Method for evaluating quality of lipid particles

Abstract

To provide a method for accurate evaluation of lipid particle quality.SOLUTION: A method for evaluating quality of lipid particles according to an embodiment includes the steps of: preparing a mixed solution containing lipid particles having a target substance encapsulated therein, and a fluorescent substance that fluoresces upon binding to the target substance; measuring fluorescence at two or more temperature points during heating of the prepared mixed solution to determine a value of change in fluorescence intensity; and comparing the determined value of change in fluorescence intensity with a preset threshold to evaluate the quality of the lipid particles under evaluation.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Embodiments of the present invention relate to a method for evaluating the quality of lipid particles.

Background Art

[0002] Conventionally, for products containing lipid particles, quality evaluation of lipid particles has been carried out as part of quality control. Lipid particles are vesicles composed of a lipid membrane, and to evaluate their quality, it is necessary to grasp the membrane state. However, in the prior art, it is difficult to directly measure the membrane state of lipid particles, and it is generally evaluated using indirect indicators such as particle size, surface charge, and the amount of encapsulated substances.

[0003] However, in many cases, it has been confirmed that the above-mentioned known indicators do not accurately reflect the quality of lipid particles. For example, even if the values of known indicators measured between individual product lots are equivalent, differences in performance may be observed when used as products. Among them, there are also undesirable cases where the values of known indicators obtained for a good product lot and a defective product lot are equivalent, making it impossible to distinguish between the two.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The problem to be solved by the present invention is to provide a method for accurately evaluating the quality of lipid particles.

Means for Solving the Problems

[0005] A method for evaluating the quality of lipid particles according to an embodiment includes a preparation step of preparing a mixed solution containing lipid particles encapsulating a target substance and a fluorescent substance that binds to the target substance and generates fluorescence, a measurement step of performing fluorescence measurement at at least two or more temperatures while heating the prepared mixed solution to obtain a change amount of fluorescence intensity, and an evaluation step of comparing the obtained value of the change amount of fluorescence intensity with a preset threshold value to determine the quality of the lipid particles to be evaluated.

Brief Description of the Drawings

[0006]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0007] Hereinafter, embodiments will be described with reference to the accompanying drawings. In each embodiment, substantially the same constituent parts are denoted by the same reference numerals, and the description thereof may be partially omitted. The drawings are schematic, and the relationship between the thicknesses of each part, the planar dimensions, the ratio of the thicknesses of each part, etc. may be different from the actual ones.

[0008] (First Embodiment) The first embodiment relates to a method for evaluating the quality of lipid particles. As shown in FIG. 1, the method includes a step of preparing a mixture containing lipid particles encapsulating a target substance and a fluorescent substance that binds to the target substance and generates fluorescence (hereinafter referred to as "preparation step (S11)"), a step of performing fluorescence measurement at at least two or more temperatures while heating the prepared mixture to obtain the amount of change in fluorescence intensity for each temperature (hereinafter referred to as "measurement step (S12)"), and a step of comparing the obtained value of the amount of change in fluorescence intensity with a preset threshold value to determine the quality of the lipid particles to be evaluated (hereinafter referred to as "evaluation step (S13)"). Hereinafter, each step will be described in detail.

[0009] In the preparation step (S11), a mixture containing lipid particles encapsulating a target substance and a fluorescent substance that binds to the target substance and generates fluorescence is prepared.

[0010] The lipid particles applied to the method of the present embodiment may be composed of any type of lipid membrane as long as they are substantially spherical hollow bodies formed by non-covalent arrangement of a plurality of lipid molecules. For example, the lipid membrane may be either a lipid monolayer or a lipid bilayer, and may be composed of a single layer of membrane or a multi-layer of membrane. More specifically, the lipid particles may be liposomes composed of a lipid bilayer composed of lipids (e.g., phospholipids).

[0011] As used herein, the "target substance" refers to a substance that is desired to be encapsulated within lipid particles. The target substance may be any type of compound or composition, regardless of its kind or use, as long as it is desired to be encapsulated within lipid particles. For example, when lipid particles are used as a means of delivery to cells, the target substance is a substance that is desired to be introduced into the cells, such as a nucleic acid or an organic compound other than a nucleic acid, and is introduced for uses such as gene editing tools, therapeutic agents, and cosmetics. Note that the target substance encapsulated within the lipid particles in the preparation step (S11) may be a single compound or composition, or may be a plurality of types of compounds or compositions. In other words, the lipid particles contained in the mixture prepared in S11 may encapsulate a plurality of types of compounds or compositions as the target substance.

[0012] As used herein, the "fluorescent substance" refers to a substance that generates fluorescence by interacting with the target substance. The interaction with the target substance may be, for example, various chemical bonds (such as covalent bonds or ionic bonds) with the target substance, or various chemical bonds that do not result in a direct chemical bond with the target substance but occur between the target substance and a complex with another substance. The reaction that generates fluorescence is preferably a reaction in which the amount of fluorescence increases according to the amount of the interacting target substance. Further, the fluorescent substance is a substance that can permeate the lipid membrane of the lipid particles during the process of increasing the temperature in the measurement step (S12). The lipid membrane of the lipid particles exhibits a certain degree of substance permeability, and as will be described later, the substance permeability tends to increase when placed in a higher temperature environment. The fluorescent substance of the present embodiment preferably exhibits physical properties such that it can permeate the lipid membrane of lipid particles whose substance permeability has increased under a high temperature environment. Conversely, a fluorescent substance that cannot permeate the lipid particles to be evaluated under any conditions (for example, an extremely high molecular weight fluorescent substance, or a fluorescent substance with extremely strong lipophilicity that stays inside the lipid membrane) is not suitable in the present embodiment.

[0013] The interaction between the target substance and the fluorescent substance is preferably more specific. However, when there is one type of target substance, or when there are multiple types and it is not necessary to distinguish between them, or when there is no substance in the measurement environment that can react with the fluorescent substance other than the target substance, it does not have to be specific. For example, when the target substance is single-stranded DNA having a specific base sequence, it may be a nucleic acid stain (e.g., an intercalator such as ethidium bromide) that can generate fluorescence by binding to a nucleic acid containing any base sequence, or it may be other organic or inorganic fluorophores that generate fluorescence by interacting with nucleic acids.

