Article pubs.acs.org/Biomac
Spray-Dried Polyelectrolyte Microparticles in Oral Antigen Delivery: Stability, Biocompatibility, and Cellular Uptake Rebecca De Smet,*,† Stephanie Verschuere,† Liesbeth Allais,† Georges Leclercq,‡ Marijke Dierendonck,§ Bruno G. De Geest,§ Isabel Van Driessche,∥ Tine Demoor,† and Claude A. Cuvelier† †
Department of Pathology, Ghent University, 5 Blok A, De Pintelaan 185, 9000 Ghent, Belgium Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, 4 blok A, De Pintelaan 185, 9000 Ghent, Belgium § Laboratory of Pharmaceutical Technology, Department of Pharmaceutics, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium ∥ Department of Inorganic and Physical Chemistry, Ghent University, Krijgslaan 281, S3, 9000 Ghent, Belgium ‡
S Supporting Information *
ABSTRACT: During the past decade, extensive research has undeniably improved the formulation and delivery of oral vaccines. Nevertheless, several factors, such as the harsh gastrointestinal environment together with tolerance induction to exogenous antigens, have thus far impeded the optimal effectiveness and clinical application of oral delivery systems. The current study encompasses an initial evaluation of the stability, biocompatibility, and cellular uptake of two promising candidate systems for oral antigen delivery, that is, calcium carbonate- (CP) and mannitol-templated (MP) porous microspheres. Both spray-dried formulations were efficiently internalized by human intestinal epithelial cells (Caco-2 and HT-29) and degraded into phagolysosomal intracellular compartments. In addition, cellular particle uptake and processing significantly up-regulated the expression of (HLA) class-II and costimulatory molecules on intestinal epithelial cells. Even though the high surface-area-tovolume ratio of the microspheres was expected to favor protease access, antigen release was remarkably limited in simulated intestinal fluid and was even absent under gastric conditions. Finally, neither CP nor MP exerted cytotoxicity upon prolonged in vitro incubation with high antigen concentration. Altogether, these data support the potential of CP and MP for oral antigen delivery and motivate the further development of these promising carrier systems in in vivo studies.
1. INTRODUCTION
improved patient compliance, are associated with oral vaccination, especially in developing countries.6,7 Therefore, there has been much interest in the development of more efficient mucosal vaccines, for example, by the use of microparticles as antigen delivery systems.8−12 In particular, the production of spray-dried microparticles has gained interest. The spray-drying technique is widely used in pharmaceutical, food, and chemical industries for different purposes.13 Spraydried formulations are less subject to antigen damage by chemical and physical processes (such as degradation,
Gastrointestinal infections are still the main cause of enteric diseases and mortality among humans and animals. Diarrheal disease is the second leading cause of death in children under five years of age and is responsible for killing 760 000 children every year.1 Oral vaccination is the only manner to acquire preventive local adaptive immune responses, required for the protection against intestinal pathogens. However, the hostile environment of the intestinal tract and oral tolerance are huge obstacles that currently challenge the successful development of new mucosal vaccines.2−5 Many socioeconomic advantages, such as the elimination of contaminated needles and adequate staffing as well as the © 2014 American Chemical Society
Received: April 11, 2014 Published: May 7, 2014 2301
dx.doi.org/10.1021/bm5005367 | Biomacromolecules 2014, 15, 2301−2309
Biomacromolecules
Article
Figure 1. Schematic representation of the synthesis of antigen-loaded porous polyelectrolyte microparticles via spray-drying technology. A mixture composed of polyelectrolytes (dextran sulfate and poly-L-arginine), sacrifial component (CaCO3 nanoparticles or mannitol), and antigen (OVA or BSA) is administered to a spray dryer, resulting in solid microspheres. Decomposition of the CaCO3 nanoparticles or mannitol following treatment with EDTA or PBS, respectively, results in porous antigen-loaded polyelectrolyte microspheres. Scanning electron microscopy images (B1 and C1) and size distribution graphs (B2 and C2) of OVA-loaded CP and MP, respectively, obtained with the cospray-drying process.
media. Subsequently, human Caco-2 and HT-29 cells were used to model the interaction between the microparticles and the intestinal epithelium and to monitor the effects on cell viability and maturation status.
