Polymer multilayer tattooing for enhanced DNA vaccination

Journal name:
Nature Materials
Volume:
12,
Pages:
367–376
Year published:
DOI:
doi:10.1038/nmat3550
Received
Accepted
Published online

Abstract

DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These ‘multilayer tattoo’ DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination.

At a glance

Figures

  1. Design of quick-release vaccine-loaded microneedle coatings.
    Figure 1: Design of quick-release vaccine-loaded microneedle coatings.

    a, Schematic view of release-layer-mediated multilayer tattooing strategy using coated microneedles: (1) PLLA microneedles are coated with PNMP release-layer films through spray deposition; (2) ultraviolet irradiation imparts pH-sensitive aqueous solubility to the PNMP film, forming a uv-PNMP ‘release-layer’; (3) Overlying multilayer films containing nucleic acids are constructed using LbL deposition at pH 5.0. b, Mechanism of action for multilayer tattooing: (1) Microneedle application to skin and exposure to interstitial fluid gives rapid release-layer dissolution, mediating overlying film delamination and retention in skin following microneedle removal; (2) Implanted films provide sustained release of nucleic acids through hydrolytic PBAE degradation and release of in situ-formed PBAE/nucleic acid polyplexes; (3) released polyplexes mediate local transfection and immune modulation in the tissue.

  2. LbL assembly of microneedle coatings carrying DNA, immunostimulatory RNA, and transfection agents.
    Figure 2: LbL assembly of microneedle coatings carrying DNA, immunostimulatory RNA, and transfection agents.

    a, Film architecture for (uv-PNMP)(PS/SPS) n(PBAE/pLUC) n multilayers. b, Growth of (poly-1/pLUC) n and (poly-2/pLUC) n multilayers assembled onto (uv-PNMP)(PS/SPS) 20 films on silicon substrates as a function of the number of deposited (PBAE/pLUC) bilayers as measured by surface profilometry. Data represent the mean±s.e.m., n  =  8. c, Representative confocal images of PLLA microneedles coated with (SAv488-bPNMP)(PS/SPS) 20(poly-1/Cy5-pLUC) 35 films (left, transverse optical sections; right, lateral sections, 100 μm z-intervals, scale bars, 200 μm; blue, Sav488-uv-bPNMP; yellow, Cy5-pLUC). d, Quantification of Cy5-pLUC and Sav488-bPNMP incorporated into (SAv488-bPNMP)(PS/SPS) 20(poly-1/Cy5-pLUC) n films on microneedles through confocal fluorescence intensity analysis (left axis, n  =  15) and measurement of total DNA recovered from dissolved films (right axis, n  =  3). Data represent the mean±s.e.m. e, Film architecture for (uv-PNMP)(PS/SPS) 20(poly-1/pLUC) n(poly-1/poly(I:C)) n multilayers. f, Representative confocal images of microneedles coated with (SAv488-uv-bPNMP)(PS/SPS) 20(poly-1/TMR-poly(I:C)) 15(poly-1/Cy5-pLUC) 15 films (left, transverse sections; right, lateral sections, 100 μm z-intervals; scale, 200 μm; blue, Sav488-uv-bPNMP; yellow, Cy5-pLUC; red, TMR-poly(I:C)). g,h, Quantification of Cy5-pLUC, TMR-poly(I:C), and SAv488-bPNMP incorporated into (SAv488-bPNMP)(PS/SPS) 20(poly-1/TMR-poly(I:C)) n(poly-1/Cy5-pLUC) n films on microneedles through confocal fluorescence intensity analysis (g, n  =  15) and measurement of total nucleic acids recovered from dissolved films (h, n  =  3). Data represent the mean±s.e.m.

