Patent Application
20190324023
The present disclosure relates to a simple method for separating and detecting extracellular vesicles. In particular, the present disclosure relates to a method for classifying exosomes into subclasses and detecting the exosomes classified into the subclasses.
Extracellular vesicles are nanometer-sized vesicles released from cells, and are classified into exosomes, microvesicles and apoptotic bodies, according to their cellular origin. Among them, exosomes, which may be possibly utilized for diagnosis and treatment, have been actively researched in recent years.
Exosomes are cell-derived vesicles of approximately 100 nm in diameter which are present on the outside of cells, and they are found in large amounts in any body fluids such as blood, saliva and urine. Exosomes have lipids and proteins derived from cell membranes on their surface and have nucleic acids such as mRNA and miRNA and proteins therein, and thus contain information derived from the cells which have released them. Therefore, the information included in exosomes can be utilized for a diagnostic marker as a biomarker.
It is also known that biological information molecules having these nucleic acids and proteins function in cells having exosomes incorporated therein, and it has been shown that intercellular communication by exchange of nucleic acids and proteins occurs through exosomes. Therefore, it has been thought that exosomes can be utilized not only for the diagnosis but also in the field of prevention and treatment (He, C et al., 2018, Theranostics, vol. 8(1), p. 237-255).
Although the biological significance of exosomes has been clarified as described above, consensus about the definition of exosomes itself has not been obtained among researchers. How exosomes having a high diversity are to be classified and defined has been an issue largely related to future exosome research.
The present disclosure provides various methods for classifying extracellular vesicles into subclasses according to their surface components and detecting the extracellular vesicles classified into the subclasses. For example, the methods include detecting an exosome subclass having a particular constituent utilizing an antigen present on the surface of the exosome or a membrane constituent of the exosome by precipitating.
Embodiments of the disclosure include a method for detecting, fractionating and/or purifying extracellular vesicles, and a kit using the same.
In an embodiment, a method for detecting, fractionating and/or purifying extracellular vesicles, comprises preparing nanoparticles coated with a ligand that specifically binds to a molecule present on the surface of the extracellular vesicles to be detected, and a sample solution in which the nanoparticles and the extracellular vesicles are mixed; providing at least one medium layer with a density higher than that of the sample solution; overlaying the sample solution on the at least one medium layer; and subjecting the at least one medium layer overlaid with the sample solution to low-speed centrifugation.
In an embodiment, a kit for detecting, classifying and/or purifying extracellular vesicles, comprises nanoparticles coated with a ligand that specifically binds to a molecule present on the surface of the extracellular vesicles to be detected; and a density gradient solution.
The foregoing summary and the following drawings and detailed description are intended to illustrate non-limiting examples but not to limit the disclosure.
The figures depict various embodiments of the present disclosure for purposes of illustration and are not intended to be limiting. Wherever practicable, similar or like reference numbers or reference labels may be used in the figures and may indicate similar or like functionality.
In order to use exosomes occurring as heterogeneous populations for diagnosis and treatment, it may be advantageous to classify exosomes into subclasses. If exosomes can be classified into subclasses, it is thought that biomarkers can be concentrated from particular cells, and it is thus possible to perform diagnosis with higher accuracy. For use in treatment, it also may be advantageous to concentrate exosomes contributing to the treatment as a tool of cellular communication. Accordingly, attempts have been made to classify exosomes into subclasses according to the antigens exposed on their surfaces, their densities, and the like.
Methods for purifying extracellular vesicles include a method for purifying them using a kit with reagents and a method for purifying them by centrifugation depending on their densities. However, the kits often contain no description of the separation principle and the details of reagents. Therefore, it may be unclear whether all of the desired extracellular vesicles can have been recovered. Since methods using ultracentrifugation such as density gradient centrifugation are time-consuming and labor-intensive, simpler methods described below are provided.
The present disclosure describes a method for purifying particular extracellular vesicles comprising: modifying nanoparticles with molecules capable of adsorbing the extracellular vesicles; and causing a plurality of nanoparticles to aggregate via extracellular vesicles to sediment aggregates. As shown in the following Examples, an extracellular vesicle having a particular surface marker can be sedimented at low-speed centrifugation, so that extracellular vesicles can be very easily fractionated. Hereinafter, exosomes will be mainly described as extracellular vesicles. However, extracellular vesicles other than exosomes, classified as microvesicles or apoptotic bodies can be detected, classified into subclasses and purified in the same manner as the exosomes.
