Method for detecting RNA encapsulation efficiency of nano-liposome by using malachite green and application
By using a method combining malachite green with recombinant MGA and detecting the fluorescence intensity of nanoliposomes with a multifunctional microplate reader, the problem of insufficient accuracy in determining the RNA encapsulation efficiency of nanoliposomes was solved. This method achieves low-cost, high-precision encapsulation efficiency measurement, which is suitable for the research and development of various RNA drugs.
Patent Information
- Application Number
- CN202511286712.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-10
- Publication Date
- 2025-12-02
AI Technical Summary
Existing methods for determining RNA encapsulation efficiency in nanoliposomes suffer from insufficient accuracy, complex operation, and high cost, making it difficult to accurately measure the encapsulation efficiency of different types of RNA.
Malachite green (MG) was used as a fluorescent marker to bind with recombinant MGA. The fluorescence intensity at 630/655 nm was detected by a multi-functional microplate reader, and the encapsulation efficiency of RNA by nanoliposomes was calculated, which simplifies the operation process and improves the measurement precision.
It enables low-cost, simple, and high-precision determination of RNA encapsulation efficiency in nanoliposomes, applicable to the research and development of various RNA drug candidates, and suitable for large-scale sample detection.
Smart Images
Figure CN121049219A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bionanomaterials technology, specifically to a method and application for detecting the RNA encapsulation efficiency of nanoliposomes using malachite green. Background Technology
[0002] Nanoparticle drug delivery systems can improve the pharmacokinetics and pharmacodynamics of drugs, enhance therapeutic efficacy, and reduce toxic side effects. Liposomes (LNPs) are biocompatible, modifiable nanoparticles, typically ranging in size from tens to hundreds of nanometers. They possess excellent biocompatibility and the ability to effectively encapsulate various hydrophobic and hydrophilic drugs, showing great potential in drug delivery.
[0003] In in vitro nanomedicine studies, drug encapsulation efficiency is a key indicator of lipid nanoparticle performance, and a satisfactory encapsulation efficiency is a prerequisite for nanomedicines to function as drug delivery systems. Encapsulation efficiency refers to the percentage of drug encapsulated within the nanoparticle relative to the initial drug dosage. A high encapsulation efficiency means that more drug can be contained within the nanoparticle, thereby improving drug utilization efficiency.
[0004] Currently, the main methods for determining the encapsulation efficiency of nanoliposomes include the following: (1) Gel electrophoresis: Based on the size and charge properties of RNA molecules, RNA migrates at a certain speed in agarose gel or polyacrylamide gel under the action of an electric field. After appropriate treatment (such as adding reagents that destroy LNPs), free RNA and encapsulated RNA can be separated in the gel. RNA bands are made visible by staining, and the encapsulation rate can be estimated by comparing the band intensities of free RNA and encapsulated RNA. However, this method is only a relatively quantitative method with relatively limited accuracy. At the same time, the results of gel electrophoresis may be affected by various factors such as RNA integrity, gel quality, and electrophoresis conditions. For RNA samples with low content, it is difficult to obtain clear bands, thus affecting the determination of encapsulation rate.
[0005] (2) Fluorescent labeling method: Fluorescent labeling groups are introduced during or after RNA synthesis. In the LNP-RNA system, since fluorescently labeled RNA exists in both free and encapsulated states, appropriate separation methods (such as dialysis, ultrafiltration, etc.) are used to separate the free fluorescently labeled RNA from the encapsulated fluorescently labeled RNA. Then, the fluorescence intensity of the two is measured separately using equipment such as a fluorescence spectrophotometer. Finally, the encapsulation rate is calculated based on the ratio of fluorescence intensity. However, fluorescent labeling may have a certain impact on the structure and function of RNA, thereby changing its encapsulation status in LNP. Errors may occur during the separation of free and encapsulated RNA, leading to inaccurate encapsulation rate measurements. In addition, some fluorescent nucleic acid dyes may enter the LNP, making it impossible to accurately measure the encapsulation rate.
