Facile Two‐Step Synthesis of Porous Antigen‐Loaded Degradable Polyelectrolyte Microspheres (2010)

Pore medicine: Porous antigen-loaded degradable polyelectrolyte microspheres are produced in a straightforward way providing high encapsulation efficiencies and efficient internalization by antigen-presenting cells. This system could find application as multilayer capsules in gene and drug delivery and for enzymatic microreactors. Without doubt, the development of vaccines constitutes one of the major breakthroughs of human medicine, allowing us to prevent numerous infectious diseases.1 Nevertheless, for major killers such as HIV, malaria, and tuberculosis, no effective vaccine is currently available. The failure of current vaccination strategies to elicit cellular immune responses, especially CD8 cytotoxic T cells (CTLs) that can recognize and eliminate infected cells, is considered to be one of the major reasons for this failure.2–4 Consequently, there is an urgent need to develop new vaccine formulations that can induce such CTL responses. One of the most promising approaches to achieve this goal is the encapsulation of antigens in particulate carriers with dimensions between 0.1 and 10 μm.5 A plethora of studies has now demonstrated that such carriers can strongly enhance antigen presentation by dendritic cells (DCs), the most potent antigen presenting cells capable of priming effector T cell responses, not only quantitatively but also qualitatively. Antigen being presented by a DC as an MHC/peptide complex (MHC=major histocompatibility complex) to the T cell receptor indeed constitutes the first step in the initiation of T cell responses. Two different pathways occur for antigen presentation by MHC I and MHC II to CD8 and CD4 T-cells, respectively. MHC I presentation is responsible for the processing and presentation of cytosolic proteins, which are cleaved by the proteasome, transported to the endoplasmatic reticulum, and subsequently loaded onto MHC I molecules. By contrast, MHC II presentation occurs for endocytosed proteins, which are degraded in endolysosomal compartments, loaded onto MHC II molecules and subsequently presented at the cell surface. The way in which antigen is internalized by a DC, however, strongly affects how the antigen is processed and presented by a DC, and consequently also the type and strength of immune response induced. Although soluble antigens are almost exclusively presented by MHC II to CD4 T cells, particulate antigens not only are far more efficiently taken up by DCs, but are also presented by MHC I to CD8 T cells,6 thus enabling the induction of CTL responses. Although particulate carriers have the capacity to evoke potent cellular immunity, their clinical application has been impeded largely by practical problems involving their generation, including a low antigen encapsulation efficiency, the use of chemical solvents and physical stresses that negatively affect antigen stability, and the involvement of complex and labor-intensive multistep processes to generate them. Polymeric multilayer capsules7–13 have emerged as promising microscopic carriers for the delivery of antigens to DCs, overcoming some of the problems described above.14–17 These capsules are based on alternate deposition of polymers (so-called layer-by-layer technology),18, 19 either through electrostatic interaction20–22 or hydrogen bonding,23 onto a sacrificial template, followed by decomposition of the template, resulting in hollow capsules and allowing efficient antigen encapsulation under non-denaturing conditions. Several papers have now demonstrated the potential of these capsules to target antigens to APCs both in vitro and in vivo, resulting in strongly enhanced antigen presentation to CD4 and CD8 T cells and the induction of broad and strong immune responses.14, 16, 17, 24–27 The major advantage for their success is presumably threefold:14, 28 1) They protect the antigen from degradation before reaching DCs; 2) Because of their size (1–10 μm), they are preferably targeted to DCs; and 3) Because of their soft thin shell, which is prone to the reductive conditions or endosomal proteases depending on the nature of the capsule shell, they allow the antigen to be readily processed upon internalization by DCs. Moreover, these capsules have been shown to be biocompatible and degradable, both in vitro and in vivo. Notwithstanding their excellent performance, polymeric multilayer capsules are fabricated in multiple steps, which is a rather cost-inefficient fashion involving the use of a large excess of polymer and several centrifugation steps during deposition of each single layer. Therefore, a simple and versatile strategy involving a minimum of process steps that mimics polymeric multilayer capsules would be of uttermost importance to allow this type of antigen carriers to reach the clinical stage. Herein, we report on the synthesis of porous antigen-loaded degradable polyelectrolyte microspheres using spray drying as a simple, yet efficient, and scalable production technique. Figure 1 shows a schematic illustration of the formation of these porous microspheres. Key in our present work is the use as a sacrificial component of calcium carbonate nanoparticles, which are