JP7800301B2 - Method for measuring the loading ratio of carrier particles - Google Patents

Description

The present disclosure relates to a method for measuring the loading rate of carrier particles. In particular, the present disclosure relates to a method for measuring the rate at which a collection of carrier particles, such as viruses, encapsulating a target substance such as nucleic acid carries the target substance (encapsulation rate).

By carrying target substances such as drug molecules or nucleic acids on carrier particles such as liposomes or viruses, the target substances can be transported to desired locations (e.g., specific organs or specific cells) in the human body, for example.

For example, in drug delivery systems (DDS), carrier particles such as liposomes that hold drug molecules inside can be used to stably control the distribution of drugs within the body.

In Non-Patent Document 1, the Japan Patent Office provides an overview of technological trends in drug delivery systems. This document describes related technology as "technology and systems that control ideal pharmacokinetics in the body" by covering active ingredients with formulation components such as liposomes or viruses.

In gene therapy, for example, nucleic acid encoding a recombinant gene is encapsulated in a virus vector, and the virus is then introduced into cells, allowing the gene to be introduced into the body. By retaining the nucleic acid inside the virus (especially the viral shell), it is possible to avoid degradation of the nucleic acid encoding the recombinant gene and increase the efficiency of introduction into cells.

Non-patent document 2 describes the history and technical challenges of virus-based gene therapy.

"Patent Application Technology Trends Survey Report (Summary) Drug Delivery Systems (DDS)" (Japan Patent Office) Current status and future prospects of clinical development of virus-based gene therapy drugs (by Fumiaki Sakurai) Drug Delivery System, 2019, Vol. 34, No. 2, pp. 99-105

When using carrier particles such as liposomes or viruses to transport target substances such as drug molecules or nucleic acids, if the proportion of carrier particles that carry the target substance (i.e., the carrier particle loading rate) is low, the desired objective may not be achieved.

For example, in gene transfer methods in which viruses carrying nucleic acid encoding recombinant genes are injected into cells, the probability that the injected viruses will successfully transfer genes is very low. Therefore, in order to increase the efficiency of gene transfer into cells, it is important to increase the proportion of nucleic acid-containing viruses.

Furthermore, the process of encapsulating target substances using viruses and other substances is not perfect, and depending on the state of the raw materials and processing conditions, not only carrier particles containing the target substance but also "empty" carrier particles that do not contain the target substance may be present.

Therefore, in order to achieve the desired objective, it is important to determine in advance the ratio of carrier particles carrying the target substance to carrier particles that do not contain the target substance (empty carrier particles). However, conventional methods for measuring the ratio of carrier particles containing the target substance have lacked convenience. Furthermore, conventional measurement principles have sometimes resulted in insufficient measurement accuracy.

Therefore, there was a need for a simple evaluation method based on a new measurement principle to determine the loading ratio.

The purpose of this disclosure is to provide a new method for easily measuring the proportion of carrier particles that carry a target substance.

The above-mentioned problems can be solved by the following aspects of the present invention:
<Aspect 1>
A method for evaluating a ratio of carrier particles not carrying a target substance to carrier particles carrying the target substance in a liquid containing a plurality of carrier particles, comprising:
measuring an impedance spectrum of the liquid;
method.
<Aspect 2>
further comprising comparing the measured impedance spectrum with a reference impedance spectrum.
The method of aspect 1.
<Aspect 3>
3. The method of claim 2, further comprising comparing the shape of the measured impedance spectrum with a shape of a reference impedance spectrum.
<Aspect 4>
4. The method of claim 2 or 3, wherein a plurality of reference impedance spectra are used as the reference impedance spectrum.
<Aspect 5>
Aspect 5. The method of any one of aspects 2 to 4, wherein the measured impedance spectrum is compared to the reference impedance spectrum in the range of 10 kHz to 100 kHz.
<Aspect 6>
Aspect 6. The method according to any one of aspects 2 to 5, wherein the comparison is performed using a COS similarity calculated from the reference impedance spectrum and the measured impedance spectrum.
<Aspect 7>
Aspect 7. The method according to any one of aspects 1 to 6, wherein the carrier particles encapsulate the target substance, thereby carrying the target substance.
<Aspect 8>
the carrier particles are viruses, and the target substance is nucleic acid.
8. The method according to aspect 7.
<Aspect 9>
Aspect 9. The method of any one of aspects 1 to 8, wherein the impedance spectrum of the liquid is measured using an interdigitated electrode.

