Exosome surface modification by using NHS-PEG-NHS and application of exosome surface modification in targeted drug delivery

Abstract

The invention belongs to the technical field of exosome modification, and particularly relates to an exosome modification method for connecting a target protein to the surface of an exosome by utilizing NHS-PEG-NHS to achieve a targeting or treatment effect, and application of the exosome modification method in targeted central nervous system demyelination disease treatment. In particular to a technology for modifying an exosome to enable the exosome to have targeting property and a preparation method of the exosome. NHS-PEG-NHS is used as a connecting substance, and protein, polypeptide and substances with primary amine groups on the surface can be connected to the surface of an exosome membrane. The modified exosome can load drugs and can be accurately delivered to tissues expressing specific proteins or polypeptides through oral administration, so that the bioavailability and the treatment effect of the drugs are remarkably improved. Taking the NG2 antibody modified cow milk exosome as an example, the NG2 antibody is connected to the surface of the cow milk exosome to target the central demyelination focus area, so that the effective therapeutic dose of the drug in the central focus area is achieved through oral delivery.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome modification, and specifically relates to exosome surface modification using NHS-PEG-NHS and application thereof in targeted drug delivery. Background Art

[0002] Myelin damage in the central nervous system (CNS) is the main pathological feature of demyelinating diseases. The harm of demyelinating diseases is mainly manifested in affecting the functions of the nervous system, including muscle control, sensation, coordination and balance. In the clinical treatment of central nervous system diseases, due to the existence of the blood-brain barrier (BBB), it is difficult for drugs to reach an effective therapeutic dose in the central lesion area, and long-term or high-dose administration is required, which is prone to cause side effects such as liver and kidney damage. Therefore, there is a lack of a targeted drug delivery system that can accurately deliver drugs to the lesion to more effectively treat and reduce drug side effects.

[0003] Common clinical drug delivery methods include oral, injection, intranasal, intraocular, and intrabuccal administration. Oral administration is more convenient and safer than other methods, especially for the treatment of chronic diseases that require long-term medication. However, orally delivered drugs need to overcome the physiological barriers of the gastrointestinal tract, such as the acidic environment, enzyme degradation, and mucus layer obstruction. These factors often lead to the inability of drugs to reach effective doses in the lesion area, poor stability, and poor therapeutic effects. Therefore, the establishment of an oral delivery system for such drugs is of great significance for the treatment of related diseases and the clinical application of drugs.

[0004] The tolerance of bovine milk exosomes to gastric acid is one of their important advantages as an oral drug delivery system. Bovine milk exosomes also have the advantages of good BBB biocompatibility, stability and permeability, and can effectively penetrate the biological barriers in the body. However, how to make exosomes effectively carry drugs for targeted delivery to the central lesion area remains a research difficulty. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for connecting other proteins to target and modify exosomes by using NHS-PEG-NHS. The present invention uses NHS-PEG-NHS to modify exosomes, which not only has simple and easy operation steps, but also further shortens the reaction time, and can simply connect related proteins to the exosome membrane.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an application of using NHS-PEG-NHS to modify the surface of exosomes.

[0008] The present invention also provides a modified body for modifying a substance containing a primary amine group, wherein the modified body comprises a connected NHS-PEG-NHS and a protein, a polypeptide and other substances having a primary amine group on the surface; wherein the NHS-ester in the NHS-PEG-NHS is connected to the primary amine group in the protein, the polypeptide and other substances having a primary amine group on the surface through a covalent bond.

[0009] Furthermore, the substance containing primary amine groups includes exosomes.

[0010] The present invention also provides a method for modifying exosomes, which comprises: dissolving NHS-PEG-NHS powder in phosphate buffered saline (PBS), vortexing thoroughly to obtain an NHS-PEG-NHS solution; performing a first mixing and incubation of the exosomes with the NHS-PEG-NHS solution to obtain an exosome linker; and performing a second mixing and incubation of the exosome linker with proteins, polypeptides and other substances having primary amine groups on the surface to obtain modified exosomes.

[0011] Furthermore, the dosage ratio of the protein, peptide and other substances with primary amine groups on the surface, NHS-PEG-NHS and exosomes is 150-200 ng: 120-140 μg: 20-40 μg; the concentration of NHS-PEG-NHS is 6 mM, and the molecular weight of PEG is 2000.

[0012] Furthermore, the pH values ​​of the first mixed incubation and the second mixed incubation are both 7.2-8.0; the temperature of the first mixed incubation is 37° C., and the time is 5 hours; the temperature of the second mixed incubation is 4° C., and the time is 12 hours.

