WO2023193665A1 - 基于细胞外囊泡酯酶响应药物递送载体及其制备方法和用途 - Google Patents

基于细胞外囊泡酯酶响应药物递送载体及其制备方法和用途 Download PDF

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WO2023193665A1
WO2023193665A1 PCT/CN2023/085668 CN2023085668W WO2023193665A1 WO 2023193665 A1 WO2023193665 A1 WO 2023193665A1 CN 2023085668 W CN2023085668 W CN 2023085668W WO 2023193665 A1 WO2023193665 A1 WO 2023193665A1
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esterase
drug
cells
engineered
extracellular vesicles
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PCT/CN2023/085668
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English (en)
French (fr)
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王浩然
满夫龙
王俊锋
侯杰
刘冬梅
周云
张馨之
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威海纽兰生物科技有限公司
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Priority claimed from CN202310092502.0A external-priority patent/CN116889632A/zh
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Publication of WO2023193665A1 publication Critical patent/WO2023193665A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biomedicine technology, and specifically relates to a preparation method of a drug delivery carrier based on extracellular vesicle esterase response and the application of the carrier in drug loading.
  • Extracellular vesicles refer to vesicle-like bodies with a double-layer membrane structure that are shed from the cell membrane or secreted by cells. They are considered to be a new generation of natural nanoscale drug delivery systems. Extracellular vesicles secreted by different types of cells carry different signaling molecules (such as various RNAs, proteins, and lipid molecules), and therefore have huge application potential in targeted drug delivery and disease treatment. In recent years, research results on the delivery of therapeutic drugs (such as proteins, RNA and other small molecule drugs) to diseased sites through extracellular vesicles to exert their therapeutic effects have been widely reported.
  • therapeutic drugs such as proteins, RNA and other small molecule drugs
  • extracellular vesicles are mainly divided into two categories.
  • One is to use genetic engineering technology to transfect the therapeutic drug gene fragments into the maternal cells at the cellular level, and endogenously produce the therapeutic drugs.
  • Extracellular vesicles but this method is not suitable for loading chemotherapeutic drugs, and the operation is time-consuming; the other type is to use physical or chemical methods (such as ultrasound and incubation) to separate extracellular vesicles from tissue cells.
  • Therapeutic drugs are loaded into extracellular vesicles, and extracellular vesicles containing therapeutic drugs are exogenously generated. This method is simple to operate, but its drug loading rate and encapsulation rate are low, which is far from the expectations for clinical application.
  • Extracellular vesicles as new drug delivery carriers, can reduce the side effects of chemotherapeutic drugs.
  • a drug delivery carrier based on extracellular vesicle esterase-responsive drug delivery and a drug loading technology platform based on this carrier were developed. This platform can significantly improve the delivery of extracellular vesicles. Drug loading capacity and encapsulation efficiency of encapsulated drugs.
  • yeast or plant expression systems have many advantages of eukaryotic expression systems: such as protein processing and folding, post-translational modification, etc.
  • the Pichia pastoris expression system has a higher expression level than the Saccharomyces cerevisiae expression system and can perform high-density culture of cells.
  • yeast extract has strong ability to resist pollution and fight free radicals, and it is already on the market.
  • Several products containing yeast extract are sold, indicating that yeast is safe for humans.
  • the plant expression system has the advantages of simple method, low cost, easy large-scale production, convenient storage and transportation.
  • Plant cells can perform post-translational folding and glycosylation modification of expressed proteins, which is similar to commonly used mammalian cells, Escherichia coli, Compared to yeast expression systems, there is no risk of pathogen contamination.
  • the present invention selects cells derived from Pichia pastoris or tobacco external vesicles as examples for constructing esterase-responsive drug delivery carriers.
  • the present invention uses engineered cells to overexpress esterase, and then encapsulates the esterase in exosomes to construct engineered extracellular vesicles containing esterase, which are used to improve the expression of esterase.
  • the drug loading amount and encapsulation rate of the active molecules corresponding to the esterase in extracellular vesicles are determined to overcome the problem of low drug loading efficiency in existing drug loading methods.
  • the present invention provides a preparation method of a drug delivery carrier based on extracellular vesicle esterase response and its application in drug loading based on the carrier.
  • the present invention solves, at least to a certain extent, one of the technical problems in existing drug delivery systems and provides at least the advantages described below.
  • the present invention is implemented through the following technical solutions:
  • the present invention provides a method for preparing a drug delivery carrier based on extracellular vesicle esterase response, including the following steps:
  • S2 Cultivate the modified engineered cells obtained in S1, and extract engineered extracellular vesicles containing the esterase therefrom;
  • S3 The engineered extracellular vesicles containing the esterase obtained in S2 are loaded with drugs that can be hydrolyzed by esterase or the drugs are converted into esters to form prodrugs that can be hydrolyzed by esterase, thereby obtaining drug-loaded extracellular vesicles, i.e. Extracellular vesicle esterase responds to drug delivery vehicles.
  • the extracellular vesicles include one or more of the following: exosomes, apoptotic bodies, microvesicles, lysosomes, endosomes, and revesicles. Bubbles or extruded cells.
  • the extracellular vesicles are exosomes.
  • the engineered cells are selected from bacterial cells, yeast cells, fungal cells, algal cells, insect cells, mammalian cells or plant cells.
  • the engineered cells are yeast cells or Aspergillus niger.
  • yeast cells are Pichia pastoris or Saccharomyces cerevisiae cells.
  • the mammalian cells include HEK293 cells, mesenchymal stem cells (MSC), Chinese hamster ovary (CHO) cells.
  • the plant includes tobacco, Arabidopsis thaliana, rice, wheat, soybean, tomato, lettuce, ginseng or American ginseng.
  • the extracellular vesicle esterase-responsive drug delivery carrier is an extracellular vesicle containing one or more of the following esterases: feruloyl esterase (Feruloyl esterase), carboxylesterase 1 (CES1), carboxylate esterase Enzyme 2 (CES2), lipase (Lipase), fatty acid amide hydrolase (Fatty acid amide hydrolase), pectinesterase (Pectinesterase), acetylcholinesterase (Acetylcholinesterase), leukocyte esterase (Leukocyte esterase), thioesterase ( Thioesterase), Sulfatase, Phosphatase, Phosphotriesterase, Phosphodiesterase, Phosphomonoesterases or Protein tyrosine phosphatase .
  • esterases feruloyl esterase
  • CES1 carboxylesterase 1
  • CES2 carboxylate esterase Enzyme 2
  • Lipase Li
  • the extracellular vesicle esterase-responsive drug delivery carrier is an extracellular vesicle containing ferulic acid esterase, carboxylesterase 1 (CES1) or carboxylesterase 2 (CES2).
  • CES1 carboxylesterase 1
  • CES2 carboxylesterase 2
  • the drug is a drug that can be hydrolyzed by the esterase, or the drug is a prodrug that can be hydrolyzed by the esterase after esterification.
  • the esterase is selected from one or more of the following: ferulic acid esterase (Feruloyl esterase), carboxylesterase 1 (CES1), carboxylesterase 2 (CES2), lipase (Lipase) , Fatty acid amide hydrolase, Pectinesterase, Acetylcholinesterase, Leukocyte esterase, Thioesterase, Sulfatase, Phosphate Phosphatase, Phosphotriesterase, Phosphodiesterase, Phosphomonoesterases or Protein tyrosine phosphatase.
  • the drug is a drug that can be hydrolyzed by the esterase, or a prodrug that can be hydrolyzed by the esterase after the drug is esterified.
  • the drug is a compound containing a structure such as ferulate, methyl carboxylate, ethyl carboxylate, propyl carboxylate, butyl carboxylate.
