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Blood-brain barrier (BBB) dysfunction is prevalent in Alzheimer’s disease and other neurological disorders. Restoring normal BBB function through RNA therapy is a potential avenue for addressing cerebrovascular changes in these disorders that may lead to cognitive decline. Although lipid nanoparticles have been traditionally used as drug carriers for RNA, bicelles have been emerging as a better alternative because of their higher cellular uptake and superior transfection capabilities. Cationic bicelles composed of DPPC/DC7PC/DOTAP at molar ratios of 63.8/25.0/11.2 were evaluated for the delivery of RNA in polarized hCMEC/D3 monolayers, a widely used BBB cell culture model. RNA-bicelle complexes were formed at five N/P ratios (1:1 to 5:1) by a thin-film hydration method. The RNA-bicelle complexes at N/P ratios of 3:1 and 4:1 exhibited optimal particle characteristics for cellular delivery. The cellular uptake of cationic bicelles laced with 1 mol% DiI-C18 was confirmed by flow cytometry and confocal microscopy. The ability of cationic bicelles (N/P ratio 4:1) to transfect polarized hCMEC/D3 with FITC-labeled control siRNA was tested vis-a-vis commercially available Lipofectamine RNAiMAX. These studies demonstrated the higher transfection efficiency and greater potential of cationic bicelles for RNA delivery to the BBB endothelium.
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A universal discoidal nanoplatform for the intracellular delivery of PNAs
Peptide nucleic acids (PNAs) have gained considerable attention due to their remarkable potential in gene editing and targeting-based strategies. However, cellular delivery of PNAs remains a challenge in developing their broader therapeutic applications. Here, we investigated a novel complex made of lipid bicelles and PNA-based carriers for the efficient delivery of PNAs. For proof of concept, PNAs targeting microRNA (miR) 210 and 155 were tested. Comprehensive evaluation of positive as well as negative charge-containing bicelles with PNA : lipid ratios of 1 : 100, 1 : 1000, and 1 : 2500 was performed. The negatively charged bicelles with a PNA : lipid molar ratio of 1 : 2500 yielded a discoidal shape with a uniform diameter of ∼30 nm and a bilayer thickness of 5 nm, while the positively charged bicellar system contained irregular vesicles after the incorporation of PNA. Small-angle X-ray scattering (SAXS) analysis was performed to provide insight into how the hydrophobic PNAs interact with bicelles. Further, flow cytometry followed by confocal microscopy analyses substantiate the superior transfection efficiency of bicelles containing dye-conjugated antimiR PNAs. Functional analysis also confirmed miR inhibition by PNA oligomers delivered by bicelles. The nanodiscoidal complex opens a new pathway to deliver PNAs, which, on their own, are a great challenge to be endocytosed into cells.
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- Award ID(s):
- 1605971
- PAR ID:
- 10296936
- Date Published:
- Journal Name:
- Nanoscale
- Volume:
- 11
- Issue:
- 26
- ISSN:
- 2040-3364
- Page Range / eLocation ID:
- 12517 to 12529
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Peptide nucleic acids (PNAs) are a promising group of synthetic analogues of DNA and RNA that offer several distinct advantages over the naturally occurring nucleic acids for applications in biosensing, drug delivery, and nanoelectronics. Because of its structural differences from DNA/RNA, methods to analyze and assess the structure, conformations, and dynamics are needed. In this work, we develop synergistic techniques for the study of the PNA conformation. We use CuQ2, a Cu(II) complex with 8-hydroxyquinoline (HQ), as an alternative base pair and as a spin label in electron paramagnetic resonance (EPR) distance methods. We use molecular dynamics (MD) simulations with newly developed force field parameters for the spin labels to interpret the distance constraints determined by EPR. We complement these methods by UV–vis and circular dichroism measurements and assess the efficacy of the Cu(II) label on a PNA duplex whose backbone is based on aminoethylglycine and a duplex with a hydroxymethyl backbone modification. We show that the Cu(II) label functions efficiently within the standard PNA and the hydroxymethyl-modified PNA and that the MD parameters may be used to accurately reproduce our EPR findings. Through the combination of EPR and MD, we gain new insights into the PNA structure and conformations as well as into the mechanism of orientational selectivity in Cu(II) EPR at X-band. These results present for the first time a rigid Cu(II) spin label used for EPR distance measurements in PNA and the accompanying MD force fields for the spin label. Our studies also reveal that the spin labels have a low impact on the structure of the PNA duplexes. The combined MD and EPR approach represents an important new tool for the characterization of the PNA duplex structure and provides valuable information to aid in the rational application of PNA at large.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1039/c9nr03667a
