Oligonucleotides (OND) represent a promising therapeutic approach. However, their instability and low intestinal permeability hamper oral bioavailability. Well-established for oral delivery, self-emulsifying drug delivery systems (SEDDS) can overcome the weakness of other delivery systems such as long-term instability of nanoparticles or complicated formulation processes. Therefore, the present study aims to prepare SEDDS for delivery of a nonspecific fluorescently labeled OND across the intestinal Caco-2 monolayer. The hydrophobic ion pairing of an OND and a cationic lipid served as an effective hydrophobization method using either dimethyldioctadecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). This strategy allowed a successful loading of OND-cationic lipid complexes into both negatively charged and neutral SEDDS. Subjecting both complex-loaded SEDDS to a nuclease, the negatively charged SEDDS protected about 16% of the complexed OND in contrast to 58% protected by its neutral counterpart. Furthermore, both SEDDS containing permeation-enhancing excipients facilitated delivery of OND across the intestinal Caco-2 cell monolayer. The negatively charged SEDDS showed a more stable permeability profile over 120 min, with a permeability of about 2 × 10-7 cm/s, unlike neutral SEDDS, which displayed an increasing permeability reaching up to 7 × 10-7 cm/s. In conclusion, these novel SEDDS-based formulations provide a promising tool for OND protection and delivery across the Caco-2 cell monolayer.

Bioneer FARMA Department of Pharmacy Faculty of Health and Medical Sciences University of Copenhagen Universitetsparken 2 2100 Copenhagen Denmark

Department of Pharmaceutical Technology Faculty of Pharmacy in Hradec Kralove Charles University Akademika Heyrovskeho 1203 500 05 Hradec Kralove Czech Republic

Department of Pharmacology Faculty of Pharmacy in Hradec Kralove Charles University Akademika Heyrovskeho 1203 500 05 Hradec Kralove Czech Republic

Department of Pharmacy Faculty of Health and Medical Sciences University of Copenhagen 2100 Copenhagen Denmark

DFM A S Kogle Alle 5 2970 Hørsholm Denmark

Faculty of Chemical Technology University of Chemistry and Technology Prague Technicka 5 166 28 Prague Czech Republic

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Gao X., Kim K., Liu D. Nonviral gene delivery: What we know and what is next. AAPS J. 2007;9:E92–E104. doi: 10.1208/aapsj0901009. PubMed DOI PMC

Lam J.K.W., Chow M.Y.T., Zhang Y., Leung S.W.S. siRNA Versus miRNA as Therapeutics for Gene Silencing. Mol. Ther. Nucleic Acids. 2015;4:1–20. doi: 10.1038/mtna.2015.23. PubMed DOI PMC

Andersson S., Antonsson M., Elebring M., Jansson-Löfmark R., Weidolf L. Drug metabolism and pharmacokinetic strategies for oligonucleotide- and mRNA-based drug development. Drug Discov. Today. 2018;23:1733–1745. doi: 10.1016/j.drudis.2018.05.030. PubMed DOI

Roth C.M. Drug Delivery: Principles and Applications. John Wiley & Sons, Inc.; Hoboken, NJ, USA: 2016. Delivery of genes and oligonucleotides; pp. 655–673.

O’Driscoll C.M., Bernkop-Schnürch A., Friedl J.D., Péat V., Jannin V. Oral delivery of non-viral nucleic acid-based therapeutics-do we have the guts for this? Eur. J. Pharm. Sci. 2019;93:206–220. doi: 10.1016/j.ejps.2019.03.027. PubMed DOI

Podolasky D.K. Inflammatory bowel disease. N. Engl. J. Med. 2002;347:417–429. doi: 10.1056/NEJMra020831. PubMed DOI

Zhang Y.Z., Li Y.Y. Inflammatory bowel disease: Pathogenesis. World J. Gastroenterol. 2014;20:91–99. doi: 10.3748/wjg.v20.i1.91. PubMed DOI PMC

Eisenstein M. Biology: A slow-motion epidemic. Nature. 2016;540:S98–S99. doi: 10.1038/540S98a. PubMed DOI