[0013] Furthermore, the method further comprises: after obtaining the modified exosomes, purifying the modified exosomes by ultracentrifugation; the centrifugal force of the ultracentrifugation is 100,000×g, and the time of the ultracentrifugation is 60 min.

[0014] The present invention further provides modified exosomes obtained by the above-mentioned exosome modification method.

[0015] The present invention further provides the use of the modified exosomes as described above as a drug targeting central demyelinating lesions.

[0016] Furthermore, NHS-PEG-NHS is used as a linking substance to connect the target protein to the bovine milk exosome membrane to obtain bovine milk exosomes with targeted therapeutic effects; the bovine milk exosomes with targeted therapeutic effects are taken in an oral delivery manner.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] The present invention provides the surface modification of bovine milk exosomes using NHS-PEG-NHS and its application in targeted oral drug delivery. In the present invention, NHS-PEG-NHS is selected as a connection between exosomes and proteins, polypeptides and other substances containing primary amines. N-hydroxysuccinimide ester (NHS-ester, NHS ester) can react with primary amines to form covalent bonds and has the ability to combine with primary amine groups (primary amines, -NH2) in structures such as cell membranes or free proteins and polypeptides. Using NHS-PEG-NHS as a connecting substance, the target protein can be connected to the exosome membrane, and the half-life can be extended by PEG. The present invention uses NHS-PEG-NHS to modify proteins or polypeptides with primary amine groups on the surface of bovine milk exosomes. After loading the drug, targeted oral delivery of drugs to tissues / cells that highly express the protein or polypeptide receptor can be achieved. Not only is the operation step simple and easy to implement, but the reaction efficiency is further improved, and the universality is also improved. Bovine milk exosomes have good stability, and their tolerance to gastric acid is one of their important advantages as an oral drug delivery system; PEG plays a role in prolonging the half-life. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following is a brief introduction to the drawings required for use in the embodiments. Take the modification of exosomes to target OPC cells as an example. The exosomes are mEVs, and the modified exosomes are mEVNs.

[0020] Figure 1 The schematic diagram of the connection between NHS-PEG-NHS and exosomes and target protein (taking NG2 antibody as an example);

[0021] Figure 2 This is a fluorescence co-localization diagram of NG2 antibody successfully modified on the surface of exosomes, and the result diagram of its modification efficiency exploration. Red is exosomes and green is NG2 antibody.

[0022] Figure 3 Electron microscopy and NTA results of mEVs / mEVNs;

[0023] Figure 4 The in vitro results show that NG2 antibody-modified exosomes effectively target OPC cells;

[0024] Figure 5 The in vivo results (physiological state) of NG2 antibody-modified exosomes effectively targeting OPC cells;

[0025] Figure 6The in vivo results (pathological state) of NG2 antibody-modified exosomes effectively targeting OPC cells;

[0026] Figure 7 The therapeutic effect of NG2-modified exosomes carrying lactate on demyelinated mice. DETAILED DESCRIPTION

[0027] The inventors found that a large number of NG2-positive oligodendrocyte progenitor cells (OPCs) aggregate in the lesion area of ​​myelin damage in demyelinating diseases, and these oligodendrocyte progenitor cells with blocked differentiation are the therapeutic targets for myelin repair. Studies on primary oligodendrocyte progenitor cells (OPCs) in mice have shown that L-lactic acid increases the expression of MBP mRNA and the proportion of MBP+ cells, and can also promote remyelination of the corpus callosum.

[0028] Bovine milk exosomes have the characteristics of low extraction cost, easy large-scale production, low immunogenicity, high biocompatibility, and can pass through multiple physiological barriers. Compared with cell-derived exosomes, the lipid bilayer membrane of bovine milk exosomes can resist degradation by intestinal digestive enzymes. This protective mechanism enables bovine milk exosomes to maintain their integrity and stability when passing through the intestine, thereby ensuring that their contents can smoothly reach the target cells and play a role. With the development of targeted drug delivery systems, bovine milk exosomes have been widely accepted as a promising carrier for transporting various biomolecules and chemotherapeutic drugs. Therefore, the inventors proposed to load lactic acid into exosomes to penetrate the blood-brain barrier to reach the lesion site. However, natural exosomes cannot accurately reach the central demyelination site, resulting in the lactic acid content in the lesion site not meeting the treatment requirements. Therefore, it is necessary to target exosomes to obtain modified exosomes with the ability to target the lesion site.

