Abstract
Lipopolyplexes present well-established nucleic acid carriers assembled from sequence-defined cationic lipo-oligomers and DNA or RNA. They can be equipped with additional surface functionality, like shielding and targeting, in a stepwise assembly method using click chemistry. Here, we describe the synthesis of the required compounds, an azide-bearing lipo-oligomer structure and dibenzocyclooctyne (DBCO) click agents as well as the assembly of the compounds with siRNA into a surface-functionalized formulation. Both the lipo-oligomer and the DBCO-equipped shielding and targeting agents are produced by solid-phase synthesis (SPS). This enables for precise variation of all functional units, like variation in the amount of DBCO attachment sites or polyethylene glycol (PEG) length. Special cleavage conditions with only 5% trifluoroacetic acid (TFA) must be applied for the synthesis of the shielding and targeting agents due to acid lability of the DBCO unit. The two-step lipopolyplex assembly technique allows for separate optimization of the core and the shell of the formulation.
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Friedmann T, Roblin R (1972) Gene therapy for human genetic disease? Science 175(4025):949–955
Fire A (1999) RNA-triggered gene silencing. Trends Genet 15(9):358–363. https://doi.org/10.1016/S0168-9525(99)01818-1
Ginn SL, Amaya AK, Alexander IE, Edelstein M, Abedi MR (2018) Gene therapy clinical trials worldwide to 2017: an update. J Gene Med 20(5). https://doi.org/10.1002/jgm.3015
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411(6836):494–498. https://doi.org/10.1038/35078107
Hannon GJ (2002) RNA interference. Nature 418(6894):244–251. https://doi.org/10.1038/418244a
Reinhard S, Wagner E (2017) How to tackle the challenge of siRNA delivery with sequence-defined oligoamino amides. Macromol Biosci 17(1). https://doi.org/10.1002/mabi.201600152
Wagner E (2012) Functional polymer conjugates for medicinal nucleic acid delivery. Polym Nanomedicine 247:1–29. https://doi.org/10.1007/12_2011_148
de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J (2007) Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 6(6):443–453. https://doi.org/10.1038/nrd2310
Wagner E (2007) Programmed drug delivery: nanosystems for tumor targeting. Expert Opin Biol Ther 7(5):587–593. https://doi.org/10.1517/14712598.7.5.587
Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, Alabi CA, Yen Y, Heidel JD, Ribas A (2010) Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464(7291):1067–1070. https://doi.org/10.1038/nature08956
Semple SC, Akinc A, Chen JX, Sandhu AP, Mui BL, Cho CK, Sah DWY, Stebbing D, Crosley EJ, Yaworski E, Hafez IM, Dorkin JR, Qin J, Lam K, Rajeev KG, Wong KF, Jeffs LB, Nechev L, Eisenhardt ML, Jayaraman M, Kazem M, Maier MA, Srinivasulu M, Weinstein MJ, Chen QM, Alvarez R, Barros SA, De S, Klimuk SK, Borland T, Kosovrasti V, Cantley WL, Tam YK, Manoharan M, Ciufolini MA, Tracy MA, de Fougerolles A, MacLachlan I, Cullis PR, Madden TD, Hope MJ (2010) Rational design of cationic lipids for siRNA delivery. Nat Biotechnol 28(2):172–U118. https://doi.org/10.1038/nbt.1602
Akinc A, Zumbuehl A, Goldberg M, Leshchiner ES, Busini V, Hossain N, Bacallado SA, Nguyen DN, Fuller J, Alvarez R, Borodovsky A, Borland T, Constien R, de Fougerolles A, Dorkin JR, Jayaprakash KN, Jayaraman M, John M, Koteliansky V, Manoharan M, Nechev L, Qin J, Racie T, Raitcheva D, Rajeev KG, Sah DWY, Soutschek J, Toudjarska I, Vornlocher HP, Zimmermann TS, Langer R, Anderson DG (2008) A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol 26(5):561–569. https://doi.org/10.1038/nbt1402
Martin B, Sainlos M, Aissaoui A, Oudrhiri N, Hauchecorne M, Vigneron JP, Lehn JM, Lehn P (2005) The design of cationic lipids for gene delivery. Curr Pharm Des 11(3):375–394. https://doi.org/10.2174/1381612053382133
Ma JB, Ye KQ, Patel DJ (2004) Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429(6989):318–322. https://doi.org/10.1038/nature02519
