No abstract

Impaired wound healing in people with diabetes has multifactorial causes, with insufficient neovascularization being one of the most important. Hypoxia-inducible factor-1 (HIF-1) plays a central role in the hypoxia-induced response by activating angiogenesis factors. As its activity is under precise regulatory control of prolyl-hydroxylase domain 2 (PHD-2), downregulation of PHD-2 by small interfering RNA (siRNA) could stabilize HIF-1α and, therefore, upregulate the expression of pro-angiogenic factors as well. Intracellular delivery of siRNA can be achieved with nanocarriers that must fulfill several requirements, including high stability, low toxicity, and high transfection efficiency. Here, we designed and compared the performance of layer-by-layer self-assembled siRNA-loaded gold nanoparticles with two different outer layers—Chitosan (AuNP@CS) and Poly L-arginine (AuNP@PLA). Although both formulations have exactly the same core, we find that a PLA outer layer improves the endosomal escape of siRNA, and therefore, transfection efficiency, after endocytic uptake in NIH-3T3 cells. Furthermore, we found that endosomal escape of AuNP@PLA could be improved further when cells were additionally treated with desloratadine, thus outperforming commercial reagents such as Lipofectamine® and jetPRIME®. AuNP@PLA in combination with desloratadine was proven to induce PHD-2 silencing in fibroblasts, allowing upregulation of pro-angiogenic pathways. This finding in an in vitro context constitutes a first step towards improving diabetic wound healing with siRNA therapy.

Near-infrared chemical imaging (NIR-CI) is an emerging tool for process monitoring because it combines the chemical selectivity of vibrational spectroscopy with spatial information. Whereas traditional near-infrared spectroscopy is an attractive technique for water content determination and solid-state investigation of lyophilized products, chemical imaging opens up possibilities for assessing the homogeneity of these critical quality attributes (CQAs) throughout the entire product. In this contribution, we aim to evaluate NIR-CI as a process analytical technology (PAT) tool for at-line inspection of continuously freeze-dried pharmaceutical unit doses based on spin freezing. The chemical images of freeze-dried mannitol samples were resolved via multivariate curve resolution, allowing us to visualize the distribution of mannitol solid forms throughout the entire cake. Second, a mannitol-sucrose formulation was lyophilized with variable drying times for inducing changes in water content. Analyzing the corresponding chemical images via principal component analysis, vial-to-vial variations as well as within-vial inhomogeneity in water content could be detected. Furthermore, a partial least-squares regression model was constructed for quantifying the water content in each pixel of the chemical images. It was hence concluded that NIR-CI is inherently a most promising PAT tool for continuously monitoring freeze-dried samples. Although some practicalities are still to be solved, this analytical technique could be applied in-line for CQA evaluation and for detecting the drying end point.

Maintaining chemical and physical stability of the product during freeze-drying is important but challenging. In addition, freeze-drying is typically associated with long process times. Therefore, mechanistic models have been developed to maximize drying efficiency without altering the chemical or physical stability of the product. Dried product mass transfer resistance ( R p ) is a critical input for these mechanistic models. Currently available techniques to determine R p only provide an estimation of the mean R p and do not allow measuring and determining essential local (i.e., intra-vial) R p differences. In this study, we present an analytical method, based on four-dimensional micro-computed tomography (4D- μ CT), which enables the possibility to determine intra-vial R p differences. Subsequently, these obtained R p values are used in a mechanistic model to predict the drying time distribution of a spin-frozen vial. Finally, this predicted primary drying time distribution is experimentally verified via thermal imaging during drying. It was further found during this study that 4D- μ CT uniquely allows measuring and determining other essential freeze-drying process parameters such as the moving direction(s) of the sublimation front and frozen product layer thickness, which allows gaining accurate process knowledge. To conclude, the study reveals that the variation in the end of primary drying time of a single vial could be predicted accurately using 4D- μ CT as similar results were found during the verification using thermal imaging.

Spin freeze-drying, as a part of a continuous freeze-drying technology, is associated with a much higher drying rate and a higher level of process control in comparison with batch freeze-drying. However, the impact of the spin freezing rate on the dried product layer characteristics is not well understood at present. This research focuses on the relation between spin-freezing and pore size, pore shape, dried product mass transfer resistance and solid state of the dried product layer. This was thoroughly investigated via high-resolution X-ray micro-computed tomography (µCT), scanning electron microscopy (SEM), thermal imaging and solid state X-ray diffraction (XRD). It was concluded that slow spin-freezing rates resulted in the formation of highly tortuous structures with a high dried-product mass-transfer resistance, while fast spin-freezing rates resulted in lamellar structures with a low tortuosity and low dried-product mass-transfer resistance.