye migration between cells. Therefore, DiR is very suitable for long-term tracking and quantitative analysis of lipid nanoparticles.

[0004] Currently, DiR, as a fluorescent label, has been widely used in cellular uptake (ACSNano. 2017, 11, 11584), tissue distribution (J Biomed Opt. 2012, 17, 126004), pharmacokinetics (Cancer Res. 2019, 79, 2985), in vivo imaging, and in vitro-in vivo correlation studies of lipid nanoparticles (J Control Release. 2014, 176, 86). The applicant's research found that the common method for quantifying DiR-containing liposomes is still to directly quantify the lipid concentration, using the amount of DiR added as the actual effective content of DIR in the liposomes. However, this method cannot guarantee the consistency of fluorescence signal intensity across batches of liposome samples, failing to reflect the differences in fluorescence attenuation of DiR-labeled liposomes under different physiological conditions, thus affecting the imaging signal-to-noise ratio and quantitative accuracy. Therefore, there is an urgent need for a simple and rapid method for the quantitative analysis of lipid nanoparticles, which can quantify the DIR content in liposomes in batches, thereby laying a consistent foundation for subsequent fluorescence intensity-based experiments. Summary of the Invention

[0005] This invention provides a quantitative method for fluorescent dyes in traceable lipid nanoparticles, enabling simple and short-time quantification of fluorescent dyes in traceable lipid nanoparticles. It can achieve batch quantification of DIR content in traceable lipid nanoparticles, providing a consistent basis for subsequent fluorescence intensity-based experiments.

[0006] In a first aspect, the present invention provides a quantitative method for tracing fluorescent dyes in lipid nanoparticles, comprising the following steps: S1. Gradually dilute the DiR fluorescent dye to obtain working solutions of various concentrations; S2. Dilute the lipid nanoparticle solution containing DiR fluorescent dye, break the membrane, centrifuge, remove the lower layer and disperse it to obtain the test solution; S3. Detect the absorbance of the working solution and the test solution at a series of concentrations; plot a standard curve based on the concentration of the working solution at a series of concentrations and its detected absorbance; substitute the absorbance of the test solution into the working curve to obtain the concentration of the test solution.

[0007] In some embodiments, in step S1, the DiR fluorescent dye stock solution is diluted to concentrations of 200 mg / L, 100 mg / L, 50 mg / L, 20 mg / L, 10 mg / L, 5 mg / L, 1 mg / L, 0.5 mg / L, and 0 mg / L.

[0008] In some embodiments, the diluent for the DiR fluorescent dye stock solution is a chloroform-methanol solution, wherein the chloroform-methanol solution is a mixture of chloroform and methanol at a volume ratio of 1:1 to 1:3, preferably at a volume ratio of 1:2.

[0009] In some embodiments, 1 to 3 times the volume of methanol is added to the DiR fluorescent dye stock solution before dilution. Preferably, 2 times the volume of methanol is added to the DiR fluorescent dye stock solution before dilution.

[0010] In some embodiments, in step S2, the lipid nanoparticle solution containing DiR fluorescent dye is synthesized using a thin-film hydration method or a microfluidic method.

[0011] In some embodiments, in step S2, the raw materials for the lipid nanoparticle solution containing DiR fluorescent dye include: DiR fluorescent dye, ionizable lipids, and cholesterol; optionally, it also includes polyethylene glycol-modified lipids. Optionally, the ionizable lipid is selected from trimethyl-2,3-dioleoyloxypropylammonium bromide, dilinoleoylphospholipid ethanolamine, or 1,2-dioleoyl-3-dimethylammonium-propane; Optionally, the polyethylene glycol-modified lipid is selected from distearylphosphatidylethanolamine-polyethylene glycol 2000, distearylphosphatidylethanolamine-polyethylene glycol 5000 or distearylphosphatidylethanolamine-polyethylene glycol 1000. Optionally, the molar ratio of ionizable lipids, cholesterol, DiR fluorescent dye and polyethylene glycol-modified lipids is 443.2~492.5:443.2~492.5:15:0~98.6.