Hua S., Marks E., Schneider J.J., Keely S. Advances in oral nano-delivery systems for colon targeted drug delivery in inflammatory bowel disease: Selective targeting to diseased versus healthy tissue. Nanomed. Nanotechnol. Biol. Med. 2015;11:1117–1132. doi: 10.1016/j.nano.2015.02.018. PubMed DOI

Youshia J., Lamprecht A. Size-dependent nanoparticulate drug delivery in inflammatory bowel diseases. Expert Opin. Drug Deliv. 2016;13:281–294. doi: 10.1517/17425247.2016.1114604. PubMed DOI

Collnot E.M., Ali H., Lehr C.M. Nano- and microparticulate drug carriers for targeting of the inflamed intestinal mucosa. J. Control. Release. 2012;161:235–246. doi: 10.1016/j.jconrel.2012.01.028. PubMed DOI

Guo J., Jiang X., Gui S. RNA interference-based nanosystems for inflammatory bowel disease therapy. Int. J. Nanomed. 2016;11:5287–5310. doi: 10.2147/IJN.S116902. PubMed DOI PMC

Ball R.L., Bajaj P., Whitehead K.A. Oral delivery of siRNA lipid nanoparticles: Fate in the GI tract. Sci. Rep. 2018;8:2178. doi: 10.1038/s41598-018-20632-6. PubMed DOI PMC

Knipe J.M., Strong L.E., Peppas N.A. Enzyme- and pH-Responsive Microencapsulated Nanogels for Oral Delivery of siRNA to Induce TNF-α Knockdown in the Intestine. Biomacromolecules. 2016;17:788–797. doi: 10.1021/acs.biomac.5b01518. PubMed DOI

Wilson D.S., Dalmasso G., Wang L., Sitaraman S.V., Merlin D., Murthy N. Orally delivered thioketal nanoparticles loaded with TNF-α-siRNA target inflammation and inhibit gene expression in the intestines. Nat. Mater. 2010;9:923–928. doi: 10.1038/nmat2859. PubMed DOI PMC

Attarwala H., Han M., Kim J., Amiji M. Oral nucleic acid therapy using multicompartmental delivery systems. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2018;10:e1478. doi: 10.1002/wnan.1478. PubMed DOI PMC

Müllertz A., Ogbonna A., Ren S., Rades T. New perspectives on lipid and surfactant based drug delivery systems for oral delivery of poorly soluble drugs. J. Pharm. Pharmacol. 2010;62:1622–1636. doi: 10.1111/j.2042-7158.2010.01107.x. PubMed DOI

Gursoy R.N., Benita S. Self-emulsifying drug delivery systems (SEDDS) for improved oral delivery of lipophilic drugs. Biomed. Pharmacother. 2004;58:173–182. doi: 10.1016/j.biopha.2004.02.001. PubMed DOI

Mahmood A., Bernkop-Schnürch A. SEDDS: A game changing approach for the oral administration of hydrophilic macromolecular drugs. Adv. Drug Deliv. Rev. 2019;142:91–101. doi: 10.1016/j.addr.2018.07.001. PubMed DOI

Meyer J.D., Manning M.C. Hydrophobic ion pairing: Altering the solubility properties of biomolecules. Pharm. Res. 1998;15:188–193. doi: 10.1023/A:1011998014474. PubMed DOI

Simberg D., Weisman S., Talmon Y., Barenholz Y. DOTAP (and other cationic lipids): Chemistry, biophysics, and transfection. Crit. Rev. Ther. Drug Carr. Syst. 2004;21:257–317. doi: 10.1615/CritRevTherDrugCarrierSyst.v21.i4.10. PubMed DOI

Lobovkina T., Jacobson G.B., Gonzalez-Gonzalez E., Hickerson R.P., Leake D., Kaspar R.L., Contag C.H., Zare R.N. In vivo sustained release of siRNA from solid lipid nanoparticles. ACS Nano. 2011;5:9977–9983. doi: 10.1021/nn203745n. PubMed DOI PMC

Wong F.M.P., Reimer D.L., Bally M.B. Cationic Lipid Binding to DNA: Characterization of Complex Formation. Biochemistry. 1996;35:5756–5763. doi: 10.1021/bi952847r. PubMed DOI