Lee ER, Marshall J, Siegel CS, Jiang CW, Yew NS, Nichols MR, Nietupski JB, Ziegler RJ, Lane MB, Wang KX, Wan NC, Scheule RK, Harris DJ, Smith AE, Cheng SH (1996) Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung. Hum Gene Ther 7(14):1701–1717. https://doi.org/10.1089/hum.1996.7.14-1701
Green JJ, Langer R, Anderson DG (2008) A combinatorial polymer library approach yields insight into nonviral gene delivery. Acc Chem Res 41(6):749–759. https://doi.org/10.1021/ar7002336
Uchida H, Miyata K, Oba M, Ishii T, Suma T, Itaka K, Nishiyama N, Kataoka K (2011) Odd-even effect of repeating aminoethylene units in the side chain of N-substituted polyaspartamides on gene transfection profiles. J Am Chem Soc 133(39):15524–15532. https://doi.org/10.1021/ja204466y
Frohlich T, Edinger D, Klager R, Troiber C, Salcher E, Badgujar N, Martin I, Schaffert D, Cengizeroglu A, Hadwiger P, Vornlocher HP, Wagner E (2012) Structure-activity relationships of siRNA carriers based on sequence-defined oligo (ethane amino) amides. J Control Release 160(3):532–541. https://doi.org/10.1016/j.jconrel.2012.03.018
Krzyszton R, Salem B, Lee DJ, Schwake G, Wagner E, Radler JO (2017) Microfluidic self-assembly of folate-targeted monomolecular siRNA-lipid nanoparticles. Nanoscale 9(22):7442–7453. https://doi.org/10.1039/c7nr01593c
Schaffert D, Troiber C, Salcher EE, Frohlich T, Martin I, Badgujar N, Dohmen C, Edinger D, Klager R, Maiwald G, Farkasova K, Seeber S, Jahn-Hofmann K, Hadwiger P, Wagner E (2011) Solid-phase synthesis of sequence-defined T-, i-, and U-shape polymers for pDNA and siRNA delivery. Angew Chem Int Ed 50(38):8986–8989. https://doi.org/10.1002/anie.201102165
Morys S, Wagner E, Lachelt U (2016) From artificial amino acids to sequence-defined targeted oligoaminoamides. Methods Mol Biol 1445:235–258. https://doi.org/10.1007/978-1-4939-3718-9_15
Lee DJ, Wagner E, Lehto T (2015) Sequence-defined oligoaminoamides for the delivery of siRNAs. Methods Mol Biol 1206:15–27. https://doi.org/10.1007/978-1-4939-1369-5_2
Dohmen C, Edinger D, Frohlich T, Schreiner L, Lachelt U, Troiber C, Radler J, Hadwiger P, Vornlocher HP, Wagner E (2012) Nanosized multifunctional polyplexes for receptor-mediated siRNA delivery. ACS Nano 6(6):5198–5208. https://doi.org/10.1021/nn300960m
Gilleron J, Querbes W, Zeigerer A, Borodovsky A, Marsico G, Schubert U, Manygoats K, Seifert S, Andree C, Stoter M, Epstein-Barash H, Zhang L, Koteliansky V, Fitzgerald K, Fava E, Bickle M, Kalaidzidis Y, Akinc A, Maier M, Zerial M (2013) Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat Biotechnol 31(7):638–646. https://doi.org/10.1038/nbt.2612
Sahay G, Querbes W, Alabi C, Eltoukhy A, Sarkar S, Zurenko C, Karagiannis E, Love K, Chen D, Zoncu R, Buganim Y, Schroeder A, Langer R, Anderson DG (2013) Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat Biotechnol 31(7):653–658. https://doi.org/10.1038/nbt.2614
Wittrup A, Lieberman J (2015) Knocking down disease: a progress report on siRNA therapeutics. Nat Rev Genet 16(9):543–552. https://doi.org/10.1038/nrg3978
Klein PM, Reinhard S, Lee DJ, Muller K, Ponader D, Hartmann L, Wagner E (2016) Precise redox-sensitive cleavage sites for improved bioactivity of siRNA lipopolyplexes. Nanoscale 8(42):18098–18104. https://doi.org/10.1039/c6nr05767e
Meyer M, Philipp A, Oskuee R, Schmidt C, Wagner E (2008) Breathing life into polycations: functionalization with pH-responsive endosomolytic peptides and polyethylene glycol enables siRNA delivery. J Am Chem Soc 130(11):3272–3273. https://doi.org/10.1021/ja710344v
Leng Q, Chou ST, Scaria PV, Woodle MC, Mixson AJ (2014) Increased tumor distribution and expression of histidine-rich plasmid polyplexes. J Gene Med 16(9-10):317–328. https://doi.org/10.1002/jgm.2807
Hughes JA, Rao GA (2005) Targeted polymers for gene delivery. Expert Opin Drug Deliv 2(1):145–157. https://doi.org/10.1517/17425247.2.1.145
Kos P, Lachelt U, Herrmann A, Mickler FM, Doblinger M, He DS, Levacic AK, Morys S, Brauchle C, Wagner E (2015) Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer. Nanoscale 7(12):5350–5362. https://doi.org/10.1039/c4nr06556e
