Application of Liposome Encapsulated Catalase and Linked to PD-L1 Antibody in the Preparation of Drugs for Tumor Therapy
A technology of catalase and PD-L1, which is 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., and achieve Promote the killing of tumor cells, enhance the effect of immunotherapy, and relieve the effect of tumor hypoxia
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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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