Marty R., N’soukpoé-Kossi C.N., Charbonneau D., Weinert C.M., Kreplak L., Tajmir-Riahi H.A. Structural analysis of DNA complexation with cationic lipids. Nucleic Acids Res. 2009;37:849–857. doi: 10.1093/nar/gkn1003. PubMed DOI PMC

Xu Y., Szoka F.C. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry. 1996;35:5616–5623. doi: 10.1021/bi9602019. PubMed DOI

Liang W.W., Lam J.K. Molecular Regulation of Endocytosis. Volume 2. InTech; London, UK: 2012. Endosomal Escape Pathways for Non-Viral Nucleic Acid Delivery Systems; p. 64.

Kiptoo P., Calcagno A.M., Siahann T.J. Drug Delivery: Principles and Applications. John Wiley & Sons, Ltd.; Hoboken, NJ, USA: 2016. Drug delivery: Principles and applications; pp. 19–34.

Maher S., Brayden D.J., Casettari L., Illum L. Application of permeation enhancers in oral delivery of macromolecules: An update. Pharmaceutics. 2019;11:1–23. doi: 10.3390/pharmaceutics11010041. PubMed DOI PMC

Hayashi M., Sakai T., Hasegawa Y., Nishikawahara T., Tomioka H., Iida A., Shimizu N., Tomita M., Awazu S. Physiological mechanism for enhancement of paracellular drug transport. J. Control. Release. 1999;62:141–148. doi: 10.1016/S0168-3659(99)00031-0. PubMed DOI

Sha X., Yan G., Wu Y., Li J., Fang X. Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells. Eur. J. Pharm. Sci. 2005;24:477–486. doi: 10.1016/j.ejps.2005.01.001. PubMed DOI

Hauptstein S., Prüfert F., Bernkop-Schnürch A. Self-nanoemulsifying drug delivery systems as novel approach for pDNA drug delivery. Int. J. Pharm. 2015;487:25–31. doi: 10.1016/j.ijpharm.2015.03.064. PubMed DOI

Mahmood A., Prüfert F., Efiana N.A., Ashraf M.I., Hermann M., Hussain S., Bernkop-Schnürch A. Cell-penetrating self-nanoemulsifying drug delivery systems (SNEDDS) for oral gene delivery. Expert Opin. Drug Deliv. 2016;13:1503–1512. doi: 10.1080/17425247.2016.1213236. PubMed DOI

Bligh E.G., Dyer W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959;37:911–917. doi: 10.1139/o59-099. PubMed DOI

Gilmore J., Deguchi K., Takeyasu K. Nanoimaging of RNA Molecules with Atomic Force Microscopy. Microsc. Imaging Sci. Pract. Approaches Appl. Res. Educ. 2017;54:300–306.

Lyubchenko Y.L., Shlyakhtenko L.S., Ando T. Imaging of nucleic acids with atomic force microscopy. Methods. 2011;54:274–283. doi: 10.1016/j.ymeth.2011.02.001. PubMed DOI PMC

Niu Z., Tedesco E., Benetti F., Mabondzo A., Montagner I.M., Marigo I., Gonzalez-Touceda D., Tovar S., Diéguez C., Santander-Ortega M.J., et al. Rational design of polyarginine nanocapsules intended to help peptides overcoming intestinal barriers. J. Control. Release. 2017;263:4–17. doi: 10.1016/j.jconrel.2017.02.024. PubMed DOI

Amara S., Patin A., Giuffrida F., Wooster T.J., Thakkar S.K., Bénarouche A., Poncin I., Robert S., Point V., Molinari S., et al. In vitro digestion of citric acid esters of mono- and diglycerides (CITREM) and CITREM-containing infant formula/emulsions. Food Funct. 2014;5:1409–1421. doi: 10.1039/C4FO00045E. PubMed DOI

Zangenberg N.H., Müllertz A., Gjelstrup K.H., Hovgaard L. A dynamic in vitro lipolysis model. II: Evaluation of the model. Eur. J. Pharm. Sci. 2001;14:237–244. doi: 10.1016/S0928-0987(01)00182-8. PubMed DOI