[0029] Exosome modification methods include: 1. Surface chemical modification: chemical groups such as carboxyl groups and primary amine groups are introduced into the surface of exosomes by chemical methods, and then surface modification is performed, such as combining with fluorescent dyes or metal nanoparticles. 2. Genetic modification: genetic modification of exosomes is performed through genetic engineering technology, such as changing protein composition, enhancing exosome secretion, etc. 3. Lipid modification: liposomes or lipid encapsulation technology are used to encapsulate and modify exosomes to enhance their stability and targeting. 5. Other modifications: also include glycosylation modification, enzymatic modification, etc.

[0030] In the chemical modification of exosome membrane, the commonly used scheme is to use NHS-PEG or DSPE-PEG solution. In the above scheme, DSPE and NHS are responsible for connecting with the hydrophobic exosome lipid membrane, and PEG can be combined with proteins, peptides and other substances due to its hydrophilicity. When chemically connected, NHS-PEG or DSPE-PEG is incubated with the target protein and peptide overnight to obtain NHS / DSPE-PEG-protein / peptide, and then this pre-connected product is co-incubated with exosomes to obtain exosomes with target proteins. Although the above method can achieve the purpose of modification, its disadvantages are: 1. The modification of PEG protein is mainly achieved by the reaction of the terminal hydroxyl group of PEG with the residue of the primary amino acid group of protein, and the activity of the terminal hydroxyl group of PEG is very poor; 2. In order to co-modify proteins under mild conditions in vivo, an activator must be used for activation; 3. The reaction system is complex and the experimental conditions are relatively harsh.

[0031] In view of this, the present invention provides an application of using NHS-PEG-NHS to modify the surface of exosomes.

[0032] The present invention also provides a modified body for modifying a primary amine group, wherein the modified body comprises a protein, a polypeptide or other substances containing a primary amine and the NHS-PEG-NHS; the primary amine group and the NHS-ester are connected via a covalent bond.

[0033] In the present invention, the primary amine group-containing substance preferably includes exosomes.

[0034] The present invention also provides a method for modifying exosomes, comprising the following steps:

[0035] Dissolve NHS-PEG-NHS powder in PBS and vortex thoroughly;

[0036] The exosomes having primary amine groups on the surface are first mixed and incubated with NHS-PEG-NHS to obtain an exosome linker;

[0037] The exosome connector is mixed and incubated for a second time with proteins, peptides and other substances having primary amine groups on the surface to obtain modified exosomes;

[0038] In the present invention, before the exosomes are mixed and incubated with NHS-PEG-NHS, it is preferred that: 13.38 mg of NHS-PEG-NHS powder is dissolved in 1 mL of PBS and vortexed sufficiently to make the concentration of NHS-PEG-NHS 6 mM, where the molecular weight of PEG is 2000; before the exosome connector is mixed and incubated with the target protein, it is preferred that: if there is sodium azide or thimerosal, impure glycerol and high concentration glycerol in the solution, it should be removed by a desalting column or a dialysis bag.

[0039] In the present invention, the pH value of the mixed incubation of the NHS-PEG-NHS solution and the exosomes is preferably 7.2-8.0, more preferably 7.2; the temperature of the mixed incubation is preferably 20-37°C, more preferably 25-37°C; the time of the mixed incubation is preferably 5h. The present invention combines NHS-PEG-NHS with the primary amine groups on the exosomes through suitable incubation conditions to obtain an exosome linker; the exosome linker is mixed with a substance such as a protein containing a primary amine group and incubated at a pH value of preferably 7.2-8.0, more preferably 7.2, the incubation temperature is preferably 4°C, and the time of the mixed incubation is preferably 12h to obtain the modified exosomes.

[0040] The present invention uses appropriate incubation conditions to react primary amine groups with NHS-PEG-NHS to form amide covalent bonds, thereby connecting proteins, polypeptides and other substances containing primary amine groups to the exosome membrane.

[0041] In the present invention, after the NHS-PEG-NHS solution and the exosomes are mixed and incubated, the method preferably further comprises: purifying the mixed solution after the mixed incubation through a Sephadex G25 column to obtain a purified exosome conjugate. In the present invention, excess NHS-PEG-NHS can be removed by elution treatment.

[0042] After obtaining the exosome linker, the present invention mixes and incubates the exosome linker with a substance such as a protein containing a primary amine group to obtain modified exosomes.

[0043] In the present invention, the ratio of the protein, polypeptide and other substances with primary amine groups on the surface, NHS-PEG-NHS and exosomes is 150-200 ng: 120-140 μg: 20-40 μg.

