Arginine-rich Peptide Coated PLGA Nanoparticles Enhance Polymeric Delivery of Antisense HIF1α-oligonucleotide to Fully Differentiated Stiff Adipocytes

  • Da Hyeon Choi
  • Yoon Shin ParkEmail author



The purpose of this study was developing the new delivery system of antisense HIF1α oligodeoxynucleotide (ASO) into the stiff adipocytes. As the adipogenesis progressed, accumulating lipid droplet in cytosol lead adipocytes membrane stiffness and difficulties in the delivery of therapeutic agents into the cytosol. Hypoxia affects a number of biological functions including angiogenesis, apoptosis, inflammation, and adipogenesis. Hypoxia-inducible transcription factor-1 alpha (HIF1α) is a major transcription factor that controls metabolic and adipogenic gene expression under hypoxia. Controlling HIFα expression can be a promising therapy for obesity treatment.


The ASO was synthesized and used in a complex with polylactic-co-glycolic acid (PLGA) nanoparticles (NP). To enhance the cell-penetrating capacity, the PLGA-ASO-NP complex was coated with arginine-rich peptide (ARP) in different N:P molar ratios (PLGA-ASO-NP:ARP = 1: 1, 2: 1, 5: 1). To examine the intracellular and intranuclear delivery, these complexes were treated to fully differentiated adipocyte.


The PLGA-ASO-NP/ARP improved the efficacy of ASO-delivery into stiff adipocytes by increasing the cell surface charge, determined by the zeta potential, and forming polyplexes with small particle size. The proper N:P molar ratio of PLGA-ASO-NP/ARP synthesis was 5:1 with significantly improved gene delivery efficiency and intracellular uptake in adipocytes. Furthermore, PLGA-ASO-NP/ARP was stable in serum for 8 h compared to naked ASO.


These results suggest that the PLGA-ASO-NP/ARP can provide an effective and serum-stable gene-delivery system, especially for cells with a stiff cell membrane.


Arginine-rich peptide Hypoxia-inducible transcription factor-1α PLGA Adipogenesis Antisense oligonucleotide 


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This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2017R1A2B4002611), and partly by the Technological innovation R&D program of SMBA (S2449311).


