Martina Vermathen

While phospholipid bicelles have long been known as membrane models, the current work focuses on their potential application as topical drug delivery systems. For this, a systematic investigation of bicelles was performed to define the conditions required to maintain the small size and disk-shaped morphology that are favorable for skin penetrating properties. Bicelles consisting of 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC, C14) and 1,2-dihexanoyl-sn-glycero-3-PC (C6) were used as starting point. PCs bearing acyl chains with either 6 or 7 carbon atoms (C6, C7) could both be mixed with C14, 1,2-dipalmitoyl-sn-glycero-3-PC (C16) or 1-palmitoyl-2-oleoyl-sn-glycero-3-PC (C16/C18:1) to fulfill the desired conditions. Size, dynamic properties, and morphology were analyzed by DLS, 1H NMR diffusion ordered spectroscopy, and cryo-TEM. Stable bicelles were formed at low q-values (<2) and total phospholipid concentrations on average above 15 mM. The phase transition temperatures Tm of C14 and C16, determined by 1H NMR spectroscopy, were significantly reduced in bicelles compared to their one-component counterparts. The capability of C14/C16—C6/C7 bicelles for encapsulating two water-insoluble porphyrinic photosensitizers (PSs), m-THPP and chlorin e4 (Ce4) in their non-aggregated form, was demonstrated by 1H NMR. The phase transition profile of bicelles with reduced Tm was largely retained in the presence of encapsulated PSs, except for C14-C6 where Tm went back up. Initial skin penetration experiments demonstrated the ability of bicelles to deliver the PS into the superficial epidermal layer of porcine skin as visualized by fluorescence microscopy. The data underline the high potential of bicelles as topical delivery systems and provide guidelines for preparing bicelles with controlled properties important for predicting and optimizing their skin interaction.

Poor treatment adherence, often referred to as the “silent epidemic”, is a growing global issue that significantly contributes to preventable illness, premature death, and rising healthcare costs. Among compliance-enhancing strategies, controlled-release depots designed for intermediate treatment cycles represent a suitable approach, particularly in contexts where frequent dosing is impractical. Current long-acting injectable formulations, though, are often hindered by challenges in injectability, delayed onset, and complex manufacturing. Here, we present a thermoresponsive, in situ forming liposomal depot (TILD) designed to modulate the release of membrane-associated drugs following subcutaneous administration. Using buprenorphine as a model analgesic, we demonstrate that TILD responds to subcutaneous divalent cations with immediate surface-drug release and to body temperature with sustained diffusion through a fluidized bilayer. Molecular simulations guided the system design, and structural and colloidal characterizations validated its responsiveness to physiological cues. In vivo, TILD formed stable depots and maintained therapeutic drug levels for up to four days in both rats and Beagle dogs. Pharmacodynamics studies in rats confirmed the delivery of prolonged analgesia.

After the Swiss ban of hexahydrocannabinol (HHC) in March 2023, other semisynthetic dibenzopyran cannabinoids emerged on the Swiss gray market. Hexahydrocannabiphorol (HHCP) was the most prominent of them due to its potent cannabimimetic effects, as anecdotal reports from recreational users suggest. In October 2023, a class wide ban of dibenzopyran cannabinoids was introduced in Switzerland to prevent new similar substances from entering the drug market. Various vendors in online shops claim that HHCP is made from CBD, even though they possess different alkyl chain lengths. An HHCP sample was analyzed by gas chromatography coupled to mass spectrometry (GC‐MS), showing that a mixture of molecules with the same or a similar molecular mass as HHCP was present. Six different substances could be isolated from this sample using column chromatography. Four phenols ((9R)‐HHCP, iso‐HHCP, cis‐HHCP, and abn‐HHCP) and two ketones (possible intermediates to (9R)‐HHCP and abn‐HHCP) were identified by various nuclear magnetic resonance spectroscopy (NMR) techniques. (9S)‐HHCP was obtained in an impure fraction. In addition, a fraction was obtained that showed characteristic molecular and fragment ions consistent with bisalkylated products from the synthesis of similar compounds. The presence of abnormal cannabinoids (abn‐HHCP) and bisalkylated cannabinoids is a confirmation that this sample was produced purely synthetically as initially suspected, as these compounds have not been reported in Cannabis. Chiral derivatization of the phenols with Mosher acid chlorides showed that only iso‐HHCP was present as a scalemic mixture, indicating a good stereocontrol of this synthetic procedure.

Porphyrinic photosensitizers (PSs) and their nano-sized polymer-based carrier systems are required to exhibit low dark toxicity, avoid side effects, and ensure high in vivo tolerability. Yet, little is known about the intracellular fate of PSs during the dark incubation period and how it is affected by nanoparticles. In a systematic study, high-resolution magic angle spinning NMR spectroscopy combined with statistical analyses was used to study the metabolic profile of cultured HeLa cells treated with different concentrations of PS chlorin e4 (Ce4) alone or encapsulated in carrier systems. For the latter, either polyvinylpyrrolidone (PVP) or the micelle-forming polyethylene glycol (PEG)-polypropylene glycol triblock copolymer Kolliphor P188 (KP) were used. Diffusion-edited spectra indicated Ce4 membrane localization evidenced by Ce4 concentration-dependent chemical shift perturbation of the cellular phospholipid choline resonance. The effect was also visible in the presence of KP and PVP but less pronounced. The appearance of the PEG resonance in the cell spectra pointed towards cell internalization of KP, whereas no conclusion could be drawn for PVP that remained NMR-invisible. Multivariate statistical analyses of the cell spectra (PCA, PLS-DA, and oPLS) revealed a concentration-dependent metabolic response upon exposure to Ce4 that was attenuated by KP and even more by PVP. Significant Ce4-concentration-dependent alterations were mainly found for metabolites involved in the tricarboxylic acid cycle and the phosphatidylcholine metabolism. The data underline the important protective role of the polymeric carriers following cell internalization. Moreover, to our knowledge, for the first time, the current study allowed us to trace intracellular PS localization on an atomic level by NMR methods.