[0006] (3) High-performance liquid chromatography (HPLC): HPLC is a technique for separation and analysis based on the difference in partition coefficients of different substances between the stationary and mobile phases. For LNP-RNA systems, free RNA and RNA encapsulated in LNPs can be separated by selecting appropriate chromatographic columns (such as reversed-phase columns or ion-exchange columns) and mobile phases. The RNA signal is detected using ultraviolet or fluorescence detectors, and RNA is quantified based on the chromatographic peak area or peak height, thereby calculating the encapsulation efficiency. However, HPLC equipment is expensive, has high operating costs, and sample processing and optimization of chromatographic conditions require certain experience and skills, making the operation relatively complex. In addition, for some complex LNP-RNA systems, there may be problems such as overlapping chromatographic peaks, which affect the determination of encapsulation efficiency. Summary of the Invention
[0007] Addressing the current research status and challenges of LNP-RNA encapsulation efficiency measurement methods, this invention prepares a recombinant MGA solution. Utilizing the proportionality between the fluorescence intensity of MGA and MG binding and the MGA content, different concentration gradients of recombinant MGA and MG are established. The fluorescence intensity emitted at 630 / 655 nm is detected using a microplate reader, and a standard curve between MGA and fluorescence intensity is calculated. This method offers high accuracy, precision, and ease of operation in RNA content determination.
[0008] The technical solution provided by this invention is as follows: The first aspect of this invention provides a method for detecting the RNA encapsulation efficiency of nanoliposomes using malachite green, characterized by comprising the following steps: S1. Prepare recombinant MGA solutions, blank sample solutions, MG solutions, and nanoliposome demulsifiers of different concentrations; S2. Preparation of nanoliposome-MGA encapsulation system; S3. After incubating recombinant MGA solutions of different concentrations and blank sample solutions with MG solution, the fluorescence intensity was detected using a multi-functional microplate reader. The theoretical concentration of different batches of recombinant MGA solutions, the accuracy and precision of the detection method were calculated based on the fluorescence intensity. S4. After incubating the nanoliposome demulsifier with the nanoliposome-MGA encapsulation system, the fluorescence intensity of the nanoliposome-MGA encapsulation system before and after demulsification was detected using a multi-functional microplate reader, and the encapsulation efficiency of the nanoliposomes on RNA was calculated based on the fluorescence intensity.
[0009] Further, in step S1, the recombinant MGA sequence in the recombinant MGA solution includes a tRNA scaffold, a miRNA precursor sequence, and a malachite green aptamer sequence; The blank sample solution was a 10mM HEPES buffer solution containing 100mM KCl and 5mM MgCl2, prepared with DEPC water, and the pH of the HEPES buffer solution was 7.4. The concentration of the MG solution is 60-100 μM; The nanoliposome demulsifier was prepared from a blank sample solution containing 5-10% Triton by volume.
[0010] Furthermore, the preparation of the nanoliposome-MGA encapsulation system includes the following steps: (1) Preparation of working solution for lipid nanoparticles Dlin-MC3-DMA, DOPE, DOTAP, and Cholesterol were dissolved in ethanol at a molar ratio of (3-5):(2.5-4.5):(8-12):(2-5) to prepare a working solution for lipid nanoparticles. (2) Preparation of RNA aqueous working solution Prepare a 10 mM sodium citrate buffer solution with a pH of 4.0 as an aqueous phase buffer. Dissolve the recombinant MGA in the sodium citrate buffer solution to prepare the RNA aqueous phase working solution. (3) Preparation of lipid nanoparticle-RNA mixture by microfluidic device The tubing and chip were rinsed with anhydrous ethanol and DEPC water respectively. The two-phase solution was injected into the two inlet channels of the preparation system. One phase of the two-phase solution was an aqueous working solution of RNA, and the other phase was a working solution of lipid nanoparticles. The lipid nanoparticle-RNA mixture was obtained by rapid mixing of the two-phase solution at the chip. (4) Removal of ethanol from lipid nanoparticle-RNA mixture The lipid nanoparticle-RNA mixture was poured into a 100kD ultrafiltration tube and centrifuged at 3000g for 20 min at room temperature. The ultrafiltration tube was removed, the liquid at the bottom was poured out, the remaining solution was added to the ultrafiltration tube and centrifuged again. This step was repeated until all the solution was ultrafiltered. All the liquid was aspirated with a pipette and transferred to a centrifuge tube, and sterilized using a 0.22μm filter membrane to obtain the nanoliposome-MGA encapsulation system.