[0044] In the present invention, the exosomes are preferably bovine milk exosomes.

[0045] In the present invention, the method for isolating exosomes preferably comprises: ultracentrifuging the mixed incubation solution to obtain a precipitate as the exosomes. In the present invention, the centrifugal force of the ultracentrifugation treatment is preferably 100,000×g, and the ultracentrifugation time is preferably 60 min.

[0046] In the present invention, after the exosome connector and the protein are mixed and incubated, ultracentrifugation is preferably further performed to obtain a precipitate as the modified exosome; the speed of the ultracentrifugation is preferably 100,000×g, and the time of the ultracentrifugation is preferably 60 min.

[0047] After obtaining the modified exosomes, it is preferred that the following steps are further included: modifying the exosomes with a fluorescent group-modified protein to obtain exosomes with a tracer effect; modifying the exosomes with a targeting protein to obtain exosomes with a targeting effect; and modifying the exosomes with a therapeutic protein to obtain exosomes with a therapeutic effect. This method has a good development prospect.

[0048] In order to further illustrate the present invention, the method for modifying exosomes provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention. All instruments and raw materials can be obtained from commercial channels or are commonly used in the industry. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

[0049] Example 1: A method and identification of NG2 antibody modified exosomes, comprising the following steps (see schematic diagram Figure 1 ):

[0050] 1) Isolate milk exosomes using chitosan and resuspend them in PBS to a final concentration of 2 mg / mL;

[0051] 2) Weigh 13.38 mg of NHS-PEG-NHS powder and dissolve it in 1 mL of PBS to a concentration of 6 mM (PEG molecular weight: 2000);

[0052] 3) Dilute the NG2 antibody 1:100 with PBS, purify with Sephadex G25 column to remove sodium azide in the solution, and collect the liquid in the collection tube;

[0053] 4) The 10 μL NHS-PEG-NHS solution and the 10 μL exosome solution were mixed evenly and incubated at 37° C. for 5 h to obtain a mixed solution;

[0054] 5) The mixed solution is purified by Sephadex G25 column to finally obtain NHS-PEG-NHS after removing excess NHS.

[0055] 6) The 20 μL mixed solution was mixed with 20 μL NG2 antibody solution and incubated at 4° C. for 12 h.

[0056] 7) The mixed solution was ultracentrifuged at 100,000×g for 60 min, and the exosome pellet was resuspended in PBS.

[0057] 8) Dil preparation: add 2 μL of Dil to 4 mL of PBS;

[0058] 9) Drop the exosomes on a glass slide and air-dry them, and draw circles with an immunohistochemical pen. Drop Dil on a glass slide and incubate at 37°C in the dark for 20 minutes;

[0059] 10) The slide was washed with PBS for 3 times, 5 min each time.

[0060] 11) Add NG2 secondary antibody (green) diluted 1:800 onto the slide and incubate in the dark for 1 hour.

[0061] 12) Seal the slide and observe.

[0062] Results Figure 2 , where the red signal is exosome staining and the green signal is NG2 staining. This result indicates that the chemical modification of exosomes is successful.

[0063] Example 2: Electron microscopic images of mEVs and mEVNs.

[0064] 1) Dilute mEVs with PBS;

[0065] 2) Pipette 20 μL of mEVs / mEVNs dilution solution and drop it onto a 200-mesh copper mesh. Allow to adsorb naturally for 5 to 10 minutes. Then use filter paper to absorb excess droplets and air dry.

[0066] 3) Take 20 μL of 2% phosphotungstic acid solution, drop it on the copper grid, and let it stand at room temperature for 3-5 minutes;

[0067] 4) Use filter paper to absorb excess droplets and dry the copper mesh under an incandescent lamp;

[0068] 5) After drying, the samples were observed using a HT7700 transmission electron microscope.

[0069] Example 3: Identification of modified exosomes by particle size analysis (NTA).

[0070] 1) Take out the decapped exosome mixture frozen in -80°C ultra-low temperature refrigerator of Experimental Example 1, thaw on ice until completely melted, add 1×PBS, and dilute 2000 times to the working concentration suspension:

[0071] 2) The mixed solution in step 1) was transferred to a flow tube, and the exosomes and vesicles with a diameter of 30-150 nm in the suspension were imaged and observed one by one in real time by a nano-flow particle size analyzer, and the particle size distribution and concentration of the exosomes in the suspension were calculated by Zeta view 8.04.02 software. The results are as follows: Figure 3 shown.

[0072] Example 4: Targeting of modified exosomes in vitro.