[0014] Here, it should be noted that in the mixture prepared in S11, the target substance and the fluorescent substance are distinguished as different substances. This means that in the mixture prepared in S11, the state where the fluorescent substance is encapsulated in lipid particles is not desired. Therefore, in the mixture prepared in S11, it is preferable that the target substance is in a state isolated from the fluorescent substance by lipid particles. In other words, the mixture prepared in S11 is preferably prepared so as not to exhibit the interaction between the target substance and the fluorescent substance.

[0015] To prevent the interaction between the target substance and the fluorescent substance from occurring, it is preferable to prepare them by a method that minimizes their contact. For example, it may be prepared by separately preparing lipid particles encapsulating the target substance and the fluorescent substance and then mixing them. Specifically, the mixture prepared in S11 may be prepared by mixing a dispersion containing lipid particles encapsulating the target substance and an aqueous solution containing a fluorescent substance that binds to the target substance to generate fluorescence. Alternatively, the mixture prepared in S11 may be prepared by mixing a dispersion containing lipid particles encapsulating the target substance and a powder of a fluorescent substance that binds to the target substance to generate fluorescence. In other words, the preparation step (S11) may include a step of mixing a dispersion containing lipid particles encapsulating the target substance and an aqueous solution containing a fluorescent substance that generates fluorescence by interacting with the target substance, or may include a step of mixing a dispersion containing lipid particles encapsulating the target substance and a powder of a fluorescent substance that generates fluorescence by interacting with the target substance.

[0016] However, even when prepared as described above, in the mixed solution prepared in S11, the fluorescent substance may be autonomously incorporated into lipid particles depending on the substance permeability of the lipid membrane. That is, even at the time of S11, when the fluorescent substance is incorporated into lipid particles encapsulating the target substance by the substance permeation of the lipid membrane, fluorescence may be generated by the interaction between the fluorescent substance and the target substance. However, in the method of the present embodiment, it is preferable from the viewpoint of accuracy that less fluorescence is generated at the time of S11. Therefore, the fluorescent substance is preferably less likely to be incorporated into lipid particles in the environment of the preparation step (S11), and preferably has physical properties (such as molecular weight) that exceed the substance permeation ability of the lipid membrane in the environment of the preparation step (S11).

[0017] Subsequently, the mixed solution prepared in the preparation step (S11) is provided to the measurement step (S12). The measurement step (S12) is a step for grasping the shape of the "fluorescence curve", heating the mixed solution, and performing fluorescence measurement at at least two or more temperatures. That is, in the measurement step (S12), fluorescence measurement is performed at least twice at different heating elapsed times to grasp the shape of the "fluorescence curve".

[0018] Here, the "fluorescence curve" refers to the change in fluorescence intensity for each temperature when heated. That is, when the mixed solution prepared in the preparation step (S11) of the present embodiment is heated, a state in which the fluorescence intensity changes due to heating is observed. Specifically, a tendency for the fluorescence intensity to increase is observed while heating up to a specific temperature, and a tendency for the fluorescence intensity to decrease is observed when heating continues beyond the same temperature. Alternatively, when using a fluorescent substance that exhibits the property of decreasing fluorescence intensity when heated, a tendency for the decrease rate of the fluorescence intensity to be relatively small (that is, the decrease rate of the fluorescence intensity is relatively slow) is observed while heating up to a specific temperature, but when heating continues beyond the same temperature, a tendency for the decrease rate of the fluorescence intensity to increase (that is, the decrease rate of the fluorescence intensity is relatively fast) is observed.

[0019] The measurement process (S12) does not matter the number of fluorescence measurements, the measurement interval, and the measurement start temperature as long as the shape of such a fluorescence curve can be grasped. Therefore, in the measurement process (S12), fluorescence measurement may be performed at least at two temperatures during the temperature rise of the mixed solution, and even a more continuous measurement interval (for example, measurement every 5°C temperature rise, or even measurement every 0.1°C temperature rise) may be used.

[0020] Hereinafter, in the mixed solution of the method of the present embodiment, the reaction mechanism in which the above-described fluorescence curve is observed will be described with reference to FIG. 2. However, the following description is based on a probable hypothesis. It has been confirmed that the method of the present embodiment has reproducibility and is applicable to a mixed solution containing lipid particles and a fluorescent substance.

[0021] First, when the temperature of the mixed solution 1 and thus the lipid particles 2 is increased, it is considered that the membrane fluidity of the lipid particles 2 increases due to the increase in molecular motion, and the structure of the lipid membrane constituting the lipid particles 2 changes. Then, the probability and the size of pores or thin parts of the membrane thickness temporarily generated in the lipid membrane increase, and the probability that the fluorescent substance 4 is incorporated into the lipid particles 2 through the pores or thin parts of the membrane thickness also increases. As a result, the interaction between the fluorescent substance 4 and the target substance 3 is enhanced, and it is considered that a tendency for the fluorescence intensity to increase with the temperature rise (or a tendency for the decrease rate of the fluorescence intensity to be relatively slow) is observed. Conversely, when a remarkable increase tendency in the fluorescence intensity (or a tendency for the decrease rate of the fluorescence intensity to be remarkably slow) is observed, it is highly presumed that a large number of or large pores or thin parts of the membrane thickness have occurred in the lipid particles 2 to be inspected, and it can be determined that the lipid particles 2 to be evaluated have high membrane fluidity and are unstable and defective lipid particles. On the contrary, when the increase tendency of the fluorescence intensity is gentle (or the decrease rate of the fluorescence intensity is relatively fast), it can be determined that the membrane fluidity of the lipid particles 2 to be evaluated is low and they are stable and high-quality lipid particles.

[0022] When the temperature continues to rise, the increasing trend of the fluorescence intensity gradually levels off and turns into a decreasing trend (when a fluorescent substance showing the property that the fluorescence intensity decreases upon heating is used, the rate of decrease of the fluorescence intensity gradually increases). Such a change is confirmed regardless of whether the lipid particles are of good quality or poor quality.

[0023] As described above, between good-quality lipid particles and poor-quality lipid particles, differences in the shape of the fluorescence curve are observed due to the different fluidities of the lipid membranes. Here, if fluorescence measurements are performed in advance on good-quality lipid particles and poor-quality lipid particles, and the values of the parameters indicating the characteristics of the shape of each fluorescence curve are obtained in advance, they can be determined as the thresholds for determining the quality of the lipid particles. By comparing this threshold value with the value of the same parameter of the lipid particles to be measured obtained by the method of this embodiment, it is possible to evaluate the quality of the lipid particles.