  1. 1.
    Barness, L. A., Opitz, J. M. & Gilbert-Barness, E. Obesity: genetic, molecular, and environmental aspects. Am. J. Med. Genet. A. 143A, 3016–3034 (2007).CrossRefGoogle Scholar
  2. 2.
    Hjartaker, A., Langseth, H. & Weiderpass, E. Obesity and diabetes epidemics: cancer repercussions. Adv. Exp. Med. Biol. 630, 72–93 (2008).CrossRefGoogle Scholar
  3. 3.
    Zhu, Y. et al. A novel type of self-assembled nanoparticles as targeted gene carriers: an application for plasmid DNA and antimicroRNA oligonucleotide delivery. Int. J. Nanomedicine 11, 399–410 (2016).CrossRefGoogle Scholar
  4. 4.
    Mykhaylyk, O. et al. Magnetic nanoparticle and magnetic field assisted siRNA delivery in vitro. Methods. Mol. Biol. 1218, 53–106 (2015).CrossRefGoogle Scholar
  5. 5.
    Le, T. D., Nakagawa, O., Fisher, M., Juliano, R. L. & Yoo, H. RGD Conjugated Dendritic Polylysine for Cellular Delivery of Antisense Oligonucleotide. J. Nanoc. 17, 2353–2357 (2017).Google Scholar
  6. 6.
    Jabs, D. A. & Griffiths, P. D. Fomivirsen for the treatment of cytomegalovirus retinitis. Am. J. Ophthalmol. 133, 552–556 (2002).CrossRefGoogle Scholar
  7. 7.
    Shoham, N. et al. Adipocyte stiffness increases with accumulation of lipid droplets. Biophys. J. 106, 1421–1431 (2014).CrossRefGoogle Scholar
  8. 8.
    Zhang, C. & Liu, P. The lipid droplet: A conserved cellular organelle. Protein & Cell 8, 796–800 (2017).CrossRefGoogle Scholar
  9. 9.
    Garcia-Chaumont, C., Seksek, O., Grzybowska, J., Borowski, E. & Bolard, J. Delivery systems for antisense oligonucleotides. Pharmacol. Ther. 87, 255–277 (2000).CrossRefGoogle Scholar
  10. 10.
    Gao, J. Q. et al. Effective tumor targeted gene transfer using PEGylated adenovirus vector via systemic administration. J. Control. Release. 122, 102–110 (2007).CrossRefGoogle Scholar
  11. 11.
    Park, K. Non-ionic polymersomes for delivery of oligonucleotides. J. Control. Release. 134, 73 (2009).CrossRefGoogle Scholar
  12. 12.
    Kim, Y. et al. Polymersome delivery of siRNA and antisense oligonucleotides. J. Control. Release. 134, 132–140 (2009).CrossRefGoogle Scholar
  13. 13.
    McClorey, G. & Banerjee, S. Cell-Penetrating Peptides to Enhance Delivery of Oligonucleotide-Based Therapeutics. Biomedicines 6 (2018).Google Scholar
  14. 14.
    Astriab-Fisher, A., Sergueev, D., Fisher, M., Shaw, B. R. & Juliano, R. L. Conjugates of antisense oligonucleotides with the Tat and antennapedia cell-penetrating peptides: effects on cellular uptake, binding to target sequences, and biologic actions. Pharm. Res. 19, 744–754 (2002).CrossRefGoogle Scholar
  15. 15.
    Dong, L. et al. Targeting delivery oligonucleotide into macrophages by cationic polysaccharide from Bletilla striata successfully inhibited the expression of TNF-alpha. J. Control. Release. 134, 214–220 (2009).CrossRefGoogle Scholar
  16. 16.
    Fisher, A. A. et al. Evaluating the specificity of antisense oligonucleotide conjugates. A DNA array analysis. J. Biol. Chem. 277, 22980–22984 (2002).CrossRefGoogle Scholar
  17. 17.
    Luten, J., van Nostrum, C. F., De Smedt, S. C. & Hennink, W. E. Biodegradable polymers as non-viral carriers for plasmid DNA delivery. J. Control. Release. 126, 97–110 (2008).CrossRefGoogle Scholar
  18. 18.
    Ropelle, E. R. et al. Inhibition of hypothalamic Foxo1 expression reduced food intake in diet-induced obesity rats. J. Phtsiol. 587, 2341–2351 (2009).CrossRefGoogle Scholar
  19. 19.
    Langhi, C. et al. Therapeutic silencing of fat-specific protein 27 improves glycemic control in mouse models of obesity and insulin resistance. J. Lipid. Res. 58, 81–91 (2017).CrossRefGoogle Scholar
  20. 20.
    Cao, Y. et al. Antisense oligonucleotide and thyroid hormone conjugates for obesity treatment. Sci. Rep. 7, 9307 (2017).CrossRefGoogle Scholar
  21. 21.
    Crooke, R. M. et al. An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis. J. Lipid. Res. 46, 872–884 (2005).CrossRefGoogle Scholar
  22. 22.
    Helsley, R. N. et al. Targeting IkappaB kinase beta in Adipocyte Lineage Cells for Treatment of Obesity and Metabolic Dysfunctions. Stem Cells 34, 1883–1895 (2016).CrossRefGoogle Scholar
  23. 23.
    Yu, X. X. et al. Peripheral reduction of FGFR4 with antisense oligonucleotides increases metabolic rate and lowers adiposity in diet-induced obese mice. PLoS One 8, e66923 (2013).CrossRefGoogle Scholar
  24. 24.
    Popov, V. B. et al. Second-generation antisense oligonucleotides against beta-catenin protect mice against diet-induced hepatic steatosis and hepatic and peripheral insulin resistance. FASEB. J. 30, 1207–1217 (2016).CrossRefGoogle Scholar
  25. 25.
    Watts, L. M. et al. Reduction of hepatic and adipose tissue glucocorticoid receptor expression with antisense oligonucleotides improves hyperglycemia and hyperlipidemia in diabetic rodents without causing systemic glucocorticoid antagonism. Diabetes 54, 1846–1853 (2005).CrossRefGoogle Scholar
  26. 26.
    Vitto, M. F. et al. Reversion of steatosis by SREBP-1c antisense oligonucleotide did not improve hepatic insulin action in diet-induced obesity mice. Horm. Metab. Res. 44, 885–890 (2012).CrossRefGoogle Scholar
  27. 27.
