Advertisement

Targeting Cancer with Peptide RNAi Nanoplexes

  • A. James MixsonEmail author
  • Qixin Leng
  • Szu-Ting Chou
  • Martin C. Woodle
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1974)

Abstract

With the recent explosion of genomic information on the root causes of disease, there is an increased interest in nucleic acid therapeutics, including siRNA and gene therapy, all of which require delivery of highly charged nucleic acids from siRNA with a molecular weight of about 1.4 × 104 to plasmids with an approximate molecular weight of 2.0–3.0 × 106. This chapter describes the delivery of shRNA via plasmid or siRNA with a peptide-based carrier. We focus on the histidine-lysine peptide which serves as an example for other peptides and polymeric carrier systems. When the HK peptide and nucleic acids are mixed together and interact with one another through ionic and nonionic interactions, nanoplexes are formed. These nanoplexes, carrying either shRNA or siRNA that target oncogenes, provide promising options for the treatment of cancer. We describe methods of preparation and characterization of these nanoplexes using dynamic light scattering, zeta potential, and gel retardation assays. We also provide protocols for transfection in vitro and in vivo for these nanoplexes.

Keywords

Peptides Polymers RNAi siRNA shRNA Tumor Polyplexes Nanoplexes 

Notes

Acknowledgments

This study was supported by the National Cancer Institute CA70394.