[0024] The value of the parameter indicating the characteristics of the shape of the fluorescence curve is, for example, the amount of change in the fluorescence intensity for each increase in temperature. The amount of change in the fluorescence intensity for each increase in temperature may be calculated, for example, as the change in the actually measured value of the fluorescence intensity for each increase in temperature. Or, for example, it may be calculated as the change for each increase in temperature of the ratio between the reference fluorescence intensity and the measured value of the fluorescence intensity (that is, the rate of change of the fluorescence intensity). For example, when a fluorescence intensity of 20,000 is observed at the start of measurement (25°C) and a fluorescence intensity of 40,000 is observed during fluorescence measurement at 45°C, the amount of change in the fluorescence intensity for each 1°C increase in temperature may be calculated as +1000, or as a change rate of +10%.

[0025] The lipid particles prepared by the method of this embodiment are lipid particles encapsulating the target substance. Therefore, for the purpose of quality evaluation only, there is no need for the step of preparing lipid particles encapsulating substances unsuitable for the use of the lipid particles. Thus, the method of this embodiment can be executed with few operations. Also, in the method of this embodiment, since the lipid particles are subjected to measurement in the form in which they are actually used, a direct evaluation in light of the use of the lipid particles is possible, which is preferable.

[0026] (Second Embodiment) The second embodiment relates to a method for evaluating the quality of lipid particles, which further includes a destruction step (S21) for destroying lipid particles, another measurement step (S22), a calculation step (S23), and an evaluation step (S24) in addition to the preparation step (S11) and the measurement step (S12) of the method of the first embodiment. This method will be described with reference to the drawings.

[0027] As shown in FIG. 3, the mixed solution obtained in the preparation step (S11) is not only subjected to the measurement step (S12), but also subjected to a step of destroying the lipid particles in the mixed solution (hereinafter referred to as the "destruction step (S21)"). The destruction treatment of lipid particles can be carried out by a general method. For example, the destruction step (S21) may include a step of adding an agent for destroying lipid particles (hereinafter referred to as a "lipid particle destroyer") to the mixed solution prepared in the preparation step (S11). As the lipid particle destroyer, for example, a lipolytic enzyme or a surfactant can be used. Alternatively, the destruction treatment of lipid particles may be performed by other physicochemical treatment methods (such as dilution of the mixed solution).

[0028] In the destruction step (S21), it is preferable that the lipid particles in the mixed solution are completely destroyed and all target substances are released from the lipid particles. Therefore, the destruction treatment of lipid particles is preferably carried out more carefully. For example, when adding a lipid particle destroyer to the mixed solution, it is preferable that the addition amount and treatment time are sufficient.

[0029] As described above, in the mixed solution obtained through the destruction step (S21), the target substance is in a state of being released from the inside of the lipid particles due to the destruction of the membrane structure of the lipid particles. Therefore, when the destruction step (S21) is in progress, the mixed solution is in a state where the target substance can come into contact with the fluorescent substance and interact to generate fluorescence.

[0030] Subsequently, the mixed solution obtained in the destruction step (S21) is subjected to a measurement step (S22). The measurement step (S22) is a step for obtaining the value of the fluorescence intensity (and thus the parameters characterizing the shape of the fluorescence curve), similar to the measurement step (S12). During the heating of the mixed solution obtained in the preparation step (S21), the fluorescence measurement of the mixed solution is performed at at least two or more measurement temperatures. That is, in the measurement step (S22), the fluorescence measurement is performed at least twice at different heating elapsed times to obtain the value of the fluorescence intensity (and thus the parameters characterizing the shape of the fluorescence curve). However, as will be described later, since the measurement step (S22) aims to correct the measurement results of the measurement step (S12), the temperature at which the measurement is performed in the measurement step (S22) and the temperature at which the measurement is performed in the measurement step (S12) are at least the same. The measurement step (S22) may be executed simultaneously with the measurement step (S12) or at different timings.

[0031] Next, in the calculation step (S23), using the value of the fluorescence intensity (and thus the parameters characterizing the shape of the fluorescence curve) obtained in the measurement step (S22), the value of the fluorescence intensity (and thus the parameters characterizing the shape of the fluorescence curve) obtained in the measurement step (S12) is corrected, and an evaluation index of the lipid particles is calculated. Finally, in the evaluation step (S24), the calculated value of the evaluation index is compared with a predetermined threshold value to evaluate the quality of the lipid particles to be evaluated. Hereinafter, the calculation step (S23) and the evaluation step (S24) will be described with reference to the drawings.

[0032] In the calculation step (S23), using the value of the parameter characterizing the shape of the fluorescence curve obtained in the measurement step (S22), the value of the same parameter obtained in the measurement step (S12) is corrected. The reason for performing such correction is that the fluorescence curve observed in the measurement step (S12) may include the influence of the change in fluorescence intensity due to factors other than the fluidity of the lipid membrane. Examples of factors other than the fluidity of the lipid membrane include the properties of the fluorescent substance and the target substance itself, and the interaction between the fluorescent substance and the target substance, which may change depending on temperature.

[0033] On the one hand, the change in fluorescence intensity observed in the measurement step (S22) should not be due to the change in the fluidity of the lipid membrane of the lipid particles caused by the temperature increase. This is because the lipid membrane of the lipid particles has been disrupted at the time of the disruption step (S21). Since the change in fluorescence intensity observed in the measurement step (S22) is considered to be due to the properties of the fluorescent substance and the target substance itself, and the interaction between the fluorescent substance and the target substance, it is considered that the same effect is also received in the measurement step (S12).

[0034] Therefore, the inventors of the present invention corrected the value of the parameter that is the characteristic of the shape of the fluorescence curve obtained in the measurement step (S12) using the value of the parameter that is the characteristic of the shape of the fluorescence curve obtained in the measurement step (S22), and designed a calculation formula for calculating an index indicating the magnitude of the fluorescence intensity change due to the fluidity of the lipid membrane. Specifically, it was found that the "relative fluorescence intensity ratio" defined by the following formula (I) is calculated as an index indicating the magnitude of the fluorescence intensity change due to the fluidity of the lipid membrane. Since the relative fluorescence intensity ratio is related to the fluidity of the lipid membrane, it can be used as an evaluation index for lipid particles.