directly spray-dried in combination with antigen and oppositely charged polyelectrolytes to form a polyelectrolyte framework that entraps the antigen and the calcium carbonate nanoparticles. Extraction of calcium carbonate subsequently leads to porous microspheres. We reasoned that a porous system exhibits a much higher surface to volume ratio, which, upon internalization by DCs, will enhance the access of proteases to the encapsulated antigen followed by antigen processing and initiation of the immune response. Schematic illustration of the encapsulation of antigen (green triangles) into porous polyelectrolyte (red and blue curves) microspheres by spray-drying a mixture of antigen, polyelectrolytes, and calcium carbonate nanoparticles (CaCO3NP; gray disk) followed by extraction of CaCO3NP by treatment with edta. Calcium carbonate nanoparticles (CaCO3NP) with an average diameter of 90 nm and a ζ-potential of 6.8±0.4 mV were mixed with dextran sulfate (DS) and ovalbumin (OVA; used as model antigen in this work) under stirring. The slightly cationic surface charge of CaCO3NP means that anionic DS and OVA will at least partly associate with CaCO3NP through electrostatic interaction and presumably also by physical adsorption. Subsequently poly-L-arginine (PLARG) was added dropwise to allow further electrostatic complexation between PLARG and DS or OVA. Hollow polyelectrolyte multilayer capsules based on CaCO3 microparticle templates coated with DS and PLARG have been shown by our group to be nontoxic and degradable upon cellular uptake, both in vitro and in vivo.29, 30 In this work, the ratio of CaCO3 to OVA, DS, and PLARG was 40:1:4:5, respectively, which tends to mimic the conditions used for the encapsulation of OVA into hollow DS/PLARG multilayer capsules templated on OVA co-precipitated CaCO3 microparticles, reported in our previous work14, 15 and by others.31 Subsequently, the mixture was spray-dried using a Buchi B290 benchtop spray-drier and collected as a dry powder comprising 80 % CaCO3. These particles are refered to as solid microspheres further in this paper. Porous microspheres were obtained by re-suspending the dry powder in an aqueous 0.2 M solution of ethylenediaminetetraacetic acid (edta), which is a potent complexing agent for calcium ions32, 33 and serves to extract CaCO3NP from the solid microspheres.31 The characteristics of the these spray-dried microspheres before (solid) and after (porous) CaCO3 dissolution are summarized in Table 1. ζ-Potential Size OVA encapsulation solid microspheres −6.8±0.4 mV 6.0±0.4 μm 94±1 % porous microspheres −18±0.7 mV 5.8±0.1 μm 86±1 % The morphologies of the CaCO3 nanoparticles, the solid microspheres, and the porous microspheres were visualized by scanning electron microscopy (SEM; Figure 2 A). CaCO3NP exhibited a primary particle size of roughly 90 nm (Figure 2 A1) and upon spray-drying are grouped into spherical clusters (Figure 2 A2) with an average size of 6 μm (Figure 2 B; note that some polydispersity is inherent to spray drying34) and a ζ-potential of −6,8 mV, as determined by laser diffraction (Figure 2 A) and dynamic light scattering, respectively, upon resuspension in aqueous medium. Most importantly, as is addressed below in more detail by confocal microscopy, the spray-dried microspheres remain stable upon resuspension in aqueous medium and do not disassemble into the initial starting components. As a control we also spray-dried a mixture of CaCO3NP and OVA, and no cluster formation was observed (data not shown), indicating the requirement of the DS/PLARG polyelectrolyte complex framework to obtain stable microspheres. Extraction of the CaCO3NP by an aqueous edta solution leads to the formation of porous microspheres (Figure 2 A3,4), which have a ζ-potential of −18 mV and are most likely stabilized through a combination of electrostatic and hydrophobic interactions together with physical entanglements of the polymer and protein chains. Short-range hydrophobic forces play a major role in the formation of polyelectrolyte complexes. These originate from contact between hydrophobic parts of the polyelectrolyte chains, excluding hydration molecules.35 The highly porous state of the microspheres is highlighted in the transmission electron microscopy of Figure 2 A4 and complete removal of the CaCO3NP by treatment with edta was confirmed by energy-dispersive X-ray (EDX) analysis. The EDX spectrum shown in Figure 2 C of the solid (C1) microspheres clearly exhibits two calcium peaks, which are lacking in the EDX spectrum of the porous (C2) microspheres. Figure 2 C2 exhibits a predominant silicon peak. This silicon peak is most likely due to the porous structure of the microspheres, which allows the electron beam to reach the underlying silicon wafer on which the microspheres were deposited upon sample preparation. Laser diffraction (Figure 2 B) did not show any significant change in mean diameter of the porous microspheres compared to the solid ones, indicating that upon CaCO3NP removal no substantial shrinkage or swelling of the polyelectrolyte framework occurs. Scanning electron microscopy images of A1) CaCO3NP (note that the aggregation is due to drying of the sample prior to SEM imaging), A2) spray-dried CaCO3NP/DS/OVA/PLARG