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Department of Dermatology, Venerology and Allergology, Charité–Universitätsmedizin Berlin, Humboldt-Universität zu Berlin and Freie Universität Berlin, Berlin, GermanyDepartment of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
Department of Dermatology, Venerology and Allergology, Charité–Universitätsmedizin Berlin, Humboldt-Universität zu Berlin and Freie Universität Berlin, Berlin, Germany
Department of Dermatology, Venerology and Allergology, Charité–Universitätsmedizin Berlin, Humboldt-Universität zu Berlin and Freie Universität Berlin, Berlin, Germany
Department of Dermatology, Venerology and Allergology, Charité–Universitätsmedizin Berlin, Humboldt-Universität zu Berlin and Freie Universität Berlin, Berlin, Germany
Needle-free vaccination, for reasons of safety, economy, and convenience, is a central goal in vaccine development, but it also needs to meet the immunological requirements for efficient induction of prophylactic and therapeutic immune responses. Combining the principles of noninvasive delivery to dendritic cells (DCs) through skin and the immunological principles of cell-mediated immunity, we developed microparticle-based topical vaccines. We show here that the microparticles are efficient carriers for coordinated delivery of the essential vaccine constituents to DCs for cross-presentation of the antigens and stimulation of T-cell responses. When applied to the skin, the microparticles penetrate into hair follicles and target the resident DCs, the immunologically most potent cells and site for induction of efficient immune responses. The microparticle vaccine principle can be applied to different antigen formats such as peptides and proteins, or nucleic acids coding for the antigens.
Abbreviations
BFA
brefeldin A
CMI
cell-mediated immunity
DC
dendritic cell
MHC
major histocompatibility complex
SiO2
silicon dioxide
TLR
Toll-like receptor
INTRODUCTION
Nearly all vaccines to date are administered by injection, a painful procedure, especially for children, which bears the risk of propagating infections when needles are re-used. Moreover, injection often delivers the vaccine into sites that are immunologically not the most active, such as muscles or subcutaneous tissue, and thus necessitates high excess of the vaccine components that add to the risks of adverse effects (
Low-dose intradermal versus intramuscular administration of recombinant hepatitis B vaccine: a comparison of immunogenicity in infants and preschool children.
). The primary target cells for induction of immune responses are dendritic cells (DCs), which are unique antigen processing and presenting cells that can efficiently initiate immune responses and induce immunological memory (
). DCs reside as sentinels of the immune system in all tissue where they sample and process antigens and where they sense dangerous situation via innate immune receptors, which triggers maturation and activation of the cells, migration to lymph nodes, and expression of costimulatory molecules for induction of T cells (
). These T cells are CD4-expressing helper cells required for both antibody and cell-mediated immunity (CMI), and CD8-expressing effector T cells for CMI. These cellular requirements define the essential constituents of vaccines for induction of CMI, which are antigens/epitopes for helper and effector T cells, and agonists for innate pattern recognition receptors for maturation of DCs. Only mature DCs are capable of inducing strong cytotoxic T-cell responses and proinflammatory cytokines, and are resistant to immunosuppressive factors as, e.g., those produced in tumor microenvironments (
). It has an unbroken network of DCs, Langerhans, and dermal DCs that can both sense danger and induce immune responses or control and silence immune responses to avoid chronic inflammation and immune pathologies (
). Ideal vaccine would be designed to target the antigens directly to these cells in the skin, best by noninvasive application. Such topical vaccination requires penetration of the physical barrier of the skin and the transfer of the vaccine components to the target DCs. We had established that particles penetrate into skin far more efficiently than soluble compounds (
Hair follicles--an efficient storage and penetration pathway for topically applied substances. Summary of recent results obtained at the Center of Experimental and Applied Cutaneous Physiology, Charité -Universitätsmedizin Berlin, Germany.
). The ports of entry are the hair follicles, and particles of different sizes penetrate to different depths in the follicle. We had tested particles with sizes ranging from 122nm to 1μm in diameter (
). With a diameter of ∼600nm, they reach deepest to ∼1,400μm into the hair root sheath area, whereas particles of ∼1,000nm diameter stay above the root sheath. To avoid disturbance of the sheath, and because the region above the sheath, the infundibulum, is lined with a particular dense network of DCs (
) and thus an immunologically most active region, we explored particle carriers of 1μm diameter for delivery of antigens to DCs cells both in vitro and in situ through human skin.
