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Introduction
Nanomedicine is a rapidly growing research area with multiple applications, including imaging [1] , vaccination [2,3] , biosensoring [4] , and drug delivery systems [5-7] . Due to the potential of nanocarriers to promote cell-specific targeting and to protect drugs on its way to the desired cell/organ/tissue, nanocarriers play an important role as drug delivery systems. There are many different preparation techniques for nanocarriers. However, miniemulsion is the preparation method of choice for many applications, which is owed to its excellent properties, including the tuning of size and size distribution, surface functionalization opportunities and the high payload efficiency. In the present study hydroxyethyl starch (HES) was chosen as biopolymer for nanocapsule synthesis, based on its biocompatible properties and its established use in various clinical applications [8] . In previous investigations we observed, that HES nanocapsules without any surface functionalization display a diminished unspecific cell uptake [9] , a liver specific deposition [10] , and excellent release properties [11] . In particular, we could demonstrate in an in vitro murine model, that hydroxyethyl starch-based nanocapsules (HES-NCs) are efficiently ingested by resident liver macrophages (Kupffer cells) and that they are able to release their content (dexamethasone) followed by a significant suppression of cytokine release [11] .
The latter observation prompted the present study focusing on the interaction of functionalized HES-NCs with human dendritic cells (DCs), which would potentially qualify HES-NCs as a vaccine platform. A critical obstacle in the development of vaccines against viral pathogens is the induction of vigorous and long-lasting, antigen-specific cellular immune responses [12] . DCs - professional antigen presenting cells - are a key cell population linking the innate with the adaptive immune system and are essential for the initiation of cellular immune responses [13] . Accordingly, DCs have become a primary target for novel vaccine formulations, such as virus-like particles, replicons, and nanoparticles [14] .
Tailor-made nanocarriers for vaccination with encapsulated antigen have a number of advantages, including (a) prevention of proteolytic degradation [15] ; (b) prolonged antigen presentation [16] ; (c) enhanced phagocytosis by antigen presenting cells (APCs) [17] ; (d) promotion of endosomal release of antigen, leading to enhanced cross-presentation [18] ; (e) co-delivery of antigen and adjuvant, promoting Th1 responses [19] ; and (f) receptor-mediated targeting of DCs by immobilization of antibodies on the polymer surface [20] .
Commonly targeted DC receptors [21] include CD205 (DEC205) [22] , CD209 (DC-SIGN) [23] , and CD40 [24] . Importantly, receptor mediated targeting not only enhances uptake but is also capable to induce activation of cells; e.g. in CD40-mediated phagocytosis.
In the present study we investigated the effects of HES-NCs functionalized with commonly used targeting receptors (anti-DEC205 and anti-CD40) and compare the latter with monophosphoryl lipid A-(MPLA) and interferon-γ-(IFNγ) coated HES-NCs. MPLA was chosen since it is a commonly used vaccine adjuvant, known to induce maturation of dendritic cells [25,26] . Notably, MPLA has been approved by US and European authorities as a vaccine adjuvant, e.g. in a Hepatitis B vaccine (Fendrix, GlaxoSmithKline). In addition, we have recently observed that coating of HES-NCs with MPLA promotes uptake by macrophages and DCs in a murine model [10] . Importantly, combining MPLA with IFNγ is a particular suitable approach to induce IL-12 secretion by human monocyte-derived DCs [27,28] . In summary, we aimed to investigate how MPLA and IFNγ immobilized on HES-NCs affects phagocytosis and maturation of human DCs, with the particular interest to induce a cytokine environment promoting Th1 T cell responses. Anti-DEC205- and anti-CD40-coated HES-NCs served as a reference.
Material and methods
Nanocapsule synthesis and characterization
Materials used for the NCs synthesis
Materials purchased included: hydroxyethyl starch (HES, Mw=200,000gmol-1 ; Fresenius Kabi), 2,4-toluene diisocyanate (TDI) and cyclohexane (>99.9%; Sigma Aldrich), sodium dodecylsulfate (SDS; Fluka), N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC), monochloroacetic acid (MCA; Aldrich), and Cy5-labeled oligonucleotides (5'-Cy5-CCACTCCTTTCCAGAAAACT-3', Thermo Scientific). The surfactant poly((ethylene-co-butylene)-b-(ethylene oxide)), P(E/B-b-EO) [29] and 4-sulfotetrafluorophenyl (STP) were synthesized at the Max-Planck Institute for Polymer Research [30] .