The present invention provides a new method for easily measuring the proportion of carrier particles that support a target substance.

FIG. 1 is a conceptual diagram for explaining the principle of the method according to the present disclosure. FIG. 2 is a schematic diagram illustrating an exemplary embodiment of a measurement device that can be used in the method according to the present disclosure. FIG. 3 is a conceptual diagram showing how the method according to the present disclosure is performed using the measurement device of FIG. FIG. 4A is a graph showing the impedance spectrum measured in Example 1. FIG. 4B is an enlarged view of a portion (enclosed in a rectangular box) of the graph in FIG. 4A. FIG. 5 is a graph showing the relationship between the COS similarity of the impedance spectra obtained for sample solutions 1 to 6 and the loading ratio (full ratio) (%) in each solution.

<Method for evaluating the loading ratio>
The method according to the present disclosure comprises:
A method for evaluating the ratio of carrier particles not carrying a target substance to carrier particles carrying a target substance in a liquid containing a plurality of carrier particles, comprising:
Measuring the impedance spectrum of the liquid (impedance measurement step);
Includes:

To date, measurements have been made using, for example, a transmission electron microscope or an absorptiometer to measure the ratio of carrier particles containing the target substance to carrier particles not containing the target substance.

The method using a transmission electron microscope calculates the percentage of viruses containing nucleic acid from the captured image based on the difference in the penetration of the electron beam due to the presence of nucleic acid in the target substance. However, this method has disadvantages such as the high cost of the equipment and the time-consuming pre-processing required, such as osmium staining.

In addition, the method using an absorptiometer (absorptiometry) is a method for calculating the percentage of viruses containing nucleic acid, based on the characteristic absorption peak of nucleic acid around 260 nm. However, due to low sensitivity and the fact that there are biological substances other than nucleic acid that also exhibit an absorption peak around 260 nm, it can be difficult to perform highly accurate measurements using absorptiometry.

Against this background, the present inventors investigated a new measurement method and found that different impedance spectra can be obtained depending on the carrier particle loading ratio, i.e., the ratio of carrier particles that do not contain the target substance to carrier particles that contain the target substance. More specifically, they found that by placing a liquid containing carrier particles between two electrodes and measuring the impedance, an impedance spectrum that corresponds to the carrier particle loading ratio can be obtained.

Figure 1 is a conceptual diagram illustrating the principle of the method disclosed herein. Note that the accompanying drawings are intended to facilitate understanding of the present invention and are not intended to limit the present invention. In Figure 1, a liquid (not shown) exists between electrodes 12 and 14, and carrier particles 16 (e.g., viruses) encapsulating a target substance 18 (e.g., nucleic acid) exist within this liquid.

Without intending to be limited by theory, when a voltage is applied between the two electrodes 12 and 14:
Transfer of electrons from the electrode to the carrier particles (charge transfer: "A" in Figure 1),
Polarization occurs near the carrier particles due to charges inside the carrier particles ("B" in Figure 1),
Movement of carrier particles by an external electric field (dielectrophoresis: "C" in Figure 1),
It is believed that phenomena such as the above occur. The impedance spectrum detected between the two electrodes reflects these phenomena ("A" to "C" in FIG. 1).

The presence or absence and/or extent of the above phenomena differ between carrier particles carrying the target substance and carrier particles not carrying the target substance, and it is therefore thought that the impedance spectrum reflecting these differences will also be different. By comparing this impedance spectrum with, for example, a reference impedance spectrum, the proportion of carrier particles carrying the target substance can be detected.

The method disclosed herein can provide a rapid and simple method. For example, the process of dropping a liquid containing carrier particles onto an electrode and measuring the impedance can usually be completed within several tens of seconds to several minutes (e.g., one minute). Furthermore, measurements can be performed without modifying the electrode. Furthermore, the process of calculating the abundance ratio in the liquid based on the measured impedance spectrum can also be performed quickly, for example, by using a computer.

