Pharmaceutical Research

, Volume 33, Issue 2, pp 384–394 | Cite as

Aerosolized Non-viral Nucleic Acid Delivery in the Vaginal Tract of Pigs

  • Katrien Remaut
  • Evelien De Clercq
  • Oliwia Andries
  • Koen Rombouts
  • Matthias Van Gils
  • Laetitia Cicchelero
  • Ian Vandenbussche
  • Sarah Van Praet
  • Juan Manuel Benito
  • José Manuel Garcia Fernandéz
  • Niek Sanders
  • Daisy Vanrompay
Research Paper

Abstract

Purpose

The human pathogen Chlamydia trachomatis is worldwide the leading cause of bacterial sexually transmitted disease. Nasal or vaginal nucleic acid vaccination is a promising strategy for controlling genital Chlamydia trachomatis infections. Since naked nucleic acids are generally not efficiently taken up by cells, they are often complexed with carriers that facilitate their intracellular delivery.

Methods

In the current study, we screened a variety of commonly used non-viral gene delivery carriers for their ability to transfect newborn pig tracheal cells. The effect of aerosolization on the physicochemical properties and transfection efficiency of the complexes was also evaluated in vitro. Subsequently, a pilot experiment was performed in which the selected complexes were aerosolized in the vaginal tract of pigs.

Results

Both mRNA and pDNA containing lipofectamine and ADM70 complexes showed promise for protein expression in vitro, before and after aerosolization. In vivo, only lipofectamine/pDNA complexes resulted in high protein expression levels 24 h following aerosolization. This correlates to the unexpected observation that the presence of vaginal mucus increases the efficiency of lipofectamine/pDNA complexes 3-fold, while the efficiency of lipofectamine/mRNA complexes and ADM70/mRNA and ADM70/pDNA complexes decreased.

Conclusions

As aerosolization was an easy and effective method to deliver complexes to the vaginal tract of pigs, we believe this application technique has future potential for both vaginal and perhaps nasal vaccination using non-viral gene delivery vectors.

Key words

plasmid DNA mRNA aerosolization vaginal administration non-viral gene delivery complexes 

Abbreviations

C.

Chlamydia

DLS

Dynamic light scattering

DMEM

Dulbecco’s modified Eagle’s medium

DMPE

1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine

DMSO

Dimethylsulfoxide

DOPE

Dioleoylphosphatidylethanolamine

DOTAP

1,2-dioleoyl-3-trimethylammonium-propane

FCS

Fetal calf serum

GFP

Green fluorescent protein

GL67

Genzyme lipid 67

Luc

Luciferase

MTT

3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide

NPTr

Newborn pig tracheal

PEG

Polyethylene glycol

PEI

Polyethyleneimine

ROI

Regions of interest

Supplementary material

11095_2015_1796_MOESM1_ESM.jpg (1.1 mb)
Suppl Fig 1Cell viability. NPTr cells were treated with different mRNA/carrier complexes as depicted in the x-axis. Blanc represents non-transfected cells and DMSO served as positive control. * Significantly different from non-treated cells (blanc) (P ≤ 0.05). (JPG 1.12 mb)

