Method for preparing heterozygous exosome based on DNA zipper mediated membrane fusion and application of heterozygous exosome in gene delivery

Abstract

The invention discloses a method for preparing a heterozygous exosome through DNA zipper mediated membrane fusion. A DNA zipper structure (ZDC / cZDC) modified by cholesterol is designed and anchored to the surface of a BMSC source exosome and the surface of a lipidosome membrane loaded with siRNA, membrane fusion is achieved through DNA complementary pairing, and after DNase I enzymolysis, the heterozygous exosome carrying siRNA and over-expressed CD146 at the same time is obtained. The method has the advantages of high fusion efficiency and uniform particle size, solves the problems of low drug loading efficiency and poor targeting property of the traditional exosome, and is suitable for gene-drug collaborative delivery.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials and nanomedicine, specifically to a method for preparing hybrid exosomes based on DNA zipper-mediated membrane fusion and its application in gene delivery. Background Technology

[0002] Exosomes, as natural delivery carriers, offer advantages such as low immunogenicity and targeting, but they also suffer from low drug loading efficiency (especially for nucleic acid drugs) and damage to membrane function during artificial drug loading. Traditional membrane fusion methods (such as PEG-induced fusion) have drawbacks such as uncontrollable fusion efficiency and toxicity from residual chemical reagents. While DNA nanotechnology can achieve precise recognition, direct modification can easily disrupt the integrity of the exosome membrane. Furthermore, current technologies struggle to simultaneously load nucleic acid drugs and functional proteins onto exosomes, limiting their application in synergistic therapy. Summary of the Invention

[0003] To address the problems of low drug loading efficiency, uncontrollable fusion efficiency, and limitations in gene-protein synergistic therapy in existing technologies, this invention discloses a method for preparing hybrid exosomes based on DNA zipper-mediated membrane fusion. This method employs serum-free culture combined with an automated exosome purification system (EXODUS H600) to extract high-purity BMSC exosomes; cholesterol-modified DNA zippers (ZDC / cZDC) are used to anchor exosomes and liposomes respectively; membrane fusion is mediated by sticky-end complementary hybridization to form hybrid exosomes; and the DNA zipper is cleaved with DNase I to obtain a dual-drug delivery system simultaneously carrying siRNA and natural functional proteins. This method overcomes the technical bottlenecks of low exosome drug loading efficiency and poor controllability of membrane fusion, providing a highly efficient delivery vector for gene-protein synergistic therapy and possessing significant clinical application prospects.

[0004] Specifically, the present invention first provides a method for preparing CD146-overexpressing exosomes, characterized in that the method includes the following steps:

[0005] 1) Select BMSCs from SD rats for passage culture. When the cell density grows to more than 80%, transfect with AAV-CD146 for 12 hours and then culture BMSCs in 10% exosome-free FBSDMEM medium to eliminate interference from serum-derived exosomes. After 24 hours, collect the cell supernatant in a 50ml centrifuge tube.

[0006] 2) Collect the cell culture medium and transfer it to a centrifuge tube for centrifugation. The precipitate obtained from this centrifugation step is the cells in the original culture medium. The centrifugation conditions are: 300g, 4℃, 10min. Discard the precipitate and keep the supernatant.

[0007] 3) Centrifuge the supernatant again. The precipitate obtained in this step is the dead cells in the original supernatant. The centrifugation conditions are: 2000g, 4℃, 15min. Discard the precipitate and keep the supernatant.

[0008] 4) Transfer the supernatant to an ultracentrifuge tube for centrifugation. The precipitate obtained from this centrifugation step is cell debris in the supernatant. Centrifugation conditions: 10,000g, 4℃, 30min.

[0009] 5) Collect the supernatant after centrifugation and filter it using a disposable 0.22μm vacuum filter to remove larger vesicles;

[0010] 6) The filtered cell supernatant was extracted and purified using the EXODUS H600 fully automated exosome extraction and purification system. After automatic extraction, the cell supernatant was filtered through a 0.22 μm filter and resuspended in 1 mL of pbs to obtain purified CD146-overexpressing exosomes from BMSCs.

