Comparing PVP and Polymeric Micellar Formulations of a PEGylated Photosensitizing Phthalocyanine by NMR and Optical Techniques.

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.

Probing the Interactions of Porphyrins with Macromolecules Using NMR Spectroscopy Techniques.

Porphyrinic compounds are widespread in nature and play key roles in biological processes such as oxygen transport in blood, enzymatic redox reactions or photosynthesis. In addition, both naturally derived as well as synthetic porphyrinic compounds are extensively explored for biomedical and technical applications such as photodynamic therapy (PDT) or photovoltaic systems, respectively. Their unique electronic structures and photophysical properties make this class of compounds so interesting for the multiple functions encountered. It is therefore not surprising that optical methods are typically the prevalent analytical tool applied in characterization and processes involving porphyrinic compounds. However, a wealth of complementary information can be obtained from NMR spectroscopic techniques. Based on the advantage of providing structural and dynamic information with atomic resolution simultaneously, NMR spectroscopy is a powerful method for studying molecular interactions between porphyrinic compounds and macromolecules. Such interactions are of special interest in medical applications of porphyrinic photosensitizers that are mostly combined with macromolecular carrier systems. The macromolecular surrounding typically stabilizes the encapsulated drug and may also modify its physical properties. Moreover, the interaction with macromolecular physiological components needs to be explored to understand and control mechanisms of action and therapeutic efficacy. This review focuses on such non-covalent interactions of porphyrinic drugs with synthetic polymers as well as with biomolecules such as phospholipids or proteins. A brief introduction into various NMR spectroscopic techniques is given including chemical shift perturbation methods, NOE enhancement spectroscopy, relaxation time measurements and diffusion-ordered spectroscopy. How these NMR tools are used to address porphyrin-macromolecule interactions with respect to their function in biomedical applications is the central point of the current review.

1H HR-MAS NMR Based Metabolic Profiling of Lung Cancer Cells with Induced and De-Induced Cisplatin Resistance to Reveal Metabolic Resistance Adaptations.

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.

Carbon source regulates polysaccharide capsule biosynthesis in Streptococcus pneumoniae.

The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon source-dependent regulation of distinct genes of WT strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose 1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is <10% that of glucose. Our findings on the effects of carbon sources on CPS production in different S. pneumoniae serotypes may contribute to a better understanding of pneumococcal diseases and could inform future therapeutic approaches.

Hepatitis C virus infection triggers a tumor-like glutamine metabolism.

Chronic infection with hepatitis C virus (HCV) is one of the main causes of hepatocellular carcinoma. However, the molecular mechanisms linking the infection to cancer development remain poorly understood. Here we used HCV-infected cells and liver biopsies to study how HCV modulates the glutaminolysis pathway, which is known to play an important role in cellular energetics, stress defense, and neoplastic transformation. Transcript levels of glutaminolytic factors were quantified in Huh7.5 cells or primary human hepatocytes infected with the Japanese fulminant hepatitis 1 HCV strain as well as in biopsies of chronic HCV patients. Nutrient deprivation, biochemical analysis, and metabolite quantification were performed with HCV-infected Huh7.5 cells. Furthermore, short hairpin RNA vectors and small molecule inhibitors were used to investigate the dependence of HCV replication on metabolic changes. We show that HCV modulates the transcript levels of key enzymes of glutamine metabolism in vitro and in liver biopsies of chronic HCV patients. Consistently, HCV infection increases glutamine use and dependence. We finally show that inhibiting glutamine metabolism attenuates HCV infection and the oxidative stress associated with HCV infection. CONCLUSION: Our data suggest that HCV establishes glutamine dependence, which is required for viral replication, and, importantly, that glutamine addiction is a hallmark of tumor cells. While HCV induces glutaminolysis to create an environment favorable for viral replication, it predisposes the cell to transformation. Glutaminolytic enzymes may be interesting therapeutic targets for prevention of hepatocarcinogenesis in chronic hepatitis C. (Hepatology 2017;65:789-803).

