Cell Permeation Research Articles

Ultrasensitive detection of thiophenol based on a water-soluble pyrenyl probe

We report a simple pyrene-based fluorescent probe, sodium 8-(2,4-dinitrophenoxy)pyrene-1,3,6-trisulfonate (HPTS-DNP), for the ultrasensitive and visual detection of thiophenol in 100% aqueous media. The sensing mechanism of this method is based on nucleophilic aromatic substitution on HPTS-DNP caused by thiophenol to afford a highly fluorescent product, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS). The extremely low fluorescence background of HPTS-DNP and high quantum yield of HPTS ensure the superior sensing performance toward thiophenol, including rapid responses, off-on detection mode and excellent sensitivity. The detection limit is as low as 0.49 nmol L−1 according to the measurements with a spectrofluorimeter. This probe also features good selectivity (response ratio of thiophenol to interferents >30), precision (2.93%) and accuracy (102%). This approach could be applied for monitoring the thiophenol concentrations in environmental water samples. HPTS-DNP also showed good cell permeation capacity and low cellular cytotoxicity, indicating further application in bioimaging studies.

Methotrexate-Loaded Extracellular Vesicles Functionalized with Therapeutic and Targeted Peptides for the Treatment of Glioblastoma Multiforme.

Despite promising in vitro evidence for effective glioblastoma treatment, most drugs are hindered from entering the central nervous system because of the presence of the blood-brain barrier (BBB). Thus, successful modification of drug delivery and novel therapeutic strategies are needed to overcome this obstacle. Extracellular vesicles (EVs), cell-derived membrane-encapsulated structures with diameters ranging from 50 to 1000 nm, have been explored as the drug delivery system to deliver their cargo to the brain tissue. Moreover, tumor targeting and selective drug delivery has been facilitated by engineering their parent cells to secrete modified EVs. However, the method suffers from many shortcomings including poor repeatability and complex and time-consuming operations. In this context, we present an easy-to-adapt and highly versatile methodology to modify EVs with an engineered peptide capable of recognition and eradication of glioma. On the basis of molecular recognition between phospholipids on EV lipid bilayer membranes and ApoA-I mimetic peptides, we have developed methotrexate (MTX)-loaded EVs functionalized with therapeutic [Lys-Leu-Ala (KLA)] and targeted [low-density lipoprotein (LDL)] peptides. In vitro experiments demonstrated that EVs decorated with LDL or KLA-LDL could obviously ameliorate their uptake by human primary glioma cell line U87 and permeation into three-dimensional glioma spheroids in contrast to blank EVs, and consequently, the treatment outcome of the payload is improved. Both ex vivo and in vivo imaging experiments revealed that peptide LDL could obviously promote EV extravasation across the BBB and distribution in the glioma site. Furthermore, compared with the mice administrated with MTX and MTX@EVs, MTX@EVs-KLA-LDL-treated mice showed the longest median survival period. In conclusion, functionalizing with the peptide onto EV surfaces may provide a substantial advancement in the application of EVs for selective target binding as well as therapeutic effects for brain tumor treatment.

Permeabilization of human stratum corneum and full-thickness skin samples by a direct dielectric barrier discharge

PurposeThe tight barrier formed by human stratum corneum (SC) has a significantly protective function but also prevents the delivery of drug substances through the skin. In order to permeabilize the SC for drug delivery we use cold atmospheric plasma (CAP). MethodsA direct ex vivo treatment of human skin with cold atmospheric plasma, specifically a dielectric barrier discharge (DBD) using skin as the ground electrode, was employed to permeabilize the stratum corneum (SC) throughout a treated area of 0.5 cm2. The permeabilization of isolated SC and full-thickness skin was evaluated through changes in transepithelial electrical resistance. Franz diffusion cell permeation using differently sized hydrophilic particles enabled an estimation of the pore size and drug transport efficiency. Furthermore, silver sheet oxidation showed the distribution of local permeabilized regions. ResultsThe transepithelial electrical resistance showed a long-term overall drop for treatments ≥90 s in isolated SC as well as full-thickness skin. Silver sheet oxidation revealed a regular pattern of local permeabilized regions greater in number than the expected number of skin appendages in treated isolated human SC. Permeation study results indicate that relatively small hydrophilic substances with Stokes’ radii up to 1.4 nm are efficiently transported through human SC subsequent to 2×90 s treatment with direct cold atmospheric plasma at a power density of about 0.2 W cm−2. A moderate permeation of particles up to 6 μm in diameter is evident for the occasional formation of large pores in the μm-range. Finally, a mechanism for DBD plasma permeabilization of skin is proposed and discussed.