  • the drug is selected from the group consisting of ethyl ferulate, methyl ferulate, ferulic acid, acetylsalicylic acid, oseltamivir, 5'-deoxy-5-fluoro-N-[(propoxy )carbonyl] cytidine, 5'-deoxy-5-fluoro-N-[(ethoxy)carbonyl] cytidine, azilsartan methyl ester, olmesartan ethyl ester or cyclopineolenol ferulate .
  • the specific steps of transforming the engineered cells in S1 include: selecting Pichia pastoris, using genetic engineering technology, introducing the esterase gene fragment, integrating it into the yeast genome, and screening A strain capable of stably expressing the esterase was obtained.
  • the specific steps of transforming Pichia pastoris in S1 include: constructing an esterase plasmid, introducing it into E. coli for amplification, linearizing the expression plasmid, electrotransfecting Pichia pastoris, and cultivating the transformed Pichia pastoris.
  • the specific steps of transforming engineered cells in S1 include: selecting plant tissues, using Agrobacterium to mediate transformation, introducing esterase gene fragments, integrating them into the plant genome, and screening to obtain Plants stably expressing the esterase.
  • the specific steps of transforming plants in S1 include: constructing an esterase plasmid, introducing it into E. coli for amplification, transforming the plasmid into Agrobacterium tumefaciens, cultivating Agrobacterium tumefaciens and allowing it to enter the plant through leaf disk method, inflorescence dip method, etc. plant cells.
  • the specific steps of transforming the engineered cells in S1 include: selecting plant leaf cells, using Agrobacterium-mediated transformation method, introducing the esterase gene fragment, and integrating it into the plant leaf cell genome. , to obtain plants capable of transiently expressing the esterase.
  • the specific steps of transforming tobacco leaf cells in S1 include: constructing an esterase plasmid, introducing it into E. coli for amplification, transforming the plasmid into Agrobacterium tumefaciens, cultivating Agrobacterium tumefaciens and injecting it into tobacco leaves.
  • the specific steps of transforming the engineered cells in S1 include: selecting Arabidopsis thaliana plants, using Agrobacterium-mediated transformation, introducing the esterase gene fragment, and integrating it into the Arabidopsis genome , screening to obtain plants that can stably express the esterase.
  • the specific steps for transforming Arabidopsis plants in S1 include: constructing an esterase plasmid, introducing it into E. coli for amplification, electroporation of the plasmid into Agrobacterium tumefaciens, cultivating Agrobacterium tumefaciens and introducing it into Arabidopsis through inflorescence dissemination. mustard cells.
  • the specific steps of extracting the engineered extracellular vesicles containing the esterase in S2 include: culturing the modified Pichia pastoris to the logarithmic growth phase, and using differential treatment on the supernatant.
  • the engineered extracellular vesicles containing the esterase are extracted by rapid centrifugation, density gradient centrifugation, molecular sieve, chromatography, filtration and other methods. Those skilled in the art can expect that the engineered extracellular vesicles have the function of hydrolyzing ester bonds.
  • the specific steps of extracting engineered extracellular vesicles containing the esterase in S2 include: culturing transgenic tobacco for 3-5 days, taking leaves in good growth status and enzymatically digesting them overnight. Differential centrifugation, filtration, ultracentrifugation and other methods are used to extract engineered extracellular vesicles containing the esterase. Those skilled in the art can expect that the engineered extracellular vesicles have the function of hydrolyzing ester bonds.
  • the specific steps of extracting engineered extracellular vesicles containing the esterase in S2 include: cultivating transgenic Arabidopsis thaliana, through three consecutive generations of antibiotic screening (seed) and genomic PCR Plants stably expressing the esterase were identified and obtained. Transgenic plants in good growth status are taken, and after overnight enzymatic hydrolysis, the engineered extracellular vesicles containing the esterase are extracted using differential centrifugation, filtration, ultracentrifugation and other methods. Those skilled in the art can expect that the engineered extracellular vesicles have the function of hydrolyzing ester bonds.
  • the drug loading method of the compound described in S3 and the engineered extracellular vesicles includes incubation method, ultrasonic method, extrusion and other methods; the conditions of the incubation method are: : 30°C-60°C; after incubation, it also includes the step of removing unloaded drugs.
  • drug-loaded extracellular vesicles i.e., extracellular vesicle esterase-responsive drug delivery carriers
  • S3 specifically includes the following steps:
  • Step 1 Dissolve ethyl ferulate in DMSO solution to obtain a drug solution
  • Step 2 Incubate the drug solution and yeast extracellular vesicle solution for 3 hours;
  • Step 3 Dilute the incubated drug-loaded system with PBS solution, and combine it with a 100kDa ultrafiltration tube to remove unloaded free drugs.
  • the concentration of the drug solution in step 1 is 0.1-0.8 mg/mL.
  • the co-incubation temperature in step 2 is 50°C.
  • This temperature parameter is the optimal temperature of the feruloyl esterase and will not damage the membrane structure of the extracellular vesicle.
  • the concentration of extracellular vesicles in step 2 is 0.4 mg/mL.
  • the mass ratio of the drug solution (including but not limited to ethyl ferulate) in step 2 to the engineered extracellular vesicles is 2:1.
  • the present invention mainly relates to a novel method for preparing a drug delivery carrier based on extracellular vesicle esterase-responsiveness.
  • Pichia pastoris or Nicotiana tabacum is selected as the engineering cell
  • ferulic acid esterase or carboxylesterase (CES1 or CES2) is selected as the esterase
  • ethyl ferulate or acetyl water is selected.
  • Cylic acid as a water-soluble chemical drug is only exemplary and not limiting.
  • the preparation method of the drug delivery carrier of the present invention can be prepared by using any type of engineered cells and any type of esterase described in the present invention, and is applicable to any drug described in the present invention.
  • the present invention provides an extracellular vesicle esterase-responsive drug delivery carrier, which is prepared by using the preparation method described in the first aspect.
  • the extracellular vesicle esterase-responsive drug delivery carrier of the present invention is a natural nanoscale drug delivery system.
  • the particle size of the drug delivery carrier is in the range of 30-800 nm, preferably in the range of 40-200 nm.
  • the present invention provides the use of the extracellular vesicle esterase-responsive drug delivery carrier described in the first or second aspect above in improving the drug-loading efficiency of extracellular vesicles, and the drug Delivery carriers improve the drug loading capacity and encapsulation efficiency of water-soluble drugs. Natural nanoparticles can be efficiently loaded with therapeutic drugs, providing a new drug-loading technology for drug delivery systems.
  • the present invention provides the use of the extracellular vesicle esterase-responsive drug delivery carrier described in the first or second aspect above in drug loading.
  • the present invention has the following beneficial effects:
  • the preparation method of the present invention can produce a large number of uniform and stable extracellular vesicles as drug carriers.
  • the engineered extracellular vesicles of the present invention have the advantages of high yield and good stability. More importantly, the engineered extracellular vesicles of the present invention contain esterases inside and have the function of hydrolyzing ester bonds and can Improve the drug loading capacity and encapsulation efficiency of the drug corresponding to the esterase.
  • Figure 1 is a schematic diagram of an exemplary yeast extracellular vesicle esterase-responsive drug loading method according to an embodiment of the present invention.
  • Figure 2 shows the construction and identification of Pichia pastoris modified by genetic technology to express ferulic acid esterase according to the embodiment of the present invention.
  • Figure 2A is the plasmid design diagram
  • Figure 2B is the PCR identification result (M is the marker, 1-5 is the modified yeast liquid group, 1'-3' is the unmodified yeast liquid group)
  • Figure 2C is the Western Blot result.