[0011] Furthermore, the tRNA scaffold includes human serine tRNA, human glutamate tRNA, human cysteine tRNA, human leucine tRNA, human lysine tRNA, human glutamine tRNA, human tyrosine tRNA, and bacterial methionine tRNA; The miRNA precursor sequence is the miR-34a precursor sequence.
[0012] Furthermore, the concentration range of the recombinant MGA solution is 1-150 ng / μL.
[0013] Further, in step S2, the ratio of nanoliposomes to MGA in the nanoliposome-MGA encapsulation system is 1:(15-25); In step S3, the excitation wavelength of the multifunctional microplate reader for detecting fluorescence is 630 nm, and the emission wavelength is 655 nm.
[0014] Further, in step S3, the ratio of the MGA solution to the MG solution is 90:10.
[0015] Furthermore, in step S4, the volume ratio of the nanoliposome demulsifier to nanoliposome-MGA is 1:1, and the incubation time is 10-30 min.
[0016] A second aspect of the present invention provides an application of the above-mentioned method for detecting the encapsulation efficiency of nanoliposomes on RNA using malachite green in detecting the encapsulation efficiency of nanoliposomes on different types of RNA products.
[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention uses malachite green (MG), which is inexpensive and a commonly used organic dye. It will not enter the LNP particles and interfere with the measurement of RNA content before demulsification. It can accurately measure the encapsulation rate of LNP, improve the precision of the measurement, and has the advantages of simple and readily available materials, low cost, and high accuracy.
[0018] 2. The invention is easy to operate, and the detection process can be completed in a short time, which is suitable for the detection needs of large-scale samples and helps to improve R&D and production efficiency.
[0019] 3. This invention is universally applicable to different types of RNA and LNP systems, and can be used for the research and development of various RNA drug candidates. Attached Figure Description
[0020] Figure 1 This is a flowchart of a method for detecting RNA encapsulation efficiency of nanoliposomes using malachite green, provided by the present invention. Figure 2 This invention utilizes a multifunctional microplate reader to measure the fluorescence emitted by MG when it binds to recombinant MGA solutions of different concentrations; Figure 3 This is a graph showing the zeta potential results of LNP-MGA prepared by microfluidic method in this invention; Figure 4 This is a diagram showing the particle size results of LNP-MGA prepared by microfluidic method according to the present invention. Detailed Implementation
[0021] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.
[0022] See Figure 1 This invention provides a method for detecting the RNA encapsulation efficiency of nanoliposomes using malachite green, comprising the following steps: S1. Prepare recombinant MGA solutions, blank sample solutions, MG solutions, and nanoliposome demulsifiers of different concentrations.
[0023] In step S1, the preparation steps for each sample are as follows: (1) Blank sample solution: Weigh 0.754g KCl, 0.0475g MgCl2, and 0.238g HEPES, dissolve them in 90mL of DEPC water, adjust the pH to 7.4 with KOH, and then make up to 100mL to prepare a HEPES-KCl-MgCl2 buffer solution containing 100mM KCl, 5mM MgCl2, and 10mM HEPES.
[0024] (2) Nanoliposome demulsifier (i.e. 5% Triton demulsifier): Mix 1 mL of Triton-100 with 19 mL of HEPES-KCL-MgCL2 buffer and stir with a magnetic stirrer for 15 minutes to obtain 5% Triton demulsifier.