Equation

[0035] Here, the "reference temperature" in formula (I) is, for example, the temperature of the mixed solution at the time when the fluorescence intensity measurement is started, or at a certain point in time after the start of the fluorescence intensity measurement. When starting to heat up the mixed solution simultaneously with the start of the fluorescence intensity measurement, the reference temperature is the temperature of the mixed solution at the time when the heating is started or immediately before that. The temperature at the start of the fluorescence intensity measurement can be set relatively arbitrarily. The reference temperature may be, for example, room temperature (25 °C). The reference temperature is preferably a temperature at which the lipid particles and fluorescent substances in the mixed solution are maintained in a stable state even if held at that temperature for a certain period of time. The stable state of the lipid particles and fluorescent substances in the mixed solution means, for example, a state where the fluorescent substance does not permeate the lipid particles (or the rate at which the fluorescent substance permeates the lipid particles is relatively slow), the target substance does not leak from the lipid particles, the lipid particles are not destroyed, and the lipid particles do not fuse with each other. Also, in order to enable the fluorescence intensity measurement at the reference temperature, it is desirable to set the reference temperature to a temperature at which the mixed solution does not freeze. For example, the reference temperature may be selected from values between 4 °C and 30 °C.

[0036] The "measurement temperature" in formula (I) refers to the temperature at any point in time after the start of the fluorescence measurement when the fluorescence intensity is measured. That is, the measurement temperature can be set to any temperature above the reference temperature. In other words, it means that the fluorescence intensity is measured for each measurement temperature.

[0037] By applying the value of the fluorescence intensity measured at each measurement temperature to formula (I), the "relative fluorescence intensity ratio" at each measurement temperature is calculated. As shown in formula (I), the "relative fluorescence intensity ratio" is a value obtained by taking the ratio of the fluorescence intensities of the mixed solution at a certain measurement temperature and the reference temperature observed in step (S12), and dividing by the value of the ratio of the fluorescence intensities calculated in the same way for step (S22) and then corrected.

[0038] The inventors have found that the change in the relative fluorescence intensity ratio observed when the measurement temperature is continuously changed (i.e., when the fluorescence measurement is continuous) is related to the quality of the lipid particles. Hereinafter, the change in the relative fluorescence intensity ratio for each measurement temperature and its relationship with the quality of the lipid particles will be described with reference to FIG. 4.

[0039] As shown in FIG. 4, when the measured temperature is equal to the reference temperature, the numerator and denominator of Equation (I) are equal, so the relative fluorescence intensity ratio is always 1. When the measured temperature is increased from the reference temperature and for a while, an increase in the relative fluorescence intensity ratio is observed regardless of whether the lipid particles are of good quality or poor quality. This is presumably due to the change in the structure of the lipid membrane caused by the temperature increase, and thus the increase in the generation rate of pores and thin portions of the membrane thickness and their sizes, as described in the first embodiment.

[0040] When the temperature increase is continued and the measured temperature reaches a certain temperature, the relative fluorescence intensity ratio levels off. When the temperature is further increased beyond this temperature, a change in which the relative fluorescence intensity ratio gradually decreases is observed. That is, the curve showing the change in the relative fluorescence intensity ratio with respect to the measured temperature (hereinafter referred to as the "relative fluorescence ratio curve") exhibits a peak shape.

[0041] Differences are seen in the shape of the relative fluorescence ratio curve depending on whether the lipid particles are of good quality or poor quality. For example, when the lipid particles are of good quality, the size of the peak of the relative fluorescence ratio curve (i.e., the maximum value of the relative fluorescence intensity ratio) tends to be lower compared to the case where the lipid particles are of poor quality. Also, when the lipid particles are of good quality, the measured temperature at which the relative fluorescence ratio curve reaches its peak tends to be lower compared to the case where the lipid particles are of poor quality. Furthermore, when the lipid particles are of good quality, the measured temperature at which the relative fluorescence intensity ratio first falls below 1 tends to be lower compared to the case where the lipid particles are of poor quality. Consequently, when the lipid particles are of good quality, the area formed by the straight line of relative fluorescence intensity ratio = 1 and the relative fluorescence ratio curve tends to be smaller compared to the case where the lipid particles are of poor quality. In addition, when the lipid particles are of good quality, the size of the area of the triangle formed by connecting three points on the relative fluorescence ratio curve, specifically (1) the reference temperature, (2) the maximum value of the relative fluorescence intensity ratio, i.e., the peak of the relative fluorescence ratio curve, and (3) the point at which the relative fluorescence intensity ratio first falls below 1, tends to be smaller compared to the case where the lipid particles are of poor quality.

[0042] Therefore, by previously measuring known high-quality lipid particles and known low-quality lipid particles, setting a threshold value that can significantly distinguish between high quality and low quality, and comparing the measured value of the lipid particles targeted by the method of this embodiment with the same threshold value, the quality can be determined. Examples of the threshold value that can significantly distinguish between high quality and low quality include, for example, any of the following (a) to (e): (a) the maximum value that the relative fluorescence intensity ratio can take; (b) the value of the measurement temperature when the relative fluorescence intensity ratio can take the maximum value; (c) the value of the measurement temperature when the relative fluorescence intensity ratio first falls below 1; (d) the size of the area enclosed by the straight line of relative fluorescence intensity ratio = 1 and the relative fluorescence ratio curve; and (e) the size of the area of the triangle formed by connecting three points on the relative fluorescence ratio curve, specifically (1) the reference temperature, (2) the maximum value of the relative fluorescence intensity ratio, that is, the peak of the relative fluorescence ratio curve, and (3) the point where the relative fluorescence intensity ratio first falls below 1. However, when the above (a) to (e) are threshold values, it is necessary to more accurately grasp the shape of the relative fluorescence ratio curve of the lipid particles to be measured, and it is necessary to continuously perform fluorescence measurement on the target lipid particles.

