KR102547446B1 - Label-free Polydiacetylene Liposome-based Method for Detection of Exosomes - Google Patents

Abstract

According to a specific embodiment of the present disclosure, a method for conveniently detecting exosomes in a sample without labeling is described using polydiacetylene liposomes in which a receptor specific for a biomarker present on the surface of exosomes is immobilized on polydiacetylene do.

Description

Label-free polydiacetylene liposome-based detection method for exosomes {Label-free Polydiacetylene Liposome-based Method for Detection of Exosomes}

The present disclosure relates to a method for detecting exosomes based on label-free polydiacetylene liposomes. More specifically, the present disclosure provides a method for easily detecting exosomes in a sample without labeling using polydiesetacetylene liposomes in which a receptor specific for a biomarker present on the surface of exosomes is immobilized (conjugated) to polydieacetylene. It's about how.

Cells are known to secrete extracellular vesicles surrounded by lipid bilayer membranes of various sizes. These extracellular vesicles are classified into exosomes, microvesicles, and large oncosomes according to their sizes. Among them, exosomes are small membrane-structured vesicles secreted from various types of cells. The diameter of exosomes has been reported to be approximately 30 to 100 nm, and in studies through electron microscopy, they originate from specific intracellular compartments called multivesicular bodies (MVBs) rather than being released directly from the plasma membrane, and are released and released outside the cell. secretion was observed. That is, when fusion between the polycystic body and the plasma membrane occurs, these vesicles are released into the environment outside the cell, which is referred to as exosomes.

Although the molecular mechanism by which the above-mentioned exosomes are formed has not yet been clearly identified, various types of immune cells, including not only red blood cells but also B-lymphocytes, T-lymphocytes, dendritic cells, platelets, macrophages, etc. It has been reported that tumor cells and the like also produce and secrete exosomes in a living state. Exosomes are known to be released from a number of different cell types in both normal and pathological conditions.

Recently, exosomes, newly emerging in the field of disease diagnosis, are in the limelight for their effectiveness as a diagnostic and drug delivery medium for various diseases. In other words, exosomes with cell-derived properties are released extracellularly and are included in various types of body fluids such as blood, saliva, and urine. method for diagnosing. In this regard, exosomes are known to contain approximately 4,500 proteins, lipids, and genetic information (eg, microRNA, mRNA, tRNA, rRNA, DNA, etc.) that are indicators of drug response. A method for diagnosing diseases by detecting the presence and amount of various microRNAs present in the body (e.g., Korean Patent Publication No. 2010-0127768, etc.), in samples originating from cancer (blood, saliva, tears, etc.) A method (for example, WO2009/015357 A, etc.) of detecting exosomes and measuring the amount of change in microRNA, predicting and diagnosing the relationship with a specific disease when increased or decreased compared to the control group has been developed.

However, since exosomes present in various body fluids have a low density and are small in size (tens of nm to 150 nm), an additional process for isolating them is required. In order to simplify the above-described separation procedure, a sensor that selectively detects or detects exosomes in bodily fluids is being studied. Most of these exosome detection methods use a chip that can flow a liquid containing exosomes through a passage. It can be detected (eg, Korean Patent Publication No. 87594, etc.).

However, the above method requires a separate chip system to be manufactured, and in the process, complicated procedures and processes must be performed. In addition, expensive equipment is required to obtain a detectable signal, and a labeling material is used for diagnosis, which is disadvantageous in terms of compatibility and versatility. In particular, since the manufacture of a chip system for detecting exosomes involves expensive manufacturing equipment and complicated procedures, its commercial use is limited and mass production is difficult. In addition, fixed-standard chips have a limited application range and are not suitable for application in various fields. In addition, the surface plasmon resonance-based device used to obtain the exosome capture signal is expensive and has low versatility.

As such, due to issues related to simplification and cost reduction of complex diagnostic systems, a simple detection principle is required for commercialization. However, in general, fluorescence and colorimetric principles related to target-specific light sources, filters, prisms, and visualization devices are used, and these methods require complicated methods and equipment, so there is a need to improve diagnostic convenience. there is.

Therefore, a method for detecting exosomes in a convenient way without using a separate label is required.

In one embodiment according to the present disclosure, unlike the prior art, exosomes in a sample can be easily and accurately detected or diagnosed based on an analysis principle that can be detected through general-purpose means without requiring the use of complicated procedures and expensive equipment We want to provide a method (or platform).

In one embodiment according to the present disclosure, it is intended to provide a method for detecting exosomes in a sample in a non-labeled manner without requiring the manufacture of a chip.

According to the first aspect of the present disclosure,

a) providing poly-acetylene liposomes modified with receptors having specific binding ability to biomarkers present on the surface of exosomes; and

b) measuring a change in optical properties induced by a ligand-receptor reaction between the exosomes in the sample and the modified polydiesel acetylene liposomes by contacting the modified polydiesel acetylene liposome with an exosome-containing sample;

A method for detecting exosomes comprising a is provided.

