CN113440610B - Liposome carrying CD73 antibody and adriamycin together, preparation method and application thereof - Google Patents
Liposome carrying CD73 antibody and adriamycin together, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a liposome carrying CD73 antibody and adriamycin together, a preparation method and application thereof, wherein the liposome carrying CD73 antibody and adriamycin together is provided with a lipid core wrapped with adriamycin in the liposome, the outside of the lipid core is modified with CD73 antibody, and the CD73 antibody is coupled on a phospholipid PEG active ester micelle and then modified on the surface of the lipid core. The liposome carrying the CD73 antibody and the adriamycin together provided by the invention reduces the drug resistance and toxic and side effects of the adriamycin in the treatment of triple negative breast cancer, improves the biological distribution of the adriamycin in vivo, improves the bioavailability, twists the immune microenvironment in triple negative breast cancer tumor, converts the immune microenvironment into heat tumor, and realizes better treatment effect on triple negative breast cancer.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to a liposome carrying a CD73 antibody and adriamycin together, and a preparation method and application thereof.
Background
Breast cancer is the most common malignancy among women worldwide, with morbidity and mortality rates leading to a high percentage of many cancers. Triple Negative Breast Cancer (TNBC) is one of the subtypes, has poor pathological and histological classification, high malignancy, and rapid growth of tumor tissues, and once the tumor develops, the tumor is easy to have early recurrence and metastasis, and has the greatest characteristic of strong invasiveness, namely, the tumor is especially easy to be transferred to other tissues of the body through blood or lymph, and mainly shows visceral metastasis (lung metastasis and liver metastasis) and brain metastasis. And the clinical prognosis is poor, the traditional Chinese medicine composition is insensitive to various treatment schemes, and the treatment effect is not ideal. The currently common local treatment is surgical treatment, but the local recurrence rate and regional lymph node metastasis rate of patients with triple-negative breast cancer after surgical treatment are higher than those of non-triple-negative breast cancer. Therefore, the prognosis of triple negative breast cancer is difficult to assess on the commonly used local scale of tumor size, regional lymph node status, etc. And the existing endocrine treatment aiming at the maturity in the malignant tumor treatment and the molecular targeted treatment aiming at the HER2 gene are not effective on the triple negative breast cancer. Therefore, more effective treatment regimens need to be sought.
Doxorubicin (DOX) belongs to an anthracycline drug, has a wide antitumor spectrum, a strong antitumor effect and a definite curative effect, can kill tumor cells and promote immunogenic death (ICD) of the tumor cells, but clinically applied chemotherapeutic doxorubicin can cause serious side effects such as alopecia, bone marrow suppression, cardiotoxicity and the like, particularly cardiotoxicity is the most serious toxic and side effect of the drug, so that the clinical application of the drug is limited, and long-term doxorubicin chemotherapy can cause drug resistance.
Disclosure of Invention
The inventors have found in their studies that the currently more effective treatment regimes are mostly a combination of immunization and chemotherapy, but are not sensitive to immune checkpoint inhibitors since the tumor microenvironment of triple negative breast cancer corresponds to a state of immune depletion (cold). Clinical application of immunotherapy is also limited due to the TNBC immune failure (cold) microenvironment. At the same time, tumor hypoxia induces a broad network of metabolic and immune changes that favor tumor growth and progression, limits the availability of energy sources, and induces the accumulation of extracellular ATP, followed by the accumulation of adenosine. Extracellular ATP is rapidly degraded to adenosine, which binds to P1 purinergic receptors (also known as adenosine receptors) to block immune cell infiltration and activation. To this end, in order to ameliorate the deficiencies of the prior art, the inventors have made the following assumptions, which include the transformation of TNBC from a cold tumor to a hot tumor state, i.e., the transformation of the environment inside the TNBC tumor from no or only few immune cells to a high content of immune cells, including T cells, macrophages, etc., to achieve the most effective anti-cancer effect, while blocking the adenosine pathway to effectively reverse the immunosuppressive effect of the tumor microenvironment.