  • Figure 3 is an electron microscope image of blank extracellular vesicles (Figure 3A), engineered extracellular vesicles (Figure 3B) and drug-loaded extracellular vesicles (Figure 3C) according to an embodiment of the present invention.
  • Figure 4 shows blank extracellular vesicles (Figure 4A before drug loading, Figure 4C after drug loading) and engineered extracellular vesicles (Figure 4B before drug loading, Figure 4D after drug loading) according to embodiments of the present invention. ) particle size distribution diagram.
  • Figure 5 shows blank extracellular vesicles (Figure 5A before drug loading, Figure 5C after drug loading) and engineered extracellular vesicles (Figure 5B before drug loading, Figure 5D after drug loading) according to embodiments of the present invention. ) zeta potential distribution diagram.
  • Figure 6 is a liquid phase separation chromatogram of ethyl ferulate and ferulic acid according to an embodiment of the present invention.
  • Figure 7 shows the effects of different addition amounts of ethyl ferulate on drug loading and encapsulation efficiency according to embodiments of the present invention.
  • Figure 8 is the results of drug loading and encapsulation efficiency of two kinds of extracellular vesicles loaded with two drugs according to the embodiment of the present invention.
  • Figure 9 is a time-varying curve of the in vitro drug release rate of drug-loaded extracellular vesicles according to an embodiment of the present invention.
  • Figure 10 is the in vitro safety evaluation results of drug-loaded extracellular vesicles according to embodiments of the present invention.
  • Figure 11 shows the construction and drug loading results of the "human carboxylesterase 1-yeast extracellular vesicle-ethyl ferulate" system.
  • Figure 11A is the plasmid design diagram
  • Figure 11B is the Western Blot result diagram
  • Figure 11C is the drug loading result diagram.
  • Figure 12 shows the construction and drug loading results of the "human carboxylesterase 2-tobacco extracellular vesicles-acetylsalicylic acid" system.
  • Figure 12A is the plasmid design diagram
  • Figure 12B is the Western Blot result diagram
  • Figure 12C It is a picture of the drug loading results (in the picture, ASA means acetylsalicylic acid
  • SA means salicylic acid
  • Figure 12D is a liquid phase separation chromatogram of aspirin and salicylic acid.
  • test materials used in the following examples are all commercially available products unless otherwise specified.
  • Primer design Based on the purchased ferulic acid esterase gene sequence (XM_025603867.1) and the multiple cloning site on the expression plasmid pPICZA, a pair of specific upstream and downstream primers were designed to amplify the cDNA encoding FaeA mature peptide, upward An EcoRI cleavage site was introduced into the downstream primer FaeA-F, and a SalI cleavage site was introduced into the downstream primer FaeA-R.
  • Upstream primer 5'-TCAAAAAACAACTAATTATTCGAAACGAG GAATTC ATGAAGCAATTCTCTGCAAAATACGC-3' (the underline is the EcoRI restriction site); the downstream primer: 5'-GCTAAAACTCAATGATGATGATGATGATG GTCGAC GCCACCGCCACCAGAC-3' (the underline is the SalI restriction site).
  • the specific steps for enzyme activity measurement are: add 0.1 mL of ethyl ferulate solution and 0.1 mL of intracellular protein solution to 0.8 mL of PBS solution, react at 50°C for 10 min, and use high-performance liquid chromatography to detect the hydrolysis products Ferulic acid production.
  • Example 2 Extraction and identification of engineered extracellular vesicles
  • Differential ultracentrifugation method Collect the culture medium after induced expression, and follow the differential ultracentrifugation method at 4°C, specifically: 500g, 10min ⁇ 2000g, 20min ⁇ 10000g, 30min ⁇ 100000g, 70min, and final precipitation.
  • the material was resuspended in PBS and filtered through a 0.22 ⁇ m filter to obtain engineered extracellular vesicles, which were stored at -80°C for later use.
  • the extraction steps for extracellular vesicles secreted by unmodified Pichia pastoris were the same and were recorded as blank extracellular vesicles.
  • Vesicles (X33@EV) were used as blank control group.
  • Enzyme activity and Western Blot analysis The specific steps for the determination of feruloyl esterase activity in engineered extracellular vesicles and the determination of intracellular protease activity are the same except that the reaction time is changed to 3h. The specific steps of Western Blot are the same as those for intracellular proteins.
  • Example 3 Studying the application of engineered extracellular vesicles in improving the drug-loading efficiency of extracellular vesicles
  • the vesicle mass ratio is 2:1.
  • the two extracellular vesicles were incubated with 2 times of ethyl ferulate in a 50°C water bath for 3 hours. After the reaction was completed, they were placed at 4°C for 0.5 hours to recover and passed through a 100kDa ultrafiltration tube. 4500g x 20min to separate drug-loaded extracellular vesicles and free drugs.
  • the drug-loaded blank extracellular vesicles are marked as X33@EV-EF, and the drug-loaded engineered extracellular vesicles are marked as FaeA@EV-EF.
  • C. Determination of drug-loading performance Determine the loading capacity of engineered extracellular vesicles by measuring the contents of ethyl ferulate (EF) and ferulic acid (FA) in extracellular vesicles after drug loading using high-performance liquid chromatography. medicinal properties.
  • EF ethyl ferulate
  • FA ferulic acid
  • the liquid phase conditions of ethyl ferulate and ferulic acid are: A (1% glacial acetic acid aqueous solution): B (methanol), 60% methanol at 0min, 50% methanol at 15min, 60% methanol at 18min, detection wavelength 325nm . Subsequently, 50 ⁇ L of drug-loaded extracellular vesicles were mixed with 950 ⁇ L of methanol, disrupted by sonication for 1 hour, and passed through a 0.45 ⁇ m filter membrane for liquid phase detection.
  • the drug loading amount the measured (EF + FA) amount / the input EV amount
  • the encapsulation rate the measured (EF + FA) amount / the input EF amount.
  • the drug-loaded extracellular vesicle release curve shows that the preparation has a certain sustained release effect.
  • the CCK-8 experiment shows that Pichia pastoris extracellular vesicles have a wide range of safe concentrations for the human body.
  • Example 4 Construction and drug loading application of engineered extracellular vesicles containing human carboxylesterase 1 (CES1)
  • CES1 human carboxylesterase 1
  • the present invention In addition to constructing the "ferulic acid esterase-yeast extracellular vesicles-ethyl ferulate" system, the present invention also constructs "human carboxylesterase 1-yeast extracellular vesicles-ethyl ferulate" system. The purpose is to further confirm the universality of the yeast extracellular vesicle esterase-responsive drug delivery carrier platform.
  • Example 4 The specific process of Example 4 is the same as the process of constructing ferulic acid esterase-containing engineered extracellular vesicles.
  • the construction process and drug loading results of the two systems are shown in Figures 11A-11C.
  • the results of Western Blot analysis and drug loading data of the two systems used in this example show that human carboxylesterase 1 (CES1) engineered extracellular vesicles were successfully constructed. And can increase the loading capacity of active drug molecules (the drug loading capacity increased from less than 10% to nearly 40%, and the encapsulation rate increased from less than 8% to nearly 25%). This result expanded the use of esterase-responsive Types of drug-loaded extracellular vesicle platforms.
  • CES1 human carboxylesterase 1
  • Example 5 Construction and drug loading application of engineered extracellular vesicles containing human carboxylesterase 2 (CES2)
  • CES2 human carboxylesterase 2
  • Primer design Based on the purchased carboxylesterase 2 gene sequence (NM_001365405.1) and the multiple cloning site on the expression plasmid pCAMBIA1300, a pair of specific upstream and downstream primers were designed to amplify cDNA encoding the mature CES2 peptide, upstream Primer CES2-F: AGAACACGGGGGACGAGCTCGGTACCATGCGGCTGCACAGACTTCG, downstream primer CES2-R: TTGCTCACCATACGCGTACGAGATCTTGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCACCACCCAGCTCTGTGTGTCTCTCT.
