Radical Stability vs. Temporal Resolution of EPR-Spectroscopy on Biological Samples

Spin-labeling active compounds is a convenient way to prepare them for EPR spectroscopy with minimal alteration of the target molecule. In this study we present the labeling reaction of dexamethasone (Dx) with either TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PCA (3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) with high yields. According to NMR data, both labels are attached at the primary hydroxy group of the steroid. In subsequent spin-stability measurements both compounds were applied onto HaCaT cells. When the signal of Dx-TEMPO decreased below the detection limit within 3 h, the signal of Dx-PCA remained stable for the same period of time

Drug distribution in nanostructured lipid particles

The targeted design of nanoparticles for efficient drug loading and defined release profiles is even after 25 years of research on lipid-based nanoparticles still no routine procedure. It requires detailed knowledge about the interaction of the drug with the lipid compounds and about its localisation and distribution in the nanoparticle. We present here an investigation on nano-sized lipid particles (NLP) composed of Gelucire and Witepsol as solid lipids, and Capryol as liquid lipid, loaded with Dexamethasone, a glucocorticoid used in topical treatment of inflammatory dermal diseases. The interactions of Dexamethasone, which was spin-labelled by 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (DxPCA), with its microenvironment are monitored by EPR spectroscopy at 94 GHz at low temperatures. The mobility of the spin-labelled drug was probed by X-band EPR at room temperature. In order to relate the magnetic and dynamic parameters deduced from EPR to the local environment of the spin probe in the NLP, investigations of DxPCA in the individual lipid compounds were carried out. The magnetic parameters reflecting the polarity of DxPCA’s environment as well as the parameters describing the mobility of the drug reveal that in the case of colloidal dispersions of the lipids and also the NLP DxPCA is attached to the surface of the nanoparticles. Although the lipophilic drug is almost exclusively associated with the NLP in aqueous solution, dilution experiments show, that it can be easily released from the nanoparticle

Nanocrystals for Improved Drug Delivery of Dexamethasone in Skin Investigated by EPR Spectroscopy

Nanocrystals represent an improvement over the traditional nanocarriers for dermal application, providing the advantages of 100% drug loading, a large surface area, increased adhesion, and the potential for hair follicle targeting. To investigate their advantage for drug delivery, compared to a base cream formulation, dexamethasone (Dx), a synthetic glucocorticoid frequently used for the treatment of inflammatory skin diseases, was covalently linked with the paramagnetic probe 3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) to DxPCA. To investigate the penetration efficiency between these two vehicles, electron paramagnetic resonance (EPR) spectroscopy was used, which allows the quantification of a spin-labeled drug in different skin layers and the monitoring of the drug release. The penetration behavior in excised healthy and barrier-disrupted porcine skin was monitored by EPR, and subsequently analyzed using a numerical diffusion model. As a result, diffusion constants and free energy values in the different layers of the skin were identified for both formulations. Dx-nanocrystals showed a significantly increased drug amount that penetrated into viable epidermis and dermis of intact (factor 3) and barrier-disrupted skin (factor 2.1) compared to the base cream formulation. Furthermore, the observed fast delivery of the spin-labeled drug into the skin (80% DxPCA within 30 min) and a successive release from the aggregate unit into the viable tissue makes these nanocrystals very attractive for clinical applications

Untersuchung von Nanoträgern als Arzneimittelabgabesysteme mittels Elektronenspin-resonanz-Spektroskopie

