WO2020029123A1

WO2020029123A1 - Method for modifying extracellular vesicles

Info

Publication number: WO2020029123A1
Application number: PCT/CN2018/099428
Authority: WO
Inventors: 王少英; 法金·哈克
Original assignee: 深圳宣泽生物医药有限公司
Priority date: 2018-08-08
Filing date: 2018-08-08
Publication date: 2020-02-13

Abstract

Provided is a method for modifying extracellular vesicles, the method comprising: isolating and purifying extracellular vesicles; then sequentially connecting a membrane insertion group, a linking group and a targeting ligand; assembling the connected membrane insertion group-linking group-targeting ligand on the surface of the purified extracellular vesicles; and after incubation for a period of time, removing the free membrane insertion group, linking group and targeting ligand, so as to obtain high-purity extracellular vesicles modified with ligands. The modified extracellular vesicles can recognize and bind to a specific receptor on the diseased cell membrane by a targeting module, and perform targeted delivery of a drug into cells, which can be widely used in the fields of treatment drugs and diagnosis of diseases.

Description

Method for modifying extracellular vesicles

[0001] Technical Field

[0002] The present application belongs to the technical field of biomedical material preparation, and relates to a method for preparing a drug carrier, and in particular, to a method for modifying extracellular vesicles.

[0003] Background Art

[0004] Extracellular Vesicles are keratinous vesicles that are automatically secreted and released by human, animal, plant, or microbial cells. They range in diameter from tens of nanometers to several micrometers. Microvesicles released by the plasma membrane, exosomes produced by the endocytosis pathway, and apoptotic bodies produced by apoptosis

body), Shedding vesicle. Extracellular vesicles were first discovered more than 30 years ago. The main role of these secretions is to remove the waste components of cells. In the past few years, the latest research found that these secretions can also be used to transmit signals. By conveying information to surrounding tissues, exosomes can transfer cell-specific proteins, lipids and genetic material to other cells, thereby altering their function

[0005] Extracellular vesicles carry biologically active proteins, lipids, messenger RNA (mRNA), micro RNA

(miRNA), non-coding RNA (ncRNA), and DNA fragments, and can transfer these active molecules to recipient cells, regulating the biological functions of target cells, tissues and organs. Certain types of cells, such as mesenchymal stem cells, produce microvesicles containing a variety of active biomolecules that can protect damaged tissues, promote growth and repair, and regulate immune functions. They can be injected into the body to repair damage, inhibit inflammation, Promote tissue regeneration and functional recovery. At the same time, the use of electroporation, chemical perforation, ultrasound, etc. can also load synthetic nucleic acid drugs, chemotherapeutic drugs, etc. into cell microvesicles, making them a drug carrier, so cell microvesicles can be used as a New delivery vehicle for biotherapy. It has the following advantages: (1) easy to extract and modify, (2) because it is derived from cells in the body, it is well tolerated in the body, (3) low immunogenicity, which can avoid immune response, (4) it can naturally deliver organisms Molecules, (5) have nanometer diameter and plastic shape, (6) can pass through biological barriers such as blood-brain barrier, (7) can avoid rapid clearance of liver and kidney, (8) can be fused directly with target cells, so No need to solve endosome escape [0006] Cell microvesicles have a wide range of applications, such as for targeted delivery of RNA therapeutic substances (miRNA, siRNA, anti-miRNA, ribozyme), targeted delivery of gene editing molecules (such as CRISPR-RNA) to target cells, and Immunotherapy, targeted delivery of chemotherapy drugs, etc. However, there are still many challenges in using exosomes for targeted drug delivery vehicles. For example, the lack of specific ligands leads to unsatisfactory therapeutic effects, non-specific fusion to healthy cells, and rapid accumulation in the liver and other healthy organs. Toxic. In addition, although cells can be modified by engineered donor cell methods to modify ligands on the surface of the vesicles, the process is complicated, poorly controllable, and inefficient, and any type of ligand cannot be modified as required. Vesicle-related applications are greatly limited.

[0007] Application Contents

[0008] To this end, the present application is to solve the above technical problems, and thus proposes a method with simple steps, high efficiency, and rapid modification of targeting ligands on the surface of extracellular vesicles.

