CN110548138A - aptamer modified alpha-Gal liposome and preparation method and application thereof - Google Patents

aptamer modified alpha-Gal liposome and preparation method and application thereof Download PDF

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CN110548138A
CN110548138A CN201810541138.0A CN201810541138A CN110548138A CN 110548138 A CN110548138 A CN 110548138A CN 201810541138 A CN201810541138 A CN 201810541138A CN 110548138 A CN110548138 A CN 110548138A
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aptamer
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gal
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gal liposome
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CN110548138B (en
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裴仁军
洪珊妮
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
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Abstract

the invention discloses an aptamer modified alpha-Gal liposome and a preparation method and application thereof. The aptamer-modified alpha-Gal liposome comprises an alpha-Gal liposome membrane and an aptamer, wherein the aptamer adopts a cholesterol-modified nucleic acid aptamer, the cholesterol modification region of the aptamer is embedded into the alpha-Gal liposome membrane, and the nucleic acid aptamer region is exposed from the surface of the alpha-Gal liposome membrane. The preparation method comprises the following steps: hydrating the α -Gal liposome membrane with a buffer comprising a cholesterol-modified aptamer such that the cholesterol-modified aptamer is partially embedded in the α -Gal liposome membrane to form an aptamer-modified α -Gal liposome. The alpha-Gal liposome modified by the aptamer has good biocompatibility and low toxicity to normal cells, and the modification of the aptamer can specifically target cancer cells and can cause the autoimmune system to kill the cancer cells.

Description

Aptamer modified alpha-Gal liposome and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an aptamer-modified alpha-Gal liposome and a preparation method thereof, a bispecific agent based on the aptamer-modified alpha-Gal liposome, and application of the bispecific agent in the field of preparation of cancer treatment drugs.
Background
With the development of tumor molecular biology and immunology, tumor-targeted therapy and immunotherapy have become hot spots for cancer treatment. The tumor targeted therapy has better molecular specificity, can effectively and selectively kill cancer cells, and has low side effect. The immunotherapy of tumor is to utilize the human body's own immune system to kill cancer cells. The tumor-targeted immunotherapy can combine the advantages of the two, and specifically causes the autoimmune system to kill cancer cells efficiently.
An α -Gal epitope (galactose- α -1,3-galactosyl- β -1,4-N-acetyl-glucosamine), which is a trisaccharide antigen, is naturally produced on glycolipid glycoproteins and is ubiquitous in various microorganisms, non-primate mammals, marsupials, and new world monkeys, but is not present in humans, apes, and old world monkeys. The location of the alpha-Gal epitope expression varies from species to species. The protein is expressed on glycolipids of red blood cell membranes of rabbits and females in a large amount, is expressed on glycoprotein on cell membranes in ehrlich ascites carcinoma cells and lymphoma cells of rats, and is expressed on thyroglobulin in bovines, canines and porcines. Approximately 1% of circulating IgG in humans is a natural antibody against α -Gal. It is produced by the persistent antigenic stimulation of B cells by intestinal colonies. The location of the alpha-Gal epitope expression varies from species to species.
The alpha-Gal liposome is usually a nano-carrier extracted from rabbit blood erythrocyte membranes, and mainly comprises alpha-Gal glycolipid, phospholipid and cholesterol. The alpha-Gal liposome is used for tumor immunotherapy and needs to be directly injected into a tumor site, and because the alpha-Gal liposome can collect anti-alpha-Gal antibodies on the surface of the liposome, the combination of antigen and antibody activates Complement Dependent Cytotoxicity (CDC) and antibody dependent cytotoxicity (ADCC), and macrophages and natural killer cells are induced to lyse cancer cells nearby the alpha-Gal liposome. However, such liposomes do not distinguish between normal and cancerous cells and are highly likely to cause autoimmune disease. Therefore, the development of the alpha-Gal liposome capable of specifically targeting cancer cells is expected to become a new strategy of tumor targeted immunotherapy.
Disclosure of Invention
The invention mainly aims to provide an aptamer modified alpha-Gal liposome and a preparation method and application thereof, so as to overcome the defects in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
The embodiment of the invention provides an aptamer modified alpha-Gal liposome, which comprises: the aptamer adopts a cholesterol modified nucleic acid aptamer, the cholesterol modified region of the aptamer is embedded into the alpha-Gal liposome membrane, and the nucleic acid aptamer region is exposed from the surface of the alpha-Gal liposome membrane.
in some embodiments, the nucleic acid aptamer comprises AS 1411.