  • Electrotransformation of Agrobacterium with recombinant expression plasmid pCAMBIA1300-CES2-eGFP was electrotransformed into Agrobacterium EHA105, and then the positive strains were identified by PCR using primers CES2-1 (CTAATTATTCGAAACGAGGAATTCATGCGGCTGCACAGACTTC) and CES2-2 (CGTCGCCGTCCAGCTCGACCAG).
  • the specific steps for enzyme activity measurement are: take 0.3g of tobacco leaves, grind them with liquid nitrogen, add 1mL of protein extract, mix and centrifuge at 10,000rpm at 4°C for 10 minutes, and suck the supernatant into a new EP tube as the protein for detection. Samples for enzyme activity determination and Western Blot analysis.
  • the specific steps for enzyme activity determination are: add 0.1 mL aspirin solution and 0.1 mL plant protein extract to 0.8 mL PBS solution, react for 10 minutes, add 1 mL acetonitrile to terminate the reaction, and use high-performance liquid chromatography to detect the hydrolysis product water Cylic acid production.
  • A. Differential ultracentrifugation method Take the tobacco leaves 5 days after instant transformation, cut them into long and thin strips with scissors, and break the cell wall; according to the differential ultracentrifugation method, specifically: 1000g, 10min ⁇ 3000g, 20 min ⁇ 5000g, 30min ⁇ 15000g, 30min ⁇ 150000g, 70min. The final precipitate is filtered with a 0.22 ⁇ m filter and resuspended in PBS buffer to remove impurities. The engineered extracellular vesicles are obtained and stored at -80°C for later use; wild type The extraction steps for tobacco extracellular vesicles are the same and are recorded as blank extracellular vesicles as a blank control group.
  • Enzyme activity and Western Blot analysis The specific steps for measuring the activity of carboxylesterase 2 in engineered extracellular vesicles are similar to those for measuring intracellular protease activity. The reaction time is extended to 2h, and the other conditions are the same. The specific steps of Western Blot are the same as those for intracellular proteins.
  • A. Esterase response drug loading experiment To explore the impact of CES2@EV (engineered extracellular vesicles of the present invention) on the drug loading capacity and encapsulation rate of acetylsalicylic acid, combine acetylsalicylic acid with CES2@EV (engineered extracellular vesicles of the present invention) Engineered extracellular vesicles) or blank EV were mixed, placed in a 37°C water bath and incubated for 2 hours. After the reaction was completed, it was placed at 4°C for 0.5h to recover, and the drug-loaded extracellular vesicles and free drugs were separated through a 100kDa ultrafiltration tube 4500g*20min. ; To explore the effects of blank extracellular vesicles and engineered extracellular vesicles on the drug loading capacity and encapsulation efficiency.
  • C. Determination of drug-loading performance Determine the drug-loading performance of engineered extracellular vesicles by measuring the contents of aspirin and salicylic acid in the extracellular vesicles after drug loading with high-performance liquid chromatography.
  • the present invention uses yeast cells or tobacco extracellular vesicles as models to develop a preparation method based on extracellular vesicle esterase-responsive drug delivery carriers.