[0012] In some embodiments, the raw materials for the lipid nanoparticle solution containing DiR fluorescent dye include: The molar ratios of trimethyl-2,3-dioleoyloxypropylammonium bromide, cholesterol, DiR fluorescent dye, distearylphosphatidylethanolamine-polyethylene glycol 2000, and distearylphosphatidylethanolamine-polyethylene glycol 5000 are 492.5:492.5:15:0:0, 467.8:467.8:15:49.4:0, 443.2:443.2:15:98.6:0, 467.8:467.8:15:0:49.4, or 443.2:443.2:15:0:98.6; Optionally, the method for preparing the lipid nanoparticle solution containing DiR fluorescent dye includes: mixing the above raw materials according to the formula, drying, then adding a solvent to obtain multilayer liposomes, and extruding; optionally, the drying step is to dry with nitrogen gas.

[0013] Optionally, the total concentration of the lipid nanoparticle solution containing DiR fluorescent dye is 5-40 mM; the total concentration is the sum of the molar concentrations of the raw materials in the lipid nanoparticle solution containing DiR fluorescent dye, that is, the sum of the molar concentrations of DiR fluorescent dye, ionizable lipids and cholesterol, or the sum of the molar concentrations of DiR fluorescent dye, ionizable lipids, cholesterol and polyethylene glycol-modified lipids.

[0014] Optionally, the solvent is water, PBS solution, Bicine buffer, HEPES buffer, EPPS buffer, or HEPPS buffer; In some embodiments, in step S2, the lipid nanoparticle solution containing DiR fluorescent dye is diluted with a solvent at a volume ratio of 1:1 to 1:10; preferably, the lipid nanoparticle solution containing DiR fluorescent dye is diluted with a solvent at a volume ratio of 1:4. Optionally, the solvent is water, PBS solution, Bicine buffer, HEPES buffer, EPPS buffer, or HEPPS buffer. And / or, in the membrane rupture step, a diluted solution of the lipid nanoparticle solution containing DiR fluorescent dye is taken and added to a chloroform-methanol solution for membrane rupture. The volume ratio of the diluted solution of the lipid nanoparticle solution containing DiR fluorescent dye to the chloroform-methanol solution is 1:1 to 1:3; the chloroform-methanol solution is a mixture of chloroform and methanol in a volume ratio of 1:1 to 1:3; preferably, the volume ratio of the diluted solution of the lipid nanoparticle solution containing DiR fluorescent dye to the chloroform-methanol solution is 1:1; preferably, the chloroform-methanol solution is a mixture of chloroform and methanol in a volume ratio of 1:2. And / or, the centrifugation conditions are: centrifugation at 300-3000×g for 2-10 minutes at 4-25°C, collecting the lower organic phase, and dispersing it with 7-10 times its volume of chloroform-methanol solution. Preferably, the centrifugation conditions are: centrifugation at 3000×g for 10 minutes at 4°C, collecting the lower organic phase, and dispersing it with 7 times its volume of chloroform-methanol solution. In some embodiments, an ELISA reader is used to detect absorbance at a wavelength of 700-770 nm. Preferably, the detection wavelength is 750 nm.

[0015] In some embodiments, when the working solution and the test solution of various concentrations are added to the well plate, the sample volume per well is 150-200 μL. Preferably, the sample volume per well is 200 μL. The well plate has a transparent bottom.

[0016] The technical solution of this invention has the following advantages 1. This invention provides a quantitative method for traceable lipid nanoparticles, comprising the following steps: S1, serially diluting DiR fluorescent dye to obtain working solutions of various concentrations; S2, diluting the lipid nanoparticle solution containing DiR fluorescent dye, breaking the membrane, centrifuging, removing the lower layer and dispersing it to obtain the test solution; S3, detecting the absorbance of the working solutions and the test solution of various concentrations; plotting a standard curve based on the concentration of the working solutions of various concentrations and their detected absorbance; substituting the absorbance of the test solution into the working curve to obtain the concentration of the test solution. The applicant's research has found that current quantitative methods for DiR-containing liposomes still tend to directly measure lipid concentration and DiR content, rather than quantifying liposomes through DiR signal intensity. However, DiR signal intensity is the key indicator for final detection in various applications. However, the current quantitative method for detecting DiR content (such as liquid chromatography or mass spectrometry) is time-consuming and cumbersome. As time progresses, the fluorescence signal intensity of DIR decays, leading to prolonged detection times for batches of liposome samples. This results in inconsistent fluorescence signal intensities across batches, affecting subsequent experiments based on fluorescence intensity consistency and hindering comparisons of the performance of different liposome drug delivery platforms. In contrast, the quantitative method for traceable lipid nanoparticles in this invention is simple and allows for rapid, batch-wise quantification of DIR content in traceable lipid nanoparticles, ensuring consistency in fluorescence signal intensity between batches of liposome samples. This provides a consistent foundation for subsequent fluorescence intensity-based experiments.