[0009] In order to solve the above technical problems, the technical solution of the present application is:

[0010] The present application provides a method for modifying extracellular vesicles, which includes the following steps:

[0011] S1. Isolating and purifying extracellular vesicles;

[2] S2, sequentially connecting the inserting group, the linking group and the targeting ligand to obtain a connected inserting group-linking group-targeting ligand composite structure, and assembling the above composite structure to the step Surface of purified extracellular vesicles obtained in S1;

[0013] S3, incubating the extracellular vesicles obtained in step S2;

[0014] S4. The free intercalating group, the linking group, and the targeting ligand that are not modified to the surface of the extracellular vesicle are removed to obtain a highly purified extracellular vesicle with a surface modified ligand.

[0015] Preferably, in the step S2, the inserting group, the linking group and the targeting ligand are connected by a click chemistry method or a fusion protein method.

[0016] Preferably, the intercalation group is a chemically synthesized group, a protein or a polypeptide group, wherein the chemically synthesized group is a hydrophobic group, which is ethyl phosphorothioate, cholesterol, and chain affinity. One of Porphyrin, Porphyrin, and Diphenylcyclooctyne.

[0017] Preferably, the protein or polypeptide group consists of a hydrophobic transmembrane or transmembrane protein.

[0018] Preferably, the hydrophobic transmembrane or transmembrane protein is TAT protein, NLS protein, penetratin protein , Transport protein, MPG protein, MAP protein, signal transduction peptide, arginine-rich, histidine, lysine sequence peptide.

[0019] Preferably, the linking group is a chemical molecule or a polypeptide amino acid sequence; the chemical molecule is one or more of tetraazabenzene, azide, alkyne, DBCO, BCN, and TCO.

[0020] Preferably, the targeting ligand is an antibody, a nucleic acid or a polypeptide ligand, specifically a VEGF antibody, a GPC3 antibody, a GRP78 antibody, an EGFR antibody or a nucleic acid aptamer, an RGD, a PSMA antibody or a nucleic acid aptamer, One of EpCAM antibody or nucleic acid, CD44 antibody or nucleic acid, CD 19 antibody or nucleic acid aptamer, AF-20 antibody, Her2 antibody or nucleic acid aptamer, Her3 antibody or nucleic acid aptamer.

[0021] Preferably, in step S1, extracellular capsule cells are separated and purified by PEG precipitation and density gradient centrifugation, first at 2000-5000.

Centrifuge at a centrifugal force of 20 g for 30 to 30 minutes, and then centrifuge the supernatant at a centrifugal force of 10,000 to 12,000 g for 30 to 60 minutes to remove large particles of impurities. Add PEG solution to the resulting supernatant and leave at 4 ° C overnight After centrifugation at 3000 ~ 4000xg for 30 ~ 60min, a pellet of tiny vesicles was obtained, and the pellet was resuspended. The sucrose density gradient method was used for ultracentrifugation at 110,000 ~ 150,000 g for 1.5 ~ 2hr to obtain purified extracellular vesicles.

[0022] Preferably, the step S3 is specifically incubating the purified extracellular vesicles-linking group-targeting ligand-purified extracellular vesicles at 15-100 ° C for 1-24h.

[0023] Preferably, in step S4, ultracentrifugation or molecular sieves are used to remove the free inserting group, the linking group and the targeting ligand; the modified extracellular vesicles are further washed with a buffer solution and the amount is 100000 Centrifuge at ~ 120,000 g for 70-90 min, or use Sepharose CL-2B column to obtain high-purity surface-modified ligand-containing extracellular vesicles.

[0024] The above technical solution of the present application has the following advantages over the prior art:

[0025] The method for modifying an extracellular vesicle described in the present application firstly isolates and purifies the extracellular vesicles, and then sequentially assembles an inserting group, a linking group, and a targeting ligand on the surface of the cellular vesicles, and incubates for a period of time By removing free intercalating groups, linking groups and targeting ligands to obtain highly purified extracellular vesicles with modified ligands, this method provides a simple, efficient, and fast modification target on the surface of extracellular vesicles The method for ligands, modified extracellular vesicles can recognize and bind to specific receptors on diseased cell membranes through the targeting module, and target drug delivery to cells, expanding the range of ligand types, In addition, the method can directly A large number of microvesicle particles similar to exosomes are extracted from edible plants, which promotes large-scale, low-cost industrial production, and can be widely used in the field of disease treatment drugs and diagnostics.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the content of the present application easier to understand, the following further describes the present application in detail based on specific embodiments of the present application and the accompanying drawings.