In this thesis, the potential of the electron paramagnetic resonance (EPR) spectroscopic technique is applied to questions at the interface of dermatology, pharmacology, chemistry, and nanotechnology. The skin as the outermost part of the body is the only organ to which drugs can be administered directly for the treatment of its diseases. However, the skin also acts as the barrier between the surrounding and the internal organs. Consequently, it protects the whole body against xenobiotics of the external environment. Thereby, the skin prevents penetration of administered drugs into its deeper layers or through it. Overcoming this intrinsic barrier is of great importance in dermatology for therapy success. Nanocarriers as drug delivery systems may be a solution for penetration through the skin barrier and are candidates for drug administration. However, efficient nanocarrier fabrication requires detailed knowledge about the interaction of drugs with these nanocarriers, in particular, with respect to the localization and distribution of the drugs within them. The aim of the study presented here is to unravel such interactions for two specific types of nanocarrier, dendritic core-multishell (CMS) nanoparticles and nanostructured lipid particles (NLP), which are both promising nanocarrier candidates. Since EPR has been established as a useful tool for probing the interaction of a spin probe with its micro-environment, it was chosen here as the method of choice for identifying these interactions. However, EPR requires paramagnetic species, while drugs typically are diamagnetic. Therefore, and in the sense of a pilot study, the small paramagnetic nitroxides 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO) and proxyl carboxylic acid (PCA) were used as model compounds for studying their interactions with their environment. The drug Dexamethasone (Dx), used in dermatology for treating inflammatory diseases, was spin labeled with PCA (DxPCA) and utilized for investigating the interactions in both, NLP and CMS nanoparticles. Using a set of continuous wave (cw) and pulsed EPR methods at X- and W-band enabled the precise extraction of the g- and A-matrices of the paramagnetic species as well as the measurement of their relaxation times. The g- and A-matrices represent specific probes for the polarity/proticity of the micro-environment. Additionally, the spin-lattice relaxation time yields complementary data on the spin probes’ micro-environment, which is independently corroborated by the mobility of the spin probes. This EPR-derived information is then used for the localization of the spin probes within the nanocarriers. Finally, comparing all obtained data enabled presenting an "association model" for the interaction between spin probes and nanocarriers. In short, in NLPs DxPCA is dispersed within the entire lipid matrix, PCA is not loaded, and TEMPO is enriched within the core. Likewise, the evidence obtained from EPR shows that DxPCA is localized at the interface between the hydrophobic and the hydrophilic shells of CMS nanoparticles. This knowledge gained may now be used in order to design more efficient nanocarriers or to select the right ones in dependence of the drug to be delivered.In der vorliegenden Arbeit wird ein breites methodisches Repertoire der Elektronenspin resonanz Spektroskopie (engl. electron paramagnetic resonance, EPR) auf Fragestellungen im Grenzbereich zwischen Dermatologie, Pharmakologie, Chemie und Nanotechnologie angewendet. Die Haut, als äußere Hülle unseres Körpers ist das einzige Organ, auf das Medikamente zur Behandlung von Krankheiten direkt aufgetragen werden können. Sie ist aber auch die Grenze zwischen der Umgebung und den inneren Organen, und schützt somit den Körper gegen Fremdstoffe aus der Umgebung. Daraus ergibt sich als Konsequenz, dass die Haut die Wanderung von Medikamenten in tiefere Schichten oder durch sie hindurch be-oder sogar verhindert. Deswegen ist es für die Dermatologie von großer Wichtigkeit diese natürliche Barriere zu überwinden. Die Verwendung von Botensystemen, z.B. in Form von Nanoträgern, bietet hierfür einen interessanten Lösungsansatz, da in ihnen Pharmaka über die Hautbarriere transportiert werden könnten. Die effiziente Verwendung von Nanoträgern erfordert jedoch ein detailliertes Wissen über die Wechselwirkung zwischen Träger und Pharmakon, insbesondere bezüglich der Verteilung und Lokalisation des Pharmakons im Träger. Das Ziel der hier vorgestellten Studie ist es, genau solche Wechselwirkungen für zwei bestimmte Klassen von Nanoträgern zu charakterisieren, die dendritischen Kern-Vielschalen Nanoträger (engl. core multi-shell, CMS) und die nanostrukturierten Lipidteilchen (engl. nanostructured lipid particles, NLP), beides vielversprechende Kandidaten für Nanoträger. Um die Wechselwirkungen zwischen Pharmakon und Nanoträger zu studieren, wurde hier die EPR-Spektroskopie ausgesucht, weil sie als Methode etabliert ist, um Wechselwirkungen zwischen einer Spinsonde und ihrer Mikroumgebung zu untersuchen. Allerdings müssen für die EPR-Spektroskopie paramagnetische Zentren vorliegen, während Pharmaka üblicherweise diamagnetisch sind. Deswegen, und im Sinne einer Pilotstudie wurden hier die kleinen paramagnetischen Nitroxide TEMPO und PCA als Modellverbindungen genutzt, um deren Wechselwirkungen mit ihrer Umgebung zu untersuchen. Dann wurde das Pharmakon Dexamethason (Dx), welches in der Dermatologie zur Behandlung von Entzündungen eingesetzt wird, mit PCA spinmarkiert (DxPCA) und genutzt, um Wechselwirkungen in NLP und CMS Nanopartikeln zu studieren. Der Einsatz verschiedener Dauerstrich (engl. continuous wave, cw) und gepulster EPR-Methoden im X- und W-Band ermöglichte die präzise Bestimmung der g- und A-Matrizen sowie der Relaxationszeiten der paramagnetischen Spezies. Die g- und A-Matrizen sind spezifische Sonden für Polarität/Protizität der Mikroumgebung. Die Spin-Gitter-Relaxationszeit liefert darüberhinaus weitere komplementäre Informationen über die Mikroumgebung, die wiederum in unabhängiger Art und Weise durch Informationen über die Mobilität der Spinsonden gestützt wird. Die so mit der EPR-Spektroskopie erhaltenen Informationen wurden dann genutzt, um die Spinsonden in den Nanoträgern zu lokalisieren. Darüberhinaus ermöglichte ein Vergleich aller Daten die Entwicklung eines “Assoziationsmodells” für die Wechselwirkung zwischen Spinsonde und Nanoträger. Kurz zusammengefasst, DxPCA ist in der gesamten Lipidmatrix der NLPs verteilt, PCA ist nicht geladen und TEMPO wird im Kern der NLP angereichert. Ganz analog zeigen die EPR-Daten, dass DxPCA an der Grenze zwischen der hydrophoben und außer hydrophilen Schalen der CMS-Nanoträger angereichert wird. Diese Erkenntnisse können nun in der Zukunft dazu verwendet werden, um Nanoträger effizienter zu machen oder in Abhängigkeit vom Pharmakon auszuwählen

Template-Mediated Formation of Colloidal Two-Dimensional Tin Telluride Nanosheets and the Role of the Ligands

We report the colloidal synthesis of 2D SnTe nanosheets through precursor hot injection in a nonpolar solvent. During the reaction, an important intermediateSn-templateis formed which defines the confined growth of SnTe. This “flake-like” structure gives the first evidence for the possible 2D morphology formation prior to the anion precursor injection (TOP-Te). Additionally, we explore the role of each ligand in the reaction process. Thus, we explain the formation and morphology evolution of 2D SnTe nanostructures from a mechanism perspective as well as the role of each ligand on the molecular scale. The interplay of ligands provides the necessary conditions for the realization of stable low-dimensional SnTe nanomaterials with tunable size and shape