In vitro and in ovo photodynamic efficacy of nebulized curcumin-loaded tetraether lipid liposomes prepared by DC as stable drug delivery system.

Lung cancer is one of the most common causes of high mortality worldwide. Current treatment strategies, e.g., surgery, radiotherapy, chemotherapy, and immunotherapy, insufficiently affect the overall outcome. In this study, we used curcumin as a natural photosensitizer in photodynamic therapy and encapsulated it in liposomes consisting of stabilizing tetraether lipids aiming for a pulmonary drug delivery system against lung cancer. The liposomes with either hydrolyzed glycerol-dialkyl-glycerol tetraether (hGDGT) in different ratios or hydrolyzed glycerol-dialkyl-nonitol tetraether (hGDNT) were prepared by dual centrifugation (DC), an innovative method for liposome preparation. The liposomes' physicochemical characteristics before and after nebulization and other nebulization characteristics confirmed their suitability. Morphological characterization using atomic force and transmission electron microscopy showed proper vesicular structures indicative of liposomes. Qualitative and quantitative uptake of the curcumin-loaded liposomes in lung adenocarcinoma (A549) cells was visualized and proven. Phototoxic effects of the liposomes were detected on A549 cells, showing decreased cell viability. The generation of reactive oxygen species required for PDT and disruption of mitochondrial membrane potential were confirmed. Moreover, the chorioallantoic membrane (CAM) model was used to further evaluate biocompatibility and photodynamic efficacy in a 3D cell culture context. Photodynamic efficacy was assessed by PET/CT after nebulization of the liposomes onto the xenografted tumors on the CAM with subsequent irradiation. The physicochemical properties and the efficacy of tetraether lipid liposomes encapsulating curcumin, especially liposomes containing hGDNT, in 2D and 3D cell cultures seem promising for future PDT usage against lung cancer.

Modern Photodynamic Glioblastoma Therapy Using Curcumin- or Parietin-Loaded Lipid Nanoparticles in a CAM Model Study.

Natural photosensitizers, such as curcumin or parietin, play a vital role in photodynamic therapy (PDT), causing a light-mediated reaction that kills cancer cells. PDT is a promising treatment option for glioblastoma, especially when combined with nanoscale drug delivery systems. The curcumin- or parietin-loaded lipid nanoparticles were prepared via dual asymmetric centrifugation and subsequently characterized through physicochemical analyses including dynamic light scattering, laser Doppler velocimetry, and atomic force microscopy. The combination of PDT and lipid nanoparticles has been evaluated in vitro regarding uptake, safety, and efficacy. The extensive and well-vascularized chorioallantois membrane (CAM) of fertilized hen's eggs offers an optimal platform for three-dimensional cell culture, which has been used in this study to evaluate the photodynamic efficacy of lipid nanoparticles against glioblastoma cells. In contrast to other animal models, the CAM model lacks a mature immune system in an early stage, facilitating the growth of xenografts without rejection. Treatment of xenografted U87 glioblastoma cells on CAM was performed to assess the effects on tumor viability, growth, and angiogenesis. The xenografts and the surrounding blood vessels were targeted through topical application, and the effects of photodynamic therapy have been confirmed microscopically and via positron emission tomography and X-ray computed tomography. Finally, the excised xenografts embedded in the CAM were analyzed histologically by hematoxylin and eosin and KI67 staining.

How to Xenograft Cancer Cells on the Chorioallantoic Membrane of a Fertilized Hen's Egg and Its Visualization by PET/CT and MRI.

The chorioallantoic membrane (CAM) of fertilized hen's eggs represents a unique and alternative model for cancer research. The CAM model provides an optimal platform for xenografting cancer cell lines and studying essential key factors. Tumor size and growth as well as angiogenesis can be investigated to evaluate the response of therapies and strategies against cancer. Preclinical imaging represented by magnetic resonance imaging and positron emission tomography/computed tomography can generate detailed anatomical and functional information and reveal excellent metabolic sensitivity. In the following, a guideline is introduced in order to find a simplified entrance to the CAM model in combination with modern preclinical imaging techniques. Finally, the presented procedures are additionally completed by histological studies in the form of hematoxylin and eosin as well as immunohistochemical staining.