According to an exemplary embodiment, step a),

a1) self-assembly of die acetylene monomers and phospholipids to form lipid vesicles having internal spaces separated by a lipid layer membrane;

a2) attaching the receptor to lipid vesicles to form modified lipid vesicles; and

a3) polymerizing the modified lipid vesicles to form a modified polydiacetylene liposome;

can include

According to an exemplary embodiment, at least one biomarker is present on the surface of the exosome, and the receptor may be a single-receptor specific to the at least one biomarker.

According to an exemplary embodiment, a plurality of biomarkers are present on the surface of the exosome, and the receptor may be a multi-receptor specific to the plurality of biomarkers.

According to an exemplary embodiment, the receptors are multiple receptors, and in this case, step a2) may include attaching any one of the multiple receptors to the lipid vesicle and then sequentially attaching the others.

According to an exemplary embodiment, the diacetylene monomer is at least one selected from the group consisting of pentacosadiyonic acid (PCDA), trocosadiyonic acid (TCDA), heneicosadiynoic acid (HCDA) and heptadecadiynoic acid (HDDA), and

The phospholipids include dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), Dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearolphosphatidylglycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), dilauroylethylphosphocholine ( DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), 1,2-bis(oleoyloxy)-3 -(trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylic acid (DMPA), dipalmitoylphosphatidylic acid (DPPA), distearoylphosphatidylic acid (DSPA), Dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), and distearoylphosphatidylinositol (DPPS) It may be at least one selected from the group consisting of thearoylphosphatidylserine (DSPS).

According to an exemplary embodiment, the molar ratio of die acetylene monomer:phospholipid in step a1) may be in the range of 2 to 10:1.

According to an exemplary embodiment, a step of activating the lipid vesicles with a chemical functional group may be further included prior to step a2).

According to an exemplary embodiment, the receptor may be at least one selected from the group consisting of an entire antibody or a partial antibody containing an antigen binding region and an aptamer.

According to an exemplary embodiment, the biomarker may be a tetraspanin protein.

According to an exemplary embodiment, the tetraspanin protein may be at least one selected from the group consisting of CD-9, CD-63, CD-81 and CD-82.

According to an exemplary embodiment, the change in optical properties may be measured by the naked eye or a measuring device.

According to an exemplary embodiment, the optical characteristic change measuring device may be a UV-Vis spectrometer or a fluorescence spectrometer.

Since the method for detecting exosomes based on unlabeled polydieacetylene liposomes according to an embodiment of the present disclosure can be detected in an aqueous medium, signals can be generated by simple mixing without complicated procedures. In particular, the amount of polydiacetylene liposome can be appropriately adjusted according to the purpose of use and the amount of sample, and the signal derived from polydiacetylene can be easily measured using a commonly used UV-Vis spectrometer, fluorescence spectrometer, etc. can Moreover, it has the advantage of providing a wide use scalability compared to surface plasmon resonance and electrical measurement devices, which are necessary devices in the existing chip method. Therefore, it is expected to be used in various diagnostic fields in the future.

1 is a diagram exemplarily illustrating a principle of detecting exosomes based on unlabeled polydieacetylene liposomes according to one embodiment of the present disclosure and a procedure for implementing the same;
Figure 2 is a diagram schematically illustrating a procedure for preparing poly-diacetylene liposomes modified with multiple receptors according to an embodiment and a procedure for detecting exosomes in a label-free manner using the same;
3 is a scanning electron microscope (SEM) photograph of the PCDA/DMPC liposome prepared in Example;
Figure 4 is a scanning electron microscope (SEM) photograph of die acetylene liposomes into which exosome biomarker receptors were introduced in Example;
5 is a scanning electron microscope (SEM) photograph of aggregates of polydieacetylene liposomes and exosomes into which receptors have been introduced in Examples;
Figure 6 is a graph showing the change in UV-Vis absorption spectrum of poly-acetylene liposomes according to the concentration of exosomes in the sample in Example;
Figure 7 is a transmission electron microscope (TEM) picture after adding exosomes to polydieacetylene liposomes modified with multiple receptors in Example;
Figure 8 is a transmission electron microscope (TEM) picture after adding albumin protein and fibrinogen to polydieacetylene liposomes modified with multiple receptors in Example;
Figure 9 is a photograph showing the color change before and after the addition of exosomes to polydieacetylene liposomes modified with multiple receptors in Example;
Figure 10 is a fluorescence photograph taken before and after the addition of an exosome solution to polydieacetylene vesicles attached on a substrate; and
Figure 11 is a graph showing the change in fluorescence intensity with time, when the polydieacetylene / phospholipid vesicles contact the exosome-containing sample according to the receptor introduced into the polydieacetylene endoplasmic reticulum.

The present invention can all be achieved by the following description with reference to the accompanying drawings. The following description should be understood to describe preferred embodiments of the present invention, and it should be understood that the present invention is not necessarily limited thereto.

In addition, the accompanying drawings may be somewhat exaggerated compared to the thickness (or height) of the actual layer or the ratio with other layers to aid understanding, the meaning of which will be properly understood by the specific purpose of the related description to be described later. can

Terms used in this specification may be defined as follows.

“Binding or binding” may mean being bound or linked to a surface in a covalent or non-covalent manner.