Antibodies and other materials for coupling onto the NCs surface were: anti-DEC205 (BD Pharmingen; clone MG38), anti-CD40 (eBioscience; clone 5C3), unspecific anti-human IgG (BD Pharmingen; clone 27-35), interferon-γ (IFNγ; Peprotech) or monophosphoryl lipid A (MPLA; Sigma Aldrich).
Preparation of nanocapsules
HES nanocapsules were synthesized by a polyaddition reaction performed at the miniemulsion droplet's interface according to previously published protocols [9,10] as shown in Fig. 1 . Nanocapsules were labeled with Cy5-oligonucleotides in order to assess NC uptake by DCs in vitro. Afterwards, HES nanocapsules were functionalized by a carboxymethylation procedure as previously published [31] . Coupling of anti-DEC205, anti-CD40, IFNγ or IgG, adsorption of MPLA onto HES nanocapsules and their subsequent characterization were performed using a method previously described [10] .
Biological analysis
Generation of human monocyte-derived dendritic cells
Adult peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats, obtained from healthy voluntary donors (blood bank of the University Medical Center Mainz), upon informed and signed consent, by centrifugation through Histopaque-1077 density gradient media (Sigma-Aldrich) for 20min at 900xg and 20°C. The interphase consisting of PBMCs were subsequently extracted and washed with Hank's balanced salt solution (Sigma-Aldrich). CD14+ monocytes were isolated from the PBMC fraction by positive selection using CD14 MicroBeads, MACS LS columns and a magnetic cell separator (MACS; Miltenyi Biotec) in accordance with the manufacturer's instructions. CD14 positive monocytes were washed in X-Vivo 15 medium (Lonza). Subsequent flow cytometric analysis (LSR II; BD Biosciences) verified a high purity of CD14+ monocytes (>98%). Purified monocytes were cultured at a concentration of 106 cells per ml in 6-well plates (Greiner Bio-One) in X-Vivo 15 medium supplemented with l-glutamine, 100Uml-1 penicillin and 100gml-1 streptomycin. Finally, GM-CSF (200Uml-1 ) and IL-4 (200Uml-1 ) was added to the medium following 6 days of culture at 37°C and 5% CO2 with addition of 1ml fresh medium at days 2 and 4. Immature moDCs were obtained by harvesting the non- or loosely adhering cell population (approx. 30% of CD14+ monocytes). Incubation of moDCs with different nanocapsule formulations was performed using X-Vivo 15 medium supplemented with antibiotics and cytokines as described above.
Confocal laser scanning microscopy (CLSM)
Nanocapsule uptake by moDCs was evaluated using a Zeiss LSM 710 NLO confocal laser scanning microscope. Immature DCs were harvested as described above and cultured with a density of 3x105 ml-1 in 8-well chamber slides (ibidi) at 37°C for 4h in the presence of 7.5gml-1 HES-IgG nanocapsules. Nuclei were stained with 2gml-1 Hoechst 33342 (Life Technologies) for 30min. Immediately before analysis 2gml-1 CellMask Orange (Life Technologies) was added for plasma membrane staining.