[0011] Furthermore, the present invention also provides a method for preparing highly loaded siRNA liposomes, characterized in that: a specific lipid ratio (DSPC / DOPC / DOPE / cholesterol = 2:1:1:1) is used in combination with high-temperature hydration (60°C PBS), freeze-thaw cycles (5-7 times), and multi-stage extrusion technology (7 times of membrane extrusion at 800nm, 400nm, 200nm, and 100nm) to achieve siRNA-loaded liposomes with an encapsulation rate of ≥50% and a uniform particle size of 153.8±5nm.

[0012] Furthermore, the present invention also provides a method for preparing heterozygous exosomes based on DNA zipper-mediated membrane fusion, characterized in that the method includes the following steps:

[0013] 1) Preparation of DNA zipper structure (ZDC) and complementary DNA zipper (cZDC) structure

[0014] (1) Biosynthesize four short nucleotide chains for DNA zipper preparation;

[0015] (2) Take SS-A and SS-B' single-stranded DNA in a 1:1 ratio and anneal them in TE buffer (10mM Tris / HCl, 150mM NaCl, 1mM EDTA) at 75°C for 15 min to form a DNA zipper structure (ZDC), with a sticky end at the 3' end and cholesterol at the 5' end. At the same time, take SS-A' and SS-B single-stranded DNA and anneal them in the same way to form a complementary DNA zipper structure (cZDC), with a sticky end at the 5' end and cholesterol at the 3' end.

[0016] 2) ZDC and cZDC are anchored on the membranes of exosomes and liposomes, respectively.

[0017] (1) Add ZDC (5 μM, 1 μL) or cZDC to the prepared BMSC-Exo and siRNA-Lipo (1 nM particle concentration determined by NTA, 50 μL) respectively, and incubate at room temperature for 1 h;

[0018] 3) DNA zipper-mediated membrane fusion for the preparation of hybrid exosomes

[0019] (1) After ZDC and cZDC are anchored on the exosome and liposome membranes respectively, the above solutions are mixed 1:1 and incubated at room temperature for 40 min to obtain hybrid exosomes.

[0020] (2) After DNA zipper-mediated membrane fusion, the anchored ZDC and cZDC were lysed using 20 U mL-1 DNase I (Thermo Scientific) and incubated at 37 °C for 15 min. The DNase I-treated fusion vesicles were centrifuged at 10,000 rpm for 15 min using a 100 kDa ultrafiltration tube, and the resulting fusion hybrid exosome vesicles (siRNA-HEV) were diluted to their original volume (100 μL) to obtain hybrid exosomes prepared by DNA zipper-mediated membrane fusion, simultaneously carrying siRNA and overexpression.

[0021] Furthermore, the present invention also provides an optimization of exosome prefiltration, wherein the supernatant after gradient centrifugation is filtered by a 0.22μm disposable vacuum filter to remove vesicle impurities >220nm, thus solving the problem of clogging of traditional ultrafiltration membranes.

[0022] Furthermore, the present invention also provides a DNA zipper directional anchoring technology, characterized by an asymmetric cholesterol modification design, wherein the DNA zipper structure (ZDC) has a sticky end at the 3' end and cholesterol at the 5' end; and the complementary DNA zipper structure (cZDC) has a sticky end at the 5' end and cholesterol at the 3' end.

[0023] Furthermore, the present invention also provides a DNA zipper-mediated membrane fusion method, characterized in that ZDC (final concentration 5 μM) is incubated with CD146-Exo (1 nM particle concentration) at room temperature for 1 h; cZDC (final concentration 5 μM) is incubated with siRNA liposomes (1 nM particle concentration) at room temperature for 1 h;

[0024] The anchored exosomes and liposomes were mixed at a 1:1 volume ratio and incubated at room temperature for 40 min to trigger fusion.

[0025] Furthermore, the present invention also provides a biosafety removal process, characterized in that DNase I is added to a final concentration of 20 U / mL after fusion, and incubated at 37°C for 15 min; centrifuged at 10,000 rpm for 15 min using a 100 kDa ultrafiltration tube; the retentate is collected and resuspended in PBS to obtain the final product.

