LNP pKa detection method based on TNS fluorescence method

Abstract

The invention provides an LNP pKa detection method based on a TNS fluorescence method, which comprises the following steps: diluting a 10 * MOPS buffer solution into a 1 * MOPS buffer solution by using purified water, and then adjusting the 1 * MOPS buffer solution into a gradient solution with the pH value of 3-11; taking a porous plate, respectively adding detection buffer solutions with different pH values into each pore, then adding an LNP encapsulated sample into each pore, uniformly mixing, then adding 300 [mu] M of a TNS solution into each pore, and uniformly mixing; detecting the fluorescence intensity by adopting a fluorescence microplate reader; and carrying out data normalization processing on the fluorescence value of the fluorescence intensity reading result, fitting a curve, and calculating to obtain pKa by adopting a fitting formula RFU = RFUmax-(RFUmax-RFUmin) / (1 + 10 (pKa-pH)). By adopting the technical scheme, the preparation process of the buffer solution is simplified, the obtained detection result is accurate, the relative standard deviation is small, and the cost is low.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a pKa detection method of LNP based on a TNS fluorescence method. Background Art

[0002] Lipid nanoparticles (LNP) are lipid vesicles with a uniform lipid core, usually around 100 nm in diameter. This unique structure and size give them unique physical and chemical properties, enabling them to effectively encapsulate and protect nucleic acid drugs, such as mRNA, siRNA, antisense oligonucleotides, etc., and deliver them to target tissues or cells through the blood circulation system. The classic LNP formulation is mainly composed of ionizable cationic lipids, polyethylene glycol-modified lipids, cholesterol, and neutral auxiliary lipids. Ionizable cationic lipids are considered to be the core of the successful development of LNP. In traditional designs, cationic lipids are continuously charged and are prone to electrostatic interactions with non-target cell membranes during circulation in the body to produce cytotoxicity. Ionizable cationic lipids are pH-sensitive. Due to their amine hydrophilic ends, they are positively charged when combined with hydrogen ions under acidic conditions (pH < 6.0), but are uncharged under neutral conditions. Therefore, the use of ionizable cationic lipids to prepare LNPs can complex negatively charged nucleic acids under low pH conditions, thereby achieving high encapsulation efficiency; they are uncharged under physiological pH conditions, which can protect the integrity of the LNP structure and reduce toxic side effects; after LNP is internalized by the cell, it will be trapped in the endosomes. After the endosomes gradually mature into lysosomes, they will decompose exogenous macromolecules, and the intracellular pH will decrease to a slightly acidic environment. The ionizable lipids will then be protonated, causing the LNP to interact electrostatically with the endosomal membrane of the cell, thereby destroying the structure and successfully releasing the functional molecules contained therein to exert their functions.

[0003] The acid dissociation constant (pKa) is defined as the pH value at which the number of ionized (protonated) and non-ionized groups in the system is equal. Therefore, the apparent pKa of LNP is the pH value at which 50% of the ionizable lipids in the LNP are protonated. Since pKa determines the ionization and surface charge of LNPs, which greatly affects their stability, efficacy and toxicity, and also affects the cellular uptake, endosomal release and biodistribution of LNPs, accurate determination of the apparent pKa of LNPs is critical for the design and optimization of LNP delivery systems for drugs.

[0004] To date, the pKa of LNPs has been generally measured using a TNS (2-p-toluidine-6-naphthalenesulfonic acid) dye-binding assay. TNS is non-fluorescent in aqueous solution, negatively charged, and shows enhanced fluorescence after binding to positively charged LNPs. In buffers covering a wide pH range, as the pH decreases, the interaction between TNS and the ionizable surface increases, resulting in a steady increase in fluorescence. When all ionizable groups are charged at a specific pH, the fluorescence value reaches a maximum, and the pKa is estimated by the pH at which half of the maximum fluorescence value is reached.