[0043] As a threshold value, it is also possible to set a measurement temperature that is higher than the measurement temperature at which the relative fluorescence intensity ratio of good lipid particles first falls below 1 and lower than the measurement temperature at which the relative fluorescence intensity ratio of poor lipid particles first falls below 1 (referred to as the "specified temperature" in this specification). When this specified temperature is used as the threshold value, the quality of the target lipid particles can be determined by fluorescence measurements at two measurement temperatures. The first measurement temperature is higher than the reference temperature and lower than the specified temperature, and for example, it may be a temperature closer to the reference temperature than the intermediate value between the reference temperature and the specified temperature. The fluorescence measurement at the first measurement temperature aims to confirm that the relative fluorescence intensity ratio can be 1 or more even for the target lipid particles, that there is an increasing trend in the relative fluorescence intensity ratio from the reference temperature to the first measurement temperature, and which of the relative fluorescence ratio curves for known good lipid particles and the relative fluorescence ratio curves for known poor lipid particles it is similar to. The second measurement temperature is the specified temperature, and the purpose is to confirm whether the relative fluorescence intensity ratio at the specified temperature is greater than 1 or less than 1. Since the relative fluorescence intensity ratio is less than 1 for good lipid particles and greater than 1 for poor lipid particles, the quality of the lipid particles can be evaluated based on the result of the fluorescence measurement at the second measurement temperature.

[0044] When the specified temperature is used as the threshold value, the quality of the target lipid particles can be determined by fluorescence measurements at the above two measurement temperatures, but fluorescence measurements may also be performed at three or more measurement temperatures including the specified temperature. That is, when the specified temperature is used as the threshold value, the relative fluorescence intensity ratio can be calculated using the results of fluorescence measurements at at least two or more measurement temperatures, and the quality of the target lipid particles can be determined based on that value. For example, the specified temperature may be selected from temperatures between, for example, 50°C and 70°C.

[0045] As described above, according to the method of the second embodiment, an evaluation method of lipid particles is provided that can more accurately evaluate the quality of lipid particles than the method of the first embodiment.

[0046] In the destruction step (S21) of the present embodiment, an example was shown in which a lipid particle-destroying agent was added to the mixed solution prepared in the preparation step (S11). However, the method of the second embodiment is not limited to this step. For example, as shown in FIG. 5, the method of the second embodiment further includes a step (S20) of separately preparing lipid particles encapsulating a target substance and a fluorescent substance that binds to the target substance to generate fluorescence. In the preparation step (S21), a mixed solution containing lipid particles encapsulating a target substance, a fluorescent substance that binds to the target substance to generate fluorescence, and a lipid particle-destroying agent is prepared.

[0047] When the method of the second embodiment includes the above-described step (S20), in the preparation step (S11), a mixed solution is prepared by using the lipid particles and the fluorescent substance prepared in the step (S20). On the other hand, in the preparation step (S21), a mixed solution is prepared by using the lipid particles, the fluorescent substance, and the lipid particle-destroying agent prepared in the step (S20). When the method of the second embodiment includes the above-described step (S20), it is preferable that the two preparation steps (S11) and (S21) be performed simultaneously. However, as long as the preparation method and preparation conditions (for example, the instruments used, the suspension speed, temperature, humidity, etc.) can be set to the same conditions, different timings are also acceptable.

[0048] [Example] The experiments that led to the discovery of the method of the present embodiment are described below.

[0049] Example 1. Cell introduction performance of lipid particles · Preparation of liposomes and measurement of various parameters Five types of liposomes A to E, which show various parameters described in Table 1, were prepared as lipid particles. The lipid composition of the lipid membrane constituting each liposome is the same for liposomes A to E. Among the parameters shown in Table 1, the particle diameter, pdI, and zeta potential were measured by the dynamic light scattering method. The encapsulated nucleic acid concentration of the lipid particles was measured using the QuantiFluor RNA system (manufactured by Promega). Triton X-100 (0.1 v / v%, manufactured by Eastman Kodak Company) was added to the lipid particle solution and mixed with a vortex mixer. Then, an equal volume of the QuantiFluor solution was added and the mixture was allowed to stand at room temperature for 10 minutes. The fluorescence intensity was measured using a single-tube fluorescence measurement device, the Quantus Fluormeter (manufactured by Promega). The concentration of the nucleic acid encapsulated in the lipid particles was calculated by subtracting the fluorescence intensity of the sample without Triton X-100 from the fluorescence intensity of the sample with Triton X-100 added.

[0050]

Table 1

[0051] Each parameter described in Table 1 is a parameter that has been conventionally known as an indirect index for evaluating the quality of lipid particles. As shown in Table 1, it can be seen that the same parameters of each of liposomes A to E are all at the same level. That is, conventionally, liposomes A to E composed of lipid membranes of the same composition were expected to exhibit the same level of quality as each other.

[0052] Therefore, it was decided to directly evaluate the performance of each of liposomes A to E and verify whether their performances match. Liposomes can be used for various applications, but in Example 1, attention was paid to the function as a means for introducing a target substance into cells. That is, as the performance of the liposome, the introduction rate of the target substance into cells was evaluated and verified.

[0053] · Preparation of cells and introduction of nucleic acid into cells The target cells were human peripheral blood mononuclear cells (PBMCs). Commercially available frozen PBMCs (manufactured by Lonza) were thawed in a 37°C thermostat, and then centrifuged to recover the cells. The cells were seeded on a culture plate coated with anti-CD3 antibody (Miltenyi) and anti-CD28 antibody (Miltenyi) for activation, and cultured in TexMACS medium containing 10 ng / mL of interleukin-2 (IL-2) in an incubator at 37°C and 5% CO2 atmosphere. After culturing for two nights, the cells were taken out of the incubator, centrifuged to recover the cells, suspended in TexMACS medium containing 10 ng / mL of interleukin-2 (IL-2), and then cultured overnight in a 48-well culture plate at 37°C and 5% CO2 atmosphere.

[0054] Liposomes A to E each encapsulating mRNA encoding GFP were added to the cell culture medium, gently mixed by pipetting, and then the culture was continued at 37°C and 5% CO2 atmosphere. Also, as a control, a cell culture medium without adding any of liposomes A to E was prepared, gently mixed by pipetting, and then the culture was continued at 37°C and 5% CO2 atmosphere.

[0055] ·Measurement of GFP expression level One day after the addition, the culture plate was taken out of the incubator, the cells were recovered by centrifugation, suspended in phosphate-buffered saline (PBS), and then the expression level of GFP was measured.