In addition, the inventors found that CD73 is expressed on the surface of various cell lines in the tumor microenvironment, such as cancer cells, DC cells, Treg cells, NK cells, MDSCs, and TAMs. And the expression of CD73 is regulated by molecules such as hypoxia inducible factor-1 (HIF-1), TGF-beta, EGFR, AKT, beta-catenin and the like. Is closely related to the poor prognosis of tumor patients. CD73 has the effects of inhibiting tumor immunity, promoting tumor growth, metastasis and angiogenesis. In addition, CD73 is also involved in the generation of anti-tumor drug resistance, which brings great challenges to tumor treatment. Based on this, the inventors tried to block the CD73 energy pathway, using CD73 antibody administered in concert with doxorubicin to try to apply to the treatment of triple negative breast cancer. Unexpectedly, however, the expected ideal effect is difficult to be exerted in the form of composition and administration prepared into conjugate, which is shown in that although the sensitivity of the immune chemotherapy can be improved, the improvement of the cardiotoxicity and the drug resistance of the adriamycin is not obvious, and the treatment effect on the triple negative breast cancer still needs to be improved.
Therefore, the inventor further provides a preparation, the co-loading of the CD73 antibody and the adriamycin is realized in the preparation, the scheme converts the triple negative breast cancer from cold tumor to hot tumor, so that the triple negative breast cancer is more sensitive to immune chemotherapy, the in vivo biological distribution of the adriamycin is improved, the toxic and side effect of the adriamycin on heart is reduced, meanwhile, the drug resistance is reduced, the preparation can deliver the adriamycin and the CD73 antibody to the triple negative breast cancer tumor part in a combined manner, the accumulation of the drug at the tumor part is promoted, the loss of the drug in the animal body is reduced, and the anti-tumor effect on the triple negative breast cancer is better exerted.
Specifically, the present invention provides the following technical features, and one or a combination of the following technical features constitutes the technical solution of the present invention.
In a first aspect of the invention, the invention provides a liposome (CDL) loaded with CD73 antibody and adriamycin together, wherein the liposome is internally provided with a lipid core wrapped with adriamycin, and the outside of the lipid core is modified with CD73 antibody.
In an embodiment of the invention, the CD73 antibody is conjugated to phospholipid PEG active ester micelles, which are then modified on the surface of the lipid core.
In some embodiments of the invention, the phospholipid PEG active ester is DSPE-PEG-NHS (distearoylphosphatidylethanolamine-polyethylene glycol-succinimidyl ester). The inventors are studyingIt was found that the reaction with the antibody is simpler when DSPE-PEG-NHS is used, only stirring is needed at a specific temperature, and the integrity and activity of the antibody can be ensured to a greater extent. If DSPE-PEG-Mal (distearoylphosphatidylethanolamine-polyethylene glycol-maleimide) is linked with antibody, the space structure of antibody is destroyed, and DSPE-PEG-NH2Or DSPE-PEG-COOH, is often required to be activated and then linked to the antibody. The inventors have demonstrated through extensive experiments that the modified antibody using DSPE-PEG3400-NHS is more stable in forming liposomes than other weight average molecular weights (e.g., Mw = 2000).
In an embodiment of the invention, the lipid core is a transparent threaded bilayer structure.
In a second aspect of the present invention, the present invention provides a method for preparing the CD73 antibody and doxorubicin-co-loaded liposome described in the first aspect above, comprising: preparing lipid core entrapping adriamycin from DOPC (1, 2-dioleoyl lecithin), DOPE (1, 2-oleoyl phosphatidylethanolamine) and CHEMS (cholesteryl succinate), coupling CD73 antibody on DSPE-PEG-NHS micelle, and modifying CD73 coupled on DSPE-PEG-NHS (distearoyl phosphatidylethanolamine-polyethylene glycol-succinimidyl ester) on the surface of the lipid core by using a post-insertion method.
In some embodiments of the invention, DOPC, DOPE and CHEMS form the lipid core, wherein the molar ratio of DOPC, DOPE and CHEMS is 1:3:2 to 1:3:0.5, preferably 1:3:1 to 1:3:0.5, and the ratio of the three affects the formation and stability of the liposome. Liposomes with better stability can be obtained within the range of the ratio of the invention, especially at a ratio of 2:6:1, and the particle size of the liposomes is optimal at this ratio.
