Abstract
Purpose
Understanding mechanisms of cellular uptake and intracellular release would enable better design of nanocarriers for delivery of nucleic acids such as siRNA and microRNA (miRNA).
Method
In this study, we investigated cellular pharmacokinetics of siRNA by co-encapsulating fluorescently labeled siRNA and molecular beacon (MB) in four different formulations of cationic lipid nanoparticles (LNPs). A miRNA mimic was also used as a probe for investigating cellular pharmacokinetics, which correlated well with RNAi activities.
Results
We tried to find the best LNP formulation based on the combination of DOTMA and DODMA. When the DOTMA/DODMA ratio was at 5/40, the LNP containing a luciferase siRNA produced the highest gene silencing activity. The superior potency of DOTMA/DODMA could be attributed to higher uptake and improved ability to facilitate siRNA release from endosomes subsequent to uptake.
Conclusions
Our findings may provide new insights into RNAi transfection pathways and have implications on cationic LNP design.
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REFERENCES
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806–11.
Yu B, Zhao X, Lee LJ, Lee RJ. Targeted delivery systems for oligonucleotide therapeutics. AAPS J. 2009;11:195–203.
Pecot CV, Calin GA, Coleman RL, Lopez-Berestein G, Sood AK. RNA interference in the clinic: challenges and future directions. Nat Rev Cancer. 2011;11:59–67.
Kim DH, Rossi JJ. Strategies for silencing human disease using RNA interference. Nat Rev Genet. 2007;8:173–84.
Love KT, Mahon KP, Levins CG, et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc Natl Acad Sci U S A. 2010;107:1864–9.
Semple SC, Akinc A, Chen J, et al. Rational design of cationic lipids for siRNA delivery. Nat Biotechnol. 2010;28:172–6.
Yu B, Hsu SH, Zhou C, et al. Lipid nanoparticles for hepatic delivery of small interfering RNA. Biomaterials. 2012;33:5924–34.
Wang X, Yu B, Ren W, Mo X, Zhou C, He H, et al. Enhanced hepatic delivery of siRNA and microRNA using oleic acid based lipid nanoparticle formulations. J Control Release. 2013;172:690–8.
Lin PJ, Tam YY, Hafez I, et al. Influence of cationic lipid composition on uptake and intracellular processing of lipid nanoparticle formulations of siRNA. Nanomedicine. 2013;9:233–46.
Wang X, Yu B, Wu Y, Lee RJ, Lee LJ. Efficient down-regulation of CDK4 by novel lipid nanoparticle-mediated siRNA delivery. Anticancer Res. 2011;31:1619–26.
Sahay G, Alakhova DY, Kabanov AV. Endocytosis of nanomedicines. J Control Release. 2010;145:182–95.
Duncan R, Richardson SC. Endocytosis and intracellular trafficking as gateways for nanomedicine delivery: opportunities and challenges. Mol Pharm. 2012;9:2380–402.
Hsu CY, Uludag H. Cellular uptake pathways of lipid-modified cationic polymers in gene delivery to primary cells. Biomaterials. 2012;33:7834–48.
Raemdonck K, Remaut K, Lucas B, Sanders NN, Demeester J, De Smedt SC. In situ analysis of single-stranded and duplex siRNA integrity in living cells. Biochemistry. 2006;45:10614–23.
Chen HH, Ho YP, Jiang X, Mao HQ, Wang TH, Leong KW. Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET. Mol Ther. 2008;16:324–32.
Abrams MT, Koser ML, Seitzer J, et al. Evaluation of efficacy, biodistribution, and inflammation for a potent siRNA nanoparticle: effect of dexamethasone co-treatment. Mol Ther. 2010;18:171–80.
Jiang S, Zhang Y. Upconversion nanoparticle-based FRET system for study of siRNA in live cells. Langmuir. 2010;26:6689–94.
Shi B, Keough E, Matter A, et al. Biodistribution of small interfering RNA at the organ and cellular levels after lipid nanoparticle-mediated delivery. J Histochem Cytochem. 2011;59:727–40.
