A method for evaluating exosomes by counting exosome nanoparticles

Abstract

This invention relates to the field of biomedical technology, and in particular to a method for evaluating exosomes by counting exosome nanoparticles. The invention proposes a nanoparticle counting scheme for evaluating exosomes. By using parameters related to the change in nanoparticle number caused by exosome vesicle rupture, the ratio of ruptured particles can be used to directly evaluate exosome samples from different batches prepared using the same method, or exosome samples from the same batch of exosome products treated by different methods. Furthermore, by measuring the particle count of exosome samples before and after treatment, and before and after rupture, and calculating the retention rate of treated exosomes based on the measurement results, the treatment effect can be quantitatively evaluated based on the retention rate of treated exosomes. This allows for more effective evaluation of treated exosomes, thereby enabling quality testing and quality control.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for evaluating exosomes by counting exosome nanoparticles. Background Technology

[0002] Extracellular vesicles are particles released by cells, encapsulated in a lipid bilayer, and unable to self-replicate (lacking a functional nucleus). In describing extracellular vesicle subsets, there is a class of terms based on hypothetical biogenesis, such as exosomes and ectosomes. Exosomes refer to extracellular vesicles originating from within the cell, released via multivesicular bodies (MVBs), while ectosomes refer to extracellular vesicles formed through cell membrane budding. Current isolation techniques struggle to enrich extracellular vesicles produced by different mechanisms, and characterizing the corresponding extracellular vesicle subsets is also difficult.

[0003] Exosomes, commonly known as small extracellular vesicles, are extremely small, generally less than 200 nm in diameter. They are primarily composed of lipids and proteins, and their contents contain various nucleic acids, including DNA, mRNA, microRNA, and ncRNA. Almost all cells can secrete exosomes, and they are naturally present in body fluids, including blood, saliva, urine, cerebrospinal fluid, and breast milk. Various cells and body fluids in the human body can secrete exosomes, including endothelial cells, immune cells, platelets, and smooth muscle cells. When exosomes are secreted from host cells into recipient cells, they can regulate the biological activity of the recipient cells through the proteins, nucleic acids, and lipids they carry.

[0004] Exosomes possess vital biological functions and are a hot topic in life science research, particularly in the study and application of stem cell exosomes. Exosome nanoparticle counting is the most commonly used method for evaluating exosomes, typically obtained by counting particles with diameters between 30-200 nm (or 30-150 nm). However, after cryopreservation or lyophilization, exosome particle counting often shows an increase in particle count instead of a decrease. This is because after preservation or other treatments, some exosome vesicles rupture and release their contents, increasing the count of particles in the 30-200 nm (or 30-150 nm) range and causing errors in the exosome counting. Current counting or evaluation methods cannot address the changes in exosomes before and after certain treatments (such as different storage conditions, lyophilization, or formulations), nor are they conducive to quantifying cross-sectional comparisons under different storage conditions or treatments. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for evaluating exosomes by counting exosome nanoparticles. The method provided by this invention enables more effective evaluation of processed exosomes, thereby facilitating quality detection and control.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for evaluating exosomes by counting exosome nanoparticles, comprising the following steps:

[0008] The exosome sample to be evaluated was permeated, and the number of exosomes before and after permeation was measured and denoted as N. t and N tr The particle size is 30-200 nm or 30-150 nm.

[0009] The perforated particle ratio of the exosome sample to be evaluated is calculated according to Equation I:

[0010] R t =N tr / N t Formula I;

[0011] Among them, R t For the ratio of membrane-breaking particles, N tr N represents the number of particles in the exosome sample after membrane rupture. t The number of particles in the exosome sample to be evaluated before membrane rupture;

[0012] The exosome samples to be evaluated include any one of 1) to 3): 1) exosome samples from different batches prepared by the same method, 2) exosome samples from the same batch of exosome products treated by different methods, and 3) treated exosome samples that need to be quantitatively evaluated.