RESULTS AND DISCUSSION
Delivery system design, conjugation of the peptides/lipopeptide to silicon dioxide (SiO2) particles, and effect on cell viability
These principles of noninvasive delivery to DCs through skin were combined with the immunological principles of CMI induction to develop a topical vaccine. The particles were prepared by a layer-by-layer technology (
) and functionalized with antigenic peptides as model vaccine antigens, and lipopeptide ligands for innate immune receptors (Figure 1). As antigens, we used influenza matrix protein M158-66 GILGFVFTL (GILG) and CMV pp65595-603 NLVPMVATV (NLVP). The activator for DCs via innate immune receptors was the diacyl-lipopeptide Pam2Cys-GDPKHPKSF (P2C). This synthetic lipopeptide represents the N-terminal sequence of the 44-kDa lipoprotein LP44 of Mycoplasma salivarium. It carries two ester-bound fatty acids and a free amino terminus. The synthetic lipopeptide FSL-1 elicits cellular responses through Toll-like receptor 2 (TLR2)/TLR6 heterodimers that involve downstream NF-κB activation and cytokine release (
). The peptides and lipopeptide were C-terminally extended by four lysine residues and immobilized on the particles via electrostatic interactions with the outermost polyanion layer of the particles, which is negatively charged at pH 8. Polymethacrylate was used as polyanion. It has a pK value of 6.2. At pH 5, it is almost completely protonated with a charge of near zero. At this pH, the polycation layer underneath the polyanion layer carries sufficient positive charges to reverse the particle surface charge (ζ-potential) to positive as validated by electrophoretic measurements (Malvern Zetasizer, Malvern Instruments, Herrenberg, Germany). As consequence, the attractive electrostatic forces at pH 8 are converted to repulsive forces and the tagged peptides were efficiently released (Figure 1). The particles were nontoxic as, among others, shown for immature DCs generated from human peripheral blood monocytes by cultivation for 5 days in medium containing IL-4 and GM-CSF and incubated with particles coated with the different components for 2 days (Supplementary Figure S1 online).
Figure 1Principle of conjugation of the peptides/lipopeptides to silicon dioxide (SiO2) particles. (a) The particles are coated with a layer-by-layer (LBL) film with a negative surface charge at pH 7.5 that binds the peptides and lipopeptides with positively charged tetralysine tags. At pH 5.5, the surface charge changes to positive, leading to an almost complete release of the peptides from the particle surface. (b) Uptake at pH 8 and release at pH 5 of GILG and Pam2Cys (P2C) as single component peptides (red, green) or in peptide mixture (blue, light blue). (c) Confocal laser scanning image of the GILG-Rho adsorbed to the 1μm LBL particles at pH 8 (left fluorescence image, right transmission image). (d) Release of the adsorbed peptides from the LBL surface by pH shift from 8 to 5. (e) Desorption of the peptide and lipopeptide from the particle surface in dependence on the pH value.
Particles are efficiently taken up and the antigen is displayed at the DC surface
The particles were efficiently taken up by human DCs in vitro regardless of whether or not peptide or lipopeptide were attached as shown by confocal fluorescence microscopy. They were found in vesicular structures where the peptides detached from the particles (Figure 2a and Supplementary Figure S2 on line). To directly demonstrate cross-presentation of the antigenic peptide by loaded DCs, we made use of recombinant TCR-like antibodies that specifically bind the major histocompatibility complex (MHC)/peptide complex HLA-A*0201/GILG (Supplementary Figure S3 online). The antibodies are designed to recognize specific human MHC-I/peptide complexes with high affinity and, thus, can be used to detect and quantify processed antigen at surfaces of the antigen-presenting cells independent from T cells (
). They do not bind the free peptides or peptides associated with other compounds but MHC-I of the correct type. Bioassays that are mostly used to monitor processed antigens cannot differentiate antigen processing and costimulatory effects on T cells and do not allow quantifying the amount of processed antigen bound to MHC molecules (
Exogenous peptides presented by transporter associated with antigen processing (TAP)-deficient and TAP-competent cells: intracellular loading and kinetics of presentation.