The method disclosed herein is a measurement method based on the dielectric properties of the target substance and carrier particles, and is based on a new measurement principle that differs from conventional methods. Therefore, it is expected that the method disclosed herein will enable measurement of samples that are difficult to accurately measure using conventional absorptiometers. Furthermore, the method disclosed herein can obtain data that is complementary to other measurement techniques.

Furthermore, because the method disclosed herein uses impedance spectra, measurement accuracy can be improved. That is, by focusing analysis on a specific frequency range (e.g., 1 kHz to 100 MHz) or specific components (phase, amplitude, etc.) of the measured impedance spectrum, differences between samples can be evaluated with high accuracy.

As described above, the method disclosed herein provides a new method for easily measuring the loading ratio of carrier particles.

Each component of the method disclosed herein is described in further detail below.

<Liquid>
The liquid according to the present disclosure contains a plurality of carrier particles. The liquid is not particularly limited, and an appropriate buffer, aqueous solution, water, etc. can be selected depending on the carrier particles. Preferably, the liquid is a liquid in which the carrier particles can exist stably and/or a liquid that allows the target substance to be well supported in the carrier particles.

When measuring the impedance spectrum of a liquid, the liquid can be pre-treated beforehand. For example, the liquid can be pre-treated to remove free target substances not supported on carrier particles (such as nucleic acids not encapsulated in vectors) and impurities. Such removal procedures include filtration, centrifugation, chromatography, and precipitation.

<Carrier particles>
The carrier particles are configured to be capable of carrying the target substance. The carrier particles may be in the form of particles, particularly fine particles. The diameter of the carrier particles is not particularly limited, but is preferably 5 nm to 750 nm, 10 nm to 500 nm, 20 nm to 250 nm, or 25 nm to 100 nm.

The carrier particles are preferably particles (hollow particles) with an internal space for holding the target substance. Examples of such carrier particles include carriers made of organic compounds, particularly liposomes, viruses, and water-soluble polymers.

(liposomes)
Liposomes are vesicles with a lipid bilayer that can store target substances such as drug molecules inside. The diameter of liposomes is not particularly limited, but may be, for example, 10 nm to 1000 nm, or 50 nm to 200 nm. Liposomes are particularly composed of phospholipids.

(virus)
Viruses include adenoviruses, adeno-associated viruses (AAV), and retroviruses.

Of these, adeno-associated virus (AAV) is a virus that is considered promising for gene therapy. AAV containing nucleic acid encoding a gene for recombination, as well as empty AAV that does not contain nucleic acid encoding a gene for recombination, are commercially available.

The diameter of the carrier particles can be determined by measuring the diameters of 20 or more carrier particles from images taken using an electron microscope or similar device and averaging the measurements. Note that if the carrier particles are not perfectly round, i.e., if they are elliptical, for example, the longest particle length can be considered to be the diameter.

<Target substances>
The target substance can be supported on carrier particles, particularly, the target substance can be encapsulated in the carrier particles and held inside the carrier particles.

The target substance is not particularly limited as long as it can be carried by carrier particles, but examples include inorganic or organic compounds, and in particular, molecules with medicinal properties or nucleic acids.

(Molecules with drug activity)
In one aspect of the method according to the present disclosure, the target substance is a molecule having a medicinal activity, which can be particularly carried by liposomes as carrier particles and retained inside the liposomes.

(nucleic acid)
In another embodiment of the method of the present disclosure, the substance of interest is a nucleic acid (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)), particularly a nucleic acid encoding a recombinant gene. The nucleic acid can be carried, in particular, inside a virus as a carrier particle (e.g., inside the outer shell in the case of AAV).

<Impedance measurement process>
In the method according to the present disclosure, the impedance spectrum of a liquid containing carrier particles is measured, in particular, the liquid containing carrier particles is placed between two electrodes, and the impedance spectrum between the two electrodes can be measured.

<Electrode>
The method according to the present disclosure can use electrodes, particularly interdigital electrodes, in which strips (tooth portions) projecting from one electrode are arranged in a staggered (alternating) parallel arrangement with strips (tooth portions) projecting from the other electrode, with the interdigital portions from different poles adjacent to each other at regular intervals.

Figure 2 is a schematic diagram illustrating an exemplary embodiment of a measurement device that can be used in the methods of the present disclosure. In the figure, L represents the length direction and W represents the width direction.

