Application of milk-derived exosome in preparation of medicine for treating malaria diseases

Abstract

The invention discloses a plasmodium falciparum proliferation inhibitor based on raw milk or pasteurized milk source exosomes (RM-EVs / CM-EVs), and the IC50 value of the plasmodium falciparum proliferation inhibitor is as low as 22.91 + / -2.7 mu g / mL and is obviously superior to that of ultrahigh-temperature sterilized milk and milk powder source EVs. The invention provides an extraction method of the EVs and application of the EVs in antimalarial drugs and functional foods, and provides a new natural and efficient choice for malaria prevention and treatment.

Description

Technical Field

[0001] This specification relates to the field of biomedicine, and particularly to the use of milk-derived exosomes in the preparation of drugs for treating malaria diseases. Background Art

[0002] Malaria is an acute infectious disease caused by Plasmodium and transmitted by mosquito bites, and it is also one of the biggest global public health problems. Plasmodium falciparum is the most lethal malaria parasite and is the most prevalent on the African continent. Traditional antimalarial drugs (such as artemisinin and its derivatives) face problems such as enhanced drug resistance and high costs, and there is an urgent need to develop new antimalarial strategies.

[0003] Exosomes are extracellular vesicles with a diameter of 30 - 150 nm, which are widely involved in intercellular communication, disease diagnosis, and drug delivery carriers for targeted disease treatment. Research has shown that milk-derived exosomes relieve intestinal diseases through multiple mechanisms such as regulating intestinal immune homeostasis, restoring the intestinal flora composition, and improving intestinal structure and integrity, but there are no reports on the antimalarial effect of milk-derived exosomes. Summary of the Invention

[0004] This application first discovers that raw milk-derived exosomes (RM-EVs) and pasteurized milk-derived exosomes (CM-EVs) have significant inhibitory activity against the proliferation of Plasmodium falciparum, while UHT milk-derived exosomes (UHT-EVs) and milk powder-derived exosomes (WMP-EVs) have no activity. This application provides a new source for the development of antimalarial drugs.

[0005] This application provides the use of milk-derived exosomes in the preparation of drugs for treating malaria diseases.

[0006] This application also provides a pharmaceutical composition, which contains the above-mentioned exosomes.

[0007] The beneficial effects brought by this application include but are not limited to: (1) High efficiency: The IC 50 values of CM-EVs and RM-EVs against the proliferation of Plasmodium falciparum are 22.9 ± 2.5 μg / mL and 63.7 ± 2.5 μg / mL, which are significantly better than those of EVs from ultra-high temperature sterilized milk and milk powder (IC 50 > 400 μg / mL). (2) Safety: Milk-derived EVs are natural components with high biocompatibility and low toxic and side effects. (3) Innovation: For the first time, it reveals the antimalarial activity of milk-derived EVs, providing a new strategy for malaria prevention and treatment. Brief Description of the Drawings

[0008] This application will be further described in the form of exemplary embodiments, and these exemplary embodiments will be described in detail through the drawings. These embodiments are not restrictive, where:

[0009] Figure 1 Analysis of the nanoparticle size and concentration of exosomes from different dairy sources as shown in some embodiments of the present application.

[0010] Figure 2 Analysis of the proportion of the exosome marker proteins CD9 and CD63 from different dairy sources as shown in some embodiments of the present application.

[0011] Figure 3 Observation of the morphology of exosomes from different dairy sources under electron microscopy as shown in some embodiments of the present application.

[0012] Figure 4 Determination of the anti - malaria activity of exosomes from different dairy sources as shown in some embodiments of the present application. Detailed implementation manners

[0013] To more clearly illustrate the technical solutions of the embodiments of this specification, the following will briefly introduce the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are only some examples or embodiments of this specification. For those of ordinary skill in the art, without creative efforts, this specification can also be applied to other similar scenarios based on these drawings. Unless obvious from the language context or otherwise stated, the same reference numerals in the figures represent the same structure or operation.

[0014] As shown in this specification and the claims, unless the context clearly indicates an exceptional situation, words such as "a", "an", "one", and / or "the" are not specifically singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of the clearly identified steps and elements, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0015] Flowcharts are used in this specification to illustrate the operations performed by the systems according to the embodiments of this specification. It should be understood that the operations before or after do not necessarily need to be executed precisely in sequence. On the contrary, they can be executed in reverse order or simultaneously. At the same time, other operations can also be added to these processes, or one or several operations can be removed from these processes.

[0016] The present application provides the use of milk - derived exosomes in the preparation of drugs for treating malaria.

[0017] In some embodiments, the exosomes can inhibit the proliferation of Plasmodium falciparum.

[0018] In some embodiments, the exosomes can be from raw cow milk or pasteurized cow milk.