Metabolic profiling of listeria rhombencephalitis in small ruminants by 1 H high-resolution magic angle spinning NMR spectroscopy.

Listeria rhombencephalitis is caused by infection with Listeria monocytogenes and is associated with a high mortality rate in humans and ruminants. Little is known about the metabolic changes associated with neurolisteriosis in particular and infectious central nervous system (CNS) diseases in general. The purpose of our study was to investigate the metabolic changes associated with listeria rhombencephalitis in small ruminants (goats and sheep) as a model for inflammatory CNS disease by 1 H high-resolution magic angle spinning nuclear magnetic resonance (1 H HR-MAS NMR) spectroscopy of brain biopsies obtained from the brainstem and thalamus. Statistical analysis revealed distinct differences in the metabolic profile of brainstem biopsies, the primary location of listeria rhombencephalitis with moderate or severe inflammatory changes. N-Acetylaspartate (NAA), N-acetylaspartylglutamate, choline, myo-inositol and scyllo-inositol were decreased, and glycine, phosphocholine, taurine and lactate were increased, in the diseased group (n = 13) in comparison with the control group (n = 12). In the thalamus, which showed no or only mild inflammatory changes in the majority of animals, no statistically significant metabolic changes were observed. However, trends for metabolic alterations were partly the same as those found in the brainstem, including NAA, choline and lactate. This may be an indicator of metabolic changes occurring in the early stages of the disease. Therefore, further research with a larger number of animals is needed to evaluate the presence of subtle metabolic changes associated with mild inflammatory changes in the thalamus. In conclusion, 1 H HR-MAS NMR investigation of listeria rhombencephalitis identified brain metabolite changes, offering new insights into the disease pathophysiology.

How Does the Encapsulation of Porphyrinic Photosensitizers into Polymer Matrices Affect Their Self-Association and Dynamic Properties?

Photodynamic therapy (PDT) with porphyrinic photosensitizers largely relies on efficient drug formulations to prevent porphyrin aggregation and to enhance water solubility and stability in physiologic environments. In this study, we compare two polymeric carrier systems, polyvinylpyrrolidone (PVP) and block copolymer micelles (BCMs) formed by the poloxamer Kolliphor P188 (KP), for their encapsulation efficiencies of porphyrin (xPP) and chlorin e6 (xCE) derivatives. Monomerization, loading efficiency, and dynamic properties were examined by 1 H NMR spectroscopy chemical shift titration, DOSY, and T2 relaxation time measurements. Binding affinity was determined by UV/Vis spectroscopy. Both PVP and KP-BCMs were well suited to disaggregate and encapsulate amphiphilic xCE, whereas they were less efficient for the xPP compounds. PVP exhibited higher monomerization efficiency than KP-BCMs. Significant differences were found in the dynamic behavior of the carriers. PVP formed rather stable complexes with the porphyrinic compounds, whereas a dynamic equilibrium between free and bound porphyrins was found to exist in the presence of KP-BCMs. This may have a considerable impact on the pharmacokinetic properties of the corresponding delivery systems.

Longitudinal investigation of the metabolome of 3D aggregating brain cell cultures at different maturation stages by 1H HR-MAS NMR.

The aim of the present study was to establish the developmental profile of metabolic changes of 3D aggregating brain cell cultures by 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. The histotypic 3D brain aggregate, containing all brain cell types, is an excellent model for mechanistic studies including OMICS analysis; however, their metabolic profile has not been yet fully investigated. Chemometric analysis revealed a clear separation of samples from the different maturation time points. Metabolite concentration evolutions could be followed and revealed strong and various metabolic alterations. The strong metabolite evolution emphasizes the brain modeling complexity during maturation, possibly reflecting physiological processes of brain tissue development. The small observed intra- and inter-experimental variabilities show the robustness of the combination of 1H-HR-MAS NMR and 3D brain aggregates, making it useful to investigate mechanisms of toxicity that will ultimately contribute to improve predictive neurotoxicology. Graphical Abstract ᅟ.

Metabolic Profiling of Cells in Response to Drug Treatment using 1H High-resolution Magic Angle Spinning (HR-MAS) NMR Spectroscopy.