Importance of Net Hydrophobicity in the Cellular Uptake of All-Hydrocarbon Stapled Peptides.

All-hydrocarbon stapled peptides make up a promising class of protein-protein interaction regulators; their potential therapeutic benefit arises because they have a high binding affinity and specificity for intracellular molecules. The cell permeation efficacy of these peptides is a critical determinant of their bioactivity. However, the factors that determine their cellular uptake remain an active area of research. In this study, we evaluated the effect of stapled (or cross-linked) formation on the cellular uptake of six known all-hydrocarbon stapled peptides. We found that the rate of cellular uptake of unstapled peptides (i.e., those bearing olefinic non-natural amino acids that are not subjected to olefin metathesis) was higher than that for the corresponding stapled peptides. Additionally, the insertion of these olefinic non-natural amino acids into peptide sequences significantly increased their rate of cellular uptake. According to the high-performance liquid chromatography retention times, the overall hydrophobicity of unstapled peptides was greater than that of stapled peptides, followed by that of the original peptides without olefinic non-natural amino acids. There was not a close correlation between helical content and the rate of cellular uptake of these peptides. Therefore, the increase in overall hydrophobicity resulting from the introduction of non-natural amino acids, rather than the structural stabilization resulting from staple formation, is the key driver promoting cellular uptake. Macropinocytosis, a form of fluid-phase endocytosis, was involved in the cellular uptake of all six peptides.

Core-shell magnetoelectric nanorobot \u2013 A remotely controlled probe for targeted cell manipulation

We have developed a remotely controlled dynamic process of manipulating targeted biological live cells using fabricated core-shell nanocomposites, which comprises of single crystalline ferromagnetic cores (CoFe2O4) coated with crystalline ferroelectric thin film shells (BaTiO3). We demonstrate them as a unique family of inorganic magnetoelectric nanorobots (MENRs), controlled remotely by applied a.c. or d.c. magnetic fields, to perform cell targeting, permeation, and transport. Under a.c. magnetic field excitation (50 Oe, 60 Hz), the MENR acts as a localized electric periodic pulse generator and can permeate a series of misaligned cells, while aligning them to an equipotential mono-array by inducing inter-cellular signaling. Under a.c. magnetic field (40 Oe, 30 Hz) excitation, MENRs can be dynamically driven to a targeted cell, avoiding untargeted cells in the path, irrespective of cell density. D.C. magnetic field (−50 Oe) excitation causes the MENRs to act as thrust generator and exerts motion in a group of cells.

MRNA-Initiated, Three-Dimensional DNA Amplifier Able to Function inside Living Cells.

DNA molecular machines show great promise in fields such as biomarker discovery and biological activity regulation, but operating DNA machines with specific functions within living systems remains extremely challenging. Although DNA machines have been engineered with exact molecular-level specifications, some intrinsic imperfections such as poor cell permeation and fragility in complex cytoplasmic milieu persist due to the well-established character of nucleic acid molecules. To circumvent these problems, we herein report a molecularly engineered, entropy-driven three-dimensional DNA amplifier (EDTD) that can operate inside living cells in response to a specific mRNA target. In particular, mRNA target/EDTD interaction can specifically initiate an autonomous DNA circuit inside living cells owing to the exclusive entropy-driven force, thus providing enormous signal amplification for ultrasensitive detection of the mRNA. Moreover, owing to molecular engineering of a unique DNA tetrahedral framework into the DNA amplifier, EDTD exhibits significantly enhanced biostability and cellular uptake efficiency, which are prerequisites for DNA machines used for in vivo applications. This programmable DNA machine presents a simple and modular amplification mechanism for the detection of intracellular biomarkers. Moreover, this study provides a potentially valuable molecular tool for understanding the chemistry of cellular systems and offers a design blueprint for further expansion of DNA nanotechnology in living systems.

Impact of Linker Length and Composition on Fragment Binding and Cell Permeation: Story of a Bisbenzimidazole Dye Fragment.

Small molecules that modulate biological functions are targets of modern day drug discovery efforts. In a common platform fragment-based drug discovery, two fragments that bind to adjacent sites on a target are identified and are then linked together using different linkers to identify the linkage for optimum activity. What are not known from these studies are the effects these linkers, which typically contain C, H, and O atoms, have on the properties of the individual fragment. Herein, we investigate such effects in a bisbenzimidazole fragment whose derivatives have a wide range of therapeutic applications in nucleic acid recognition, sensing, and photodynamic therapy and as cellular probes. We report a dramatic effect of linker length and composition of alkynyl (clickable) Hoechst 33258 derivatives in target binding and cell uptake. We show that the binding of Hoechst 33258-modeled bisbenzimidazoles (1-9) that contain linkers of varying lengths (3-21 atoms) display length- and composition-dependent variation in B-DNA stabilization using a variety of spectroscopic methods. For a dodecamer DNA duplex, the thermal stabilization varied from 0.3 to 9.0 °C as the linker length increased from 3 to 21 atoms, respectively. Compounds with linker lengths of ≤11 atoms (such as compounds 1 and 5) are localized in the nucleus, while compounds with long linkers (such as compounds 8 and 9) are distributed in the extranuclear space, as well, with possible interactions with extranuclear targets. These findings provide insights into future drug design by revealing how linkers can influence the biophysical and cellular properties of individual drug fragments.