References

  1. 1.
    Hayashi Y, Yamauchi J, Khalil IA, Kajimoto K, Akita H, Harashima H (2011) Cell penetrating peptide-mediated systemic siRNA delivery to the liver. Int J Pharm 419:308–313CrossRefPubMedGoogle Scholar
  2. 2.
    Shukla RS, Qin B, Cheng K (2014) Peptides used in the delivery of small noncoding RNA. Mol Pharm 11:3395–3408CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Subramanian N, Kanwar JR, Kanwar RK, Sreemanthula J, Biswas J, Khetan V, Krishnakumar S (2015) EpCAM aptamer-siRNA chimera targets and regress epithelial cancer. PLoS One 10:e0132407CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G, Molema G, Lu PY, Scaria PV, Woodle MC (2004) Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res 32:e149CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Chou ST, Hom K, Zhang D, Leng Q, Tricoli LJ, Hustedt JM, Lee A, Shapiro MJ, Seog J, Kahn JD, Mixson AJ (2014) Enhanced silencing and stabilization of siRNA polyplexes by histidine-mediated hydrogen bonds. Biomaterials 35:846–855CrossRefPubMedGoogle Scholar
  6. 6.
    Leng Q, Mixson AJ (2005) Small interfering RNA targeting Raf-1 inhibits tumor growth in vitro and in vivo. Cancer Gene Ther 12:682–690CrossRefPubMedGoogle Scholar
  7. 7.
    Leng Q, Scaria P, Lu P, Woodle MC, Mixson AJ (2008) Systemic delivery of HK Raf-1 siRNA polyplexes inhibits MDA-MB-435 xenografts. Cancer Gene Ther 15:485–495CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    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 77:98–110CrossRefPubMedGoogle Scholar
  9. 9.
    Bartlett DW, Davis ME (2008) Impact of tumor-specific targeting and dosing schedule on tumor growth inhibition after intravenous administration of siRNA-containing nanoparticles. Biotechnol Bioeng 99:975–985CrossRefPubMedGoogle Scholar
  10. 10.
    Takeshita F, Minakuchi Y, Nagahara S, Honma K, Sasaki H, Hirai K, Teratani T, Namatame N, Yamamoto Y, Hanai K, Kato T, Sano A, Ochiya T (2005) Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo. Proc Natl Acad Sci U S A 102:12177–12182CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Villares GJ, Zigler M, Wang H, Melnikova VO, Wu H, Friedman R, Leslie MC, Vivas-Mejia PE, Lopez-Berestein G, Sood AK, Bar-Eli M (2008) Targeting melanoma growth and metastasis with systemic delivery of liposome-incorporated protease-activated receptor-1 small interfering RNA. Cancer Res 68:9078–9086CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Yano J, Hirabayashi K, Nakagawa S, Yamaguchi T, Nogawa M, Kashimori I, Naito H, Kitagawa H, Ishiyama K, Ohgi T, Irimura T (2004) Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer. Clin Cancer Res 10:7721–7726CrossRefPubMedGoogle Scholar
  13. 13.
    Chen Y, Wu JJ, Huang L (2010) Nanoparticles targeted with NGR motif deliver c-myc siRNA and doxorubicin for anticancer therapy. Mol Ther 18:828–834CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    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 464:1067–1070CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Strumberg D, Schultheis B, Traugott U, Vank C, Santel A, Keil O, Giese K, Kaufmann J, Drevs J (2012) Phase I clinical development of Atu027, a siRNA formulation targeting PKN3 in patients with advanced solid tumors. Int J Clin Pharmacol Ther 50:76–78CrossRefPubMedGoogle Scholar
  16. 16.
    Alsina M, Tabernero J, Shapiro G, Burris H, Infante JR, Weiss GJ, Cervantes-Ruiperez C, Gounder MM, Paz-Ares L, Falzone R, Hill J, Cehelsky J, Vaishnaw A, Gollob J, LoRusso P (2012) Open-label extension study of the RNAi therapeutic ALN-VSP02 in cancer patients responding to therapy. ASCO annual meeting. American Society of Clinical Oncology, Chicago, IL, 2012Google Scholar
  17. 17.
    Brower V (2010) RNA interference advances to early-stage clinical trials. J Natl Cancer Inst 102:1459–1461CrossRefPubMedGoogle Scholar
  18. 18.
    Zuckerman JE, Davis ME (2015) Clinical experiences with systemically administered siRNA-based therapeutics in cancer. Nat Rev Drug Discov 14:843–856CrossRefPubMedGoogle Scholar
  19. 19.
    Chou ST, Leng Q, Scaria P, Kahn JD, Tricoli LJ, Woodle M, Mixson AJ (2013) Surface-modified HK:siRNA nanoplexes with enhanced pharmacokinetics and tumor growth inhibition. Biomacromolecules 14:752–760CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Chou ST, Leng Q, Scaria P, Woodle M, Mixson AJ (2011) Selective modification of HK peptides enhances siRNA silencing of tumor targets in vivo. Cancer Gene Ther 18:707–716CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Leng Q, Mixson AJ (2016) The neuropilin-1 receptor mediates enhanced tumor delivery of H2K polyplexes. J Gene Med 18:134–144CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Leng Q, Chou ST, Scaria PV, Woodle MC, Mixson AJ (2014) Increased tumor distribution and expression of histidine-rich plasmid polyplexes. J Gene Med 16:317–328CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    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 411:494–498CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Amarzguioui M, Rossi JJ, Kim D (2005) Approaches for chemically synthesized siRNA and vector-mediated RNAi. FEBS Lett 579:5974–5981CrossRefPubMedGoogle Scholar
  25. 25.
    Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296CrossRefPubMedGoogle Scholar
  26. 26.
    Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293:1146–1150CrossRefPubMedGoogle Scholar
  27. 27.
    Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Schlee M, Uematsu S, Noronha A, Manoharan M, Akira S, de Fougerolles A, Endres S, Hartmann G (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med 11:263–270CrossRefPubMedGoogle Scholar
  28. 28.
    Robbins M, Judge A, Liang L, McClintock K, Yaworski E, MacLachlan I (2007) 2′-O-methyl-modified RNAs act as TLR7 antagonists. Mol Ther 15:1663–1669CrossRefGoogle Scholar
  29. 29.
    Schlee M, Hornung V, Hartmann G (2006) siRNA and isRNA: two edges of one sword. Mol Ther 14:463–470CrossRefPubMedGoogle Scholar
  30. 30.
    Hansmann L, Groeger S, von Wulffen W, Bein G, Hackstein H (2008) Human monocytes represent a competitive source of interferon-alpha in peripheral blood. Clin Immunol 127:252–264CrossRefPubMedGoogle Scholar