[0013] When the exosome sample to be evaluated is 1), the higher the ratio of perforated particles, the higher the purity or the better the integrity of the exosomes.

[0014] When the number of exosome samples to be evaluated is 2), the larger the ratio of permeabilized particles, the lower the loss rate of exosomes after treatment, and the better the treatment effect.

[0015] When the number of exosome samples to be evaluated is 3), the following steps are also included:

[0016] The exosome sample before treatment was perforated, and the number of particles in the exosome sample before and after perforation was determined; the particle size was 30-200 nm or 30-150 nm; the exosome sample before treatment was from the same batch as the exosome sample to be evaluated before treatment.

[0017] The preservation rate of the processed exosome samples was calculated according to Equations II and III:

[0018] P=(N t-cal -N r ) / (NN r Formula II: (100%) × 100%

[0019] N t-cal =N t ×(N tr / N r Formula III;

[0020] Where P is the retention rate of the processed exosome sample, and N is... t-cal For calibrated N t Value, N t N represents the number of exosomes in the sample before membrane rupture, and N represents the number of exosomes in the sample before membrane rupture. r To process the particle count after membrane rupture in pre-exosome samples, N tr The number of particles in the exosome sample to be evaluated after membrane rupture;

[0021] The protective effect of treatment on exosomes is quantitatively evaluated based on the preservation rate of the treated exosome samples. The higher the preservation rate of treated exosomes and the lower the loss rate of treated exosomes, the better the protective effect of treatment on exosomes.

[0022] Preferably, the treatment includes one or more of the following: preservation under different conditions, freeze-drying, and purification.

[0023] Preferably, the method for determination includes exosome nanoparticle counting.

[0024] Preferably, the method for counting exosome nanoparticles is NTA nanoparticle counting.

[0025] Preferably, the reagents used for membrane perforation include tissue and / or cell lysis buffers.

[0026] Preferably, the tissue and / or cell lysis buffer is RIPA lysis buffer.

[0027] Preferably, the membrane rupture time is ≥25 min.

[0028] Preferably, when there are ≥2 treatment methods, the preservation rate of exosomes after different treatments is calculated using Formula II and Formula III respectively.

[0029] Preferably, the loss rate is 100% - P.

[0030] This invention provides the application of the method described above in exosome quality detection and / or quality control.

[0031] Beneficial effects:

[0032] This invention proposes a nanoparticle counting scheme for evaluating exosomes. By using parameters related to the change in nanoparticle number caused by exosome vesicle rupture, the scheme allows for direct evaluation of exosome samples from different batches prepared using the same method, or exosome samples from the same batch treated with different methods, through the ratio of ruptured particles. Furthermore, by measuring the particle number of exosome samples before and after treatment, as well as before and after rupture, and calculating the retention rate of treated exosomes based on the measurement results, the scheme enables quantitative evaluation of the treatment effect based on the retention rate of treated exosomes. This allows for more effective evaluation of treated exosomes, thereby facilitating quality testing and quality control. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0034] Figure 1 These are the particle size and concentration distribution results of exosome NSCE240612F1 in Example 1;

[0035] Figure 2 These are the results of the permeabilized particle size and concentration distribution of exosome NSCE240612F1 in Example 1;

[0036] Figure 3 These are the particle size and concentration distribution results of exosome NSCE240612F1DG in Example 1;

[0037] Figure 4 The results show the permeabilized particle size and concentration distribution of exosome NSCE240612F1DG in Example 1. Detailed Implementation

[0038] This invention provides a method for evaluating exosomes by counting exosome nanoparticles, comprising the following steps:

[0039] The exosome sample to be evaluated was permeated, and the number of exosomes before and after permeation was measured and denoted as N. t and N tr The particle size is 30-200 nm or 30-150 nm.

