Novel Lipid-Oligonucleotide Conjugates Containing Long-Chain Sulfonyl Phosphoramidate Groups: Synthesis and Biological Properties
Total Page：16
File Type：pdf, Size：1020Kb
applied sciences Article Novel Lipid-Oligonucleotide Conjugates Containing Long-Chain Sulfonyl Phosphoramidate Groups: Synthesis and Biological Properties Alina Derzhalova 1,2,†, Oleg Markov 2,† , Alesya Fokina 1,3,† , Yasuo Shiohama 4, Timofei Zatsepin 5,6 , Masayuki Fujii 7 , Marina Zenkova 2 and Dmitry Stetsenko 1,3,* 1 Department of Physics, Novosibirsk State University, 630090 Novosibirsk, Russia; [email protected] (A.D.); [email protected] (A.F.) 2 Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia; [email protected] (O.M.); [email protected] (M.Z.) 3 Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 63090 Novosibirsk, Russia 4 Tropical Biosphere Research Center, University of the Ryukyus, Okinawa 903-0213, Japan; [email protected] 5 Skolkovo Institute of Science and Technology, Innovation Centre Skolkovo, 121205 Moscow, Russia; [email protected] 6 Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia 7 Department of Biological & Environmental Chemistry, Kindai University, Iizuka, Fukuoka 820-8555, Japan; [email protected] * Correspondence: [email protected]; Tel.: +7-383-363-4963 † These authors contributed equally to the manuscript. Citation: Derzhalova, A.; Markov, O.; Abstract: New lipid conjugates of DNA and RNA incorporating one to four [(4-dodecylphenyl)sulfonyl] Fokina, A.; Shiohama, Y.; Zatsepin, T.; phosphoramidate or (hexadecylsulfonyl)phosphoramidate groups at internucleotidic positions near Fujii, M.; Zenkova, M.; Stetsenko, D. 0 0 Novel Lipid-Oligonucleotide the 3 or 5 -end were synthesized and characterized. Low cytotoxicity of the conjugates and their Conjugates Containing Long-Chain ability to be taken up into cells without transfection agents were demonstrated. Lipid-conjugated Sulfonyl Phosphoramidate Groups: siRNAs targeting repulsive guidance molecules a (RGMa) have shown a comparable gene silencing Synthesis and Biological Properties. activity in PK-59 cells to unmodified control siRNA when delivered into the cells via Lipofectamine Appl. Sci. 2021, 11, 1174. https:// mediated transfection. doi.org/10.3390/app11031174 Keywords: therapeutic nucleic acid; drug delivery; nanoparticles; cytotoxicity; macrophages; cellular Academic Editor: Tamaki Endoh, uptake; small interfering RNA; multiple sclerosis; repulsive guidance molecule a Eriks Rozners and Takashi Ohtsuki Received: 8 December 2020 Accepted: 24 January 2021 Published: 27 January 2021 1. Introduction Publisher’s Note: MDPI stays neutral Nucleic acid derivatives such as antisense oligonucleotides or small interfering RNAs with regard to jurisdictional claims in (siRNAs) are extensively studied as therapeutic agents that target biologically important published maps and institutional affil- RNAs; either mRNAs or noncoding RNAs such as micro-RNAs via either enzyme-mediated iations. RNA cleavage [1–3] or steric blocking of RNA function [4,5]. While the former may be applicable to a wide range of diseases including some genetic disorders [6], cancer [7], viral [8] and bacterial [9] infections, where the target RNA can be eliminated, the latter is often employed where a fine tuning of the RNA activity is required such as in splice switching [10]. The progress in the development of oligonucleotide-based therapies in the Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. past few years has resulted in the recent approval of several antisense and siRNA drugs by This article is an open access article the FDA and EMA [11]. However, widespread application of therapeutic oligonucleotides distributed under the terms and still suffers from their relatively inefficient cellular uptake and often poor bioavailabil- conditions of the Creative Commons ity [12,13]. The ways of promoting cellular delivery of oligonucleotides and their analogues Attribution (CC BY) license (https:// include their structural modification [14,15], noncovalent binding [16] or covalent conjuga- creativecommons.org/licenses/by/ tion [17,18] to the molecules that could improve translocation through the outer membrane 4.0/). and direct intracellular trafficking of nucleic acids. Among these, lipids in a wider sense Appl. Sci. 2021, 11, 1174. https://doi.org/10.3390/app11031174 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, x FOR PEER REVIEW 2 of 16 the outer membrane and direct intracellular trafficking of nucleic acids. Among these, li- pids in a wider sense including biomolecules such as cholesterol, tocopherol (vitamin E) or fatty acids, and their synthetic analogues such as, e.g., DOTMA [19] have been shown to improve cellular uptake of oligonucleotides, most relevantly, in the