3D Printed Hydrogel Multiassay Platforms for Robust Generation of Engineered Contractile Tissues.

We present a method for reproducible manufacture of multiassay platforms with tunable mechanical properties for muscle tissue strip analysis. The platforms result from stereolithographic 3D printing of low protein-binding poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Contractile microtissues have previously been engineered by immobilizing suspended cells in a confined hydrogel matrix with embedded anchoring cantilevers to facilitate muscle tissue strip formation. The 3D shape and mechanical properties of the confinement and the embedded cantilevers are critical for the tissue robustness. High-resolution 3D printing of PEGDA hydrogels offers full design freedom to engineer cantilever stiffness, while minimizing unwanted cell attachment. We demonstrate the applicability by generating suspended muscle tissue strips from C2C12 mouse myoblasts in a compliant fibrin-based hydrogel matrix. The full design freedom allows for new platform geometries that reduce local stress in the matrix and tissue, thus, reducing the risk of tissue fracture.

Microcontainers for protection of oral vaccines, in vitro and in vivo evaluation.

Oral vaccines are highly desirable due to simple logistics, mass vaccination potential and for mucosal immunity. Subunit vaccines are preferred due to high safety, but are inherently difficult to deliver orally, thus providing motivation for the use of advanced oral delivery systems. Polymeric devices in micrometer size (microcontainers) were tested here for this purpose. Microcontainers were loaded with a vaccine consisting of spray dried cubosomes with OVA and Quil-A, and coated with a pH-sensitive lid for oral delivery to C57Bl/6 mice. The microcontainers were explored in vitro and in vivo for their potential as oral vaccine delivery system in an oral prime-boost setting and as an oral booster after a subcutaneously injected prime. The residence time of microcontainers in the small intestine was less than one hour. Eudragit® L100-55 was therefore chosen as lid material on the microcontainers as it remained stable in vitro at pH 4.7, which simulated the maximal pH of the stomach, and allowed release of the cubosomes within 30-60 min at pH 6.6, which simulated the mean pH of the distal half of the small intestine. In vitro small angle X-ray scattering showed that cubosomes dissolved in small intestinal fluid when not confined in microcontainers but when loaded into microcontainers they were released as hexosomes. However, while microcontainers could protect and release particles with OVA and Quil-A within relevant time frames in vitro, an immune response was not elicited in vivo after oral administration. Nonetheless, some effect was observed when the microcontainers were used to deliver oral boosters following a subcutaneous prime. This work indicates that oral vaccination with subunit vaccines has potential when combined with a parenteral prime and that oral delivery systems like microcontainers may be used to increase the potency of vaccines with low oral immunogenicity.

Evaluation of the effects of spray drying parameters for producing cubosome powder precursors.

Vaccines provide great benefit for global health, but are insufficiently distributed in developing countries due to high costs of manufacturing and limited storage stability. Spray drying formulations of peptide-based vaccines offer a promising strategy to reduce production costs and improve unrefrigerated storage stability. This design of experiments investigated how adjusting spray drying parameters (inlet temperature, atomization pressure, feed rate and aspiration rate) affects residual moisture and reconstitution properties of the powder product, and morphology and size of the rehydrated particles. An emulsion capable of forming cubosomes with the protein ovalbumin as model antigen was used as vaccine formulation and spray dried. The produced powders had low residual moisture content, independent of the spray drying parameters within the range investigated. Cryogenic transmission electron microscopy revealed that cubosomes were produced after rehydration regardless of the spray drying parameters. Furthermore, dynamic light scattering showed that the average diameter of the rehydrated particles consistently was close to 230 nm at any combination of spray drying parameters as long as the outlet temperature was kept below a threshold of 115 °C. The process thus appears to be robust allowing optimization to be focused on yield, dry particle engineering or outlet temperature considerations.

Spray dried cubosomes with ovalbumin and Quil-A as a nanoparticulate dry powder vaccine formulation.

Subunit vaccine formulations are often produced as liquid dispersions through complicated processes. It is desirable, however, to have simple, cheap and up-scalable methods to produce nanoparticulate subunit vaccines in powder form. Here, a simple single-step spray drying process for production of powder cubosome precursors with the model antigen ovalbumin (OVA) and the adjuvant Quil-A is presented. The cubosomes were characterized in vitro and evaluated in vivo by subcutaneous and oral administration for their potential as a vaccine formulation. Hydrated cubosomes had average particle size of 257 ±â€¯8 nm and zeta potential of -18.0 ±â€¯0.6 mV. The powder contained 10.6 ±â€¯0.7% w/w OVA prior to hydration, of which 65 ±â€¯1% was released within the first 20 min in 9.5 mM PBS at pH 7.3, with the remaining OVA gradually released over the following 24 h. Immunization with cubosomes resulted in significantly stronger antigen-specific serum IgG responses (p < 0.01), CD8+ T cell expansion (p < 0.0001) and target T cell killing compared to controls when given s.c., and was ineffective orally. This study shows that spray drying is a suitable method for producing nanoparticulate vaccine formulations in dry powder form.