As shown in the Examples, nanoparticles function as an anchor to sediment extracellular vesicles including exosomes and also function as an indicator for visually indicating the sedimentation state of the extracellular vesicles. For functioning as an anchor, it can be preferable to use materials with high density, and it is better to use materials containing a sixth periodic element of the periodic table. Examples include gold (19.32 g/cm3) or platinum (21.45 g/cm3) in an amount of 50% by weight or more and 100% by weight or less. Among sixth periodic elements, tantalum (Ta), tungsten (W), rhenium (Re), osmium (Os), iridium (Ir), platinum (Pt) and gold (Au) can be used due to their high density exceeding 15 g/cm3. The materials for fine particles may be composed of a single element, but may be composed of a plurality of elements such as an alloy, solid solution or a compound such as an oxide.
As an indicator for visually indicating the presence of exosomes, light absorption by plasmon resonance of a noble metal such as gold or platinum can be utilized. Since gold nanoparticles exhibit a red color and nanoparticles with gold-platinum core-shell structure exhibit a black color, sedimentation of nanoparticles can be clearly confirmed visually.
It is also known that new plasmon mode appears by causing a plurality of noble metal nanoparticles having plasmon resonance to aggregate, and the light absorption spectrum, that is, known for color changes (Tatsumoto, E. et al. 2012, the Second Meeting in Heisei 24, Kansai Analysis Sciety; Cha, H. et al., 2014, ACS Nano, vol. 8, No. 8, p. 8554-8563). It is also possible to use fine particles composed of such nanoparticle aggregates. It is possible to perform detection and quantification not only by visual indicators but also by producing alloy nanoparticles with magnetic metal such as iron to perform detection and quantification with magnetism.
The fine particles may be composed of a single particle, but may be clusters composed of a plurality of particles. The fine particle refers to particle having a diameter of 1 nm to 10 μm, and a nanometer sized particle, nanoparticle, is preferably used. The size of nanoparticles in the longest axis may be 500 nm or less and 5 nm or more, preferably 200 nm or less and 20 nm or more, and most preferably 100 nm or less and 30 nm or more. Depending on the density of the materials, if the size is too large, the fine particles themselves sediment, whereas if it is too small, sedimentation by centrifugation requires a long time or a very high rotation speed.
As shown in the following Examples, embodiments of methods include: modifying nanoparticles with molecules capable of binding to molecules present on the surface of extracellular vesicles; causing the nanoparticles to bind to the molecules present on the surface of the extracellular vesicles; and by utilizing formation of aggregates of the nanoparticles and the extracellular vesicles due to binding, precipitating the formed aggregates via the low-speed centrifugation by density gradient centrifugation. The density gradient may be a continuous density gradient or a step density gradient.
The density gradient may be a step density gradient or a continuous density gradient. The step density gradient can be obtained by layering several media with different densities and then layering thereon a mixture of nanoparticles and a sample containing extracellular vesicles (
For the density of the medium, the density of the high density layer is a factor under which only the nanoparticles bound to extracellular vesicles are sedimented and the nanoparticles not bound to extracellular vesicles are not sedimented. The high density layer varies depending on the type and size of the particles to be used, but when using nanoparticles of 100 nm or less and 30 nm or more in the longest axis comprising a noble metal with a density exceeding 15 g/cm3, the high density layer have a density of 1.053 g/mL (10% in the case of iodixanol) or more and 1.32 g/mL (60% in the case of iodixanol) or less. The above-mentioned density is a density when iodixanol is mixed with pure water. When mixing iodixanol with a buffer solution such as physiological saline, the density can be about 0.5% higher than the above-mentioned value. Solutes other than iodixanol whose density can be set at the same level as iodixanol may be also used.
In the following Example, the density gradient is created by iodixanol, but it can be created by using any compound conventionally used in density gradient centrifugation, including salts such as cesium chloride or sodium bromide; sugars such as sucrose, sorbitol or glycerol; triiodobenzene-based compounds such as Metrizamide or Nycodenz; polymer-based compounds such as Ficoll; and colloidal silica-based compounds such as Percoll.