). Applying the antibodies to particle-loaded DCs, specific peptides carried by the particles were detected at the cell surface associated with the MHC-I. The amounts of cross-presented peptide on the DCs were about the same for particles loaded with peptides alone and those with peptides plus P2C (Figure 2b and c). The presentation of antigenic peptides by MHC-I molecules is thus not dependent on the TLR ligand on the cargo as it had been suggested to be the case for MHC class II presentation (
). As the peptides were adsorbed to the surface of the particles, it could be argued that they might be released from the particles into the medium and in this free soluble form would bind to MHC-I molecules without intracellular processing. To address this issue, we inhibited the phagocytosis by treating the DCs with cytochalasin D before adding the particles to the cell cultures. Inhibition of phagocytosis completely abrogated the presentation of peptides bound to particles (Figure 2d) but had no effect on the presentation of soluble peptides. Particle-bound peptides, thus, follow the intracellular route of antigen cross-processing to get to the cell surface.
Figure 2Conjugation of peptide GILG to silicon dioxide (SiO2) particles resulted in efficient cross-presentation of the GILG antigen. (a) Confocal images of dendritic cells (DCs) loaded with particle carrying GILG (green particles and red peptide) and Pam2Cys (P2C; blue particles). The cell surface was stained with FITC-labeled anti-MHC-I antibodies. The confocal analysis shows that internalized GILG was inside the cells in both particle-bound form and free form in intracellular compartments of the DCs. Images were originally obtained at × 1,000 total magnification and electronically zoomed. Flow cytometric analysis showing cross-presentation of particle-bound GILG by DCs (b, c). The cross-presentation of the particle-bound GILG was blocked by treatment with (d) cytochalasin D and (f) brefeldin A, and slightly increased by (e) chloroquine treatment. MFI, mean fluorescence intensity; MHC-I, major histocompatibility complex I; w/o, without. *P<0.05; **P<0.01; ***P<0.001.
Cross-presentation of the antigen delivered by SiO2 particles does not rely on endosomal acidification and involves newly synthesized MHC-I molecules from the endoplasmic reticulum
Upon uptake, antigens enter an endocytic pathway and progressively pass through increasingly acidic compartments. Raising the endosomal pH reduces the proteolytic activity of most endosomal proteases and interferes with fusion of vesicles (
Mechanisms of exogenous antigen presentation by MHC class I molecules in vitro and in vivo: implications for generating CD8+ T cell responses to infectious agents, tumors, transplants, and vaccines.
). We examined whether acidification of the endosomal compartment of human DCs was required for cross-presentation of antigenic peptides when delivered on particles. For that, DCs were preincubated with chloroquine (Figure 2e) or ammonium chloride (Supplementary Figure S4 online), and then incubated with peptide-carrying particle with or without P2C. Surprisingly, the neutralization of endosomal pH with chloroquine did not decrease the cross-presentation, but slightly increased it (Figure 2e). Cross-presentation of the particle-bound peptides apparently does not require endosomal functions dependent on acidification such as proteolysis by proteases or endosomal fusion for maximum efficiency. Similar results were obtained when endosomal acidification was inhibited with ammonium chloride (Supplementary Figure S4 online).