[0019] In some embodiments, the pasteurization conditions for the pasteurized milk can be heat treatment at 65 - 75°C for 10 - 20 seconds.

[0020] In some embodiments, the pasteurization conditions for the pasteurized milk can be heat treatment at 66 - 74°C for 11 - 19 seconds.

[0021] In some embodiments, the pasteurization conditions for the pasteurized milk can be heat treatment at 67 - 73°C for 12 - 18 seconds.

[0022] In some embodiments, the pasteurization conditions for the pasteurized milk can be heat treatment at 68 - 72°C for 13 - 17 seconds.

[0023] In some embodiments, the pasteurization conditions for the pasteurized milk can be heat treatment at 69 - 71°C for 14 - 16 seconds.

[0024] In some embodiments, the pasteurization conditions for the pasteurized milk can be heat treatment at 70 - 71°C for 15 - 16 seconds.

[0025] In some embodiments, preferably, the pasteurization conditions for the pasteurized milk can be heat treatment at 72°C for 15 seconds.

[0026] In some embodiments, the exosomes can be obtained by extraction through differential centrifugation combined with ultrafiltration centrifugation.

[0027] In some embodiments, the particle size of the exosomes can be 60 - 65 nm.

[0028] In some embodiments, the concentration of the exosomes can be greater than 1.0*10 12 / mL.

[0029] The present application also provides a pharmaceutical composition, and the pharmaceutical composition contains the above-mentioned exosomes.

[0030] In some embodiments, the pharmaceutical composition further contains a pharmaceutically acceptable carrier or excipient.

[0031] As used generally herein, "pharmaceutically acceptable" means such compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of reasonable medical judgment, for contact with the tissues, organs, and / or body fluids of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0032] The excipients include various excipients and diluents, which are not essential active ingredients and have no excessive toxicity after administration. The excipients include sterile water or physiological saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.) or binders. The excipients also include other low-molecular-weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol or sorbitol. The excipients for aqueous solutions for injection are selected from physiological saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sorbitol isotonic solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG) and / or nonionic surfactants (Tween 80 or HCO-50).

[0033] In the pharmaceutical composition, the exosomes can be a single active ingredient or can be combined with one or more other active components useful for the treatment of diseases to form a combined preparation.

[0034] The content of the active component in the pharmaceutical composition is a safe and effective amount, and the safe and effective amount should be adjustable for those skilled in the art. For example, the dosage of the exosomes and the active ingredients of the pharmaceutical composition depends on the patient's body weight, the type of application, the condition and severity of the disease.

[0035] In some embodiments, the administration mode of the pharmaceutical composition can be selected from any one of oral administration, intraperitoneal injection, intravenous injection, subcutaneous injection or intramuscular injection.

[0036] In the following examples, the experimental methods are conventional methods unless otherwise specified. The test materials used in the following examples are obtained from regular biochemical reagent companies unless otherwise specified. In the following examples, the quantitative tests are all set up with three repeated experiments, and the results are averaged.

[0037] Example 1 Preparation of milk-derived exosomes

[0038] 1. Material preparation

[0039] (1) Experimental products: Raw milk is heat-treated at 72°C ± 2°C for 15 seconds and then stored in a low-temperature refrigerator at 4°C for standby, ensuring that its quality meets relevant standards (raw milk should meet the requirements of GB 19301, being a milky white or slightly yellow homogeneous liquid, without clots, precipitation, and visible foreign objects under normal vision, with a protein content of not less than 2.8 g / 100 g; fat content of not less than 3.1 g / 100 g).

[0040] 2. Pretreatment of Dairy Product Samples and Preparation of Exosomes

[0041] (1) Homogenization treatment: According to the sample formula table, by calculation, samples with the same protein amount are taken for standby.

[0042] (2) Low-speed centrifugation to remove milk fat and impurities: The samples in step (1) are centrifuged at a low speed. Centrifuge at 3000g for 30 minutes to remove large particle impurities and milk fat, and select the upper layer of whey after low-speed centrifugation for the next step of treatment.

[0043] (3) Coagulating casein with chymotrypsin: Take the supernatant in step (2), add chymotrypsin (12.5 mg / mL) at a ratio of 1:100 (w / w) of the whey protein mass. After thorough mixing, incubate at 36.5 °C for 50 minutes to agglutinate the milk protein.

[0044] (4) High-speed centrifugation to obtain whey: The dairy products treated with chymotrypsin are subjected to low-temperature high-speed centrifugation. Centrifuge at 4 °C, 14000 rpm (20817g) for 35 minutes, and take the supernatant after low-temperature high-speed centrifugation.

[0045] (5) Filtering with two layers of micron-sized filter membranes to obtain impurity-free and sterile supernatant: The supernatant obtained in step (4) is filtered successively with 0.45 μm and 0.22 μm filters and then reserved for use.

