RESUMO
The challenge in effective delivery of mRNA has been a major hurdle in their development as therapeutics. Herein, we present that the incorporation of cationic nanogels as the condensing material for mRNA into liposomes enables stable and enhanced mRNA delivery to cells in vitro. We prepared dextran-based nanogel particles, which were surface functionalized with oligoarginine peptide (DNPR9) and complexed with mRNA for incorporation into liposomes (LipoDNPR9). The use of DNPR9 with the liposomes resulted in enhanced internalization, as well as a 4-fold increase in transfection of luciferase mRNA when treated with A549 cells in vitro, compared to control liposomes. The enhancement in transfection efficiency was also observed in various cell lines while causing low cytotoxicity. The versatility of the strategy was also investigated by applying DNPR9 for mRNA condensation to ionizable lipid particles, which resulted in an â¼55% increase in transfection. The current development based on nanogel-incorporated liposomes introduces an effective platform for mRNA delivery, while the condensation strategy using DNPR9 can be widely applied for various lipid-based formulations to enhance their efficacy.
Assuntos
Lipídeos , Lipossomos , Nanogéis , RNA Mensageiro/genética , TransfecçãoRESUMO
Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well-characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO-SPR) bioassay. In this context, EV binding on the FO-SPR probes was achieved only with EV-specific antibodies (e.g. anti-CD9 and anti-CD63) but not with non-specific anti-IgG. To increase detection sensitivity, we tested six different combinations of EV-specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti-CD9/Banti-CD81 and anti-CD63/Banti-CD9), resulting in 103 and 104 times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti-CD63/Banti-CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti-EpCAM antibody on the FO-SPR surface. The obtained results combined with FO-SPR real-time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis.