Entwicklung neuer präklinischer Testsysteme für das Dünndarmkarzinom

Die Diagnose Dünndarmkarzinom wird meist von einer schlechten Prognose begleitet, da bereits etwa 59 % der Patienten Metastasen im Körper gebildet haben. Die Behandlung erfolgt in diesen Fällen meist nur palliativ, durch den Einsatz von chirurgischer Resektion in Kombination mit einer Chemotherapie. Zur Erhöhung der Heilungschancen des Patienten, werden dringend neue Therapieformen benötigt. Eine Möglichkeit ist der Einsatz der photodynamischen Therapie bei der ein photoaktivierbarer Arzneistoff (Photosensibilisator) in Kombination mit Licht einer spezifischen Wellenlänge zur Behandlung von Krebszellen eingesetzt wird. Photosensibilisatoren haben im allgemeinen meist stark hydrophobe Eigenschaften, die zu einer schlechten Bioverfügbarkeit führen. Zum Transport des Photosensibilisators zum Wirkort wurden daher unterschiedliche nanopartikuläre Arzneistoffträgersysteme auf Basis von Polymilch-co-glykolsäure (PLGA) und Liposomen entwickelt. Diese wurden zunächst mit standardisierten In-vitro-Testsystemen unter Verwendung von 2D-Monolayer-Zellkulturen und 3D-Tumorsphäoriden auf ihre Wirksamkeit und auf unerwünschte toxische Effekte untersucht. Zudem ist die Akkumulation des Photosensibilisators in den Zellen ein wichtiger Faktor, da diese ausschlaggebend für eine effektive anti-tumorale Wirksamkeit ist. Um diese zu quantifizieren, wurde eine analytische HPLC-Methode entwickelt und nach den Angaben der ICH-Guideline validiert. Im nächsten Schritt lag der Fokus der Untersuchungen darauf, die mukoadhäsiven und mukopermeablen Eigenschaften der zu entwickelnden Arzneistoffträgersysteme zu testen. Diese sind nötig, um ein gezieltes Tumor-Targeting zu erreichen. Zur Untersuchung dieser spezifischen Eigenschaften wurden neue präklinische Testmodelle entwickelt: (i) Ein mikrofluidischer Mukus-Chip zur Untersuchung der Mukopermeation; (ii) ein mikrofluidisches Darmtumor-Modell zur Erforschung der mukoadhäsiven Eigenschaften in einem peristaltischen Fluss und (iii) ein vaskularisiertes Darmtumor-Modell zur Überprüfung der Arzneistoff-Akkumulation und der biologischen Wirksamkeit. Anhand dieser Testsysteme war es möglich, wirksame Photosensibilisatoren und deren nanopartikuläre Formulierungen zu identifizieren, die ein hohes Potential zur Behandlung von Dünndarmkarzinomen aufweisen. Zudem stellen die entwickelten spezifischen präklinischen Testmodelle ein hilfreiches Werkzeug zur Untersuchung der Eigenschaften von Nanopartikeln in dynamischen fluidischen Systemen dar.The diagnosis of small intestine carcinoma is usually followed by a poor prognosis as about 59 % of patients have already developed metastases in the body. In these cases, treatment is usually palliative, using surgical resection in combination with chemotherapy. To increase the patient's chances of recovery, new therapies are needed. One option is the use of photodynamic therapy in which a photoactivatable drug (photosensitizer) is used in combination with light of a specific wavelength to treat cancer cells. In general, photosensitizers have highly hydrophobic properties which lead to poor bioavailability. Therefore different nanoparticulate drug delivery systems based on poly(lactic-co-glycolic acid) (PLGA) and liposomes were developed to transport the photosensitizer to the site of action. The efficacy and undesired toxic effects of these were first investigated with standardized in vitro test systems using 2D monolayer cell cultures and 3D tumor spheroids. In addition, the accumulation of the photosensitizer in the cells is an important factor, as it is decisive for effective anti-tumoral efficacy. To quantify this an analytical HPLC method was developed and validated according to the ICH guideline. In the next step the focus of the investigations was on testing the mucoadhesive and mucopermeable properties of the drug delivery systems to be developed. These are necessary to achieve targeted tumor targeting. New preclinical test models were developed to investigate these specific properties: (i) a microfluidic mucus chip to study mucopermeation; (ii) a microfluidic intestinal tumor model to study mucoadhesive properties in a peristaltic flow; and (iii) a vascularized intestinal tumor model to test drug accumulation and biological efficacy. With these test systems it was possible to identify effective photosensitizers and their nanoparticulate formulations with a high potential for the treatment of small intestine carcinoma. In addition, the specific preclinical test models developed represent a useful tool for investigating the properties of nanoparticles in dynamic fluidic systems

