Quality by Design as a Tool in the Optimisation of Nanoparticle Preparation-A Case Study of PLGA Nanoparticles.

Nanoparticles can be used as drug carriers in various applications (e.g., in pulmonary drug delivery and mucosal vaccination). For further investigations, such as drug release studies, as well as for cell and tissue targeting, particles with defined properties are needed. The purpose of the study was to show a multi-step systematic method utilising quality by design to ensure the quality of ovalbumin loaded polylactic-co-glycolic acid nanoparticles (OVA-PLGA-NP), which can be delivered to the lung, and to gain knowledge of the preparation method (double-emulsion solvent evaporation method) in an early development process. Within a definitive screening design, several process parameters (OVA, PLGA and stabiliser concentrations, stirring time and stirring speed of inner emulsion and stirring time and stirring speed of double emulsion) were varied to analyse their impact on resulting properties (z-average, PDI, loading efficiency and loading capacity). The results showed that the preparation of the inner emulsion mainly influenced the drug loading, while the parameters of the second emulsifying step controlled the size. Then a central composite response surface design was used to achieve a predictable OVA-PLGA-NP with an average particle size of 700 nm and high drug-loading. This also enabled the demonstration of curvature and interaction of the stabiliser and the PLGA concentration.

Preparation of Albatrellus ovinus ß-Glucan Microparticles with Dectin-1a Binding Properties.

Fungal ß-glucans are compounds with the potential to activate the innate immune system, in part through binding to the receptor dectin-1. In the present study, small-scale methods for preparing dectin-1a binding microparticles from Albatrellus ovinus alkali-soluble ß-glucans were investigated. Mechanical milling was time-consuming and yielded large particles with wide size distributions. Precipitation was more successful: the ß-glucan was dissolved in 1 M NaOH, diluted, and precipitated in 1:1 mol equiv HCl. This yielded particles in sizes ranging from 0.5-2 µm. The dectin-1a binding activity was determined using HEK-Blue reporter cells. The prepared particles were able to bind to dectin-1a to the same extent as baker's-yeast-derived ß-glucan particles. The precipitation method was convenient as a quick method for small-scale preparation of ß-glucan microparticle dispersions from mushroom ß-glucans.

Chitosan nanoparticles as antigen vehicles to induce effective tumor specific T cell responses.

Cancer vaccinations sensitize the immune system to recognize tumor-specific antigens de novo or boosting preexisting immune responses. Dendritic cells (DCs) are regarded as the most potent antigen presenting cells (APCs) for induction of (cancer) antigen-specific CD8+ T cell responses. Chitosan nanoparticles (CNPs) used as delivery vehicle have been shown to improve anti-tumor responses. This study aimed at exploring the potential of CNPs as antigen delivery system by assessing activation and expansion of antigen-specific CD8+ T cells by DCs and subsequent T cell-mediated lysis of pancreatic ductal adenocarcinoma (PDAC) cells. As model antigen the ovalbumin-derived peptide SIINFEKL was chosen. Using imaging cytometry, intracellular uptake of FITC-labelled CNPs of three different sizes and qualities (90/10, 90/20 and 90/50) was demonstrated in DCs and in pro- and anti-inflammatory macrophages to different extents. While larger particles (90/50) impaired survival of all APCs, small CNPs (90/10) were not toxic for DCs. Internalization of SIINFEKL-loaded but not empty 90/10-CNPs promoted a pro-inflammatory phenotype of DCs indicated by elevated expression of pro-inflammatory cytokines. Treatment of murine DC2.4 cells with SIINFEKL-loaded 90/10-CNPs led to a marked MHC-related presentation of SIINFEKL and enabled DC2.4 cells to potently activate SIINFEKL-specific CD8+ OT-1 T cells finally leading to effective lysis of the PDAC cell line Panc-OVA. Overall, our study supports the suitability of CNPs as antigen vehicle to induce potent anti-tumor immune responses by activation and expansion of tumor antigen-specific CD8+ T cells.