Multifunctional carrier based on cytogenic vesicle in body fluid, preparation method and application

Abstract

The invention discloses a multifunctional carrier based on cytogenic vesicle in body fluid, a preparation method and an application. The method comprises the following steps: (1) collecting human body fluid, performing gradient ultra-centrifugation, removing supernatant and using sterile PBS heavy suspension for precipitating and (2) adding the cytogenic membrane vesicle from the body fluid, ultra-small luminescent material and electroporation buffer solution into an electric shock cup, uniformly mixing, placing onto an electroporation instrument and treating, placing under room temperature and repairing after the ending of electroporation, performing ultra-centrifugation and then using sterile PBS heavy suspension for precipitating, thereby acquiring the multifunctional carrier based on the cytogenic membrane vesicle from the body fluid and marked with the ultra-small luminescent material. The carrier is used for preparing a chemotherapeutic drug or gene therapeutic drug for treating or preventing tumor, has high biocompatibility, is convenient to carry various therapeutic drugs, is capable of effectively restraining tumor growth, is suitable for diagnosis and treatment of various innocent and malignant tumors and especially supplies a choice for the diagnosis and treatment of deep tumors in skull, thoracic cavity and abdominal cavity.

Description

technical field

[0001] The invention relates to the technical field of new dosage forms and preparations of biology and medicine, and more specifically relates to a multifunctional carrier based on cell-derived membranous vesicles in body fluids. The carrier has good biocompatibility and superior, stable and durable living fluorescence Imaging capability and magnetic resonance imaging capability, efficient drug loading and safe tumor-targeted delivery capability; more specifically, it relates to a preparation method of a multifunctional carrier based on cell-derived membrane vesicles in body fluids, which is simple and efficient, and produces High yield, uniform and stable product quality; also relates to the use of a multifunctional carrier based on cell-derived membrane vesicles in body fluids, using the multifunctional carrier based on cell-derived membrane vesicles in body fluids to realize therapeutic gene fragments Or the specific targeted delivery of chemotherapy drugs ...

Examples

Embodiment 1

[0114] A method for preparing a multifunctional carrier labeled with ultra-small manganese magnetic near-infrared quantum dots (Mn-NIR-QDs) based on cell-derived vesicles in body fluids, the steps of which are:

[0115] 1. Use vacuum blood collection tubes containing EDTA-K2 anticoagulant to collect peripheral blood samples, transfer them to centrifuge tubes, and centrifuge at 1800 or 1900 or 2000 or 2100 or 2200 g for 18 or 19 days at 3 or 4 or 5 °C Or 20 or 21 or 22min, take the supernatant, and centrifuge again at 1800 or 1900 or 2000 or 2100 or 2200g at 3 or 4 or 5°C for 18 or 19 or 20 or 21 or 22min, get the supernatant, and then Continue to centrifuge at 48000 or 49000 or 50000 or 51000 or 52000 g for 60 min at 4 or 5°C, resuspend the obtained pellet with sterile PBS, and freeze the resuspended liquid at -80°C to obtain blood-derived microparticles.

[0116] 2. Add 20 μg (50 μL) blood-derived microparticles / 50 μL ultra-small manganese magnetic near-infrared quantum dots ...

Embodiment 2

[0124] Application of a multifunctional carrier labeled with ultra-small manganese magnetic near-infrared quantum dots (Mn-NIR-QDs) based on cell-derived vesicles in body fluids in the preparation of gene therapy drugs and chemotherapy drugs for the treatment or prevention of oral cancer and oral cavity The application in the early diagnosis of cancer, its steps are as follows:

[0125] 1. Use vacuum blood collection tubes containing EDTA-K2 anticoagulant to collect peripheral blood samples, transfer them to centrifuge tubes, and centrifuge at 1800 or 1900 or 2000 or 2100 or 2200 g for 18 or 19 days at 3 or 4 or 5 °C Or 20 or 21 or 22min, take the supernatant, and centrifuge again at 1800 or 1900 or 2000 or 2100 or 2200g at 3 or 4 or 5°C for 18 or 19 or 20 or 21 or 22min, get the supernatant, and then Continue to centrifuge at 48000 or 49000 or 50000 or 51000 or 52000 g for 60 min at 4 or 5°C, resuspend the obtained pellet with sterile PBS, and freeze the resuspended liquid at...