In FIG. 2, the impedance analyzer 20 is connected to a measuring device 28. The measuring device 28 includes a substrate 22 and two interdigital electrodes 24 and 26 on the substrate. A plurality of strip-shaped comb teeth protruding from interdigital electrode 24 and a plurality of strip-shaped comb teeth protruding from interdigital electrode 26 are arranged in a staggered, parallel fashion, adjacent to each other at regular intervals (e.g., 1 μm to 10 μm).

The interdigital electrodes 24 and 26 are connected to the terminals HP and LP of the impedance analyzer 20, respectively. Furthermore, the terminal HC of the impedance analyzer 20 is connected to the terminal HP and the interdigital electrode 24, and the terminal LC is connected to the terminal LP and the interdigital electrode 26.

In the embodiment shown in FIG. 2, terminals HC and LC are connected between the impedance analyzer 20 and the measuring instrument 28, but they may also be connected inside the impedance analyzer 20 or on the substrate 22 of the measuring instrument 28.

When performing the method of the present disclosure using the measurement device of Figure 2, for example, a liquid containing carrier particles to be measured is dropped between the interdigital electrodes 24 and 26, so that the liquid is present at least between one interdigital portion of the interdigital electrode 26 and one interdigital portion of the adjacent interdigital electrode 24.

After the liquid is dripped, a voltage is applied and the impedance can be measured by measuring the current value between terminals LC and HC and the voltage value between terminals LP and HP.

Figure 3 is a conceptual diagram showing how the method disclosed herein is performed using the measurement device of Figure 2. Figure 3 shows a cross section taken along the cutting line A'-A' in Figure 2. In the figure, H represents the height direction, and W represents the width direction.

In Figure 3, the comb teeth 162 and 164 of the interdigital electrode 26 and the interdigital electrode 24's interdigital tooth portions 142 and 144, which are arranged alternately with these, are arranged on the substrate 22 of the measuring device 28. A droplet 37 of sample solution containing carrier particles 35 to be detected is dropped onto these interdigital tooth portions so as to cover them.

The carrier particles 35 may be present in the following forms: attached to the electrode's comb-teeth, present between the electrode's comb-teeth, or present away from the electrode's comb-teeth.

Adjacent comb tooth portions (for example, comb tooth portion 164 and comb tooth portion 144) serve as opposite poles, and the impedance between them is measured.

(comb-shaped electrode)
The comb-shaped electrode may have a comb width of 0.5 μm to 25 μm, or 1 μm to 10 μm. The comb-shaped electrode may have 10 to 200 comb-shaped teeth, or 30 to 100 comb-shaped teeth. The interval between the comb-shaped teeth of a comb-shaped electrode formed by a pair of electrodes may be 0.5 μm to 25 μm, or 1 μm to 10 μm. The comb-shaped electrode may be formed of, for example, gold.

The number of pairs of alternatingly parallel comb teeth is not particularly limited, but may be 1 to 200 pairs, 2 to 100 pairs, 5 to 90 pairs, 10 to 80 pairs, or 20 to 60 pairs.

<Evaluation of loading ratio>
In one embodiment of the method according to the present disclosure, the loading of support particles in a particular sample liquid is assessed (particularly estimated or calculated) by comparing the impedance spectrum obtained for that sample liquid with a reference impedance spectrum, in particular by comparing the shape (waveform) of the impedance spectra.

(Reference Impedance Spectrum)
The reference impedance spectrum is in particular an impedance spectrum previously acquired in a liquid having a known loading of carrier particles.

For example, the reference impedance spectrum may be an impedance spectrum obtained in a liquid in which the proportion of carrier particles containing the target substance is zero (liquid with a 0% loading proportion), or it may be an impedance spectrum obtained in a liquid in which the proportion of carrier particles containing the target substance is 100% (liquid with a 100% loading proportion).

By comparing such a reference impedance spectrum, previously obtained for a liquid with a known loading ratio, with an impedance spectrum obtained for any liquid (particularly by detecting differences between the shapes of the impedance spectra), the loading ratio of the liquid can be evaluated (particularly estimated or calculated).

(Multiple Reference Impedance Spectra)
Preferably, a plurality of reference impedance spectra are used as the reference impedance spectrum. For example, by comparing an impedance spectrum obtained for a given liquid with reference impedance spectra previously obtained for liquids exhibiting various loading ratios, the loading ratio of carrier particles in the given liquid can be evaluated (particularly estimated or calculated) with higher accuracy.