[0028] FIG. 1 is an exosomal diameter distribution diagram derived from grapefruit according to Example 1 of the present application;

[0029] FIG. 2 is a schematic diagram of a modification process of an extracellular vesicle according to an embodiment of the present application;

[0030] FIG. 3 is a diameter distribution diagram of lemon-derived exosomes derived from Example 2 of the present application.

[0031] The reference numerals in the figure are represented as: 1-extracellular vesicles; 2-intercalating group; 3-connecting group; 4-targeting ligand

[0032] Specific embodiments

Example 1

[0034] This embodiment provides a method for modifying extracellular vesicles, which includes the following steps:

[0035] S1. Extracellular vesicles 1 are isolated and purified. The extracellular vesicles can be extracted from edible plants. In this embodiment, the edible plants are grapefruit. The extraction process is specifically:

[0036] a. Squeeze out juice from grapefruit.

[0037] b. Put grapefruit juice at 3000

Centrifuge for 25 minutes at a centrifugal force of g, take the supernatant, and then centrifuge the supernatant for 50 min at a centrifugal force of 1,000 g, to initially remove large particles of impurities.

[0038] c. Add PEG solution (final concentration of 8% PEG solution, containing 0.5M sodium chloride) to the supernatant obtained after centrifugation in step b, and place at 4 ° C overnight.

[0039] d. The sample obtained in step c is centrifuged under a centrifugal force of 3200 × g for one hour to obtain a precipitate of microvesicles.

[0040] e. Resuspend the pellet, place it on a sucrose density gradient and perform ultracentrifugation for 1.8 hours under the conditions of 120,000 g. Due to the difference in density, the microvesicles after centrifugation will be enriched at the interface between 30% and 45% sucrose to separate them from impurities of different densities in the plant juice. The particle size distribution of the purified microvesicles is shown in Figure 1.

S2. Ethyl phosphorothioate inserting group 2, linking group 3 and VEGF antibody targeting ligand 4 are sequentially combined together, and the combined inserting group-linking group -Assembly of the targeting ligand composition to step S1 The resulting structure of the purified extracellular vesicle surface is shown in Figure 2. This step is specifically: using click chemistry to connect, first insert the membrane group 2 modified with Tetmzine or Azide (linking group 3), and Targeting ligand 4 modified with Alkene, Alkyne or DBCO at the end is reacted under certain chemical conditions, so that insert membrane group 2 and targeting ligand 4 are connected by forming a covalent bond. Alternatively, as a transformable embodiment, a fusion protein method can also be used to combine the inserting membrane group 2 and the targeting ligand 4, and the basic steps are: combining the inserting protein gene sequence, connecting the group gene sequence, and the targeting ligand. The gene sequence of the sequence is constructed under the same promoter as the expression of the same protein. Under certain conditions, the insert protein 2, the linker group 3, and the targeting ligand 4 protein will be expressed in a fusion form.

S3. Incubate the inserted membrane group-linking group-targeting ligand-purified extracellular vesicles obtained in step S2 at 15 ° C. for 24 h.

[0043] S4. The free intercalating group, the linking group and the targeting ligand that are not modified to the surface of the extracellular vesicle are removed to obtain a highly purified extracellular vesicle with a surface modified ligand: using ultracentrifugation precipitation method The modified extracellular vesicles were further washed with a buffer solution and centrifuged under a centrifugal force of 110,000 g for 80 minutes to obtain high-purity extracellular vesicles with a surface modification membrane-linking group-targeting ligand.

Example 2

[0045] This embodiment provides a method for modifying extracellular vesicles, which includes the following steps:

[0046] S1. Extracellular vesicles 1 are isolated and purified. The extracellular vesicles can be extracted from edible plants. In this embodiment, the edible plants are lemons, and the extraction process is specifically:

[0047] a. Squeeze out the juice from the lemon.

[0048] b. The squeezed lemon juice is centrifuged at a centrifugal force of 2000 g for 30 min, the supernatant is taken, and then the supernatant is centrifuged at a centrifugal force of 10,000 g for 60 min, and large particles of impurities are initially removed.

[0049] c. Add PEG solution (final concentration of 8% PEG solution, containing 0.5M sodium chloride) to the supernatant obtained after centrifugation in step b, and place at 4 ° C overnight.