  3. PNMP release-layers promote rapid implantation of multilayer films at microneedle penetration sites in vivo.
    Figure 3: PNMP release-layers promote rapid implantation of multilayer films at microneedle penetration sites in vivo.

    a, Optical micrograph of ear skin stained with trypan blue to reveal epidermal penetration following PLLA microneedle application (scale bar, 500 μm). b, Representative confocal images of (SAv488-bPNMP)(PS/SPS) 20(poly-1/Cy5-pLUC) 35-coated PLLA microneedles with or without ultraviolet sensitization of the PNMP layer (blue, Sav488-bPNMP; yellow, Cy5-pLUC), before application, or after 15 min application to murine ear skin (lateral sections, 100 μm z-intervals, scale bar, 200 μm). c, Quantitation of confocal fluorescence intensities (n  =  15) showing loss of Sav488-uv-bPNMP and Cy5-pLUC films from coated microneedles on application to skin, dependent on ultraviolet-induced photo-switching of the PNMP layer solubility. Data represent the mean±s.e.m., ***,p < 0.0001, analysed by the unpaired t-test. d, Representative confocal image of treated murine skin showing film implantation after 15 min (green, MHC II-GFP; yellow, Cy5-pLUC; penetration site outlined; scale bar, 100 μm). ex-y/x-z/y-z confocal images showing depth of Cy5-pLUC film deposition after 15 min microneedle application (green, MHC II-GFP; yellow, Cy5-pLUC; penetration sites outlined; scale bar, 200 μm). f, Representative confocal image of treated murine skin showing TMR-poly(I:C) film implantation after 15 min microneedle application (green, MHC II-GFP; red, TMR-poly(I:C); penetration site outlined; scale bar, 100 μm). g, Colocalization and uptake of TMR-poly(I:C) by MHC II-GFP+ LCs at the microneedle insertion site 24 h following film implantation (green, MHC II-GFP; red, TMR-poly(I:C); yellow, overlay; scale bar, 50 μm).

  4. Implanted films control the physical and functional persistence of pDNA and poly(I:C)
in vivo.
    Figure 4: Implanted films control the physical and functional persistence of pDNA and poly(I:C) in vivo.

    a, Representative whole-animal fluorescence images showing TMR-poly(I:C) retention at the application site and quantitative analysis of normalized total fluorescence R(t)relative to initial fluorescence Ro from groups of animals (n  =  3) over time following 15 min application of PLLA microneedles coated with (uv-PNMP)(PS/SPS) 20(PBAE/TMR-poly(I:C)) 35 multilayers containing poly-1 or poly-2 as the PBAE component. Data represent the mean±s.e.m. b, Representative whole-animal luminescent images and quantitative analysis of luminol signal from MPO-dependent oxidative burst in activated phagocytes at the treatment site over time following intradermal injection of 10 μg poly(I:C) or 15 min application of PLLA microneedles coated with (uv-PNMP)(PS/SPS) 20(poly-2/poly(I:C)) 35 multilayers. Data represent the mean±s.e.m., n  =  4. c, Representative whole-animal bioluminescence images of pLUC expression at the application site and mean bioluminescence intensity over time following 15 min application of microneedles coated with (uv-PNMP)(PS/SPS) 20(PBAE/pLUC) 35 multilayers containing poly-1 or poly-2 as the PBAE component. Data represent the mean±s.e.m., n  =  4. d, Mean bioluminescent intensity on day 2 following 15 min application of microneedles coated with (uv-PNMP)(PS/SPS) 20(Poly-1/pLUC) 35 multilayers stored dry at 25 °C for 0, 14 or 28 days. Data represent the mean±s.e.m., n  =  4.

