CN110548138B - 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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CN110548138B
CN110548138B CN201810541138.0A CN201810541138A CN110548138B CN 110548138 B CN110548138 B CN 110548138B CN 201810541138 A CN201810541138 A CN 201810541138A CN 110548138 B CN110548138 B CN 110548138B
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aptamer
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gal
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gal liposome
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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, a cholesterol modified region of the aptamer is embedded into the alpha-Gal liposome membrane, and a nucleic acid aptamer region is exposed from the surface of the alpha-Gal liposome membrane. The preparation method comprises the following steps: hydrating an alpha-Gal liposome membrane with a buffer comprising cholesterol-modified aptamer such that cholesterol-modified aptamer is partially intercalated into the alpha-Gal liposome membrane to form an aptamer-modified alpha-Gal liposome. The aptamer modified alpha-Gal liposome has good biocompatibility and low toxicity to normal cells, and the aptamer modified alpha-Gal liposome 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 in particular relates to an aptamer modified alpha-Gal liposome and a preparation method thereof, a bispecific reagent based on the aptamer modified alpha-Gal liposome and application thereof in the field of preparing cancer treatment medicines.
Background
With the development of tumor molecular biology and immunology, tumor targeted therapies and immunotherapy have become hot spots for cancer therapy. The tumor targeted therapy has better molecular specificity, can effectively and selectively kill cancer cells, and has low side effect. Whereas the immunotherapy of tumors is to kill cancer cells by using the human autoimmune system. Whereas tumor-targeted immunotherapy combines the advantages of both, specifically causing the autoimmune system to kill cancer cells with high efficiency.
The α -Gal epitope (galactose- α -1, 3-galactose- β -1, 4-N-acetyl-glucosamine), an antigen of trisaccharides, naturally occurring on glycolipid glycoproteins, is ubiquitous in a variety of microorganisms, non-primate mammals, marsupials and new world monkeys, but not in humans, apes and old world monkeys. The location of the expression of the α -Gal epitope varies from species to species. Expressed in large amounts on glycolipids of the red blood cell membranes of rabbits and females, expressed in large amounts on cell membranes in murine Ehrlich carcinoma cells and lymphoma cells, and expressed in thyroglobulin in bovine, canine and porcine animals. About 1% of human circulating IgG is a natural antibody against a-Gal. It is due to the constant antigen stimulation of B cell production by intestinal colonies. The location of the expression of the α -Gal epitope varies from species to species.
The alpha-Gal liposome is usually a nano-carrier extracted from rabbit red blood cell membrane, and the main components are alpha-Gal glycolipid, phospholipid and cholesterol. Currently, α -Gal liposomes are used in tumor immunotherapy and need to be injected directly into tumor sites, since α -Gal liposomes can recruit anti- α -Gal antibodies to the surface of the liposomes, antigen-antibody binding activates Complement Dependent Cytotoxicity (CDC) and antibody dependent cytotoxicity (ADCC), inducing macrophages and natural killer cells to lyse cancer cells in the vicinity of α -Gal liposomes. However, such liposomes are not able to distinguish between normal cells and cancer cells, and are highly likely to cause autoimmune diseases. Therefore, the development of alpha-Gal liposome capable of specifically targeting cancer cells is expected to become a new strategy for 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 aim of the invention, the invention adopts the following technical scheme:
the embodiment of the invention provides an aptamer modified alpha-Gal liposome, which comprises the following components: the aptamer comprises an alpha-Gal liposome membrane and an aptamer, wherein the aptamer adopts a cholesterol modified nucleic acid aptamer, a cholesterol modified region of the aptamer is embedded into the alpha-Gal liposome membrane, and a nucleic acid aptamer region is exposed from the surface of the alpha-Gal liposome membrane.
In some embodiments, the nucleic acid aptamer comprises AS1411.