The centrifugation speed and centrifugation time also can vary depending on the density of the medium to be used, but considering that examination of the sample may be performed promptly after centrifugation, sedimentation can be performed in a centrifugation time of 30 minutes or less, preferably 10 minutes or less and more preferably 5 minutes or less. The centrifugation speed for precipitating extracellular vesicles bound to nanoparticles in a short time can be set in the range of 1 g to 2000 g, preferably 200 g to 1500 g, more preferably 500 g to 1,500 g.
The method for producing fine particles may be either a chemical method or a physical method. However, it has been reported that since the fine particles prepared by the in-liquid laser ablation method used in the following Examples contain neither a surfactant nor a reaction by-product in a colloidal solution, the surfaces of the fine particles are chemically-bare and in particular, ligand molecules are passively adsorbed the surface with high efficiency (Cederquist, K. B. et al., 2017, Colloids and Surface B: Biointerfaces, vol. 149, p. 351-357). Therefore, the in-liquid laser ablation is a particularly preferable production method in some embodiments.
The surface of the nanoparticle is modified with a ligand that specifically binds to a molecule present on the extracellular vesicle. As described above, since the nanoparticles bind to the extracellular vesicles via the ligand on the surface of the nanoparticles and sediments, the extracellular vesicles binding to the ligand can be fractionated and purified. Since the extracellular vesicles binding to particular ligands can be selectively sedimented, extracellular vesicles can be classified into subclasses by the binding to ligands.
As molecules exposed on the surface of extracellular vesicles, for example, on an exosome which is one of extracellular vesicles, various marker molecules are known such as CD9, CD13, CD31, CD44, CD63, CD81, Rab5b, MHC, α2-macrogloblin, LAMP1/2, ICAM-1, Flotilin 1, PSMA, Tetraspanin-1, SLC44A4, PROM2, CD133, CD14, LRRC26, integrin, ceramide, cholesterol, phosphatidylserine, EpCAM and sugar chains. Also, researches on exosomes may proceed and new surface markers may be found. Exosome surface markers that are newly found and surface markers other than the above mentioned surface markers can be utilized. Although examples of the surface markers for exosomes are described here, for other extracellular vesicles such as microvesicles and apoptotic bodies, molecules that specifically bind to marker molecules such as proteins, lipids and sugar chains can be used.
Molecules (ligands) that function to specifically bind to target surface markers include antibodies, proteins, peptides and nucleic acids. Examples of the antibodies specific to the surface markers for exosomes includes antibodies to CD9, CD63 and CD81, known as so-called general exosome markers, and antibodies to proteins exposed on the surfaces of the exosomes. Examples of the proteins include annexin V and Lactadherin (or milk fat globule-EGF factor 8 protein, MFGE8) which specifically bind to phosphatidylserine, and lectins which bind to sugar chains. Examples of the peptides include a peptide aptamer that binds to EpCAM, a protein present on the surface of the exosome (Yoshida, M et al. 2017, Biotechnol. Bioeng. doi: 10.1002/bit.26489). Examples of the nucleic acids include a single-stranded DNA aptamer that binds to CD63 (Jiang, Y et al., 2017, Angew. Chem. Int. Ed. vol. 56, 11916-11920).
For the binding between the fine particle and the ligand molecule, a method by chemical bonding can be utilized in addition to or as an alternative to the above-mentioned physical adsorption. For example, ligand molecules having thiol (—SH) or disulfide (—S—S—) can be chemically bound to the surface of metals such as gold. Instead of directly binding molecules that function to specifically bind to target molecules on extracellular vesicles to the surfaces of the nanoparticles, it is also possible to bind the molecules via linkers to the surfaces of the nanoparticles.
In some embodiments, it may be desirable to perform blocking of the surfaces of the nanoparticles not bound by the ligands in order to reduce or avoid nonspecific binding of nontarget molecules on the surfaces of extracellular vesicles. Examples of the blocking molecules include proteins such as BSA, surfactants such as polysorbate 80 (Tween-80) or polysorbate 20 (Tween-20), polymers such as polyvinylpyrrolidone (PVP), and mixtures thereof. Blocking may be performed after coating the nanoparticles with the ligands or before use. Blocking may be also performed using a buffer solution containing the above-mentioned blocking molecule as a solution for storing the nanoparticles.