Liposome Photosensitizer Formulations for Effective Cancer Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising non-invasive strategy in the fight against that which circumvents the systemic toxic effects of chemotherapeutics. It relies on photosensitizers (PSs), which are photoactivated by light irradiation and interaction with molecular oxygen. This generates highly reactive oxygen species (such as 1O2, H2O2, O2, ·OH), which kill cancer cells by necrosis or apoptosis. Despite the promising effects of PDT in cancer treatment, it still suffers from several shortcomings, such as poor biodistribution of hydrophobic PSs, low cellular uptake, and low efficacy in treating bulky or deep tumors. Hence, various nanoplatforms have been developed to increase PDT treatment effectiveness and minimize off-target adverse effects. Liposomes showed great potential in accommodating different PSs, chemotherapeutic drugs, and other therapeutically active molecules. Here, we review the state-of-the-art in encapsulating PSs alone or combined with other chemotherapeutic drugs into liposomes for effective tumor PDT.

Effects of different air change rates on cleanroom 'in operation' status.

The objective of this experimental study is to analyze non-viable and viable particle loads in a pharmaceutical cleanroom under 'in operation' conditions using different air change rates (ACRs). Regulatory guidelines give limit values for particles/m3 and colony forming units (CFUs)/m3. A widely used ACR is 20 h-1 as this value is recommended by the Food and Drug Administration (FDA) in its guidance for industry on sterile drug products. However, this value may be too high, resulting in increased costs for energy. A typical pharmaceutical cleanroom was used for this study, and operations were simulated with a process unit and two operators in the room. The experiments were conducted twice with four different ACRs and four different types of operator garments, resulting in 32 trials in total. Particle load and CFUs were measured by calibrated particle counters and microbial air samplers. The results give evidence that an ACR of 20 h-1 is not required. ACR 10 h-1 is sufficient without compromising the demanded air quality. Furthermore, it was found that regulatory agencies should reevaluate the expected limits as these currently give a high buffer between the required and actual values, which potentially cover up problems in aseptic manufacturing.

In Ovo Testing Method for Inhalants on a Chorio-Allantoic Membrane.

Solid tumors and metastasis rely on angiogenesis for sufficient supply as they grow, making antiangiogenic treatment a promising option in the combat of cancer. Testing of inhalants on the chorio-allantoic membrane offers a simple but precise method to assess the impact on angiogenesis. The in ovo testing method can be used to directly determine the effect of inhaled formulations solely or in the context of photodynamic therapy. In this study curcumin liposomes served as a model for testing of pulmonary application and revealed an excellent antiangiogenetic effect. This efficacy of a model inhalant illustrates the suitability of the method.

Atomic force microscopy and analytical ultracentrifugation for probing nanomaterial protein interactions.

Upon contact with the human body, nanomaterials are known to interact with the physiological surroundings, especially with proteins. In this context, we explored analytical methods to provide biologically relevant information, in particular for manufactured nanomaterials as produced by the chemical industry. For this purpose, we selected two batches of SiO(2) nanoparticles as well as four batches of CeO(2) nanoparticles, each of comparably high chemical purity and similar physicochemical properties. Adsorption of serum proteins and bovine serum albumin (BSA) was quantified by SDS-PAGE in combination with densitometry and further investigated by atomic force microscopy (AFM) and analytical ultracentrifugation (AUC). The protein adsorption to SiO(2) nanoparticles was below the limit of detection, regardless of adjusting pH or osmolality to physiological conditions. In contrast, the four CeO(2) nanomaterials could be classified in two groups according to half-maximal protein adsorption. Measuring the work of adhesion and indention by AFM for the BSA-binding CeO(2) nanomaterials revealed the same classification, pointing to alterations in shape of the adsorbed protein. The same trend was also reflected in the agglomeration behavior/dispersibility of the four CeO(2) nanomaterials as revealed by AUC. We conclude that even small differences in physicochemical particle properties may nevertheless lead to differences in protein adsorption, possibly implicating a different disposition and other biological responses in the human body. Advanced analytical methods such as AFM and AUC may provide valuable additional information in this context.