RU2741776C1 - Method for plasma exosome release - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology and diagnostic medicine, namely to a method for extracting exosomes from blood plasma. Method for extracting exosomes from blood plasma involves taking peripheral blood, separating plasma from cell mass by centrifugation, adding to plasma mixture of dextran polymers with molecular weight of 450–650 kDa and polyethylene glycol with molecular weight of 20 kDa is dissolved in one volume of blood plasma to concentration of 1.5 % and -3.5 % respectively followed by incubation for 1 hour at temperature +4 °C and centrifugation at acceleration 1000 G, +4 °C for 10 minutes, obtained precipitate is dissolved in polymer mixture of dextran and polyethylene glycol in phosphate-salt buffer in the same concentration, repeatedly centrifuged in the same mode until the end product is obtained.

EFFECT: method described above enables to extract directly from blood plasma maximum number of exosomes with minimal contamination of the preparation with plasma proteins and lipoproteins, is easy to implement and has low cost, which increases efficiency of subsequent analysis of exosomal fraction of circulating micro-RNA molecules, for example, for diagnosis or monitoring of oncological diseases.

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Description

The invention relates to the field of biotechnology and diagnostic medicine, namely to oncology and can be used to isolate exosomes from blood plasma.

Exosomes are membrane nano-vesicles (80-130 nm), which are secreted by almost all cells of multicellular organisms into the extracellular space. The biochemical composition of exosomes is close to the composition of the cells that secrete them. Exosomes secreted by tumor cells also have peculiarities of biochemical composition, in particular, the composition of short regulatory molecules RNA - 2 microRNA. Based on these data, it is suggested that the analysis of microRNAs of circulating exosomes may underlie methods of screening or early diagnosis of cancer [Gorji-Bahri G, Hashemi A, Moghimi HR: ExomiRs: A Novel Strategy in Cancer Diagnosis and Therapy. Current gene therapy 2018, 18 (6): 336-350.].

The development of such methods is actively carried out by many scientific laboratories by biotechnological companies, but the introduction of such developments into practice is hampered by the lack of a reliable technology for the isolation of exosomes from plasma. This task is not trivial due to the complex composition of the plasma, the various components of which have dimensions, density, surface charge, hydrophilicity, and other characteristics similar to exosomal ones. If a subsequent analysis of the exosomal fraction of circulating microRNAs is necessary, two factors are of critical importance: 1. the efficiency of exosome isolation; 2. the possibility of separating exosomes from the fraction of proteins and plasma lipoproteins that bind diagnostically insignificant microRNA molecules.

A known method for the isolation of exosomes from blood plasma using super-hydrophilic polymers, in which the addition to serum, previously purified from large vesicles and cellular debris, a concentrated (30-60%) solution of a hydrophilic polymer of polyethylene glycol (PEG) relatively low mol. mass (5-10 kDa) "displaces" less soluble components from solution, including exosomes, causing them to aggregate. The resulting aggregates are precipitated by low-speed centrifugation. This principle is implemented in a number of commercial kits for the isolation of exosomes from various biological media, including plasma: ExoQuick (System Bioscience), ExoPrep (HansaBioMed), Total exosomes isolation kit (Invitrogen).

The main disadvantages of this approach are the high cost of commercial reagents and the insufficient "purity" of the isolation - the precipitate usually contains plasma proteins and other components that complicate the subsequent analysis of exosomal microRNAs.

There is a modification of this technique that optimizes the procedure and reduces the cost of isolation (patent RU 2678988, publ. 02/05/2019), but does not solve the problem of contamination of the exosome preparation with plasma proteins.

A known method for the isolation of exosomes from blood plasma, based on the phenomenon of separation or formation of two separate phases in a solution of two polymers, for example, polyethylene glycol (PEG) and dextran. Due to the physicochemical features of the interaction of polymers with the vesicle membrane, the latter are predominantly concentrated in one of the phases - the dextran solution, while the proteins pass into the PEG solution. For a more complete purification of vesicles from proteins, the PEG-containing phase can be changed several times. In original studies (Shin H, Han C, Labuz JM, Kim J, Kim J, Cho S, Gho YS, Takayama S, Park J: High-yield isolation of extracellular vesicles using aqueous two-phase system. Scientific reports 2015, 5 : 13103.) It was proposed to use a system of polymers PEG (35 kDa) and dextran (450-650 kDa), which form two phases of an aqueous solution. On a model mixture of exosomes and proteins, the authors of the method proved the efficiency of separation of the mixture components. This method can be considered as the closest analogue.