[0026] Furthermore, the present invention also provides a dual-function synergistic drug delivery system, characterized by using CD146-overexpressing exosomes and liposomes to encapsulate siRNA to overexpress and inhibit different genes, respectively.

[0027] The advantages of this invention are as follows: This invention obtains CD146-overexpressing exosomes by transfecting BMSCs with AAV-CD146, combines them with liposomes loaded with siRNA, utilizes cholesterol-modified DNA zippers (ZDC / cZDC) to mediate membrane fusion, and after DNase I enzymatic digestion, obtains hybrid exosomes carrying both the target protein CD146 and therapeutic siRNA. This significantly solves the technical bottlenecks of low drug loading efficiency and poor membrane fusion controllability of exosomes, providing a highly efficient delivery vector for gene-protein synergistic therapy and showing significant clinical application prospects. Attached Figure Description

[0028] Figure 1 TEM image of hybrid exosomes.

[0029] Figure 2 Distribution map of NTA in heterozygous exosomes.

[0030] Figure 3 DNA zipper-mediated membrane fusion anchoring status. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0032] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0033] Example 1

[0034] A method for preparing CD146-overexpressing exosomes, characterized in that the method comprises the following steps:

[0035] 1) Select BMSCs from SD rats for passage culture. When the cell density grows to more than 80%, transfect with AAV-CD146 for 12 hours and then culture BMSCs in 10% exosome-free FBSDMEM medium to eliminate interference from serum-derived exosomes. After 24 hours, collect the cell supernatant in a 50ml centrifuge tube.

[0036] 2) Collect the cell culture medium and transfer it to a centrifuge tube for centrifugation. The precipitate obtained from this centrifugation step is the cells in the original culture medium. The centrifugation conditions are: 300g, 4℃, 10min. Discard the precipitate and keep the supernatant.

[0037] 3) Centrifuge the supernatant again. The precipitate obtained in this step is the dead cells in the original supernatant. The centrifugation conditions are: 2000g, 4℃, 15min. Discard the precipitate and keep the supernatant.

[0038] 4) Transfer the supernatant to an ultracentrifuge tube for centrifugation. The precipitate obtained from this centrifugation step is cell debris in the supernatant. Centrifugation conditions: 10,000g, 4℃, 30min.

[0039] 5) Collect the supernatant after centrifugation and filter it using a disposable 0.22μm vacuum filter to remove larger vesicles;

[0040] 6) The filtered cell supernatant was extracted and purified using the EXODUS H600 fully automated exosome extraction and purification system. After automatic extraction, the cell supernatant was filtered through a 0.22 μm filter and resuspended in 1 mL of pbs to obtain purified CD146-overexpressing exosomes from BMSCs.

[0041] Example 2

[0042] A liposome loaded with siRNA and a method for preparing the same, characterized in that the method comprises the following steps:

[0043] 1) Accurately weigh DSPC, DOPC, DOPE, and cholesterol samples, and dissolve them in chloroform or anhydrous ethanol according to the reagent instructions to achieve a final concentration of 10 mg / mL.

[0044] 2) Dissolve the reagents DSPC, DOPC, DOPE, and cholesterol from step 1) in an organic solvent (chloroform:methanol = 3:1) at a mass ratio of 2:1:1:1 to form a lipid mixture;

[0045] 3) Place the lipid mixture from step 2) into a round-bottom flask and connect it to a rotary evaporator to evaporate at 40°C and 160 rpm for 40 min, so that a uniform liposome film is formed at the bottom of the flask.

[0046] 4) Place the liposome membrane formed by rotary evaporation in step 3) in a vacuum drying oven and dry it at 40°C for 4 hours to remove residual chloroform from the liposome membrane;

[0047] 5) Filter the PBS using a 0.22μm filter membrane, preheat it in a 60℃ water bath, and then add it to the round-bottom flask in step 4) and stir magnetically for 1 hour to hydrate it;

[0048] 6) Add 20 μm of siRNA annealed at 70 °C to step 5) for further hydration;

[0049] 7) The hydrated liposomes from step 6) are subjected to freeze-thaw cycles 5-7 times to promote the formation of a monolayer membrane structure.