According to the canonical model for cross-presentation, endocytosed or phagocytosed antigens are translocated into the cytosol where they are degraded into antigenic peptides by proteasomes. Epitopes generated in this process are transported into the lumen of the endoplasmic reticulum by the transporter associated with antigen processing loaded onto nascent MHC-I molecules for presentation at the cell surface (
). To examine whether the cross-presentation of the particle-bound peptide involved MHC-I molecules recycled from the cell surface or requires access to nascent MHC-I in the endoplasmic reticulum, we treated DCs with brefeldin A (BFA) before adding particle-bound peptides. BFA is a fungal metabolite that disassembles the Golgi apparatus and thereby blocks the transport of newly synthesized MHC-I molecules from the endoplasmic reticulum to the cell surface (
). Treatment of DCs with BFA nearly abolished the presentation of particle-bound peptide but not of soluble peptides (Figure 2f). Vesicle transport via the Golgi apparatus is, thus, required for cross-presentation of particle-bound peptide. This suggests that binding of the epitope to MHC-I molecules occurs in intracellular compartments involved in export pathways that contain newly synthesized MHC molecules, and not in recycling vesicles. Interestingly, BFA prevented the cross-presentation irrespective of P2C on the particles. The preference for this canonical route of cross-presentation even when the antigen was administered together with a TLR ligand was surprising, as a recent study by
demonstrated that in the presence of TLR ligand the endosomic route was favored. The authors had observed that, following signaling via TLR and MyD88, transporter associated with antigen processing was selectively recruited to endocytic organelles and antigen processing for cross-presentation directed to endosomal compartments (
), we examined whether the particles could induce DC maturation. Particles carrying only peptides were not able to (Figure 3a–d). However, as expected because the DCs had been shown to express TLR2 (Supplementary Figure S5 online), when the P2C was attached to the particles, maturation of the cells was triggered and detectable by the upregulation of the costimulatory molecules CD80 and CD86, the expression of the activation marker CD83, and production of IL-12 (Figure 3a–d).
Figure 3Particle-bound Pam2Cys (P2C) induced maturation of dendritic cells (DCs) and increased the efficiency of T-cell stimulation. Particles carrying P2C induced upregulation of (a) CD80, (b) CD83, (c) CD86 expression, and (d) higher levels of IL-12 secretion by DCs compared with DCs incubated with particles without P2C. Flow cytometry profiles showing that DCs exposed to particle-bound P2C were more efficient in inducing (e) GILG- and (f) NLVP-specific CD8+ T cells than DCs exposed to particles without P2C. The analyses shown in e and f were performed on cells from different donors. (g) The activated T cells also produce high amounts of the immune-regulating and effector cytokine IFN-γ. MFI, mean fluorescence intensity; w/o, without.
To test the immune-stimulatory capacity of DCs charged with particle-bound peptides with or without P2C, the cells were cultured with T cells from the same donor for 7 days. Proliferation of peptide-specific CD8+ T cells was then quantified by flow cytometry using peptide/HLA-A*0201 tetramers. Only fully matured DCs were able to stimulate proliferation of the peptide-specific CD8+ T cells (Figure 3e and f). DCs that had not been exposed to P2C and therefore remained immature were not capable of inducing T-cell proliferation despite carrying the same levels of MHC-I/peptide complexes as the mature DCs. Matured DCs also induced strong IFN-γ secretion by the T cells (Figure 3g). Interestingly, for some donors better expansion of peptide-specific CD8+ T cells was observed when the cognate peptide was coupled with TLR ligand to the same particles, whereas for other donors better stimulation was achieved when peptide and P2C were on separate particles.
SiO2 particles reach the hair follicles and deliver the antigen to the resident DCs
To test skin penetration of the particle vaccines, we labeled the particles with Cy5 and loaded them with Rhodamine-labeled peptides. The particles were massaged onto porcine ears by standardized conditions and left for 1 and 24hours, respectively. After this time, punch biopsies were taken and cryofixed. Sections of 10μm were prepared and then analyzed by laser scan fluorescence microscopy for the penetration depths of the blue fluorescence (Cy5) of the particles and red fluorescence (Rhodamine) associated with the peptides. After 1hour, the particles were detected in the hair follicles up to a depth of 151.1±47.1μm, the peptide penetrated further to 509.1±144.2μm. After 24hours, an average penetration depth of 259.1±92.7μm was found for the particles and 631.2±182.4μm for the peptide (Figure 4a). The results show that the peptides reach the target region at the infundibulum between 400 and 600μm (
) already after 1hour. Detection of peptides and particles at different depths in the hair follicles indicated release of the peptide in the follicle presumably triggered by a lower pH compared with the carrier gel into which the particles had been formulated.