Morys S, Levacic AK, Urnauer S, Kempter S, Kern S, Radler JO, Spitzweg C, Lachelt U, Wagner E (2017) Influence of defined hydrophilic blocks within oligoaminoamide copolymers: compaction versus shielding of pDNA nanoparticles. Polymers 9(4). https://doi.org/10.3390/polym9040142
Tockary TA, Osada K, Motoda Y, Hiki S, Chen QX, Takeda KM, Dirisala A, Osawa S, Kataoka K (2016) Rod-to-globule transition of pDNA/PEG-poly(L-lysine) polyplex micelles induced by a collapsed balance between DNA rigidity and PEG crowdedness. Small 12(9):1193–1200. https://doi.org/10.1002/smll.201501815
Troiber C, Edinger D, Kos P, Schreiner L, Klager R, Herrmann A, Wagner E (2013) Stabilizing effect of tyrosine trimers on pDNA and siRNA polyplexes. Biomaterials 34(5):1624–1633. https://doi.org/10.1016/j.biomaterials.2012.11.021
Muller K, Kessel E, Klein PM, Hohn M, Wagner E (2016) Post-PEGylation of siRNA lipo-oligoamino amide polyplexes using tetra-glutamylated folic acid as ligand for receptor-targeted delivery. Mol Pharm 13(7):2332–2345. https://doi.org/10.1021/acs.molpharmaceut.6b00102
Muller K, Klein PM, Heissig P, Roidl A, Wagner E (2016) EGF receptor targeted lipo-oligocation polyplexes for antitumoral siRNA and miRNA delivery. Nanotechnology 27(46):464001. https://doi.org/10.1088/0957-4484/27/46/464001
Morys S, Urnauer S, Spitzweg C, Wagner E (2018) EGFR targeting and shielding of pDNA lipopolyplexes via bivalent attachment of a sequence-defined PEG agent. Macromol Biosci 18(1). https://doi.org/10.1002/mabi.201700203
Zhang W, Muller K, Kessel E, Reinhard S, He D, Klein PM, Hohn M, Rodl W, Kempter S, Wagner E (2016) Targeted siRNA delivery using a lipo-oligoaminoamide nanocore with an influenza peptide and transferrin shell. Adv Healthc Mater 5(12):1493–1504. https://doi.org/10.1002/adhm.201600057
Klein PM, Kern S, Lee DJ, Schmaus J, Hohn M, Gorges J, Kazmaier U, Wagner E (2018) Folate receptor-directed orthogonal click-functionalization of siRNA lipopolyplexes for tumor cell killing in vivo. Biomaterials 178:630–642. https://doi.org/10.1016/j.biomaterials.2018.03.031
Klein PM, Klinker K, Zhang W, Kern S, Kessel E, Wagner E, Barz M (2018) Efficient shielding of polyplexes using heterotelechelic polysarcosines. Polymers 10(6). https://doi.org/10.3390/polym10060689
Lee DJ, Kessel E, Edinger D, He D, Klein PM, Voith von Voithenberg L, Lamb DC, Lachelt U, Lehto T, Wagner E (2016) Dual antitumoral potency of EG5 siRNA nanoplexes armed with cytotoxic bifunctional glutamyl-methotrexate targeting ligand. Biomaterials 77:98–110. https://doi.org/10.1016/j.biomaterials.2015.11.004
Lee DJ, Kessel E, Lehto T, Liu X, Yoshinaga N, Padari K, Chen YC, Kempter S, Uchida S, Radler JO, Pooga M, Sheu MT, Kataoka K, Wagner E (2017) Systemic delivery of folate-PEG siRNA lipopolyplexes with enhanced intracellular stability for in vivo gene silencing in leukemia. Bioconjug Chem 28(9):2393–2409. https://doi.org/10.1021/acs.bioconjchem.7b00383
Schaffert D, Badgujar N, Wagner E (2011) Novel Fmoc-polyamino acids for solid-phase synthesis of defined polyamidoamines. Org Lett 13(7):1586–1589. https://doi.org/10.1021/ol200381z
Kaiser E, Colescott RL, Bossinger CD, Cook PI (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem 34(2):595–598
Reinhard S, Zhang W, Wagner E (2017) Optimized solid-phase-assisted synthesis of oleic acid containing siRNA nanocarriers. ChemMedChem 12(17):1464–1470. https://doi.org/10.1002/cmdc.201700350
Acknowledgments
This work was supported by DFG SFB1032 B4 (P.K. and E.W.), SFB1066 B5 (P.K. and E.W.), and DFG Excellence Cluster Nanosystems Initiative Munich (E.W.).
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Klein, P.M., Wagner, E. (2019). Click-Shielded and Targeted Lipopolyplexes. In: Gissberg, O., Zain, R., Lundin, K. (eds) Oligonucleotide-Based Therapies. Methods in Molecular Biology, vol 2036. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9670-4_8
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DOI: https://doi.org/10.1007/978-1-4939-9670-4_8
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