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Current Pharmaceutical Biotechnology

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ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

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Research Article

Intracellular Trafficking Kinetics of Nucleic Acid Escape from Lipid Nanoparticles via Fluorescence Imaging

Author(s): Christina M. Bailey-Hytholt*, Gregory Ulinski, Julia Dugas, Mohammed Haines, Mihael Lazebnik, Peter Piepenhagen, Isidro E. Zarraga and Amey Bandekar*

Volume 25, Issue 1, 2024

Published on: 22 May, 2023

Page: [102 - 112] Pages: 11

DOI: 10.2174/1389201024666230403094238

Price: $65

Abstract

Background/Introduction: Lipid nanoparticles (LNPs) are one of the most clinically advanced candidates for delivering nucleic acids to target cell populations, such as hepatocytes. Once LNPs are endocytosed, they must release their nucleic acid cargo into the cell cytoplasm. For delivering messenger RNA (mRNA), delivery into the cytosol is sufficient; however, for delivering DNA, there is an added diffusional barrier needed to facilitate nuclear uptake for transcription and therapeutic effect.

Objective: The objective of the presented study was to use fluorescence microscopy to identify nucleic acid localization to endosomes and lysosomes after escape from lipid nanoparticles in order to optimize lipid nanoparticle formulations.

Methods: Here, we use fluorescence microscopy to investigate the intracellular fate of different LNP formulations to determine the kinetics of localization to endosomes and lysosomes. LNPs used in the studies were prepared via self-assembly using a NanoAssemblr for microfluidic mixing. As the content of polyethylene glycol (PEG) within the LNP formulation influences cellular uptake by hepatocyte cells, the content and hydrocarbon chain length within the formulation were assessed for their impact on intracellular trafficking. Standard LNPs were then formed using three commercially available ionizable lipids, Dlin-MC3-DMA (MC3), Dlin-KC2-DMA (KC2), and SS-OP. Plasmid DNA (pDNA) and mRNA were used, more specifically with a mixture of Cyanine 3 (Cy3)-labeled and green fluorescence protein (GFP) producing plasmid DNA (pDNA) as well as Cy5-labeled GFP producing mRNA. After formulation, LNPs were characterized for the encapsulation efficiency of the nucleic acid, hydrodynamic diameter, polydispersity, and zeta potential. All standard LNPs were ~100 nm in diameter and had neutral surface charge. All LNPs resulted in encapsulation efficiency greater than 70%. Confocal fluorescence microscopy was used for the intracellular trafficking studies, where LNPs were incubated with HuH-7 hepatocyte cells at times ranging from 0-48 h. The cells were antibody-stained for subcellular components, including nuclei, endosomes, and lysosomes.

Results: Analysis was performed to quantify localization of pDNA to the endosomes and lysosomes. LNPs with 1.5 mol% PEG and a hydrocarbon chain C14 resulted in optimal endosomal escape and GFP production. Results from this study demonstrate that a higher percentage of C14 PEG leads to smaller LNPs with limited available phospholipid binding area for ApoE, resulting in decreased cellular uptake. We observed differences in the localization kinetics depending on the LNP formulation type for SS-OP, KC2, and MC3 ionizable lipids. The results also demonstrate the technique across different nucleic acid types, where mRNA resulted in more rapid and uniform GFP production compared to pDNA delivery.

Conclusion: Here, we demonstrated the ability to track uptake and the sub-cellular fate of LNPs containing pDNA and mRNA, enabling improved screening prior to in vivo studies which would aid in formulation optimization.