[0025] (3) 100µM MG solution: Weigh 185.406mg of MG and dissolve it in 50mL of DEPC water to prepare a 100µM MG solution.
[0026] (4) Recombinant MGA stock solution: Prepare a 1.6 μg / μL recombinant MGA stock solution using sterile DEPC water.
[0027] (5) Recombinant MGA solution: The recombinant MGA stock solution was diluted with HEPES-KCL-MgCL2 buffer to prepare a series of recombinant MGA solutions with the following concentrations: 1.6 ng / μL, 3.2 ng / μL, 6.4 ng / μL, 8 ng / μL, 16 ng / μL, 25.6 ng / μL, 32 ng / μL, 40 ng / μL, 64 ng / μL, and 80 ng / μL.
[0028] S2. Preparation of nanoliposome-MGA encapsulation system.
[0029] In step S2, the preparation of the nanoliposome-MGA encapsulation system includes the following steps: (1) Preparation of working solution for lipid nanoparticles Dlin-MC3-DMA (4-(N,N-dimethylamino) acid (dilinoleyl) methyl ester), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine), DOTAP (1,2-dioleoyl-3-trimethylpropylammonium chloride), and Cholesterol were dissolved in 30 mL of ethanol at a molar ratio of 4:3:9:4 to prepare an 8 mM lipid nanoparticle working solution.
[0030] (2) Preparation of RNA aqueous working solution Prepare a 10mM sodium citrate buffer solution with a pH of 4.0 as the aqueous phase buffer. Weigh 240μg of recombinant MGA and dissolve it in 3mL of sodium citrate buffer solution to prepare an 80μg / mL recombinant small RNA sodium citrate working solution, which is the RNA aqueous phase working solution.
[0031] (3) Preparation of lipid nanoparticle-RNA mixture by microfluidic device The tubing and chip were rinsed with anhydrous ethanol and DEPC water, respectively, and the two-phase solution was injected into the two inlet channels of the preparation system. One phase of the two-phase solution was an aqueous working solution of RNA, and the other phase was a working solution of lipid nanoparticles. The lipid nanoparticle-RNA mixture was obtained by rapid mixing of the two-phase solution at the chip.
[0032] The microfluidic parameters for sample injection were as follows: lipid phase injection volume: 1 mL, lipid phase flow rate: 5 mL / min; RNA phase injection volume: 3 mL, RNA phase flow rate: 15 mL / min; flow rate ratio: 1:3, total flow rate: 20 mL / min; the outflowing mixture was collected in the collection tube and characterized for particle size, potential, etc. in the LNP-RNA (i.e., lipid nanoparticle-RNA) solution.
[0033] (4) Removal of ethanol from lipid nanoparticle-RNA mixture The lipid nanoparticle-RNA mixture was poured into a 100kD ultrafiltration tube and centrifuged at 3000g for 20 min at room temperature. The ultrafiltration tube was removed, the liquid at the bottom was poured out, the remaining solution was added to the ultrafiltration tube and centrifuged again. This step was repeated until all the solution was ultrafiltered. All the liquid was aspirated with a pipette and transferred to a 10mL centrifuge tube. The tube was then sterilized using a 0.22μm filter membrane to obtain the nanoliposome-MGA encapsulation system.
[0034] S3. After incubating recombinant MGA solutions of different concentrations and blank sample solutions with MG solution, the fluorescence intensity was detected using a multi-functional microplate reader. The theoretical concentration of different batches of recombinant MGA solutions, as well as the accuracy and precision of the detection method, were calculated based on the fluorescence intensity.
[0035] S4. After incubating the nanoliposome demulsifier with the nanoliposome-MGA encapsulation system, the fluorescence intensity of the nanoliposome-MGA encapsulation system before and after demulsification was detected using a multi-functional microplate reader, and the encapsulation efficiency of the nanoliposomes on RNA was calculated based on the fluorescence intensity.
