CICECO-Aveiro Institute of Materials

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Multimodal ionic liquid-based chromatographic supports for an effective RNA purification

Publication . Carapito, Ana Rita Mugeiro; Bernardo, Sandra C.; Pereira, Matheus M.; Neves, Márcia C.; Freire, Mara G.; Sousa, Fani

Nucleic acids have been considered interesting molecules to be used as biopharmaceuticals for the treatment of various diseases, in gene therapy strategies. In particular, RNA arises as the most promising approach because it does not require access to the nucleus of cells to exert its function; however, it is quite challenging due to its labile nature. To increase the possibility of translating RNA-based technology to clinical protocols, the bio- manufacturing of RNAs has been intensively exploited in the last few years. However, the standard RNA puri- fication processes remain time-consuming and present limitations regarding recovery yield and purity. This work describes the functionalization of chromatographic silica-based supports with four ionic liquids (ILs) composed of functional moieties that can promote distinct interactions with nucleic acids. After an initial screening to evaluate the binding and elution behavior of nucleic acids in the IL-based supports, SSi[C3C3NH2Im]Cl has shown to be the most promising for further purification assays. This support was studied for the RNA purification from different samples (clarified or more complex) and has shown to be highly effective, for all the conditions studied. Generally, it is here presented a new method for RNA isolation in a single step, using an IL-based chromato- graphic support, able to eliminate the usage of hazardous compounds often included in standard RNA extraction protocols.

2023Journal article

Open access

Brain-Targeted Delivery of Pre-miR-29b Using Lactoferrin-Stearic Acid-Modified-Chitosan/Polyethyleneimine Polyplexes

Publication . Pereira, Patrícia; Barreira, Maria; Cruz, Carla; Tomás, Joana; Luís, Ângelo; Pedro, Augusto; Queiroz, João; Sousa, Fani

The efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.

2020Journal article

Open access