The embodiment of the invention also provides a preparation method of the aptamer modified alpha-Gal liposome, which comprises the following steps:
Providing an α -Gal liposome membrane;
Hydrating the α -Gal liposome membrane with a buffer comprising a cholesterol-modified aptamer such that the cholesterol-modified aptamer is partially embedded in the α -Gal liposome membrane to form an aptamer-modified α -Gal liposome.
In some embodiments, the method of making further comprises: and hydrating the alpha-Gal liposome membrane with a buffer solution containing a cholesterol-modified aptamer, and then performing extrusion treatment to obtain the aptamer-modified alpha-Gal liposome with uniform particle size.
Embodiments of the invention also provide a bispecific agent comprising an aptamer-modified α -Gal liposome as described above.
The embodiment of the invention also provides the application of the aptamer modified alpha-Gal liposome or the dual-specific reagent in preparing a cancer targeted immunotherapy medicament.
Further, the aptamer-modified α -Gal liposome can specifically target cancer cells to provoke the body's own immune system to attack the targeted cancer cells.
embodiments of the invention also provide a cancer-targeted immunotherapeutic drug comprising the aforementioned aptamer-modified α -Gal liposome or bispecific agent.
Compared with the prior art, the invention has the beneficial effects that:
The aptamer modified bispecific alpha-Gal liposome provided by the invention is a novel bispecific agent for tumor immunotherapy. The aptamer modified by cholesterol is used as a main targeting agent, has good biocompatibility and low toxicity to normal cells, can specifically target cancer cells by modification of the aptamer, and has the key point of treatment that an alpha-Gal epitope on a liposome can also be attached to tumor cells, so that anti-Gal antibodies existing in a large amount in a human body are recruited to the surfaces of the cancer cells, the antigen-antibody combination activates an autoimmune system, macrophages and natural killer cells are induced to crack the cancer cells nearby the alpha-Gal liposome, and the treatment effect of the alpha-Gal liposome is improved; meanwhile, the preparation method of the invention is simple, and is different from the conventional liposome in that the main raw material of the bispecific liposome is derived from cell membranes, the preparation method is easy to obtain, the preparation cost is low, and the process is easy to control.
Drawings
FIG. 1 is a schematic diagram of aptamer-modified α -Gal liposomes for tumor-targeted immunotherapy in an exemplary embodiment of the invention.
FIG. 2 is a schematic diagram of the structure of an aptamer-modified α -Gal liposome in an exemplary embodiment of the invention.
FIG. 3 is a TEM image of aptamer-modified α -Gal liposomes (Apt-Lip) obtained according to an exemplary embodiment of the present invention.
FIG. 4a is a diagram showing the analysis of the hydrated particle size of an aptamer-modified α -Gal liposome (Apt-Lip) and an unmodified α -Gal liposome (Lip) according to an exemplary embodiment of the present invention.
FIG. 4b is a graph showing a comparison of zeta potentials of aptamer-modified α -Gal liposomes (Apt-Lip) and unmodified α -Gal liposomes (Lip) according to an exemplary embodiment of the present invention.
FIG. 5 is a graph comparing the recognition of cancer cells and anti- α -Gal antibodies by aptamer-modified α -Gal liposomes obtained according to an exemplary embodiment of the present invention.
FIG. 6 is a schematic diagram showing the results of cytotoxicity assay of aptamer-modified α -Gal liposomes in MCF-7 cells, according to an exemplary embodiment of the present invention.
FIG. 7 is a graph comparing cell lysis in PBMC and MCF-7 cells using PBS, α -Gal liposomes and aptamer-modified α -Gal liposomes, respectively, at different ratios.
FIG. 8a is a schematic representation of the effect of anti- α -Gal antibodies on aptamer-modified α -Gal liposome-induced cell lysis.
FIG. 8b is a graph comparing the application of the aptamer-modified α -Gal liposomes of the present invention to normal and cancer cell lysis.
FIG. 9 is a comparison of the cell lysis of aptamer-modified α -Gal liposomes obtained according to an exemplary embodiment of the present invention in whole blood.
Detailed Description
In view of the defects of the prior art, the inventors of the present invention have made extensive studies and practice to propose a technical solution of the present invention, which is to extract liposome components from cell membranes, and hydrate the liposome membranes by adding a buffer containing an aptamer, thereby obtaining the aptamer-modified α -Gal liposome. The technical solution, its implementation and principles, etc. will be further explained as follows. It is to be understood, however, that within the scope of the present invention, each of the above-described features of the present invention and each of the features described in detail below (examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
As an aspect of the technical solution of the present invention, it relates to an aptamer-modified α -Gal liposome comprising: the aptamer adopts a cholesterol modified nucleic acid aptamer, the cholesterol modified region of the aptamer is embedded into the alpha-Gal liposome membrane, and the nucleic acid aptamer region is exposed from the surface of the alpha-Gal liposome membrane.
In some embodiments, the nucleic acid aptamer may preferably be AS1411, but is not limited thereto.
In some embodiments, the aptamer-modified α -Gal liposomes are relatively uniform in size, about 129.1 to 171.1nm, and have a significantly reduced surface zeta potential, e.g., about-16.26 to-12.14 mV.
Further, the content of the aptamer in the aptamer-modified alpha-Gal liposome is 3.25-4.16 wt%.
Further, the concentration of the modified aptamer on the α -Gal liposome membrane at 1mg/mL was about 294 nM.
In a preferred embodiment, the aptamer-modified α -Gal liposome has the structure shown in fig. 2.
Further, the main components of the α -Gal liposome membrane include α -Gal glycolipids, phospholipids, cholesterol and the like.
Further, the aptamer-modified α -Gal liposome has a bilayer structure.
As another aspect of the technical solution of the present invention, it relates to a method for preparing an aptamer-modified α -Gal liposome, comprising:
Providing an α -Gal liposome membrane;
Hydrating the α -Gal liposome membrane with a buffer comprising a cholesterol-modified aptamer such that the cholesterol-modified aptamer is partially embedded in the α -Gal liposome membrane to form an aptamer-modified α -Gal liposome.
In some embodiments, the method of making further comprises: and hydrating the alpha-Gal liposome membrane with a buffer solution containing a cholesterol-modified aptamer, and then performing extrusion treatment to obtain the aptamer-modified alpha-Gal liposome with uniform particle size.
Further, the aperture of the PC filter membrane adopted by the extrusion treatment is 200 nm.
Furthermore, the number of times of the extrusion treatment is 30-50.
Further, the preparation method may specifically include: dissolving the cholesterol modified aptamer (including AS1411) in a buffer, adding the buffer to the alpha-Gal liposome membrane for hydration, violently shaking to dissolve the alpha-Gal liposome membrane in the buffer, and extruding to obtain the aptamer modified alpha-Gal liposome with uniform particle size. Finally, removing non-specific binding or free aptamer through ultrafiltration.
in a preferred embodiment, the preparation method further comprises: the liposome is extruded by an extruder to obtain the aptamer-modified alpha-Gal liposome with relatively uniform particle size (such as about 129.1-171.1 nm), and the zeta potential of the surface of the liposome after aptamer modification is obviously reduced (such as about-16.26-12.14 mV).
In some embodiments, the method of making can comprise: and uniformly mixing the alpha-Gal glycolipid, the phospholipid, the cholesterol and the solvent, reacting for 10-16h at room temperature, and removing the solvent to obtain the alpha-Gal liposome membrane.
In a preferred embodiment, the method for producing an α -Gal liposome membrane from a human cell further comprises: using a human cell line as a raw material, and obtaining cell membranes from the enriched cells through hypotonic centrifugation, wherein the cell membranes, methanol and chloroform are mixed in a ratio of 1: 1: 2, extracting liposome membrane components phospholipid and cholesterol in the cell membrane, adding alpha-Gal glycolipid, mixing uniformly, and performing rotary evaporation to obtain the alpha-Gal liposome membrane.
In another preferred embodiment, the method for preparing α -Gal liposome membrane from fresh rabbit blood further comprises: fresh rabbit blood is used as a raw material, rabbit blood erythrocyte membranes are obtained from the fresh rabbit blood through a hypotonic centrifugation method, then the rabbit blood erythrocyte membranes are mixed with methanol and chloroform (1: 1: 2), main components of alpha-Gal liposome membranes, namely alpha-Gal glycolipids, phospholipids and cholesterol, are obtained through extraction, the reaction time is 10-16h at room temperature, residual erythrocyte membranes, proteins and the like are removed through filtration, and finally the alpha-Gal liposome membranes are obtained through rotary evaporation.