(standardization)
When comparing the impedance spectrum measured for a given liquid with a reference impedance spectrum, the impedance spectrum can be normalized. The impedance of a liquid may fluctuate due to absorption of oxygen, carbon dioxide, etc. from the ambient atmosphere. Therefore, normalization can sometimes further improve the accuracy of the comparison process.

Normalization can be performed as appropriate depending on the carrier particles and target substance used. For example, the absolute values of impedance at a specific frequency (e.g., 1 kHz) can be normalized to the absolute values of other frequency ranges.

(component)
The comparison of impedance spectra can be performed using the components (amplitude, phase, real part, and imaginary part) constituting the impedance spectra. For example, the comparison of impedance spectra can be performed by focusing on the component with the largest difference among the components constituting the impedance spectra.

In addition, impedance spectra can be compared based on features used in waveform, signal analysis, and vector analysis.

(COS similarity)
For example, the difference (or similarity) in a specific wavelength region of the impedance spectrum can be expressed by COS similarity (also called "cosine similarity" or "cos similarity"). By using COS similarity, it is possible to improve the quantitativeness of the comparison process in some cases. COS similarity is an index used to evaluate the similarity of vectors, and expresses the similarity in terms of the direction of two vectors using cosine (COS). The value of COS similarity approaches 1 as the two vectors are in the same direction (high similarity), and approaches 0 as the two vectors are in different directions (low similarity).

For example, the COS similarity S can be calculated using the following formula based on data in the range of 10 kHz to 100 kHz. Formula (1) below is used to calculate the COS similarity when an impedance spectrum obtained from a liquid with a loading ratio of 100% is used as the reference impedance spectrum.

In the above formula (1), S is the cosine similarity (COS similarity), _f100 (ω) is a vector representing the impedance spectrum of a sample with a loading ratio of 100% (Full 100%), and f(ω) is a vector representing the impedance spectrum of the target sample. When another impedance spectrum is used as the reference impedance spectrum, the COS similarity S can be calculated by replacing _f100 (ω) in the above formula (1) with the vector fs(ω) representing the reference impedance spectrum.

(Standard curve)
When evaluating the carrier particle loading rate in an arbitrary liquid, the carrier particle loading rate in the arbitrary liquid can also be estimated (or calculated) using a graph (standard curve) or equation showing the relationship between the carrier particle loading rate and the impedance spectrum.

Such a standard curve can be created, for example, using the COS similarity described above. That is, a specific wavelength region (particularly the region between 10 kHz and 100 kHz) of the impedance spectra of multiple liquids with known and varying loading rates can be expressed as a COS similarity to a reference impedance spectrum with a 100% loading rate, and a standard curve can be created by graphing the relationship between this COS similarity and the loading rate (see Figure 5).

The present invention will be described below based on examples. The examples are intended to illustrate exemplary embodiments of the present invention and are not intended to limit the scope of the present invention.

Example 1
In Example 1, nucleic acid (DNA) encoding a GFP (Green fluorescent protein) gene was used as the target substance, and adeno-associated virus (AAV) was used as the carrier particle.

<Preparation of liquid>
(Sample solution 1)
A solution containing an adeno-associated virus in which nucleic acid encoding the GFP gene was packaged (product name: AAV2-CMV-GFP, manufactured by Applied Viromics) was subjected to a filter concentration treatment (filter diameter: 50 kDa; Amicon® Ultra, manufactured by Merck Millipore) three times to replace the solution with an aqueous solution of 0.025 μM Tris-HCl, pH 8.0, and 0.001% Pluronic®, thereby obtaining sample solution 1 from which nucleic acid not encapsulated in the virus had been removed.

(Sample solution 2)
The solution containing empty adeno-associated virus (AAV2-Empty, manufactured by Applied Viromics) was replaced with an aqueous solution of 0.025 μM Tris-HCl pH 8.0 containing 0.001% Pluronic (registered trademark), and the resulting solution was used as sample solution 2.

(Sample solutions 3 to 6)
Sample solutions 3 to 6 were prepared by mixing the above sample solutions 1 and 2 in the proportions shown in Table 1 below.