A “sample” is not limited to a specific type or form, as long as it can contain the target pathogen to be detected. Illustratively, the sample may be a biological sample, for example, a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, and amniotic fluid. Biological tissue is a collection of cells, usually connective tissue, epithelial tissue, muscle tissue and nerves, as well as certain types of collections of intracellular substances that form one of the structural substances of human, animal, plant, bacterial, fungal or viral structures. organizations, etc., may correspond to this. Examples of biological tissues may also include organs, tumors, lymph nodes, arteries, and individual cell(s).

It can be understood that the expressions "on" and "above" are used to refer to the concept of relative position. Therefore, not only when other components or layers are directly present in the mentioned layers, other layers (intermediate layers) or components may be interposed or present therebetween. Similarly, the expressions “under”, “under” and “below” and “between” may also be understood as relative concepts of position. In addition, the expression “sequentially” may also be understood as a relative positional concept.

full disclosure

Figures 1a and 1b respectively show the detection principle and implementation procedure of the label-free polydieacetylene liposome-based exosome detection system according to one embodiment by way of example.

Referring to the above figure, as a platform for detecting exosomes in a sample, polydieacetylene liposomes that cause color changes in response to external stimuli are generally used. As a polymer, it is a material whose optical properties can be changed without a separate label. Polydieacetylene may be prepared in various shapes and structures, such as crystals, films, and liposomes, depending on the purpose of use. Poly die acetylene forms a stable structure having an internal space isolated by a lipid layer membrane (specifically, a bilayer) in an aqueous solution, which may be referred to as "poly die acetylene liposome".

Die acetylene monomers used in poly die acetylene synthesis maintain a sufficiently close distance by non-polar bonds in an aqueous solution. At this time, triple bonds are converted to double bonds under 254 nm UV irradiation, and other monomers and specific sites of the polymer backbone is polymerized in such a way that Before irradiation with ultraviolet rays, it does not express color, but after polymerization, it expresses blue color with a maximum absorption wavelength of 640 nm. Thereafter, the absorption spectrum is changed by external stimuli such as temperature, pH, solvent, molecular recognition, etc., and color transition may occur to red with a maximum absorption wavelength of 540 nm.

In this embodiment, a receptor is introduced into polydiacetylene in order to impart specific binding characteristics or selectivity to a specific molecule (specifically, a biomarker present on the surface of exosomes, which is a mediator of disease diagnosis or drug delivery). Such a receptor may be a molecular group or receptor capable of recognizing a specific substance, and by introducing it, a unique optical property change due to a conjugated structure of polydiacetylene may be induced.

As such, the change in the optical properties of the polydieacetylene liposome can be measured with the naked eye or by a measuring means or device commonly used in the art to qualitatively and/or quantitatively detect the exosomes in the sample.

polydiacetylene liposomal manufacturing

Referring to Figures 1a and 1b, first, a lipid (specifically, a phospholipid) is combined with a diacetylene monomer in a solution (eg, an organic solvent such as chloroform, tetrahydrofuran, etc.), self-assembly ), it is possible to form lipid vesicles having an internal space isolated by a lipid layer membrane. In this way, by combining phospholipids with diacetylene monomers, charge stability and membrane flexibility of polydiacetylene (PDA) liposomes prepared in subsequent steps can be enhanced.

According to an exemplary embodiment, the diacetylene monomer may be a compound containing a diacetylene group, for example, PCDA (pentacosadiyonic acid), TCDA (trocosadiyonic acid), HCDA (heneicosadiynoic acid) and HDDA (heptadecadiynoic acid). It may be at least one selected from the group. Specifically, the diacetylene monomer may be PCDA and/or TCDA, and more specifically PCDA. In this regard, in an exemplary embodiment, it may contain at least one terminal selected from a carboxy group, an amine group, a sulfone group, etc., which allows a specific receptor to bind to PCDA, and may also be used for EDC/NHS coupling reaction, sulfone- It may be advantageous to easily attach the receptor to the diacetylene monomer through a maleimide coupling reaction, an avidin-biotin coupling reaction, or the like.

In addition, phospholipids, for example, dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidyl Glycerol (DSPG), dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearolphosphatidylglycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), dilauroyl Ethylphosphocholine (DLEP), Dimyristoyl Ethylphosphocholine (DMEP), Dipalmitoyl Ethylphosphocholine (DPEP) and Distearoyl Ethylphosphocholine (DSEP), 1,2-bis(Ole Oiloxy)-3-(trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylic acid (DMPA), dipalmitoylphosphatidylic acid (DPPA), distearoylphosphatidyl Acid (DSPA), Dimyristoylphosphatidylinositol (DMPI), Dipalmitoylphosphatidylinositol (DPPI), Distearoylphosphatidylinositol (DSPI), Dimyristoylphosphatidylserine (DMPS), Dipalmitoylphosphatidylserine ( DPPS) and distearoylphosphatidylserine (DSPS). Specifically, it may be at least one selected from the group consisting of DMPC and DMPA, and more specifically, it may be DMPC.