[0050] d. Centrifuging the sample obtained in step c under a centrifugal force of 3000 x g for 50 min to obtain a precipitate of microvesicles

[0051] e. Resuspend the pellet, place it on a sucrose density gradient and perform ultracentrifugation at 110,000 g for 2 hours. Due to the difference in density, the microvesicles after centrifugation will be enriched at the interface between 30% and 45% sucrose to separate them from impurities of different densities in the plant juice. The particle size distribution of the purified microvesicles is shown in Figure 3. [0052] S2, a protein or polypeptide intercalating group 2, a linking group 3 and a GPC3 antibody targeting ligand 4 are sequentially combined together, the linking group and the intercalating group may be covalently linked or non- Covalently attach and assemble the combined intercalating group-linking group-targeting ligand composition on the surface of the purified extracellular vesicle obtained in step S1. The structure formed is shown in FIG. 2. This step is specific To: click the chemical method to connect, first insert Tetrazine or BCN (linking group 3) modified membrane group 2 and terminally modified Alkene, A1 kyne or DBCO targeting ligand 4, under certain chemical conditions A reaction is performed to connect the intercalation membrane group 2 and the targeting ligand 4 by forming a covalent bond. Alternatively, as a transformable embodiment, a fusion protein method can also be used to combine the inserting membrane group 2 and the targeting ligand 4, and the basic steps are: combining the inserting protein gene sequence, connecting the group gene sequence, and the targeting ligand. The gene sequence of the sequence is constructed under the same promoter as the expression of the same protein. Under certain conditions, the insert protein 2, the linker group 3, and the targeting ligand 4 protein will be expressed in a fusion form.

[0053] In this embodiment, the protein or polypeptide insertion group is a hydrophobic transmembrane or transmembrane protein, where the TAT protein sequence 歹 IJ is GRKKRRQRRRPQ, the NLS protein sequence 歹 ij is KRPAAIKKAGQAKKKK, Penetr atin protein The sequence is

The RQIKIWFQNRRMKWKK transport protein sequence is GWTLNSAGYLLGKINLKALAALA KKIL.

[0054] S3. Incubate the inserted membrane group-linking group-targeting ligand-purified extracellular vesicle obtained in step S2 at 100 ° C for 1 h.

[0055] S4. The free intercalating group, the linking group and the targeting ligand that are not modified to the surface of the extracellular vesicle are removed to obtain a highly purified extracellular vesicle with surface modified ligand: using ultracentrifugation precipitation method The modified extracellular vesicles were further washed with a buffer solution and centrifuged under a centrifugal force of 120,000 g for 70 minutes to obtain a high-purity extracellular vesicle with a surface-modified membrane-linking group-targeting ligand.

Example 3

[0057] This embodiment provides a method for modifying extracellular vesicles, which includes the following steps:

[0058] S1. Extracellular vesicles 1 are prepared by isolation and purification. The extracellular vesicles can be extracted from edible plants. In this embodiment, the edible plants are grapes. The extraction process is specifically:

[0059] a. Extract the juice from the grapes.

[0060] b, centrifuging the squeezed grape juice for 20 min under a centrifugal force of 5000 g, taking the supernatant, and then the supernatant Centrifuge at 12000g for 30min to remove large particles.

[0061] c. Add PEG solution (final concentration of 8% PEG solution, containing 0.5M sodium chloride) to the supernatant obtained after centrifugation in step b, and place at 4 ° C overnight.

[0062] d. Centrifuging the sample obtained in step c under a centrifugal force of 4000 x g for 30 min to obtain a pellet of microvesicles

[0063] e. Resuspend the pellet, place it on a sucrose density gradient and perform ultracentrifugation at 150,000 g for 1.5 hours. Due to the difference in density, microvesicles will be enriched at the interface between 30% and 45% sucrose after centrifugation, so as to be separated from impurities of different densities in the plant juice to obtain purified microcellular extracellular vesicles.