The disadvantage of this method is that it is not optimized for working with plasma, which has a complex composition, and does not effectively separate exosomes and proteins in its composition. In addition, the original implementation of the method is too laborious for use in laboratory or clinical practice.

The technical result of the invention is the isolation directly from blood plasma of the maximum number of exosomes with minimal contamination of the drug with plasma proteins and lipoproteins, ease of implementation and low cost, increased efficiency of subsequent analysis of the exosomal fraction of circulating microRNA molecules in the context of, for example, solving problems of diagnostics or monitoring of oncological diseases.

The specified technical result is achieved in a method for isolating exosomes from blood plasma, including taking peripheral blood, separating plasma from the cell mass by centrifugation, adding a mixture of dextran and polyethylene glycol polymers to the plasma, incubating the polymer mixture and precipitating the target product by centrifugation, in which the polymers dextran and polyethylene glycol are dissolved in blood plasma to a concentration of 1.5% and 3.5%, respectively, followed by incubation for 1 hour at a temperature of + 4 ° C and centrifugation at an acceleration of 1000 G, + 4 ° C for 10 minutes, the resulting precipitate is dissolved in a polymer mixture dextran and polyethylene glycol phosphate-buffered saline in the same concentration, re-centrifuged in the same mode to obtain the desired product.

It is advisable to use the obtained target product for the isolation of exosomal microRNA with subsequent RT-PCR analysis.

The optimal ratio of molecular weights and concentrations of polymers, determined experimentally, as well as an original technique for dissolving polymers directly in plasma, make it possible to achieve the maximum number of exosomes with minimal drug contamination with plasma proteins and lipoproteins.

Obtaining the target product directly from blood plasma simplifies the method and reduces economic costs.

The method is illustrated by Figures 1-4, which presents the characteristics of the isolated vesicles:

fig. 1 - laser correlation spectroscopy of the sediment, dilution in 500 μl of PBS; fig. 2 - nano-track analysis of the sediment, dilution in 2000 μl of PBS; fig. 3 - atomic force microscopy; fig. 4 - dot blot with antibodies against CD63, where: 1 - sediment diluted in 500 μl of PBS; 2 - sediment, diluted in 200 μl of RIPA buffer, 3 - supernatant after the first centrifugation; 4 - initial plasma.

Estimation of the size and concentration of vesicles isolated from plasma of healthy donors was carried out using laser correlation spectroscopy (see Fig. 1) and nano-track analysis (see Fig. 2).

Evaluation of the exosomal nature of the isolated vesicles and analysis of the content of exosomes in the supernatant ("wash" PEG solution) were carried out using dot blotting (see Fig. 3,) and atomic force microscopy (see Fig. 4).

The method is carried out, for example, as follows.

Collect peripheral blood in vacutainers with EDTA and within 5-10 minutes the plasma is separated from the cell mass by centrifugation at 300 G for 15 minutes. In 2 centrifuge tubes with a volume of 2 ml add 0.0225 g of dextran M = 450-650 kDa and 0.0525 g of polyethylene glycol M = 20 kDa. Plasma (1.5 ml) is added to one of the tubes, and phosphate-buffered saline (1.5 ml) is added to the other. The indicated amounts of dextran and PEG provide a final polymer concentration of 1.5% and 3.5%, respectively. The incubation is carried out for one hour with vigorous stirring (vortex) at a temperature of + 4 ° C. Dissolution efficiency is monitored visually. Separation of the polymer mixture in a test tube with plasma is carried out by centrifugation at 1000 G for 10 minutes at a temperature of + 4 ° C. The supernatant is removed, 1.4 ml of the contents of the tube with a solution of PEG and dextran in phosphate-buffered saline are added to the sediment, the sediment is stirred, the mixture is centrifuged under the same conditions (1000 G for 10 minutes, at + 4 ° C), the supernatant is removed ... The sediment is a mixture of exosomes with dextran with a minimum content of plasma proteins. The pellet can be resuspended in phosphate buffered saline to analyze the physical characteristics of exosomes, or lysed in an appropriate lysis buffer for protein or microRNA analysis.