In vitro loading of moDCs with nanocapsules and flow cytometric analysis
In vitro uptake of nanocapsules by moDCs and maturation analysis were performed by coincubation of immature moDCs in a density of 106 ml-1 with 7.5gml-1 of Cy5-labeled nanocapsules (HES-IgG, HES-CD40, HES-DEC205, HES-IFNγ, HES-MPLA, HES-IgG-MPLA, HES-CD40-MPLA, HES-DEC205-MPLA, HES-IFNγ-MPLA) or without nanocapsules (control) for 4 and/or 24h in 48-well plates (Nunclon Surface; Nunc--Thermo Scientific). Cell culture supernatants were collected after 24h of incubation at 37°C and stored at -20°C for subsequent cytokine analysis. Uptake, maturation and toxicity were analyzed by flow cytometry using the multi-channel cytometer BD LSR II (BD Biosciences) equipped with FACSDiva software (BD Biosciences). Data analysis was performed with FlowJo software (Tree Star). Approximately 5x105 moDCs were incubated with excess human polyvalent IgG antibody (Sandoglobulin Liquid; CSL Behring) in order to block the Fc receptor and avoid unspecific binding of primary antibodies, followed by a 30min incubation with fluorochrome-conjugated antibodies CD14 (PerCP; clone MφP9), CD40 (PE; clone 5C3), CD80 (FITC; clone BB1), CD83 (PE; clone HB15e), CD86 (FITC; clone MMRI-7), HLA-A,B,C (FITC; clone G46-2.6), HLA-DR (PerCP; clone G46-6), or CD11c (V450; clone B-ly6).
Measurement of cytokine secretion
In order to quantify the secretion of IL-6, TNF and IL-12p70, cell culture supernatants collected after 24h incubation in the presence of nanocapsules were analyzed using a commercial enzyme-linked immunosorbent assay kit (IL-12p70; eBiosciences) and a Cytometric Bead Array Kit (Human Th1/Th2 Cytokine Kit II; BD Biosciences) in accordance with the manufacturer's instructions followed by data analysis using FCAP Array software (Soft Flow).
Toll-like receptor 4 blocking
The involvement of the Toll-like receptor 4 (TLR4) in the activation of moDCs induced by MPLA-coated nanocapsules was evaluated through antibody-mediated blocking. Briefly, moDCs (106 ml-1 ) were preincubated with different concentrations of a human TLR4 antibody (Invivogen; clone W7C11) for one hour followed by incubation with 7.5gml-1 HES-IFNγ, HES-MPLA, HES-IFNγ-MPLA capsules or 4gml-1 MPLA. After 24h incubation, moDCs were harvested and analyzed for nanocapsule uptake and maturation marker expression by flow cytometry. Furthermore, culture supernatants were obtained and analyzed using a Cytometric Bead Array Kit (Human Th1/Th2 Cytokine Kit II; BD Biosciences).
Statistical analysis
Experiments were performed in triplicates and analyzed using SigmaPlot 11 software (Systat Software Inc). When more than two groups were compared with each other, a one way ANOVA test was performed followed by a Holm-Sidak test to determine which groups differed significantly (* p<0.05; ** p<0.01; *** p<0.001). For comparisons between two groups only a non-paired Student's t-test was performed (* p<0.05; ** p<0.01; *** p<0.001).
Results
Nanocapsule synthesis
HES-NCs were synthesized by interfacial polyaddition resulting in a core-shell structure as confirmed by SEM images (Fig. 1 ). The polymeric shell consists of HES and inside the aqueous core, Cy5 oligonucleotides were encapsulated for fluorescently labeling of NCs.
Carboxymethylation and antibody binding including its effect on size, polydispersity and zeta potential has been recently described by our group [10] . The characteristics of the synthesized NCs are summarized in Table 1 . The average size of NCs was between 220nm and 240nm. The percentage of MPLA adsorbed to hydroxyethyl starch nanocapsules was calculated based on the quantities applied in the adsorption procedure. Therefore, a percentage of 2.4% could be calculated assuming optimal conditions with an adsorption efficacy of 100%. Thus, 7.5g HES-MPLA-NCs carried a maximum load of 0.18g MPLA corresponding to an average of 760 molecules per nanocapsule.