The following Examples are intended to illustrate, but not to limit, aspects of the technology.
A colloidal solution of a laser-fabricated gold nanoparticle, i-colloid Au 40 nm (Product No. icAu 40-1-100, manufactured by IMRA America, Inc.; zeta potential <−60 mV; colloid conductivity <10 μS/cm), having an average particle diameter of approximately 40 nm and OD of 1 at its plasmon peak wavelength, was prepared. The colloidal solution of 14.1 mL was placed in a 15 mL centrifuge tube, 0.6 mL of a 0.1 M borate buffer solution (pH 8.2) is added thereto, and the mixture was stirred.
In another 15 mL centrifuge tube is placed 0.1 mL of deionized water and 0.1 mL of a 0.5 mg/mL Bovine Lactadherin (MFGE8) solution (manufactured by Haematologic Technologies, Inc.), and 4.8 mL of the above-prepared mixture of the borate buffer solution (pH 8.2) and the gold nanoparticle colloidal solution is poured thereinto. The mixture was stirred with a vortex mixer, and incubated on a shaker at room temperature at 100 rpm for 30 minutes. MFGE8 is a protein that binds to phosphatidylserine (PS) on the surfaces of exosomes.
The reaction mixture was subjected to centrifugation at 4,000 g for 30 minutes at room temperature to sediment the gold nanoparticles. The supernatant was removed, 0.3 mL of a 4 mM borate buffer solution (pH 8.2) was added to the precipitate, and the mixture was stirred with a vortex mixer to redisperse the gold nanoparticle.
The absorption spectrum of the MFGE8-modified gold nanoparticle colloidal solution thus prepared was measured with an ultraviolet-visible spectrophotometer (UV-2700, manufactured by Shimadzu Corporation) and normalized with its plasmon absorption peak wavelength, and compared with the absorption spectrum of an unmodified gold nanoparticle colloidal solution. The results are shown in
A colloidal solution of a laser-fabricated gold-platinum alloy nanoparticle, i-colloid AuPt 40 nm (Product No. icAuPt 40-1-100, manufactured by IMRA America, Inc.; zeta potential <−50 mV; colloid conductivity <10 μS/cm), having an average particle diameter of approximately 40 nm and OD of 1 at a wavelength of 400 nm is prepared. The colloidal solution of 14.1 mL was placed in a 15 mL centrifuge tube, 0.6 mL of a 0.1 M borate buffer solution (pH 8.2) was added thereto, and the mixture was stirred.
In a 1.7 mL centrifuge tube was placed 0.1 mL of a 0.5 mg/mL MFGE8 solution, and 0.9 mL of the above-prepared mixture of the borate buffer solution (pH 8.2) and the gold-platinum alloy nanoparticle colloidal solution was poured thereinto. The mixture was stirred with a vortex mixer, incubated on a shaker at 200 rpm for two hours at room temperature, and then allowed to stand at 4° C. overnight.
The reaction mixture contained in the tube was subjected to centrifugation at 4,000 g for 30 minutes at room temperature to sediment gold-platinum alloy nanoparticles. The supernatant was removed to leave a pellet on the bottom of the tube. To the tube was added 0.5 mL of a 4 mM borate buffer solution (pH 8.2), and the mixture was stirred with a vortex mixer to redisperse the gold-platinum alloy nanoparticles.
The absorption spectrum of the MFGE8-modified gold-platinum alloy nanoparticles (40 nm) thus prepared was measured in the same manner as in Example 1. The results are shown in
In addition, the particle diameter of the nanoparticle was measured by a dynamic light scattering method (Zetasizer Nano ZS-90, manufactured by Malvern Instruments Ltd.). The measured hydrodynamic diameter was 74.61 nm, which was confirmed to have increased by approximately 19 nm in size compared with the hydrodynamic diameter of 55.44 nm before MFGE8 modification.
One mL of the above-prepared MFGE8-modified gold-platinum alloy nanoparticle (40 nm) colloidal solution was subjected to centrifugation at 4,000 g for 30 minutes at room temperature, 0.9 mL of the supernatant was removed to concentrate the MFGE8-modified gold-platinum alloy nanoparticles by 10 times, and the concentrate was stored at 4° C. until use.