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 cholesterol-modified aptamer such that cholesterol-modified aptamer is partially intercalated into 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 cholesterol-modified aptamer, and then performing extrusion treatment to obtain the aptamer-modified alpha-Gal liposome with uniform particle size.
The embodiment of the invention also provides a bispecific agent, which comprises the aptamer modified alpha-Gal liposome.
The embodiment of the invention also provides application of the aptamer modified alpha-Gal liposome or the bispecific agent in preparing cancer targeted immunotherapy medicaments.
Further, the aptamer-modified α -Gal lipids are capable of specifically targeting cancer cells to provoke an attack of the body's autoimmune system against the targeted cancer cells.
The embodiment of the invention also provides a cancer targeted immunotherapeutic drug, which comprises the aptamer modified alpha-Gal liposome or a 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 reagent for tumor immunotherapy. The cholesterol modified nucleic acid aptamer is used as a main targeting agent, the biocompatibility is good, the toxicity to normal cells is low, the aptamer is modified to specifically target cancer cells, the key of treatment is that an alpha-Gal epitope on a liposome can be attached to tumor cells, a large amount of anti-Gal antibodies existing in a human body are recruited to the surfaces of the cancer cells, the antigen antibodies are combined to activate an autoimmune system, macrophages and natural killer cells are induced to lyse the cancer cells near the alpha-Gal liposome, and therefore 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 materials of the bispecific liposome are derived from cell membranes, and the bispecific liposome is easy to obtain, low in preparation cost and easy to control the process.
Drawings
FIG. 1 is a schematic diagram of an aptamer modified α -Gal liposome in accordance with an exemplary embodiment of the invention for use in tumor-targeted immunotherapy.
FIG. 2 is a schematic representation of the structure of an aptamer modified α -Gal liposome in an exemplary embodiment of the invention.
FIG. 3 is a TEM profile of aptamer modified α -Gal liposomes (Apt-Lip) obtained according to an exemplary embodiment of the invention.
FIG. 4a is a graph of hydrated particle size analysis of aptamer modified α -Gal liposomes (Apt-Lip), unmodified α -Gal liposomes (Lip) obtained according to an exemplary embodiment of the invention.
FIG. 4b is a graph comparing zeta potential of aptamer modified α -Gal liposomes (Apt-Lip) and unmodified α -Gal liposomes (Lip) obtained according to an exemplary embodiment of the invention.
FIG. 5 is a graph showing comparison of recognition of cancer cells and anti-alpha-Gal antibodies by aptamer-modified alpha-Gal liposomes obtained according to an exemplary embodiment of the invention.
FIG. 6 is a schematic representation of cytotoxicity assays in MCF-7 cells of aptamer modified α -Gal liposomes obtained according to an exemplary embodiment of the invention.
FIG. 7 is a graph comparing cell lysis in PBMC and MCF-7 cells using PBS, alpha-Gal liposomes and aptamer modified alpha-Gal liposomes, respectively, in different proportions.
FIG. 8a is a schematic of the effect of anti- α -Gal antibodies on aptamer-modified α -Gal liposome induced cell lysis.
FIG. 8b is a graph showing comparison of aptamer modified α -Gal liposomes of this invention for use in normal and cancer cell lysis.
FIG. 9 is a graph showing comparison of cell lysis of aptamer modified α -Gal liposomes obtained according to an exemplary embodiment of the invention in whole blood.
Detailed Description
Aiming at a plurality of defects in the prior art, the inventor of the present invention has provided a technical scheme of the present invention through long-term research and a large amount of practice, wherein liposome components are firstly extracted from cell membranes, and the liposome membranes are hydrated by adding buffer solution containing an aptamer, so as to obtain the aptamer modified alpha-Gal liposome. The technical scheme, the implementation process, the principle and the like are further explained as follows. It should be understood, however, that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described in the following (embodiments) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
As one aspect of the present invention, it relates to an aptamer-modified α -Gal liposome comprising: the aptamer comprises an alpha-Gal liposome membrane and an aptamer, wherein the aptamer adopts a cholesterol modified nucleic acid aptamer, a cholesterol modified region of the aptamer is embedded into the alpha-Gal liposome membrane, and a 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 particle size, on the order of 129.1-171.1 nm, and the surface zeta potential is significantly reduced, e.g., on the order of-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 was about 294nM at 1 mg/mL.