High-resolution magic angle spinning (HR-MAS) is an NMR technique that provides access to well resolved liquid-like 1H NMR spectra of semi-solid samples. Therefore, 1H HR-MAS NMR spectroscopy has become an important tool for the direct analysis of biological samples such as tissues and cells in a mostly non-destructive way. Here, we focus on the application of HR-MAS NMR combined with multivariate statistical methods used for metabolic profiling of cells and in particular for the study of cellular metabolic responses to drug exposure. The principles of HR-MAS and the metabolomic approach are briefly described. As an example, a study on the metabolic response of different cell types towards treatment with a highly cytotoxic hexacationic ruthenium metallaprism as potential anti-cancer drug is presented. Specific metabolites and metabolic pathways are suggested to be associated with the cellular response. The study demonstrates the potential of HR-MAS metabolomics applied to cells for addressing the intracellular processes involved in the treatment with organometallic drugs.

Direct determination of phosphate sugars in biological material by (1)H high-resolution magic-angle-spinning NMR spectroscopy.

The study aim was to unambiguously assign nucleotide sugars, mainly UDP-X that are known to be important in glycosylation processes as sugar donors, and glucose-phosphates that are important intermediate metabolites for storage and transfer of energy directly in spectra of intact cells, as well as in skeletal muscle biopsies by (1)H high-resolution magic-angle-spinning (HR-MAS) NMR. The results demonstrate that sugar phosphates can be determined quickly and non-destructively in cells and biopsies by HR-MAS, which may prove valuable considering the importance of phosphate sugars in cell metabolism for nucleic acid synthesis. As proof of principle, an example of phosphate-sugar reaction and degradation kinetics after unfreezing the sample is shown for a cardiac muscle, suggesting the possibility to follow by HR-MAS NMR some metabolic pathways. Graphical abstract Glucose-phosphate sugars (Glc-1P and Glc-6P) detected in muscle by 1H HR-MAS NMR.

Isolation and characterization of synthesis intermediates and side products in hexahydrocannabiphorol.

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.

Fluorescence lifetime imaging and phasor analysis of intracellular porphyrinic photosensitizers applied with different polymeric formulations.

The fluorescence lifetime of a porphyrinic photosensitizer (PS) is an important parameter to assess the aggregation state of the PS even in complex biological environments. Aggregation-induced quenching of the PS can significantly reduce the yield of singlet oxygen generation and thus its efficiency as a medical drug in photodynamic therapy (PDT) of diseased tissues. Hydrophobicity and the tendency to form aggregates pose challenges on the development of efficient PSs and often require carrier systems. A systematic study was performed to probe the impact of PS structure and encapsulation into polymeric carriers on the fluorescence lifetime in solution and in the intracellular environment. Five different porphyrinic PSs including chlorin e6 (Ce6) derivatives and tetrakis(m-hydroxyphenyl)-porphyrin and -chlorin were studied in free form and combined with polyvinylpyrrolidone (PVP) or micelles composed of triblock-copolymers or Cremophor. Following incubation of HeLa cells with these systems, fluorescence lifetime imaging combined with phasor analysis and image segmentation was applied to study the lifetime distribution in the intracellular surrounding. The data suggest that for free PSs, the structure-dependent cell uptake pathways determine their state and emission lifetimes. PS localization in the plasma membrane yielded mostly monomers with long fluorescence lifetimes whereas the endocytic pathway with subsequent lysosomal deposition adds a short-lived component for hydrophilic anionic PSs. Prolonged incubation times led to increasing contributions from short-lived components that derive from aggregates mainly localized in the cytoplasm. Encapsulation of PSs into polymeric carriers led to monomerization and mostly fluorescence emission decays with long fluorescence lifetimes in solution. However, the efficiency depended on the binding strength that was most pronounced for PVP. In the cellular environment, PVP was able to maintain monomeric long-lived species over prolonged incubation times. This was most pronounced for Ce6 derivatives with a logP value around 4.5. Micellar encapsulation led to faster release of the PSs resulting in multiple components with long and short fluorescence lifetimes. The hydrophilic hardly aggregating PS exhibited a mostly stable invariant lifetime distribution over time with both carriers. The presented data are expected to contribute to optimized PDT treatment protocols and improved PS-carrier design for preventing intracellular fluorescence quenching. In conclusion, amphiphilic and concurrent hydrophobic PSs with high membrane affinity as well as strong binding to the carrier have best prospects to maintain their photophysical properties in vivo and serve thus as efficient photodynamic diagnosis and PDT drugs.