  • the use of engineered extracellular vesicles with the acquired function of hydrolyzing ester bonds can improve the drug loading capacity and encapsulation efficiency of active pharmaceutical molecules, providing new ideas for improving the efficient loading of therapeutic drugs by extracellular vesicles.

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Abstract

本发明涉及一种基于细胞外囊泡酯酶响应药物递送载体的制备方法及所述载体在药物装载中的应用。所述制备方法包括以下步骤：S1：通过基因工程手段在工程细胞中导入酯酶基因表达质粒，并表达酯酶蛋白；S2：培养S1中得到的改造后工程细胞，并从中提取含有所述酯酶的工程化细胞外囊泡；S3：将S2中得到的含有所述酯酶的工程化细胞外囊泡装载药物，进而获得细胞外囊泡酯酶响应药物递送载体。采用本发明方法制备得到的工程化细胞外囊泡能够显著提高药物活性分子的装载效率和药物稳定性。

Description

基于细胞外囊泡酯酶响应药物递送载体及其制备方法和用途
相关申请的交叉引用
本申请要求2022年04月07日提交的中国申请号2022103653485的权益和2023年02月03日提交的中国申请号2023100925020的权益。所述申请号2022103653485和申请号2023100925020据此全文以引用方式并入本文。
技术领域
本发明属于生物医药技术领域，具体涉及基于细胞外囊泡酯酶响应药物递送载体的制备方法及基于该载体在药物装载中的应用。
背景技术
细胞外囊泡(Extracellular Vesicles,EVs)是指从细胞膜上脱落或者由细胞分泌的双层膜结构的囊泡状小体，被认为是新一代天然纳米级药物递送系统。不同类型细胞分泌的细胞外囊泡携带不同的信号分子(如多种RNA和蛋白质及脂质分子)，因此在靶向药物传递和疾病治疗方面具有巨大的应用潜力。近年来，通过细胞外囊泡输送治疗药物(如蛋白、RNA及其他小分子药物)到病变部位，进而发挥其治疗作用的研究成果已经被广泛报道。
目前，利用细胞外囊泡包裹治疗药物的方法主要分为两类，一类是借助基因工程技术，在细胞水平上将治疗药物基因片段转染进母本细胞，内源性产生含有治疗药物的细胞外囊泡，但该方法不适合化学治疗药物的装载，且操作耗时；另一类是从组织细胞中分离出细胞外囊泡后，利用物理或化学方法(如超声和孵育)，将治疗药物加载到细胞外囊泡中，外源性产生含有治疗药物的细胞外囊泡，该方法操作简单，但其载药率和包封率较低，远未达到临床应用的预期。当前很多疾病的治疗依然离不开化学治疗药物，细胞外囊泡作为新型药物递送载体，能够降低化学治疗药物带来的副作用。
为解决现有载药方法存在载药效率低的问题，开发一种基于细胞外囊泡酯酶响应药物递送载体及基于该载体所搭建的载药技术平台，该平台能够显著提高细胞外囊泡包裹药物的载药量和包封率。
作为真核表达系统的代表，酵母或植物表达系统具有真核表达系统的许多优点：如蛋白加工折叠、翻译后修饰等。例如，作为酵母表达系统的代表，毕赤酵母表达系统比酿酒酵母表达系统表达水平更高且可进行细胞的高密度培养。研究表明，酵母提取物具有强大的抵御污染和抗自由基的能力，市场上已 经有多款包含酵母提取物的产品销售，表明酵母对人体是安全的。植物表达系统则具有方法简单、成本低廉、易于规模化生产、储藏和运输方便等优点，植物细胞既能对表达蛋白进行翻译后折叠和糖基化修饰，与通常使用的哺乳细胞、大肠杆菌、酵母表达系统相比，又没有病原体污染的风险。基于以上这些优点，本发明选择毕赤酵母来源的细胞或烟草外囊泡作为实例，用于构建酯酶响应药物递送载体。受固定化酶和前药设计原理的启发，本发明利用工程细胞过表达酯酶，进而将酯酶包裹在外泌体中，构建出含有酯酶的工程化细胞外囊泡，用于提高与所述酯酶对应的活性分子在细胞外囊泡中的载药量和包封率，以克服现有载药方法存在载药效率低的问题。
发明内容
为了解决上述技术问题，本发明提供了一种基于细胞外囊泡酯酶响应药物递送载体的制备方法及其基于该载体在药物装载中的应用。本发明至少在一定程度上解决了现有药物递送系统中的技术问题之一，并提供至少后面将说明的优点。
具体地，通过以下几个方面的技术方案实现了本发明：
在第一个方面中，本发明提供了一种基于细胞外囊泡酯酶响应药物递送载体的制备方法，包括以下的步骤：
S1：通过基因工程手段在工程细胞中导入酯酶基因表达质粒，并表达酯酶蛋白；
S2：培养S1中得到的改造后工程细胞，并从中提取含有所述酯酶的工程化细胞外囊泡；
S3：将S2中得到含有所述酯酶的工程化细胞外囊泡装载酯酶能水解的药物或药物成酯后形成可被酯酶水解的前药，进而获得载药细胞外囊泡，即细胞外囊泡酯酶响应药物递送载体。
作为可选的方式，在上述制备方法中，所述细胞外囊泡包括以下一种或多种：外泌体、凋亡小体、微泡、溶酶体、内体、再囊泡化囊泡或者挤出的细胞。
优选地，所述细胞外囊泡是外泌体。
所述工程细胞选自细菌细胞、酵母细胞、真菌细胞、藻类细胞、昆虫细胞、哺乳动物细胞或植物细胞。
优选地，所述工程细胞是酵母细胞或黑曲霉菌。
更优选地，所述酵母细胞是巴斯德毕赤酵母(Pichia pastoris)或者面包酵母(Saccharomyces cerevisiae)细胞。
优选地，所述哺乳动物细胞，包括HEK293细胞、间充质干细胞(MSC)、 中国仓鼠卵巢(CHO)细胞。优选地，所述植物包括烟草、拟南芥、稻米、小麦、大豆、西红柿、生菜、人参或西洋参。所述细胞外囊泡酯酶响应药物递送载体为含有以下一种或多种酯酶的细胞外囊泡：阿魏酸酯酶(Feruloyl esterase)、羧酸酯酶1(CES1)、羧酸酯酶2(CES2)、脂肪酶(Lipase)，脂肪酸酰胺水解酶(Fatty acid amide hydrolase)，果胶酯酶(Pectinesterase)、乙酰胆碱酯酶(Acetylcholinesterase)、白细胞酯酶(Leukocyte esterase)，硫酯酶(Thioesterase)、硫酸酯酶(Sulfatase)、磷酸酶(Phosphatase)、磷酸三酯酶(Phosphotriesterase)、磷酸二酯酶(Phosphodiesterase)、磷酸单酯酶(Phosphomonoesterases)或酪氨酸磷酸酶(Protein tyrosine phosphatase)。
优选地，所述细胞外囊泡酯酶响应药物递送载体为含有阿魏酸酯酶、羧酸酯酶1(CES1)或羧酸酯酶2(CES2)的细胞外囊泡。
作为可选的方式，在上述制备方法中，所述药物为可被所述酯酶水解的药物，或者所述药物为成酯后形成可被所述酯酶水解的前药。
任选地，所述酯酶选自以下一种或多种：阿魏酸酯酶(Feruloyl esterase)、羧酸酯酶1(CES1)、羧酸酯酶2(CES2)、脂肪酶(Lipase)，脂肪酸酰胺水解酶(Fatty acid amide hydrolase)，果胶酯酶(Pectinesterase)、乙酰胆碱酯酶(Acetylcholinesterase)、白细胞酯酶(Leukocyte esterase)，硫酯酶(Thioesterase)、硫酸酯酶(Sulfatase)、磷酸酶(Phosphatase)、磷酸三酯酶(Phosphotriesterase)、磷酸二酯酶(Phosphodiesterase)、磷酸单酯酶(Phosphomonoesterases)或酪氨酸磷酸酶(Protein tyrosine phosphatase)。
与所述酯酶对应地，任选地，所述药物为可被所述酯酶水解的药物，或者为药物成酯后形成可被所述酯酶水解的前药。例如，所述药物为含有阿魏酸酯、羧酸甲酯、羧酸乙酯、羧酸丙酯、羧酸丁酯等结构的化合物。