[0005] At present, different experimenters use different formulations of wide pH range buffers to perform pKa tests for TNS fluorescence detection of pKa. The test results show that buffers with different formulations can all produce test results, but some buffers have more individual components, high procurement costs, and complex preparation processes and long preparation times, such as buffers with HEPES as the main component; some buffers have limited pH buffering capacity, resulting in large deviations between pKa measurement results and theoretical values, and cannot reflect the true pKa value of LNP, such as TBS buffer. At present, there is no fixed buffer formulation for TNS fluorescence detection, which makes the pKa detection process of LNP more complicated. Summary of the invention

[0006] In view of the above technical problems, the present invention discloses a pKa detection method of LNP based on TNS fluorescence method, which has accurate detection results, good repeatability and low cost.

[0007] To this end, the technical solution adopted by the present invention is:

[0008] A method for detecting the pKa of LNP based on TNS fluorescence method comprises the following steps:

[0009] Step S1, diluting 10×MOPS buffer with purified water to 1×MOPS buffer, and then adjusting the 1×MOPS buffer to a gradient solution of pH 3-pH 11 with NaOH or HCl as a detection buffer; wherein the main component of the MOPS buffer is 3-(N-morpholino)propanesulfonic acid; it is a zwitterionic buffer with the characteristics of being non-toxic, having good chemical stability, and having a stable pH value between 6.5 and 7.9.

[0010] Step S2, taking a multi-well plate, adding detection buffers of different pH values ​​to each well, then adding LNP encapsulated samples to each well and mixing, then adding 300 μM TNS solution to each well and mixing;

[0011] Step S3, using a fluorescence microplate reader to detect the fluorescence intensity;

[0012] Step S4, normalize the fluorescence intensity reading result and fit the curve using the fitting formula RFU=RFU max -(RFU max -RFU min ) / (1+10 pKa-pH ), calculate the pKa;

[0013] Among them, RFU is the fluorescence intensity at different pH values ​​during the measurement;

[0014] RFU max is the maximum fluorescence intensity of the detection;

[0015] RFU min is the minimum fluorescence intensity of the detection.

[0016] With this technical solution, compared with the detection method using HEPES buffer, the preparation of the buffer is more convenient, and compared with TBS buffer, it has a smaller relative standard deviation; and the results are highly accurate and reproducible.

[0017] As a further improvement of the present invention, in step S1, the gradient solution includes one solution for each pH value from pH 3 to pH 11, for a total of 9 solutions.

[0018] As a further improvement of the present invention, in step S2, the volume ratio of the detection buffer, the LNP encapsulated sample, and the 300 μM TNS solution added is 97:1:2.

[0019] As a further improvement of the present invention, in step S3, the excitation light wavelength of the fluorescence microplate reader is 325 nm, and the emission light wavelength is 435 nm.

[0020] As a further improvement of the present invention, the 300 μM TNS solution is prepared by dissolving 1 mg of 6-(p-toluidine)-2-naphthalenesulfonic acid sodium salt in 10 mL of DMSO.

[0021] The invention also discloses the application of a MOPS buffer in the pKa detection of LNP, wherein the MOPS buffer comprises a 1×MOPS gradient solution of pH 3-pH 11.

[0022] The invention also discloses the use of a MOPS buffer in preparing a pKa detection kit for LNP, wherein the MOPS buffer comprises a 1×MOPS gradient solution of pH 3-pH 11.

[0023] The present invention also discloses a pKa detection kit for LNP based on TNS fluorescence method. The kit is provided with a multi-porous plate, and the multi-porous plate contains gradient solutions of pH 3-pH 11 of 1×MOPS buffer with different pH values.

[0024] As a further improvement of the present invention, the pH 3-pH 11 gradient solution of the 1×MOPS buffer solution is prepared by the following steps: diluting the 10×MOPS buffer solution with purified water to form a 1×MOPS buffer solution, and then adjusting the 1×MOPS buffer solution with NaOH or HCl to form a gradient solution with one portion for each pH value from pH 3 to pH 11.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The technical solution of the present invention simplifies the buffer preparation process, saves preparation time, optimizes the buffer used for detecting pKa by TNS fluorescence method, and has low cost; the detection method is simple, the obtained detection result is accurate, the relative standard deviation is small, the repeatability is good, and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 1 is a fitting curve obtained by using MOPS buffer for detection in Example 1 of the present invention; wherein (a), (b) and (c) are the results of three repeated detections.