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[1]
Mitchell, M.J.; Billingsley, M.M.; Haley, R.M.; Wechsler, M.E.; Peppas, N.A.; Langer, R. Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov., 2021, 20(2), 101-124.
[http://dx.doi.org/10.1038/s41573-020-0090-8] [PMID: 33277608]
[2]
Cullis, P.R.; Hope, M.J. Lipid nanoparticle systems for enabling gene therapies. Mol. Ther., 2017, 25(7), 1467-1475.
[http://dx.doi.org/10.1016/j.ymthe.2017.03.013] [PMID: 28412170]
[3]
Schoenmaker, L.; Witzigmann, D.; Kulkarni, J.A.; Verbeke, R.; Kersten, G.; Jiskoot, W.; Crommelin, D.J.A. mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm., 2021, 601, 120586.
[http://dx.doi.org/10.1016/j.ijpharm.2021.120586] [PMID: 33839230]
[4]
Shin, M.D.; Shukla, S.; Chung, Y.H.; Beiss, V.; Chan, S.K.; Ortega-Rivera, O.A.; Wirth, D.M.; Chen, A.; Sack, M.; Pokorski, J.K.; Steinmetz, N.F. COVID-19 vaccine development and a potential nanomaterial path forward. Nat. Nanotechnol., 2020, 15(8), 646-655.
[http://dx.doi.org/10.1038/s41565-020-0737-y] [PMID: 32669664]
[5]
Thanh Le, T.; Andreadakis, Z.; Kumar, A.; Gómez Román, R.; Tollefsen, S.; Saville, M.; Mayhew, S. The COVID-19 vaccine development landscape. Nat. Rev. Drug Discov., 2020, 19(5), 305-306.
[http://dx.doi.org/10.1038/d41573-020-00073-5] [PMID: 32273591]
[6]
Chen, C.Y.; Tran, D.M.; Cavedon, A.; Cai, X.; Rajendran, R.; Lyle, M.J.; Martini, P.G.V.; Miao, C.H. Treatment of hemophilia A using factor VIII messenger RNA lipid nanoparticles. Mol. Ther. Nucleic Acids, 2020, 20, 534-544.
[http://dx.doi.org/10.1016/j.omtn.2020.03.015] [PMID: 32330871]
[7]
Koide, H. Engineering of lipid nanoparticles by the multifunctionalization of the surface with amino acid derivatives for the neutralization of a target toxic peptide. Adv. Funct. Mater., 2021, 31(3), p. 2005641.
[http://dx.doi.org/10.1002/adfm.202005641]
[8]
Pattni, B.S.; Chupin, V.V.; Torchilin, V.P. New developments in liposomal drug delivery. Chem. Rev., 2015, 115(19), 10938-10966.
[http://dx.doi.org/10.1021/acs.chemrev.5b00046] [PMID: 26010257]
[9]
Allen, T.M.; Cullis, P.R. Liposomal drug delivery systems: From concept to clinical applications. Adv. Drug Deliv. Rev., 2013, 65(1), 36-48.
[http://dx.doi.org/10.1016/j.addr.2012.09.037] [PMID: 23036225]
[10]
Evers, M.J.W.; Kulkarni, J.A.; van der Meel, R.; Cullis, P.R.; Vader, P.; Schiffelers, R.M. State-of-the-art design and rapid-mixing production techniques of lipid nanoparticles for nucleic acid delivery. Small Methods, 2018, 2(9), 1700375.
[http://dx.doi.org/10.1002/smtd.201700375]
[11]
Kulkarni, J.A.; Cullis, P.R.; van der Meel, R. Lipid nanoparticles enabling gene therapies: from concepts to clinical utility. Nucleic Acid Ther., 2018, 28(3), 146-157.
[http://dx.doi.org/10.1089/nat.2018.0721] [PMID: 29683383]
[12]
Adams, D.; Gonzalez-Duarte, A.; O’Riordan, W.D.; Yang, C.C.; Ueda, M.; Kristen, A.V.; Tournev, I.; Schmidt, H.H.; Coelho, T.; Berk, J.L.; Lin, K.P.; Vita, G.; Attarian, S.; Planté-Bordeneuve, V.; Mezei, M.M.; Campistol, J.M.; Buades, J.; Brannagan, T.H., III; Kim, B.J.; Oh, J.; Parman, Y.; Sekijima, Y.; Hawkins, P.N.; Solomon, S.D.; Polydefkis, M.; Dyck, P.J.; Gandhi, P.J.; Goyal, S.; Chen, J.; Strahs, A.L.; Nochur, S.V.; Sweetser, M.T.; Garg, P.P.; Vaishnaw, A.K.; Gollob, J.A.; Suhr, O.B. Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis. N. Engl. J. Med., 2018, 379(1), 11-21.