Embodiment 3

[0133] A multifunctional carrier based on cell-derived vesicles in body fluids, labeled with ultra-small manganese magnetic near-infrared quantum dots (Mn-NIR-QDs) and loaded with gene therapy fragments (survivin-siRNA) in the preparation of treatment or prevention of oral cavity The application of cancer chemotherapy drugs or gene therapy drugs, the steps are:

[0134] 1. Use vacuum blood collection tubes containing EDTA-K2 anticoagulant to collect peripheral blood samples, transfer them to centrifuge tubes, and centrifuge at 1800 or 1900 or 2000 or 2100 or 2200 g for 18 or 19 days at 3 or 4 or 5 °C or 20 or 21 or 22min, take the supernatant, centrifuge again at 1800 or 1900 or 2000 or 2100 or 2200g at 3 or 4 or 5°C for 18 or 19 or 20 or 21 or 22min, get the supernatant, and then Continue to centrifuge at 48000 or 49000 or 50000 or 51000 or 52000 g for 60 min at 4 or 5°C, resuspend the obtained pellet with sterile PBS, and freeze the resuspended liquid at -80°C to obtain bloo...

technical field

[0001] The invention relates to the technical field of new dosage forms and preparations of biology and medicine, and more specifically relates to a multifunctional carrier based on cell-derived membranous vesicles in body fluids. The carrier has good biocompatibility and superior, stable and durable living fluorescence Imaging capability and magnetic resonance imaging capability, efficient drug loading and safe tumor-targeted delivery capability; more specifically, it relates to a preparation method of a multifunctional carrier based on cell-derived membrane vesicles in body fluids, which is simple and efficient, and produces High yield, uniform and stable product quality; also relates to the use of a multifunctional carrier based on cell-derived membrane vesicles in body fluids, using the multifunctional carrier based on cell-derived membrane vesicles in body fluids to realize therapeutic gene fragments Or the specific targeted delivery of chemotherapy drugs ...

Examples

Embodiment 1

[0114] A method for preparing a multifunctional carrier labeled with ultra-small manganese magnetic near-infrared quantum dots (Mn-NIR-QDs) based on cell-derived vesicles in body fluids, the steps of which are:

[0115] 1. Use vacuum blood collection tubes containing EDTA-K2 anticoagulant to collect peripheral blood samples, transfer them to centrifuge tubes, and centrifuge at 1800 or 1900 or 2000 or 2100 or 2200 g for 18 or 19 days at 3 or 4 or 5 °C Or 20 or 21 or 22min, take the supernatant, and centrifuge again at 1800 or 1900 or 2000 or 2100 or 2200g at 3 or 4 or 5°C for 18 or 19 or 20 or 21 or 22min, get the supernatant, and then Continue to centrifuge at 48000 or 49000 or 50000 or 51000 or 52000 g for 60 min at 4 or 5°C, resuspend the obtained pellet with sterile PBS, and freeze the resuspended liquid at -80°C to obtain blood-derived microparticles.

[0116] 2. Add 20 μg (50 μL) blood-derived microparticles / 50 μL ultra-small manganese magnetic near-infrared quantum dots ...

Embodiment 2

[0124] Application of a multifunctional carrier labeled with ultra-small manganese magnetic near-infrared quantum dots (Mn-NIR-QDs) based on cell-derived vesicles in body fluids in the preparation of gene therapy drugs and chemotherapy drugs for the treatment or prevention of oral cancer and oral cavity The application in the early diagnosis of cancer, its steps are as follows:

[0125] 1. Use vacuum blood collection tubes containing EDTA-K2 anticoagulant to collect peripheral blood samples, transfer them to centrifuge tubes, and centrifuge at 1800 or 1900 or 2000 or 2100 or 2200 g for 18 or 19 days at 3 or 4 or 5 °C Or 20 or 21 or 22min, take the supernatant, and centrifuge again at 1800 or 1900 or 2000 or 2100 or 2200g at 3 or 4 or 5°C for 18 or 19 or 20 or 21 or 22min, get the supernatant, and then Continue to centrifuge at 48000 or 49000 or 50000 or 51000 or 52000 g for 60 min at 4 or 5°C, resuspend the obtained pellet with sterile PBS, and freeze the resuspended liquid at...

Embodiment 3

[0133] A multifunctional carrier based on cell-derived vesicles in body fluids, labeled with ultra-small manganese magnetic near-infrared quantum dots (Mn-NIR-QDs) and loaded with gene therapy fragments (survivin-siRNA) in the preparation of treatment or prevention of oral cavity The application of cancer chemotherapy drugs or gene therapy drugs, the steps are:

[0134] 1. Use vacuum blood collection tubes containing EDTA-K2 anticoagulant to collect peripheral blood samples, transfer them to centrifuge tubes, and centrifuge at 1800 or 1900 or 2000 or 2100 or 2200 g for 18 or 19 days at 3 or 4 or 5 °C or 20 or 21 or 22min, take the supernatant, centrifuge again at 1800 or 1900 or 2000 or 2100 or 2200g at 3 or 4 or 5°C for 18 or 19 or 20 or 21 or 22min, get the supernatant, and then Continue to centrifuge at 48000 or 49000 or 50000 or 51000 or 52000 g for 60 min at 4 or 5°C, resuspend the obtained pellet with sterile PBS, and freeze the resuspended liquid at -80°C to obtain bloo...

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