Figure 4Penetration of the particles into hair follicle. (a) After application of fluorochrome Cy5-labeled particles coated with Rhodamine-labeled peptides to porcine skin, the fluorescence associated with the particles (left panel) and peptides (right panel) was detected by confocal laser scanning microscopy. The penetration depths are indicated with arrows. (b) Fluorochrome-labeled particles were applied to human calf skin and the fluorescence measured at the indicated time points. The values were normalized to the fluorescence determined after 30minutes as starting value. (c) Illustration of the distribution of dendritic cells in the skin and hair follicles of human skin and of the experiments to determine delivery to cutaneous dendritic cells of antigenic peptides administered on particles. (d) After applying particles carrying Rhodamine-labeled peptides to excised human skin, the skin was dissociated and the dendritic cells isolated by magnetic bead separation. The isolated cells were then analyzed for the fluorescence associated with the peptides by confocal laser scanning microscopy. The left panel shows bright field image of isolated cells and the right panel shows fluorescence detection on the cells.
The follicles serve as depots for the particle vaccines. Using human calf skin in situ, fluorescein-labeled particles were applied and the fluorescence monitored over 10 days in the follicles and in stratum corneum. Already after 1 day, the particles at the stratum corneum were nearly gone, likely shed off through contact with the fabric of the cloths. In contrast, in the hair follicles a considerable fraction of the particles still prevailed after 10 days (Figure 4b). To finally test the delivery of particle-bound antigenic peptides to DCs of the skin, we prepared particles with Rhodamine-labeled GILG peptide and applied them to excised human skin of a HLA-A*0201-positive donor. HLA-A*0201 is the specific antigen-presenting MHC-I molecule for GILG. After thoroughly cleansing the skin to remove surface-associated particles and peptide, the skin was minced and dissociated by trypsin digestion. The resulting suspension was filtered and DCs isolated with magnetic beads with anti-CD1c antibodies (Figure 4c). The isolated DCs were analyzed by confocal laser scanning microscopy for the fluorescence associated with the vaccine peptide. A high proportion of the DCs (77.7%) was found to be positive for the peptide (Figure 4d), demonstrating an efficient delivery of the peptide to the DCs of the skin. The calf region used in this study was chosen because of its large hair follicles, which favors the application of the vaccine formulation onto this area. However, the scalp could also be considered for that, once it displays a high follicular density. The validation of the application area is an important issue that will be addressed in further studies.
Taken together, the experiments reported here demonstrate that layer-by-layer–coated SiO2 particles are efficient carriers for the delivery of vaccine antigens into the cross-presentation pathway in human DCs. When the antigen is administered together with TLR agonists as adjuvant, the DCs undergo full maturation and efficiently trigger specific CD8+ T-cell activation. Application of peptide carrying particles to the skin results in their penetration into hair follicles to a depth controlled by the size of the particles. The particles are retained in the follicle as a depot for several days. Antigenic peptides coupled to the particles are efficiently delivered to DCs of the skin. Microparticles appear to be suitable carriers for topical vaccines for induction of CMI.
MATERIALS AND METHODS
Peptides, lipopeptides, and peptide-loaded particles
The peptides influenza matrix protein M158–66 GILGFVFTL-KKKK (GILG) and CMV pp65595-603 NLVPMVATV-KKKK (NLVP) as well as diacyl-lipopeptide Pam2Cys-GDPKHPKSF-KKKK (P2C) were synthesized with C-terminal tetralysine tags. Fully automated solid-phase peptide synthesis and Fmoc/tBu chemistry on TCP resin were applied. For coupling of amino acids, a 7-fold molar excess of single Fmoc-L-amino acids was used. The peptide resin carrying the sequence GDPKHPKSFKKKK was elongated with the unusual amino acid Fmoc-Dhc (Dhc: S-2,3-dihydroxy-2-(R,S)-propyl-(R)-cysteine) followed by O-palmitoylation and Fmoc deprotection (