In some embodiments of the invention, the mass ratio of doxorubicin to lipid core is 1:20 to 1: 10; preferably 1:15 to 1:10, under which conditions the liposome has the highest drug loading, and a too high ratio may cause instability of the liposome, especially when the ratio is 1:10, the liposome is stable and the drug loading is the highest.
In some embodiments of the invention, the molar ratio of CD73 antibody to DSPE-PEG-NHS is 1: 3. Within the range, the utilization rate of the DSPE-PEG-NHS is the highest, ideal connection can be realized, the connection rate is not lower than 80%, and a better treatment effect can be realized.
Further, in some embodiments of the present invention, the method comprises dissolving DOPC, DOPE, CHEMS in an organic solvent (such as chloroform) to prepare a phospholipid solution, performing rotary evaporation to form a lipid membrane with a transparent spiral thread shape, adding an ammonium sulfate solution, performing ultrasonic treatment in a water bath, and mixing to obtain a liposome solution; removing free ammonium sulfate by dialysis, adding adriamycin solution into liposome solution, heating in water bath under stirring, and removing unencapsulated adriamycin by dialysis to obtain lipid core solution coated with adriamycin;
dispersing phospholipid PEG active ester in solvent (such as chloroform), evaporating under reduced pressure to form phospholipid PEG active ester micelle, adding PBS solution, performing ultrasonic treatment in water bath, adding CD73 antibody into the solution, stirring in water bath, adding glycine to stop reaction to obtain antibody solution, adding the antibody solution into lipid core solution coated with adriamycin, stirring in water bath, and removing free antibody to obtain liposome carrying CD73 antibody and adriamycin.
In some embodiments of the invention, the rotary evaporation is performed at a rotational speed of 60 to 100rpm/min, at a temperature of 20 to 40 ℃, preferably 20 to 23 ℃; the concentration of the ammonium sulfate solution is 100-250 mmol/L.
In some embodiments of the present invention, the doxorubicin solution is a PBS solution of doxorubicin, and the temperature of the water bath after the doxorubicin solution is added is 35-45 ℃ (40 ℃ is more preferred), and the stirring rate is 500-.
In some embodiments of the invention, the CD73 antibody is reacted with phospholipid PEG active ester micelle in a water bath at a temperature of 20 ℃ to 50 ℃ for a stirring time of 3h to 5 h.
In a third aspect of the invention, the invention provides a pharmaceutical composition comprising a liposome co-loaded with the CD73 antibody and doxorubicin as described in the first aspect above.
In a fourth aspect of the present invention, the present invention provides a pharmaceutical preparation comprising the CD73 antibody-co-loaded liposome described in the first aspect above and doxorubicin, and at least one pharmaceutically acceptable excipient.
In a fifth aspect of the present invention, the present invention provides a use of the liposome co-loaded with CD73 antibody and doxorubicin described in the first aspect above, or the pharmaceutical composition described in the third aspect above, or the pharmaceutical preparation described in the fourth aspect above, for the preparation of a medicament for the treatment of triple negative breast cancer.
Compared with the prior art, the invention has the advantages that: the invention provides a liposome carrying CD73 antibody and adriamycin together, which can specifically aim at triple negative breast cancer, convert the triple negative breast cancer from cold tumor to hot tumor, improve the immune exhaustion state in the tumor microenvironment, reverse the immune suppression in the tumor, the environment in the tumor of the triple negative breast cancer is changed from no or few immune cells to more immune cells (including T cells, macrophages and the like), so that the tumor is more sensitive to the immune chemotherapy, and improves the biological distribution of the adriamycin in vivo, reduces the toxic and side effect of the adriamycin on the heart, meanwhile, drug resistance is reduced, and the liposome can jointly deliver the adriamycin and the CD73 antibody to a triple negative breast cancer tumor part, promote the accumulation of the drug at the tumor part, reduce the loss of the drug in an animal body, and better exert the anti-tumor effect on triple negative breast cancer.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a graph showing the results of the CDL in vitro release behavior experiment in example 1 of the present invention.
FIG. 2 shows the killing effect of CDL on triple negative breast cancer cells (4T1 cells) at 24 hours (left panel) and 48 hours (right panel) in example 2 of the present invention, whereinp<0.001，**p<0.01，*p<0.05。
FIG. 3 is a graph showing the in vivo distribution of fluorescence at various times after tail vein injection of Free DOX, DOXLIP, and CDL in the in vivo distribution study of example 3.