优选地，所述药物选自阿魏酸乙酯、阿魏酸甲酯、阿魏酸、乙酰水杨酸、奥司他韦、5'-脱氧-5-氟-N-[(丙氧基)羰基]胞苷、5'-脱氧-5-氟-N-[(乙氧基)羰基]胞苷、阿齐沙坦甲酯、奥美沙坦乙酯或环木菠萝烯醇阿魏酸酯。
作为可选的方式，在上述制备方法中，S1中改造工程细胞的具体步骤包括：选取巴斯德毕赤酵母，利用基因工程技术，导入酯酶基因片段，使其整合到酵母基因组中，筛选得到能够稳定表达所述酯酶的菌株。
优选地，S1中改造毕赤酵母的具体步骤包括：酯酶质粒构建，导入大肠杆菌中扩增，表达质粒线性化，电转毕赤酵母，培养改造后毕赤酵母。
作为可选的方式，在上述制备方法中，S1中改造工程细胞的具体步骤包括：选取植物组织，利用农杆菌介导转化，导入酯酶基因片段，使其整合到植物基因组中，筛选获得能够稳定表达所述酯酶的植株。
优选地，S1中改造植物的具体步骤包括：酯酶质粒构建，导入大肠杆菌中扩增，质粒转化到根癌农杆菌，培养根癌农杆菌并通过叶盘法、花序浸染法等使其进入植物细胞。
作为可选的方式，在上述制备方法中，S1中改造工程细胞的具体步骤包括：选取植物叶片细胞，利用农杆菌介导转化法，导入酯酶基因片段，使其整合到植物叶片细胞基因组中，获得能够瞬时表达所述酯酶的植株。
优选地，S1中改造烟草叶片细胞的具体步骤包括：酯酶质粒构建，导入大肠杆菌中扩增，质粒转化到根癌农杆菌，培养根癌农杆菌并将其注射到烟草叶片。
作为可选的方式，在上述制备方法中，S1中改造工程细胞的具体步骤包括：选取拟南芥植株，利用农杆菌介导转化法，导入酯酶基因片段，使其整合到拟南芥基因组中，筛选获得能够稳定表达所述酯酶的植株。
优选地，S1中改造拟南芥植株的具体步骤包括：酯酶质粒构建，导入大肠杆菌中扩增，质粒电转至根癌农杆菌，培养根癌农杆菌并通过花序浸染法使其进入拟南芥细胞。
作为可选的方式，在上述制备方法中，S2中提取含有所述酯酶的工程化细胞外囊泡的具体步骤包括：培养改造后毕赤酵母至对数生长期，对上清液采用差速离心、密度梯度离心、分子筛、层析、过滤等方法提取含有所述酯酶的工程化细胞外囊泡。本领域技术人员能够预期的是，所述工程化细胞外囊泡具有水解酯键的功能。
作为可选的方式，在上述制备方法中，S2中提取含有所述酯酶的工程化细胞外囊泡的具体步骤包括：培养转基因烟草3-5天，取生长状态良好的叶片过夜酶解后采用差速离心、过滤、超速离心等方法提取含有所述酯酶的工程化细胞外囊泡。本领域技术人员能够预期的是，所述工程化细胞外囊泡具有水解酯键的功能。
作为可选的方式，在上述制备方法中，S2中提取含有所述酯酶的工程化细胞外囊泡的具体步骤包括：培养转基因拟南芥，通过连续三代的抗生素筛选(种子)和基因组PCR鉴定获得稳定表达所述酯酶的植株。取生长状态良好的转基因植株，过夜酶解后采用差速离心、过滤、超速离心等方法提取含有所述酯酶的工程化细胞外囊泡。本领域技术人员能够预期的是，所述工程化细胞外囊泡具有水解酯键的功能。
作为可选的方式，在上述载药方法中，S3中所述化合物与所述工程化细胞外囊泡的载药方法包括孵育法、超声法、挤压等方法；所述孵育法的条件为：30℃-60℃；孵育后，还包括去除未装载药物的步骤。
仅作为一个细胞外囊泡载药的代表性实例，S3中获得载药细胞外囊泡(即细胞外囊泡酯酶响应药物递送载体)，具体包括以下步骤：
步骤1：将阿魏酸乙酯溶于DMSO溶液，得到药物溶液；
步骤2：将药物溶液与酵母细胞外囊泡溶液共孵育3h；
步骤3：用PBS溶液稀释孵育后的载药体系，结合100kDa超滤管除去未载入的游离药物。
优选的是，步骤1中药物溶液浓度为0.1-0.8mg/mL。
优选的是，步骤2中共孵育的温度是50℃，该温度参数为所述阿魏酸酯酶的最适温度，且不会破坏细胞外囊泡的膜结构。
优选的是，步骤2中所述细胞外囊泡浓度为0.4mg/mL。
优选的是，步骤2中所述药物溶液(包括但不限于阿魏酸乙酯)，所述药物与工程化细胞外囊泡的质量比为2：1。
本领域技术人员能够理解的是，本发明主要涉及一种新型的基于细胞外囊泡酯酶响应药物递送载体的制备方法。在上述示例性制备方法中选择使用毕赤酵母或烟草作为工程细胞，以及选择使用阿魏酸酯酶或羧酸酯酶(CES1或CES2)作为酯酶和选择使用阿魏酸乙酯或乙酰水杨酸作为水溶性化学药物均仅是示例性的，而非限制性的。
本领域技术人员能够预期的是，本发明所述药物递送载体的制备方法可以采用本发明所述的任何种类的工程细胞和任何种类的酯酶制备，并且适用于本发明所述的任何药物。
在第二个方面中，本发明提供了一种细胞外囊泡酯酶响应药物递送载体，所述药物递送载体是采用上述第一个方面所述制备方法制备得到的。本发明细胞外囊泡酯酶响应药物递送载体是天然纳米级药物递送系统。所述药物递送载体的粒径在30-800nm范围，优选地，在40-200nm范围。
在第三个方面中，本发明提供了上述第一个方面或第二个方面所述的细胞外囊泡酯酶响应药物递送载体在提高细胞外囊泡载药效率中的用途，所述药物递送载体提高水溶性药物的载药量和包封率。可以实现天然纳米颗粒高效装载治疗药物，为药物递送系统提供一种新的载药技术。
在第四个方面中，本发明提供了上述第一个方面或第二个方面所述细胞外囊泡酯酶响应药物递送载体在药物装载中的用途。
本发明相对于现有技术，具有以下有益效果：
采用本发明制备方法可以产出大量、均一、稳定的细胞外囊泡作为药物载体。本发明工程化细胞外囊泡具有产率高、稳定性好等优点。更重要的是，本发明所述工程化细胞外囊泡由于其内部含有酯酶，具有水解酯键的功能，能够 提高所述酯酶对应的药物的载药量和包封率。
本发明的其他优点、目标和特征将部分通过下面的说明体现，部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。在附图中：
图1是根据本发明实施例的示例性酵母细胞外囊泡酯酶响应载药方法的示意图。
图2是根据本发明实施例的基因技术改造毕赤酵母表达阿魏酸酯酶的构建与鉴定。其中，图2A是质粒设计图、图2B是PCR鉴定结果(M为marker，1-5为改造后酵母菌液组，1’-3’为未改造酵母菌液组)和图2C是Western Blot结果。
图3是根据本发明实施例的空白细胞外囊泡(图3A)、工程化细胞外囊泡(图3B)与载药细胞外囊泡(图3C)的电镜图。
图4是根据本发明实施例的空白细胞外囊泡(图4A为载药前，图4C为载药后)、工程化细胞外囊泡(图4B为载药前，图4D为载药后)的粒径大小分布图。
图5是根据本发明实施例的空白细胞外囊泡(图5A为载药前，图5C为载药后)、工程化细胞外囊泡(图5B为载药前，图5D为载药后)的zeta电位分布图。
图6是根据本发明实施例的阿魏酸乙酯与阿魏酸的液相分离色谱图。
图7是根据本发明实施例的不同阿魏酸乙酯加入量对载药量及包封率的影响。
图8是根据本发明实施例的两种细胞外囊泡装载两种药物的载药量及包封率的结果。
图9是根据本发明实施例的载药细胞外囊泡的体外药物释放率随时间变化曲线。
图10是根据本发明实施例的载药细胞外囊泡的体外安全性评价结果。
图11是“人羧酸酯酶1-酵母细胞外囊泡-阿魏酸乙酯”体系的构建及载药结果。其中，图11A为质粒设计图，图11B为Western Blot结果图，图11C为载药结果图。
图12是“人羧酸酯酶2-烟草细胞外囊泡-乙酰水杨酸”体系的构建示及载药结果。其中，图12A为质粒设计图，图12B为Western Blot结果图，图12C 为载药结果图(图中，ASA表示乙酰水杨酸，SA表示水杨酸)，图12D为阿司匹林与水杨酸液相分离色谱图。
具体实施方式
以下通过特定的具体实例说明本发明的技术方案和应用。应理解，本发明提到的一个或多个方法步骤并不排斥在所述组合步骤前后还存在其他方法步骤或在这些明确提到的步骤之间还可以插入其他方法步骤；还应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。而且，除非另有说明，各方法步骤的编号仅为鉴别各方法步骤的便利工具，而非为限制各方法步骤的排列次序或限定本发明可实施的范围，其相对关系的改变或调整，再无实质变更技术内容的情况下，当亦视为本发明可实施的范畴。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道购买得到的常规产品。