). The coupling was carried out in DMF/CH2Cl2 (1:1) with DIC/HOBt in 3-fold excess within 3hours and was monitored by Kaiser assay. The peptides and the lipopeptide were cleaved off the resin by treatment of the resin with trifluoroacetic acid/phenol/ethanedithiol/thioanisole (96:2:1:1) for 3hours. Trifluoroacetic acid was removed in vacuum and the products were precipitated from cold diethylether. The precipitate was washed three times with cold diethylether and the pellets were dissolved in tert-butyl alcohol/water 4:1, lyophilized, and purified by preparative HPLC. The structural characterization of the products was carried out by HPLC/electrospray ionization mass spectrometry. Peptides and lipopeptide were attached to layer-by-layer-coated silica beads of 1μm diameter via negative charges on the outermost polymer layer, as previously described (
with minor modifications. Briefly, peripheral blood mononuclear cells from healthy donors were isolated from buffy coats by Ficoll gradient centrifugation. Cells of the interface were collected and plated in six-well plates. Monocytes were allowed to adhere. After 2hours, non-adherent cells were washed off and the adherent cells cultured for 7 days in RPMI-1640 culture medium (Invitrogen, Carlsbad, CA) with 10% heat-inactivated fetal calf serum (Biochrom, Berlin, Germany). The cells were supplied with the cytokines GM-CSF (50ngml−1; R&D Systems, Minneapolis, MN) and IL-4 (50ngml−1; R&D Systems) on days 0 and 4 of the culture. The cultures were kept in a CO2 incubator in a humidified atmosphere with 8% CO2 at 37°C. On day 5, particle-bound peptides as indicated or an inflammatory cytokine cocktail of IL-1β (10ngml−1), IL-6 (25ngml−1), tumor necrosis factor-α (10ngml−1; all from Strathmann Biotech, Hamburg, Germany) were added and the culture continued (Supplementary Table S1 online). In some experiments, soluble peptides were used as indicated in the respective figures. Where inhibitors were used, cells were incubated for 2hours at 37°C with the respective inhibitor before addition of particle-bound or soluble peptides. The specific inhibitors used were cytochalasin D at 5μM, chloroquine at 50μM (both from Sigma-Aldrich, Steinheim, Germany), ammonium choride at 50nM (Merck, Darmstadt, Germany), BFA at 10μM (Sigma-Aldrich). Cells were harvested on day 7 and analyzed by flow cytometry or used for co-cultures with T cells.
The use of human cells for the reported study was reviewed and approved by the institutional ethics committee of the Charité–Universitätsmedizin Berlin (EA1/148/08).
Confocal microscopy for the uptake of particles by DCs
To examine the uptake of the particles by DCs and the intracellular localization of the peptides, cells were transferred to 5ml plastic tubes and fixed in 1% paraformaldehyde solution for 20minutes. The cell membrane was stained with FITC-labeled anti-β-microglobulin (BD Pharmingen, Heidelberg, Germany) for 25minutes at 4°C. The intracellular localization of the particles or peptides carried by the particles was analyzed by scanning confocal microscopy (Leica TCS SP2, Leica, Wetzlar, Germany). All images were analyzed and processed using the Leica Confocal Software Version 2.5 Build 1227.
Flow cytometry
To determine DC states and activation, the cell suspensions were labeled with fluorescent monoclonal antibodies against Lin (cocktail of FITC-conjugated antibodies against the molecules CD3, CD14, CD19, and CD56) for exclusion of non-DCs, CD11c, CD80, CD83, CD86, HLA-DR (BD Bioscience, Chicago, IL), and TLR2 (BioLegend, San Diego, CA). For the detection and quantification of the complexes HLA-A*0201/GILG at the DC surface, the cells were labeled with the purified Fab against such a complex (clone M1D12) (
) followed by staining with FITC-labeled secondary polyclonal antibody against human F(ab)2 (AbCam, Cambridge, UK). T lymphocytes were labeled with antibody for CD8 and HLA-A*0201/GILG or HLA-A*0201/NLVP tetramers (both from Beckman Coulter, Fullerton, CA). Proliferation of the specific CD8+ T cells was determined by flow cytometry after incubation of these cells with the differently treated DCs. All stained cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). The data were processed and displayed using the CellQuest software (Becton Dickinson) or WinMDi (Purdue University, West Lafayette, IN; www.purdue.edu).
ELISA for IL-12p70 and IFN-γ
The supernatants of the DC cultures were collected after 48hours of incubation with the indicated stimuli and stored at -20°C. The supernatants of the cocultures of DCs and CD8+ T cells were collected after 7 days of culture. IL-12p70 levels from the DC cultures and IFN-γ from the cocultures were measured by ELISA Max Standard kit (BioLegend) in 96-well microtiter plates according to the manufacturer’s instructions. Results are expressed as pgml−1.