[0040] The perforated particle ratio of the exosome sample to be evaluated is calculated according to Equation I:

[0041] R t =N tr / N t Formula I;

[0042] Among them, R t For the ratio of membrane-breaking particles, Ntr N represents the number of particles in the exosome sample after membrane rupture. t The number of particles in the exosome sample to be evaluated before membrane rupture;

[0043] The exosome samples to be evaluated include any one of 1) to 3): 1) exosome samples from different batches prepared by the same method, 2) exosome samples from the same batch of exosome products treated by different methods, and 3) treated exosome samples that need to be quantitatively evaluated.

[0044] When the exosome sample to be evaluated is 1), the higher the ratio of perforated particles, the higher the purity or the better the integrity of the exosomes.

[0045] When the number of exosome samples to be evaluated is 2), the larger the ratio of permeabilized particles, the lower the loss rate of exosomes after treatment, and the better the treatment effect.

[0046] When the number of exosome samples to be evaluated is 3), the following steps are also included:

[0047] The exosome sample before treatment was perforated, and the number of particles in the exosome sample before and after perforation was determined; the particle size was 30-200 nm or 30-150 nm; the exosome sample before treatment was from the same batch as the exosome sample to be evaluated before treatment.

[0048] The preservation rate of the processed exosome samples was calculated according to Equations II and III:

[0049] P=(N t-cal -N r ) / (NN r Formula II: (100%) × 100%

[0050] N t-cal =N t ×(N tr / N r Formula III;

[0051] Where P is the retention rate of the processed exosome sample, and N is... t-cal For calibrated N t Value, N t N represents the number of exosomes in the sample before membrane rupture, and N represents the number of exosomes in the sample before membrane rupture. r To process the particle count after membrane rupture in pre-exosome samples, N tr The number of particles in the exosome sample to be evaluated after membrane rupture;

[0052] The protective effect of treatment on exosomes is quantitatively evaluated based on the preservation rate of the treated exosome samples. The higher the preservation rate of treated exosomes and the lower the loss rate of treated exosomes, the better the protective effect of treatment on exosomes.

[0053] In this invention, the number of exosome nanoparticles is N, and the average number of nanoparticles after membrane rupture treatment for each exosome is kN, where k is the membrane rupture coefficient. Generally, exosomes in the same batch have the same k value, so the number of nanoparticles after membrane rupture is the same for the same number of exosomes in the same batch.

[0054] In one implementation method, when the exosome samples to be evaluated are exosome samples from different batches prepared by the same method, the present invention performs membrane rupture treatment on the exosome samples to be evaluated, thereby destroying the membrane structure of the exosomes and releasing their contents. By counting the nanoparticles before and after membrane rupture and calculating the membrane rupture particle ratio, the number of nanoparticles after membrane rupture is the same for the same batch and the same number of exosomes. If some exosome vesicles have already ruptured before membrane rupture, and the more exosomes that rupture, the more nanoparticles are released, the smaller the membrane rupture particle ratio will be. Thus, the treatment effect of exosomes can be evaluated based on the membrane rupture coefficient.

[0055] As one implementation, the reagents used for membrane perforation include: tissue and / or cell lysis buffer.

[0056] In one embodiment, the tissue and / or cell lysis buffer is RIPA lysis buffer.

[0057] As one implementation method, the membrane rupture time is ≥25 min.

[0058] In one embodiment, the determination method can be exosome nanoparticle counting; in another embodiment, the exosome nanoparticle counting method is NTA nanoparticle counting. In one embodiment, a Nanosight NS300 nanoparticle tracking analyzer can be used to count nanoparticles and obtain the concentration of particles with exosome diameters within a specified range (30-200 nm or 30-150 nm). The total exosome count result is then calculated as nanoparticle concentration × exosome sample volume.