[0073] 1) Extract mixed neural cells and seed them into 24-well plates. Divide them into two groups, 2*10 per well. 4cells, cultured for 2 days. The culture medium formula is: 10% FBS + 1% L-glutamine + 1% Penni + DMEM / F12, 1 ml per well.

[0074] 2) Add 1*10 8 Particles-modified exosomes (mEVN) and unmodified exosomes (mEV) were co-cultured for 2 days.

[0075] 3) Observation. Red is mEVs / mEVNs, green is OPCs, and blue is cell nuclei. Figure 4 shown.

[0076] Example 5: Targeting of modified exosomes in vivo 1.

[0077] 1) 6-8 week old C57BL / 6 mice were divided into two groups and each mouse was given 1*10 9 The mEVs / mEVNs of particles were expressed, and the brains were harvested 5 days later and sliced ​​and stained.

[0078] 2) Observation. Red is mEVs / mEVNs, green is OPCs, and blue is cell nuclei. Figure 5 shown.

[0079] Example 6: Targeting of modified exosomes in vivo 2.

[0080] 1) 6-8 week old C57BL / 6 mice were divided into two groups and LPC model was established. 5 days after model establishment, each mouse was given 1*10 10 The mEVs / mEVNs of particles were expressed, and the brains were harvested 5 days later and sliced ​​and stained.

[0081] 2) Observation. Red is mEVs / mEVNs, green is OPCs, and blue is cell nuclei. Figure 6 shown.

[0082] Example 7: Treatment of demyelinated mice with modified lactate-loaded exosomes.

[0083] 1) 6-8 week old C57BL / 6 mice were divided into two groups for LPC modeling. Three days before modeling, each mouse was given 1*10 10 The mEVs / mEVNs of particles were expressed, and the brains were harvested 14 days later and sliced ​​and stained.

[0084] 2) Observation. Green is the myelin sheath in the corpus callosum, and blue is the cell nucleus. Figure 7 shown.

[0085] The preferred specific implementation modes and embodiments of the present invention are described in detail above, but the present invention is not limited to the above implementation modes and embodiments, and various changes can be made within the knowledge scope of those skilled in the art without departing from the concept of the present invention.

Claims

1. Application of NHS-PEG-NHS for exosome surface modification.

2. A modified substance for modifying a substance containing a primary amine group, characterized in that: The modified body includes connected NHS-PEG-NHS and proteins, peptides and other substances with primary amine groups on the surface; The NHS-ester in the NHS-PEG-NHS is connected to the primary amine groups in the proteins, peptides and other substances with primary amine groups on the surface through covalent bonds.

3. The modified substance for modifying a substance containing a primary amine group according to claim 1, characterized in that: The primary amine group-containing substance includes exosomes.

4. A method for modifying exosomes, characterized in that: The method includes: Dissolve NHS-PEG-NHS powder in phosphate buffered saline and vortex thoroughly to obtain NHS-PEG-NHS solution; The exosomes are first mixed and incubated with the NHS-PEG-NHS solution to obtain an exosome conjugate; The exosome connector is mixed and incubated for a second time with proteins, peptides and other substances having primary amine groups on the surface to obtain modified exosomes.

5. The method according to claim 4, characterized in that The dosage ratio of the protein, peptide and other substances with primary amine groups on the surface, NHS-PEG-NHS and exosomes is 150-200 ng: 120-140 μg: 20-40 μg; the concentration of NHS-PEG-NHS is 6 mM, and the molecular weight of PEG is 2000.

6. The method according to claim 4, characterized in that The pH values ​​of the first mixed incubation and the second mixed incubation are both 7.2-8.0; the temperature of the first mixed incubation is 37° C., and the time is 5 hours; the temperature of the second mixed incubation is 4° C., and the time is 12 hours.

7. The method according to claim 4, characterized in that The method further comprises: after obtaining the modified exosomes, purifying the modified exosomes by ultracentrifugation; the centrifugal force of the ultracentrifugation is 100,000×g, and the time of the ultracentrifugation is 60 minutes.

8. The modified exosomes obtained by the method for modifying exosomes according to any one of claims 4 to 8.

9. Use of the modified exosomes as claimed in claim 8 as a drug targeting central demyelinating lesions.

10. The use according to claim 9, characterized in that: NHS-PEG-NHS is used as a linking substance to connect the target protein to the bovine milk exosome membrane to obtain bovine milk exosomes with targeted therapeutic effects; the bovine milk exosomes with targeted therapeutic effects are taken in an oral delivery manner.

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