Siqueira J.S.D., Al Sawaf M., Graeser K., Mu H., Müllertz A., Rades T. The ability of two in vitro lipolysis models reflecting the human and rat gastro-intestinal conditions to predict the in vivo performance of SNEDDS dosing regimens. Eur. J. Pharm. Biopharm. 2018;124:116–124. doi: 10.1016/j.ejpb.2017.12.014. PubMed DOI

Berthelsen R., Klitgaard M., Rades T., Müllertz A. In vitro digestion models to evaluate lipid based drug delivery systems; present status and current trends. Adv. Drug Deliv. Rev. 2019;142:35–49. doi: 10.1016/j.addr.2019.06.010. PubMed DOI

Michaelsen M.H., Siqueira J.S.D., Abdi I.M., Wasan K.M., Rades T., Müllertz A. Fenofibrate oral absorption from SNEDDS and super-SNEDDS is not significantly affected by lipase inhibition in rats. Eur. J. Pharm. Biopharm. 2019;142:258–264. doi: 10.1016/j.ejpb.2019.07.002. PubMed DOI

Fountain K.J., Gilar M., Budman Y., Gebler J.C. Purification of dye-labeled oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2003;783:61–72. doi: 10.1016/S1570-0232(02)00490-7. PubMed DOI

Schön P. Atomic force microscopy of RNA: State of the art and recent advancements. Semin. Cell Dev. Biol. 2018;73:209–219. doi: 10.1016/j.semcdb.2017.08.040. PubMed DOI

Seeman N.C. An Overview of Structural DNA Nanotechnology. Mol. Biotechnol. 2007;37:246–257. doi: 10.1007/s12033-007-0059-4. PubMed DOI PMC

Berg J., Tymoczko J., Stryer L. Biochemistry. 5th ed. WH Freeman; New York, NY, USA: 2002. Fatty Acids Are Key Constituents of Lipids.

Banyay M., Sarkar M., Gräslund A. A library of IR bands of nucleic acids in solution. Biophys. Chem. 2003;104:477–488. doi: 10.1016/S0301-4622(03)00035-8. PubMed DOI

Choosakoonkriang S., Wiethoff C.M., Anchordoquy T.J., Koe G.S., Smith J.G., Middaugh C.R. Infrared Spectroscopic Characterization of the Interaction of Cationic Lipids with Plasmid DNA. J. Biol. Chem. 2001;276:8037–8043. doi: 10.1074/jbc.M010592200. PubMed DOI

Coates J. Interpretation of Infrared Spectra, A Practical Approach. Encycl. Anal. Chem. 2004:1–23.

Taillandier E., Liquier J. Handbook of Vibrational Spectroscopy. John Wiley & Sons, Ltd.; Hoboken, NJ, USA: 2006. Vibrational Spectroscopy of Nucleic Acids.

Wu Y., Manna S., Petrochenko P., Koo B., Chen L., Xu X., Choi S., Kozak D., Zheng J. Coexistence of oil droplets and lipid vesicles in propofol drug products. Int. J. Pharm. 2020;577:118998. doi: 10.1016/j.ijpharm.2019.118998. PubMed DOI

de Ilarduya C.T., Sun Y., Düzgüneş N. Gene delivery by lipoplexes and polyplexes. Eur. J. Pharm. Sci. 2010;40:159–170. doi: 10.1016/j.ejps.2010.03.019. PubMed DOI

Bouxsein N.F., McAllister C.S., Ewert K.K., Samuel C.E., Safinya C.R. Structure and gene silencing activities of monovalent and pentavalent cationic lipid vectors complexed with siRNA. Biochemistry. 2007;46:4785–4792. doi: 10.1021/bi062138l. PubMed DOI

Lv H., Zhang S., Wang B., Cui S., Yan J. Toxicity of cationic lipids and cationic polymers in gene delivery. J. Control. Release. 2006;114:100–109. doi: 10.1016/j.jconrel.2006.04.014. PubMed DOI