[0028] Figure 2 It is the fitting curve obtained by using HEPES buffer solution for detection in Comparative Example 1 of the present invention; wherein (a), (b) and (c) are the results of three repeated detections.

[0029] Figure 3 It is the fitting curve obtained by using purified water for detection in Comparative Example 2 of the present invention; wherein (a), (b) and (c) are the results of three repeated detections.

[0030] Figure 4 It is the fitting curve obtained by using TBS buffer solution for detection in Comparative Example 3 of the present invention; wherein (a), (b) and (c) are the results of three repeated detections. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present invention are described in further detail below.

[0032] Example 1

[0033] A method for detecting the pKa of LNP based on TNS fluorescence method comprises the following steps:

[0034] (1) Solution preparation:

[0035] ① 300 μM TNS: Weigh 1 mg of 6-(p-toluidine)-2-naphthalenesulfonic acid sodium salt (TNS) and dissolve it in 10 mL of DMSO.

[0036] ②MOPS buffer: Take 20mL of 10×MOPS buffer (Shenggong / C516042-0001), add 180mL of purified water, and stir to mix. Dispense into 9 tubes with 10mL each, and mark pH3-pH11. Then use NaOH or HCl to adjust to the corresponding pH gradient point to obtain MOPS buffer with pH3-pH11.

[0037] (2) Detection steps:

[0038] ① Take a 96-well plate (transparent), add 97 μL of each pH buffer solution (pH3-pH11) to each well, then add 1 μL of LNP encapsulated sample to each well, mix well, then add 2 μL of 300 μM TNS and mix well.

[0039] ② Read the fluorescence using a fluorescence microplate reader (excitation light 325nm, emission light 435nm).

[0040] ③ The fluorescence intensity reading results were normalized and the curve was fitted using Matlab software. The fitting formula was RFU = RFU max -(RFU max -RFU min ) / (1+10 pKa-pH ), calculate the pKa.

[0041] The same sample was tested three times.

[0042] (3) Results analysis

[0043] The experiment was repeated three times using MOPS buffer. Figure 1 shown.

[0044] Results of the first test: RFU max =1,RFU min =0.03542, pKa =6.169 (6.099, 6.239);

[0045] Results of the second test: RFU max =1,RFU min =0.03457, pKa = 6.603 (6.496, 6.71);

[0046] Results of the third test: RFU max =1,RFU min =0.04190, resulting in pKa=6.549 (6.439, 6.658).

[0047] The pKa values ​​of three replicates were 6.169, 6.603, and 6.549, respectively, with an average pKa of 6.44. The correlation coefficient R2 They are 0.9981, 0.9964 and 0.9965 respectively.

[0048] Comparative Example 1

[0049] Based on Example 1, the difference of this comparative example is that the buffer solution is different, and specifically comprises the following steps:

[0050] (1) Solution preparation:

[0051] ① 300 μM TNS: Weigh 1 mg of 6-(p-toluidine)-2-naphthalenesulfonic acid sodium salt (TNS) and dissolve it in 10 mL of DMSO.

[0052] ②HEPES buffer: Weigh 0.19 g of ammonium acetate, 0.53 g of 2-(N-morpholino)ethanesulfonic acid (MES), 0.6 g of HEPES, and 1.9 g of sodium chloride, dissolve in 240 mL of purified water, then dispense into 9 tubes (24 mL each), marked with pH 3-pH 11, and then adjust to the corresponding pH gradient point with NaOH or HCl, and then adjust the volume to a final volume of 25 mL with purified water.

[0053] (2) Detection steps:

[0054] ① Take a 96-well plate (transparent), add 97 μL of each pH buffer solution (pH3-pH11) to each well, then add 1 μL of LNP encapsulated sample to each well, mix well, then add 2 μL of 300 μM TNS and mix well.