denaturation) and avoid the cold-chain-constrained settings (for instance, the use of refrigeration for storage and transport of vaccines), a very important aspect for vaccination campaigns in third-world countries. Furthermore, the spray-drying procedure allows a good production yield, reproducibility, and scalability and offers the adjustment of specific parameters of the dry formulation such as enhanced stability and controlled particle size ranges.14−16 A novel encapsulation method yielding spray-dried porous polyelectrolyte microspheres could be of great interest. The resulting calcium carbonate- (CP)17 or mannitol-templated (MP) porous microspheres consist of a polyelectrolyte framework that can encapsulate a cospray-dried protein (for instance ovalbumin (OVA) or bovine serum albumin (BSA)) in combination with CaCO3 nanoparticles or mannitol. The latter serves as a sacrificial template that is extracted upon EDTA or PBS treatment, respectively (Figure 1A). In comparison with layer-by-layer synthesized polyelectrolyte microcapsules, previously shown to induce cellular uptake and processing of antigen,18−23 the spray-dried microspheres allow more stable and higher antigen encapsulation. Furthermore, spray-drying is more efficient, inexpensive, and less time-consuming. Finally, both spray-dried calcium carbonate- and mannitol-templated porous polyelectrolyte particles have been shown to promote successful uptake, processing , and cross-presentation of encapsulated OVA by bone-marrow-derived dendritic cells (BMDCs).24−26 Altogether, these features motivate additional research on the use of spray-dried microspheres for the vaccination of animals and humans. The current study thus aimed to evaluate spray-dried CP and MP as candidate antigen carriers for oral vaccination. First, we characterized the antigen-loaded CP and MP microparticles and studied their stability in simulated gastric and intestinal
2. MATERIAL AND METHODS 2.1. Synthesis of Spray-Dried Microspheres and Protein Encapsulation. The spray-dried CP and MP polyelectrolyte particles were synthesized, as previously described.24,25 In brief, CaCO3 nanoparticles (Plasmachem, Berlin, Germany) or mannitol (Cargill, Minneapolis, MN) were mixed with dextran sulfate (DEXS; 10 kDa), OVA or BSA as model antigen, and poly-L-arginine (PLARG; Mw > 70 kDa) (all from Sigma-Aldrich, Diegem, Belgium) in a 40/4/1/5 ratio in water, with a resulting solids concentration of 1% w/w and an antigen load of 2% w/w. PLARG was added dropwise from a 0.5% w/ w solution in water to the stirring CaCO3 nanoparticles or mannitol/ DEXS/antigen dispersion. The mixture was spray-dried on a lab-scale spray-dryer, type B-290, equipped with a B-296 dehumidifier (Buchi Labortechnik AG, Flawil, Switzerland). The mixture was fed to a twofluid nozzle (orifice diameter: 0.7 mm) at the top of the spray-dryer, which operated in a cocurrent nitrogen flow mode. Applied process parameters are summarized in Table 1. 2.2. Characterization of Spray-Dried Microspheres. Particle size distribution was determined by laser diffraction, using a Mastersizer 2000 apparatus (Malvern, Worcestershire, U.K.). Image
Table 1. Spray-Drying Process Parameters
2302
process parameters
actual range
inlet drying nitrogen temperature (°C) outlet drying nitrogen temperature (°C) drying nitrogen aspirator (%) atomising nitrogen pressure (mm) feed spray rate (g/min)
117−130 54−63 80−90 45 2.8−2.9
dx.doi.org/10.1021/bm5005367 | Biomacromolecules 2014, 15, 2301−2309
Biomacromolecules
Article
Table 2. Analytical Results of Microparticle Characteristics
a
concept
size (μm)
surface area (m2/g)
mean pore diameter (nm)
pore volume (cm3/g)
S/V (μm−1)a
EEb
CP-OVA MP-OVA
5.1 ± 0.9 3.2 ± 0.3
16.8 2.3
25.8 13.8
0.108 0.008
2.8 ± 1.2 4.2 ± 2.1
86 ± 1%24 99 ± 1%25
S/V: surface area to volume ratio. bEE: encapsulation efficiency of OVA. 2.6. Intestinal Epithelial Cell Uptake Assessment. Following cell detachment by Accutase (eBioscience, Vienna, Austria) and FcR blocking with human FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany), single-cell suspensions of Caco-2 and HT-29 cells were stained with CD324-APC (clone 67A4) (E-Cadherin) (Miltenyi Biotec), and dead cells were excluded by propidium iodide (0.1 mg/mL) (Invitrogen, Ghent, Belgium). Fluorescent microparticle uptake was quantified using a LSR II cytometer equipped with FACSDiva software version 6.1.2 (both BD Biosciences, Erembodegem, Belgium). Furthermore, the expression of costimulatory molecules was analyzed on intestinal epithelial cells (live CD324+ cells) using anti-CD86 (clone IT2.2) and anti-CD40 (Clone 5C3) (all eBioscience). MHC class-II molecule expression was analyzed with anti-HLA-DR (Clone LN3) (eBioscience). For confocal microscopy analysis, Caco-2 cells and HT-29 cells were cultured in a Lab Tek II eight-well glass chamber slide (Nunc, Roskilde, Denmark). Upon reaching confluence, the cell monolayers were incubated with BSA-FITC-loaded microparticle solutions for 24 h. Subsequently, cells were washed with PBS and fixed in an aqueous 4% formaldehyde solution overnight, followed by nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) and cell membrane staining with wheat germ agglutinin (WGA)-Texas Red-X conjugate (20 μg/ mL) (Invitrogen). Confocal images of 3D cultures were made with a Leica TCS SP5 AOBS confocal laser scanning microscope. For TEM, cell culture samples are embedded in Epon medium. Semithin sections of 1 μm were cut and stained with toluidine blue to select the most appropriate area. Ultrathin sections of 60 nm were cut and contrasted with uranyl acetate and lead citrate, followed by imaging with a Zeiss TEM900 transmission electron microscope (TEM, Carl Zeiss, Oberkochen, Germany) at 50 kV. 2.7. Statistical Analyses. Reported values are expressed as mean ± SEM. Statistical analysis was performed by SPSS 20 Software (SPSS 20, Chicago, IL) using nonparametric tests (Mann−Whitney U). For comparison of more than two groups, use was made of one-way analysis of variance (ANOVA), followed by post hoc Bonferroni tests or nonparametric Kruskall−Wallis tests if conditions for ANOVA were not met. A P value