[0064] S2, a protein or polypeptide intercalating group 2, a linking group 3 and a GRP78 antibody targeting ligand 4 are sequentially combined together, the linking group and the intercalating group may be covalently linked or non- Covalently attach and assemble the combined intercalating group-linking group-targeting ligand composition on the surface of the purified extracellular vesicle obtained in step S1. The structure formed is shown in FIG. 2. This step is specific To: Use click chemistry to connect. First, insert membrane group 2 modified with Tetrazine or Azide (linking group 3) and targeting ligand 4 modified with Alkene, Alkyne or DBCO at the end, under certain chemical conditions. The reaction allows the intercalation group 2 and the targeting ligand 4 to be connected by forming a covalent bond. Alternatively, as a transformable embodiment, a fusion protein method can also be used to combine the inserting membrane group 2 and the targeting ligand 4, and the basic steps are: combining the inserting protein gene sequence, connecting the group gene sequence, and the targeting ligand. The gene sequence of the sequence is constructed under the same promoter as the expression of the same protein. Under certain conditions, the insert protein 2, the linker group 3, and the targeting ligand 4 protein will be expressed in a fusion form.

[0065] In this embodiment, the protein or polypeptide insertion group is an arginine-rich sequence peptide, such as the TAT protein sequence 歹 IJ is GRKKRRQRRRPQ, the NLS protein sequence 歹 ij is the KRPAAIKKAGQAKKKK, Penetratin protein sequence

RQIKIWFQNRRMKWKK transport protein sequence is GWTLNSAGYLLGKINLKALAALA KKIL

[0066] S3. Incubate the inserted membrane group-linking group-targeting ligand-purified extracellular vesicles obtained in step S2 at 15 ° C for 24 hours.

[0067] S4. The free intercalating group, the linking group and the targeting ligand that are not modified to the surface of the extracellular vesicle are removed to obtain a highly purified extracellular vesicle with surface modified ligand: using ultracentrifugation precipitation method The modified fine The extracellular vesicles were further washed with a buffer solution and centrifuged for 90 min under a centrifugal force of 10,000 g to obtain high-purity extracellular vesicles with a surface-modified intercalating group-linking group-targeting ligand.

Example 4

[0069] This embodiment provides a method for modifying extracellular vesicles, which includes the following steps:

[0070] S1. Extracellular vesicles 1 are prepared by isolation and purification. The extracellular vesicles can be extracted from edible plants. In this embodiment, the edible plants are oranges. The extraction process is as follows:

[0071] a. Extract the juice from the oranges.

[0072] b, the squeezed orange juice at 2500

Centrifuge for 23 min at a centrifugal force of g, take the supernatant, and then centrifuge the supernatant at a centrifugal force of 11200 g for 45 min to initially remove large particles of impurities.

[0073] c. Add PEG solution (final concentration of 8% PEG solution, containing 0.5M sodium chloride) to the supernatant obtained after centrifugation in step b, and place at 4 ° C overnight.

[0074] d. Centrifuging the sample obtained in step c under a centrifugal force of 3600 x g for 55 min to obtain a precipitate of microvesicles

[0075] e. Resuspend the pellet, place it on a sucrose density gradient, and perform ultracentrifugation at 130,000 g for 1.7 hours. Due to the difference in density, microvesicles will be enriched at the interface between 30% and 45% sucrose after centrifugation, so as to be separated from impurities of different densities in the plant juice to obtain purified microcellular extracellular vesicles.

[0076] S2. Porphyrin inserting group 2, linking group 3 and PSMA antibody or nucleic acid aptamer targeting ligand 4 are sequentially combined together, and the linking group and the inserting group can be Valence or non-covalent attachment, and the combined intercalating group-linking group-targeting ligand composition is assembled on the surface of the purified extracellular vesicle obtained in step S1, and the structure formed is shown in FIG. 2 . Alternatively, as a transformable embodiment, the targeting ligand may be one of EpCAM antibody or nucleic acid, CD44 antibody or nucleic acid, CD19 antibody or nucleic acid aptamer, and AF-20 antibody.

[0077] S3. Incubate the inserted membrane group-linking group-targeting ligand-purified extracellular vesicles obtained in step S2 at 75 ° C for 10 h.

[0078] S4. The free intercalating group, the linking group and the targeting ligand that are not modified to the surface of the extracellular vesicle are removed to obtain a highly purified extracellular vesicle with a surface modified ligand: using a molecular sieve method to remove Free intercalation group, linking group and targeting ligand were treated with Sepharose CL-2B column to obtain high-purity surface modification Extracellular vesicles decorated with ligands.