Characterization of human monocyte-derived dendritic cells
CD14+ monocytes were isolated from PBMCs and subsequently cultured for six days in the presence of IL-4 and GM-CSF resulting in the generation of immature monocyte-derived dendritic cells (moDCs) (with a yield of 25% to 35%). MoDCs were characterized by flow cytometry and phase contrast microscopy displaying the typical morphology of immature moDCs which appeared round, non- or slightly-adherent with few cytoplasmic projections (Fig. 2 A). Non-moDCs appeared flat and strongly adherent; therefore, only non-adherent cells were harvested and used for further experiments. Flow cytometric analysis was performed to verify the phenotype of immature moDCs which was CD14- , CD11c+ , CD40+ , CD205+ , CD80- , CD83- , CD86intermediate , MHC Iintermediate and MHC IIintermediate (Fig. 2 B and C).
Uptake of nanocapsules by moDCs
In order to confirm nanocapsule uptake rather than adherence to the plasma membrane, confocal laser scanning microscopy was performed. Fig. 3 A clearly documents the intracellular uptake of Cy5-labeled nanocapsules (red), which appear to be compartmentalized. Furthermore, the uptake behavior of different nanocapsule formulations (HES-IgG, HES-CD40, HES-DEC205, HES-IFNγ, HES-MPLA, HES-IgG-MPLA, HES-CD40-MPLA, HES-CD205-MPLA and HES-IFNγ-MPLA) was quantified with HES-IgG serving as a control. Fig. 3 B shows a significantly increased uptake of moDCs when HES-NCs were coated with the targeting antibody anti-CD40 compared to capsules coupled to the isotype control (IgG). However, coupling of anti-DEC205 or IFNγ to HES-NCs exhibited only a minute and non-significant effect with respect to the uptake intensity of moDCs in vitro. Interestingly, coating with MPLA induced the most pronounced effect on the ingestion of nanocapsules with up to 58% of moDCs being NC positive after 24h of coincubation. Notably, a combined coating with IgG, anti-DEC205 or IFNγ rather reduced the effects of MPLA-coating regarding the uptake capacity.
Nanocapsule-induced maturation and cytokine secretion
Nanocapsules were cocultured with moDCs and analyzed for their potential to induce maturation and secretion of pro-inflammatory cytokines. Flow cytometric analysis revealed a significant increase in CD83 and CD80 expression induced by MPLA-coated HES-NCs from a baseline expression of 8% to 32.5% (Fig. 4 A) and from 1.2 to 3.9% (Fig. 4 B), respectively. Nanocapsules without MPLA did not trigger any increase in CD83 or CD80 expression, even when anti-CD40 or IFNγ was coupled to the surface. MPLA in solution and a combination of MPLA and IFNγ served as positive controls and lead to a comparable upregulation of CD83 and CD80 expression when compared to MPLA-coated nanocapsules. Supernatants of HES-MPLA capsules after the adsorption procedure served as a control and showed no activation compared to HES-IgG (data not shown). In addition to the phenotypic maturation status of moDCs, cytokine secretion analysis was performed using a multiplex assay. In parallel to the maturation experiments, HES-MPLA-NCs induced a massive secretion of the pro-inflammatory cytokines IL-6 and TNF, whereas nanocapsules without MPLA-coating did not induce any cytokine response by moDCs (Fig. 5 A and B). In order to evaluate whether the different MPLA nanocapsules elicit a Th1-directed cytokine secretion, IL-12p70 levels were measured after coincubation with moDCs (Fig. 5 C). Similar to analysis of IL-6 and TNF, only MPLA-coated nanocapsules induced remarkable levels of IL-12. Interestingly, HES-MPLA capsules showed a 4.5 fold increased IL-12 secretion compared to HES-IgG-MPLA capsules, whereas a coincubation with HES-IFNγ-MPLA-NCs led to an increase of 30%. However, HES-CD40-MPLA and HES-DEC205-MPLA capsules reduced the IL-12 secretion by 33% and 41%, respectively.
Notably, MPLA in solution did not elicit a significant effect on IL-12 secretion, contrasted by the overwhelming IL-12 secretion caused by MPLA and IFNγ in solution.