[http://dx.doi.org/10.1056/NEJMoa1716153] [PMID: 29972753]
[13]
Urits, I.; Swanson, D.; Swett, M.C.; Patel, A.; Berardino, K.; Amgalan, A.; Berger, A.A.; Kassem, H.; Kaye, A.D.; Viswanath, O. A Review of patisiran (ONPATTRO®) for the treatment of polyneuropathy in people with hereditary transthyretin amyloidosis. Neurol. Ther., 2020, 9(2), 301-315.
[http://dx.doi.org/10.1007/s40120-020-00208-1] [PMID: 32785879]
[14]
Stanton, M.; Manganiello, M. Generation Bio Co, 2022. Non-active lipid nanoparticles with non-viral, capsid free dna. U.S. Patent Application 17/435,416.
[15]
Bailey-Hytholt, C.M.; Ghosh, P.; Dugas, J.; Zarraga, I.E.; Bandekar, A. Formulating and characterizing lipid nanoparticles for gene delivery using a microfluidic mixing platform. J. Vis. Exp., 2021, 2021(168), 1-16.
[PMID: 33720139]
[16]
Durymanov, M.; Reineke, J. Non-viral delivery of nucleic acids: Insight into mechanisms of overcoming intracellular barriers. Front. Pharmacol., 2018, 9, 971.
[http://dx.doi.org/10.3389/fphar.2018.00971] [PMID: 30186185]
[17]
Akinc, A.; Querbes, W.; De, S.; Qin, J.; Frank-Kamenetsky, M.; Jayaprakash, K.N.; Jayaraman, M.; Rajeev, K.G.; Cantley, W.L.; Dorkin, J.R.; Butler, J.S.; Qin, L.; Racie, T.; Sprague, A.; Fava, E.; Zeigerer, A.; Hope, M.J.; Zerial, M.; Sah, D.W.Y.; Fitzgerald, K.; Tracy, M.A.; Manoharan, M.; Koteliansky, V.; Fougerolles, A.; Maier, M.A. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther., 2010, 18(7), 1357-1364.
[http://dx.doi.org/10.1038/mt.2010.85] [PMID: 20461061]
[18]
Chen, S.; Tam, Y.Y.C.; Lin, P.J.C.; Sung, M.M.H.; Tam, Y.K.; Cullis, P.R. Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA. J. Control. Release, 2016, 235, 236-244.
[http://dx.doi.org/10.1016/j.jconrel.2016.05.059] [PMID: 27238441]
[19]
Suzuki, Y.; Ishihara, H. Structure, activity and uptake mechanism of siRNA-lipid nanoparticles with an asymmetric ionizable lipid. Int. J. Pharm., 2016, 510(1), 350-358.
[http://dx.doi.org/10.1016/j.ijpharm.2016.06.124] [PMID: 27374199]
[20]
Kumar, V.; Qin, J.; Jiang, Y.; Duncan, R.G.; Brigham, B.; Fishman, S.; Nair, J.K.; Akinc, A.; Barros, S.A.; Kasperkovitz, P.V. Shielding of lipid nanoparticles for siRNA delivery: Impact on physicochemical properties, cytokine induction, and efficacy. Mol. Ther. Nucleic Acids, 2014, 3(11), e210.
[http://dx.doi.org/10.1038/mtna.2014.61] [PMID: 25405467]
[21]
Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C.; Stöter, M.; Epstein-Barash, H.; Zhang, L.; Koteliansky, V.; Fitzgerald, K.; Fava, E.; Bickle, M.; Kalaidzidis, Y.; Akinc, A.; Maier, M.; Zerial, M. Image-based analysis of lipid nanoparticle–mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol., 2013, 31(7), 638-646.
[http://dx.doi.org/10.1038/nbt.2612] [PMID: 23792630]
[22]
Donahue, N.D.; Acar, H.; Wilhelm, S. Concepts of nanoparticle cellular uptake, intracellular trafficking, and kinetics in nanomedicine. Adv. Drug Deliv. Rev., 2019, 143, 68-96.
[http://dx.doi.org/10.1016/j.addr.2019.04.008] [PMID: 31022434]
[23]
Kowalski, P.S.; Rudra, A.; Miao, L.; Anderson, D.G. Delivering the messenger: advances in technologies for therapeutic mRNA Delivery. Mol. Ther., 2019, 27(4), 710-728.