In this regard, when inserting phospholipids, it may be advantageous to combine them in an appropriate ratio in consideration of the bar that may affect the sensitivity of polydiacetylene (PDA). Illustratively, the molar ratio of die acetylene monomer:phospholipid may be, for example, about 2 to 10:1, specifically about 3 to 8:1, and more specifically about 4 to 6:1. Illustratively, when the diacetylene monomer and the phospholipid are combined, the temperature may be, for example, about 20 to 40 °C, specifically about 25 to 37 °C, but this may be understood as an example.

According to an exemplary embodiment, after formation of lipid vesicles, for example, a drying step (eg, under an inert gas atmosphere) may be performed, after which water is added and a solution (or dispersion) containing vesicles is formed. At this time, the concentration (total concentration) of lipids in the endoplasmic reticulum-containing solution is, for example, about 0.1 to 2 mM, specifically about 0.4 to 1.5 mM, more specifically about 0.5 to 1.2 mM within the range. Adjustable. In addition, it is possible to induce the formation of uniform vesicles by optionally performing ultrasound irradiation prior to the subsequent polymerization reaction. In this regard, when preparing a homogeneous vesicle-containing solution (or dispersion), the temperature may be adjusted, for example, in the range of about 60 to 90 ° C, specifically about 70 to 80 ° C, but this is understood as an example. It can be.

According to an exemplary embodiment, the size of the lipid vesicles formed (measured by dynamic light scattering (DLS)) is, for example, about 50 to 500 nm, specifically about 100 to 300 nm, more specifically about 200 to 250 nm. range, which can be understood for illustrative purposes.

Then, the lipid vesicles can be modified by attaching (immobilizing) or conjugating a receptor having a specific binding ability to the exosome (specifically, the target exosome), specifically, a biomarker on the surface of the exosome to the lipid vesicle. can

Optionally, the surface of the lipid vesicle may be activated with a chemical functional group prior to attaching the receptor to the lipid vesicle. These chemical functional groups can be attached to or bonded to the surface of lipid vesicles and can be selected from various types capable of binding or coupling (eg, covalent bonds) with receptors. Representatively, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (NHS/EDC) can be used as the above-mentioned chemical functional group, and the receptor is fixed on the lipid vesicle. useful for bonding or

According to an exemplary embodiment, the receptor is typically at least selected from whole antibodies or partial antibodies (F _ab , F _{(ab') 2} , scFv, etc.) containing an antigen binding region, aptamers, and the like. can be one At this time, the aptamer can function as a nucleic acid-based receptor that can accept proteins such as CD-9, CD-63, CD-81, and CD-82, which are biomarkers of exosomes. More typically it may be an antibody and/or an aptamer.

Meanwhile, according to an exemplary embodiment, the receptor may be a single receptor that corresponds to a specific biomarker in exosomes or includes a specific biomarker receptor.

According to an alternative embodiment, the receptor may be a multi-receptor comprising two or more different biomarker receptors that correspond to or are specific for a plurality of biomarkers in the exosome. To this end, after attaching any one of the multiple receptors to the lipid vesicle, the step of sequentially attaching the rest may be included. In this way, when multiple receptors are introduced, the polydieacetylene liposome modified with multiple receptors prepared in the subsequent procedure not only exhibits specificity for exosomes in the mixed solution, but also induces a more efficient ligand-receptor reaction to obtain a measurement signal. Since it can amplify, it can be advantageous compared to the case where a single receptor is applied. However, the range of receptors applicable to the specific embodiments of the present disclosure may be understood as a concept including both single receptors and multiple receptors.

According to an exemplary embodiment, the amount of modification or immobilization of the receptor (i.e., single receptor or multiple receptors) is about 0.0001 to 0.05, specifically about 0.0005 to 0.01, more than Specifically, it may be set in the range of about 0.001 to 0.005, but this may be understood as an example.

According to an exemplary embodiment, after forming the modified lipid vesicles, a polymerization reaction may be performed to form a modified poly-diacetylene liposome (ie, poly-diacetylene (PDA) liposome complex). In this case, the polymerization reaction may be a photopolymerization reaction by irradiation of ultraviolet rays or a polymerization reaction by irradiation of gamma-rays, and more specifically, it may be a polymerization reaction using ultraviolet rays irradiation.

According to a specific embodiment, the polymerization reaction may be a photopolymerization reaction by irradiation of ultraviolet rays, and the wavelength of the irradiated ultraviolet rays is, for example, about 100 to 400 nm, specifically about 150 to 350 nm, and more specifically about 200 to 400 nm. It may have a wavelength in the range of 300 nm. In addition, the intensity of ultraviolet light may be adjusted within a range of, for example, about 200 to 600 μW/cm 2 , specifically about 300 to 500 μW/cm 2 , and more specifically about 350 to 450 μW/cm 2 . In addition, the UV irradiation time may be selected within the range of, for example, about 10 to 60 minutes, specifically about 20 to 50 minutes, and more specifically about 25 to 40 minutes. However, polymerization conditions by irradiation of such ultraviolet rays may be understood as exemplary purposes.