[0055] ② Read the fluorescence using a fluorescence microplate reader (excitation light 325nm, emission light 435nm).

[0056] ③ The fluorescence intensity reading results were normalized and the curve was fitted using Matlab software. The fitting formula was RFU = RFU max -(RFU max -RFU min ) / (1+10 pKa-pH ), calculate the pKa.

[0057] The assay was performed in triplicate.

[0058] (3) Result analysis:

[0059] The experiment was repeated three times using HEPES buffer. Figure 2 shown.

[0060] Results of the first test: RFU max =1,RFU min =0.04379, pKa=6.518 (6.457, 6.579);

[0061] Results of the second test: RFU max =1,RFU min =0.03314, pKa=6.176 (6.022, 6.33);

[0062] Results of the third test: RFU max =1,RFU min =0.04781, resulting in pKa=6.61 (6.512, 6.707).

[0063] The pKa values ​​of three replicates were 6.518, 6.176, and 6.610, respectively, with an average pKa of 6.43. The correlation coefficient R 2 They are 0.9987, 0.9895 and 0.9966 respectively.

[0064] Comparative Example 2

[0065] Based on Example 1, the difference of this comparative example is that purified water is used instead of buffer solution, and specifically comprises the following steps:

[0066] (1) Solution preparation:

[0067] ① 300 μM TNS: Weigh 1 mg of 6-(p-toluidine)-2-naphthalenesulfonic acid sodium salt (TNS) and dissolve it in 10 mL of DMSO.

[0068] ②Purified water: Take an appropriate amount of purified water and dispense it into 10 mL tubes, a total of 9 tubes, and mark pH3-pH11. Then use NaOH or HCl to adjust to the corresponding pH gradient point (pH3-pH11).

[0069] (2) Detection steps:

[0070] The detection steps are the same as those in Example 1. The detection is repeated three times.

[0071] (3) Results analysis

[0072] The experiment was repeated three times using purified water. Figure 3 shown.

[0073] Results of the first test: RFU max =1,RFU min =0.028607, pKa =5.174 (4.558, 5.791);

[0074] Results of the second test: RFU max =1,RFU min=0.028192, pKa =4.993 (4.417, 5.569);

[0075] Results of the third test: RFU max =1,RFU min =0.021151, resulting in pKa=4.705 (4.236, 5.173).

[0076] The pKa values ​​of three replicates were 5.174, 4.993, and 4.705, respectively. The correlation coefficient R 2 They are 0.756, 0.7993 and 0.8588 respectively.

[0077] Comparative Example 3

[0078] This comparative example is different from Example 1 in that TBS buffer is used for detection. Specifically, the following steps are included:

[0079] (1) Solution preparation:

[0080] ① 300 μM TNS: Weigh 1 mg of 6-(p-toluidine)-2-naphthalenesulfonic acid sodium salt (TNS) and dissolve it in 10 mL of DMSO.

[0081] ②TBS buffer: Take 10 mL of 20×TBS buffer (Shenggong / B548105-0500), add 190 mL of purified water, stir and mix. Then use NaOH or HCl to adjust to the corresponding pH gradient point (pH3-pH11).

[0082] (2) Detection steps:

[0083] The detection steps are the same as those in Example 1. The detection is repeated three times.

[0084] (3) Results analysis

[0085] The experiment was repeated three times using TBS buffer. Figure 4 shown.

[0086] Results of the first test: RFU max =1,RFU min =0.02583, pKa=5.551 (5.449, 5.653);

[0087] Results of the second test: RFU max =1,RFU min =0.02904, pKa =5.202 (5.166, 5.237);

[0088] Results of the third test: RFU max=1,RFU min =0.02873, resulting in pKa=5.836 (5.665, 6.006).

[0089] The pKa values ​​of three replicates were 5.551, 5.202, and 5.836, respectively, with an average pKa of 5.53. The correlation coefficient R 2 They are 0.9957, 0.9995 and 0.9886 respectively.