[0056] ·Results The measurement results of the GFP expression level are shown in Figure 6. Since the results using liposomes A to C showed comparable and relatively high expression levels to each other, liposomes A to C were shown to have good quality. On the other hand, since the results using liposomes D and E showed comparable but relatively low expression levels to each other, liposomes D and E were shown to be lipid particles of poor quality. Therefore, it became clear that even when known indicators are comparable, there are differences in the quality of lipid particles.

[0057] Example 2: Measurement of the fluorescence curve of lipid particles For each of liposomes A to E used in the verification described in Example 1, it was verified whether there was a relationship between the performance as lipid particles and the shape of the fluorescence curve.

[0058] · Preparation of the mixed solution Dispersions of liposomes A to E encapsulating nucleic acid as the target substance were prepared respectively, and five kinds of mixed solutions were prepared by adding an aqueous solution containing QuantiFluor (registered trademark) RNA Dye, a fluorescent substance, to each dispersion. The nucleic acid as the target substance was mRNA of about 1000 bases, and its base sequence was the same for liposomes A to E. QuantiFluor RNA Dye is a reagent that specifically binds to mRNA, absorbs blue light, and emits green fluorescence, and its fluorescence intensity is proportional to the abundance of mRNA.

[0059] · Destruction treatment of lipid particles For each of the five kinds of mixed solutions prepared, an experimental system in which the liposomes were further subjected to a destruction treatment (hereinafter referred to as the "liposome destruction system") and an experimental system in which the destruction treatment was not performed (hereinafter referred to as the "liposome non-destruction system") were prepared. That is, a total of 10 kinds of mixed solutions were prepared, including five kinds of mixed solutions subjected to the liposome destruction treatment and five kinds of mixed solutions not subjected to the destruction treatment. The destruction treatment of the liposomes was carried out by adding a surfactant (1% Triton-X-100) to each mixed solution and allowing it to stand at about 25°C for about 10 minutes.

[0060] · Heating of the mixed solution and measurement of fluorescence intensity Next, for each of the 10 kinds of mixed solutions, while heating each mixed solution, the fluorescence intensity generated during the process was measured. Each of the mixed solutions started heating from room temperature (25°C), and the fluorescence measurement was carried out continuously. A real-time PCR device (manufactured by Applied Biosystems) was used for heating and fluorescence intensity measurement.

[0061] · Results of fluorescence measurement The results of the non-destructive fluorescence measurement of liposomes are shown in Fig. 7, and the results of the destructive fluorescence measurement of liposomes are shown in Fig. 8. Note that (a) in Fig. 7 shows the measured values of the fluorescence intensity of the non-destructive liposome system at each measurement temperature, and (b) shows the ratio of the measured value of the fluorescence intensity of the non-destructive liposome system at each measurement temperature to the fluorescence intensity at the reference temperature. (a) in Fig. 8 shows the measured values of the fluorescence intensity of the destructive liposome system at each measurement temperature, and (b) shows the ratio of the measured value of the fluorescence intensity of the destructive liposome system at each measurement temperature to the fluorescence intensity at the reference temperature. In other words, (a) in Fig. 7 shows X(Tevaluate) in the above formula (I), (b) in Fig. 7 shows X(Tevaluate) / X(Tstandard), (a) in Fig. 8 shows Y(Tevaluate), and (b) in Fig. 8 shows Y(Tevaluate) / Y(Tstandard).

[0062] Comparing (a) in Fig. 7 and (a) in Fig. 8, it can be seen that the fluorescence intensity of the destructive liposome system was observed to be about 6 to 8 times that of the non-destructive liposome system. This indicates that when the liposome is not destroyed, the interaction between QuantiFluor RNA Dye and mRNA is suppressed compared to the case where the liposome is destroyed. Conversely, it was shown that by increasing the degree of destruction of the liposome membrane structure, the interaction between QuantiFluor RNA Dye and mRNA increases and the fluorescence intensity increases.

[0063] Referring to (a) and (b) in Fig. 8, it can be seen that the measured values of the fluorescence intensity of the destructive liposome system were different for each liposome, but the reduction rate of the measured values for each temperature increase was almost the same for liposomes A to E. This means that the destruction treatment of liposomes A to E in the destructive liposome system had proceeded sufficiently.

[0064] Referring to FIGS. 7(a) and 8(a) respectively, it can be commonly seen that the fluorescence intensity tends to decrease with the increase in temperature. As described above, in the liposome disruption system, the membrane structures of liposomes A to E were sufficiently disrupted. Therefore, the tendency of the fluorescence intensity to decrease with the increase in temperature was considered to be caused by factors other than the fluidity of the lipid membrane of the liposome, for example, due to QuantiFluor RNA Dye itself or mRNA itself, or the interaction between QuantiFluor RNA Dye and mRNA. For whatever reason, it was found that under the experimental conditions of Example 1, when QuantiFluor RNA Dye and mRNA were used with the increase in temperature, a tendency for the fluorescence intensity to decrease was observed.

[0065] Referring to FIG. 7(a), although the tendency for the fluorescence intensity to decrease with the increase in temperature is common among liposomes A to E, it can be seen that for defective liposomes D and E, overall higher measured fluorescence intensity values were observed compared to good liposomes A to C. For example, when the measured temperature reached 60°C with the increase in temperature, the fluorescence intensities of liposomes D and E were approximately twice those of liposomes A to C. Also, referring to FIG. 7(b), for defective liposomes D and E and good liposomes A to C, the changes in the rate of decrease in fluorescence intensity for each increase in temperature were clearly different. Therefore, it is effective to compare the shapes of the fluorescence curves of the two to distinguish between good lipid particles and defective lipid particles, and the magnitude of the measured fluorescence intensity value and / or the rate of decrease in fluorescence intensity for each increase in temperature were revealed as characteristic parameters indicating the difference in the shape of the fluorescence curve.

[0066] Example 3: Calculation of relative fluorescence intensity ratio · Calculation and change of relative fluorescence intensity ratio Based on the measurement results of Example 2, the relative fluorescence intensity ratio was calculated using the aforementioned formula (I). Here, the reference temperature was set at 26°C.