The size of the polydiacetylene (or modified polydiacetylene) liposome prepared in this way (measured by dynamic light scattering (DLS)) is, for example, about 100 to 300 nm, specifically about 120 to 200 nm, more specifically It may range from about 130 to 160 nm, but this can be understood as an example. As an example, the size of polydiacetylene liposomes modified with receptors and formed through polymerization is, for example, at least about 10%, specifically at least about 12%, more specifically about 15 to 20% compared to lipid vesicles before modification may have an increased size.

In addition, the polydiacetylene liposome modified by the receptor may exhibit a different charge from that of the unmodified case. It may exhibit a large negative charge (eg, about -30 to -20 mV), whereas in its acceptor modified form it may exhibit a relatively small negative charge (eg, about -10 to 0 mV).

polydiacetylene liposomes used exosome detection

According to one embodiment of the present disclosure, poly-acetylene liposomes can be used to detect exosomes contained in a sample, specifically an aqueous sample.

Although the present disclosure is not bound by any particular theory, the aforementioned optical property change can be explained as follows.

As the biomarker present on the surface of the exosome and the receptor immobilized on polydiacetylene specifically bind or bind (ie, ligand-receptor interaction), a physical stimulus may be applied to the surface of the polydiacetylene polymer. These physical stimuli are transmitted to the main chain of the solid-liquid polymer, and the conjugated chain is twisted to induce a change in absorption spectrum and fluorescence. At this time, larger clusters can be formed by inducing a chain reaction through ligand-receptor interaction.

According to an exemplary embodiment, the size of the cluster formed by binding of the exosome and the modified polydieacetylene liposome is, for example, about 200 to 3000 nm, specifically about 300 to 1500 nm, and more specifically about 500 to 1000 nm. It may be in the nm range, and may also be, for example, at least about 50%, specifically about 100 to 1000%, more specifically about 300 to 500% higher than the size of the PDA liposome before binding with the exosome. . However, the present disclosure is not limited to these numerical ranges, and may vary according to the properties and types of exosomes.

According to an exemplary embodiment, the biomarker on an exosome may typically be a tetraspanin protein, which is a group consisting of CD-9, CD-63, CD-81 and CD-82. It may be at least one selected from. These biomarkers correspond to ligands and can specifically bind to receptors immobilized on the modified polydieacetylene liposome through a ligand-receptor reaction.

As described above, the receptor may be a single receptor or multiple receptors, and when fixed in the form of multiple receptors, binding to a plurality of biomarkers present in exosomes is induced, thereby increasing the number of ligands between polydiacetylene liposomes and exosomes. -Can induce a receptor response to provide an amplification effect of a signal.

This specific reaction can typically be performed in an aqueous medium, and as an example, a modified polydieacetylene liposome aqueous solution and a sample (ie, an exosome-containing aqueous solution) may be prepared and combined. According to an exemplary embodiment, the concentration of the modified polydieacetylene liposome aqueous solution ranges, for example, from about 0.05 to 0.5 mg/mL, specifically from about 0.1 to 0.4 mg/mL, and more specifically from about 0.2 to 0.3 mg/mL. can be adjusted within In addition, the ligand-receptor reaction may be appropriately controlled for a temperature and time so that the reaction may occur effectively, and the temperature is exemplarily about 25 to 50°C, specifically about 30 to 45°C, and more specifically about 35 to 40°C. It can be adjusted within the range, and the time can be adjusted, for example, in the range of about 10 to 60 minutes, specifically about 15 to 50 minutes, and more specifically about 20 to 40 minutes. However, the above temperature and time conditions are provided as examples, and the present disclosure is not limited thereto.

However, when the sample contains a significant amount of residues (debris, flakes, etc.) in the sample, such as when the sample is human fluid, specifically blood, only exosomes can be selectively isolated prior to detection. This separation process may be performed, for example, by centrifugation or filtering.

As described above, in the exosome detection method or system according to the present embodiment, exosomes present in the sample can be detected or detected with high selectivity by adding the modified polydiacetylene solution to the sample, such as body fluid or liquid mixture. . Alternatively, the sample may be an exosome solution in which an isolation process has been performed.

In addition, it is useful as an exosome sensor because it can provide a simple and intuitive signal compared to a chip-based system, which is a conventional exosome detection technology. In particular, the presence or absence of exosomes (qualitative analysis) can be confirmed by measuring the color change and / or fluorescence expressed by simply mixing in an aqueous solution, and furthermore, the concentration of exosomes in the sample can be predicted (quantitative analysis), etc. , it is simple and provides good reliability. As an example, in applying the above exosome detection method, the detection limit of exosomes in a sample (ie, exosome-containing solution) is, for example, about 1×10 ¹² vesicles/mL or less, specifically about 1× 10 ¹¹ vesicles/mL or less, more specifically about 3×10 ⁸ vesicles/mL or less.

According to an alternative embodiment, the modified polydieacetylene vesicles may be subjected to qualitative/quantitative analysis of the sample by contacting the sample (or exosome-containing solution) while being attached to or immobilized on the substrate. In this regard, the substrate is not particularly limited as long as it can detect a signal generated from a ligand-receptor reaction, and may be, for example, a material such as silicon, quartz, glass, ceramic, or transparent plastic. It may be a slide glass.