Further, the cell membrane is preferably rabbit blood erythrocyte membrane, but is not limited thereto.
Yet another aspect of the embodiments of the invention provides a bispecific agent comprising an aptamer-modified α -Gal liposome as described above.
In another aspect of the embodiments of the present invention, there is also provided a use of the aptamer-modified α -Gal liposome or bispecific agent described above for the preparation of a medicament for cancer-targeted immunotherapy.
Further, the aptamer-modified α -Gal liposome can specifically target cancer cells to provoke the body's own immune system to attack the targeted cancer cells, the principle of which can be seen in fig. 1. The aptamer-modified alpha-Gal liposome and the bispecific agent adopt the aptamer as a main targeting agent, and the aptamer has good biocompatibility, low biotoxicity and high molecular specificity and can selectively target cancer cells. The modification of the aptamer enables the alpha-Gal liposome to become a dual-specific reagent, can specifically target cancer cells, and is enriched near the cancer cells to cause an autoimmune system to crack the cancer cells, so that the therapeutic effect of the alpha-Gal liposome is improved. Tests show that the capacity of the bispecific agent for cracking cancer cells is obviously higher than that of alpha-Gal liposome without aptamer modification.
For example, another aspect of the embodiments of the present invention also provides a cancer-targeting immunotherapeutic agent comprising the aforementioned aptamer-modified α -Gal liposome or bispecific agent.
By the technical scheme, the aptamer modified alpha-Gal liposome adopts the aptamer as a main targeting agent, has good biocompatibility and low toxicity to normal cells, can specifically target cancer cells by modifying the aptamer, and can cause the autoimmune system to kill the cancer cells.
The technical solutions of the present invention will be described in further detail below with reference to several preferred embodiments and accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The conditions used in the following examples may be further adjusted as necessary, and the conditions used in the conventional experiments are not generally indicated.
Example 1 a preparation method of an aptamer-modified α -Gal liposome, using rabbit blood erythrocytes as a raw material as an example, comprises the following specific steps:
(1) Diluting fresh rabbit blood in 1 × PBS, centrifuging for several times to remove serum, lymphocyte, etc., and centrifuging at 3000rpm/min to obtain erythrocyte.
(2) Diluting in 0.25 × PBS for hypotonic lysis of erythrocyte, centrifuging for many times to remove hemoglobin, etc., and centrifuging at 13000rpm/min to obtain erythrocyte membrane.
(3) Erythrocyte membrane, methanol, chloroform in a 1: 1: 2, mixing, stirring at room temperature for 10h, filtering to remove residual erythrocyte membranes, proteins and the like, obtaining an extracting solution which is alpha-Gal liposome membrane components, namely alpha-Gal glycolipids, phospholipids and cholesterol, and removing the organic solvent by rotary evaporation to obtain the alpha-Gal liposome membrane.
(4) weighing the membrane weight, adding an aptamer buffer solution modified by cholesterol into the alpha-Gal liposome membrane, fully oscillating, hydrating to obtain the aptamer-modified alpha-Gal liposome, wherein the final concentration is 8mg/mL, and the corresponding aptamer concentration is 8 mM. Similarly, the alpha-Gal liposome without modification is hydrated by using a buffer solution.
(5) The obtained liposomes were subjected to multiple extrusion using a 200nm filter and an extruder to obtain aptamer-modified α -Gal liposomes (hereinafter, they may be abbreviated as Apt-Lip) having a uniform particle diameter.
(6) And (3) carrying out centrifugal ultrafiltration on the obtained Apt-Lip by using a 100KDa ultrafiltration tube to remove redundant unmodified free aptamer, and finally obtaining the aptamer modified alpha-Gal liposome.
Example 2 taking human cells as raw materials, the method for preparing the aptamer-modified alpha-Gal liposome comprises the following steps:
(1) Enriching cells, diluting in 1 × PBS, centrifuging for many times to wash out the culture medium, and obtaining the cells at the centrifugation speed of 3000 rpm/min.
(2) Then diluting the cell in 0.25 XPBS for hypotonic lysis, and centrifuging and cleaning for multiple times at the speed of 13000rpm/min to obtain cell membranes.