[0067] The changes in the relative fluorescence intensity ratio with increasing temperature, i.e., the relative fluorescence ratio curve, are shown in Fig. 9. It can be seen that there are clearly differences in the shape of the relative fluorescence ratio curve between liposomes A - C, which are good-quality lipid particles, and liposomes D and E, which are poor-quality lipid particles. Conversely, in order to clearly show the differences in the shape of the relative fluorescence ratio curve between lipid particles A - C and lipid particles D and E, it is found that the reference temperature should be set at 40°C or lower under the experimental conditions.

[0068] Also, referring to Fig. 9, when the measurement temperature is 60°C, it can be seen that the relative fluorescence intensity ratio is less than 1 for good-quality liposomes A - C, and greater than 1 for poor-quality liposomes D and E. Therefore, when the value of the relative fluorescence intensity ratio (=1) is set as the threshold, and the evaluation index of the lipid particles is set as the value of the relative fluorescence intensity ratio at the measurement temperature (i.e., the specified temperature) where the relative fluorescence intensity ratio is less than 1 for good-quality lipid particles and greater than 1 for poor-quality lipid particles, it is shown that when the evaluation index is higher than the threshold, the quality of the lipid particles to be evaluated is evaluated as poor, and when the evaluation index is lower than the threshold, the lipid particles to be evaluated can be evaluated as good-quality.

[0069] Also, as shown in Fig. 9, it can be seen that there are clearly differences in the maximum values of the relative fluorescence intensity ratios between liposomes A to C, which are high-quality lipid particles, and liposomes D and E, which are poor-quality lipid particles. Specifically, the maximum value of the relative fluorescence intensity ratio observed for the poor liposomes D and E was approximately 1.3, and the maximum value of the relative fluorescence intensity ratio observed for the good liposomes A to C was approximately 1.17. From this, it can be understood that liposomes showing a relative fluorescence intensity ratio with a maximum value higher than 1.2 can be determined as poor lipid particles, and liposomes showing a relative fluorescence intensity ratio lower than 1.2 can be determined as good lipid particles. Therefore, when setting the value of the relative fluorescence intensity ratio (hereinafter referred to as the specified intensity ratio) that exceeds the maximum value of the relative fluorescence intensity ratio of good lipid particles and does not exceed the maximum value of the relative fluorescence intensity ratio of poor lipid particles as the threshold, and setting the evaluation index of lipid particles as the maximum value of the relative fluorescence intensity ratio, it was shown that when the evaluation index is higher than the threshold, the quality of the lipid particles to be evaluated is evaluated as poor, and when the evaluation index is lower than the threshold, the lipid particles to be evaluated can be evaluated as having good quality. The specified intensity ratio may be selected from values, for example, between 1.1 and 1.3.

[0070] Also, as shown in Fig. 9, upon heating from the reference temperature (26°C), the maximum value of the relative fluorescence intensity ratio (about 1.3) was observed at a measurement temperature of about 50 to 55°C for defective liposomes D and E. On the other hand, upon heating from the reference temperature (26°C), the maximum value of the relative fluorescence intensity ratio (about 1.17) was observed at a measurement temperature of about 50 to 55°C for good liposomes A to C. That is, the increase rate of the relative fluorescence intensity ratio per temperature increase for defective lipid particles was (1.3 - 1) / (52.5 - 26) ≈ 0.012 / °C, and the increase rate of the relative fluorescence intensity ratio per temperature increase for good lipid particles was (1.17 - 1) / (52.5 - 26) ≈ 0.007 / °C. From this, it can be seen that liposomes with an increase rate of the relative fluorescence intensity ratio per temperature increase higher than 0.01 / °C can be determined as defective lipid particles, and liposomes showing a relative fluorescence intensity ratio lower than 0.01 / °C can be determined as good lipid particles. Therefore, the evaluation index of lipid particles was set as the increase rate of the relative fluorescence intensity ratio calculated by dividing the value obtained by subtracting 1 from the maximum value of the relative fluorescence intensity ratio by the temperature difference between the measurement temperature and the reference temperature at the maximum value of the relative fluorescence intensity ratio. By setting a value (hereinafter referred to as the specified increase rate), which is larger than the increase rate of the relative fluorescence intensity ratio observed in good lipid particles and smaller than the increase rate of the relative fluorescence intensity ratio observed in defective lipid particles, as the threshold value, it was shown that when the evaluation index is higher than the threshold value, the quality of the lipid particles to be evaluated is determined to be defective, and when the evaluation index is lower than the threshold value, the lipid particles to be evaluated can be determined to be of good quality. For example, the reference temperature may be selected from values between 4°C and 30°C, and the specified increase rate may be selected from values between 0.005 and 0.02.

[0071] Also, as shown in Fig. 9, for defective liposomes D and E, the maximum value of the relative fluorescence intensity ratio (about 1.3) was observed, and the relative fluorescence intensity ratio first dropped below 1 at about 63°C from the start of measurement (reference temperature (26°C)). On the other hand, for good liposomes A to C, the maximum value of the relative fluorescence intensity ratio (about 1.17) was observed, and the relative fluorescence intensity ratio first dropped below 1 at about 55 - 60°C from the start of measurement (reference temperature (26°C)). Here, when calculating the area of the triangle formed by connecting the three points: the reference temperature, the maximum value of the relative fluorescence intensity ratio, and the point where the relative fluorescence intensity ratio first drops below 1, the area of the aforementioned triangle for defective liposomes D and E was (63 - 26)×(1.3 - 1)÷2 ≒ 5.55, and the area of the aforementioned triangle for good liposomes A to C was (57.5 - 26)×(1.17 - 1)÷2 ≒ 2.68. From this, it can be seen that lipid particles with a triangle area larger than 4.0 can be determined as defective lipid particles, and lipid particles with a triangle area smaller than 4.0 can be determined as good lipid particles. Therefore, by setting the evaluation index of lipid particles as the area of the triangle formed by connecting three different points on the relative fluorescence ratio curve (specifically, (1) the reference temperature, (2) the maximum value of the relative fluorescence intensity ratio, i.e., the peak of the relative fluorescence ratio curve, and (3) the point where the relative fluorescence intensity ratio first drops below 1), and setting a value (hereinafter referred to as the "specified area") that is larger than the area of the same triangle observed for good-quality lipid particles and smaller than the area of the same triangle observed for defective lipid particles as the threshold value, it was shown that when the evaluation index is higher than the threshold value, the quality of the lipid particles to be evaluated is determined to be defective, and when the evaluation index is lower than the threshold value, the lipid particles to be evaluated can be determined to be of good quality. For example, the specified area may be selected from values between 42.0 and 6.0.