The gold nanoparticle colloidal solution used in Example 1 was prepared and 14.1 mL of the solution was placed in a 15 mL centrifuge tube, 0.6 mL of a 0.1 M phosphate buffer solution (pH 7.0) was added thereto, stirred with a vortex mixer, and allowed to stand.
Two hundred μL of an anti-CD81 monoclonal antibody solution (mouse monoclonal CD 81, M 38 (IgG1), manufactured by EXBIO Praha, a.s.) and 50 μL of a 0.1 M phosphate buffer solution were placed in a 1.5 mL centrifuge tube, then mixed.
In another 1.5 mL centrifuge tube was placed 20 μL of the anti-CD81 monoclonal antibody-phosphate buffer solution thus prepared, and 980 μL of the above-prepared mixture of the phosphate buffer (pH 7.0) and the gold nanoparticle colloidal solution was poured thereinto. The mixture was stirred with a vortex mixer, and incubated on a rotary shaker at room temperature for two hours.
Then, the half volume, 0.5 mL out of 1.0 mL of the above mixed solution was taken out, and placed in a new 1.5 mL centrifuge tube. 0.5 mL of 4 mM phosphate buffer solution (pH 7.0) containing 10 mg/mL of BSA was added thereto, stirred with a vortex mixer, and incubated overnight at 4° C. After incubated, the mixture was stirred again with a vortex mixer, subjected to centrifugation at 2,500 g for 15 minutes at room temperature to sediment gold nanoparticles. Approximately 0.95 mL of the supernatant was removed to leave a pellet on the bottom of the tube.
The absorption spectrum of the anti-CD81 monoclonal antibody-modified gold nanoparticle colloidal solution thus prepared was adjusted by dilution with a 4 mM phosphate buffer solution (pH 7.0) containing 5 mg/mL of BSA so that the OD is approximately 20 at the final plasmon absorption peak wavelength as measured in the same manner as in Example 1.
The absorption spectrum measured with an ultraviolet-visible spectrophotometer was normalized with the plasmon absorption peak wavelength, and compared with the absorption spectrum of an unmodified gold nanoparticle colloidal solution. The results are shown in
Using the MFGE8-modified gold nanoparticles (40 nm) prepared in Example 1 (hereinafter referred to as M40), exosomes were detected utilizing the binding between MFGE8 and phosphatidylserine on the surface of the exosomes.
An exosome sample was prepared from a culture medium of MiaPaca-2 human pancreatic cancer cell line. Crude exosomes obtained by ultracentrifugation were fractionated by density gradient centrifugation into ten fractions each having an equal volume with the fraction on the top of the tube (having the lowest density) designated as fraction 1. For proteins expressed in each fraction which was confirmed by Western blotting, the number of particles in each fraction was analyzed with nanoparticle analyzer (NanoSight LM 10, manufactured by Malvern Instruments).
Each of the above-mentioned exosome fractions 1, 2, 3, 7 and 8 was mixed with M40 in PBS buffer (10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) containing 0.01% BSA and were allowed to stand at room temperature for 30 minutes. For comparison, a sample in which PBS alone instead of exosomes and M40 were mixed, or a sample in which BSA-modified gold nanoparticles (hereinafter referred to as B40) and fraction 3 in which exosome particles having CD63 and CD81 exposed on its surface were concentrated was prepared in the same manner. Each solution was prepared so that the number of particles contained in the exosome fraction was 4.4×1010, and approximately 3.6 times more gold nanoparticles than the particles were added thereto.
