PLGA Microparticle Synthesis: Polyvinyl alcohol (PVA)-stabilized PLGA microparticles were prepared by a single/double emulsion/solvent evaporation approach. Briefly, 80 mg PLGA (50:50 lactide:glycolide ratio, IV 0.35 dL/g, Lakeshore Biomaterials or Resomer RG 755 S, IV 0.70 dL/g) was dissolved in 5 mL dichloromethane. The PLGA solution was then added to 40 ml 0.5% PVA (MW 150,000 Da, MP Biomedicals) while homogenizing at 12,000rpm using a T-25 Digital Homogenizer (IKA). Homogenization was performed for 3 min following PLGA addition, and solvent was subsequently removed by stirring overnight. For fluorescently labeled particles 160 ug 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) or 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD, Invitrogen) were codissolved with PLGA. For poly(I:C) loaded particles, 3mg Cy3-labeled poly(I:C) (Invivogen, labeled using Mirus LabelIT, according to the manufacturer’s instructions) was dissolved in distilled water and added to the polymer-containing organic phase while sonicating at 12W with a Microson XL probe tip (Microson). After sonication for 30 s the resulting emulsion was added to the PVA solution and homogenized as before. The resulting particles were collected by centrifugation, washed, and resuspended in distilled water before lyophilization and storage under dessication at 4 °C until use. Particle size and zeta potential was determined using a BIC 90+ light scattering instrument (Brookhaven Instruments Corp).
Microneedle Fabrication: Microneedles encapsulating free PLGA microparticles or bulk PLGA implants were fabricated from PDMS molds (Sylgard 184, Dow Corning) machined using laser ablation (Clark-MXR, CPA-2010 micromachining system) to create patterns of micron-scale surface-cavities. First, PLGA microparticles were deposited into PDMS molds through addition of aqueous particle suspensions to the mold surface. Following PLGA microparticle addition (0.2–3.0 mg/array), molds were centrifuged for 20 min at rcf ≈ 2 to compact particles into mold cavities. Following removal of residual material from the mold surface, molds were dried at 25 °C. For microneedles encapsulating free microparticles, addition of 35% poly(acrylic acid) (PAA, 250kDa) to the mold surface was followed by centrifugation (20 min, rcf ≈ 2) and drying at 25 °C (48 h on the benchtop, followed by 2–14 days under dessication), before removal. Microneedles encapsulating multiple distinct particle populations were fabricated similarly through addition of mixed particle suspensions. For microneedles encapsulating bulk PLGA implants, PDMS molds encapsulating free microparticles were heated under vacuum (–25 in. Hg) at 145 °C for 40 min, and then cooled at –20 °C before addition of 35% PAA, centrifugation, and drying as previously described. For microneedles encapsulating layered PLGA implants, sequential microparticle deposition, drying, and melting was performed as previously described. All microneedles were stored under dessication at 25 °C until use. Microneedle arrays were characterized by scanning electron microscopy (SEM) using a JEOL 6700F FEG-SEM and confocal microscopy using a Zeiss LSM 510.
Characterization of Microparticle and Implant Delivery: Microparticle and bulk implant release was characterized in vitro through brief (<30 s) exposure of fabricated arrays to PBS. Microparticles and bulk implants were then collected through centrifugation and washed in PBS before application of aqueous suspensions to glass coverslips. After drying, microparticles and implants were imaged by confocal microscopy. Similar delivery was measured in vivo following array application to the skin of mice. Animals were cared for in the USDA-inspected MIT Animal Facility under federal, state, local, and NIH guidelines for animal care. Microneedle application experiments were performed on anesthetized C57BL/6 mice (Jackson Laboratories) at the flank or dorsal ear skin. Skin was rinsed briefly with PBS and dried before application of microneedle arrays by gentle pressure. Following application mice were euthanized at subsequent time points and the application site and draining lymph nodes were dissected. Excised skin was stained with trypan blue before imaging for needle penetration. In separate experiments treated skin and applied microneedle arrays were imaged by confocal microscopy to assess transcutaneous delivery of encapsulated microparticles and bulk implants. In some cases, treated skin was excised and fixed in 3.7% formaldehyde for 18 h, then incubated in 30% sucrose/PBS for 2 h before embedding in optimal cutting temperature (OCT) medium (Tissue-Tek) for histological sectioning on a cryotome. Lymph nodes were similarly embedded in OCT and sectioned. Histological sections were then imaged using confocal microscopy.
In Vivo Imaging: Live whole animal imaging was performed using a Xenogen IVIS Spectrum (Caliper Life Sciences) on anesthetized mice. Fluorescence data was processed using region of interest (ROI) analysis with background subtraction and internal control ROI comparison to untreated skin using the Living Image 4.0 software package (Caliper).
Immunizations: All animal studies were approved by the MIT IUCAC and animals were cared for in the USDA-inspected MIT Animal Facility under federal, state, local, and NIH guidelines for animal care. Groups of 4 C57Bl/6 mice were immunized on days 0 and 35 with 15 μg ovalbumin and 50 ng poly(I:C) by intramuscular injection (15 μL in the quadriceps) intradermal injection (15 μL in the dorsal caudal ear skin) or by microneedle array (5 min application). Frequencies of OVA-specific CD8+ T-cells and their phenotypes elicited by immunization were determined by flow cytometry analysis of peripheral blood mononuclear cells at selected time points following staining with DAPI (to discriminate live/dead cells), anti-CD8α, anti-CD44, anti-CD62L (BD Biosciences), and phycoerythrin-conjugated SIINFEKL/H-2Kb peptide-MHC tetramers (Beckman Coulter). To assess the functionality of primed CD8+ T-cells peripheral blood mononuclear cells were stimulated ex vivo with 10 ug/mL OVA-peptide SIINFEKL for 6 h with Brefeldin-A (Invitrogen), fixed, permeabilized, stained with anti-IFNγ, anti-TNFα, and anti-CD8α (BD Biosciences), and analyzed by flow cytometry. Anti-Ovalbumin IgG titers, defined as the dilution of sera at which 450 nm OD reading was 0.25, were determined by ELISA analysis of sera from immunized mice. Animals were cared for following NIH, state, and local guidelines.