[0072] Although several embodiments of the present invention have been described, these embodiments are presented by way of example and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and their modifications are included in the scope and gist of the invention, and are included in the invention described in the claims and its equivalent scope.

Claims

1. (S11) A step of preparing a lipid particle encapsulating a target substance and a mixed solution containing a fluorescent substance that binds to the target substance and generates fluorescence; (S12) A step of performing fluorescence measurement at at least two or more temperatures while heating the prepared mixed solution, and obtaining the amount of change in fluorescence intensity for each temperature; and (S13) A step of comparing the obtained amount of change in fluorescence intensity with a preset threshold value to determine the quality of the lipid particles to be evaluated A method for evaluating the quality of lipid particles, comprising:

2. The method according to claim 1, wherein the amount of change in fluorescence intensity is the rate of decrease in fluorescence intensity for each temperature.

3. (S11) A step of preparing a lipid particle encapsulating a target substance and a mixed solution containing a fluorescent substance that binds to the target substance and generates fluorescence; (S12) A step of performing fluorescence measurement at at least two or more temperatures while heating the mixed solution prepared in the step (S11), and obtaining the amount of change in fluorescence intensity for each temperature; (S21) A step of subjecting the lipid particles in the mixed solution prepared in the step (S11) to a treatment for destroying the lipid particles; (S22) A step of performing fluorescence measurement at at least two or more temperatures while heating the mixed solution subjected to the treatment in the step (S21), and obtaining the amount of change in fluorescence intensity for each temperature; (S23) A step of correcting the amount of change in fluorescence intensity obtained in the step (S12) using the amount of change in fluorescence intensity obtained in the step (S22), and calculating an evaluation index for the lipid particles; and (S24) A step of comparing the evaluation index calculated in the step (S23) with a preset threshold value to determine the quality of the lipid particles to be evaluated A method for evaluating the quality of lipid particles, comprising:

4. The fluorescence measurement in the step (S22) is performed at least at a reference temperature and a measurement temperature, The reference temperature is the temperature of the mixed solution at the start of the fluorescence measurement or at a certain point in time after the start of the fluorescence measurement, The measurement temperature is the temperature of the mixed solution at any point in time during the fluorescence measurement and is a temperature equal to or higher than the reference temperature. The method according to claim 3.

5. The step (S23) includes a step of calculating a relative fluorescence intensity ratio defined by the following formula (I), The evaluation index is determined using the relative fluorescence intensity ratio. The method according to claim 4. 【Number 1】 In the formula, Z refers to the relative fluorescence intensity ratio, X(Tevaluation) refers to the fluorescence intensity of the mixed solution observed at the measurement temperature in the step (S12), X(Tstandard) refers to the fluorescence intensity of the mixed solution observed at the reference temperature in the step (S12), Y(Tevaluation) refers to the fluorescence intensity of the mixed solution observed at the measurement temperature in the step (S22), Y(Tstandard) refers to the fluorescence intensity of the mixed solution observed at the reference temperature in the step (S22).

6. The threshold value is a value of the relative fluorescence intensity ratio and is 1, The evaluation index is a value of the relative fluorescence intensity ratio when the measurement temperature is the specified temperature, The specified temperature is a temperature at which the relative fluorescence intensity ratio is less than 1 for good lipid particles and greater than 1 for poor lipid particles, In the step (S24), when the value of the evaluation index is higher than the threshold value, it is evaluated that the quality of the lipid particles is poor, and when the evaluation index is lower than the threshold value, it is evaluated that the lipid particles are of good quality. The method according to claim 5.

7. The specified temperature is selected from temperatures between 50°C and 70°C. The method according to claim 6.

8. The fluorescence measurement in the step (S22) is performed at a plurality of the measurement temperatures, The step (S23) includes a step of calculating the maximum value of the relative fluorescence intensity ratio, Obtaining a curve showing the change of the relative fluorescence intensity ratio with respect to the measurement temperature. The method according to claim 5.

9. The evaluation index is the maximum value of the relative fluorescence intensity ratio, The threshold value is a specified intensity ratio, The specified intensity ratio is a value greater than the maximum value of the relative fluorescence intensity ratio observed for good lipid particles and less than the maximum value of the relative fluorescence intensity ratio observed for poor lipid particles, In the step (S24), when the evaluation index is higher than the threshold value, it is evaluated that the quality of the lipid particles is poor, and when the evaluation index is lower than the threshold value, it is evaluated that the lipid particles are of good quality. The method according to claim 8.

10. The specified intensity ratio is selected from values between 1.1 and 1.

3. The method according to claim 9.

11. The evaluation index is the increase rate of the relative fluorescence intensity ratio calculated by dividing the value obtained by subtracting 1 from the maximum value of the relative fluorescence intensity ratio by the temperature difference between the measurement temperature and the reference temperature at the maximum value of the relative fluorescence intensity ratio, The threshold value is a specified increase rate, and the specified increase rate is a value greater than the increase rate of the relative fluorescence intensity ratio observed for good lipid particles and smaller than the increase rate of the relative fluorescence intensity ratio observed for poor lipid particles. The method according to claim 8, wherein in the step (S24), when the evaluation index is higher than the threshold value, it is evaluated that the quality of the lipid particles is poor, and when the evaluation index is lower than the threshold value, it is evaluated that the lipid particles are of good quality.

12. The method according to claim 11, wherein the reference temperature is selected from values between 4°C and 30°C, and the specified increase rate is selected from values between 0.005 and 0.

02.

13. The evaluation index is the area of a triangle formed by connecting three different points on the curve, and the three points are the reference temperature, the measurement temperature at the maximum value of the relative fluorescence intensity ratio, and the measurement temperature at which the relative fluorescence intensity ratio is less than 1. The threshold value is a specified area, and the specified area is a value greater than the area of the triangle observed for good lipid particles and smaller than the area of the triangle observed for poor lipid particles. The method according to claim 8, wherein in the step (S24), when the evaluation index is higher than the threshold value, it is evaluated that the quality of the lipid particles is poor, and when the evaluation index is lower than the threshold value, it is evaluated that the lipid particles are of good quality.

14. The method according to claim 13, wherein the specified area is selected from between 2.0 and 6.0.

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