Porphyrinic photosensitizers (PSs) and their nano-sized polymer-based carrier systems are required to exhibit low dark toxicity, avoid side effects, and ensure high in vivo tolerability. Yet, little is known about the intracellular fate of PSs during the dark incubation period and how it is af-fected by nanoparticles. In a systematic study, high resolution magic angle spinning NMR spectroscopy combined with statistical analyses was used to study the metabolic profile of cultured HeLa cells treated with different concentrations of the PS chlorin e4 (Ce4) alone or encapsulated in carrier systems. For the latter, either polyvinylpyrrolidone (PVP) or the micelle forming polyethylene glycol (PEG)-polypropylene glycol triblock copolymer Kolliphor P188 (KP) were used. Diffusion edited spectra indicated Ce4 membrane localization evidenced by Ce4 concentration dependent chemical shift perturbation of the cellular phospholipid choline resonance. The effect was also visible in the presence of KP and PVP, but less pronounced. The appearance of the PEG resonance in the cell spectra pointed towards cell internalization of KP whereas no conclusion could be drawn for PVP that remained NMR-invisible. Multivariate statistical analyses of the cell spectra (PCA, PLS-DA and oPLS) revealed a concentration-dependent metabolic response upon exposure to Ce4 that was attenuated by KP and even more by PVP. Significant Ce4-concentration dependent alterations were mainly found for metabolites involved in the tricarboxylic acid cycle and the phosphatidylcholine metabolism. The data underline an important protective role of the polymeric carriers following cell internalization. Moreover, the current study allowed - to our knowledge for the first time - to trace the intracellular PS localization on an atomic level by NMR methods.

Phthalocyanines are ideal candidates as photosensitizers for photodynamic therapy (PDT) of cancer due to their favorable chemical and photophysical properties. However, their tendency to form aggregates in water reduces PDT efficacy and poses challenges in obtaining efficient forms of phthalocyanines for therapeutic applications. In the current work, polyvinylpyrrolidone (PVP) and micellar formulations were compared for encapsulating and monomerizing a water-soluble zinc phthalocyanine bearing four non-peripheral triethylene glycol chains (Pc1). ¹H NMR spectroscopy combined with UV-vis absorption and fluorescence spectroscopy revealed that Pc1 exists as a mixture of regioisomers in monomeric form in dimethyl sulfoxide but forms dimers in an aqueous buffer. PVP, polyethylene glycol castor oil (Kolliphor RH40), and three different triblock copolymers with varying proportions of polyethylene and polypropylene glycol units (termed P188, P84, and F127) were tested as micellar carriers for Pc1. ¹H NMR chemical shift analysis, diffusion-ordered spectroscopy, and 2D nuclear Overhauser enhancement spectroscopy was applied to monitor the encapsulation and localization of Pc1 at the polymer interface. Kolliphor RH40 and F127 micelles exhibited the highest affinity for encapsulating Pc1 in the micellar core and resulted in intense Pc1 fluorescence emission as well as efficient singlet oxygen formation along with PVP. Among the triblock copolymers, efficiency in binding and dimer dissolution decreased in the order F127 > P84 > P188. PVP was a strong binder for Pc1. However, Pc1 molecules are rather surface-attached and exist as monomer and dimer mixtures. The results demonstrate that NMR combined with optical spectroscopy offer powerful tools to assess parameters like drug binding, localization sites, and dynamic properties that play key roles in achieving high host-guest compatibility. With the corresponding adjustments, polymeric micelles can offer simple and easily accessible drug delivery systems optimizing phthalocyanines' properties as efficient photosensitizers.

Cisplatin (cisPt) is an important drug that is used against various cancers, including advanced lung cancer. However, drug resistance is still a major ongoing problem and its investigation is of paramount interest. Here, a high-resolution magic angle spinning (HR-MAS) NMR study is presented deciphering the metabolic profile of non-small cell lung cancer (NSCLC) cells and metabolic adaptations at different levels of induced cisPt-resistance, as well as in their de-induced counterparts (cells cultivated in absence of cisPt). In total, fifty-three metabolites were identified and quantified in the ¹H-HR-MAS NMR cell spectra. Metabolic adaptations to cisPt-resistance were detected, which correlated with the degree of resistance. Importantly, de-induced cell lines demonstrated similar metabolic adaptations as the corresponding cisPt-resistant cell lines. Metabolites predominantly changed in cisPt resistant cells and their de-induced counterparts include glutathione and taurine. Characteristic metabolic patterns for cisPt resistance may become relevant as biomarkers in cancer medicine.