  31. 31.
    Sioud M (2005) Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization. J Mol Biol 348:1079–1090CrossRefPubMedGoogle Scholar
  32. 32.
    Leng Q, Chou ST, Scaria PV, Woodle MC, Mixson AJ (2012) Buffering capacity and size of siRNA polyplexes influence cytokine levels. Mol Ther 20:2282–2290CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Kurreck J (2003) Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem 270:1628–1644CrossRefPubMedGoogle Scholar
  34. 34.
    Janas MM, Jiang Y, Schlegel MK, Waldron S, Kuchimanchi S, Barros SA (2017) Impact of oligonucleotide structure, chemistry, and delivery method on in vitro cytotoxicity. Nucleic Acid Ther 27:11–22CrossRefPubMedGoogle Scholar
  35. 35.
    Scheule RK (2000) The role of CpG motifs in immunostimulation and gene therapy. Adv Drug Deliv Rev 44:119–134CrossRefPubMedGoogle Scholar
  36. 36.
    Yew NS, Zhao H, Przybylska M, Wu IH, Tousignant JD, Scheule RK, Cheng SH (2002) CpG-depleted plasmid DNA vectors with enhanced safety and long-term gene expression in vivo. Mol Ther 5:731–738CrossRefPubMedGoogle Scholar
  37. 37.
    Lachelt U, Wittmann V, Muller K, Edinger D, Kos P, Hohn M, Wagner E (2014) Synthetic polyglutamylation of dual-functional MTX ligands for enhanced combined cytotoxicity of poly(I:C) nanoplexes. Mol Pharm 11:2631–2639CrossRefPubMedGoogle Scholar
  38. 38.
    Miyata K (2016) Smart polymeric nanocarriers for small nucleic acid delivery. Drug Discov Ther 10:236–247CrossRefPubMedGoogle Scholar
  39. 39.
    Muratovska A, Eccles MR (2004) Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells. FEBS Lett 558:63–68CrossRefPubMedGoogle Scholar
  40. 40.
    Jarver P, Coursindel T, Andaloussi SE, Godfrey C, Wood MJ, Gait MJ (2012) Peptide-mediated cell and in vivo delivery of antisense oligonucleotides and siRNA. Mol Ther Nucleic Acids 1:e27CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Wagner E, Plank C, Zatloukal K, Cotten M, Birnstiel ML (1992) Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proc Natl Acad Sci U S A 89:7934–7938CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Chen QR, Zhang L, Luther PW, Mixson AJ (2002) Optimal transfection with the HK polymer depends on its degree of branching and the pH of endocytic vesicles. Nucleic Acids Res 30:1338–1345CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Palm-Apergi C, Lonn P, Dowdy SF (2012) Do cell-penetrating peptides actually “penetrate” cellular membranes? Mol Ther 20:695–697CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Hirose H, Takeuchi T, Osakada H, Pujals S, Katayama S, Nakase I, Kobayashi S, Haraguchi T, Futaki S (2012) Transient focal membrane deformation induced by arginine-rich peptides leads to their direct penetration into cells. Mol Ther 20:984–993CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Kim B, Tang Q, Biswas PS, Xu J, Schiffelers RM, Xie FY, Ansari AM, Scaria PV, Woodle MC, Lu P, Rouse BT (2004) Inhibition of ocular angiogenesis by siRNA targeting vascular endothelial growth factor pathway genes: therapeutic strategy for herpetic stromal keratitis. Am J Pathol 165:2177–2185CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    An DS, Qin FX, Auyeung VC, Mao SH, Kung SK, Baltimore D, Chen IS (2006) Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors. Mol Ther 14:494–504CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Cabral H, Matsumoto Y, Mizuno K, Chen Q, Murakami M, Kimura M, Terada Y, Kano MR, Miyazono K, Uesaka M, Nishiyama N, Kataoka K (2011) Accumulation of sub-100 nm polymeric micelles in poorly permeable tumours depends on size. Nat Nanotechnol 6:815–823CrossRefPubMedGoogle Scholar
  48. 48.
    Talmadge JE, Singh RK, Fidler IJ, Raz A (2007) Murine models to evaluate novel and conventional therapeutic strategies for cancer. Am J Pathol 170:793–804CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Caysa H, Hoffmann S, Luetzkendorf J, Mueller LP, Unverzagt S, Mader K, Mueller T (2012) Monitoring of xenograft tumor growth and response to chemotherapy by non-invasive in vivo multispectral fluorescence imaging. PLoS One 7:e47927CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Baklaushev VP, Grinenko NF, Yusubalieva GM, Abakumov MA, Gubskii IL, Cherepanov SA, Kashparov IA, Burenkov MS, Rabinovich EZ, Ivanova NV, Antonova OM, Chekhonin VP (2015) Modeling and integral X-ray, optical, and MRI visualization of multiorgan metastases of orthotopic 4T1 breast carcinoma in BALB/c mice. Bull Exp Biol Med 158:581–588CrossRefPubMedGoogle Scholar
  51. 51.
    Martin I, Dohmen C, Mas-Moruno C, Troiber C, Kos P, Schaffert D, Lachelt U, Teixido M, Gunther M, Kessler H, Giralt E, Wagner E (2012) Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery. Org Biomol Chem 10:3258–3268CrossRefPubMedGoogle Scholar
  52. 52.
    Krhac Levacic A, Morys S, Wagner E (2017) Solid-phase supported design of carriers for therapeutic nucleic acid delivery. Biosci Rep 37:BSR20160617CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Leng Q, Woodle MC, Mixson AJ (2017) Targeted delivery of siRNA therapeutics to malignant tumors. J Drug Deliv 2017:6971297CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Tiffen JC, Bailey CG, Ng C, Rasko JE, Holst J (2010) Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo. Mol Cancer 9:299CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Brutkiewicz S, Mendonca M, Stantz K, Comerford K, Bigsby R, Hutchins G, Goebl M, Harrington M (2007) The expression level of luciferase within tumour cells can alter tumour growth upon in vivo bioluminescence imaging. Luminescence 22:221–228CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • A. James Mixson
    • 1
    • 2
    Email author
  • Qixin Leng
    • 1
  • Szu-Ting Chou
    • 3
  • Martin C. Woodle
    • 4
  1. 1.Department of PathologyUniversity of Maryland School of MedicineBaltimoreUSA
  2. 2.Greenebaum Cancer CenterUniversity of Maryland School of MedicineBaltimoreUSA
  3. 3.Five Prime Therapeutics, Inc.South San FranciscoUSA
  4. 4.AparnaBio, Inc.GaithersburgUSA

Personalised recommendations