[0036] Example 1 This embodiment verifies the relationship between the concentration of recombinant MGA solution and fluorescence intensity through the following steps: (1) Eight concentration gradients of recombinant MGA solution were selected, namely: blank sample solution, 1.6 ng / μL, 3.2 ng / μL, 8 ng / μL, 16 ng / μL, 32 ng / μL, 40 ng / μL, and 80 ng / μL. 90 μL of sample was added to each well in a black 96-well plate, and each concentration was duplicated in three wells.
[0037] (2) Add MG solution Add 10 µL of 100 µM MG solution to each well, for a total reaction volume of 100 µL.
[0038] (3) Parameter reading of multi-functional microplate reader Incubate at 37℃ with shaking for 30 seconds, and then detect the fluorescence value at 630 / 655nm using an ELISA reader.
[0039]
[0040] (4) Plotting the standard curve A standard curve was plotted with the concentration of the recombinant MGA solution on the x-axis and the corresponding fluorescence intensity on the y-axis. The curve equation was determined by linear regression analysis.
[0041] Figure 2The linear curve for detecting the fluorescence value of recombinant MGA and MG binding by the microplate reader was obtained. The regression equation was y = 43.43x + 47.73 (x is the concentration; y is the fluorescence value), and the correlation coefficient r = 0.998. This indicates that the standard curve has a good linear relationship with the corresponding fluorescence value in the concentration range of 0-80 ng / μL, and the good fit is high.
[0042] (5) Repeat the above experimental steps three times.
[0043] Example 2 This embodiment tests the accuracy and precision of the method provided by the present invention through the following steps, specifically including: (1) Eight recombinant MGA concentration gradients were selected: blank sample solution, 1.6 ng / μL, 3.2 ng / μL, 8 ng / μL, 16 ng / μL, 32 ng / μL, 40 ng / μL, and 80 ng / μL. 90 μL of sample was added to each well in a black 96-well plate, and each concentration was duplicated in triplicate.
[0044] (2) Select recombinant MGA samples (concentrations of 1.6 ng / μL, 6.4 ng / μL, 25.6 ng / μL, and 64 ng / μL), add 90 μL of sample to each well in a 96-well plate, and repeat the process for each concentration in three wells.
[0045] (3) Add MG solution Add 10 µL of 100 µM MG solution to each well, for a total reaction volume of 100 µL.
[0046] (4) Reading parameters of multi-functional microplate reader Incubate at 37℃ with shaking for 30 seconds, then detect the fluorescence values at 630 / 655nm using a microplate reader.
[0047] (5) Calculation of accuracy and precision Based on the detection results of the multi-functional microplate reader, the linear regression equation and the measured value of the sample to be tested are obtained.
[0048] Accuracy (accuracy describes how close the measured value of an analyte is to the true concentration, expressed as a percentage of the measured value) and precision (precision refers to the consistency between measured values when an analyte is repeatedly measured under the same conditions, expressed as a relative standard deviation, RSD %) were calculated. The sample was tested three times on the same day to determine intra-day accuracy and precision. This process was repeated three times on different days to determine inter-day accuracy and precision. The results are shown in Tables 1 (intra-day) and 2 (inter-day). The linear regression method between MGA concentration and fluorescence intensity met the requirements for both accuracy and precision in detecting the concentration of recombinant MGA solution.
[0049]
[0050]
[0051] Example 3 This embodiment provides a method for preparing an LNP-MGA, including the following steps: (1) Preparation of working solution for lipid nanoparticles Dlin-MC3-DMA (4-(N,N-dimethylamino) acid (dilinoleyl) methyl ester), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine), DOTAP (1,2-dioleoyl-3-trimethylpropylammonium chloride), and Cholesterol were dissolved in 30 mL of ethanol at a molar ratio of 4:3:9:4 to prepare an 8 mM lipid nanoparticle working solution.