[0059] As one implementation method, when the exosome samples to be evaluated are exosome samples from the same batch of exosome products treated by different methods, since the exosomes in the same batch have the same membrane rupture coefficient, after different treatments, the more exosomes are lost and the more nanoparticles are released, the higher the nanoparticle count of the exosome sample before membrane rupture after treatment, and the smaller the membrane rupture particle ratio (according to Formula I, the membrane rupture particle ratio is equal to the ratio of the nanoparticle count after membrane rupture after treatment to the nanoparticle count before membrane rupture after treatment). Therefore, the exosome sample with a smaller membrane rupture particle ratio has a higher exosome loss rate after treatment, and the treatment method has a worse protective effect on exosomes. Thus, the protective effect of different treatment methods on exosomes can be evaluated based solely on the membrane rupture coefficient.

[0060] As one implementation, the loss rate = 100% - P.

[0061] As one implementation, the process includes one or more of the following: preservation under different conditions, freeze-drying, and purification.

[0062] As one implementation method, the present invention can also quantitatively evaluate the processed exosome samples, that is, quantitatively evaluate the preservation rate or loss rate of the processed exosome samples. For example, it can evaluate the effect of freezing the same exosome product at -80°C for a certain period of time. Freezing at -80°C may cause the exosome vesicles to rupture and the exosomes to be lost. In this case, simply counting the nanoparticles of the exosomes cannot evaluate the preservation effect of the exosomes at this temperature.

[0063] Calculate the preservation rate or loss rate of exosomes under this treatment condition: Let N be the number of exosome nanoparticles before treatment (cryopreservation), and N be the number of exosomes after membrane rupture. r ;

[0064] After processing (cryopreservation), the exosome nanoparticle count was N. t After exosomes rupture, they become N. tr ;

[0065] Let the preservation rate of exosomes be P. Then, according to the definition of the membrane rupture coefficient k, k is equal to the ratio of the total number of nanoparticles in the same exosome before and after membrane rupture, i.e., k = N. r / N;

[0066] After processing (cryopreservation), the total number of exosome particles preserved without rupture of vesicles is N×P;

[0067] After processing (cryopreservation), the exosomes that have ruptured from the vesicles, i.e. the lost exosomes, are N(1-P).

[0068] After processing (cryopreservation), the total number of nanoparticles in the exosomes after vesicle rupture is N(1-P)×k; therefore, after processing (cryopreservation), the total number of exosome nanoparticles N t= N×P+N(1-P)×k;

[0069] Calculations yielded: P = (N) t -N r ) / (N-kN)=(N t -N r ) / (NN r )×100%.

[0070] Generally, for exosomes of the same batch, volume, or quantity, the nanoparticle counting results should be consistent before and after membrane rupture during treatment, i.e., N... tr =N r At this point, it is not necessary to obtain N. tr However, due to the existence of errors, N may be... tr ≠N r At this point, an approximate evaluation can be performed as needed (ignoring N). tr The result can also be expressed as N. tr To calibrate N t The value of N after calibration. t The value is N t-cal Considering factors such as exosome volume error, or differences in the volume or quantity of exosomes from the same batch used in different treatments, N t-cal =N t ×(N tr / N r The formula obtained after calculation is: P = (N) t-cal -N r ) / (N-kN)=(N t-cal -N r ) / (NN r )×100%.

[0071] Therefore, the exosome loss rate is 100% - P.

[0072] The method provided by this invention is mainly applicable to evaluating the destructive effects or protective effects of different treatments (such as preservation, freeze-drying, or purification) on exosomes.

[0073] As one implementation method, when there are ≥2 treatment methods, the preservation rate of exosomes after different treatments can be calculated using Formula II and Formula III respectively, thereby quantitatively evaluating the effects of different treatments.

[0074] Based on the above advantages, the present invention provides the application of the method described in the above technical solution in exosome quality detection and / or quality control.