FIG. 4 is a graph showing in vitro organ distribution profiles at various times following tail vein injection of Free DOX, DOXLIP, and CDL in the in vivo distribution study of example 3, wherein heart (H), liver (L), spleen (S), lung (L), kidney (K), and tomor (T).
FIG. 5 is a graph showing fluorescence quantitation at different times after tail vein injection of Free DOX, DOXLIP, CDL in example 3 in an in vivo distribution study, where n =3, and results are expressed as mean. + -. SDp<0.01，*p<0.05。
FIG. 6 is a graph showing the inhibitory effect of CDL on triple negative breast cancer tumors in example 4 of the present invention ((***p<0.001）。
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Preparation example 1
Preparation of immunochemotherapeutic liposomes (CDL) co-loaded with CD73 antibody and DOX
The CD73 antibody used in the examples of the present invention is a commercial product, and is purchased from BioXcell biotechnology limited, usa.
1. Preparation of DOX-Supported liposomes (DOX Lip)
First, DOPC at an appropriate concentration was mixed in proportion: DOPE: CHEMS = mole ratio 2:6:1 was dissolved in 3mL chloroform, rotary evaporated, water bath temperature 22 ℃, rotation speed 80rpm/min, finally formed with transparent spiral-grained lipid membrane, then thoroughly dried to remove solvent.
2. A solution of ammonium sulfate (250 mmoL, pH = 5.5) suitably preheated to 40 ℃ was added to the mixture in a water bath and sonicated, and the extrusion was repeated several times using a miniliposome extruder. Putting the obtained solution into treated dialysis bag (3000 Da), dialyzing in PBS solution at constant speed for 4h, changing dialysate every 2h, and removing free ammonium sulfate to obtain blank liposome. In the form of liposomes; doxorubicin = 10: 1, heating to 40 ℃ in a water bath, rotationally stirring at the speed of 800rpm for 1h, and removing unencapsulated adriamycin by a dialysis method to finally obtain the purified adriamycin liposome.
3. Preparing immunochemotherapeutic liposome (CDL) carrying CD73 antibody and DOX together by using a post-insertion method, dispersing DSPE-PEG3400-NHS into chloroform, and evaporating to dryness in vacuum by using a rotary evaporator to form a film. Adding PBS solution and performing water bath ultrasound. The antibody was added in a molar ratio of CD73 antibody to DSPE-PEG3400-NHS =1:3, stirred in a water bath at 50 ℃ for 4h, and the reaction was stopped by the addition of glycine. The obtained antibody solution was added to a doxorubicin liposome solution, stirred in a water bath at 40 ℃ for 1 hour (800rpm), and free antibody was removed by dialysis to obtain the final CDL. Purifying and storing at 4 ℃.
The physical and chemical properties of the prepared CDL are measured, the particle size is 216.3 +/-4.40 nm, the PDI is 0.215 +/-0.006, the entrapment rate is 99.8 +/-0.140%, and the drug-loading rate is 9.8 +/-0.147%. The particle size, the encapsulation efficiency and the drug loading rate are good.
Example 1 CDL in vitro Release behavior study
The experimental method comprises the following steps: the same volume of free doxorubicin (free DOX), CDL formulation (preparation 1) was placed in a dialysis bag, all samples were thermostatically shaken in PBS to simulate an in vivo environment at 37 ℃, 100 r/min, and the sample solutions were sampled at different time points (0.25, 0.5, 1,2, 4, 6, 8, 10, 12, 24, 48, 72h, 96 h) and added again to PBS medium of the same pH. The sample solution was passed through a 0.22 μm organic filter and the in vitro release behaviour of the sample was analysed by recording its peak area using HPLC detection. The results of the in vitro release behavior of CDL are shown in fig. 1.
As can be seen from FIG. 1, there is a significant decrease in the release rate of doxorubicin from CDL. At pH =7.4, the cumulative release rate of the formulation at 72h was 23.15 + -0.58%, showing significant sustained release effect, at pH =5.0, the formulation was kept at a fast release rate, and by 72h, the cumulative release rate had reached 99.13 + -0.42%, indicating that CDL has pH sensitivity and has more desirable release effect.