form of lipid-oligo- nucleotide conjugates (LONs) [20–22]. In particular, lipid conjugates of antisense oligonucleotides and siRNAs have im- proved transfection ability and extended half-life in the bloodstream with beneficial im- Appl. Sci. 2021, 11, 1174 pact on therapeutic activity in vivo [23–25]. We describe herein a series of novel DNA2 ofand 15 RNA lipid conjugates, which have been chemically modified by one to four long-chain N- (sulfonyl)phosphoramidate groups, namely, [(4-dodecylphenyl)sulfonyl]-phosphorami- date group (δ) or (hexadecylsulfonyl)phosphoramidate group (η) at the internucleotidic including biomolecules such as cholesterol, tocopherol (vitamin E) or fatty acids, and theirposition(s) synthetic near analogues the 3′- or such5′-end as, of e.g., the DOTMAoligonucleotide [19] have chain been (Figure shown 1). to The improve oligonucleo- cellular uptaketides were of oligonucleotides, obtained by an mostautomated relevantly, phosphoramidite in the form of lipid-oligonucleotidesolid-phase synthesis conjugates protocol (LONs)that employs [20–22 the]. Staudinger reaction between the support-bound phosphite triester and the respectiveIn particular, sulfony lipidl azide conjugates instead ofof antisenseusual aqueous oligonucleotides iodine oxidation and to siRNAs introduce have either im- provedof the lipophilic transfection modifications ability and extendedfollowing half-lifea previously in the developed bloodstream procedure with beneficial [26–28]. impact on therapeuticThe obtained activity lipid inconjugates vivo [23– of25 ].oligodeoxyribonucleotides We describe herein a series with of one novel or two DNA of andthe RNArespective lipid conjugates,hydrophobic which groups have were been nearly chemically non-cytotoxic modified for by oneeither to fourmurine long-chain or humanN- (sulfonyl)phosphoramidatemacrophages up to 20 μM concentration. groups, namely, Both [(4-dodecylphenyl)sulfonyl]-phosphoramidate types of lipid conjugates were able to enter groupthe cells (δ )in or a (hexadecylsulfonyl)phosphoramidatecarrier free manner. Both features are beneficial group (η) for at thepotential internucleotidic therapeutic posi- ap- tion(s)plication. near The the corresponding 30- or 50-end of lipid the oligonucleotideconjugates of siRNAs chain (Figure with either1). The two oligonucleotides or four hydro- werephobic obtained groups bywere an designed automated to downregulate phosphoramidite a validated solid-phase target synthesis in multiple protocol sclerosis, that employsnamely, repulsive the Staudinger guidance reaction molecule between a (RGMa) the support-bound [29]. The conjugates phosphite with triester two andgroups the respectivewere shown sulfonyl to possess azide silencing instead ofability usual comparable aqueous iodine to the oxidation unmodified to introduce siRNA when either de- of thelivered lipophilic by lipofectamine. modifications This following type of alipid previously conjugate developed may represent procedure a useful [26–28 addition]. to the inventory of therapeutic oligonucleotide derivatives. Figure 1. Structures andand positionspositions ofof hydrophobichydrophobic groups groups in in lipid-oligonucleotide lipid-oligonucleotide conjugates conjugates used used in thisin this study: study: oligodeoxynucleotide oligodeoxynucleotide (A );(A small); small interfering interfering RNA RNA (siRNA) (siRNA (B) ).(B). 2. MaterialsThe obtained and Methods lipid conjugates of oligodeoxyribonucleotides with one or two of the respective2.1. Oligonucleotide hydrophobic Synthesis groups were nearly non-cytotoxic for either murine or human macrophages up to 20 µM concentration. Both types of lipid conjugates were able to enter Oligodeoxynucleotides and siRNAs containing ((4-dodecylphenyl)sulfonyl)phos- the cells in a carrier free manner. Both features are beneficial for potential therapeutic application.phoramidate The(δ) or corresponding (hexadecylsulfonyl)phosphoramidate lipid conjugates of siRNAs (η) withgroups either were two assembled or four hy-by drophobicautomated groupssolid-phase were synthesis designed on to downregulatea Mermaid MM a- validated12 DNA/RNA target synthesizer in multiple according sclerosis, namely,to a modified repulsive β-cyanoethyl guidance phosphoramidite molecule a (RGMa) protocol [29]. The replacing conjugates aqueous with twoiodine groups oxidation were shownby a Staudinger to possess reaction silencing with ability either comparable 4-dodecylbenzenesulfonyl to the unmodified azide siRNA or when 1-hexadecanesul- delivered by lipofectamine.fonyl azide (0.5 This M in type acetonitrile of lipid conjugate for (δ) or mayin acetonitrile represent— a usefulTHF (2:1 addition v/v) for to (η), the 120 inventory min at of therapeutic oligonucleotide derivatives. 2.