[http://dx.doi.org/10.1016/j.ymthe.2019.02.012] [PMID: 30846391]
[24]
Tam, Y.; Chen, S.; Cullis, P. Advances in lipid nanoparticles for siRNA delivery. Pharmaceutics, 2013, 5(4), 498-507.
[http://dx.doi.org/10.3390/pharmaceutics5030498] [PMID: 24300520]
[25]
Schlich, M.; Palomba, R.; Costabile, G.; Mizrahy, S.; Pannuzzo, M.; Peer, D.; Decuzzi, P. Cytosolic delivery of nucleic acids: The case of ionizable lipid nanoparticles. Bioeng. Transl. Med., 2021, 6(2), e10213.
[http://dx.doi.org/10.1002/btm2.10213] [PMID: 33786376]
[26]
Akita, H.; Ishiba, R.; Hatakeyama, H.; Tanaka, H.; Sato, Y.; Tange, K.; Arai, M.; Kubo, K.; Harashima, H. A neutral envelope-type nanoparticle containing pH-responsive and SS-cleavable lipid-like material as a carrier for plasmid DNA. Adv. Healthc. Mater., 2013, 2(8), 1120-1125.
[http://dx.doi.org/10.1002/adhm.201200431] [PMID: 23386367]
[27]
Ryals, R.C.; Patel, S.; Acosta, C.; McKinney, M.; Pennesi, M.E.; Sahay, G. The effects of PEGylation on LNP based mRNA delivery to the eye. PLoS One, 2020, 15(10), e0241006.
[http://dx.doi.org/10.1371/journal.pone.0241006] [PMID: 33119640]
[28]
Bao, Y.; Jin, Y.; Chivukula, P.; Zhang, J.; Liu, Y.; Liu, J.; Clamme, J.P.; Mahato, R.I.; Ng, D.; Ying, W.; Wang, Y.; Yu, L. Effect of PEGylation on biodistribution and gene silencing of siRNA/lipid nanoparticle complexes. Pharm. Res., 2013, 30(2), 342-351.
[http://dx.doi.org/10.1007/s11095-012-0874-6] [PMID: 22983644]
[29]
Akinc, A.; Goldberg, M.; Qin, J.; Dorkin, J.R.; Gamba-Vitalo, C.; Maier, M.; Jayaprakash, K.N.; Jayaraman, M.; Rajeev, K.G.; Manoharan, M.; Koteliansky, V.; Röhl, I.; Leshchiner, E.S.; Langer, R.; Anderson, D.G. Development of lipidoid-siRNA formulations for systemic delivery to the liver. Mol. Ther., 2009, 17(5), 872-879.
[http://dx.doi.org/10.1038/mt.2009.36] [PMID: 19259063]
[30]
Cheng, Q.; Wei, T.; Farbiak, L.; Johnson, L.T.; Dilliard, S.A.; Siegwart, D.J. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol., 2020, 15(4), 313-320.
[http://dx.doi.org/10.1038/s41565-020-0669-6] [PMID: 32251383]
[31]
Mui, B.L.; Tam, Y.K.; Jayaraman, M.; Ansell, S.M.; Du, X.; Tam, Y.Y.C.; Lin, P.J.C.; Chen, S.; Narayanannair, J.K.; Rajeev, K.G.; Manoharan, M.; Akinc, A.; Maier, M.A.; Cullis, P.; Madden, T.D.; Hope, M.J. Influence of polyethylene glycol lipid desorption rates on pharmacokinetics and pharmacodynamics of siRNA lipid nanoparticles. Mol. Ther. Nucleic Acids, 2013, 2(12), e139.
[http://dx.doi.org/10.1038/mtna.2013.66] [PMID: 24345865]
[32]
Sago, C.D.; Lokugamage, M.P.; Islam, F.Z.; Krupczak, B.R.; Sato, M.; Dahlman, J.E. Nanoparticles that deliver RNA to bone marrow identified by in vivo directed evolution. J. Am. Chem. Soc., 2018, 140(49), 17095-17105.
[http://dx.doi.org/10.1021/jacs.8b08976] [PMID: 30394729]
[33]
Bohdanowicz, M.; Grinstein, S. Role of phospholipids in endocytosis, phagocytosis, and macropinocytosis. Physiol. Rev., 2013, 93(1), 69-106.
[http://dx.doi.org/10.1152/physrev.00002.2012] [PMID: 23303906]
[34]
Eskelinen, E.L. Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy. Mol. Aspects Med., 2006, 27(5-6), 495-502.
[http://dx.doi.org/10.1016/j.mam.2006.08.005] [PMID: 16973206]

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