[0090] The pKa results of Example 1 and Comparative Examples 1-3 are compared, and the results are shown in Table 1. It can be seen from the experimental results of the four solutions that the pKa value measured by MOPS buffer in Example 1 is not much different from the average value of the HEPES test results in the literature, with a relative standard deviation (RSD) of 0.11%, and the R of the fitting curve is 2 All of them are greater than 0.98, indicating that MOPS buffer can replace the complex HEPES buffer to detect pKa. 2 The relative standard deviation (RSD) of the commercial TBS buffer and HEPES test results is 10.64%, which is quite different. By comparison, it can be seen that the results of TNS fluorescence method using MOPS buffer are highly accurate, with a small relative standard deviation, and the buffer preparation steps are simple and easy to use.

[0091] Table 1 Summary of experimental results of embodiments and comparative examples

[0092] Buffer pKa-1 pKa-2 pKa-3 Average pKa <![CDATA[R 2 -1]]> <![CDATA[R 2 -2]]> <![CDATA[R 2 -3]]> HEPES 6.518 6.176 6.610 6.43 0.9987 0.9895 0.9966 MOPS 6.169 6.603 6.549 6.44 0.9981 0.9964 0.9965 Purified water 5.174 4.993 4.705 4.96 0.756 0.7993 0.8588 TBS 5.551 5.202 5.836 5.53 0.9957 0.9995 0.9886

[0093] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A method for detecting the pKa of LNP based on TNS fluorescence method, characterized in that: The steps include: Step S1, diluting 10×MOPS buffer with purified water to 1×MOPS buffer, and then adjusting the 1×MOPS buffer with NaOH or HCl to a gradient solution of pH 3-pH 11 as a detection buffer; Step S2, taking a multi-well plate, adding detection buffers of different pH values ​​to each well, then adding LNP encapsulated samples to each well and mixing, then adding 300 μM TNS solution to each well and mixing; Step S3, using a fluorescence microplate reader to detect the fluorescence intensity; Step S4, normalize the fluorescence intensity reading result and fit the curve using the fitting formula RFU=RFU max -(RFU max -RFU min ) / (1+10 (pKa-pH) ), calculate the pKa; Among them, RFU is the fluorescence intensity at different pH values ​​during the measurement; RFU max is the maximum fluorescence intensity of the detection; RFU min is the minimum fluorescence intensity of the detection.

2. The method for detecting the pKa of LNP based on TNS fluorescence method according to claim 1, characterized in that: In step S2, the volume ratio of the detection buffer, LNP encapsulated sample, and 300 μM TNS solution added was 97:1:

2.

3. The method for detecting the pKa of LNP based on TNS fluorescence method according to claim 1, characterized in that: In step S3, the excitation light wavelength of the fluorescence microplate reader is 325 nm, and the emission light wavelength is 435 nm.

4. The method for detecting the pKa of LNP based on TNS fluorescence method according to claim 1, characterized in that: The 300 μM TNS solution was prepared by dissolving 1 mg of 6-(p-toluidine)-2-naphthalenesulfonic acid sodium salt in 10 mL of DMSO.

5. The application of MOPS buffer in the pKa detection of LNP is characterized by: The MOPS buffer includes a 1×MOPS gradient solution of pH 3-pH 11.

6. The use of MOPS buffer in the preparation of a pKa detection kit for LNP, characterized in that: The MOPS buffer includes a 1×MOPS gradient solution of pH 3-pH 11.

7. A pKa detection kit for LNP based on TNS fluorescence method, characterized in that: The kit is provided with a multi-well plate, and the multi-well plate contains gradient solutions of pH 3-pH 11 of 1×MOPS buffer with different pH values.

8. The pKa detection kit of LNP based on TNS fluorescence method according to claim 7, characterized in that: The pH 3-pH 11 gradient solution of the 1×MOPS buffer solution is prepared by the following steps: diluting the 10×MOPS buffer solution with purified water to form a 1×MOPS buffer solution, and then adjusting the 1×MOPS buffer solution with NaOH or HCl to form a gradient solution with one portion for each pH value from pH 3 to pH 11.

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