Alabi CA, Love KT, Sahay G, et al. FRET-labeled siRNA probes for tracking assembly and disassembly of siRNA nanocomplexes. ACS Nano. 2012;6:6133–41.
Hirsch M, Strand D, Helm M. Dye selection for live cell imaging of intact siRNA. Biol Chem. 2012;393:23–35.
Zhou C, Zhang Y, Yu B, Phelps MA, Lee LJ, Lee RJ. Comparative cellular pharmacokinetics and pharmacodynamics of siRNA delivery by SPANosomes and by cationic liposomes. Nanomedicine. 2013;9:504–13.
Jarve A, Muller J, Kim IH, et al. Surveillance of siRNA integrity by FRET imaging. Nucleic Acids Res. 2007;35:e124.
Schneider S, Lenz D, Holzer M, Palme K, Suss R. Intracellular FRET analysis of lipid/DNA complexes using flow cytometry and fluorescence imaging techniques. J Control Release. 2010;145:289–96.
Shaheen SM, Akita H, Yamashita A, et al. Quantitative analysis of condensation/decondensation status of pDNA in the nuclear sub-domains by QD-FRET. Nucleic Acids Res. 2011;39:e48.
Shin S, Kwon HM, Yoon KS, Kim DE, Hah SS. FRET-based probing to gain direct information on siRNA sustainability in live cells: Asymmetric degradation of siRNA strands. Mol Biosyst. 2011;7:2110–3.
Hayashi Y, Noguchi Y, Harashima H. Non-linear pharmacokinetics of octaarginine-modified lipid nanoparticles: barriers from in vitro to in vivo. J Control Release. 2012;161:757–62.
Yang X, Koh CG, Liu S, et al. Transferrin receptor-targeted lipid nanoparticles for delivery of an antisense oligodeoxyribonucleotide against Bcl-2. Mol Pharm. 2009;6:221–30.
Hsu SH, Yu B, Wang X, et al. Cationic lipid nanoparticles for therapeutic delivery of siRNA and miRNA to murine liver tumor. Nanomedicine. 2013;9:1169–80.
Wu Y, Ho YP, Mao Y, et al. Uptake and intracellular fate of multifunctional nanoparticles: a comparison between lipoplexes and polyplexes via quantum dot mediated Forster resonance energy transfer. Mol Pharm. 2011;8:1662–8.
Wang B, Hsu SH, Wang X, Kutay H, Bid HK, Yu J, et al. Reciprocal regulation of miR-122 and c-Myc in hepatocellular cancer: Role of E2F1 and TFDP2. Hepatology. 2014;59:555–66.
Bai S, Nasser MW, Wang B, et al. MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib. J Biol Chem. 2009;284:32015–27.
Hsu SH, Wang B, Kota J, et al. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest. 2012;122:2871–83.
Whitehead KA, Sahay G, Li GZ, et al. Synergistic silencing: combinations of lipid-like materials for efficacious siRNA delivery. Mol Ther. 2011;19:1688–94.
Dominska M, Dykxhoorn DM. Breaking down the barriers: siRNA delivery and endosome escape. J Cell Sci. 2010;123:1183–9.
Stanton MG, Colletti SL. Medicinal chemistry of siRNA delivery. J Med Chem. 2010;53:7887–901.
Garzon R, Marcucci G, Croce CM. Targeting microRNAs in cancer: rationale, strategies and challenges. Nat Rev Drug Discov. 2010;9:775–89.
ACKNOWLEDGMENTS AND DISCLOSURES
This work was supported by NSF Nanoscale Science and Engineering Center (NSEC) grant EEC-0914790 and the Chinese National 863 Project (No. 2012AA020804).
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Bo Yu and Xinmei Wang contributed equally to this work.
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Yu, B., Wang, X., Zhou, C. et al. Insight into Mechanisms of Cellular Uptake of Lipid Nanoparticles and Intracellular Release of Small RNAs. Pharm Res 31, 2685–2695 (2014). https://doi.org/10.1007/s11095-014-1366-7
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DOI: https://doi.org/10.1007/s11095-014-1366-7