Releasable layer-by-layer assembly of stabilized lipid nanocapsules on microneedles for enhanced transcutaneous vaccine delivery

Abstract

Here we introduce a new approach for transcutaneous drug delivery, using microneedles coated with stabilized lipid nanocapsules, for delivery of a model vaccine formulation. Poly(lactide-co-glycolide) microneedle arrays were coated with multilayer films via layer-by-layer assembly of a biodegradable cationic poly(β-amino ester) (PBAE) and negatively charged interbilayer-cross-linked multilamellar lipid vesicles (ICMVs). To test the potential of these nanocapsule-coated microneedles for vaccine delivery, we loaded ICMVs with a protein antigen and the molecular adjuvant monophosphoryl lipid A. Following application of microneedle arrays to the skin of mice for 5 min, (PBAE/ICMV) films were rapidly transferred from microneedle surfaces into the cutaneous tissue and remained in the skin following removal of the microneedle arrays. Multilayer films implanted in the skin dispersed ICMV cargos in the treated tissue over the course of 24 h in vivo, allowing for uptake of the lipid nanocapsules by antigen presenting cells in the local tissue and triggering their activation in situ. Microneedle-mediated transcutaneous vaccination with ICMV-carrying multilayers promoted robust antigen-specific humoral immune responses with a balanced generation of multiple IgG isotypes, whereas bolus delivery of soluble or vesicle-loaded antigen via intradermal injection or transcutaneous vaccination with microneedles encapsulating soluble protein elicited weak, IgG(1)-biased humoral immune responses. These results highlight the potential of lipid nanocapsules delivered by microneedles as a promising platform for noninvasive vaccine delivery applications.

Figure 1

(a) Schematic illustration of (Poly-1/ICMV) multilayers deposited onto PLGA microneedle surfaces. ICMV lipid nanocapsules are prepared with inter-bilayer covalent crosslinks between maleimide head groups (M) of adjacent phospholipid lamellae in the walls of multi-lamellar vesicles. (Poly-1/ICMV) PEMs were constructed on microneedles after (PS/SPS) base layer deposition. (b) Microneedles transfer (Poly-1/ICMV) coatings into the skin as cutaneous depots at microneedle insertion points. (c) Hydrolytic degradation of Poly-1 leads to PEM disintegration and ICMV release into the surrounding tissue. (d) ICMV delivery to skin-resident APCs provides coincident antigen exposure and immunostimulation, leading to initiation of adaptive immunity.

Figure 2

Shown are poly-1/lipid film thicknesses determined by profilometry for deposited ICMVs or MLVs varying (a) concentration and (b) deposition time (N = 12). (c) CLSM image of (PS/SPS)₂₀(Poly-1/ICMV)₂₀ or (PS/SPS)₂₀(Poly-1/MLV)₂₀ multilayers deposited on silicon (scale bar ~ 20μm). ICMVs and MLVs contained AF647-OVA (pink) and were labeled with DiI (red). (d–g) AFM imaging of a dried (Poly-1/ICMV)₅(PS/SPS)₂₀ multilayers built on silicon (scale bar 100 nm). Shown are (d) phase, (e) height, and (f) 3-D rendered AFM height micrograph data for a (Poly-1/ICMV)₅(PS/SPS)₂₀ multilayer (scale bar 100 nm). (g) Height trace data (trace shown in panel (e) for a single embedded ICMV in a (PS/SPS)₂₀(Poly-1/ICMV)₅ multilayer.

Figure 3

(a) Representative confocal images of PLGA microneedles coated with (PS/SPS)₂₀(Poly-1/ICMV)₃₅ films (left, transverse optical sections; right, lateral sections; 100 μm interval; scale bar 100 μm; Red, DiI-ICMVs; Pink, AF647-OVA). (b) Quantification of DiI-ICMV and AF647-OVA incorporation into (PS/SPS)₂₀(Poly-1/ICMV)_n films on microneedles. Analysis was performed using Image J measurement of total fluorescent signal intensity in confocal z-stacks collected along the length of microneedles, normalized to the total intensity obtained for 30 bilayer films (results shown are averaged from N = 15 individual microneedles per condition). (c) SEM micrographs of (PS/SPS)₂₀(Poly-1/ICMV)₃₅ multilayer-coated PLGA microneedles (scale bars: left 200 μm, right 50 μm).

Figure 4

(a–b) Representative confocal images of PLGA microneedles coated with (PS/SPS)₂₀(Poly-1/ICMV)₃₅ films before application (a) and after a 5 minute application to murine skin IN VIVO (b) (lateral sections, 100 μm Z-interval; scale bar 100 μm; Red, DiI-ICMVs; Pink, AF647-OVA). (c) Quantitation of confocal fluorescence intensities (N = 15) showing loss of DiI-ICMV and AF647-OVA films from coated microneedles upon application to skin. (d–g) Representative confocal images of mouse skin treated for 5 minutes with (PS/SPS)₂₀(Poly-1/ICMV)₃₅ multilayer-coated PLGA microneedles after (d) 6 hr or (e) 24 hr showing ICMV delivery at microneedle insertion sites (outlined). Shown is fluorescent signal from (top to bottom): MHC II-GFP (green), DiI-ICMVs (red), AF647-OVA (pink), and overlay (yellow) at low (left) and high (right) magnification (scale bars 100 μm). (f) High magnification CLSM image (field location highlighted by box in panel (e)) showing colocalization of ICMVs and OVA with APCs in the skin (scale bar - 20μm). (g) High magnification CLSM image showing APC phagocytosis of ICMVs with OVA after 24 hr (scale bar 20 μm).

Figure 5

Representative CLSM images of MHC II-GFP⁺ cells in skin dissected (a) 6 or (b) 24 hours after (PS/SPS)₂₀(Poly-1/ICMV)₃₅–coated microneedle treatment for 5 minutes (insertion points outlined); ICMVs were loaded with MPLA and polyI:C was added to the skin directly before treatment (scale bar 200 μm). (c) Results of quantitative CLSM image analysis to determine total cell number per field, individual cell area and perimeter, and MHC II-GFP MFI, expressed as fold change relative to uncoated microneedle treated mice. Mice were either treated with uncoated microneedles (MN), or microneedles coated with (PS/SPS)₂₀(Poly-1/ICMV)₃₅ multilayers with or without added MPLA and polyI:C (MN+ICMV and MN+ICMV+TLRa, respectively). Data was analyzed for significance using two-way ANOVA (* - p<0.05, ** - p<0.01, *** - p<0.001).

Figure 6

(a) Schematic representation of vaccine treatments tested. (b) Anti-OVA serum IgG titers were measured over time with immunizations on days 0, 28, and 56 with OVA-ICMVs or soluble antigen administered VIA either microneedle-based multilayers or intradermal bolus injection at dorsal auricular skin. (c, d) Quantification of anti-OVA IgG₁ (b) and IgG_2c (c) subtypes in sera at day 107. ^#, P < 0.05 and ^##, P < 0.01, compared to OVA-ICMV ID, and ***, P < 0.001, compared to OVA-MN or OVA ID, as analyzed by two-way ANOVA, followed by Tukey’s HSD. *, P < 0.05, as analyzed by one-way ANOVA, followed by Tukey’s HSD.