(3) cell membrane, methanol, chloroform in a 1: 1: mixing the two solutions in volume 2, stirring at room temperature for 10h, filtering to remove residual cell membranes, proteins and the like to obtain an extracting solution containing phospholipid and cholesterol, adding alpha-Gal glycolipid, fully stirring and uniformly mixing, and performing rotary evaporation to remove the organic solvent to obtain the alpha-Gal liposome membrane embedded with the alpha-Gal glycolipid.
(4) Weighing the membrane weight, adding an aptamer buffer solution modified by cholesterol into the alpha-Gal liposome membrane, fully oscillating, hydrating to obtain the aptamer-modified alpha-Gal liposome, wherein the final concentration is 8mg/mL, and the corresponding aptamer concentration is 8 mM. Similarly, the unmodified α -Gal liposome (Lip) is hydrated by using a buffer.
(5) The obtained liposome was subjected to multiple extrusion using a 200nm filter and an extruder to obtain an aptamer-modified α -Gal liposome (Apt-Lip) having a uniform particle size.
(6) And (3) carrying out centrifugal ultrafiltration on the obtained Apt-Lip by using a 100KDa ultrafiltration tube to remove redundant unmodified free aptamer, and finally obtaining the aptamer modified alpha-Gal liposome.
The test results of the aptamer-modified alpha-Gal liposome obtained in a typical case of the present invention are as follows:
Firstly, TEM topography of aptamer-modified alpha-Gal liposome (Apt-Lip) is determined, as shown in FIG. 3.
Secondly, the Apt-Lip and Lip hydrated particle sizes were measured as shown in FIG. 4a, and the zeta potential was measured as shown in FIG. 4 b. This test, using a Malvern particle sizer for particle size analysis, yielded Apt-Lip particle sizes of 181.9 + -0.178 nm, while the Lip particle size without aptamer was 191.4 + -0.123 nm. The particle sizes of the two are relatively close. Since the DNA is negatively charged, the potential of Apt-Lip of the aptamer-linked liposome (-14.2 + -2.06 mV) is lower than the potential of Lip (-6.56 + -0.8 mV), indicating that the liposome has successfully modified the aptamer.
And thirdly, detecting the recognition of the aptamer modified alpha-Gal liposome to cancer cells and anti-alpha-Gal antibodies.
MCF-7 cells (human breast cancer cell line) were trypsinized into suspension cells, and Apt-Lip, Lip and PBS were added in an amount of 1mg/mL, respectively, to a centrifuge tube diluted to 5X 10 5 cells to 1.5mL, and after culturing at 37 ℃ for 1h using a centrifuge tube rotary incubator, ice 1 XPBS was centrifuged 2 times to wash out liposomes nonspecifically adsorbed to MCF-7 cells, human IgG was diluted at 10. mu.g/mL in ice 1 XPBS, after incubating with cells at 4 ℃ for 1h, ice 1 XPBS containing 10. mu.g/mLFITC-modified anti-IgG antibody was added, and the cells were incubated at 4 ℃ for 1h, finally FL-1 fluorescence intensity was detected using a flow cytometer, and fluorescence of FITC-modified anti-IgG antibody bound was analyzed, 4 replicates per experimental and control groups, Apt-Lip-treated MCF-7 cells of the present invention showed significantly higher fluorescence than Lip-treated cells (FIG. 5), and it was more easily demonstrated that the surface of the present invention's Apt-Lip-7 cells could be bound to the target the MCF-7 cells specifically to the MCF-7 cells.
And fourthly, detecting the cytotoxicity of the aptamer modified alpha-Gal liposome.
The cytotoxicity of the bispecific liposomes of the present invention in MCF-7 cells was determined by the CCK-8 method (FIG. 5). A96-well plate was seeded at a density of 7000 cells per well, placed in a CO 2 incubator, incubated at 37 ℃ to a cell density of 60-70%, liposomes (0.1-4mg/mL) at different concentrations were diluted in complete medium, sterilized by filtration, added to the 96-well plate, 100. mu.L per well, 100. mu.L of complete medium was added to the control group, incubation was continued for 24h, finally the medium was replaced with fresh medium, 10. mu.L of CCK-8 was added to each well, the incubator was followed for 2h, absorbance at 450nm was determined using a microplate reader. 4 replicates per concentration gradient and control group.
