Preparation of exosomes for siRNA delivery to cancer cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 °C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 ± 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 ± 0.22 × 1012 particle/mL and 8.3 ± 1.7 × 1010 particle/µg, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells

Trichinella spiralis secretes abundant unencapsulated small RNAs with potential effects on host gene expression

Many organisms, including parasitic nematodes, secrete small RNAs into the extracellular environment, largely encapsulated within small vesicles. Parasite-secreted material often contains microRNAs (miRNAs), raising the possibility that they might regulate host genes in target cells. Here we characterise secreted RNAs from the parasitic nematode Trichinella spiralis at two different life stages. We show that adult T. spiralis, which inhabit intestinal mucosa, secrete miRNAs within vesicles. Unexpectedly, T. spiralis muscle stage larvae, which live intracellularly within skeletal muscle cells, secrete miRNAs that appear not to be encapsulated. Notably, secreted miRNAs include a homologue of mammalian miRNA-31, which has an important role in muscle development. Our work therefore suggests that RNAs may be secreted without encapsulation in vesicles, with implications for the biology of T. spiralis infection

Bioinspired Polymerization of Quercetin to Produce a Curcumin-Loaded Nanomedicine with Potent Cytotoxicity and Cancer-Targeting Potential in Vivo

Nanomedicine has had a profound impact on the treatment of many diseases, especially cancer. However, synthesis of multifunctional nanoscale drug carriers often requires multistep coupling and purification reactions, which can pose major scale-up challenges. Here, we leveraged bioinspired oxidation-triggered polymerization of catechols to synthesize nanoparticles (NPs) from the plant polyphenol quercetin (QCT) loaded with a hydrophobic anticancer drug, curcumin, and functionalized with poly(ethylene glycol) (PEG) for steric stabilization in one reaction step. NPs were formed by base-catalyzed oxidative self-polymerization of QCT in the presence of curcumin and thiol-terminated PEG upon mixing in a universal solvent (dimethyl sulfoxide), followed by self-assembly with the gradual addition of water. Dynamic light scattering and X-ray photoelectron spectroscopy were used to confirm NP PEGylation. Drug loading was verified by UV–vis spectroscopy. Curcumin-loaded NPs were efficiently internalized by CT26 murine colon cancer cells as determined by flow cytometry and confocal microscopy. NPs also demonstrated sustained release and potent cytotoxicity in vitro. Moreover, in vivo imaging of CT26 tumor-bearing Balb/c mice following tail vein injection of DiR-labeled QCT NPs showed steady tumor accumulation of the NPs up to 24 h. This was further supported by significant tumor uptake of curcumin-loaded QCT NPs as measured by flow cytometry analysis of tumor homogenates. Our findings present a greener synthetic route for the fabrication of drug-loaded surface-functionalized NPs from poorly water-soluble plant polyphenols such as QCT as promising anticancer delivery systems