[0046] (6) Ultra-high-speed centrifugation to obtain a crude extract of bovine milk exosomes: The liquid filtered in step (5) is subjected to low-temperature and ultra-high-speed centrifugation. Centrifuge at 4 °C, 40000 rpm (117524g) for 80 minutes, discard the upper layer of liquid, and the lower layer of precipitate obtained is the preliminarily separated bovine milk exosomes.

[0047] (7) Exosome purification I: Add 26 mL of 1×PBS pre-cooled at 4 °C to the precipitate obtained in step (6), resuspend the precipitate, and centrifuge again at 4 °C, 40000 rpm (117524g) for 60 minutes, and take the precipitate after centrifugation.

[0048] (8) Exosome purification II: Resuspend the precipitate obtained in (7) in 1 mL of pre-cooled PBS, purify it through a 100Kda ultrafiltration tube, centrifuge at 4 °C, 5000 rpm (2655g) for 40 minutes, discard the lower layer of filtered liquid, and aspirate the upper layer of retentate to obtain the purified bovine milk exosomes.

[0049] (9) The exosomes obtained in step (8) are used immediately or stored in a -80 °C ultra-low temperature refrigerator.

[0050] Comparative Example 1

[0051] Raw milk was not sterilized, and the remaining steps were the same as in Example 1.

[0052] Comparative Example 2

[0053] Raw cow milk was treated by ultra-high temperature treatment (UHT, 135°C / 4 s), and the remaining steps were the same as those in Example 1.

[0054] Comparative Example 3

[0055] The milk was reconstituted milk of whole milk powder (spray drying (180°C, 0.2 s)), and the remaining steps were the same as those in Example 1.

[0056] Example 2 - Physicochemical Characterization of Exosomes

[0057] The exosomes extracted in Example 1 were identified and characterized by means of exosome flow cytometry particle size analysis and transmission electron microscopy (TEM) to confirm their particle size distribution, protein concentration, and the presence of specific marker proteins, ensuring that the extracted exosomes meet the quality requirements.

[0058] 1. Exosome Flow Cytometry Particle Size Analysis

[0059] (1) Dilute the sample: Dilute the exosome-PBS mixture to the working concentration (about 2000-fold to 10000-fold).

[0060] (2) Real-time detection: Use flow cytometry to detect the number of EV particles to select the most appropriate dilution concentration (average flow rate less than 10,000 particles / s). At the appropriate dilution factor, perform exosome determination with the same number of particles or the same volume as the termination condition.

[0061] (3) Marker protein analysis: After selecting the appropriate dilution factor, add 1 μg of CD9 antibody and CD63 antibody to the sample and mix well. Incubate in the dark at room temperature for 15 min, and then use flow cytometry to detect the molecular expression.

[0062] 2. BCA Protein Concentration Determination

[0063] (1) According to the instructions, use buffer to serially dilute bovine serum albumin (BSA) standards to prepare protein standards of 25 μg / mL - 2000 μg / mL.

[0064] (2) In the 96-well microplate protocol, 200 μL of working solution is required for each sample. Therefore, first prepare the BCA working solution, and calculate the total volume of the working solution required according to the following formula. Formula: (Number of standards + Number of protein samples to be measured) X (Number of experimental repetitions) X (Volume of working solution for each sample). Mix 50 parts of BCA reagent A with 1 part of BCA reagent B (reagent A:reagent B ratio = 50:1) to prepare the working solution.

[0065] (3) Sample addition: Add 10 μL of the sample to both the standard well and the well to be tested. Use a multichannel pipette to add 200 μL of the working solution to each well, and shake for 30 seconds to mix thoroughly. Seal the 96-well microplate and incubate at 37 °C for 30 minutes.

[0066] (4) After the 96-well microplate has cooled to room temperature, use a microplate reader to measure the absorbance at 562 nm.

[0067] (5) Plot the standard curve and calculate the concentration of the protein sample. Specifically: First, subtract the average absorbance value of the blank standard at 562 nm from the absorbance values of each standard and the protein sample to be tested at 562 nm; Second, plot the average absorbance value of the BSA standard at 562 nm after blank correction against its concentration (μg / mL) to draw the standard curve; Third, use this standard curve to determine the protein concentration of each protein sample to be tested.

[0068] 3. Observation and identification by transmission electron microscopy (TEM)

[0069] (1) Take 10 μL of the exosome + PBS mixture separated in the aforementioned examples and comparative examples, drop it onto a 100-mesh copper grid, let it stand for 1 min, and use filter paper to suck away the floating liquid;

[0070] (2) On the copper grid in step (1) above, add 10 μL of phosphotungstic acid again, let it stand for 1 min, and use filter paper to suck away the floating liquid;

[0071] (3) Dry at room temperature for 10 min;

[0072] (4) Select a voltage of 100 kV for transmission electron microscopy imaging detection and take pictures for preservation;

[0073] Example 3: Antimalarial activity test

[0074] 1. Synchronize the Plasmodium falciparum 3D7 strain to the ring stage and inoculate it into a 96-well plate (2% red blood cells, 0.5% hematocrit, final volume 200 μL).