References

  1. 1.
    WHO. Global incidence and prevalence of selected curable sexually transmitted infections – 2008. Geneva: World Health Organization; 2012.Google Scholar
  2. 2.
    Peipert JF. Genital chlamydial infections. N Engl J Med. 2003;349:2424–30.CrossRefPubMedGoogle Scholar
  3. 3.
    Brunham RC, Rey-Ladino J. Immunology of Chlamydia infection: implications for a Chlamydia trachomatis vaccine. Nat Rev Immunol. 2005;5(2):149–61.CrossRefPubMedGoogle Scholar
  4. 4.
    Manavi K. A review on infection with Chlamydia trachomatis. Best Pract Res Clin Obstet Gynaecol. 2006;20(6):941–51.CrossRefPubMedGoogle Scholar
  5. 5.
    Cunningham KA, Beagley KW. Male genital tract chlamydial infection: implications for pathology and infertility. Biol Reprod. 2008;79(2):180–9.CrossRefPubMedGoogle Scholar
  6. 6.
    Westrom L, Joesoef R, Reynolds G, Hagdu A, Thompson SE. Pelvic inflammatory disease and fertility. A cohort study of 1,844 women with laparoscopically verified disease and 657 control women with normal laparoscopic results. Sex Transm Dis. 1992;19(4):185–92.CrossRefPubMedGoogle Scholar
  7. 7.
    Haggerty CL, Gottlieb SL, Taylor BD, Low N, Xu F, Ness RB. Risk of sequelae after Chlamydia trachomatis genital infection in women. J Infect Dis. 2010;201 Suppl 2:S134–55.CrossRefPubMedGoogle Scholar
  8. 8.
    Plummer FA, Simonsen JN, Cameron DW, Ndinya-Achola JO, Kreiss JK, Gakinya MN, et al. Cofactors in male–female sexual transmission of human immunodeficiency virus type 1. J Infect Dis. 1991;163(2):233–9.CrossRefPubMedGoogle Scholar
  9. 9.
    Koskela P, Anttila T, Bjørge T, Brunsvig A, Dillner J, Hakama M, et al. Chlamydia trachomatis infection as a risk factor for invasive cervical cancer. Int J Cancer. 2000;85(1):35–9.CrossRefPubMedGoogle Scholar
  10. 10.
    Samoff E, Koumans EH, Markowitz LE, Sternberg M, Sawyer MK, Swan D, et al. Association of Chlamydia trachomatis with persistence of high-risk types of human papillomavirus in a cohort of female adolescents. Am J Epidemiol. 2005;162(7):668–75.CrossRefPubMedGoogle Scholar
  11. 11.
    Stagg AJ. Vaccines against Chlamydia: approaches and progress. Mol Med Today. 1998;4(4):166–73.CrossRefPubMedGoogle Scholar
  12. 12.
    Hafner L, Beagley K, Timms P. Chlamydia trachomatis infection: host immune responses and potential vaccines. Mucosal Immunol. 2008;1(2):116–30.CrossRefPubMedGoogle Scholar
  13. 13.
    Kanazawa T, Takashima Y, Shibata Y, Tsuchiya M, Tamura T, Okada H. Effective vaginal DNA delivery with high transfection efficiency is a good system for induction of higher local vaginal immune responses. J Pharm Pharmacol. 2009;61(11):1457–63.CrossRefPubMedGoogle Scholar
  14. 14.
    Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat Med. 2005;11(4 Suppl):S45–53.CrossRefPubMedGoogle Scholar
  15. 15.
    Wassén L, Schön K, Holmgren J, Jertborn M, Lycke N. Local intravaginal vaccination of the female genital tract. Scand J Immunol. 1996;44(4):408–14.CrossRefPubMedGoogle Scholar
  16. 16.
    Johansson EL, Wassén L, Holmgren J, Jertborn M, Rudin A. Nasal and vaginal vaccinations have differential effects on antibody responses in vaginal and cervical secretions in humans. Infect Immun. 2001;69(12):7481–6.PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Symens N, Soenen SJ, Rejman J, Braeckmans K, De Smedt SC, Remaut K. Intracellular partitioning of cell organelles and extraneous nanoparticles during mitosis. Adv Drug Deliv Rev. 2012;64(1):78–94.CrossRefPubMedGoogle Scholar
  18. 18.
    Tavernier G, Andries O, Demeester J, Sanders NN, De Smedt SC, Rejman J. mRNA as gene therapeutic: how to control protein expression. J Control Release. 2011;150(3):238–47.CrossRefPubMedGoogle Scholar
  19. 19.
    Remaut K, Sanders NN, De Geest BG, Braeckmans K, Demeester J, De Smedt SC. Nucleic acid delivery: where material sciences and bio-sciences meet. Mater Sci Eng R Rep. 2007;58(3–5):117–61.CrossRefGoogle Scholar
  20. 20.
    Symens N, Méndez-Ardoy A, Díaz-Moscoso A, Sánchez-Fernández E, Remaut K, Demeester J, et al. Efficient transfection of hepatocytes mediated by mRNA complexed to galactosylated cyclodextrins. Bioconjug Chem. 2012;23(6):1276–89.CrossRefPubMedGoogle Scholar
  21. 21.
    Ferrari M, Scalvini A, Losio MN, Corradi A, Soncini M, Bignotti E, et al. Establishment and characterization of two new pig cell lines for use in virological diagnostic laboratories. J Virol Methods. 2003;107(2):205–12.CrossRefPubMedGoogle Scholar
  22. 22.
    Geall AJ, Mandl CW, Ulmer JB. RNA: the new revolution in nucleic acid vaccines. Semin Immunol. 2013;25(2):152–9.CrossRefPubMedGoogle Scholar
  23. 23.
    Longbottom D, Livingstone M. Vaccination against chlamydial infections of man and animals. Vet J. 2006;171(2):263–75.CrossRefPubMedGoogle Scholar