In a preferred embodiment, the aptamer modified α -Gal liposome has the structure shown in FIG. 2.
Further, the main components of the alpha-Gal liposome membrane comprise alpha-Gal glycolipid, phospholipid, cholesterol and the like.
Further, the aptamer-modified α -Gal liposome has a bilayer structure.
As another aspect 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 cholesterol-modified aptamer such that cholesterol-modified aptamer is partially intercalated into 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 cholesterol-modified aptamer, and then performing extrusion treatment to obtain the aptamer-modified alpha-Gal liposome with uniform particle size.
Further, the PC filter membrane adopted in the extrusion treatment has a pore size of 200nm.
Further, the number of times of the pressing treatment is 30 to 50 times.
Further, the preparation method may specifically include: cholesterol-modified aptamer (including AS 1411) is dissolved in a buffer, added to the α -Gal liposome membrane for hydration, and vigorously shaken to dissolve the α -Gal liposome membrane in the buffer, and extrusion is performed to obtain aptamer-modified α -Gal liposome with uniform particle size. Finally removing non-specific binding or free aptamer through ultrafiltration.
In a preferred embodiment, the method of preparation further comprises: the liposome is extruded by an extruder to obtain the aptamer modified alpha-Gal liposome (such as about 129.1-171.1 nm) with relatively uniform particle size, and the zeta potential of the surface of the aptamer modified liposome can be obviously reduced (such as about-16.26 mV to-12.14 mV).
In some embodiments, the method of making may include: uniformly mixing alpha-Gal glycolipid, phospholipid, cholesterol and a solvent, reacting for 10-16 hours at room temperature, and removing the solvent to obtain the alpha-Gal liposome membrane.
In a preferred embodiment, the method for preparing an α -Gal liposome membrane from a human cell further comprises: using human cell line as raw material, the enriched cells were subjected to hypotonic centrifugation to obtain cell membranes, which were then subjected to hypotonic centrifugation with methanol and chloroform at 1:1:2, mixing and extracting liposome membrane components of phospholipid and cholesterol in cell membranes according to the volume ratio, adding alpha-Gal glycolipid, fully mixing, and performing rotary evaporation to obtain the alpha-Gal liposome membrane.
In another preferred embodiment, the method for preparing an α -Gal liposome membrane from fresh rabbit blood further comprises: fresh rabbit blood is taken as a raw material, rabbit red blood cell membranes are obtained from the fresh rabbit blood by a hypotonic centrifugation method, then the rabbit red blood cell membranes are mixed with methanol and chloroform (1:1:2), main components of the alpha-Gal liposome membrane, namely alpha-Gal glycolipid, phospholipid and cholesterol are obtained through extraction, the room temperature reaction time is 10-16 hours, residual red blood cell membranes, proteins and the like are removed through filtration, and finally the alpha-Gal liposome membrane is obtained through rotary evaporation.
Further, the cell membrane is preferably a rabbit red blood cell membrane, but is not limited thereto.
In another aspect of embodiments of the invention there is provided a bispecific agent comprising an aptamer-modified α -Gal liposome as described above.
In another aspect, the embodiments of the present invention further provide the use of the aptamer-modified α -Gal liposomes or bispecific agents described above for the preparation of a cancer targeted immunotherapeutic agent.