  5. Microneedle tattooing with multilayer films carrying pDNA and poly(I:C) generates potent cellular and humoral immunity against a model HIV antigen.
    Figure 5: Microneedle tattooing with multilayer films carrying pDNA and poly(I:C) generates potent cellular and humoral immunity against a model HIV antigen.

    a, C57Bl/6 mice (n  =  4 mice per group) were immunized with 20 μg pGag and 10 μg poly(I:C) on days 0 and 28 intramuscularly (with or without electroporation (EP)) in the quadriceps, intradermally in the dorsal ear skin (with free pGag or pGag/poly-1 polyplexes, ID±Polyplex), or by 15 min application of (PNMP)(PS/SPS) 20(poly-1/poly(I:C)) 35(poly-1/pLUC) 35–coated microneedles with or without ultraviolet-priming of the PNMP release-layer (MN±UV) to the dorsal ear skin. bd, Frequency of Gag-specific CD8+ T-cells in peripheral blood assessed by flow cytometry analysis of tetramer+CD8+ T-cells. Shown are mean±s.e.m. tetramer+ values from day 14 (b), representative cytometry plots from individual mice (c), and mean±s.e.m. tetramer+ values from day 42 (d). e,f, Analysis of T-cell effector/central memory phenotypes in peripheral blood by CD44/CD62L expression of tetramer+cells from peripheral blood. Shown are representative cytometry plots from individual mice at day 49 (e) and mean±s.e.m. percentages of tetramer+CD44+CD62L+ among CD8+ T-cells at day 98 (f). g, Mice immunized with microneedles were recalled on day 105 by IM injection of 50 μg pGag, and assessed for cytokine production on ex vivo restimulation with AL11 peptide on day 112. Shown is representative flow cytometry analysis of IFN- γ/TNFα-producing CD8+ T-cells. h, Enzyme-linked immunosorbent assay analysis of total Gag-specific IgG in sera at day 42. Data represent the mean±s.e.m., **P < 0.005, analysed by two-way ANOVA.

  6. Multilayer tattooing enhances transfection in non-human primate skin.
    Figure 6: Multilayer tattooing enhances transfection in non-human primate skin.

    a, Optical micrograph of macaque quadriceps skin showing microneedle penetration pattern stained using trypan blue (scale bar, 500 μm). b, Histological section of microneedle-treated macaque skin showing epidermal disruption at microneedle insertion sites (boxed, left, scale bar, 500 μm; right, scale bar, 100 μm). c, Bioluminescence images of luciferase expression 2 days after pLUC delivery by ID injection or microneedle tattooing with (PS/SPS) 20(poly-1/pLUC) 35 films from either uv-PNMP- or non-irradiated PNMP-coated microneedles following a 15 min application. d, Quantification of total bioluminescent signal in cultured skin tissue explants 1, 2 and 3 days after treatment. Data represent the mean±s.e.m., n  =  3. ***,p < 0.0001, analysed by an unpaired t-test.

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Author information

Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA

    • Peter C. DeMuth,
    • Bonnie Huang &
    • Darrell J. Irvine
  2. Department of Chemical Engineering, MIT, Cambridge, Massachusetts 02139, USA

    • Younjin Min &
    • Paula T. Hammond
  3. New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772, USA

    • Joshua A. Kramer &
    • Andrew D. Miller
  4. Division of Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA

    • Dan H. Barouch
  5. Ragon Institute of MGH, MIT, and Harvard, Charlestown, Massachusetts 02129, USA

    • Dan H. Barouch &
    • Darrell J. Irvine
  6. Institute for Soldier Nanotechnologies, MIT, Cambridge, Massachusetts 02139, USA

    • Paula T. Hammond &
    • Darrell J. Irvine
  7. Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts 02139, USA

    • Paula T. Hammond &
    • Darrell J. Irvine
  8. Department of Materials Science and Engineering, MIT, Cambridge, Massachusetts 02139, USA

    • Darrell J. Irvine
  9. Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA

    • Darrell J. Irvine

Contributions

P.C.D., P.T.H. and D.J.I. designed the experiments. P.C.D., D.H.B. and D.J.I. designed macaque skin studies. P.C.D. carried out the experiments; Y.M. performed in vitro nucleic acid release studies. B.H. synthesized the PNMP polymers. A.D.M. and J.A.K. collected the macaque skin. P.C.D., P.T.H. and D.J.I. analysed the data and wrote the paper.

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The authors declare no competing financial interests.

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