[0017] Furthermore, the method of the present invention does not have the disadvantages of traditional lipid quantification methods, such as only quantifying the nano-drug delivery platform and not being able to directly quantify the effective concentration of DIR, high quantification cost, and long quantification time. It can also provide reliable concentration quantification for subsequent biological exposure injection concentration.

[0018] Furthermore, in the above method, the present invention indirectly quantifies lipid nanoparticles by quantifying the liposome tracer DiR in a solution of lipid nanoparticles containing DiR fluorescent dye. The amount of DiR calibrated can represent the drug loading capacity of liposomes, which has a guiding role in the development of liposome drug carrier platforms. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1This is a particle size distribution diagram of each liposome particle solution P0, P1, P2, P5, and P6 in Example 1 of the present invention; Figure 2 This is a diagram showing the average particle size of each liposome particle solution P0, P1, P2, P5, and P6 in Example 1 of the present invention. Figure 3 This is the standard curve diagram in Embodiment 2 of the present invention; Figure 4 This is the correlation curve diagram in Embodiment 3 of the present invention; Figure 5 It refers to the fluorescence signal intensity of each sample at the quantitative target concentration level in Example 3 of this invention. Detailed Implementation

[0021] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0022] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0023] The fluorescent dye for 1,1'-dioctyl-3,3,3',3'-tetramethylindole iodide (DiR) used in the following examples was purchased from MCE.

[0024] Trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP) was purchased from Maclean's. Cholesterol (Chol) was purchased from Maclean's. Distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2k) was purchased from Maclean's. Distearate phosphatidylethanolamine-polyethylene glycol 5000 (DSPE-PEG 5k) was purchased from Maclean's.

[0025] Example 1: Preparation of lipid nanoparticle solution containing DiR fluorescent dye Fluorescent dye-traced lipid nanoparticles were prepared according to the following steps: 244 mg of DOTAP powder was dissolved in 17.46 mL of chloroform to obtain a 20 mM DOTAP stock solution, which was named solution X.

[0026] 63.1 mg of Chol powder was dissolved in 8.15 mL of chloroform to obtain a 20 mM Chol stock solution.

[0027] 250 mg of DSPE-PEG 2k powder was dissolved in 4.46 mL of chloroform to obtain a 20 mM DSPE-PEG 2k stock solution.

[0028] Dissolve 250 mg of DSPE-PEG 5k powder in 2.15 mL of chloroform to obtain a 20 mM DSPE-PEG 5k stock solution.

[0029] Dissolve 10 mg of DiR powder in 0.494 mL of chloroform to obtain a 20 mM DiR stock solution.

[0030] Taking sample P1 as an example, according to the sample transfer volume, 467.8 μL of X solution, 467.8 μL of ChoL stock solution, 15 μL of DiR stock solution, and 49.4 μL of DSPE-PEG 2k stock solution were transferred to a brown glass bottle and mixed. The mixture was then dried under nitrogen to obtain a mixed lipid film. 1 mL of PBS buffer at 25°C was added to the mixed lipid film, and after simple pipetting, multilayer liposomes were obtained. Subsequently, the multilayer liposomes were extruded 20 times using a manual extruder carrying a 0.1 μm 19 mm polycarbonate membrane to obtain lipid nanoparticle solution P1 containing DiR fluorescent dye. When preparing samples P0, P2, P5, and P6, only the transfer volumes of X solution, ChoL stock solution, DiR stock solution, DSPE-PEG 2k stock solution, and DSPE-PEG 5k stock solution were changed, while other preparation conditions remained unchanged. The specific transfer volumes for each sample are as follows: P0 sample: X solution 492.5 μL, ChoL stock solution 492.5 μL, DiR stock solution 15 μL; P1 sample: X solution 467.8 μL, ChoL stock solution 467.8 μL, DiR stock solution 15 μL, DSPE-PEG 2k stock solution 49.4 μL; P2 sample: X solution 443.2 μL, ChoL stock solution 443.2 μL, DiR stock solution 15 μL, DSPE-PEG 2k stock solution 98.6 μL; P5 sample: X solution 467.8 μL, ChoL stock solution 467.8 μL, DiR stock solution 15 μL, DSPE-PEG 5k stock solution 49.4 μL; P6 sample: X solution 443.2 μL, ChoL stock solution 443.2 μL, DiR stock solution 15 μL, DSPE-PEG 5k stock solution 98.6 μL.