[0075] To further illustrate the present invention, a method for evaluating exosomes by counting exosome nanoparticles provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0076] Example 1

[0077] This invention provides a method for evaluating exosomes by counting exosome nanoparticles, comprising the following steps:

[0078] The exosomes were processed to obtain the processed exosomes;

[0079] The exosomes before and after treatment were subjected to membrane rupture operation to obtain four types of samples: exosomes before treatment, exosomes after treatment, exosomes with membrane rupture before treatment, and exosomes with membrane rupture after treatment.

[0080] The number of particles in the four samples was determined by the exosome nanoparticle counting method, and the particle size was 30-200 nm or 30-150 nm.

[0081] The preservation rate of exosomes after treatment was calculated according to Equations II and III:

[0082] P=(N t-cal -N r ) / (NN r Formula II: (100%) × 100%

[0083] N t-cal =N t ×(N tr / N r Formula III;

[0084] Where P is the preservation rate of exosomes after treatment, and N t-cal For calibrated N t Value, N t N represents the number of particles in the exosome sample after treatment, and N represents the number of particles in the exosome sample before treatment. r To process the particle count of pre-exosome rupture samples, N tr This represents the particle count in the exosome perforation sample after processing.

[0085] The effectiveness of treatment is evaluated based on the preservation rate of exosomes after treatment. The higher the preservation rate of exosomes after treatment, the lower the loss rate of exosomes after treatment, and the better the protective effect of treatment on exosomes.

[0086] Example 2

[0087] 1. Preparation of exosome concentrate: Collect 10L of human neural stem cell culture supernatant (i.e., neural stem cell conditioned medium, provided by Shanghai Anji Xiekang Biotechnology Co., Ltd.), and use 3D... The vivaEXO exosome harvesting system (manufacturer: Huakan Biotechnology) is used to concentrate and purify exosome concentrate, which is then stored at -80℃.

[0088] 2. Preparation of exosome lyophilized product NSCE240612F1DG:

[0089] Prepare exosome concentrate according to step 1, add lyophilization protectant (PBS solution containing 3% trehalose and 12% mannitol to a final concentration), mix well, and dispense into vials, 2 ml / vial. Take one vial labeled NSCE240612F1 for sampling and testing (store the rest at -80℃); freeze-dry the remaining vials using a vacuum freeze dryer (Boyikang Pilot5-8ES), cap and remove the vials after freeze-drying, label them NSCE240612F1DG, and send them for testing. Store the product at 2-8℃.

[0090] 3. Exosome membrane rupture treatment

[0091] 1) Reagents used: High-efficiency RIPA tissue / cell lysis buffer, with one tube of PMSF (0.3mL / 1.5mL), brand: Solarbio, catalog number: R0010.

[0092] 2) Reagent preparation: According to the amount to be used, add 10 μL PMSF to each 1 mL RIPA to make the final concentration of PMSF 1 mM, mix well and set aside.

[0093] 3) Membrane breaking and sample delivery:

[0094] Take NSCE240612F1 prepared in step 2, and take 2 vials at 100 μL / vial, and process them as follows:

[0095] Take one vial and add PBS buffer (NSCE240612F1 sample) at a 1:1 volume ratio. Take another vial and add 100 μL of the prepared high-efficiency RIPA tissue / cell lysis buffer at a 1:1 volume ratio. Mix well and lyse on ice for 25 min (NSCE240612F1 lysed sample). Send the samples to Heyuan Biotechnology (Shanghai) Co., Ltd. for NTA nanoparticle counting.

[0096] Take the exosome lyophilized powder NSCE240612F1DG prepared in step 2, reconstitute it with 2 mL of sterile water for injection, and mix thoroughly to obtain an exosome lyophilized powder solution. Take two vials (100 μL / vial) and process them as follows:

[0097] Take one vial and add PBS buffer (NSCE240612F1DG sample) at a 1:1 volume ratio. Take another vial and add 100 μL of the prepared high-efficiency RIPA tissue / cell lysis buffer at a 1:1 volume ratio. Mix well and lyse on ice for 25 min (NSCE240612F1DG ruptured sample). Send the samples to Heyuan Biotechnology (Shanghai) Co., Ltd. for NTA nanoparticle counting.

