A tumor-targeted delivery carrier based on cell-derived microvesicles and its preparation method and application

Abstract

The invention discloses a tumor-targeted delivery carrier based on cell-derived micro-vacuoles, a preparation method and an application. The preparation method comprises the following steps of: (A) preparing a conditioned medium: supplementing fetal bovine serum, antibiotics, DSPE-PEG-Biotin and DSPE-PEG-Folate into a basal medium; (B) using the obtained conditioned medium in cell culture, and collecting cell culture supernatant for subsequent separation; (C) carrying out low-speed centrifugation on the obtained culture supernatant to remove cell debris and apoptotic bodies, then adding SA-IONPs, mixing uniformly, incubating, then separating by a magnet, using PBS for re-suspension, eluting for multiple times to obtain the cell-derived micro-vacuoles with membrane surfaces modified by folic acid and iron oxide nano-particles, and freezing for storage; and (D) loading chemotherapeutic drugs or therapeutic genes into the functionalized micro-vacuoles doubly-modified by an electroporation mode, and carrying out re-suspension after separation with the magnet. The tumor-targeted delivery carrier based on cell-derived micro-vacuoles, the preparation method and the application disclosed by the invention are applicable to specific targeting delivery of multiple chemotherapeutic drugs and therapeutic genes, and have the advantages of enhancing the anti-tumor effect, reducing the systemic toxicity and improving the clinical effect of the current therapeutic selection, so that a new hope is brought for clinical therapy of tumors.

Description

technical field

[0001] The present invention relates to the technical field of new dosage forms and preparations of biology and medicine, more specifically to a tumor-targeted delivery carrier based on cell-derived microvesicles, and also to a tumor-targeted delivery carrier based on cell-derived microvesicles The preparation method is convenient and efficient, and simultaneously realizes the folic acid and magnetic dual labeling on the surface of cell-derived microvesicles; it also involves a targeted delivery carrier based on cell-derived microvesicles in the preparation of local chemotherapy drugs for treating or preventing tumors and The application of gene therapy vectors is suitable for the use of this tumor-targeted delivery vector in scientific research and clinical practice for the specific targeted delivery of drugs or therapeutic genes to prevent or treat the following diseases: 1. Cervical cancer, ovarian cancer , endometrial cancer, testicular cancer, gastric canc...

Examples

Embodiment 1

[0089] A method for preparing a tumor-targeted delivery carrier based on cell-derived microvesicles, the steps of which are:

[0090] 1. Conditioned medium: basal medium (HyClone, Waltham, MA, USA) was supplemented with 10% (v / v) fetal bovine serum (fetal bovine serum, FBS) (HyClone, Waltham, MA, USA) and 1% ( v / v) Antibiotics, add DSPE-PEG-Biotin (50ug / mL) and DSPE-PEG-Folate (5ug / mL) at the same time, store at 4°C to obtain conditioned medium.

[0091] 2. Use the conditioned medium obtained in step 1 for cell culture, culture under standard conditions for about 48 hours, replace the medium when the cells grow to a confluence of about 80%, and continue to culture with serum-free basal medium Cells, to promote the release of microvesicles from the cells, collect the cell culture supernatant after 46-50 hours of culture, and use for subsequent isolation to obtain functionalized microvesicles.

[0092] 3. Centrifuge the culture supernatant obtained in step 2 at 2000g for 20min ...

Embodiment 2

[0096] In vivo and in vitro biosafety testing of a tumor-targeted delivery carrier based on cell-derived microvesicles, the steps of which are as follows:

[0097] 1. Conditioned medium: basal medium (HyClone, Waltham, MA, USA) was supplemented with 10% (v / v) fetal bovine serum (fetal bovine serum, FBS) (HyClone, Waltham, MA, USA) and 1% ( v / v) Antibiotics, add DSPE-PEG-Biotin (50ug / mL) and DSPE-PEG-Folate (5ug / mL) at the same time, store at 4°C to obtain conditioned medium.

[0098] 2. Use the conditioned medium obtained in step 1 for cell culture, culture under standard conditions for about 48 hours, replace the medium when the cells grow to a confluence of about 80%, and continue to culture with serum-free basal medium Cells, to promote the release of microvesicles from the cells, collect the cell culture supernatant after 46-50 hours of culture, and use for subsequent isolation to obtain functionalized microvesicles.

[0099] 3. Centrifuge the collected supernatant obtain...

Embodiment 3

[0105]A tumor-targeted detection in vivo and in vitro of a cell-derived microvesicle-based tumor-targeted delivery carrier, the steps of which are as follows:

[0106] 1. Conditioned medium: basal medium (HyClone, Waltham, MA, USA) was supplemented with 10% (v / v) fetal bovine serum (fetal bovine serum, FBS) (HyClone, Waltham, MA, USA) and 1% ( v / v) Antibiotics, DSPE-PEG-Biotin (50 μg / mL) and DSPE-PEG-Folate (5 μg / mL) were added at the same time, stored at 4° C. to obtain conditioned medium.