Further, the aptamer modified alpha-Gal lipid can specifically target cancer cells to excite the body autoimmune system to attack the targeted cancer cells, and the principle can be seen in FIG. 1. The aptamer-modified alpha-Gal liposome and the bispecific reagent adopt the aptamer as a main targeting agent, and the aptamer has good biocompatibility, low biotoxicity, high molecular specificity and capability of selectively targeting cancer cells. The modification of the aptamer enables the alpha-Gal liposome to be a bispecific reagent, can specifically target cancer cells and enrich near the cancer cells, and causes an autoimmune system to lyse the cancer cells, thereby improving the treatment effect of the alpha-Gal liposome. Experiments show that the capacity of the bispecific reagent for cracking cancer cells is obviously higher than that of alpha-Gal liposome without aptamer modification.
For example, another aspect of embodiments of the invention also provides a cancer targeted immunotherapeutic agent comprising the aptamer-modified α -Gal liposome or bispecific agent described above.
By means of the technical scheme, the aptamer modified alpha-Gal liposome disclosed by the invention adopts the aptamer as a main targeting agent, has good biocompatibility and low toxicity to normal cells, can specifically target cancer cells through modification of the aptamer, and can cause the autoimmune system to kill the cancer cells.
The technical solution of the present invention will be described in further detail below with reference to a number of preferred embodiments and accompanying drawings, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The implementation conditions used in the following examples may be further adjusted according to actual needs, and the implementation conditions not specified are generally those in routine experiments.
Example 1 taking rabbit red blood cells as raw materials, the preparation method of the aptamer-modified alpha-Gal liposome comprises the following specific steps:
(1) Fresh rabbit blood is diluted in 1 XPBS, serum, lymphocyte and the like are removed by centrifugation for a plurality of times, and the centrifugation speed is 3000rpm/min, so that red blood cells are obtained.
(2) Then diluting the red blood cells in 0.25 XPBS for hypotonic lysis, centrifuging for many times to wash out hemoglobin and the like, wherein the centrifuging speed is 13000rpm/min, and obtaining the red blood cell membrane.
(3) Erythrocyte membrane, methanol, chloroform at 1:1: mixing 2 volumes, stirring at room temperature for 10h, filtering to remove residual erythrocyte membrane and protein, etc., to obtain an extract which is alpha-Gal glycolipid, phospholipid and cholesterol which are components of the alpha-Gal liposome membrane, and removing the organic solvent by rotary evaporation to obtain the alpha-Gal liposome membrane.
(4) And (3) after weighing the membrane, adding an aptamer buffer solution containing cholesterol modification into the alpha-Gal liposome membrane, sufficiently oscillating, and hydrating to obtain the aptamer modified alpha-Gal liposome, wherein the final concentration is 8mg/mL, and the corresponding aptamer concentration is 8mM. Similarly, unmodified α -Gal liposomes are hydrated using buffers.
(5) The obtained liposome was subjected to multiple extrusion using a 200nm filter membrane and an extruder to obtain an aptamer-modified α -Gal liposome (hereinafter, abbreviated as Apt-Lip) having a uniform particle size.
(6) And (3) performing 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 preparation method of the aptamer-modified alpha-Gal liposome comprises the following specific steps:
(1) Enriching cells, diluting in 1 XPBS, centrifuging for several times to wash out the culture medium, and obtaining cells at a centrifugation speed of 3000 rpm/min.
(2) Cells were lysed by hypotonic dilution in 0.25 XPBS, washed by centrifugation several times at 13000rpm/min to obtain cell membranes.
(3) Cell membrane, methanol, chloroform at 1:1: mixing 2 volumes, stirring at room temperature for 10h, filtering to remove residual cell membrane and protein, obtaining extract which is phospholipid and cholesterol, adding alpha-Gal glycolipid, stirring thoroughly, mixing well, and removing organic solvent by rotary evaporation to obtain alpha-Gal liposome membrane embedded with the alpha-Gal glycolipid.
(4) And (3) after weighing the membrane, adding an aptamer buffer solution containing cholesterol modification into the alpha-Gal liposome membrane, sufficiently oscillating, and hydrating to obtain the aptamer modified alpha-Gal liposome, wherein the final concentration is 8mg/mL, and the corresponding aptamer concentration is 8mM. Similarly, unmodified α -Gal liposomes (Lip) are hydrated using buffers.