下面实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为市售产品。
实施例1：基因技术改造毕赤酵母
A.引物设计：根据购得阿魏酸酯酶基因序列(XM_025603867.1)和表达质粒pPICZA上的多克隆位点，设计出一对扩增编码FaeA成熟肽cDNA的特异性上下游引物，向上游引物FaeA-F中引入EcoRⅠ酶切位点，向下游引物FaeA-R中引入SalⅠ酶切位点。
上游引物：5’-TCAAAAAACAACTAATTATTCGAAACGAGGAATTCATGAAGCAATTCTCTGCAAAATACGC-3’(下划线为EcoRⅠ酶切位点)；下游引物：5’-GCTAAAACTCAATGATGATGATGATGATGGTCGACGCCACCGCCACCAGAC-3’(下划线为SalⅠ酶切位点)。
B.克隆重组表达质粒：经EcoRⅠ和SalⅠ双酶切，将目的基因连接至表达质粒pPICZA，转化E.coli DH5α，经测序鉴定，重组表达质粒命为pPICZA-FaeA。
C.重组表达质粒电转化毕赤酵母：pPICZA-FaeA用SacⅠ线性化，电转化毕赤酵母X33，随后利用通用引物5’-AOX和3’-AOX进行PCR鉴定出重组子。
D.FaeA成熟肽编码基因在毕赤酵母中表达：对测序正确的重组子保种，并取出部分菌体在BMGY培养基中扩大培养，待OD值为1，转入BMMY培养基(含5‰甲醇)中诱导表达48h。将诱导表达的菌体经超声破碎仪裂解，提取胞内蛋白，用于酶活测定和Western Blot分析。空白毕赤酵母所得胞内蛋 白记为X33胞内蛋白，工程化毕赤酵母所得胞内蛋白记为FaeA胞内蛋白。其中酶活测定的具体步骤为：向0.8mL的PBS溶液中，加入0.1mL的阿魏酸乙酯溶液和0.1mL的胞内蛋白溶液，在50℃反应10min，利用高效液相色谱检测水解产物阿魏酸生成量。
结果分析：引物设计的质粒图谱如图2A所示；PCR验证重组子正确(图2B)，并且基因组测序结果正确，表明阿魏酸酯酶基因已经整合到毕赤酵母基因组中；Western Blot分析结果表明改造后的酵母能够表达出阿魏酸酯酶(图2C)。
实施例2：工程化细胞外囊泡提取与鉴定
A.差速超速离心法：收集诱导表达后的培养基，在4℃条件下，按照差速超速离心法，具体为：500g，10min→2000g，20min→10000g，30min→100000g，70min，最终沉淀物用PBS重悬并通过0.22μm滤膜过滤，得到工程化细胞外囊泡，-80℃下保存备用；未改造的毕赤酵母分泌的细胞外囊泡提取步骤同理，记为空白细胞外囊泡(X33@EV)，作空白对照组。
B.透射电镜、粒径大小及zeta电位分析：得到空白细胞外囊泡和工程化细胞外囊泡后，通过透射电子显微镜、福流纳米流式检测仪和马尔文粒度仪对其形态、粒径大小和zeta电位进行检测。
C.酶活性和Western Blot分析：工程化细胞外囊泡中阿魏酸酯酶的活性测定的具体步骤和胞内蛋白酶活测定除反应时间更改为3h，其余条件相同。Western Blot的具体步骤和胞内蛋白相同。
结果分析：形态粒径酶活力结果细胞外囊泡形态、粒径分布及zeta电位分布如图3A-3C、图4A-4D及图5A-5D所示，结果表明细胞外囊泡呈茶托状，粒径符合40-150nm范围，状态稳定；此外，工程化细胞外囊泡中的阿魏酸酯酶经Western Blot验证存在，如图2C所示。
实施例3：研究工程化细胞外囊泡在提高细胞外囊泡载药效率中的应用
A.酯酶响应载药实验：探究不同阿魏酸乙酯加入量对载药量和包封率的影响，将阿魏酸乙酯(EF)与FaeA@EV(本发明工程化细胞外囊泡)质量比为1：4-2：1共5组，置于50℃水浴中孵育3h，反应结束后置于4℃0.5h恢复，经100kDa超滤管4500g x 20min分离载药细胞外囊泡和游离药物；探究空白细胞外囊泡和工程化细胞外囊泡对两种药物的载药量和包封率的影响，由上述结果确定最佳投料比为阿魏酸乙酯与细胞外囊泡质量比为2：1，将两种细胞外囊泡分别和2倍阿魏酸乙酯置于50℃水浴中孵育3h，反应结束后置于4℃0.5h恢复，经100kDa超滤管4500g x 20min分离载药细胞外囊泡和游离药物。载药空白细胞外囊泡记为X33@EV-EF，载药工程化细胞外囊泡载药记为 FaeA@EV-EF。
B.载药后透射电镜、粒径大小及zeta电位分析：得到载药细胞外囊泡后，通过透射电子显微镜、福流纳米流式检测仪和马尔文粒度仪对其形态、粒径大小和zeta电位进行检测，与未载药细胞外囊泡进行比较。
C.载药性能的测定：通过高效液相色谱仪测定载药后细胞外囊泡中阿魏酸乙酯(EF)和阿魏酸(FA)的含量来确定工程化细胞外囊泡的载药性能。
阿魏酸乙酯和阿魏酸的液相条件为：A(1％冰醋酸水溶液)：B(甲醇)，在0min甲醇60％，在15min甲醇50％，在18min甲醇60％，检测波长325nm。随后，将50μL载药细胞外囊泡与950μL甲醇混合，经过超声破碎1h，过0.45μm滤膜进液相检测。
以nEF：nFA＝1：1换算成阿魏酸来计算载药量和包封率。其中载药量＝测得(EF+FA)量/投入EV量；包封率＝测得(EF+FA)量/投入EF量。
实验数据采用平均值±标准差表示,以GraphPad Prism 8软件进行统计学分析,组间比较采用单因素方差分析，p＜0.05表示差异有统计学意义(其中*p＜0.05、**p＜0.01)。
D.载药细胞外囊泡体外释放度和体外安全性考察：采用动态透析技术测定载药细胞外囊泡的体外释药百分率，用不同的方程对其释药百分率进行拟合，具体步骤为：分子量8kDa透析袋中装入1mL载药细胞外囊泡，32±0.5℃下悬浮于100mL PBS中并置于磁力搅拌器上；采用CCK-8试剂盒评估载药细胞外囊泡的治疗安全性。
结果分析：分别如图6-8所示，利用酯酶响应进行阿魏酸乙酯的装载，工程化细胞外囊泡能够大幅度提高阿魏酸的载药量(从原来的不足10％提高到接近55％)和包封率(从原来的不足8％提高到接近32％)载药前后，粒径有所增加，稳定性不变。
如图9所示，载药细胞外囊泡释放曲线表明该制剂具有一定的缓释作用。
如图10所示，CCK-8实验表明毕赤酵母细胞外囊泡对人体具有较宽的安全浓度。
实施例4：含有人羧酸酯酶1(CES1)工程化细胞外囊泡构建及载药应用
除构建“阿魏酸酯酶-酵母细胞外囊泡-阿魏酸乙酯”体系之外，本发明还构建了“人羧酸酯酶1-酵母细胞外囊泡-阿魏酸乙酯”体系。目的是进一步证实基于酵母细胞外囊泡酯酶响应药物递送载体平台的普适性。
实施例4的具体过程同构建含阿魏酸酯酶工程化细胞外囊泡过程。两种体系的构建过程及载药结果见图11A-11C。该实施例所用两种体系Western Blot分析和载药数据结果表明，人羧酸酯酶1(CES1)工程化细胞外囊泡构建成功， 并能够提高活性药物分子的装载量(载药量从原来的不足10％提高到接近40％，包封率从原来的不足8％提高到接近25％)，该结果扩展了以酯酶响应型载药细胞外囊泡平台的类型。
实施例5：含有人羧酸酯酶2(CES2)工程化细胞外囊泡构建及载药应用
5.1工程化烟草细胞的制备
A.引物设计：根据购得羧酸酯酶2基因序列(NM_001365405.1)和表达质粒pCAMBIA1300上的多克隆位点，设计出一对扩增编码CES2成熟肽cDNA的特异性上下游引物，上游引物CES2-F：AGAACACGGGGGACGAGCTCGGTACCATGCGGCTGCACAGACTTCG，下游引物CES2-R：TTGCTCACCATACGCGTACGAGATCTTGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCACCACCCAGCTCTGTGTGTCTCTCT。