Lobo B.A., Davis A., Koe G., Smith J.G., Middaugh C.R. Isothermal Titration Calorimetric Analysis of the Interaction between Cationic Lipids and Plasmid DNA. Arch. Biochem. Biophys. 2001;386:95–105. doi: 10.1006/abbi.2000.2196. PubMed DOI

Reimer D.L., Zhang Y.P., Kong S., Wheeler J.J., Graham R.W., Bally M.B. Formation of Novel Hydrophobic Complexes between Cationic Lipids and Plasmid DNA. Biochemistry. 1995;34:12877–12883. doi: 10.1021/bi00039a050. PubMed DOI

Belletti D., Tonelli M., Forni F., Tosi G., Vandelli M.A., Ruozi B. AFM and TEM characterization of siRNAs lipoplexes: A combinatory tools to predict the efficacy of complexation. Colloids Surfaces A Physicochem. Eng. Asp. 2013;436:459–466. doi: 10.1016/j.colsurfa.2013.07.021. DOI

Scholz C., Wagner E. Therapeutic plasmid DNA versus siRNA delivery: Common and different tasks for synthetic carriers. J. Control. Release. 2012;161:554–565. doi: 10.1016/j.jconrel.2011.11.014. PubMed DOI

Kubackova J., Zbytovska J., Holas O. Nanomaterials for direct and indirect immunomodulation: A review of applications. Eur. J. Pharm. Sci. 2020;142:105–139. doi: 10.1016/j.ejps.2019.105139. PubMed DOI

Tran T., Xi X., Rades T., Müllertz A. Formulation and characterization of self-nanoemulsifying drug delivery systems containing monoacyl phosphatidylcholine. Int. J. Pharm. 2016;502:151–160. doi: 10.1016/j.ijpharm.2016.02.026. PubMed DOI

Fatouros D.G., Bergenstahl B., Mullertz A. Morphological observations on a lipid-based drug delivery system during in vitro digestion. Eur. J. Pharm. Sci. 2007;31:85–94. doi: 10.1016/j.ejps.2007.02.009. PubMed DOI

Armand M. Lipases and lipolysis in the human digestive tract: Where do we stand? Curr. Opin. Clin. Nutr. Metab. Care. 2007;10:156–164. doi: 10.1097/MCO.0b013e3280177687. PubMed DOI

McCartney F., Jannin V., Chevrier S., Boulghobra H., Hristov D.R., Ritter N., Miolane C., Chavant Y., Demarne F., Brayden D.J. Labrasol® is an efficacious intestinal permeation enhancer across rat intestine: Ex vivo and in vivo rat studies. J. Control. Release. 2019;310:115–126. doi: 10.1016/j.jconrel.2019.08.008. PubMed DOI

Liu J., Werner U., Funke M., Besenius M., Saaby L., Fanø M., Mu H., Müllertz A. SEDDS for intestinal absorption of insulin: Application of Caco-2 and Caco-2/HT29 co-culture monolayers and intra-jejunal instillation in rats. Int. J. Pharm. 2019;560:377–384. doi: 10.1016/j.ijpharm.2019.02.014. PubMed DOI

Maher S., Geoghegan C., Brayden D.J. Intestinal permeation enhancers to improve oral bioavailability of macromolecules: Reasons for low efficacy in humans. Expert Opin. Drug Deliv. 2020;18:273–300. doi: 10.1080/17425247.2021.1825375. PubMed DOI

Li P., Nielsen H.M., Müllertz A. Impact of Lipid-Based Drug Delivery Systems on the Transport and Uptake of Insulin Across Caco-2 Cell Monolayers. J. Pharm. Sci. 2016;105:2743–2751. doi: 10.1016/j.xphs.2016.01.006. PubMed DOI

Swain S., Patra C.N., Rao M.E.B. Pharmaceutical Drug Delivery Systems and Vehicles. Woodhead Publishing India; New Delhi, India: 2016.

van Hoogevest P. Review–An update on the use of oral phospholipid excipients. Eur. J. Pharm. Sci. 2017;108:1–12. doi: 10.1016/j.ejps.2017.07.008. PubMed DOI

Fröhlich E. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int. J. Nanomed. 2012;7:5577–5591. doi: 10.2147/IJN.S36111. PubMed DOI PMC