Relative cell survival rate (%) -100% × (OD experimental group-OD blank)/(OD control group-OD blank)
As shown in FIG. 6, the survival rate of MCF-7 cells incubated with the inventive bispecific liposomes was concentration dependent, approaching 100% below 0.2mg/mL and still above 90% at 4 mg/mL. This indicates that the aptamer-modified α -Gal liposomes of the invention have low biotoxicity.
And fifthly, detecting the lysis of the MCF-7 cells by the bispecific liposome in different proportions of PBMC and MCF-7 cells.
Using a human peripheral blood mononuclear cell separation solution kit, extracting human peripheral blood mononuclear cells (PBMC cells) from fresh human blood, labeling the MCF-7 cells by a CFDA-SE cell proliferation tracer probe, diluting the labeled MCF-7 cells in a 1.5mL centrifuge tube by 5 × 10 5, adding PBMC cells containing human IgG with different density gradients and Apt-Lip, rotating at a low speed for 4h at 37 ℃, finally adding Propidium Iodide (PI) with a final concentration of 5 μ g/mL into each tube, incubating for 10min to label dead cells, and analyzing the cell lysis rate by using a flow cytometer, wherein each concentration gradient and a control group are in 4 parallels.
Cell lysis rate (%). 100% × (SL-STCL-SECL)/(ML-STCL)
SL indicates the cell lysis rate of the experimental group, STCL indicates the spontaneous cell lysis rate, SECL indicates the spontaneous effector cell lysis rate, and ML indicates the highest cell lysis rate.
As shown in FIG. 7, Apt-Lip treated MCF-7 cells of the invention, when mixed with PBMCs of varying proportions, all exhibited higher lysis rates than Lip without aptamer modification. Immune effector cells PBMC cells (E) to MCF-7 cells (T) ratio of 20:1 and 40: 1, the cell lysis rate by Apt-Lip was significantly increased. This indicates that Apt-Lip of the present invention can efficiently target cancer cells and cause cancer cell killing by PBMC cells.
And sixthly, detecting the influence of the anti-alpha-Gal antibody on the cell lysis caused by the aptamer-modified alpha-Gal liposome, and comparing the cell lysis caused by the aptamer-modified alpha-Gal liposome and the cell lysis caused by normal cells and cancer cells.
MCF-7 cells and HEVEC cells (human umbilical vein endothelial cells) are labeled with CFDA-SE respectively, the labeled cells are diluted with 5 × 10 5 cells respectively in a 1.5mL centrifuge tube, PBMC cells (E: T ═ 20:1) containing human IgG and Apt-Lip, Lip or PBS are added, the centrifuge tube is rotated at a medium and low speed for 4h at 37 ℃, a group of experiments are simultaneously set, the MCF-7 cells are mixed with PBMC not containing the IgG and liposome, and the influence of the existence of the antibody on the lysis efficiency is detected.
And seventhly, determining the cracking rate of the aptamer-modified alpha-Gal liposome to cancer cells in whole blood.
CFDA-SE labeled MCF-7 cells were previously incubated with Apt-Lip and Lip, respectively, for 15 min. To simulate a tumor patient sample, the above pretreated MCF-7 cells were mixed with healthy human blood, spun at medium and low speeds at 37 ℃, blood samples were taken at different time points, and red blood cells were lysed using red blood cell lysate to eliminate interference of red blood cells. The addition of PI to dead MCF-7 cells staining, flow cytometry analysis of cell lysis rate. The above results indicate that Apt-Lip binds MCF-7 cells more efficiently (FIG. 9), and that due to the high amount of IgG contained in human whole blood, it is recruited by liposomes bound to the cell surface of MCF-7 to bind to α -Gal antigen, causing ADCC effect. Thus Apt-Lip can effectively stimulate the lysis of MCF-7 cells.
It should be noted that, in the present context, an element defined by the phrase "comprising … …" does not exclude the presence of other identical elements in steps, processes, methods or experimental facilities including the element.
It should be understood that the above-mentioned examples are only for illustrating the technical idea and features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the content of the present invention and to implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims (15)

1. An aptamer-modified α -Gal liposome, comprising an α -Gal liposome membrane and an aptamer, wherein the aptamer employs a cholesterol-modified nucleic acid aptamer, the cholesterol-modified region of the aptamer is embedded in the α -Gal liposome membrane, and the nucleic acid aptamer region is exposed from the surface of the α -Gal liposome membrane.