Each of the solutions that were allowed to stand at room temperature for 30 minutes exhibited a red color derived from gold nanoparticles, but no noticeable color difference was observed depending on the fractions or the presence or absence of exosomes (
The mixture of the exosome of each fraction or PBS and M40, or the mixture of the exosome of fraction 3 and B40 was layered on an iodixanol density gradient solution, and subjected to low-speed centrifugation. Specifically, 20 mM HEPES (pH 7.4) solutions containing 14%, 12% and 10% iodixanol were prepared, they are layered in a glass tube (Micro tube No. 1, manufactured by Maruemu Corporation) in the following order from the bottom: 200 μL of a 14% iodixanol solution (density: 1.079 g/mL), 100 μL of a 12% iodixanol solution (density: 1.069 g/mL) and 100 μL of a 10% iodixanol solution (density: 1.058 g/mL) to prepare a density gradient solution with three density layers. The mixture of the exosomes of each fraction or PBS and M40, or the mixture of the exosomes of fraction 3 and B40 (each 40 μL) was layered on the density gradient solution, and subjected to centrifugation at 1,500 g for five minutes in a centrifuge (centrifuge: H-3R, rotor: RF-110, both manufactured by KOKUSAN Co. Ltd.) set at 4° C.
A photograph of the tube after centrifugation and a schematic view illustrating the state in which gold nanoparticles and exosomes were present are shown in
It was determined in the following procedure that the amount of phosphatidylserine exposed on the exosome surface was more in fraction 1 than in fraction 3. Using annexin V the most commonly used for the detection of phosphatidylserine on the surface of a cell membrane, phosphatidylserine on the surface of an exosome membrane was labelled with a gold particle, and was observed with an atomic force microscope (AFM).
Specifically, a drop of each of exosome fractions 1 and 3 was put on an aminated mica substrate and allowed to stand for one hour so as to adsorb the exosome on the surface of the substrate. After washing the substrate with PBS, a PBS solution containing 5% BSA was put on the substrate and allowed to stand at room temperature for five minutes so as to perform blocking, and washed once with PBS. Biotinylated annexin V was added thereto and allowed to stand at room temperature for two hours for binding and washed with PBS. Thereafter, an anti-biotin antibody conjugated with a 20 nm gold particle was added as a secondary antibody thereto and allowed to stand overnight at 4° C. After washing with PBS, it was subjected to fixation with glutaraldehyde and osmium tetroxide. It was washed three times with PBS and five times with water, allowed to stand at room temperature for drying, and observed in the air at room temperature with AFM (Asylum MFP-3D, manufactured by Oxford Instruments).
AFM images of fractions 1 and 3 are shown in
It was also confirmed by AFM observation that M40 binds to a MiaPaca-2 exosome. Instead of using annexin V and anti-biotin antibody, M40 in PBS solution containing 1% BSA was put and allowed to stand at room temperature for two hours. It was washed three times with PBS and five times with water, and was observed with AFM in the same manner as above. As shown in
Using the 40 nm alloy nanoparticle comprising platinum and gold modified with MFGE8 (hereinafter referred to as MAP40) prepared in Example 2, an exosome was detected utilizing the binding between MFGE8 and phosphatidylserine on the surface of the exosome.
Each of exosome fractions 1, 3, 4 and 6 fractionated from a culture medium of MiaPaca-2 in the same manner as above was mixed with MAP40 in PBS containing 0.1% BSA, and was allowed to stand at room temperature for 30 minutes. For comparison, a sample in which PBS alone instead of exosomes and MAP40 were mixed, or a sample in which BSA-modified alloy nanoparticles (hereinafter referred to as BAP40) and fraction 1 were mixed was prepared in the same manner. The number of particles contained in the exosome fraction was 4×1010 in each solution, and a certain amount of alloy particles (having a concentration at which the absorbance at 400 nm was 2.4) was added thereto so that the ratio of the number of alloy particles to the number of particles contained in each exosome fraction was the same.
Twenty μL of the mixture of the exosomes and MAP40 after allowed to stand was layered on a density gradient solution containing 40 μL of 10% iodixanol overlaid on 100 μL of 14% iodixanol and subjected to low-speed centrifugation. The centrifugation was performed at 600 g for five minutes in a centrifuge (centrifuge: Model 5500, rotor: ST-722M, both manufactured by KUBOTA CORPORATION) set at 20° C.
Photographs of the tube after centrifugation are shown in
The amount of precipitate varied depending on the number of exosome particles contained in the samples (
It was then confirmed by adding MFGE8 to the samples that the precipitation was derived from the specific binding of molecules on the exosome surface and nanoparticles. MFGE8 (0.56 μM) was mixed with fraction 1 and allowed to stand at room temperature for 40 minutes, and then subjected to mixing with MAP40 and centrifugation in the same manner as above. When fraction 1 and MFGE8 were mixed before mixing with MAP40, no precipitate was observed (
Exosomes were detected using gold nanoparticles prepared in Example 3 (hereinafter referred to as 81A40).