Digital research data: from analysis of existing standards to a scientific foundation for a modular metadata schema in nanosafety

Background: Assessing the safety of engineered nanomaterials (ENMs) is an interdisciplinary and complex process producing huge amounts of information and data. To make such data and metadata reusable for researchers, manufacturers, and regulatory authorities, there is an urgent need to record and provide this information in a structured, harmonized, and digitized way. Results: This study aimed to identify appropriate description standards and quality criteria for the special use in nanosafety. There are many existing standards and guidelines designed for collecting data and metadata, ranging from regulatory guidelines to specific databases. Most of them are incomplete or not specifically designed for ENM research. However, by merging the content of several existing standards and guidelines, a basic catalogue of descriptive information and quality criteria was generated. In an iterative process, our interdisciplinary team identified deficits and added missing information into a comprehensive schema. Subsequently, this overview was externally evaluated by a panel of experts during a workshop. This whole process resulted in a minimum information table (MIT), specifying necessary minimum information to be provided along with experimental results on effects of ENMs in the biological context in a flexible and modular manner. The MIT is divided into six modules: general information, material information, biological model information, exposure information, endpoint read out information and analysis and statistics. These modules are further partitioned into module subdivisions serving to include more detailed information. A comparison with existing ontologies, which also aim to electronically collect data and metadata on nanosafety studies, showed that the newly developed MIT exhibits a higher level of detail compared to those existing schemas, making it more usable to prevent gaps in the communication of information. Conclusion: Implementing the requirements of the MIT into e.g., electronic lab notebooks (ELNs) would make the collection of all necessary data and metadata a daily routine and thereby would improve the reproducibility and reusability of experiments. Furthermore, this approach is particularly beneficial regarding the rapidly expanding developments and applications of novel non-animal alternative testing methods

A novel microfluidic mucus-chip for studying the permeation of compounds over the mucus barrier

A major challenge of drug discovery is to overcome the mucus barrier, which is lined the surface of the gastrointestinal tract. The ability to overcome this barrier must be tested in preclinical in vitro models, prior to in vivo studies. Therefore, a microfluidic chip was developed to examine the possibility of compounds to overcome a mucus barrier. Native porcine intestinal mucus was used as a mucus model. The mucus was loaded inside the chip comprising of a microporous membrane and a fluidic compartment. To validate the system, caffeine as a hydrophilic positive compound, fluorescein isothiocyanate–dextran (FITC-dextran) as high molecular negative compound and diclofenac sodium as poorly permeating compound were tested. Test samples were applied on the surface of the mucus and samples were collected every 30 min from the fluidic compartment underneath the membrane. The permeation was measured over 3 h and the permeated compound was detected by high-pressure liquid chromatography (HPLC) and a microplate fluorescence reader. The results show that caffeine and diclofenac sodium are permeating over the mucus layer proportionally to the time. FITC-dextran, chosen as non-permeating compound, displays equivalently no permeation. The validation provides the functionality of the developed mucus-chip system

A HET-CAM based vascularized intestine tumor model as a screening platform for nano-formulated photosensitizers

The development of new tumor models for anticancer drug screening is a challenge for preclinical research. Conventional cell-based in vitro models such as 2D monolayer cell cultures or 3D spheroids allow an initial assessment of the efficacy of drugs but they have a limited prediction to the in vivo effectiveness. In contrast, in vivo animal models capture the complexity of systemic distribution, accumulation, and degradation of drugs, but visualization of the individual steps is challenging and extracting quantitative data is usually very difficult. Furthermore, there are a variety of ethical concerns related to animal tests. In accordance with the 3Rs principles of Replacement, Reduction and Refinement, alternative test systems should therefore be developed and applied in preclinical research. The Hen's egg test on chorioallantoic membrane (HET-CAM) model provides the generation of vascularized tumor spheroids and therefore, is an ideal test platform which can be used as an intermediate step between in vitro analysis and preclinical evaluation in vivo. We developed a HET-CAM based intestine tumor model to investigate the accumulation and efficacy of nano-formulated photosensitizers. Irradiation is necessary to activate the phototoxic effect. Due to the good accessibility of the vascularized tumor on the CAM, we have developed a laser irradiation setup to simulate an in vivo endoscopic irradiation. The study presents quantitative as well as qualitative data on the accumulation and efficacy of the nano-formulated photosensitizers in a vascularized intestine tumor model