Cell viability assay
Cell viability was assessed by staining the cells with a commercial kit for discrimination of live and dead cells (Invitrogen) by flow cytometry. The staining procedures were carried out following the manufacturer’s instruction. Briefly, the cell suspension containing DCs was labeled with calcein-AM and ethidium homodimer-1 and the cells were then analyzed by flow cytometry. Cells positive for calcein were considered live and those positive for ethidium were considered dead.
CD8+ T lymphocyte proliferation assay
Total CD8+ T lymphocytes were isolated from peripheral blood by magneto sorting. First, CD8+ T cells were isolated from the peripheral blood mononuclear cells by positive selection using a commercial kit (Invitrogen) following the manufacturer’s instruction. Then, the cells were washed twice in phosphate-buffered saline and cultured together with the autologous DCs in 96-well plates of 1 × 104 DCs with 2 × 105 T lymphocytes for 7 days, and then harvested and analyzed by flow cytometry.
Porcine skin samples, particle application, and penetration measurement
The follicular penetration depth of the particles was assessed in porcine ear skin. The porcine ears used in this study were obtained directly from the butcher from freshly slaughtered pigs. Before the experiments were carried out, the porcine ears were rinsed with cold water, dried with paper towels, and then the bristles were carefully shortened. The experiments with the animal samples were performed with approval by the Governmental Office of Veterinary Medicine in Berlin-Treptow, Germany.
The application of the particles onto the porcine skin and the penetration measurement were carried out as described previously (
). Briefly, 2mgcm−2 of the preparation containing the Cy5-labeled particles carrying the Rhodamine-labeled peptide (GILGFVFTL-KKKK) was applied homogeneously onto a defined areas of the porcine ear skin using a massage appliance for 3minutes (Massage Gerät PC60, Petra electric, Burgau, Germany). At least five porcine ears were used. Following a penetration time of 1 or 24hours, 3mm punch biopsies were taken. The biopsies were shock-frozen in liquid nitrogen and 10μm thick cryosections were prepared. The sections were analyzed by laser scanning confocal fluorescence microscopy for the penetration depths of the blue fluorescence (Cy5) associated with the particles and red fluorescence (rhodamine) associated with the peptides. A laser scanning microscope LSM 2000 (Carl Zeiss, Jena, Germany) was used for the analyses. By overlapping the images, the penetration depths of the fluorescent particles and peptides were determined. For statistically sound data, at least 25 hair follicles were investigated.
Investigation of follicular penetration of the particles in vivo in the human skin by “differential stripping”
The follicular penetration of fluorescein-labeled particles was investigated at different time points. Skin samples were taken at 30minutes, and 1, 4, 8, and 10 days after topical application of the particles. Five adjacent skin areas of 4 × 4 cm (A–E) of the calf region of volunteers were marked with a permanent marker. Volunteers had signed an informed consent form approved by the Institutional Ethics Committee of the Medical Faculty of the Charité–Universitätsmedizin Berlin (Germany) and in accordance with the ethical rules stated in the Declaration of Helsinki Principles. The hair were removed with scissors and the skin was rinsed with water and carefully dried with paper towels. Subsequently, 2mgcm−2 of an aqueous solution containing 1% (wt/v) fluorescein-labeled particles was applied onto the marked skin area. Skin sampling was performed from skin area A at 30minutes after application and from skin areas B, C, D, and E at 1, 4, 8, and 10 days, respectively, after application by differential stripping as previously described (
). Differential stripping combines the tape stripping method with the cyanoacrylate skin surface biopsy and affords selective determination of intercellular and follicular penetration in vivo. Tape stripping removes the part of the stratum corneum that contained the topically applied dye. First, the stratum corneum was removed layer by layer by tape stripping with an adhesive film (teas No. 5529, Beiersdorf, Hamburg, Germany). Each tape strip was pressed onto the skin with a roller to minimize the influence of skin furrows and wrinkles. The procedure was repeated until no more fluorescein was detectable in the stratum corneum as controlled by laser scanning microscope (LSM 2000, Carl Zeiss). Subsequent to the removal of the necessary tape strips, a drop of cyanoacrylate superglue (UHU, Brühl, Germany) was placed on the stripped skin areas. The glue was covered with a glass slide under slight pressure. After 5minutes, the polymerized cyanoacrylate was removed together with the glass slide with one quick movement. These cyanoacrylate skin biopsies contained the content of the hair follicles.