[0079] Obviously, the foregoing embodiment is merely an example for clear description, and is not a limitation on the implementation. For a person of ordinary skill in the art, other different forms of changes can be made based on the foregoing description. Or change. There is no need and cannot be exhaustive for all implementations. However, the obvious changes or changes introduced thereby are still within the protection scope created by this application.

Claims

Claim

[Claim 1] A method for modifying extracellular vesicles, comprising the following steps:

51. Isolate and purify extracellular vesicles;

52. The inserting group, the linking group, and the targeting ligand are sequentially connected to obtain a connected inserting group-linking group-targeting ligand composite structure, and the above composite structure is assembled into step S1. Purify the surface of extracellular vesicles;

53. Incubate the extracellular vesicles obtained in step S2;

54. The free intercalating group, the linking group, and the targeting ligand that are not modified to the surface of the extracellular vesicle are removed to obtain a highly purified extracellular vesicle with a surface modified ligand.

[Claim 2] The method for modifying extracellular vesicles according to claim 1, wherein in the step S2, the intercalating group, the linking group and the targeting ligand are obtained by a click chemical method or Fusion protein approach.

[Claim 3] The method for modifying extracellular vesicles according to claim 2, characterized in that the intercalation group is a chemically synthesized group, a protein or a polypeptide group; and the chemically synthesized group is hydrophobic The sex group is one of ethyl phosphorothioate, cholesterol, streptavidin, porphyrin, and diphenylcyclooctyne.

[Claim 4] The method for modifying extracellular vesicles according to claim 3, wherein the protein or polypeptide group is composed of a hydrophobic transmembrane or transmembrane protein.

[Claim 5] The method for modifying extracellular vesicles according to claim 4, wherein the hydrophobic transmembrane or transmembrane protein is TAT protein, NLS protein, penetratin protein, transporter protein, MPG protein, One of MAP protein, signal transduction peptide, arginine-rich, histidine, and lysine sequence peptide.

[Claim 6] The method for modifying extracellular vesicles according to claim 5, characterized in that the linking group is a chemical molecule or a polypeptide amino acid sequence; the chemical molecule is tetraazabenzene or azide Or alkyne, DBCO, BCN, TCO.

[Claim 7] The method for modifying extracellular vesicles according to claim 6, wherein the targeting ligand is an antibody, a nucleic acid, or a peptide ligand, and specifically a VEGF antibody, a GPC3 antibody, or a G RP78 antibody , EGFR antibody or nucleic acid aptamer, RGD, PSMA antibody or nucleic acid adapter Body, EpCAM antibody or nucleic acid, CD44 antibody or nucleic acid, CD 19 antibody or nucleic acid aptamer, AF-20 antibody, Her2 antibody or nucleic acid aptamer, Her3 antibody or nucleic acid aptamer.

[Claim 8] The method for modifying extracellular vesicles according to any one of claims 1-7, characterized in that, in step S1, PEG precipitation and density gradient centrifugation are used to separate and purify extracellular cells, first Centrifuge at a centrifugal force of 2000 to 5000 g for 20 to 30 minutes, and then centrifuge the supernatant at a centrifugal force of 10,000 to 12,000 g for 30 to 60 minutes to remove large particles of impurities. Add PEG solution to the resulting supernatant at 4 ° It was left at C overnight, centrifuged at 3000 ~ 4000 xg centrifugal force for 30 ~ 60min to obtain the precipitation of microvesicles, and the pellet was resuspended. The sucrose density gradient method was used for ultracentrifugation at 110000-150000 g centrifugal force for 1.5 ~ 2hr to obtain purified cells Outer vesicle.

[Claim 9] The method for modifying an extracellular vesicle according to claim 8, characterized in that the step S3 is specifically: connecting the inserted membrane group-linking group-targeting ligand- Purified extracellular vesicles were incubated at 15-100 ° C for 1-24h.

[Claim 10] The method for modifying extracellular vesicles according to claim 9, characterized in that, in step S4, a method of ultracentrifugation or molecular sieve is used to remove free intercalating group, linking group and targeting Ligand; The modified extracellular vesicles are further washed with a buffer solution and centrifuged at a centrifugal force of 100,000 to 120,000 g for 70 to 90 minutes, or treated with a Sepharose CL-2B column to obtain a high-purity extracellular surface modified with a ligand Vesicles.