Example 2 killing effect of CDL on triple negative breast cancer cells
Cell killing experiment: triple negative breast cancer cells (4T1 cells) at 4X 104cells/mL, 200. mu.L/well were plated in 96-well plates and incubated overnight for adherence. The experimental components were as follows: blank liposome, adriamycin liposome (DOX Lip), liposome (CDL) carrying adriamycin and CD73 antibody together, adding complete culture medium into control group, respectively converting into DOX concentration (0.002, 0.02, 0.2, 2, 20 μ g/mL), and adding into corresponding 96-well plate; the blank liposome set was added to the corresponding wells at a concentration of (0.02, 0.2, 2, 20, 200 μ g/mL) and a volume of 100 μ L of drug was added per well. After the operation is finished, the culture box is placed for 24h/48 h. The prepared CCK-8 solution (10. mu.L/well) was added to each sample well and placed in an incubator for 1 hour. The maximum absorbance value of the micropore microplate reader is set at 450nm, and the sample holes are scanned to collect data for further analysis. Relative Cell Viability (RCV) (%) was calculated using the following formula:
RCV% = average absorbance of experimental group/average absorbance of control group × 100%
Each preparation group is provided with 5 multiple holes.
The killing effect of CDL on triple negative breast cancer (4T1 cells) is shown in fig. 2.
As can be seen from the results in fig. 2, CDL has stronger cell killing effect compared to DOX Lip of the doxorubicin preparation group, and the advantage is more obvious as the acting time is prolonged.
Example 3 in vivo distribution study
In the experiment, the tissue distribution of the preparation in a mouse body is explored through a small animal living body imaging instrument, and the distribution of the preparation can be observed in real time and recorded and analyzed. Doxorubicin itself is fluorescent and its distribution can be observed by in vivo imaging using this property. Prepared Free DOX, DOX Lip and CDL (preparation example 1) were first sterilized through a 0.22 μm organic filter, and each preparation was diluted 10-fold with physiological saline and injected through the tail vein of mice at a dose of 5 mg/kg. The hair on the abdomen of the mouse is removed through a depilation operation, the mouse is anesthetized through intraperitoneal injection of 1% pentobarbital sodium (80 mg/kg), the medicine is injected into the body through the tail vein of the mouse for 4h, 12 h and 24h, and then the photographing observation is carried out on the mouse by using a mouse living body imaging instrument. After completion of the photograph, the mice were sacrificed by removing their necks and the tumors and organs were collected for photograph analysis. The results of the experiments are shown in FIGS. 3-5.
The in vivo imaging results in mice (fig. 3) show that the fluorescence signal of the prepared CDL is the strongest in tumor tissues at 24h compared with free doxorubicin and doxorubicin liposomes. Ex vivo results (fig. 4) show that in tumor tissues, the drug accumulation in the free doxorubicin group was lower than in the doxorubicin liposome group, making it more distributed in tumor tissues; the drug accumulation of the CDL group is higher than that of the adriamycin liposome group, and more CDL enrichment is shown by quantitative analysis, so that the effect of actively targeting and promoting the drug accumulation in tumor tissues is also proved, and the CDL has good treatment effect. Meanwhile, DOX has significant cardiotoxicity and is prone to accumulate in the heart. From the quantitative analysis, it can be seen that the fluorescence signal of DOX in the CDL group is the lowest in the heart and is 0.3 times that of the free doxorubicin group, which confirms that the free doxorubicin loaded in the liposome can significantly reduce the cardiotoxicity of DOX, thereby significantly reducing the damage of doxorubicin to normal tissues. As can be seen from the fluorescence quantification results (fig. 5), doxorubicin in the CDL group was more accumulated in the liver of the metabolic organ, while doxorubicin in the doxorubicin liposome group was more accumulated in the kidney, which may be due to the different metabolic profiles caused by the targeting effect of the CD73 antibody; the result shows that the metabolism efficiency of the adriamycin encapsulated in the carrier is lower than that of free adriamycin, which indicates that the liposome can prolong the existence time of the adriamycin in vivo and improve the accumulation of the adriamycin at a tumor part, thereby better exerting the anti-tumor effect.