For quantification, the tape strips and the cyanoacrylate skin surface biopsies were dissolved separately in ethanol (Uvasol, Merck, Darmstadt, Germany) in an ultrasonic bath (Sonorex Super RK 102H, Bandelin Electronic, Berlin, Germany). The concentration of fluorescein was determined by fluorescence spectrometry (Luminescent LS 50B, PerkinElmer, Überlingen, Germany). Excitation was at 450nm and the fluorescence signal measured with a spectral filter covering the wavelength between 480 and 600nm. The maximum fluorescence signal was detected at 510nm. The intensity of the fluorescence signal was used as a measure of the concentration of the fluorescent dye. The intensity is given in relative units normalized to the values for the first time point at 30minutes.
Human skin samples and topical particle application
Human skin (retroauricular region, breast, and abdomen) was obtained from healthy volunteers undergoing plastic surgery within 4–24hours after surgical excision. Volunteers had signed an informed consent form approved by the Institutional Ethics Committee of the Medical Faculty of the Charité–Universitätsmedizin Berlin (Germany) and in accordance with the ethical rules stated in the Declaration of Helsinki Principles. Skin samples were examined macroscopically and microscopically for tissue damage. Only intact skin explants were used for transcutaneous particle penetration experiments. Skin samples were stretched on expanded polystyrene sheets, previously covered with aluminum foil and laboratory packaging film, and placed in a humidified chamber that consisted of a box provided with wet napkin paper and a cover in order to prevent skin dehydration. A volume of 20μlcm2 containing particles coated with Rhodamine-labeled peptide (GILGFVFTL-KKKK) was applied to the excised human skin and the chamber was placed for 16hours in an incubator at 37°C, 5% CO2, and 100% humidity. This experimental setup efficiently prevented the evaporation of the particle dispersing phase and drying of the skin. After incubation, adhesive tape stripping was performed five times to remove the particles that had not penetrated. The skin samples were then processed to isolate the Langerhans DCS as described below.
Isolation of epidermal Langerhans DCs from the skin explants
Skin samples (16cm2) were cut into 3mm2 slices and were digested with Dispase (2.4Uml−1 Dispase I, Roche, Mannheim, Germany) for 2.5hours at 37°C to detach the epidermis layers. Trypsin digestion (0.025% trypsin, 1.5mM CaCl2 in phosphate-buffered saline) of the epidermis was then performed over 15minutes to isolate epidermal cells. After filtration, the cells were collected by centrifugation. Subsequently, MACS separation with anti-CD1c antibodies was performed on the isolated epidermal cell according to the manufacturer’s instructions (Dendritic Cell Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany). The presence of the peptide (Rhodamine-labeled) at the cell surface was then analyzed by laser scan fluorescence microscopy using a laser scanning microscope LSM 2000 (Carl Zeiss).
Statistical analysis
Results were tested for normality by Kolmogorov–Smirnov tests. Comparisons of the results obtained from the different experimental groups were done by t-test. Differences with a P<0.05 were considered significant. All statistical analyses were performed using the Graphpad Software Prism 2.01 for Windows (GraphPad Software, La Jolla, CA).
ACKNOWLEDGMENTS
This study was supported by grants from the Bundesministerium für Bildung und Forschung (BMBF, German Ministry for Education and Research) WING Programme for Nanobiotechnology grants 13N9196 and 13N9197, Coordenação de aprimoramento de pessoal de nível superior (Capes), Deutscher Akademischer Austausch Dienst (DAAD), and the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP).
Low-dose intradermal versus intramuscular administration of recombinant hepatitis B vaccine: a comparison of immunogenicity in infants and preschool children.
Hair follicles--an efficient storage and penetration pathway for topically applied substances. Summary of recent results obtained at the Center of Experimental and Applied Cutaneous Physiology, Charité -Universitätsmedizin Berlin, Germany.
Exogenous peptides presented by transporter associated with antigen processing (TAP)-deficient and TAP-competent cells: intracellular loading and kinetics of presentation.
Mechanisms of exogenous antigen presentation by MHC class I molecules in vitro and in vivo: implications for generating CD8+ T cell responses to infectious agents, tumors, transplants, and vaccines.
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