As such, the prepared polydieacetylene liposome is advantageous in terms of scalability as it can be applied in various forms by attaching to substrates of various materials and structures depending on the use.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but the following examples are provided to more easily understand the present invention, but the present invention is not limited thereto.

Example

The materials used in this example are as follows:

- 10,12-pentacosadiyonic acid (PCDA) was purchased from GFS Chemicals (Powell, OH).

- 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) were purchased from Tokyo Chemical Industry (Tokyo, Japan).

- N-hydroxysuccinimide (NHS) and ethanolamine were purchased from Sigma-Aldrich (St. Louis, MO).

- Monoclonal antibodies (Anti CD-9, CD-63, CD-81 and CD-82) were purchased from Abcam (Cambridge, UK).

- PBS buffer (pH 7.4) was supplied by Welgene, Inc. (Gyeongsan-si, Korea).

Example 1

Detection of polydiacetylene exosomes with a single receptor

Following the procedure shown in FIG. 1B, a polydieacetylene liposome into which a single receptor was introduced was prepared, and the exosome was detected using a polydieacetylene/phospholipid vesicle (liposome) exosome sensor in which a polydieacetylene liposome was dispersed in a solution.

Preparation of PCDA/DMPC lipid vesicles (liposomes)

A solution obtained by dissolving PCDA and DMPC in chloroform was put into a glass vial and mixed well to adjust the molar ratio of PCDA: DMPC to 4: 1. Then, chloroform was evaporated in a nitrogen atmosphere to form a lipid layer in the form of a thin film. (lipid vesicles).

After completely drying the lipid layer, distilled water was added to adjust the lipid concentration to 1.0 mM, the temperature was raised by heating in a water bath at 80 ° C for 5 minutes, and ultrasonic waves were irradiated for 20 minutes at a power of 150 W using a tip sonicator. By doing so, the lipid in the form of a film was uniformly dispersed in the aqueous medium. The lipid vesicle solution, in which dispersion was completed using ultrasonic waves, was immediately filtered through a syringe filter having a size of 0.45 μm. After cooling the filtered aqueous solution at room temperature, it was left in a refrigerator at 4° C. for at least 6 hours to form PCDA/DMPC lipid vesicles (liposomes).

SEM pictures of the prepared PCDA/DMPC vesicles (liposomes) are shown in FIG. 3 . According to the figure, it can be seen that lipid vesicles form a crystalline phase.

- Preparation of modified PCDA/DMPC lipid vesicles (liposomes)

EDC/NHS was added to the cold PCDA/DMPC lipid vesicles to adjust the EDC/NHS concentration to 100 mM, followed by reaction at room temperature for 2 hours to NHS-activate the lipid vesicles. The activated liposomes were centrifuged at 4° C. at 25000 g for 15 minutes to remove the supernatant, and the precipitate was redispersed by injecting the same amount of distilled water. The same process was repeated three times, and the third solution was filled with 5 times the volume of PBS instead of distilled water. The antibody concentration was adjusted to 0.1 mg/mL by adding 100 uL of 1 mg/mL anti-CD-63 antibody to 1 mL of the lipid vesicle solution.

Antibodies were introduced into NHS-active lipid vesicles while stirring for 4 hours in a refrigerator at 4°C.

An ethanolamine-PBS solution was added to the antibody-introduced lipid vesicle solution to adjust the concentration to 2 mM, followed by stirring at 4° C. for 2 hours to inactivate residual NHS-activated lipid vesicles.

The obtained solution was centrifuged at 20000 g at 4 ° C. for 15 minutes to remove the supernatant, and the same amount of PBS was added to the precipitate and the process of redispersion was repeated three times to obtain residual antibodies and ethanolamine. has been removed. The SEM picture of the lipid vesicle into which the antibody was introduced is shown in FIG. 4 . As shown in the figure, it can be confirmed that the antibody, the receptor, was uniformly immobilized on the lipid vesicles.

- Preparation of poly die acetylene (or modified poly die acetylene) liposomes

On the other hand, before adding the exosomes, the modified lipid vesicles were irradiated with UV light of 254 nm wavelength at an intensity of 400 μW/cm 2 for 30 minutes until they turned blue, thereby producing PDA liposomes.

- Detection of exosomes in samples

After mixing the blue PDA liposome solution (concentration: 0.4 mg/mL, 1.0 mM) and the exosome solution to be measured (concentration: 3 × 10 ⁸ vesicles/mL or more) in equal volumes, leave at 37 ° C. for 30 minutes. A ligand-receptor reaction between exosomes and polydiacetylene liposomes was maximized. SEM pictures of aggregates formed by the reaction between PDA liposomes and exosomes are shown in FIG. 5 . As shown, the exosomes are firmly immobilized between the PDA liposome particles.

- Absorbance analysis

Absorbance at 540 nm and 640 nm of the liposome solution to which PBS was added and the liposome solution to which exosome solution was added were measured using a UV-Vis spectrometer to prepare for comparison.

Color change (%) was calculated by substituting the absorbance at 540 nm and 640 nm using Equation 1 below.