[0075] 2. Add different concentrations of EVs (quantified by BCA, 0 - 100 μg / mL). The final concentrations of exosomes in the 96-well plate are 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5628 μg / mL, 0.78125 μg / mL respectively. There are at least 3 replicates for each concentration. Use PBS as the negative control. After culturing for 48 hours, detect the Plasmodium DNA content by fluorescence labeling method (such as SYBR Green I).

[0076] 3. Calculate the inhibition rate and IC 50 value: The IC 50They were 22.9±2.5 μg / mL and 63.7±2.5 μg / mL respectively. The exosomes from Comparative Example 2 and Comparative Example 3 had no significant activity (IC 50 >400 μg / mL).

[0077] Experimental results

[0078] (I) Physiological and biochemical characteristics of exosomes from milk sources

[0079] 1. NanoFCM showed that the hydrated particle size of exosomes from different bovine dairy products was about 60 - 65 nm, and the number of particles exceeded 10 11 / mL. Among them, the exosome concentrations of Example 1 and Comparative Example 1 were significantly higher than those of the exosomes from Comparative Example 2 and Comparative Example 3 ( Figure 1 ). The proportion of CD9 and CD63 positive particles in exosomes from different bovine dairy products was relatively low ( Figure 2 ).

[0080] 2. Morphology of exosomes from different dairy sources

[0081] As Figure 3 showed, exosomes from different sources had a typical extracellular vesicle structure under the electron microscope, that is, a double - membrane structure, saucer - shaped or hemispherical with one - side depression.

[0082] (II) The exosomes of Example 1 have strong anti - malaria activity

[0083] Both Example 1 and Comparative Example 1 had a rapid killing effect on asexual - stage Plasmodium falciparum Pf3D7. Taking the solvent control as a reference, the half - inhibitory concentration (IC 50 ) of Example 1 against Plasmodium falciparum 3D7 strain was 22.9 μg / mL. In contrast, the exosomes from Comparative Example 2 and Comparative Example 3 had no anti - malaria activity ( Figure 4 ).

[0084] The basic concepts have been described above. Obviously, for those skilled in the art, the above - mentioned detailed disclosure is only an example and does not constitute a limitation to this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are proposed in this specification, so such modifications, improvements, and corrections still fall within the spirit and scope of the exemplary embodiments of this specification.

[0085] Meanwhile, this specification uses specific terms to describe the embodiments of this specification. For example, "an embodiment", "one embodiment", and / or "some embodiments" mean a certain feature, structure, or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "an embodiment" or "one embodiment" or "an alternative embodiment" mentioned twice or more at different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0086] In some embodiments, numbers are used to describe components and the quantities of attributes. It should be understood that such numbers used to describe embodiments are, in some examples, modified by the modifiers "about", "approximately", or "substantially". Unless otherwise specified, "about", "approximately", or "substantially" indicate that the said numbers allow a variation of ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, and such approximate values can change according to the characteristics required by individual embodiments. In some embodiments, the numerical parameters should consider the specified significant digits and adopt the method of retaining the general number of digits. Although the numerical ranges and parameters used in some embodiments of this specification to confirm the breadth of their scope are approximate values, in specific embodiments, such numerical settings are made as precise as possible within the feasible range.

[0087] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification can be considered to be in accordance with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. Use of milk-derived exosomes in the preparation of a drug for treating malaria.

2. The use according to claim 1, characterized in that, The exosomes inhibit the proliferation of Plasmodium falciparum.

3. The use according to claim 1, characterized in that, The exosomes are derived from raw milk or pasteurized milk.

4. The use according to claim 3, characterized in that, The pasteurization conditions of the pasteurized milk are heat treatment at 65-75 °C for 10-20 seconds.

5. The use according to claim 3, characterized in that The pasteurization conditions of the pasteurized milk are heat treatment at 72 °C for 15 seconds.

6. The use according to claim 1, wherein The exosomes are obtained by differential centrifugation combined with ultrafiltration centrifugation.

7. The use according to claim 1, characterized in that, The exosome particle size is 60-65 nm.

8. The use according to claim 1, characterized in that, The concentration of the exosomes is greater than 1.0*10 12 / mL.

9. A pharmaceutical composition, wherein the pharmaceutical composition contains the exosomes according to any one of claims 1-8.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition further contains a pharmaceutically acceptable carrier or excipient.

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