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Method for entrapment of chemotherapeutic drug by using autologous organoid-derived microvesicles of tumor patient and application of chemotherapeutic drug

A technology for tumor patients and chemotherapeutic drugs, applied in the field of tumor biology, can solve problems such as organoid-derived microvesicles derived from patients' tumors that have not yet been found, and achieve the effects of extensive human tumor targeting, strong tumor cells, and prolonged half-life.

Pending Publication Date: 2022-07-12
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there has been no report about patient-derived organoid-derived microvesicles encapsulating chemotherapy drugs to achieve individualized targeted therapy

Method used

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  • Method for entrapment of chemotherapeutic drug by using autologous organoid-derived microvesicles of tumor patient and application of chemotherapeutic drug
  • Method for entrapment of chemotherapeutic drug by using autologous organoid-derived microvesicles of tumor patient and application of chemotherapeutic drug
  • Method for entrapment of chemotherapeutic drug by using autologous organoid-derived microvesicles of tumor patient and application of chemotherapeutic drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Construction of drug-loaded organoid microvesicle MVs org-DOX , all experimental operations were carried out in a biological safety cabinet ( figure 1 ).

[0059] (1) The pancreatic cancer tissue derived from pancreatic cancer patients (tissue size is about 1 mm) 3 ) cut into pieces, and use 5ml of pre-prepared digestion solution (the formula of the digestion solution is Advanced containing collagenase III (LS004182, final concentration 5mg / ml), Y27632 (Y0503, final concentration 10.5μM), DNAseⅠ (10μg / ml) DMEM / F12) digested tissue (37°C for 50 min), the digested tissue solution was filtered through a 70 μm filter, centrifuged at 200 g to collect tumor cells, and then the tumor cells were dispersed in Matrigel (25 μl of pellet was resuspended with 800 μl of Matrigel) and It was fixed on a 24-well plate for 3D culture of tumor organoids. After the Matrigel solidified, the organoid culture medium prepared in advance was added.

[0060] (2) At 37℃, 5%(v / v)CO 2 Cultivate...

Embodiment 2

[0063] Construction of drug-loaded organoid microvesicle MVs org-Gem , all experimental operations were performed in a biological safety cabinet.

[0064] (1) The pancreatic cancer tissue derived from pancreatic cancer patients (tissue size is about 1 mm) 3 ) cut into pieces, and use 5ml of pre-prepared digestion solution (the formula of the digestion solution is Advanced containing collagenase III (LS004182, final concentration 5mg / ml), Y27632 (Y0503, final concentration 10.5μM), DNAseⅠ (10μg / ml) DMEM / F12) digested the tissue (37°C for 50 min), the digested tissue solution was filtered through a 70 μm filter, and the tumor cells were collected by centrifugation at 200 g. The 24-well plate is used for 3D culture of tumor organoids. After the matrigel is solidified, the organoid medium prepared in advance is added.

[0065] (2) At 37°C, 5% (v / v) CO 2 Cultivated for 7-10 days under conditions, and when the organoids grow to a suitable size (200-400 μm in diameter is acceptabl...

Embodiment 3

[0070] Determine the optimal experimental conditions by changing the UV irradiation time.

[0071] (a) With reference to the method of Example 1, the organoid suspension in step (2) was placed in an HL-2000 HybridizationOven combined molecular hybridization box (inner chamber size 356×273×273mm, built-in 8W, 254nm ultraviolet light source 5 MVs), irradiated under UV light for 30 min, taken out, added DOX (final concentration 10 mM), incubated for 24 h, and centrifuged to collect MVs org-DOX -1.

[0072] (b) With reference to the method of Example 1, place the organoid suspension in step (2) in a HL-2000 HybridizationOven combined molecular hybridization box (inner chamber size 356×273×273mm, built-in 8W, 254nm ultraviolet light source 5 MVs), irradiated under UV light for 60 min, taken out, added DOX (final concentration 10 mM), incubated for 24 h, and centrifuged to collect MVs org-DOX -2.

[0073] (c) With reference to the method of Example 1, the organoid suspension in s...

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Abstract

The invention discloses a method for entrapment of a chemotherapeutic drug by using autologous organoid derived microbubbles of a tumor patient and application of the chemotherapeutic drug. The method comprises the following steps: (1) shearing autologous tumor tissues of a tumor patient, then carrying out enzyme digestion, then filtering digested tumor cells by using a filter screen, carrying out centrifugal collection, then uniformly mixing with matrigel, then adding an organoid culture medium for culturing, and collecting to obtain an organoid when the diameter of the organoid is 200-400 [mu] m; (2) performing ultraviolet stimulation on the organoid, adding a chemotherapeutic drug, incubating, collecting a culture solution, centrifuging to remove cells and fragments, and centrifuging and collecting to obtain drug-loaded organoid microbubbles; wherein the ultraviolet stimulation condition is that an ultraviolet lamp with the power being 8 W and the wavelength being 254 nm is used for irradiating for 30-90 min. The drug-loaded organoid microbubble constructed in the invention has a tumor targeting effect and a stronger tumor cell killing effect, and can be used for treating tumors.

Description

technical field [0001] The present invention relates to the field of tumor biology, in particular to a method for encapsulating chemotherapeutic drugs using autologous organoid-derived microvesicles derived from tumor patients and applications thereof. Background technique [0002] Due to the characteristics of high metastasis, rapid development, chemotherapy resistance and clinical occultation in patients with triple-negative breast cancer, non-small cell lung cancer and pancreatic ductal adenocarcinoma, there is still no effective individualized treatment method. Although chemotherapy drugs such as Doxorubicin (DOX) and Gemcitabine (Gem) can improve the survival rate of the above cancer patients to a certain extent, the low blood vessels in the tumor area and poor drug targeting lead to the low efficiency of single-agent chemotherapy. . Therefore, it is urgent to construct a drug delivery system to improve the tumor delivery efficiency of chemotherapeutic drugs, thereby i...

Claims

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Application Information

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IPC IPC(8): A61K47/46A61K31/704A61K31/7068A61P35/00A61K49/00
CPCA61K47/46A61K31/704A61K31/7068A61P35/00A61K49/0013A61K49/0091Y02A50/30
Inventor 黄炳培江雪尚立焕赵新保孔祥展
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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