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Exosome, method for preparing same and application of exosome to preparing medicines for skin superficial tumor

A technology of exosomes and drugs, applied in the field of biomedicine, can solve the problems of lack of preparation methods to treat superficial tumors, achieve efficient and rapid preparation, solve body inflammation, and reduce immunogenicity

Active Publication Date: 2018-09-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the shortcomings of the existing preparation methods and the lack of existing methods for treating superficial tumors, and provide a method for preparing exosomes that is quick and convenient to produce, simple and efficient in drug loading and its application in the preparation of skin The application of drugs in superficial tumors, the prepared exosomes are combined with photothermal therapy to treat superficial tumors after transdermal administration, and it is the first time that exosomes are applied to transdermal drug delivery, and it is the first time to propose transdermal drugs Novel strategy for synergistic treatment of superficial tumors with photothermal therapy and biotherapy after administration

Method used

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  • Exosome, method for preparing same and application of exosome to preparing medicines for skin superficial tumor
  • Exosome, method for preparing same and application of exosome to preparing medicines for skin superficial tumor
  • Exosome, method for preparing same and application of exosome to preparing medicines for skin superficial tumor

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Effect test

Embodiment 1

[0039] The frozen and transfected Escherichia coli were thawed overnight. The collected culture fluid was centrifuged at 5000g for 5 minutes at 4°C to remove dead cells and large debris. Filter the supernatant with a 0.22 μm filter membrane to further remove impurities such as bacteria. Transfer the supernatant to a sterile ultracentrifuge tube, centrifuge at 100,000 g for 2 hours at 4°C, and discard the supernatant. Wash with PBS, and ultracentrifuge at 100,000 g for 2 hours at 4°C, and the obtained precipitates are exosomes. Depending on the volume of the initially collected culture medium, add physiological saline for injection to resuspend as appropriate, use a BCA kit to detect the total protein concentration, and store in -80°C.

[0040] Add lysozyme (2mg / mL) to the exosomes, place in a shaker at 33°C, shake at 100rpm for 1h. Then add the polypeptide iRGD (25 μg / mL) and the polypeptide PEP (25 μg / mL) targeting melanoma respectively, wherein, the polypeptide iRGD is th...

Embodiment 2

[0042] The frozen and transfected Escherichia coli were thawed overnight. The collected culture fluid was centrifuged at 5000g for 5 minutes at 4°C to remove dead cells and large debris. Filter the supernatant with a 0.22 μm filter membrane to further remove impurities such as bacteria. Transfer the supernatant to a sterile ultracentrifuge tube, centrifuge at 100,000 g for 2 hours at 4°C, and discard the supernatant. Wash with PBS, and ultracentrifuge at 100,000 g for 2 hours at 4°C, and the obtained precipitates are exosomes. Depending on the volume of the initially collected culture medium, add physiological saline for injection to resuspend as appropriate, use a BCA kit to detect the total protein concentration, and store in -80°C.

[0043] Add lysozyme (1mg / mL) to the exosomes, place in a shaker at 20°C, shake at 80rpm for 1h. Then, the melanoma-targeted polypeptide iRGD (10 μg / mL) and the polypeptide PEP (10 μg / mL) were added respectively, placed in a shaker at 20° C.,...

Embodiment 3

[0045] The frozen and transfected Escherichia coli were thawed overnight. The collected culture fluid was centrifuged at 5000g for 5 minutes at 4°C to remove dead cells and large debris. Filter the supernatant with a 0.22 μm filter membrane to further remove impurities such as bacteria. Transfer the supernatant to a sterile ultracentrifuge tube, centrifuge at 100,000 g for 2 hours at 4°C, and discard the supernatant. Wash with PBS, and ultracentrifuge at 100,000 g for 2 hours at 4°C, and the obtained precipitates are exosomes. Depending on the volume of the initially collected culture medium, add physiological saline for injection to resuspend as appropriate, use a BCA kit to detect the total protein concentration, and store in -80°C.

[0046] Add lysozyme (8mg / mL) to the exosomes, place in a shaker at 33°C, shake at 200rpm for 3h. Then add peptide iRGD (30 μg / mL) and peptide PEP (30 μg / mL) targeting melanoma respectively, place in a shaker at 33 °C, shake at 200 rpm for 3 ...

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Abstract

The invention discloses an exosome, a method for preparing the same and application of the exosome to preparing medicines for skin superficial tumor. The method particularly includes carrying out Trail gene transformation on Escherichia coli so as to express Trail proteins; extracting the exosome of the Trail proteins; carrying out lysozyme treatment, then modifying nano-particles by the aid of target peptides and loading a photosensitizer indocyanine green (ICG). The ICG can be triggered by near-infrared light to emit heat and release singlet oxygen after the nano-particles are delivered by means of skin penetration, and tumor cells can be synergistically killed by the nano-particles and the Trail proteins. The exosome, the method and the application have the advantages that the shortcomings of the traditional methods can be overcome by the aid of the method for preparing the exosome, the method is high in yield and medicine-loading rate, short in cycle and low in cost, and the medicines are easy and convenient to load; reports of application of exosomes to delivery by means of skin penetration are unavailable at present, the exosome can efficiently penetrate skin, and accordinglynovel strategies can be provided to treating skin diseases; in addition, therapy for curing melanoma is clinically available, the exosome can be combined with photo-thermal and biological therapy after penetrating the skin, accordingly, development and metastasis of the melanoma can be synergistically suppressed, and excellent in-vivo treatment effects can be realized.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an exosome, a preparation method of the exosome and its application in preparing medicine for superficial skin tumors. Background technique [0002] Transdermal drug delivery refers to a drug delivery method that directly acts on the skin surface, allowing the drug to penetrate the skin and enter the blood circulation of the human body, or act on the local skin of the patient, thereby exerting a therapeutic effect. For skin diseases, especially superficial tumors, it is an ideal route of administration. Because this transdermal drug delivery method can directly deliver the drug to the lesion, enhance the targeting of drug delivery, and reduce the toxic and side effects of the whole body; avoid the first-pass effect of the liver and improve the bioavailability of the drug; the drug delivery method is simple, It has the advantages of less harm to the human body, and the admi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70A61K41/00A61K38/17A61K47/46A61K47/42A61P35/00C12R1/19
CPCA61K38/177A61K41/0042A61K41/0052A61K41/0057A61K47/42A61K47/46A61P35/00C07K14/70575C12N15/70A61K2300/00
Inventor 彭丽华褚阳
Owner ZHEJIANG UNIV
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