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Application of catalase-entrapped liposome connected with PD-L1 antibody in preparation of tumor treatment drugs

A catalase, PD-L1 technology, applied in the application field of immunoliposomes in the preparation of tumor therapeutic drugs, can solve the problems of inability to improve the targeting of free aPDL1, reduce toxic side effects, increase the number of administrations, etc. Promote the killing of tumor cells, enhance the effect of immunotherapy, and relieve the effect of tumor hypoxia

Active Publication Date: 2020-04-10
PEKING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, most of the domestic existing technologies use catalase-loaded liposomes in combination with free aPDL1, which not only increases the number of administrations, but also makes clinical application more difficult, and at the same time cannot improve the targeting of free aPDL1. In turn, it is impossible to increase its effectiveness and reduce toxic side effects
The combined use of liposomes, PD-L1 and catalase has not been reported in China, nor has the application of the corresponding combination substances in the preparation of tumor therapeutic drugs been reported.

Method used

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  • Application of catalase-entrapped liposome connected with PD-L1 antibody in preparation of tumor treatment drugs
  • Application of catalase-entrapped liposome connected with PD-L1 antibody in preparation of tumor treatment drugs
  • Application of catalase-entrapped liposome connected with PD-L1 antibody in preparation of tumor treatment drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 liposome preparation

[0062] Ordinary long-circulating liposomes (SSL) are prepared by thin-film dispersion method.

[0063] Raw material SPC (soybean lecithin), cholesterol, DSPE-PEG2000, dissolved in 1ml chloroform, the molar ratio is 100:50:8. Afterwards, the chloroform solution was suspended to dryness using a rotary evaporator, and the chloroform solution was removed to obtain a lipid film. The lipid film was hydrated with 2 ml of phosphate buffered saline (PBS). The final SSL was obtained after filtration through a 200nm polycarbonate membrane.

[0064] Multifunctional immunoliposomes (CAT@aPDL1-SSL) were prepared by thin-film dispersion / post-insertion method.

[0065] First, the raw material-directed compound DSPE-Hyd-PEG2000-NHS is mixed with aPDL1 at a molar ratio of 10:1, and the aPDL1-directed compound DSPE-Hyd-PEG2000 is synthesized by reacting the amino group of the antibody aPDL1 with the succinimide group of the raw material-directed co...

Embodiment 2

[0068] Example 2 Characterization

[0069] 2.1 Characterization of liposomes

[0070] The particle size and dispersion of CAT@aPDL1-SSL were measured by a dynamic light scattering particle sizer (DLS) (Zetasizer Nano ZS90; Malvern; UK).

[0071] After the liposome solution was stained with 2% phosphotungstic acid, the morphology of the particles was observed with a transmission electron microscope (TEM) (JEM-1400Plus, JEOL, Japan).

[0072] The entrapment efficiency of CAT was measured by BCA protein quantification.

[0073] Enzyme activity of free CAT and CAT entrapped in liposomes was determined by standard Goth's method.

[0074] First, 0.5 mL of H 2 o 2 The solution (30% aqueous solution) was added to 1.5 mL of Eppendorf (EP) tubes, then 1 mL of free CAT and 1 mL of CAT@aPDL1-SSLs were added to each EP tube and incubated at 37 °C with H 2 o 2 React for 1 minute. Subsequently, 0.5 mL of ammonium molybdate (32.4 mM) was added to the reaction solution. Ammonium moly...

Embodiment 3

[0087] Example 3 In vitro cell uptake experiment

[0088] C6-loaded liposomes C6@aPDL1-SSL were prepared using the thin film dispersion method described above.

[0089] B16-F10 cells in 1 × 10 6 Cells / well density were seeded into six-well plates and incubated overnight. The medium was then removed, and the cells were washed 3 times with PBS. Add PBS, free C6, C6@aPDL1-SSL, C6@aPDL1-SSL and free aPDL1 (pH 7.4) to each well of the plate (C6, 150ng / mL) and mix with B16-F10 cells at 37°C and 5% CO 2 Incubate for 2 hours under conditions. After 2 hours, the sample solution was removed, and 200 μL of trypsin and ethylenediaminetetraacetic acid (EDTA) were added to each well to digest the cells. The digested cell suspension was centrifuged at 1,000rpm for 5 minutes, and then the pellet was washed at 1×10 6 Resuspend in PBS at a density of cells / mL.

[0090] To study the cellular uptake of liposomes under low pH conditions, the medium was adjusted to pH 6.5 using citrate buf...

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Abstract

The invention discloses an application of a catalase-entrapped liposome connected with a PD-L1 antibody in preparation of a tumor treatment drug. The liposome is a multifunctional immune liposome which takes soyabean lecithin and cholesterol as skeletons, encapsulates catalase and is connected with aPDL1 on the surface. The liposome is prepared by a film dispersion / post-insertion method, wherein the catalase is entrapped in the liposome; the aPDL1 is an aPDL1 guide compound synthesized by reaction, and is inserted into a liposome phospholipid bilayer; and the surface of the liposome is also connected with a hydrazone bond. The liposome is of a spherical structure, the particle size of the liposome is 118.2 + / -1.763 nm, and the polydispersity index of the liposome is 0.223 + / -0.007; and theencapsulation efficiency of catalase is 30-36%. The drug obtained by the invention can effectively relieve hypoxia in a tumor region and block a tumor inhibitory signal path (PD-1 / PD-L1), has a goodtreatment effect on melanoma, and has a wide research prospect in the aspect of tumor immunotherapy.

Description

technical field [0001] The invention relates to the application of a liposome in the preparation of tumor treatment drugs, which belongs to the field of immunoliposome drug delivery system, and in particular to the application of an immunoliposome carrying catalase and connected to the surface of PD-L1 antibody in the preparation of tumor treatment drug application. Background technique [0002] Tumor cells can suppress the function of the immune system through a variety of mechanisms to escape the recognition and killing of the immune system. There are usually two ways to block tumor suppression of the immune system, namely blocking tumor suppressive signaling pathways and improving tumor suppressive microenvironment. The immunosuppressive pathway mediated by immune checkpoint proteins inhibits the recognition of tumor cells by the immune system by inhibiting the function of T lymphocytes, which is a representative immunosuppressive mechanism. Due to inflammation and anti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61K9/127A61K47/28A61K47/24A61P35/00A61K38/44
CPCA61K9/127A61K38/44A61K39/39558A61K47/24A61K47/28A61P35/00C12Y111/01006A61K2300/00
Inventor 魏世成黑玉滕彬宏熊春阳陈庆林
Owner PEKING UNIV
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