How Structured Metadata Acquisition Contributes to the Reproducibility of Nanosafety Studies: Evaluation by a Round-Robin Test

It has been widely recognized that nanosafety studies are limited in reproducibility, caused by missing or inadequate information and data gaps. Reliable and comprehensive studies should be performed supported by standards or guidelines, which need to be harmonized and usable for the multidisciplinary field of nanosafety research. The previously described minimal information table (MIT), based on existing standards or guidelines, represents one approach towards harmonization. Here, we demonstrate the applicability and advantages of the MIT by a round-robin test. Its modular structure enables describing individual studies comprehensively by a combination of various relevant aspects. Three laboratories conducted a WST-1 cell viability assay using A549 cells to analyze the effects of the reference nanomaterials NM101 and NM110 according to predefined (S)OPs. The MIT contains relevant and defined descriptive information and quality criteria and thus supported the implementation of the round-robin test from planning, investigation to analysis and data interpretation. As a result, we could identify sources of variability and justify deviating results attributed to differences in specific procedures. Consequently, the use of the MIT contributes to the acquisition of reliable and comprehensive datasets and therefore improves the significance and reusability of nanosafety studies

How Structured Metadata Acquisition Contributes to the Reproducibility of Nanosafety Studies: Evaluation by a Round-Robin Test

It has been widely recognized that nanosafety studies are limited in reproducibility, caused by missing or inadequate information and data gaps. Reliable and comprehensive studies should be performed supported by standards or guidelines, which need to be harmonized and usable for the multidisciplinary field of nanosafety research. The previously described minimal information table (MIT), based on existing standards or guidelines, represents one approach towards harmonization. Here, we demonstrate the applicability and advantages of the MIT by a round-robin test. Its modular structure enables describing individual studies comprehensively by a combination of various relevant aspects. Three laboratories conducted a WST-1 cell viability assay using A549 cells to analyze the effects of the reference nanomaterials NM101 and NM110 according to predefined (S)OPs. The MIT contains relevant and defined descriptive information and quality criteria and thus supported the implementation of the round-robin test from planning, investigation to analysis and data interpretation. As a result, we could identify sources of variability and justify deviating results attributed to differences in specific procedures. Consequently, the use of the MIT contributes to the acquisition of reliable and comprehensive datasets and therefore improves the significance and reusability of nanosafety studie

Digital research data: from analysis of existing standards to a scientific foundation for a modular metadata schema in nanosafety

BACKGROUND: Assessing the safety of engineered nanomaterials (ENMs) is an interdisciplinary and complex process producing huge amounts of information and data. To make such data and metadata reusable for researchers, manufacturers, and regulatory authorities, there is an urgent need to record and provide this information in a structured, harmonized, and digitized way. RESULTS: This study aimed to identify appropriate description standards and quality criteria for the special use in nanosafety. There are many existing standards and guidelines designed for collecting data and metadata, ranging from regulatory guidelines to specific databases. Most of them are incomplete or not specifically designed for ENM research. However, by merging the content of several existing standards and guidelines, a basic catalogue of descriptive information and quality criteria was generated. In an iterative process, our interdisciplinary team identified deficits and added missing information into a comprehensive schema. Subsequently, this overview was externally evaluated by a panel of experts during a workshop. This whole process resulted in a minimum information table (MIT), specifying necessary minimum information to be provided along with experimental results on effects of ENMs in the biological context in a flexible and modular manner. The MIT is divided into six modules: general information, material information, biological model information, exposure information, endpoint read out information and analysis and statistics. These modules are further partitioned into module subdivisions serving to include more detailed information. A comparison with existing ontologies, which also aim to electronically collect data and metadata on nanosafety studies, showed that the newly developed MIT exhibits a higher level of detail compared to those existing schemas, making it more usable to prevent gaps in the communication of information. CONCLUSION: Implementing the requirements of the MIT into e.g., electronic lab notebooks (ELNs) would make the collection of all necessary data and metadata a daily routine and thereby would improve the reproducibility and reusability of experiments. Furthermore, this approach is particularly beneficial regarding the rapidly expanding developments and applications of novel non-animal alternative testing methods