  24. 24.
    Devoldere J, Dewitte H, De Smedt SC, Remaut K. Evading innate immunity in nonviral mRNA delivery: don’t shoot the messenger. Drug Discov Today. 2015. doi:10.1016/j.drudis.2015.07.009.PubMedGoogle Scholar
  25. 25.
    Andries O, De Filette M, De Smedt SC, Demeester J, Van Poucke M, Peelman L, et al. Innate immune response and programmed cell death following carrier-mediated delivery of unmodified mRNA to respiratory cells. J Control Release. 2013;167(2):157–66.CrossRefPubMedGoogle Scholar
  26. 26.
    Andries O, Cafferty SM, De Smedt SC, Weiss R, Sanders NN, Kitada T. N1-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control release. 2015;217:337–44.Google Scholar
  27. 27.
    Schautteet K, Stuyven E, Beeckman DS, Van Acker S, Carlon M, Chiers K, et al. Protection of pigs against Chlamydia trachomatis challenge by administration of a MOMP-based DNA vaccine in the vaginal mucosa. Vaccine. 2011;29(7):1399–407.CrossRefPubMedGoogle Scholar
  28. 28.
    Schautteet K, De Clercq E, Jönsson Y, Lagae S, Chiers K, Cox E, et al. Protection of pigs against genital Chlamydia trachomatis challenge by parenteral or mucosal DNA immunization. Vaccine. 2012;30(18):2869–81.CrossRefPubMedGoogle Scholar
  29. 29.
    Iwasaki A. Antiviral immune responses in the genital tract: clues for vaccines. Nat Rev Immunol. 2010;10(10):699–711.PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Bettinger T, Carlisle RC, Read ML, Ogris M, Seymour LW. Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells. Nucleic Acids Res. 2001;29(18):3882–91.PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther. 2008;16(11):1833–40.PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Capecchi MR. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980;22(2 Pt 2):479–88.CrossRefPubMedGoogle Scholar
  33. 33.
    Mirzayans R, Aubin RA, Paterson MC. Differential expression and stability of foreign genes introduced into human fibroblasts by nuclear versus cytoplasmic microinjection. Mutat Res. 1992;281(2):115–22.CrossRefPubMedGoogle Scholar
  34. 34.
    Thorburn AM, Alberts AS. Efficient expression of miniprep plasmid DNA after needle micro-injection into somatic cells. Biotechniques. 1993;14(3):356–8.PubMedGoogle Scholar
  35. 35.
    Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem. 1995;270(32):18997–9007.CrossRefPubMedGoogle Scholar
  36. 36.
    Martens TF, Remaut K, Demeester J, De Smedt SC, Braeckmans K. Intracellular delivery of nanomaterials: How to catch endosomal escape in the act. Nano Today. 2014;9(3):344–64.CrossRefGoogle Scholar
  37. 37.
    Zu R, Zuhorn IS, Hoekstra D. How cationic lipids transfer nucleic acids into cells and across cellular membranes: recent advances. J Control Release. 2013;166(1):46–56.CrossRefGoogle Scholar
  38. 38.
    Zu R, Hoekstra D, Zuhorn IS. Mechanism of polyplex- and lipoplex-mediated delivery of nucleic acids: real-time visualization of transient membrane destabilization without endosomal lysis. ACS Nano. 2013;7(5):3767–77.CrossRefGoogle Scholar
  39. 39.
    Andries O, De Filette M, Rejman J, De Smedt SC, Demeester J, Van Poucke M, et al. Comparison of the gene transfer efficiency of mRNA/GL67 and pDNA/GL67 complexes in respiratory cells. Mol Pharm. 2012;9(8):2136–45.PubMedGoogle Scholar
  40. 40.
    Ponsaerts P, Van Tendeloo VFI, Berneman ZN. Cancer immunotherapy using RNA-loaded dendritic cells. Clin Exp Immunol. 2003;134(3):378–84.PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    Gilboa E, Vieweg J. Cancer immunotherapy with mRNA-transfected dendritic cells. Immunol Rev. 2004;199:251–63.CrossRefPubMedGoogle Scholar
  42. 42.
    Ulmer JB, Mason PW, Geall A, Mandl CW. RNA-based vaccines. Vaccine. 2012;30(30):4414–8.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Katrien Remaut
    • 1
  • Evelien De Clercq
    • 2
  • Oliwia Andries
    • 3
  • Koen Rombouts
    • 1
  • Matthias Van Gils
    • 2
  • Laetitia Cicchelero
    • 3
  • Ian Vandenbussche
    • 1
  • Sarah Van Praet
    • 1
  • Juan Manuel Benito
    • 4
  • José Manuel Garcia Fernandéz
    • 4
  • Niek Sanders
    • 3
  • Daisy Vanrompay
    • 2
    • 5
  1. 1.Laboratory of General Biochemistry and Physical PharmacyGhent UniversityGhentBelgium
  2. 2.Laboratory of Immunology and Animal Biotechnology, Department of Molecular Biotechnology, Faculty of Bioscience EngineeringGhent UniversityGhentBelgium
  3. 3.Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary MedicineGhent UniversityMerelbekeBelgium
  4. 4.Institute for Chemical Research, CSICUniversity of SevillaSevillaSpain
  5. 5.Department of Animal Production, Faculty of Bioscience EngineeringGhent UniversityGhentBelgium

Personalised recommendations