Blocking of Toll-like receptor 4 (TLR4)
Preincubation with a TLR4-blocking-antibody showed no effect regarding the uptake of MPLA-coated nanocapsules by moDCs in vitro (Fig. 6 A). However, an antibody concentration-dependent decrease in CD83 expression was observed after incubation with HES-MPLA and HES-IFNγ-MPLA capsules (Fig. 6 B). MPLA in solution served as a positive control and led to a similar reduction after blocking of TLR4. Cytokine analysis of culture supernatants paralleled these findings with a pronounced statistically significant decrease in IL-6 and TNF secretion even after incubation with low blocking antibody concentrations (Fig. 6 C).
Discussion
The lack of efficient vaccines against pathogens such as HCV or HIV underlines the need for novel, tailor-made vaccine delivery platforms. Particulate vaccines like virus-like particles (VLPs) have proven to be successful in inducing protective immunity against Human Papilloma and Hepatitis B virus [32,33] . Other innovative vaccine formulations include liposomes, immunostimulating complexes (ISCOMs), polymeric nanocapsules and nanogels [34,35] . Nanocapsules are of particular interest because they combine all essential components needed for a successful vaccine like adjuvants, antigen and antibodies for targeting purposes [35] . Targeting antigen to antigen-presenting cells (APCs), especially dendritic cells is crucial for the induction of antigen-specific immune responses [20] . Several studies show, that a broad intrahepatic CD8+ and CD4+ T cell response is needed for the eradication of HCV [36,37] , whereas a missing CD4+ T cell help is associated with a chronification of the infection [38] . In particular, a Th1-directed immune response was observed in HCV-infected patients who displayed a sustained virological response and eliminated the virus [39] . The liver is prone to tolerance, which in part explains the susceptibility to chronic infections with HBV and HCV [40] . Thus, formulating vaccine nanoparticles containing antigens in combination with adjuvants overcoming the state of tolerance is a promising approach for the development of therapeutic/prophylactic vaccines.
Therefore, the aim of the present study was the development of a nanocapsule delivery platform composed of hydroxyethyl starch characterized by an efficient uptake, and maturation of human moDCs in vitro, as well as the induction of Th1-related cytokine secretion patterns. We made use of monocyte-derived dendritic cells as a model system in order to obtain sufficient numbers of human dendritic cells [41] . Phenotypic characterization of generated immature moDCs displayed a CD14- CD11c+ CD40+ CD205+ CD80- CD83- CD86intemediate phenotype and a round morphology with few small dendrites. In a first step, the uptake of hydroxyethyl starch nanocapsules by moDCs was documented by confocal microscopy, evidencing intracellular uptake of HES-NCs rather than attachment.
Quantification of the overall uptake by FACS analysis revealed an enhanced phagocytosis when NCs were equipped with the targeting antibody anti-CD40 (Fig. 3 B). To our surprise anti-DEC205-coating had only a minute effect on the uptake behavior of human DCs. Interestingly, NCs coated with the TLR4 ligand MPLA were taken up by moDCs to a significant higher level compared to all other capsule formulations. This finding is in parallel to our previous study where murine liver DCs expanded in vivo with hFlt3-ligand were preferentially ingesting MPLA-coated nanocapsules [10] . Remarkably, murine DCs showed an even higher uptake when HES-NCs were additionally coupled with targeting antibodies anti-CD40 or anti-DEC205, which was not the case for human moDCs.
Besides an enhanced uptake of HES-NCs, activation and maturation of moDCs reflected by the expression of the maturation marker CD83 and the costimulatory molecule CD80 could also be induced by MPLA-coated NCs. HES-CD40-MPLA-NCs appeared as the best inducer of CD83 expression.
In a preliminary dose finding study an optimal concentration of 7.5gml-1 in terms of neglectable toxicity caused by nanocapsules was determined (data not shown). MPLA-coated nanocapsules at this concentration feature just a small fraction of MPLA related to the weight of the capsule (max. 2.4%) that is at least 22 times lower than the concentration of the applied positive control with 4gml-1 pure MPLA in solution. Stimulation of DCs with 4gml-1 MPLA in solution was determined in a dose finding study to be optimal in terms of the induction of IL-12p70 secretion and thus used as positive control (Supplementary Fig. 1). Remarkably, the effect of MPLA-NCs upon maturation and especially upon the secretion of pro-inflammatory cytokines was even higher compared to the positive control (MPLA in solution). This finding documents the benefit of particle-bound MPLA in contrast to MPLA in solution. Furthermore, targeting dendritic cells with antigen- and adjuvant-loaded nanoparticles provides the opportunity to reduce the amount of adjuvant up to 100 times [42] . Such dose-sparing strategies are essential for the development of novel vaccines, reducing systemic and/or local side-effects caused by MPLA. The size of nanocapsules in combination with the presentation of the TLR4 ligand resembles pathogen-like structures inducing antigen-specific immunity [43] .