[0031] The obtained liposome particle solutions P0, P1, P2, P5, and P6 were analyzed using a Malvern laser particle size analyzer. The results are as follows: Figure 1 and Figure 2 This indicates that lipid nanoparticle solutions P0, P1, P2, P5, and P6 containing DiR fluorescent dye were successfully prepared.

[0032] Example 2: Quantitative method for traceable lipid nanoparticles 1. Preparation of working solutions of various concentrations Preparation of chloroform-methanol solution: Add 5 mL of chloroform to 10 mL of methanol and mix.

[0033] Working solution of 200 mg / L DiR fluorescent dye: Take 30 μL of 20 mM 1,1'-dioctyl-3,3,3',3'-tetramethylindole iodide (DiR) fluorescent dye stock solution, add 60 μL of methanol to obtain a chloroform-methanol solution of DiR fluorescent dye. Take 50 μL of the DiR fluorescent dye chloroform-methanol solution, add 1639 μL of chloroform-methanol solvent, and you will get a working solution with a concentration of 200 mg / L.

[0034] 100 mg / L DiR fluorescent dye working solution: Take 700 μL of 200 mg / L DiR fluorescent dye working solution and add 700 μL of chloroform-methanol solution. 50 mg / L DiR fluorescent dye working solution: Take 400 μL of 100 mg / L DiR fluorescent dye working solution and add 400 μL of chloroform-methanol solution. 20 mg / L DiR fluorescent dye working solution: Take 240 μL of 100 mg / L DiR fluorescent dye working solution and add it to 960 μL of chloroform-methanol solution. 10 mg / L DiR fluorescent dye working solution: Take 400 μL of 20 mg / L DiR fluorescent dye working solution and add 400 μL of chloroform-methanol solution. 5 mg / L DiR fluorescent dye working solution: Take 80 μL of 50 mg / L DiR fluorescent dye working solution and add it to 720 μL of chloroform-methanol solution. 1 mg / L DiR fluorescent dye working solution: Take 80 μL of 10 mg / L DiR fluorescent dye working solution and add it to 720 μL of chloroform-methanol solution. 0.5 mg / L DiR fluorescent dye working solution: Take 80 μL of 5 mg / L DiR fluorescent dye working solution and add it to 720 μL of chloroform-methanol solution; 0 mg / L DiR fluorescent dye working solution: Take 500 μL of chloroform-methanol solution.

[0035] 2. Preparation of the sample to be tested Take 100 μL of the lipid nanoparticle solution containing DiR fluorescent dye prepared in Example 1 (P0, P1, P2, P5, or P6), then dilute it with 400 μL of PBS buffer (pH=7), and then add 500 μL of chloroform-methanol solution (prepared by mixing 5 mL of chloroform with 10 mL of methanol in step 1). Mix well by pipetting, let stand at room temperature for 10 minutes, and then centrifuge at 3000×g for 10 minutes at 4°C. After centrifugation, gently aspirate the upper aqueous solution, gently mix the lower organic phase, take the lower organic phase (about 100 μL), add 700 μL of chloroform-methanol solution and mix well to obtain the sample to be tested (P0, P1, P2, P5, or P6).

[0036] 3. Draw the standard curve Take a 96-well black microplate, add the working solution of a series of concentrations and the sample to be tested at 200 μL / well, and measure in parallel three times. Then quickly put it into the microplate reader (Thermo) and select the detection wavelength of 750 nm to measure its absorbance.