<Impedance spectrum measurement>
(electrode)
The electrode used was a comb-shaped electrode (tooth width 10 μm, tooth spacing 5 μm, length 2 mm, 65 tooth sets, gold electrode, manufactured by BAS Co., Ltd.).

(measurement)
20 μL of the above sample solution 1 was dropped onto the above interdigital electrode. Then, using an impedance analyzer (ZA57630, manufactured by NF Corporation), the impedance of the above interdigital electrode was measured at an applied voltage of 1 V and at 2000 points obtained by logarithmically dividing the frequency range from 1 kHz to 36 MHz.

Impedance measurements were also performed for sample solutions 2 to 6 in the same manner as for sample solution 1.

(Waveform analysis)
The impedance spectra obtained as a result of the measurement were normalized with the value at 1 kHz set to 1. The normalized impedance spectra for sample solutions 1 to 6 are shown in FIG. 4A. FIG. 4B shows an enlarged view of a portion of the graph in FIG. 4A (the portion surrounded by a square frame). In FIGS. 4A and 4B, "Full" represents the mixing ratio of a solution with a loading ratio of 100% (sample solution 1), and "Empty" represents the mixing ratio of a solution with a loading ratio of 0% (sample solution 2).

As can be seen in Figures 4A and 4B, impedance spectra with different waveforms were obtained for sample solutions 1 to 6, which had different loading ratios. The normalized impedance spectra changed continuously, and significant differences were observed between the sample solutions, particularly in the range of 10 kHz to 100 kHz (Figure 4B).

(COS similarity)
The COS similarity was calculated for the impedance spectra of sample solutions 1 to 6. Specifically, for the waveforms of the impedance spectra in the range of 10 kHz to 100 kHz, the COS similarity to the waveform of the impedance spectrum at a loading ratio of 100% was calculated according to the following formula (1). The results are shown in Figure 5.

In the above formula (1), S is the cosine similarity (COS similarity), f ₁₀₀ (ω) is a vector representing the impedance spectrum of a full 100% sample, and f(ω) is a vector representing the impedance spectrum of the target sample.

Figure 5 is a graph showing the relationship between the COS similarity of the impedance spectra of sample solutions 1 to 6 and the proportion of carrier particles carrying the target substance in each solution (loading fraction, loading rate, or full fraction). As can be seen in Figure 5, the COS similarity of the impedance spectra increases in proportion to the loading rate, confirming a linear relationship between the loading rate and COS similarity. This demonstrates that the loading rate of carrier particles can be calculated with high accuracy by measuring the impedance spectrum of a liquid according to the method disclosed herein.

For example, when evaluating an arbitrary solution whose loading ratio is unknown, the loading ratio in the solution can be calculated by measuring the impedance spectrum of the solution, calculating the COS similarity with the reference impedance, and applying this to the graph (calibration curve) in Figure 5.

12, 14 Electrodes 16, 35 Carrier particles 18 Target substance 20 Impedance analyzer 22 Substrate 24, 26 Interdigital electrodes 28 Measuring instrument 37 Droplet 142, 144 Teeth of interdigital electrode 24 162, 164 Teeth of interdigital electrode 26 A'-A' Cutting line L Length direction W Width direction H Height direction

Claims (9)

A method for evaluating a ratio of carrier particles not carrying a target substance to carrier particles carrying the target substance in a liquid containing a plurality of carrier particles, comprising:
measuring an impedance spectrum of the liquid;
method. further comprising comparing the measured impedance spectrum with a reference impedance spectrum.
The method of claim 1. The method of claim 2, wherein the shape of the measured impedance spectrum is compared with the shape of a reference impedance spectrum. The method of claim 2 or 3, wherein multiple reference impedance spectra are used as the reference impedance spectrum. The method of claim 2 or 3, wherein the measured impedance spectrum is compared with the reference impedance spectrum in the range of 10 kHz to 100 kHz. The method of claim 2 or 3, wherein the comparison is performed using a COS similarity calculated from the reference impedance spectrum and the measured impedance spectrum. The method of claim 1 or 2, wherein the carrier particles support the target substance by encapsulating the target substance. the carrier particles are viruses, and the target substance is nucleic acid.
The method of claim 7. The method of claim 1 or 2, wherein the impedance spectrum of the liquid is measured using an interdigitated electrode.