2. The aptamer-modified α -Gal liposome of claim 1, wherein: the nucleic acid aptamer comprises AS 1411.
3. The aptamer-modified α -Gal liposome of claim 1, wherein: the particle size of the aptamer-modified alpha-Gal liposome is 129.1-171.1 nm, and the surface zeta potential is-16.26-12.14 mV.
4. The aptamer-modified α -Gal liposome of claim 1, wherein: the content of the aptamer in the aptamer-modified alpha-Gal liposome is 3.25-4.16 wt%.
5. The aptamer-modified α -Gal liposome of claim 1, wherein: the α -Gal liposome membrane comprises an α -Gal glycolipid, a phospholipid and cholesterol.
6. The aptamer-modified α -Gal liposome of claim 5, wherein: the aptamer-modified alpha-Gal liposome has a bilayer structure.
7. a method for preparing an aptamer-modified α -Gal liposome, comprising:
Providing an α -Gal liposome membrane;
hydrating the α -Gal liposome membrane with a buffer comprising a cholesterol-modified aptamer such that the cholesterol-modified aptamer is partially embedded in the α -Gal liposome membrane to form an aptamer-modified α -Gal liposome.
8. The method of claim 7, further comprising: hydrating the alpha-Gal liposome membrane with a buffer solution containing a cholesterol-modified aptamer, and then performing extrusion treatment to obtain an aptamer-modified alpha-Gal liposome with uniform particle size; preferably, the pore diameter of the filter membrane adopted by the extrusion treatment is 200 nm; preferably, the number of times of the extrusion treatment is 30 to 50.
9. The production method according to claim 7, characterized by comprising: and uniformly mixing the alpha-Gal glycolipid, the phospholipid, the cholesterol and the solvent, reacting for 10-16h at room temperature, and removing the solvent to obtain the alpha-Gal liposome membrane.
10. the method according to claim 9, comprising: obtaining cell membrane from human cell line by hypotonic centrifugation method, mixing the cell membrane, methanol and chloroform uniformly, extracting phospholipid and cholesterol in the cell membrane, adding alpha-Gal glycolipid, mixing uniformly, and rotary evaporating to obtain alpha-Gal liposome membrane.
11. The method according to claim 9, comprising: obtaining rabbit blood erythrocyte membranes from fresh rabbit blood by a hypotonic centrifugation method, uniformly mixing the rabbit blood erythrocyte membranes, methanol and chloroform, extracting alpha-Gal glycolipids, phospholipids and cholesterol, reacting at room temperature for 10-16h, and then performing rotary evaporation to obtain the alpha-Gal liposome membranes.
12. A bispecific agent characterized by comprising an aptamer-modified α -Gal liposome of any one of claims 1-6.
13. Use of the aptamer-modified α -Gal liposome of any one of claims 1-6 or the bispecific agent of claim 12 for the preparation of a medicament for cancer-targeted immunotherapy.
14. Use according to claim 13, characterized in that: the aptamer-modified alpha-Gal liposome can specifically target cancer cells to stimulate the body's own immune system to attack the targeted cancer cells.
15. A cancer-targeting immunotherapeutic drug characterized by comprising the aptamer-modified α -Gal liposome of any one of claims 1 to 6 or the bispecific agent of claim 12.
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CN112190715A (en) * 2020-10-21 2021-01-08 中国人民解放军军事科学院军事医学研究院 Nano medicine, its preparation method and medicinal application
CN114410636A (en) * 2021-12-31 2022-04-29 郑州大学 Improved AS1411 aptamers and EVs conjugated therewith

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ABDEL-MOTAL UM 等: "Mechanism for increased immunogenicity of vaccines that form in vivo immune complexes with the natural anti-Gal antibody", 《VACCINE》 *
XING H等: "Selective Delivery of an Anticancer Drug with Aptamer-Functionalized Liposomes to Breast Cancer Cells in Vitro and in Vivo", 《J MATER CHEM B》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112190715A (en) * 2020-10-21 2021-01-08 中国人民解放军军事科学院军事医学研究院 Nano medicine, its preparation method and medicinal application
CN112190715B (en) * 2020-10-21 2022-09-30 中国人民解放军军事科学院军事医学研究院 Nano medicine, its preparation method and medicinal application
CN114410636A (en) * 2021-12-31 2022-04-29 郑州大学 Improved AS1411 aptamers and EVs conjugated therewith

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