[0098] The results are shown in Table 1 and... Figures 1-4 .

[0099] Table 1. NTA nanoparticle counting results for different samples.

[0100] batch number Particle size, percentage concentration NSCE240612F1 30-200nm, 94.5% <![CDATA[4.36×10 8 particles / mL]]> NSCE240612F1 membrane breakage 30-200nm, 98.7% <![CDATA[7.73×10 9 particles / mL]]> NSCE240612F1DG 30-200nm, 90.1% <![CDATA[8.25×10 8 particles / mL]]> NSCE240612F1DG membrane breakage 30-200nm, 91.5% <![CDATA[6.11×10 9 particles / mL]]>

[0101] Calculated according to the method in Example 1, the calibrated N of exosomes after lyophilization... t The value is N t-cal :

[0102] N t-cal =N t ×(N tr / N r ) = 6.52 × 10 8 ;

[0103] Exosome preservation rate P = (N t-cal -N r ) / (NN r ) × 100% = 97%;

[0104] Exosome loss rate 100% - P = 3%.

[0105] In summary, the method provided by this invention can more effectively process exosome evaluation, thereby enabling quality detection and quality control.

[0106] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for evaluating exosomes by counting exosome nanoparticles, characterized in that, Includes the following steps: The exosome sample to be evaluated was permeated, and the number of exosomes before and after permeation was measured and denoted as N. t and N tr ; Exosome samples were perforated before treatment, and the number of exosomes before and after perforation was measured; these numbers were denoted as N and N, respectively. r ; The particle size is 30-200 nm; The exosome samples to be evaluated are processed exosome samples that require quantitative assessment; The exosome sample before treatment is an exosome sample from the same batch as the exosome sample to be evaluated before treatment; The perforated particle ratio of the exosome sample to be evaluated is calculated according to Equation I: R t =N tr / N t Formula I; Among them, R t For the ratio of membrane-breaking particles, N tr N represents the number of particles in the exosome sample after membrane rupture. t The number of particles in the exosome sample to be evaluated before membrane rupture; The preservation rate of the processed exosome samples was calculated according to Equations II and III: P=(N t-cal -N r ) / (NN r Formula II (100%) N t-cal =N t ×R t Formula III; Where P is the retention rate of the processed exosome sample, and N is... t-cal For calibrated N t The value, N, represents the number of particles in the exosome sample before membrane rupture. r To process the particle count after membrane rupture in pre-exosome samples, R t The ratio of membrane-breaking particles; The protective effect of treatment on exosomes is quantitatively evaluated based on the preservation rate of the treated exosome samples. The higher the preservation rate of treated exosomes and the lower the loss rate of treated exosomes, the better the protective effect of treatment on exosomes. The reagent used for membrane lysis was RIPA lysis buffer; the volume ratio of the exosome sample to RIPA lysis buffer was 1:

1.

2. The method according to claim 1, characterized in that, The processing includes one or more of the following: preservation under different conditions, freeze-drying, and purification.

3. The method according to claim 1, characterized in that, The method used for the determination includes exosome nanoparticle counting.

4. The method according to claim 1 or 3, characterized in that, The method for counting exosome nanoparticles is NTA nanoparticle counting.

5. The method according to claim 1, characterized in that, The membrane rupture time is ≥25 min.

6. The method according to claim 1, characterized in that, When there are ≥2 treatment methods, the preservation rate of exosomes after different treatments is calculated using Equation II and Equation III respectively.

7. The method according to claim 1, characterized in that, The loss rate = 100% - P.

8. The method according to claim 1, characterized in that, The particle size is 30-150 nm.

9. The application of the method according to any one of claims 1 to 8 in the quality control of exosomes.

10. The application according to claim 9, characterized in that, The quality control includes quality inspection.

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