Targeted anti-malignant-melanoma DTIC (dacarbazine) and miR-205 co-carrying exosome as well as preparation method and application thereof

A malignant melanoma, mir-205 technology, applied in the field of biomedicine, to achieve the effect of improving the effect, improving the sensitivity, and improving the comprehensive curative effect

Active Publication Date: 2017-11-24
SHANGHAI DERMATOLOGY HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no reports on using exosomes as co-carriers of chemotherapy drugs and gene drugs, combining them wi

Method used

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  • Targeted anti-malignant-melanoma DTIC (dacarbazine) and miR-205 co-carrying exosome as well as preparation method and application thereof
  • Targeted anti-malignant-melanoma DTIC (dacarbazine) and miR-205 co-carrying exosome as well as preparation method and application thereof
  • Targeted anti-malignant-melanoma DTIC (dacarbazine) and miR-205 co-carrying exosome as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Inhibitory effect of DTIC and miR-205 in combination on the activity of human melanoma A375 cells in vitro

[0046] Human melanoma cell line A375 (purchased from the Cell Bank of the Chinese Academy of Sciences) was cultured in vitro, and divided into DTIC group (0.5 μg / ml), miR-205 group (100 nmol / L), DTIC+miR-205 group, 2×DTIC group ( 1 μg / ml), 2×miR-205 group (200nmol / L) were intervened on A375 cells, and the cell viability was detected by MTT assay after 24 hours of treatment. The wells of cells not treated with drugs were used as control wells, and the cell viability thereof was set as 100%. from the result( figure 1 ) concluded that the combined use of DTIC and miR-205 had a more significant inhibitory effect on A375 cells than that of DTIC and miR-205 alone (p<0.01), and more importantly, the effect of the DTIC+miR-205 group was stronger than that of the DTIC+miR-205 group. 2×DTIC group (p<0.05), which suggests that DTIC and miR-205 have a synergistic...

Embodiment 2

[0047] Example 2 Preparation and drug loading performance evaluation of milk-derived exosomes

[0048] ①Preparation of exosomes: The milk produced by dairy cows in the mid-lactation period of Shanghai Milk Group was collected, and exosomes were prepared by ultracentrifugation. Specific steps: centrifuge at 13,000g at low temperature for 30min at 4°C, take the supernatant and continue to centrifuge at 100,000g at low temperature at 4°C for 60min, carefully separate the supernatant and centrifuge again at 135,000g at low temperature at 4°C for 90min, discard the supernatant, collect the precipitate, and obtain exosomes . Exosomes were resuspended in PBS, sterilized by filtration with a 0.22 μm microporous membrane, and stored at -80°C for later use.

[0049] Micro-volume spectrophotometer The protein content of exosomes was measured, and the protein concentration was adjusted to 1-6mg / ml; the particle size and Zeta potential were measured by dynamic laser scattering method, t...

Embodiment 3

[0053] Example 3 Preparation of Ap16-exosomes / (DTIC / miR-205) co-loaded nano drug delivery system and evaluation of synergistic anti-tumor effect in vitro

[0054] (1) Human malignant melanoma cell culture

[0055] Human MM cell line A375 cells and A2058 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in MEM medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin, at 37.0°C and 5% CO 2 Cultured in an incubator, observed cell morphology and growth every day, replaced the culture medium every 2 days, digested and passaged as usual.

[0056] (2) Preparation of exosomes

[0057] Concrete operation is with embodiment 2.

[0058] (3) Construction and targeting performance evaluation of Ap16-exosomes

[0059] ① Synthesis of Ap16 aptamer: refer to [Wang CY, et al. Int J Cancer, 2016, 138(4): 918-26] reported sequence 5'-AGCACTCAATATTCCC-3' (SEQ ID NO: 2), provided by Guangzhou City Ruibo Biotechnology Co., Ltd. synthesized ch...

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Abstract

The invention relates to a targeted anti-malignant-melanoma DTIC (dacarbazine) and miR-205 co-carrying exosome as well as a preparation method and application thereof. The condition that DTIC and miR-205 achieve a cooperated inhibition effect on the human melanoma through being used in a combined way is discovered; a cholesterol modified DNA aptamer Ap16 is used as a targeted head; exosomes from the middle lactation period of milk are coated and carried with DTIC and miR-205; an Ap16-exosome/(DTIC/miR-205) nanometer medication system is successfully built by a freeze thawing method; the cooperated anti-tumor effect of the DTIC and miR-205 is proved; the aptamer Ap16 mediated co-carrying exosomes improve the feasibility of the miR-205 for enhancing the anti-malignant-melanoma effect of the DTIC. The invention provides a novel path for building the anti-malignant-melanoma nanometer medication system, and also provides the theoretical basis for the combined use of gene therapy and chemotherapy.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to a drug delivery system, in particular to a co-loaded exosome of dacarbazine and miR-205 targeting anti-malignant melanoma and its preparation method and application. Background technique [0002] Malignant melanoma (MM) is a highly malignant tumor derived from melanocytes, which mostly occurs on the surface of the skin, accounting for 7% to 20% of malignant skin tumors, and is mainly caused by mutations in nerve sheath cells. , caused by abnormalities in tyrosine metabolism and pigment production. Early malignant melanoma patients can be cured by surgery, but there are also many patients with uncontrollable invasion and metastasis, which is not only the main factor affecting the survival of melanoma patients, but also the biggest obstacle to the treatment of malignant melanoma. Once distant metastasis of malignant melanoma occurs, its prognosis is very poor, and the ...

Claims

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Application Information

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IPC IPC(8): A61K47/69A61K47/54A61K31/655A61K31/7088A61K47/46A61P35/00
CPCA61K31/655A61K31/7088A61K2300/00
Inventor 朱全刚陈中建李莹信如娟刘业强沈翠娥张红敏安多朋潘会君
Owner SHANGHAI DERMATOLOGY HOSPITAL
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