[0050] 3) The lipid solution in step 2) is subjected to probe sonication and then extruded through a polycarbonate membrane at 800nm, 400nm, 200nm and 100nm in sequence 7 times using a liposome extruder to obtain liposomes with uniform particle size and liposomes loaded with siRNA.

[0051] Example 3

[0052] A liposome loaded with siRNA and a method for preparing the same, characterized in that the method comprises the following steps:

[0053] 1) Preparation of DNA zipper structure (ZDC) and complementary DNA zipper (cZDC) structure

[0054] (1) Biosynthesize four short nucleotide chains for DNA zipper preparation;

[0055] (2) Take SS-A and SS-B' single-stranded DNA in a 1:1 ratio and anneal them in TE buffer (10mM Tris / HCl, 150mM NaCl, 1mM EDTA) at 75°C for 15 min to form a DNA zipper structure (ZDC), with a sticky end at the 3' end and cholesterol at the 5' end. At the same time, take SS-A' and SS-B single-stranded DNA and anneal them in the same way to form a complementary DNA zipper structure (cZDC), with a sticky end at the 5' end and cholesterol at the 3' end.

[0056] 2) ZDC and cZDC are anchored on the membranes of exosomes and liposomes, respectively.

[0057] (1) Add ZDC (5 μM, 1 μL) or cZDC to the prepared BMSC-Exo and siRNA-Lipo (1 nM particle concentration determined by NTA, 50 μL) respectively, and incubate at room temperature for 1 h;

[0058] 3) DNA zipper-mediated membrane fusion for the preparation of hybrid exosomes

[0059] (1) After ZDC and cZDC are anchored on the exosome and liposome membranes respectively, the above solutions are mixed 1:1 and incubated at room temperature for 40 min to obtain hybrid exosomes.

[0060] (2) After DNA zipper-mediated membrane fusion, the anchored ZDC and cZDC were lysed using 20 U mL-1 DNase I (Thermo Scientific) and incubated at 37 °C for 15 min. The DNase I-treated fusion vesicles were centrifuged at 10,000 rpm for 15 min using a 100 kDa ultrafiltration tube, and the resulting fusion hybrid exosome vesicles (siRNA-HEV) were diluted to their original volume (100 μL) to obtain hybrid exosomes prepared by DNA zipper-mediated membrane fusion, simultaneously carrying siRNA and overexpression.

Claims

1. A method for preparing heterozygous exosomes based on DNA zipper-mediated membrane fusion and its application in gene delivery, characterized in that... Includes the following steps: 1) Preparation of CD146 overexpressing exosomes: AAV-CD146 was transfected into BMSCs, cultured in exosome-free medium, and CD146 overexpressing exosomes were obtained by gradient centrifugation and EXODUS H600 purification. 2) Preparation of siRNA-loaded liposomes: Using a lipid ratio of DSPC / DOPC / DOPE / cholesterol = 2:1:1:1, siRNA liposomes with an encapsulation efficiency ≥50% and a particle size of 153.8±5nm were prepared by hydration with PBS at 60℃, 5-7 freeze-thaw cycles and multi-stage extrusion technology. 3) Constructing DNA zippers: Annealing SS-A and SS-B' to form 5' cholesterol-modified ZDCs, and annealing SS-A' and SS-B to form 3' cholesterol-modified cZDCs; 4) Anchor ZDC to the exosome membrane of step (1) and cZDC to the liposome membrane of step (2), and incubate them together to achieve membrane fusion; 5) The DNA zipper was digested with DNase I and purified by ultrafiltration to obtain a hybrid exosome carrying both CD146 and siRNA.

2. The method as described in claim 1, characterized in that: In step (1), the transfection conditions for AAV-CD146 are MOI = 50000, and after 12 hours of transfection, the medium is replaced with exosome-free medium and cultured for 24 hours.

3. The method as described in claim 1, characterized in that: In step (3), the DNA annealing buffer is 10mM Tris / HCl, 150mM NaCl, and 1mM EDTA, and the annealing conditions are 75℃ for 15min.

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