Feasibility of 1H-high resolution-magic angle spinning NMR spectroscopy in the analysis of viscous cosmetic and pharmaceutical formulations.

The feasibility of (1)H-High Resolution-Magic Angle Spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy for the direct analysis of viscous cosmetic and pharmaceutical formulations such as creams, gels, and pastes is presented. Three examples are described: (i) the detection of chitosan in toothpaste, (ii) the analysis of dexamethasone acetate (DMA) in a cream, and (iii) the analysis of the local anesthetics, lidocaine and prilocaine, in a gel and a cream. All active components could be directly detected in their original commercial formulations without the need for laborious sample preparation steps. In addition, the possibility for HR-MAS-based quantifications and the analysis of dynamic properties of active components in different formulations applying HR-MAS diffusion-ordered NMR spectroscopy are shown.

Intracellular Fate of the Photosensitizer Chlorin e4 with Different Carriers and Induced Metabolic Changes Studied by 1H NMR Spectroscopy.

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.

Liposomal aggregates sustain the release of rapamycin and protect cartilage from friction.

Liposomes show promise as biolubricants for damaged cartilage, but their small size results in low joint and cartilage retention. We developed a zinc ion-based liposomal drug delivery system for local osteoarthritis therapy, focusing on sustained release and tribological protection from phospholipid lubrication properties. Our strategy involved inducing aggregation of negatively charged liposomes with zinc ions to extend rapamycin (RAPA) release and improve cartilage lubrication. Liposomal aggregation occurred within 10 min and was irreversible, facilitating excess cation removal. The aggregates extended RAPA release beyond free liposomes and displayed irregular morphology influenced by RAPA. At nearly 100 µm, the aggregates were large enough to exceed the previously reported size threshold for increased joint retention. Tribological assessment on silicon surfaces and ex vivo porcine cartilage revealed the system's excellent protective ability against friction at both nano- and macro-scales. Moreover, RAPA was shown to attenuate the fibrotic response in human OA synovial fibroblasts. Our findings suggest the zinc ion-based liposomal drug delivery system has potential to enhance OA therapy through extended release and cartilage tribological protection, while also illustrating the impact of a hydrophobic drug like RAPA on liposome aggregation and morphology.

Interactions between selected photosensitizers and model membranes: an NMR classification.

Membrane interactions of porphyrinic photosensitizers (PSs) are known to play a crucial role for PS efficiency in photodynamic therapy (PDT). In the current paper, the interactions between 15 different porphyrinic PSs with various hydrophilic/lipophilic properties and phospholipid bilayers were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. PS-membrane interactions were deduced from analysis of the main DOPC 1H-NMR resonances (choline and lipid chain signals). Initial membrane adsorption of the PSs was indicated by induced changes to the DOPC choline signal, i.e. a split into inner and outer choline peaks. Based on this parameter, the PSs could be classified into two groups, Type-A PSs causing a split and the Type-B PSs causing no split. A further classification into two subgroups each, A1, A2 and B1, B2 was based on the observed time-dependent changes of the main DOPC NMR signals following initial PS adsorption. Four different time-correlated patterns were found indicating different levels and rates of PS penetration into the hydrophobic membrane interior. The type of interaction was mainly affected by the amphiphilicity and the overall lipophilicity of the applied PS structures. In conclusion, the NMR data provided valuable structural and dynamic insights into the PS-membrane interactions which allow deriving the structural constraints for high membrane affinity and high membrane penetration of a given PS.