(5) The obtained liposome was subjected to multiple extrusion using a 200nm filter membrane and an extruder to obtain aptamer-modified α -Gal liposome (Apt-Lip) having a uniform particle size.
(6) And (3) performing 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 aptamer modified α -Gal liposomes obtained in a typical case of the invention were tested and the results were as follows:
1. TEM topography of aptamer-modified alpha-Gal liposomes (Apt-Lip) was determined and shown in FIG. 3.
2. The hydrated particle sizes of Apt-Lip and Lip were measured as shown in FIG. 4a, and the zeta potential was measured as shown in FIG. 4 b. The test used a Markov particle sizer for particle size analysis, resulting in a particle size of Apt-Lip of 181.9 + -0.178 nm and a particle size of Lip without adapter of 191.4+ -0.123 nm. The particle sizes of the two are relatively close. Because of the negative charge of the DNA, the aptamer-linked liposome Apt-Lip potential (-14.2.+ -. 2.06 mV) was lower than that of Lip (-6.56.+ -. 0.8 mV), indicating that the liposome has successfully modified the aptamer.
3. Identification and detection of cancer cells and anti-alpha-Gal antibodies by aptamer-modified alpha-Gal liposomes.
MCF-7 cells (human breast cancer cell line) were trypsinized into suspension cells and diluted 5X 10 5 The individual cells were placed in 1.5mL centrifuge tubes, 1mg/mL of Apt-Lip, lip and PBS were added, and after 1 hour incubation at 37℃using a centrifuge tube spin incubator, the cells were centrifuged 2 times in ice 1 XPBS to wash out liposomes that had nonspecifically adsorbed to MCF-7 cells. Human IgG was diluted at 10. Mu.g/mL in iced 1 XPBS, incubated with cells at 4℃for 1h, iced 1 XPBS containing 10. Mu.g/mL FITC-modified anti-IgG antibodies was added, incubation with cells at 4℃was continued for 1h, and finally the fluorescence intensity of FL-1 was detected using a flow cytometer, and the bound FITC-modified anti-IgG antibodies were analyzed for fluorescence. Each experimental and control group was run in 4 replicates. The Apt-Lip treated MCF-7 cells of the invention fluoresced significantly more than Lip treated cells (FIG. 5), indicating that the Apt-Lip of the invention can specifically target MCF-7 cells, and IgG in solution more readily binds to liposomes on the surface of cancer cells.
4. Cytotoxicity detection of aptamer-modified α -Gal liposomes.
The cytotoxicity of the bispecific liposomes of the invention in MCF-7 cells was determined using the CCK-8 method (FIG. 5). Planting into 96-well plate at 7000 cells per well, and placing into CO 2 CulturingCulturing at 37deg.C to cell density of 60-70%, diluting liposomes (0.1-4 mg/mL) with different concentrations to complete culture medium, and filtering for sterilization. Then, 100. Mu.L of the total medium was added to each well of a 96-well plate, and 100. Mu.L of the total medium was added to the control group, and the culture was continued for 24 hours. Finally, the medium was replaced with fresh medium, 10. Mu.L of CCK-8 was added to each well, followed by culturing in an incubator for 2 hours, and the absorbance at 450nm was measured using an microplate reader. 4 replicates were made for each concentration gradient and control. The relative viability of the cells was calculated from the absorbance values. The blank group was without the addition of cell and liposome solution, and the control group was without the addition of liposome solution.
Cell relative survival (%) = 100% × (OD experiment group-OD blank)/(OD control group-OD blank)
As shown in FIG. 6, the survival rate of MCF-7 cells after incubation with the bispecific liposomes of the present invention was concentration dependent, approaching 100% below 0.2mg/mL, and still above 90% to 4 mg/mL. This indicates that the aptamer modified alpha-Gal liposomes of this invention are low in biotoxicity.