In order to determine whether the applied nanocapsules induce a Th1-type response we analyzed the IL-12p70 secretion levels after incubation with NCs and identified a reasonable secretion for all MPLA-coated nanocapsule formulations. However, in contrast to IL-6 or TNF secretion and expression of maturation markers, HES nanocapsules solely coated with MPLA induced a massive, 4.5x increased IL-12 secretion as compared to NCs additionally coated with targeting antibodies or interferon-γ. This could be due to a reduced coating efficacy caused by prior coupling of antibodies or IFNγ on the nanocapsule surface resulting in a diminished surface area available for MPLA adsorption. Furthermore, we hypothesize that an equipment of nanocapsules with antibodies or proteins hinders the TLR4 ligand to bind to its receptor on the cell plasma membrane, therefore, leading to a decreased IL-12 secretion.
Monophosphoryl lipid A as an agonist of the Toll-like receptor 4 mediates through TRIF-based pathway leading to a Th1-directed response [44] . Blocking of TLR4 led to a significant reduction in the maturation state of moDCs and their secretion of pro-inflammatory cytokines after treatment with MPLA-coated nanocapsules. This finding indicates that the activation of moDCs by MPLA-NCs is mediated through TLR4 ligation. Furthermore, Toll-like receptor 4 seems to be involved in the phagocytic process induced by MPLA-coated nanocapsules as has been described for murine macrophages [45] and bone marrow-derived dendritic cells [46] . As shown in Fig. 3 , the uptake of HES nanocapsules by moDCs could be increased when MPLA was coated onto the capsule surface. However, the uptake intensity could not be reduced by blocking TLR4 (Fig. 6 ). Therefore, we hypothesize that other receptors like scavenger receptors could have been responsible for the compensation of TLR4 blockade leading to an unaltered uptake behavior by moDCs. The class A scavenger receptors type I and II are capable of recognition of lipopolysaccharide moieties like lipid A and thus could be the target of MPLA-coated nanocapsules [47,48] .
Conclusion
In conclusion, this publication presents a nanocarrier platform based on hydroxyethyl starch nanocapsules with a diameter between 220 and 240nm. Coating with the vaccine adjuvant MPLA effectively enhanced nanocapsule phagocytosis by dendritic cells and led to maturation and the induction of IL-12 secretion as an indicator for a Th1-directed response. Notably, the amount of MPLA attached to the HES-NCs was at least 20 fold lower than soluble MPLA used for maturation of DCs evidencing the intense dose-sparing potential of particle-bound MPLA.
Funding
Deutsche Forschungsgemeinschaft (DFG) grant DFG GE1193--2/1.
Conflict of interest statement
The authors have no conflict of interest to disclose.
Supplementary data
Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2014.12.072.
Supplementary data
The following are the supplementary data to this article:
Samples | Average diameter /standard deviation [%] | Zeta potential (pH 7) | ||
In water phase | After antibody coupling | After MPLA adsorption | ||
HES | 230/28 | - | - | -24 |
HES-MPLA | - | 235/32 | -32 | |
HES-DEC205 | 240/30 | - | -14 | |
HES-DEC205-MPLA | 240/32 | -30 | ||
HES-CD40 | 220/29 | - | -18 | |
HES-CD40-MPLA | 235/33 | -30 | ||
HES-IgG | 235/30 | - | -18 | |
HES-IgG-MPLA | 235/30 | -32 | ||
HES-IFNγ | 220/31 | - | -17 | |
HES-IFNγ-MPLA | - | 235/30 | -32 |
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