A liposome carrying catalase and linked to PD-L1 antibody and its preparation method

A catalase and liposome technology, applied in biochemical equipment and methods, oxidoreductase, liposome delivery and other directions, can solve the problems of increasing difficulties in clinical application, reducing toxic and side effects, and increasing the number of administrations, etc. To achieve the effect of promoting the killing of tumor cells, enhancing the effect of immunotherapy, and relieving tumor hypoxia

Active Publication Date: 2021-08-10
PEKING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, domestic existing technologies mostly 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 , which in turn fails to increase its effectiveness and reduce toxic side effects
The combined use of liposomes, PD-L1 and catalase has not been reported in China

Method used

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  • A liposome carrying catalase and linked to PD-L1 antibody and its preparation method
  • A liposome carrying catalase and linked to PD-L1 antibody and its preparation method
  • A liposome carrying catalase and linked to PD-L1 antibody and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1 liposome preparation

[0056] Ordinary long-circulating liposomes (SSL) are prepared by thin-film dispersion method.

[0057] 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.

[0058] Multifunctional immunoliposomes (CAT@aPDL1-SSL) were prepared by thin-film dispersion / post-insertion method.

[0059] 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

[0061] The characterization of embodiment 2 liposome

[0062] 1. Experimental process

[0063] The particle size and dispersion of CAT@aPDL1-SSL were measured by a dynamic light scattering particle sizer (DLS) (Zetasizer Nano ZS90; Malvern; UK).

[0064] After the liposome solution in Example 1 was stained with 2% phosphotungstic acid, the morphology of the particles was observed with a transmission electron microscope (TEM) (JEM-1400Plus, JEOL, Japan).

[0065] The entrapment efficiency of CAT was measured by BCA protein quantification.

[0066] Enzyme activity of free CAT and CAT entrapped in liposomes was determined by standard Goth's method.

[0067] 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.

[0068] Subsequently, 0.5 mL of ammonium molybdate (32.4 mM) was added to the re...

Embodiment 3

[0083] Example 3 In vitro cell uptake experiment

[0084] 1. Prepare C6-loaded liposome C6@aPDL1-SSL using the above-mentioned film dispersion method.

[0085] 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.

[0086] To study the cellular uptake of liposomes under low pH conditions, the medium was adjusted to pH 6.5 using citrate buffer (pH 6...

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Abstract

The invention discloses a liposome carrying catalase and linking PD-L1 antibody and a preparation method thereof. The liposome is a kind of multifunctional immunoliposome with soybean phospholipid and cholesterol as the backbone, carrying catalase and connecting aPDL1 on the surface. The liposome is prepared by film dispersion / post-insertion method; the catalase is contained in the liposome; the aPDL1 is synthesized by a reaction to guide the aPDL1 compound, and inserted into the liposome phospholipid bilayer; The surface of the liposome is also connected with a hydrazone bond. The liposome has a spherical structure, the particle diameter is 118.2±1.763nm, and the polydispersity coefficient is 0.223±0.007; the encapsulation efficiency of catalase is 30-36%. The liposome can effectively relieve hypoxia in the tumor area and block the tumor suppressive signaling pathway (PD-1 / PD-L1), has a good therapeutic effect on melanoma, and has broad research prospects in tumor immunotherapy.

Description

technical field [0001] The invention relates to a liposome, which belongs to the field of multifunctional immunoliposome delivery system, in particular to an immunoliposome carrying catalase and connecting PD-L1 antibody on the surface and a preparation method thereof. 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 antigenic stimulation, tumor cells overexpress inhibitory receptors on the surface, which facilitates th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K9/127A61K47/24A61K47/28A61K47/68A61K38/44A61K39/395A61P35/00
CPCA61K9/127A61K38/44A61K39/39558A61K47/24A61K47/28A61K47/6843A61P35/00C12Y111/01006A61K2300/00
Inventor 魏世成黑玉滕彬宏熊春阳陈庆林
Owner PEKING UNIV
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