[0107] 2. Use the conditioned medium obtained in step 1 for cell culture, culture under standard conditions for about 48 hours, replace the medium when the cells grow to a confluence of about 80%, and continue to culture with serum-free basal medium Cells, to promote the release of microvesicles from the cells, collect the cell culture supernatant after 46-50 hours of culture, and use for subsequent isolation to obtain functionalized microvesicles.

[0108] 3. Centrifuge the collected su...

technical field

[0001] The present invention relates to the technical field of new dosage forms and preparations of biology and medicine, more specifically to a tumor-targeted delivery carrier based on cell-derived microvesicles, and also to a tumor-targeted delivery carrier based on cell-derived microvesicles The preparation method is convenient and efficient, and simultaneously realizes the folic acid and magnetic dual labeling on the surface of cell-derived microvesicles; it also involves a targeted delivery carrier based on cell-derived microvesicles in the preparation of local chemotherapy drugs for treating or preventing tumors and The application of gene therapy vectors is suitable for the use of this tumor-targeted delivery vector in scientific research and clinical practice for the specific targeted delivery of drugs or therapeutic genes to prevent or treat the following diseases: 1. Cervical cancer, ovarian cancer , endometrial cancer, testicular cancer, gastric canc...

Examples

Embodiment 1

[0089] A method for preparing a tumor-targeted delivery carrier based on cell-derived microvesicles, the steps of which are:

[0090] 1. Conditioned medium: basal medium (HyClone, Waltham, MA, USA) was supplemented with 10% (v / v) fetal bovine serum (fetal bovine serum, FBS) (HyClone, Waltham, MA, USA) and 1% ( v / v) Antibiotics, add DSPE-PEG-Biotin (50ug / mL) and DSPE-PEG-Folate (5ug / mL) at the same time, store at 4°C to obtain conditioned medium.

[0091] 2. Use the conditioned medium obtained in step 1 for cell culture, culture under standard conditions for about 48 hours, replace the medium when the cells grow to a confluence of about 80%, and continue to culture with serum-free basal medium Cells, to promote the release of microvesicles from the cells, collect the cell culture supernatant after 46-50 hours of culture, and use for subsequent isolation to obtain functionalized microvesicles.

[0092] 3. Centrifuge the culture supernatant obtained in step 2 at 2000g for 20min ...

Embodiment 2

[0096] In vivo and in vitro biosafety testing of a tumor-targeted delivery carrier based on cell-derived microvesicles, the steps of which are as follows:

[0097] 1. Conditioned medium: basal medium (HyClone, Waltham, MA, USA) was supplemented with 10% (v / v) fetal bovine serum (fetal bovine serum, FBS) (HyClone, Waltham, MA, USA) and 1% ( v / v) Antibiotics, add DSPE-PEG-Biotin (50ug / mL) and DSPE-PEG-Folate (5ug / mL) at the same time, store at 4°C to obtain conditioned medium.

[0098] 2. Use the conditioned medium obtained in step 1 for cell culture, culture under standard conditions for about 48 hours, replace the medium when the cells grow to a confluence of about 80%, and continue to culture with serum-free basal medium Cells, to promote the release of microvesicles from the cells, collect the cell culture supernatant after 46-50 hours of culture, and use for subsequent isolation to obtain functionalized microvesicles.

[0099] 3. Centrifuge the collected supernatant obtain...

Embodiment 3

[0105]A tumor-targeted detection in vivo and in vitro of a cell-derived microvesicle-based tumor-targeted delivery carrier, the steps of which are as follows:

[0106] 1. Conditioned medium: basal medium (HyClone, Waltham, MA, USA) was supplemented with 10% (v / v) fetal bovine serum (fetal bovine serum, FBS) (HyClone, Waltham, MA, USA) and 1% ( v / v) Antibiotics, DSPE-PEG-Biotin (50 μg / mL) and DSPE-PEG-Folate (5 μg / mL) were added at the same time, stored at 4° C. to obtain conditioned medium.

[0107] 2. Use the conditioned medium obtained in step 1 for cell culture, culture under standard conditions for about 48 hours, replace the medium when the cells grow to a confluence of about 80%, and continue to culture with serum-free basal medium Cells, to promote the release of microvesicles from the cells, collect the cell culture supernatant after 46-50 hours of culture, and use for subsequent isolation to obtain functionalized microvesicles.

[0108] 3. Centrifuge the collected su...

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