5. Lysis assay of MCF-7 cells by bispecific liposomes in different proportions of PBMC and MCF-7 cells.
Human peripheral blood mononuclear cells (PBMC cells) were extracted from fresh human blood using a human peripheral blood mononuclear cell isolate kit. CFDA-SE cell proliferation tracer probe labeled MCF-7 cells and labeled MCF-7 cells were labeled 5X 10 5 PBMC cells containing human IgG at different density gradients were diluted in 1.5mL centrifuge tubes, and Apt-Lip, lip were added and spun at low speed at 37 ℃ for 4h, and finally Propidium Iodide (PI) was added to each tube to incubate for 10min at a final concentration of 5 μg/mL to label dead cells, and the cell lysis ratio was analyzed using a flow cytometer. 4 replicates were run for each concentration gradient and control.
Cell lysis ratio (%) =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, the Apt-Lip treated MCF-7 cells of the invention were mixed with different proportions of PBMC, and the lysis rates were higher than those of Lip without aptamer modification. The ratio of immune effector cells PBMC cells (E) to MCF-7 cells (T) was 20:1 and 40:1, the cell lysis rate caused by Apt-Lip is obviously improved. This suggests that the Apt-Lip of the present invention is highly effective in targeting cancer cells and causing killing of cancer cells by PBMC cells.
6. The effect of an anti-alpha-Gal antibody on the lysis of cells by aptamer-modified alpha-Gal liposomes was examined, and the lysis of normal cells and cancer cells by aptamer-modified alpha-Gal liposomes was compared.
MCF-7 cells, HEVEC cells (human umbilical vein endothelial cells) were labeled with CFDA-SE, respectively, and the labeled cells were labeled with 5X 10, respectively 5 Individual cells were diluted in 1.5mL centrifuge tubes, PBMC cells containing human IgG (E: t=20:1) were added, and Apt-Lip, lip or PBS were spun at 37 ℃ for 4h at low speed, while a set of experiments were set up, MCF-7 cells were mixed with PBMCs without IgG and liposomes, and the effect of the presence or absence of antibodies on lysis efficiency was examined. Finally, dead cells were labeled by adding Propidium Iodide (PI) at a final concentration of 5. Mu.g/mL to each tube and incubating for 10min, and the cell lysis ratio was analyzed using a flow cytometer. 4 replicates were run for each concentration gradient and control. The results showed that the absence of IgG in the system resulted in a significant decrease in cell lysis rate (fig. 8 a), indicating that the presence of IgG and Apt-Lip is a bridge between MCF-7 cells and PBMC cells, stimulating ADCC effects, leading to lysis of MCF-7 cells. Comparison with human normal HEVEC cells (FIG. 8 b) shows that the Apt-Lip of the present invention specifically targets cancer cells.
7. The lysis rate of aptamer-modified α -Gal liposomes on cancer cells in whole blood was determined.
CFDA-SE labeled MCF-7 cells were pre-incubated with Apt-Lip, respectively, for 15min. To simulate tumor patient samples, the pretreated MCF-7 cells were mixed with healthy human blood, spun at low speed at 37℃and blood samples were taken at various time points and red blood cells were lysed using red blood cell lysates to exclude interference from red blood cells. Dead MCF-7 cells were stained with PI and analyzed for cell lysis by flow cytometry. The above results indicate that Apt-Lip binds MCF-7 cells more effectively (fig. 9), and that it is recruited by liposomes bound to the surface of MCF-7 cells to bind α -Gal antigen, resulting in ADCC effects, due to the large amount of IgG contained in human whole blood. Thus Apt-Lip was effective in stimulating lysis of MCF-7 cells.
It should be noted that, in this document, an element defined by the phrase "including … …" generally does not exclude that there are additional identical elements in a step, a process, a method or an experimental apparatus including the element.
It should be understood that the above examples are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and implement the same according to the present invention without limiting the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.