microspheres, and A3) porous DS/OVA/PLARG microspheres after treatment with edta. A4) Transmission electron microscopy image of porous DS/OVA/PLARG microspheres after treatment with edta. B) Size distribution of solid spray-dried CaCO3NP/DS/OVA/PLARG microspheres (•) and porous DS/OVA/PLARG microspheres obtained after treatment with edta (○). C) Energy-dispersive X-ray (EDX) spectra of C1) solid spray-dried CaCO3NP/DS/OVA/PLARG microspheres and C2) porous DS/OVA/PLARG microspheres obtained after treatment with edta. Confocal microscopy was used to visualize the microspheres in a hydrated state. For visualization purposes, green fluorescent OVA-alexa488 was mixed with native OVA in a 50:1 ratio. The confocal micrographs in Figure 3 A confirm that the microspheres remain stable in aqueous medium. When the microspheres were resuspended in 0.2 M edta, the dark shine in the transmission channel (Figure 3 A2) vanished and transparent (Figure 3 B2) “sponge-like” porous microspheres were obtained (Figure 3 B). Importantly, the green fluorescent OVA-alexa488 was retained within the polyelectrolyte framework of the porous microspheres rather than being spontaneously released into the surrounding aqueous medium. Confocal microscopy images of A) spray-dried solid CaCO3NP/DS/OVA/PLARG microspheres and B) porous DS/OVA/PLARG microspheres obtained after treatment with edta. The green fluorescence is due to OVA-alexa488. Quantification of the encapsulation efficiency was determined by resuspending dry solid microspheres in phosphate-buffered saline (PBS; pH 7.4) and in an aqueous 0.2 M edta solution. Subsequently, the microspheres were centrifuged, and the OVA concentration in the supernatant was determined. An encapsulation efficiency (defined as the amount of protein that is retained within the microspheres upon resuspension relative to the amount of protein in the dry microspheres) of 94±1 % upon resuspension in PBS and of 85±1 % upon resuspension in aqueous 0.2 M edta was observed. These results show that upon resuspension in PBS only 6±1 % of the OVA is released from the solid microsphere whereas 15±1 % of the OVA is released upon extraction of the CaCO3NP from the microspheres. The higher amount of released OVA from the porous microspheres relative to that from the solid microspheres is attributed to the higher surface area of the porous ones, allowing OVA that is weakly bound to the surface to be released. However, an encapsulation efficiency of 85 % into the porous polyelectrolyte spheres is considerable higher than the value of approximately 50 % that we reported earlier for encapsulation of OVA into hollow polyelectrolyte multilayer capsules. In that case, OVA was co-precipitated into calcium carbonate microparticles by mixing it with calcium chloride and sodium carbonate. After alternate coating of these microparticles with dextran sulfate and poly-L-arginine and subsequent dissolution of the CaCO3 microparticles approximately 50 % of the OVA was lost by diffusion through the polyelectrolyte membrane. As it is our aim to use the porous polyelectrolyte microspheres as antigen carriers for vaccination purposes, it is important to assess whether these porous microspheres are able to deliver their payload to DCs. Murine DCs were differentiated from bone marrow and incubated with OVA-alexa488 loaded porous microspheres followed by confocal microscopy imaging. To discriminate whether the microspheres were internalized by DCs, we counterstained the cell nuclei blue fluorescent with Hoechst stain and the cell membrane red fluorescent with Cy5 conjugated cholera toxin B (Figure 4 A,B). As the green fluorescent microspheres, cell nuclei, and cell membrane were visible in the same confocal plane and the microspheres were clearly situated within the boundaries of the cell membrane, we could unambiguously conclude that the microspheres were effectively internalized by the DCs. The Supporting Information contains a z-stack of the zoomed image, which supports our conclusion. In a subsequent series of experiments to assess the intracellular localization of the porous microspheres, we counterstained with red fluorescence the cellular cytoplasm (CellTracker Red; Figure 4 C) and the intracellular acidic vesicles, such as endosomes and lysosomes (LysoTracker Red; Figure 4 D). Colocalization between green and red fluorescence, yielding a yellow/orange signal indicated by orange arrows in Figure 4 D1, shows the presence of the porous microspheres in that specific compartment. Porous microspheres that do not colocalize with the endosomes only yield a green signal and are indicated in Figure 4 D1 with a green arrow. When comparing the images in Figure 4 C and D, one can unambiguously conclude that the porous microspheres are located in intracellular acidic vesicles. This result is in agreement with literature data on microparticles with similar sizes and composition.29, 36, 37 Confocal microscopy images of bone marrow dendritic cells incubated with green fluorescent OVA-alexa488 loaded porous microspheres. The cell nuclei in all panels were stained blue fluorescent with Hoechst stain. Panels (A) and (B), taken at different zoom levels, show images where Cy5-conjugated cholera toxin B was used to stain