Difference between revisions of "Part:BBa K1942002"

Revision as of 11:49, 13 October 2016

A coding sequence of iRGD peptide and position it outside the membrane

iRGD, a tumor-penetrating peptide, can increase vascular and tissue permeability. Importantly, this effect did not require the drugs to be chemically conjugated to the peptide. To enhance the accuracy of drug delivery system and improve targeting index of drugs, iRGD peptide was displayed on the surface of the exosome containing our previously designed siRNA, allowing us to target recipient cells in vivo efficiently.
The tumor targeting capability of exosomes was conferred by engineering the immature dendritic cells (imDCs) to express lysosome-associated membrane glycoprotein 2b (Lamp2b), a well-characterized exosomal membrane protein, fused with iRGD (CRGDKGPDC) targeting peptide for av integrin. Lamp-2b is a protein found specifically abundant in exosomal membranes. So we connect iRGD with Lamp2b by a glycine-linker, and promote the expression using cmv promoter. In that way, the iRGD exosomes (iRGD-Exos) are endowed with site-specific recognition ability and were purified from cell culture supernatants and loaded with Dox by electroporation.

USAGE AND BIOLOGY

iRGD is a tumor-penetrating peptide that can increase vascular and tissue permeability. Importantly, this effect did not require the drugs to be chemically conjugated to the peptide. To enhance the accuracy of drug delivery system and improve targeting index of drugs, iRGD peptide was displayed on the surface of the exosome containing our previously designed siRNA, allowing us to target recipient cells in vivo efficiently.
The tumor targeting capability of exosomes was conferred by engineering the immature dendritic cells (imDCs) to express lysosome-associated membrane glycoprotein 2b (Lamp2b), a well-characterized exosomal membrane protein, fused with iRGD (CRGDKGPDC) targeting peptide for αν integrin. Immature DCs were transfected with the vector expressing iRGD-Lamp2b fusion proteins using Lipofectamine 2000 transfection reagent. Lamp-2b is a protein found specifically abundant in exosomal membranes. So we connect iRGD with Lamp2b by a glycine-linker, and promote the expression using cmv promoter. We engineered our chassis, human embryonic kidney 293 (HEK293) cells, to express iRGD-Lamp2b fusion protein. Therefore, the iRGD exosomes (iRGD-Exos) are endowed with site-specific recognition ability and were purified from cell culture supernatants and loaded with Dox by electroporation.

CHARACTERIZATION

The iRGD-Lamp2b expressing vector was thoroughly described in Tian’s article (Yanhua Tian, et al, Biomaterials, 2013). He showed that exosomes, endogenous nano-sized membrane vesicles secreted by most cell types, could deliver chemotherapeutics such as doxorubicin (Dox) to tumor tissue in BALB/c nude mice. To reduce immunogenicity and toxicity, mouse immature dendritic cells (imDCs) were used for exosome production. Tumor targeting was facilitated by engineering the imDCs to express a well-characterized exosomal membrane protein (Lamp2b) fused to αν integrin-specific iRGD peptide (CRGDKGPDC). Purified exosomes from imDCs were loaded with Dox via electroporation, with an encapsulation efficiency of up to 20%. IRGD exosomes showed highly efficient targeting and Dox delivery to αν integrin-positive breast cancer cells in vitro as demonstrated by confocal imaging and flow cytometry. Intravenously injected targeted exosomes delivered Dox specifically to tumor tissues, leading to inhibition of tumor growth without overt toxicity. The results suggested that exosomes modified by targeting ligands could be used therapeutically for the delivery of Dox to tumors, thus having great potential value for clinical applications in our project.

Sequence and Features