Claims (10)

1. The application of an aptamer modified alpha-Gal liposome in preparing a cancer targeted immunotherapy drug, wherein the aptamer modified alpha-Gal liposome comprises an alpha-Gal liposome membrane and an aptamer, the aptamer is a cholesterol modified nucleic acid aptamer, a cholesterol modified region of the aptamer is embedded into the alpha-Gal liposome membrane, a nucleic acid aptamer region is exposed out of the surface of the alpha-Gal liposome membrane, the particle size of the aptamer modified alpha-Gal liposome is 129.1-171.1 nm, the surface zeta potential is-16.26-12.14 mV, and the content of the aptamer in the aptamer modified alpha-Gal liposome is 3.25-4.16wt%; the aptamer modified alpha-Gal lipid can specifically target cancer cells to excite the body autoimmune system to attack the targeted cancer cells.
2. Use according to claim 1, characterized in that: the aptamer is AS1411.
3. Use according to claim 1, characterized in that: the alpha-Gal liposome membrane comprises alpha-Gal glycolipid, phospholipid and cholesterol.
4. Use according to claim 3, characterized in that: the aptamer modified alpha-Gal liposome has a bilayer structure.
5. The use according to claim 1, wherein the method for preparing the aptamer modified α -Gal liposome comprises:
providing an α -Gal liposome membrane;
hydrating the α -Gal liposome membrane with a buffer comprising cholesterol-modified aptamer such that cholesterol-modified aptamer is partially intercalated into the α -Gal liposome membrane to form an aptamer-modified α -Gal liposome.
6. The use according to claim 5, wherein the preparation method further comprises: hydrating the alpha-Gal liposome membrane with a buffer solution containing cholesterol-modified aptamer, and then extruding to obtain aptamer-modified alpha-Gal liposome with uniform particle size; the aperture of a filter membrane adopted in the extrusion treatment is 200nm; the number of times of extrusion treatment is 30-50 times.
7. The use according to claim 5, wherein the preparation method comprises: uniformly mixing the alpha-Gal glycolipid, the phospholipid, the cholesterol and the solvent, reacting for 10-16 hours at room temperature, and removing the solvent to obtain the alpha-Gal liposome membrane.
8. The use according to claim 7, characterized in that the preparation method comprises in particular: obtaining a cell membrane from a human cell line by a hypotonic centrifugation method, uniformly mixing the cell membrane, methanol and chloroform, extracting phospholipid and cholesterol in the cell membrane, adding alpha-Gal glycolipid, uniformly mixing, and performing rotary evaporation to obtain the alpha-Gal liposome membrane.
9. The use according to claim 7, characterized in that the preparation method comprises in particular: and obtaining rabbit red blood cell membranes from fresh rabbit blood by a hypotonic centrifugation method, uniformly mixing the rabbit red blood cell membranes, methanol and chloroform, extracting alpha-Gal glycolipid, phospholipid and cholesterol, reacting for 10-16 hours at room temperature, and then performing rotary evaporation to obtain the alpha-Gal liposome membrane.
10. The cancer targeted immunotherapy drug is characterized by comprising an aptamer modified alpha-Gal liposome, wherein the aptamer modified alpha-Gal liposome comprises an alpha-Gal liposome membrane and an aptamer, the aptamer is a cholesterol modified nucleic acid aptamer, a cholesterol modified region of the aptamer is embedded into the alpha-Gal liposome membrane, a nucleic acid aptamer region is exposed from the surface of the alpha-Gal liposome membrane, the particle size of the aptamer modified alpha-Gal liposome is 129.1-171.1 nm, the surface zeta potential is-16.26-12.14 mV, and the content of the aptamer in the aptamer modified alpha-Gal liposome is 3.25-4.16 wt%; the aptamer modified alpha-Gal lipid can specifically target cancer cells to excite the body autoimmune system to attack the targeted cancer cells.
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