Exosome samples were prepared in the same manner as in Example 4, and each of exosome fractions 1, 3, 4 and 6 obtained by fractionation was mixed with 81A40 in PBS containing 0.1% BSA, and the mixture was allowed to stand at room temperature for two hours. For comparison, a sample in which PBS alone and 81A40 were mixed, or a sample in which gold nanoparticles modified with an isotype control antibody (hereinafter referred to as ctA40) and fraction 3 were mixed was prepared in the same manner. Each solution was prepared so that the number of particles contained in the exosome fraction was 3.3×1010, and approximately 4.9 times more gold nanoparticles than the particles were added thereto.
The mixture of the exosomes and the gold nanoparticles after allowed to stand was layered on a density gradient solution of iodixanol and subjected to low-speed centrifugation. Specifically, 20 mM HEPES (pH 7.4) solutions containing 14% and 10% iodixanol were prepared, they are layered in a 0.2 mL plastic tube (a PCR tube manufactured by Greiner Bio-One International GmbH) in the following order from the bottom: 100 μL of 14% iodixanol solution and 40 μL of 10% iodixanol solution to prepare two layers of density gradient. Twenty μL of the mixture of the exosomes of each fraction and gold nanoparticles was layered on the density gradient, and subjected to centrifugation at 1,000 g for five minutes in a centrifuge (centrifuge: Model 5500, rotor: ST-722M, both manufactured by KUBOTA CORPORATION) set at 20° C.
Photographs of the tube after centrifugation are shown in
Next, it will be shown that even when a sample is layered on a single density layer, exosomes can be detected. In the same manner as in Example 4, the exosome sample was prepared from MiaPaca-2, and the exosome fraction 3 obtained by fractionation or PBS was mixed with 81A40. Twenty μL of the mixed solution was mixed with 20 μL of 20% iodixanol (iodixanol final concentration: 10%) and allowed to stand at room temperature, and then overlaid on 160 μL of 30% iodixanol.
The centrifugation was performed at 1,000 g for five minutes in a centrifuge (centrifuge: Model 5500, rotor: ST-722M, both manufactured by KUBOTA CORPORATION) set at 20° C. As shown in
Changing the density of a medium, a study was performed for the density at which extracellular vesicles bound to nanoparticles could be fractionated. Using BSA-modified gold nanoparticles, the density of the medium in which the gold nanoparticles did not precipitate was studied. As shown in the table of
The presence of the gold nanoparticles is indicated by arrows in
In addition, the density gradient was studied. Twenty μL of ctA40 alone was layered on a density gradient solution with graded two densities obtained by layering iodixanol solutions in a tube in the following order from the tube bottom: 100 μL of 14% iodixanol and 40 μL of 10% iodixanol, or on 140 μL of 14% iodixanol alone, and subjected to centrifugation at 1,000 g for five minutes in the same manner as above (centrifuge: Model 5500, rotor: ST-722M, both manufactured by KUBOTA CORPORATION) (
These results show that, in this Example, in a single layer, the nanoparticles diffuse, thus making it more challenging to separate them, but by providing two or more layers, heavy complexes of nanoparticles and exosomes formed by their binding can be separated. Accordingly, it was concluded that at least two layers with different densities, including a sample layer, can be used for precipitating exosomes, in some embodiments.
The above results show that, when a solution containing extracellular vesicles and nanoparticles modified with a ligand that binds to a molecule on the surface of the extracellular vesicles is prepared, so that the concentration of an iodixanol solution is 10%, that is, its density is 1.053 g, and layered on a medium with the higher density, the nanoparticles bound to the extracellular vesicles precipitate but the nanoparticles not bound thereto do not precipitate, and the extracellular vesicles can be fractionated depending on their surface molecules.
If exosomes can be detected without purification, it will be very useful in clinical examinations and the like. Accordingly, a detection study was performed in the same manner using a crude exosome fraction.