Example 4 tumor inhibition experiment
Treatment experiment of triple negative breast cancer tumor: selecting healthy and age-appropriate Balb/c mice, inoculating the treated 4T1 cell suspension on a fourth pair of mammary gland fat pads of the mice, observing and measuring every day until the tumor volume on the mammary gland fat pads of the mice reaches 50mm3The administration was carried out in experimental groups. Tumor volume and body weight were measured after dosing and mice were considered dead when tumor volume was greater than 1000 mm3 or when body weight continued to drop more than 10%. Mouse body weight and tumor volume were measured and recorded every two days.
The experiments were grouped into 6 groups. Grouping experiments:
(1) PBS group: phosphate buffer is only given during the treatment process;
(2) antibody control group (Isotype): isotype control of antibody alone during treatment;
(3) free doxorubicin group (Free DOX): DOX alone during treatment;
(4) free CD73 antibody panel (Free CD73 Ab): CD73 antibody alone during treatment;
(5) free combination group (Free Combo): (ii) co-administering DOX and CD73 antibodies during treatment;
(6) doxorubicin liposome group (DOX Lip): only DOX Lip was given during treatment;
(7) CD73 Ab liposome group (Ab Lip): ab Lip during treatment;
(8) combination dosing group (CDL): CDL was given during the course of treatment.
Dose and mode of administration: the dosing interval was two days, 5 times, and each group was given 100 μ L/time/tail vein injection, with a dose of 5mg/kg (as DOX) and 100 μ g/time as CD73 antibody.
The results of the experiment are shown in FIG. 6.
The experimental results show that the CDL group shows particularly excellent tumor inhibition activity, the tumor inhibition rate of the CDL group is obviously better than that of other all groups such as DOX Lip group, CD73 antibody group, CD73 and DOX mixed group and the like, which shows that the CDL has better killing effect on adriamycin, and the CD73 antibody can block the generation of adenosine in the tumor microenvironment, so that the immunosuppression of the tumor microenvironment is twisted, the combined application can well inhibit the tumor growth, and the survival period is prolonged.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A liposome carrying CD73 antibody and adriamycin together is characterized in that a lipid core wrapped with adriamycin is arranged in the liposome, and the outside of the lipid core is modified with CD73 antibody; wherein, the CD73 antibody is coupled on the phospholipid PEG active ester micelle and then modified on the surface of the lipid core;
the lipid core has a transparent thread-shaped bilayer structure;
the phospholipid PEG active ester is DSPE-PEG 3400-NHS;
the preparation method comprises the following steps: dissolving DOPC, DOPE and CHEMS in an organic solvent according to a certain proportion to prepare a phospholipid solution, performing rotary evaporation to form a transparent spiral-thread-shaped lipid membrane, adding an ammonium sulfate solution, performing water bath ultrasound, and uniformly mixing to obtain a liposome solution; removing free ammonium sulfate by dialysis, adding adriamycin solution into liposome solution, heating in water bath under stirring, and removing unencapsulated adriamycin by dialysis to obtain lipid core solution coated with adriamycin;
dispersing phospholipid PEG active ester in a solvent, evaporating under reduced pressure to form phospholipid PEG active ester micelles, adding a PBS solution, performing ultrasonic treatment in a water bath, adding a CD73 antibody into the solution, stirring in the water bath, adding glycine to stop the reaction to obtain an antibody solution, adding the antibody solution into a lipid core solution coated with adriamycin, stirring in the water bath, and removing free antibodies to obtain a liposome carrying the CD73 antibody and the adriamycin together;
a lipid core formed by DOPC, DOPE and CHEMS, wherein the molar ratio of DOPC, DOPE and CHEMS is 2:6: 1; the mass ratio of the adriamycin to the lipid core is 1: 10;
the molar ratio of CD73 antibody to DSPE-PEG-NHS was 1: 3.
2. A pharmaceutical composition comprising the CD73 antibody-co-loaded liposome of claim 1 and doxorubicin.
3. A pharmaceutical formulation comprising the CD73 antibody and doxorubicin-co-loaded liposome of claim 1, and at least one pharmaceutically acceptable excipient.
4. Use of the liposome co-loaded with CD73 antibody and doxorubicin of claim 1 or the pharmaceutical composition of claim 2 or the pharmaceutical formulation of claim 3 for the preparation of a medicament for the treatment of triple negative breast cancer.
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