[Equation 1]

In addition, the results of measuring the UV-Vis absorption spectrum while varying the exosome concentration are shown in FIG. 6 . According to the figure, it can be seen that the absorbance in the long-wavelength band (red band) increases as the exosome concentration in the sample increases.

The above results suggest that the PDA vesicle cluster was formed by the ligand-receptor interaction between the antibody, which is a receptor conjugated on the PDA, and the exosome, and as a result, the color change of the PDA vesicle was induced.

- Fluorescence intensity analysis

Fluorescence intensity of poly-acetylene was measured using a fluorescence microscope from ZEISS. Specifically, the fluorescence intensity of the liposome solution to which PBS was added and the liposome mixture solution to which exosome solution was added was measured at an excitation wavelength of 500 nm around 640 nm, and the fluorescence intensity in the control (PBS solution) was subtracted to obtain fluorescence. Changes in intensity were measured. As a result, it was confirmed that the fluorescence intensity in the liposome solution to which the exosome solution was added was significantly higher than that of the control group.

Example 2

According to the procedure shown in FIG. 2, poly-acetylene liposomes into which multiple receptors were introduced were prepared to detect exosomes.

Preparation of polydiacetylene (PDA) liposomes

- Manufacturing of PCDA/DMPC lipid vesicles (liposomes)

Diacetylene suspension (PCDA: DMPC molar ratio adjusted to 4: 1) was prepared by injecting a solution in which PCDA and DMPC were dissolved in tetrahydrofuran, respectively, into a vial containing distilled water with a syringe. At this time, the total concentration of PCDA and phospholipid was adjusted to be 1.0 mM.

The prepared lipid vesicle suspension was irradiated with ultrasonic waves at a power of 150 W for 15 minutes to form lipid vesicles (liposomes) and homogenization was performed. Immediately after the ultrasonic dispersion was completed, the lipid vesicle solution was filtered through a 0.45 μm syringe filter. After cooling the filtered aqueous solution at room temperature, it was left in a refrigerator at 4° C. for at least 6 hours to form PCDA/DMPC lipid vesicles (liposomes).

Preparation of modified PCDA/DMPC lipid vesicles (liposomes)

After adjusting the total concentration to 100 mM by adding EDC/NHS to the cold PCDA/DMPC lipid vesicles, the lipid vesicles were NHS-activated by reacting at room temperature for 2 hours. The activated liposomes were centrifuged at 4° C. at 25000 g for 15 minutes to remove the supernatant, and the precipitate was redispersed by injecting the same amount of distilled water. The same process was repeated three times, and the third solution was filled with 5 times the volume of PBS instead of distilled water. An antibody solution having specific binding ability to CD-9, CD-63, CD-81 and CD-82 was added to 1 mL of the activated lipid vesicle solution. At this time, the total concentration of the added antibody was adjusted to be 10 uM. The concentration ratio of each antibody was added equally according to the number of antibody solution types to be added.

Residual NHS-activated lipid vesicles were inactivated by adding 2 mM ethanolamine-PBS to the antibody-introduced lipid vesicle solution or by stirring in 1 mg/mL BSA at 4° C. for 2 hours.

The obtained solution was centrifuged at 20000 g at 4 ° C. for 15 minutes to remove the supernatant, and the same amount of PBS was added to the precipitate and the process of redispersion was repeated three times to obtain residual antibodies and ethanolamine. has been removed.

Then, polydieacetylene (or modified polydieacetylene) liposomes were prepared according to the same procedure as in Example 1.

Detection of exosomes in samples

After mixing the previously prepared solution containing the PDA liposomes and the exosome solution used in Example 1 in equal volumes, they were left at 37 ° C. for 30 minutes to maximize the ligand-receptor reaction between the exosomes and polydiacetylene liposomes. did After adding the exosomes, a transmission electron microscope (JEOL, product name: JEM-3010) photograph of the PDA liposomes is shown in FIG. 7 .

According to the figure, as the exosomes were added to the PDA liposomes, aggregates were formed by mutually binding due to the interaction between tetraspanin and multiple receptors on the surface of the exosomes.

On the other hand, in order to confirm the selectivity of the receptor-introduced PDA liposomes for exosomes, the same experiment was performed on albumin protein and fibrinogen, respectively, which are abundant in bodily fluids or plasma, and analyzed using a transmission electron microscope. The results are shown in Figure 8 (left figure: albumin protein, right figure: fibrinogen). According to the figure, unlike the case where exosomes were added, it was confirmed that aggregates were not formed.

In addition, when the exosome solution was added to the solution containing PDA liposomes, the color change of the solution was visually observed. The results are shown in FIG. 9 .

As can be seen in the figure, the color of the solution changed from blue to light purple, which is due to the ligand-receptor interaction between the multireceptor conjugated on the PDA liposome and tetraspanin on the surface of the exosome. This indicates that a structural change has occurred and has affected the color of the solution.

Example 3

In this example, exosomes were detected using a sensor modified with a single receptor and multiple receptors, based on a sensor in which polydiacetylene/phospholipid endoplasmic reticulum (liposome) exosomes were immobilized on a substrate.