Exploring high-resolution magic angle spinning (HR-MAS) NMR spectroscopy for metabonomic analysis of apples.

Classical liquid-state high-resolution (HR) NMR spectroscopy has proved a powerful tool in the metabonomic analysis of liquid food samples like fruit juices. In this paper the application of (1)H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to apple tissue is presented probing its potential for metabonomic studies. The (1)H HR-MAS NMR spectra are discussed in terms of the chemical composition of apple tissue and compared to liquid-state NMR spectra of apple juice. Differences indicate that specific metabolic changes are induced by juice preparation. The feasibility of HR-MAS NMR-based multivariate analysis is demonstrated by a study distinguishing three different apple cultivars by principal component analysis (PCA). Preliminary results are shown from subsequent studies comparing three different cultivation methods by means of PCA and partial least squares discriminant analysis (PLS-DA) of the HR-MAS NMR data. The compounds responsible for discriminating organically grown apples are discussed. Finally, an outlook of our ongoing work is given including a longitudinal study on apples.

pH-dependent distribution of chlorin e6 derivatives across phospholipid bilayers probed by NMR spectroscopy.

The pH-dependent membrane adsorption and distribution of three chlorin derivatives, chlorin e6 (CE), rhodin G7 (RG), and monoaspartyl-chlorin e6 (MACE), in the physiological pH range (pH 6-8) were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. The chlorin derivatives were characterized with respect to their aggregation behavior, the pK(a) values of individual carboxylate groups, the extent of membrane adsorption, and their flip-flop rates across the bilayer membrane for pH 6-8. External membrane adsorption was found to be lower for RG than for CE and MACE. Both electrostatic interactions and the extent of aggregation seemed to be the main determinants of membrane adsorption. Rate constants for chlorin transfer across the membrane were found to correlate strongly with the pH of the surrounding medium, in particular, for CE and RG. In acidic solution, CE and RG transfer across the membrane was strongly accelerated, and in basic solution, all compounds were retained, mostly in the outer monolayer. In contrast, MACE flip-flop across the membrane remained very low even at pH 6. The protonation of ionizable groups is suggested to be a major determinant of chlorin transfer rates across the bilayer. pK(a) values of CE and RG were found to be between 6 and 8, and two of the carboxylate groups in MACE had pK(a) values below 6. For CE and RG, the kinetic profiles at acidic pH indicated that the initial fast membrane distribution was followed by secondary steps that are discussed in this article.

Interactions of Cationic Diruthenium Trithiolato Complexes with Phospholipid Membranes Studied by NMR Spectroscopy.

To apprehend the possible mechanisms involved in the cellular uptake and the membrane interactions of cytotoxic dinuclear p-cymene trithiolato ruthenium(II) complexes, the interactions of the complexes [(η6-p-MeC6H4Pri)2Ru2(R1)2(R2)]+ (R1 = R2 = SC6H4-m-Pri:1; R1 = SC6H4-p-OMe, R2 = SC6H4-p-OH:2; R1 = SCH2C6H4-p-OMe, R2 = SC6H4-p-OH:3) with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles were studied using nuclear magnetic resonance (NMR) spectroscopy. 1H NMR, nuclear Overhauser effect (NOE), diffusion ordered spectroscopy (DOSY), and T1 and T2 relaxation data provided information on interactions between the complexes and the model membranes and on the submolecular localization of the complexes at the membrane interface. The results suggest that (a) the interaction takes place without new covalent adduct formation, (b) the cationic diruthenium complexes interact with DOPC head groups most likely involving electrostatic interactions while remaining structurally unchanged, (c) the changes indicating interactions are more pronounced for the most lipophilic complex 1, and (d) the diruthenium complexes remain at the exterior vesicle surface and are unlikely inserted between the phospholipid chains. The complexes also interact with micellar/free DHPC and seem to induce micellization or aggregation in solutions below critical micelle concentration (CMC). Our study suggests high affinity of the Ru complexes for the membrane surface that likely plays a key role in cellular uptake and possibly also in redistribution in mitochondria.