[0037] The absorbance values ​​obtained from a series of working solutions at different concentrations were statistically analyzed. A standard curve was plotted with the measured absorbance on the ordinate and the corresponding concentration on the abscissa. Figure 3 As shown, the linear curve is Y = 0.4103X + 0.05183, R... 2 =0.9987. Substituting the absorbance of the sample into the standard curve above, the concentration of DiR in the sample (DiR-C1) was calculated. The concentration of DiR in the lipid nanoparticle solution containing DiR fluorescent dye (DiR-C2) was then obtained by conversion. The conversion formula is DiR-C... 2= DiR-C1×8.

[0038] Example 3 Application Case The lipid nanoparticle solutions containing DiR fluorescent dye (P0, P1, P2, P5, or P6) were prepared according to Example 1. The concentration of DiR fluorescent dye in the stock solution was 303.9 ppm (theoretical concentration, i.e., the target quantitative concentration). The lipid nanoparticle solutions containing DiR fluorescent dye were diluted with PBS buffer to the theoretical concentrations of DiR fluorescent dye, which were 160 mg / L, 120 mg / L, 80 mg / L, and 40 mg / L, respectively.

[0039] Following the procedure in Example 2, the lipid nanoparticle solution (stock solution) containing DiR fluorescent dye prepared in Example 1 and the diluent were used to prepare the test samples (P0, P1, P2, P5 or P6), wherein the theoretical concentration values ​​of DiR fluorescent dye (i.e. the target quantitative concentration) were 37.99 mg / L, 20 mg / L, 15 mg / L, 10 mg / L, 5 mg / L and 0 mg / L (chloroform-methanol solution).

[0040] The absorbance of the sample prepared with the above dilution was measured. The measured absorbance was substituted into the standard curve to obtain the concentration of DiR in the sample. This concentration was then converted to obtain the DiR concentration in the lipid nanoparticle solution containing the DiR fluorescent dye. A correlation curve was plotted with the measured DiR concentration (actual value) as the ordinate and the corresponding target quantitative concentration as the abscissa. The results are shown below. Figure 4 As shown, the correlation between the measured values ​​and the target quantitative concentration values ​​of the five sample dilutions (P0, P1, P2, P5, or P6) is R. 2 >0.97 or higher. Further, the fluorescence signal intensity of the diluted solutions of the above 5 samples was detected (using a Thermo microplate reader, detection conditions: absorption light 700-765nm, emission light (signal acquisition) 770-800nm), and the results are as follows: Figure 5 As shown, after multiple dilutions, the fluorescence signal intensity of the samples remained consistent at the same target quantitative concentration of the DiR fluorescent dye, demonstrating high uniformity.

[0041] The above demonstrates that, for lipid nanoparticle solutions containing DiR fluorescent dye, the gradient change of the concentration measurement value obtained using the quantitative method of this invention is consistent with the target quantitative concentration value. For lipid nanoparticle solution samples containing DiR fluorescent dye, the quantitative method of this invention allows for rapid quantification, ensuring that when the target concentration value of DiR fluorescent dye in the lipid nanoparticle solution is the same, consistent fluorescence signal intensities can be obtained in different or identical samples within the same batch. This provides a consistent basis for subsequent fluorescence intensity-based experiments, avoiding inconsistencies in fluorescence signal intensities that would hinder comparisons of the performance of different liposome samples.

[0042] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A quantitative method for fluorescent dyes in traceable lipid nanoparticles, characterized in that, Includes the following steps: S1. Gradually dilute the DiR fluorescent dye to obtain working solutions of various concentrations; S2. Dilute the lipid nanoparticle solution containing DiR fluorescent dye, break the membrane, centrifuge, remove the lower layer and disperse it to obtain the test solution; S3. Detect the absorbance of the working solution and the test solution at a series of concentrations; plot a standard curve based on the concentration of the working solution at a series of concentrations and its detected absorbance. Substitute the absorbance of the test solution into the working curve to obtain the concentration of the test solution.

2. The quantitative method for traceable lipid nanoparticle fluorescent dyes according to claim 1, characterized in that, In step S1, the DiR fluorescent dye stock solution was diluted to concentrations of 200 mg / L, 100 mg / L, 50 mg / L, 20 mg / L, 10 mg / L, 5 mg / L, 1 mg / L, 0.5 mg / L, and 0 mg / L.