    Park, Y. S. et al. Specific down regulation of 3T3-L1 adipocyte differentiation by cell-permeable antisense HIF1alpha-oligonucleotide. J. Control. Release. 144, 82–90 (2010).CrossRefGoogle Scholar
  28. 28.
    Takashima, Y. et al. Spray-drying preparation of microparticles containing cationic PLGA nanospheres as gene carriers for avoiding aggregation of nanospheres. Int. J. Pharm. 343, 262–269 (2007).CrossRefGoogle Scholar
  29. 29.
    Kolte, A., Patil, S., Lesimple, P., Hanrahan, J. W. & Misra, A. PEGylated composite nanoparticles of PLGA and polyethylenimine for safe and efficient delivery of pDNA to lungs. Int. J. Pharm. 524, 382–396 (2017).CrossRefGoogle Scholar
  30. 30.
    Hosogai, N. et al. Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation. Diabetes 56, 901–911 (2007).CrossRefGoogle Scholar
  31. 31.
    Fleischmann, E. et al. Tissue oxygenation in obese and non-obese patients during laparoscopy. Obes. Surg. 15, 813–819 (2005).CrossRefGoogle Scholar
  32. 32.
    Malcolm, D. W., Varghese, J. J., Sorrells, J. E., Ovitt, C. E. & Benoit, D. S. W. The Effects of Biological Fluids on Colloidal Stability and siRNA Delivery of a pH-Responsive Micellar Nanoparticle Delivery System. ACS. nano. 12, 187–197 (2018).CrossRefGoogle Scholar
  33. 33.
    Yang, C. et al. Theranostic poly (lactic-co-glycolic acid) nanoparticle for magnetic resonance/infrared fluorescence bimodal imaging and efficient siRNA delivery to macrophages and its evaluation in a kidney injury model. Nanomedicine 13, 2451–2462 (2017).CrossRefGoogle Scholar
  34. 34.
    Brock, R. The uptake of arginine-rich cell-penetrating peptides: putting the puzzle together. Bioconjug. Chem. 25, 863–868 (2014).CrossRefGoogle Scholar
  35. 35.
    Park, Y. J. et al. Nontoxic membrane translocation peptide from protamine, low molecular weight protamine (LMWP), for enhanced intracellular protein delivery: in vitro and in vivo study. FASEB. J. 19, 1555–1557 (2005).CrossRefGoogle Scholar
  36. 36.
    Li, Y. T. et al. Preliminary in vivo evaluation of the protein transduction domain-modified ATTEMPTS approach in enhancing asparaginase therapy. J. Biomed. Mater. Res. A. 91, 209–220 (2009).CrossRefGoogle Scholar
  37. 37.
    Ahn, S., Seo, E., Kim, K. & Lee, S. J. Controlled cellular uptake and drug efficacy of nanotherapeutics. Sci. Rep. 3, 1997 (2013).CrossRefGoogle Scholar
  38. 38.
    Cho, E. C., Xie, J., Wurm, P. A. & Xia, Y. Understanding the role of surface charges in cellular adsorption versus internalization by selectively removing gold nanoparticles on the cell surface with a I2/KI etchant. Nano Letters 9, 1080–1084 (2009).CrossRefGoogle Scholar
  39. 39.
    Pindiprolu, S., Chintamaneni, P. K., Krishnamurthy, P. T. & Ratna Sree Ganapathineedi, K. Formulation-optimization of solid lipid nanocarrier system of STAT3 inhibitor to improve its activity in triple negative breast cancer cells. Drug Dev. Ind. Pharm. 1–10 (2018).Google Scholar
  40. 40.
    dos Santos, T., Varela, J., Lynch, I., Salvati, A. & Dawson, K. A. Quantitative assessment of the comparative nanoparticle-uptake efficiency of a range of cell lines. Small 7, 3341–3349 (2011).CrossRefGoogle Scholar
  41. 41.
    Harush-Frenkel, O., Debotton, N., Benita, S. & Altschuler, Y. Targeting of nanoparticles to the clathrin-mediated endocytic pathway. Biochem. Biophys. Res. Commun. 353, 26–32 (2007).CrossRefGoogle Scholar
  42. 42.
    Verma, A. & Stellacci, F. Effect of surface properties on nanoparticle-cell interactions. Small 6, 12–21 (2010).CrossRefGoogle Scholar
  43. 43.
    Midoux, P. & Monsigny, M. Efficient gene transfer by histidylated polylysine/pDNA complexes. Bioconjug. Chem. 10, 406–411 (1999).CrossRefGoogle Scholar
  44. 44.
    Junghans, M., Kreuter, J. & Zimmer, A. Antisense delivery using protamine-oligonucleotide particles. Nucleic Acids Res. 28, E45 (2000).CrossRefGoogle Scholar
  45. 45.
    Harush-Frenkel, O., Rozentur, E., Benita, S. & Altschuler, Y. Surface charge of nanoparticles determines their endocytic and transcytotic pathway in polarized MDCK cells. Biomacromolecules 9, 435–443 (2008).CrossRefGoogle Scholar
  46. 46.
    Cu, Y., LeMoellic, C., Caplan, M. J. & Saltzman, W. M. Ligand-modified gene carriers increased uptake in target cells but reduced DNA release and transfection efficiency. Nanomedicine 6, 334–343 (2010).CrossRefGoogle Scholar
  47. 47.
    Verma, A. et al. Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles. Nat. Mater. 7, 588–595 (2008).CrossRefGoogle Scholar
  48. 48.
    Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R. & Brock, R. A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8, 848–866 (2007).CrossRefGoogle Scholar
  49. 49.
    Park, Y. S. et al. Controlled release of simvastatin from in situ forming hydrogel triggers bone formation in MC3T3-E1 cells. AAPS. J. 15, 367–376 (2013).CrossRefGoogle Scholar

Copyright information

© The Korean Society of Environmental Risk Assessment and Health Science and Springer 2019

Authors and Affiliations

  1. 1.Major in Microbiology, School of Biological Sciences, College of Natural SciencesChungbuk National UniversityCheongjuRepublic of Korea

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