Twenty μL of a sample in which crude exosomes from MiaPaca-2 concentrated by ultracentrifugation and MAP40 or BAP40 were mixed was layered on density gradient layers in the following order from the tube bottom: 100 μL of 14% iodixanol, 40 μL of 10% iodixanol, and subjected to centrifugation at 800 g for five minutes at 4° C. with a centrifuge manufactured by KOKUSAN Co. Ltd. (
Even in the case of not using any purified sample, this method can be used to separate exosomes of a particular subclass and is very useful method for examination or research.
As shown in the above Examples, a nanoparticle coated with a ligand that binds to a molecule present on the surface of exosomes can be used to precipitate the exosomes by low-speed centrifugation, for example, in the range of 500 g to 1,500 g. Since exosomes can be classified into subclasses utilizing molecules present on the surface thereof, they can be classified into subclasses and thereafter subjected to analysis of exosomes.
Example, non-limiting experimental data are included in this specification to illustrate results achievable by various embodiments of the systems and methods described herein. All ranges of data and all values within such ranges of data that are shown in the figures or described in the specification are expressly included in this disclosure. The example experiments, experimental data, tables, graphs, plots, figures, and processing and/or operating parameters (e.g., values and/or ranges) described herein are intended to be illustrative of operating conditions of the disclosed systems and methods and are not intended to limit the scope of the operating conditions for various embodiments of the methods and systems disclosed herein. Additionally, the experiments, experimental data, calculated data, tables, graphs, plots, figures, and other data disclosed herein demonstrate various regimes in which embodiments of the disclosed systems and methods may operate effectively to produce one or more desired results. Such operating regimes and desired results are not limited solely to specific values of operating parameters, conditions, or results shown, for example, in a table, graph, plot, or figure, but also include suitable ranges including or spanning these specific values. Accordingly, the values disclosed herein include the range of values between any of the values listed or shown in the tables, graphs, plots, figures, etc. Additionally, the values disclosed herein include the range of values above or below any of the values listed or shown in the tables, graphs, plots, figures, etc. as might be demonstrated by other values listed or shown in the tables, graphs, plots, figures, etc. Also, although the data disclosed herein may establish one or more effective operating ranges and/or one or more desired results for certain embodiments, it is to be understood that not every embodiment need be operable in each such operating range or need produce each such desired result. Further, other embodiments of the disclosed systems and methods may operate in other operating regimes and/or produce other results than shown and described with reference to the example experiments, experimental data, tables, graphs, plots, figures, and other data herein. Thus, the invention has been described in several non-limiting embodiments. It is to be understood that the embodiments are not mutually exclusive, and elements described in connection with one embodiment may be combined with, rearranged, or eliminated from other embodiments in suitable ways to accomplish desired design objectives. No single feature or group of features is necessary or required for each embodiment. All possible combinations and sub-combinations of elements are included within the scope of this disclosure.
For purposes of summarizing the present invention, certain aspects, advantages and novel features of the present invention are described herein. It is to be understood, however, that not necessarily all such advantages may be achieved in accordance with any particular embodiment. Thus, the present invention may be embodied or carried out in a manner that achieves one or more advantages without necessarily achieving other advantages as may be taught or suggested herein.
As used herein any reference to “one embodiment” or “some embodiments” or “an embodiment” means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment. Conditional language used herein, such as, among others, “can,” “could,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and/or steps. In addition, the articles “a” or “an” or “the” as used in this application and the appended claims are to be construed to mean “one or more” or “at least one” unless specified otherwise.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, are open-ended terms and intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), or both A and B are true (or present). As used herein, a phrase referring to “at least one of” a list of items refers to any combination of those items, including single members. As an example, “at least one of: A, B, or C” is intended to cover: A, B, C, A and B, A and C, B and C, and A, B, and C. Conjunctive language such as the phrase “at least one of X, Y and Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be at least one of X, Y or Z. Thus, such conjunctive language is not generally intended to imply that certain embodiments require at least one of X, at least one of Y, and at least one of Z to each be present.
Thus, while only certain embodiments have been specifically described herein, it will be apparent that numerous modifications may be made thereto without departing from the spirit and scope of the invention. Further, acronyms are used merely to enhance the readability of the specification and claims. It should be noted that these acronyms are not intended to lessen the generality of the terms used and they should not be construed to restrict the scope of the claims to the embodiments described therein.