B.克隆重组表达质粒：经KpnⅠ和BglⅡ双酶切，将目的基因无缝克隆至表达质粒pCAMBIA1300，转化E.coli DH5α，经测序鉴定，重组表达质粒命为pCAMBIA1300-CES2-eGFP。
C.重组表达质粒电转化农杆菌：pCAMBIA1300-CES2-eGFP电转化农杆菌EHA105，随后利用引物CES2-1(CTAATTATTCGAAACGAGGAATTCATGCGGCTGCACAGACTTC)和CES2-2(CGTCGCCGTCCAGCTCGACCAG)进行PCR鉴定出阳性菌株。
D.CES2成熟肽编码基因在烟草中表达：对测序正确的阳性菌株保种，并取出部分菌体在LB(Kan+Rif)培养基中培养，48h后，按照1:100(1mL菌液+100mL LB抗性培养基)扩大培养。将过夜培养的菌液经离心收集菌体，配制烟草侵染液，重悬菌体，使OD600＝0.4，室温静置2-3h，使用注射器经烟草叶片下表皮注射，3天后提取叶片蛋白，用于酶活测定和Western Blot分析。其中酶活测定的具体步骤为：取0.3g烟草叶片，液氮研磨后加1mL蛋白提取液，混匀后4℃10000rpm离心10min，将上清吸至新的EP管中，作为检测用的蛋白样品，用于酶活测定和Western Blot分析。其中酶活测定的具体步骤为：向0.8mL的PBS溶液中，加入0.1mL的阿司匹林溶液和0.1mL的植物蛋白提取液，反应10min后加入1mL乙腈终止反应,利用高效液相色谱检测水解产物水杨酸生成量。
结果分析：引物设计的质粒图谱如图12A所示；测序验证重组子正确，表明羧酸酯酶2基因已经整合到烟草基因组中。
5.2工程化细胞外囊泡提取与鉴定
A.差速超速离心法：取瞬时转化后5天的烟草叶片，用剪刀剪成细长条状，破掉细胞壁；按照差速超速离心法，具体为：1000g，10min→3000g，20 min→5000g，30min→15000g，30min→150000g，70min，最终沉淀物用0.22μm滤膜过滤除杂后的PBS缓冲液重悬，得到工程化细胞外囊泡，-80℃下保存备用；野生型烟草的细胞外囊泡提取步骤同理，记为空白细胞外囊泡，作空白对照组。
B.粒径大小分析：得到空白细胞外囊泡和工程化细胞外囊泡后，通过福流纳米流式检测仪粒径大小检测。
C.酶活性和Western Blot分析：工程化细胞外囊泡中羧酸酯酶2的活性测定的具体步骤和胞内蛋白酶活测定相似，反应时间延长至2h，其余条件相同。Western Blot的具体步骤和胞内蛋白相同。
结果分析：如图12B所示，结果表明细胞外囊泡，粒径符合40-150nm范围，状态稳定；此外，工程化细胞外囊泡中的羧酸酯酶2经Western Blot验证存在。
5.3研究工程化细胞外囊泡在提高细胞外囊泡载药效率中的应用
A.酯酶响应载药实验：探究CES2@EV(本发明工程化细胞外囊泡)对乙酰水杨酸载药量和包封率的影响，将乙酰水杨酸与CES2@EV(本发明工程化细胞外囊泡)或空白EV混合，置于37℃水浴中孵育2h，反应结束后置于4℃0.5h恢复，经100kDa超滤管4500g*20min分离载药细胞外囊泡和游离药物；探究空白细胞外囊泡和工程化细胞外囊泡对药物的载药量和包封率的影响。
B.载药后粒径大小分析：得到载药细胞外囊泡后，通过福流纳米流式检测仪对其粒径大小进行检测，与未载药细胞外囊泡进行比较。
C.载药性能的测定：通过高效液相色谱仪测定载药后细胞外囊泡中阿司匹林和水杨酸的含量来确定工程化细胞外囊泡的载药性能。
结果分析：如图12C-12D所示，利用酯酶响应进行乙酰水杨酸的装载，工程化细胞外囊泡能够提高乙酰水杨酸的载药量(从原来不足10％提高到接近20％)和包封率(从原来不足7％提高到接近15％)。
综上所述，本发明以酵母细胞或烟草细胞外囊泡为模型，开发了一种基于细胞外囊泡酯酶响应药物递送载体的制备方法。利用这种被后天赋予的水解酯键功能的工程化细胞外囊泡，能够提高药物活性分子的载药量和包封率，为提高细胞外囊泡高效装载治疗药物提供新的思路。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。

Claims (10)

  1. 一种基于细胞外囊泡酯酶响应药物递送载体的制备方法，其特征在于：包括以下步骤：
    S1：通过基因工程手段在工程细胞中导入酯酶基因表达质粒，并表达酯酶蛋白；
    S2：培养S1中得到的改造后工程细胞，并从中提取含有所述酯酶的工程化细胞外囊泡；
    S3：将S2中得到的含有所述酯酶的工程化细胞外囊泡装载药物，进而获得载药细胞外囊泡，即细胞外囊泡酯酶响应药物递送载体。
  2. 根据权利要求1所述的制备方法，其特征在于：所述细胞外囊泡包括以下一种或多种：外泌体、凋亡小体、微泡、溶酶体、内体、再囊泡化囊泡或者挤出的细胞，优选地，所述细胞外囊泡是外泌体。
  3. 根据权利要求1所述的制备方法，其特征在于：所述细胞外囊泡酯酶响应药物递送载体为含有以下一种或多种酯酶的细胞外囊泡：阿魏酸酯酶(Feruloyl esterase)、羧酸酯酶1(CES1)、羧酸酯酶2(CES2)、脂肪酶(Lipase)，脂肪酸酰胺水解酶(Fatty acid amide hydrolase)，果胶酯酶(Pectinesterase)、乙酰胆碱酯酶(Acetylcholinesterase)、白细胞酯酶(Leukocyte esterase)，硫酯酶(Thioesterase)、硫酸酯酶(Sulfatase)、磷酸酶(Phosphatase)、磷酸三酯酶(Phosphotriesterase)、磷酸二酯酶(Phosphodiesterase)、磷酸单酯酶(Phosphomonoesterases)或酪氨酸磷酸酶(Protein tyrosine phosphatase)。
  4. 根据权利要求1所述的制备方法，其特征在于：所述工程细胞选自酵母细胞、细菌细胞、藻类细胞、昆虫细胞、哺乳动物细胞或植物细胞，优选地，所述工程细胞是酵母细胞或黑曲霉菌；所述哺乳动物细胞是HEK293细胞、间充质干细胞(MSC)或中国仓鼠卵巢(CHO)细胞；或者，所述植物是烟草、拟南芥、稻米、小麦、大豆、西红柿、生菜、人参或西洋参。
  5. 根据权利要求1所述的制备方法，其特征在于：所述药物为可被所述酯酶水解的药物，或者所述药物为成酯后形成可被所述酯酶水解的前药。
  6. 根据权利要求5所述的制备方法，其特征在于：所述药物含有酯结构，优选所述药物含有阿魏酸酯、羧酸甲酯、羧酸乙酯、羧酸丙酯或羧酸丁酯。
  7. 根据权利要求1所述的制备方法，其特征在于：S1中改造工程细胞的具体步骤包括：选取酵母细胞，利用基因工程技术，导入酯酶基因片段，使其整合到酵母基因组中，筛选得到能够稳定表达所述酯酶的菌株；或者，S1中改造工程细胞的具体步骤包括：选取植物组织，利用农杆菌介导转化，导入酯酶基因片段，使其整合到植物基因组中，筛选获得能够稳定表达所述酯酶的植株； 或者，S1中改造工程细胞的具体步骤包括：选取植物叶片细胞，利用农杆菌介导转化法，导入酯酶基因片段，使其整合到植物叶片细胞基因组中，获得能够瞬时表达所述酯酶的植株。
  8. 采用权利要求1至7中任一项所述的制备方法制备得到的基于细胞外囊泡酯酶响应药物递送载体。
  9. 权利要求8所述的细胞外囊泡酯酶响应药物递送载体在提高外泌体载药效率中的用途，其特征在于：所述药物递送载体提高药物的载药量和包封率。
  10. 权利要求8所述的细胞外囊泡酯酶响应药物递送载体在药物装载中的应用。
PCT/CN2023/085668 2022-04-07 2023-03-31 基于细胞外囊泡酯酶响应药物递送载体及其制备方法和用途 WO2023193665A1 (zh)

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