Specifically, a solution containing an antibody having specific binding ability to CD-63 (single receptor), an antibody having specific binding ability to CD-81 (single receptor), and a combination thereof (multiple receptor) as receptors Modified PCDA/DMPC lipid vesicles (liposomes) were prepared according to the same procedure as in the previous example except for the use. Then, a modified liposome array (using a manual microarrayer from Labnext) of about 0.3 mm was formed on a slide glass (substrate) coated with amine in a thermo-hygrostat in which a humidity of 70% or more was maintained at room temperature. After forming the array, it was reacted at room temperature for about 4 hours. The substrate to which the polydieacetylene vesicles were attached was washed with 0.1% TWEEN 20 solution, then washed twice with distilled water, dried to remove surface moisture, and exosome solution was added dropwise to each array part. did After 1 hour, the fluorescence intensity of polydieacetylene vesicles was measured using a fluorescence microscope. The results are shown in FIGS. 10 and 11, respectively.

10 is a fluorescence photograph taken before and after addition of an exosome solution to polydieacetylene vesicles attached on a substrate.

According to the figure, the modified polydieacetylene vesicles exhibit fluorescence due to ligand-receptor interaction when an exosome-containing sample is contacted in a state attached to a substrate, and polydieacetylene endoplasmic reticulum showing low fluorescence before introduction It was confirmed that gradually became fluorescent due to interaction with exosomes.

Figure 11 is a graph showing the change in fluorescence intensity with time when the polydieacetylene/phospholipid vesicles contact the exosome-containing sample according to the receptor introduced into the polydieacetylene endoplasmic reticulum.

Referring to the figure, rather than introducing a single receptor (antibody having specific binding ability to CD-63 or CD-81), a plurality of receptors (antibody having specific binding ability to CD-63 and CD-81) It was confirmed that a higher fluorescence signal could be obtained from the polydieacetylene endoplasmic reticulum into which a combination of antibodies having specific binding ability) was introduced. This indicates that more receptor-binding sites can be secured in a sample of the same exosome concentration through the introduction of a plurality of receptors.

Simple modifications or changes of the present invention can be easily used by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (15)

a) providing poly-acetylene liposomes modified with receptors having specific binding ability to biomarkers present on the surface of exosomes; and
b) measuring a change in optical properties induced by a ligand-receptor reaction between the exosomes and the modified polydieacetylene liposomes in the sample as the modified polydieacetylene liposome is brought into contact with the exosome-containing aqueous sample;
Including,
Here, the step b) is a method for detecting exosomes performed by combining a modified poly-diacetylene liposome aqueous solution and an exosome-containing aqueous sample. The method of claim 1, wherein step a),
a1) self-assembly of die acetylene monomers and phospholipids to form lipid vesicles having internal spaces separated by a lipid layer membrane;
a2) attaching the receptor to lipid vesicles to form modified lipid vesicles; and
a3) polymerizing the modified lipid vesicles to form a modified polydiacetylene liposome;
A method for detecting exosomes, characterized in that it comprises a. The method of claim 2, wherein at least one biomarker is present on the surface of the exosome, and the receptor is a single receptor specific for the at least one biomarker. The method of claim 2, wherein a plurality of biomarkers are present on the surface of the exosome, and the receptor is a multi-receptor specific to the plurality of biomarkers. The exosome according to claim 4, wherein the receptors are multiple receptors, wherein step a2) comprises attaching any one of the multiple receptors to the lipid vesicle and then sequentially attaching the others. detection method. The method of claim 2, wherein the die acetylene monomer is at least one selected from the group consisting of PCDA (pentacosadiyonic acid), TCDA (trocosadiyonic acid), HCDA (heneicosadiynoic acid) and HDDA (heptadecadiynoic acid), and
The phospholipids include dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), Dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearolphosphatidylglycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), dilauroylethylphosphocholine ( DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), 1,2-bis(oleoyloxy)-3 -(trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylic acid (DMPA), dipalmitoylphosphatidylic acid (DPPA), distearoylphosphatidylic acid (DSPA), Dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), and distearoylphosphatidylinositol (DPPS) A method for detecting exosomes, characterized in that at least one selected from the group consisting of thearoylphosphatidylserine (DSPS). The method for detecting exosomes according to claim 2, wherein the molar ratio of diacetylene monomer:phospholipid in step a1) is in the range of 2 to 10:1. The method of claim 2, further comprising activating the lipid vesicles with a chemical functional group prior to step a2). The method of claim 1, wherein the receptor is at least one selected from the group consisting of a whole antibody or a partial antibody containing an antigen binding region, and an aptamer. The method of claim 1, wherein the biomarker is a tetraspanin protein. 11. The method of claim 10, wherein the tetraspanin protein is at least one selected from the group consisting of CD-9, CD-63, CD-81 and CD-82. The method for detecting exosomes according to claim 1, wherein the exosome-containing aqueous sample is a bodily fluid or a liquid from which residues have been previously separated. The method of claim 1, wherein the change in optical properties is measured with the naked eye or with a measuring device. 14. The method of claim 13, wherein the device for measuring the change in optical properties is a UV-Vis spectrometer or a fluorescence spectrometer. The method of claim 1, wherein the method is performed without a label.

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