[0052] (2) Preparation of RNA aqueous working solution Prepare a 10mM sodium citrate buffer solution with a pH of 4.0 as the aqueous phase buffer. Weigh 240μg of recombinant MGA and dissolve it in 3mL of sodium citrate buffer solution to prepare an 80μg / mL recombinant small RNA sodium citrate working solution, which is the RNA aqueous phase working solution.
[0053] (3) Preparation of lipid nanoparticle-RNA mixture by microfluidic device The tubing and chip were rinsed with anhydrous ethanol and DEPC water, respectively, and the two-phase solution was injected into the two inlet channels of the preparation system. One phase of the two-phase solution was an aqueous working solution of RNA, and the other phase was a working solution of lipid nanoparticles. The lipid nanoparticle-RNA mixture was obtained by rapid mixing of the two-phase solution at the chip.
[0054] The microfluidic parameters for sample injection were as follows: lipid phase injection volume: 1 mL, lipid phase flow rate: 5 mL / min; RNA phase injection volume: 3 mL, RNA phase flow rate: 15 mL / min; flow rate ratio: 1:3, total flow rate: 20 mL / min; the outflowing mixture was collected in the collection tube and characterized for particle size, potential, etc. in the LNP-RNA (i.e., lipid nanoparticle-RNA) solution.
[0055] (4) Removal of ethanol from lipid nanoparticle-RNA mixture The lipid nanoparticle-RNA mixture was poured into a 100kD ultrafiltration tube and centrifuged at 3000g for 20 min at room temperature. The ultrafiltration tube was removed, the liquid at the bottom was poured out, and the remaining solution was added to the ultrafiltration tube and centrifuged again. This step was repeated until all the solution was ultrafiltered. All the liquid was aspirated with a pipette and transferred to a 10mL centrifuge tube, and sterilized using a 0.22μm filter membrane to obtain the nanoliposome-MGA encapsulation system.
[0056] like Figure 3 and Figure 4 The figures show the Zeta potential and particle size results for the LNP-MGA solution, respectively. The results indicate that the LNP-MGA solution has a Zeta potential of 30-40 mV, is positively charged, has an average particle size of 104.8 nm, and an average PDI of 0.191. Furthermore, both the particle size range and the potential distribution range exhibit narrow peak shapes, meeting the requirements for particle size and Zeta potential in nucleic acid delivery.
[0057] Example 4 This embodiment provides a method for measuring the encapsulation efficiency of LNP-MGA, including the following steps: (1) Eight recombinant MGA concentration gradients were selected: blank sample solution, 1.6 ng / μL, 3.2 ng / μL, 8 ng / μL, 16 ng / μL, 32 ng / μL, 40 ng / μL, and 80 ng / μL. 90 μL of sample was added to each well in a black 96-well plate, and each concentration was duplicated in triplicate.
[0058] The MGA sequence in the recombinant MGA sample includes a tRNA scaffold, a miRNA precursor sequence, and a malachite green aptamer sequence. The tRNA scaffold includes human serine tRNA, human glutamate tRNA, human cysteine tRNA, human leucine tRNA, human lysine tRNA, human glutamine tRNA, human tyrosine tRNA, and bacterial methionine tRNA. The miRNA precursor sequence is the miR-34a precursor sequence.
[0059] (2) Preparation of LNP-MGA test samples For LNP-MGA samples to be tested, add 10 μL of sample to each well of a black 96-well plate, and then add HEPES-KCL-MgCL2 buffer to make up to 90 μL. Repeat for three wells. LNP-MGA demulsification sample: 30 μL LNP-MGA + 30 μL 5% Triton demulsifier, vortex to mix, incubate for 20 minutes, add to black 96-well plates, 20 μL per well, add HEPES-KCL-MgCL2 buffer to make up to 90 μL, repeat in triplicate.