In order to evaluate the claimed technical result of the invention, a direct comparison of the efficiency of RT-PCR analysis of several microRNA molecules obtained from exosome preparations isolated by the claimed method and the analogs described above from 1.5 ml of donor plasma (formula n. 2) was carried out.

Example. Isolation of plasma exosomes and RT-PCR analysis of exosomal microRNAs. Venous blood samples were obtained from five healthy donors in EDTA vacutainers. Immediately after receiving the material, blood samples were centrifuged at 300 G for 15 minutes at 4 ° C. Plasma was carefully separated and transferred to new tubes. To isolate exosomes by the claimed method, 1.5 ml of plasma was transferred into a test tube containing 0.0225 g of dextran (molecular weight 450-650 kDa) and 0.0525 g of PEG (molecular weight 20 kDa). In parallel, the same amounts of polymers were dissolved in 1.5 ml of phosphate-buffered saline. The dissolution was carried out for an hour, with vigorous stirring (vortex), at a temperature of + 4 ° C. After complete dissolution of the polymers, the contents of the first tube were centrifuged at 1000 G, 10 minutes, at + 4 ° C. The supernatant was removed, 1.4 ml of the contents of the second tube (a solution of PEG and dextran in phosphate buffered saline) was added to the sediment. The precipitate was resuspended, the mixture was centrifuged at 1000 G, 10 minutes, at + 4 ° C. After removing the supernatant, the pellet containing exosomes was dissolved in lysis buffer (Lira reagent, Biolabmix / Novosibirsk). Next, miRNA was isolated using columns and a reagent as part of an isolation kit (LRU-100-50, Biolabmix / Novosibirsk) according to the manufacturer's protocol. In parallel, exosomes were isolated from similar plasma samples (1.5 ml) using commercial kits (closest analogues), the closest in principle to the claimed method: ExoPrep (Hansa Bio Med) and Tota lexosomes isolation kit (Invitrogen). Isolation of exosomes was carried out in accordance with the manufacturer's protocols, microRNA was isolated in the same way using the Lira reagent and an isolation kit (LRU-100-50, Biolabmix / Novosibirsk). In all three cases, RNA was eluted with 50 μL of buffer (EB); for subsequent analysis of microRNA, 2 μL of solution was used. Using RT-PCR, the relative concentration of three microRNA molecules (miR-29b-1, miR-451a, and miR-375) was analyzed using the previously described “two-tailed reverse transcription” technology [19, 20]. All reactions were carried out in volumes of 20 μl, standard kits from Biolabmix were used: OT-M-MuLV-RH for reverse transcription and HS-qPCRSYBR Blue (2 ×) for PCR.

The sequences of the primers for RT and PCR are presented in table. one.

The specificity of the reaction was assessed by the character of the melting curve of the final HSC product. The results are presented in table. 2.

Thus, the claimed method allows to isolate directly from blood plasma the maximum number of exosomes with minimal drug contamination with plasma proteins and lipoproteins, is easy to implement and has a low cost. The implementation of the method increases the efficiency of the subsequent analysis of the exosomal fraction of circulating microRNA molecules, for example, for the diagnosis or monitoring of oncological diseases.

Claims (2)

1. A method for isolating exosomes from blood plasma, including taking peripheral blood, separating plasma from the cell mass by centrifugation, adding a mixture of dextran and polyethylene glycol polymers to the plasma, incubating the polymer mixture and precipitating the target product by centrifugation, characterized in that dextran polymers with a molecular weight of 450 -650 kDa and polyethylene glycol with a molecular weight of 20 kDa are dissolved in one volume of blood plasma to a concentration of 1.5% and -3.5%, respectively, followed by incubation for 1 hour at a temperature of + 4 ° C and centrifugation at an acceleration of 1000 G, + 4 ° C for 10 minutes, the resulting precipitate is dissolved in a polymer mixture of dextran and polyethylene glycol in phosphate-buffered saline at the same concentration, re-centrifuged in the same mode to obtain the target product. 2. The method according to claim 1, characterized in that the obtained target product is lysed to isolate exosomal microRNA, followed by RT-PCR analysis.

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