3. The quantitative method for traceable lipid nanoparticle fluorescent dyes according to claim 1 or 2, characterized in that, The diluent for the DiR fluorescent dye stock solution is a chloroform-methanol solution, which is a mixture of chloroform and methanol in a volume ratio of 1:1 to 1:

3.

4. The quantitative method for traceable lipid nanoparticle fluorescent dyes according to any one of claims 1-3, characterized in that, Before diluting the DiR fluorescent dye stock solution, 1 to 3 times its volume of methanol is added.

5. The quantitative method for traceable lipid nanoparticle fluorescent dyes according to any one of claims 1-4, characterized in that, In step S2, the lipid nanoparticle solution containing DiR fluorescent dye is synthesized using a thin-film hydration method or a microfluidic method.

6. The quantitative method for traceable lipid nanoparticle fluorescent dyes according to any one of claims 1-5, characterized in that, In step S2, the raw materials for the lipid nanoparticle solution containing DiR fluorescent dye include: DiR fluorescent dye, ionizable lipids, and cholesterol; optionally, it also includes polyethylene glycol-modified lipids. Optionally, the ionizable lipid is selected from trimethyl-2,3-dioleoyloxypropylammonium bromide, dilinoleoylphospholipid ethanolamine, or 1,2-dioleoyl-3-dimethylammonium-propane; Optionally, the polyethylene glycol-modified lipid is selected from distearylphosphatidylethanolamine-polyethylene glycol 2000, distearylphosphatidylethanolamine-polyethylene glycol 5000 or distearylphosphatidylethanolamine-polyethylene glycol 1000. Optionally, the molar ratio of ionizable lipids, cholesterol, DiR fluorescent dye and polyethylene glycol-modified lipids is 443.2~492.5:443.2~492.5:15:0~98.

6.

7. The quantitative method for traceable lipid nanoparticle fluorescent dyes according to claim 6, characterized in that, The raw materials for the lipid nanoparticle solution containing DiR fluorescent dye include: The molar ratios of trimethyl-2,3-dioleoyloxypropylammonium bromide, cholesterol, DiR fluorescent dye, distearylphosphatidylethanolamine-polyethylene glycol 2000, and distearylphosphatidylethanolamine-polyethylene glycol 5000 are 492.5:492.5:15:0:0, 467.8:467.8:15:49.4:0, 443.2:443.2:15:98.6:0, 467.8:467.8:15:0:49.4, or 443.2:443.2:15:0:98.6; Optionally, the method for preparing the lipid nanoparticle solution containing DiR fluorescent dye includes: mixing the above raw materials according to the formula, drying, then adding a solvent to obtain multilayer liposomes, and extruding. Optionally, the total concentration of the lipid nanoparticle solution containing DiR fluorescent dye is 5-40 mM; Optionally, the solvent is water, PBS solution, Bicine buffer, HEPES buffer, EPPS buffer, or HEPPS buffer.

8. The quantitative method for traceable lipid nanoparticle fluorescent dyes according to any one of claims 1-7, characterized in that, In step S2, the lipid nanoparticle solution containing DiR fluorescent dye is mixed and diluted with a solvent at a volume ratio of 1:1 to 1:10; optionally, the solvent is water, PBS solution, Bicine buffer, HEPES buffer, EPPS buffer or HEPPS buffer. And / or, in the membrane rupture step, a diluted solution of lipid nanoparticles containing DiR fluorescent dye is taken and a chloroform-methanol solution is added to rupture the membrane. The volume ratio of the diluted solution of lipid nanoparticles containing DiR fluorescent dye to the chloroform-methanol solution is 1:1 to 1:

3. The chloroform-methanol solution is a solution of chloroform and methanol mixed in a volume ratio of 1:1 to 1:

3. And / or, the centrifugation conditions are 4~25℃, centrifugation at 300~3000×g for 2~10 minutes, taking the lower organic phase, and adding 7~10 times the volume of chloroform-methanol solution for dispersion.

9. The quantitative method for traceable lipid nanoparticle fluorescent dyes according to any one of claims 1-8, characterized in that, The absorbance was detected using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 700–770 nm.

10. The quantitative method for traceable lipid nanoparticle fluorescent dyes according to any one of claims 1-9, characterized in that, When adding the working solution and the test solution of various concentrations into the well plate, the sample volume per well is 150~200μL.

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