[0060] (3) Add MG solution Add 10 µL of 100 µM MG solution to each well, for a total reaction volume of 100 µL.
[0061] (4) Reading parameters of multi-functional microplate reader Incubate at 37℃ with shaking for 30 seconds, then detect the fluorescence values at 630 / 655nm using a microplate reader.
[0062] (5) Plotting the standard curve A standard curve was plotted with the concentration of MG on the x-axis and the corresponding fluorescence intensity on the y-axis. The curve equation was determined by linear regression analysis.
[0063] (6) Calculate the MGA content in LNP-MGA. After measuring the fluorescence intensity of the sample, the average fluorescence intensity value was substituted into the standard curve equation to calculate the content of unencapsulated MGA in the sample.
[0064] (7) Calculate the encapsulation ratio The encapsulation efficiency is calculated based on the total amount of nucleic acid added during the initial preparation of LNP. The encapsulation efficiency is calculated using the following formula: Encapsulation efficiency = (Total initial MGA - Amount of unencapsulated MGA) / Total initial MGA × 100%.
[0065] The results are shown in Table 3. The encapsulation efficiency of LNP-MGA measured by malachite green was 95.9%. From the above, it can be concluded that the encapsulation efficiency of LNP is >95%, which is good and meets the requirements for nucleic acid delivery.
[0066] Table 3 Encapsulation efficiency of LNP-MGA
[0067] The method for detecting RNA encapsulation efficiency of liposome nanoparticles provided by this invention is based on the principle that the fluorescence intensity of malachite green (MG) and its aptamer (MGA) is proportional to the amount of MGA. LNP-MGA is obtained by encapsulating MGA in lipid nanoparticles. MG is then mixed with LNP-MGA before and after demulsification, and the MGA content before and after demulsification is measured. The difference between the two values represents the MGA content encapsulated within the LNP particles, thus allowing for the calculation of the RNA encapsulation efficiency of the LNPs. Malachite green does not enter the LNP particles and interfere with the measurement of RNA content before demulsification, therefore, the RNA encapsulation efficiency of the LNPs can be accurately measured. This method has advantages such as readily available materials, low cost, and high accuracy.
[0068] This invention prepares recombinant MGA and utilizes the fact that the fluorescence intensity of MGA and MG after binding is proportional to the MGA content. Different concentration gradients of recombinant MGA and MG are set up, and the fluorescence intensity emitted at 630 / 655 nm is detected using a microplate reader. A standard curve of MGA versus fluorescence intensity is calculated based on the fluorescence intensity, demonstrating that this method has high accuracy, precision, and ease of operation in RNA content determination. Furthermore, this method is used to detect the amount of RNA free outside LNP particles in LNP-RNA solution and the total amount of RNA released into the external solution after treatment with 5% Triton solution. The difference between these two values yields the amount of RNA encapsulated inside the LNP particles, thus obtaining the encapsulation rate.
[0069] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.
Claims
1. A method for detecting the RNA encapsulation efficiency of nanoliposomes using malachite green, characterized in that, Includes the following steps: S1. Prepare recombinant MGA solutions, blank sample solutions, MG solutions, and nanoliposome demulsifiers of different concentrations; S2. Preparation of nanoliposome-MGA encapsulation system; S3. After incubating recombinant MGA solutions of different concentrations and blank sample solutions with MG solution, the fluorescence intensity was detected using a multi-functional microplate reader. The theoretical concentration of different batches of recombinant MGA solutions, the accuracy and precision of the detection method were calculated based on the fluorescence intensity. S4. After incubating the nanoliposome demulsifier with the nanoliposome-MGA encapsulation system, the fluorescence intensity of the nanoliposome-MGA encapsulation system before and after demulsification was detected using a multi-functional microplate reader, and the encapsulation efficiency of the nanoliposomes on RNA was calculated based on the fluorescence intensity.