the cell membrane. Panels (C) show images in which CellTracker Red (red fluorescence) was used to stain the cellular cytoplasm and panels (D) show images in which LysoTracker Red (red fluorescence) was used to stain intracellular acid vesicles. In panel (D1), colocalization between red and green fluorescence (a yellow/orange signal indicates cellular uptake of the porous microspheres) is annotated with an orange arrow. Porous microspheres that are not yet taken up only exhibit a green signal and are annotated in panel (D1) with a green arrow. Panels (1) show the overlay of blue, green, and red fluorescence channels, panels (2) show the differential interference contrast (DIC) channel, and panels (3) show the overlay of blue, green, red, and DIC channels. In conclusion, we have demonstrated a facile method to encapsulate antigen into porous degradable polyelectrolyte microspheres. The major advantage of this approach is the possibility to produce these microspheres on a large scale involving a minimum of process steps combined with a high encapsulation efficiency to minimize waste of expensive antigen and polyelectrolytes. The conceptual simplicity of the porous microspheres makes it possible to further tailor the microsphere surface with specific ligands with immune-potentiating properties such as oligonucleotides containing unmethylated CpG motifs.6 Furthermore, we have demonstrated that the porous microspheres are efficiently taken up by DCs, which are the most potent antigen presenting cells. These types of microspheres could be promising vaccine delivery systems, and we are performing in vitro and in vivo DC activation and T-cell proliferation assays to elucidate the potential of the porous microspheres to efficiently target and activate the immune system. The presented approach for polyelectrolyte microsphere formation is not restricted only to antigen encapsulation/delivery but could find the same broad applications as multilayer capsules, such as enzymatic microreactors,38, 39 gene delivery,40, 41 and low molecular weight drug delivery.42 Materials: Calcium carbonate nanoparticles (CaCO3NP) were obtained from Plasmachem. Dextran sulfate (DEXS; 10 kDa), poly-L-arginine (PLARG, 100 kDa), ovalbumin (OVA; grade VII), and Hoechst 33258 were obtained from Sigma–Aldrich. OVA-alexa488, Cy5 conjugated cholera toxin B, LysoTracker Red, and CellTracker Red were obtained from Invitrogen. All water used in the experiments was of Milli-Q grade. Synthesis of (porous) microspheres: CaCO3NP, DS, OVA, and PLARG were mixed in water in a 40:4:1:5 ratio at a total solid concentration of 1 %. In detail, 200 mg CaCO3NP, 20 mg DS, and 5 mg OVA were dissolved in 20 mL water. Subsequently 25 mg PLARG was dissolved in 5 mL water and added dropwise to the stirring CaCO3NP/DS/OVA dispersion. Spray-drying of this mixture was performed in a lab-scale Bucchi B290 spray-dryer. The mixture was fed to a two-fluid nozzle (diameter 0.7 mm) at the top of the spray-dryer. In addition, the spray-dryer operated in cocurrent air flow at 35 m3 h−1 with an inlet drying air temperature of 130 °C and an outlet drying air temperature of 65 °C. Owing to evaporation of the liquid, the temperature in the droplet itself is significantly lower than that in the air stream.34 Particle characterization: Laser diffraction was performed on a Malvern Mastersizer equipped with an RF300 objective. ζ-Potential measurements were performed on a Malvern Nanosizer ZS. Confocal microscopy images were recorded on a Leica SP5 AOBS confocal microscope. Scanning electron microscopy images and EDX-spectra were recorded on a quanta FEG FEI 200 apparatus. Samples were dried on a silicon wafer and sputtered with an ultrathin layer of palladium/gold. Transmission electron microscopy images of ultrathin microtomed sections were recorded on a JEOL 1010 electron microscope. DC uptake assessment: Female C57BL/6 mice were purchased from Janvier and housed in a specified pathogen-free facility in micro-isolator units. Dendritic cells were generated using a modified Inaba protocol. Two- to six-month-old C57BL/6 mice were sacrificed and bone marrow was flushed from their femurs and tibias. After lysis of red blood cells with ACK lysis buffer (BioWhittaker), granulocytes and B cells were depleted using Gr-1 (Pharmingen) and B220 (Pharmingen) antibodies, respectively, along with low-toxicity rabbit were at a of cells in in DC medium medium containing 5 % 1 % 1 % and 50 containing 10 and 10 from After 2 and after 4 of the cells were centrifuged, resuspended in and to the same the cells were and medium containing 10 and 5 was of cells were and in The porous microspheres were in PBS at a concentration of of mg OVA, and 10 of this was added to the DCs. After 2 the cells were in an aqueous 4 % solution Subsequently, the cells were with PBS and stained with Cy5-conjugated cholera toxin B and Hoechst 33258 For the of the intracellular localization of the porous microspheres, cells were not with and the cellular cytoplasm and acidic were stained by CellTracker Red and LysoTracker Red respectively, and visualized directly by confocal of importance to are as are but not or They are as by the The is not responsible for the or of any by the than be to the for the