2. The method for detecting RNA encapsulation efficiency of nanoliposomes using malachite green according to claim 1, characterized in that: In step S1, the recombinant MGA sequence in the recombinant MGA solution includes a tRNA scaffold, a miRNA precursor sequence, and a malachite green aptamer sequence. The blank sample solution was a 10mM HEPES buffer solution containing 100mM KCl and 5mM MgCl2, prepared with DEPC water, and the pH of the HEPES buffer solution was 7.
4. The concentration of the MG solution is 60-100 μM; The nanoliposome demulsifier was prepared from a blank sample solution containing 5-10% Triton by volume.
3. The method for detecting the RNA encapsulation efficiency of nanoliposomes using malachite green according to claim 1, characterized in that, The preparation of the nanoliposome-MGA encapsulation system includes the following steps: (1) Preparation of working solution for lipid nanoparticles Dlin-MC3-DMA, DOPE, DOTAP, and Cholesterol were dissolved in ethanol at a molar ratio of (3-5):(2.5-4.5):(8-12):(2-5) to prepare a working solution for lipid nanoparticles. (2) Preparation of RNA aqueous working solution Prepare a 10 mM sodium citrate buffer solution with a pH of 4.0 as an aqueous phase buffer. Dissolve the recombinant MGA in the sodium citrate buffer solution to prepare the RNA aqueous phase working solution. (3) Preparation of lipid nanoparticle-RNA mixture by microfluidic device The tubing and chip were rinsed with anhydrous ethanol and DEPC water respectively. The two-phase solution was injected into the two inlet channels of the preparation system. One phase of the two-phase solution was an aqueous working solution of RNA, and the other phase was a working solution of lipid nanoparticles. The lipid nanoparticle-RNA mixture was obtained by rapid mixing of the two-phase solution at the chip. (4) Removal of ethanol from lipid nanoparticle-RNA mixture The lipid nanoparticle-RNA mixture was poured into a 100kD ultrafiltration tube and centrifuged at 3000g for 20 min at room temperature. The ultrafiltration tube was removed, the liquid at the bottom was poured out, the remaining solution was added to the ultrafiltration tube and centrifuged again. This step was repeated until all the solution was ultrafiltered. All the liquid was aspirated with a pipette and transferred to a centrifuge tube. The solution was then sterilized using a filter membrane to obtain the nanoliposome-MGA encapsulation system.
4. The method for detecting RNA encapsulation efficiency of nanoliposomes using malachite green according to claim 2, characterized in that: The tRNA scaffold includes human serine tRNA, human glutamate tRNA, human cysteine tRNA, human leucine tRNA, human lysine tRNA, human glutamine tRNA, human tyrosine tRNA, and bacterial methionine tRNA; The miRNA precursor sequence is the miR-34a precursor sequence.
5. The method for detecting RNA encapsulation efficiency of nanoliposomes using malachite green according to any one of claims 1-4, characterized in that: The concentration range of the recombinant MGA solution is 1-150 ng / μL.
6. The method for detecting RNA encapsulation efficiency of nanoliposomes using malachite green according to claim 5, characterized in that: In step S2, the ratio of nanoliposomes to MGA in the nanoliposome-MGA encapsulation system is 1:(15-25); In step S3, the excitation wavelength of the multifunctional microplate reader for detecting fluorescence is 630 nm, and the emission wavelength is 655 nm.
7. The method for detecting RNA encapsulation efficiency of nanoliposomes using malachite green according to claim 5, characterized in that: In step S3, the ratio of the MGA solution to the MG solution is 90:
10.
8. The method for detecting RNA encapsulation efficiency of nanoliposomes using malachite green according to claim 6 or 7, characterized in that: In step S4, the volume ratio of the nanoliposome demulsifier to nanoliposome-MGA is 1:1, and the incubation time is 10-30 min.
9. The application of the method for detecting the RNA encapsulation efficiency of nanoliposomes using malachite green as described in any one of claims 1-8 in detecting the encapsulation efficiency of nanoliposomes for different types of RNA products.