Galactosylated Pro-Drug of Ursodeoxycholic Acid: Design, Synthesis, Characterization, and Pharmacological Effects in a Rat Model of Estrogen-Induced Cholestasis.

Ursodeoxycholic acid (UDCA) is considered the first-choice therapy for cholestatic disorders. To enhance solubility and exploit specific transporters in liver, we synthesized a new galactosyl pro-drug of UDCA (UDCAgal). Ethinylestradiol (EE)-induced cholestasis was used to study and compare the effects of UDCAgal with UDCA on bile flow, hepatic canalicular efflux transporter expression, and inflammation. UDCAgal resulted quite stable both at pH 7.4 and 1.2 and regenerated the parent drug after incubation in human plasma. Its solubility, higher than UDCA, was pH- and temperature-independent. UDCAgal displayed a higher cell permeation compared to UDCA in liver HepG2 cells. Moreover, in cholestatic rats, UDCAgal showed a higher potency compared to UDCA in reducing serum biomarkers (AST, ALT, and ALP) and cytokines (TNF-α and IL-1β). The higher effect of UDCAgal on the increase in bile salt export pump and multidrug resistance-associated protein 2 transcription indicated an improved spillover of bile acids from the liver. UDCAgal showed a reduction in CCL2, as well as TNF-α, IL-1β, and cyclooxygeanse-2 mRNAs, indicating a reduction in hepatic neutrophil accumulation and inflammation. Moreover, UDCAgal, similarly to UDCA, heightens bile flow and modulates biliary acids secretion. These results indicate that UDCAgal has a potential in the treatment of cholestatic disease.

Stable LSM/LSTM bi-layer interconnect for flat-tubular solid oxide fuel cells

A bi-layer interconnect with La0.8Sr0.2MnO3 and La0.4Sr0.6Ti0.6Mn0.4O3 (LSM/LSTM) is applied to anode-supported button cells and flat-tubular cells. Using a button cell, SEM images and gas permeation tests confirm that the bi-layer possesses a dense microstructure. The area specific resistance (ASR) of the LSM/LSTM remains nearly constant under oxidizing/reducing atmospheres with varying gas concentrations. For comparison, an LSM/LST with the same thickness is prepared; an increase in the ASR is observed as the concentration of H2 feed to the LST side decreases. The difference in the ASR of LSM/LST can be explained by exposure to a relatively high oxygen partial pressure and partial destruction of the interfacial LST layer region where oxygen diffuses from the LSM layer. Flat-tubular cells with the LSM/LSTM bi-layer interconnect achieve a maximum power density (MPD) of 463 mW cm−2 using humidified H2 fuel and air at 800 °C. With decreasing H2 concentration in the fuel, the polarization resistance increases rather than the ohmic resistance, implying that the LSM/LSTM interconnect provides stable conduction property. In comparison with the conventional LSM/LST interconnect cell, it shows improved stability and performance as the concentration of H2 in the fuel decreases.

Enhanced formation of hydroxyapatites in gelatin/imogolite macroporous hydrogels

HypothesisGelatin is widely investigated for the fabrication of synthetic scaffolds in bone tissue engineering. Practical limitations to its use are mainly due to the fast dissolution rate in physiological conditions and to the lack of pores with suitable dimensions for cell permeation. The aim of this work is to exploit imogolite clays as nucleation sites for the growth of calcium phosphates in gelatin-based hydrogels and to take advantage of a cryogenic treatment to obtain pores of ∼100µm. ExperimentsWe evaluated the effect of imogolites and a biocompatible cross-linker on the gelatin network in terms of morphology, thermal and rheological behavior. The hydrogels were cryogenically-treated and characterized to investigate the modification of the polymer network, both at the micro- and nano-scale. The samples were mineralized to investigate the effect of imogolites on the formation of calcium phosphates. FindingsThe interaction between gelatin, imogolite and cross-linker leads to the modification of the hydrogel structure at the micro-scale, while minor effects are detected at the nano-scale. The cryogenic procedure is successful in generating pores with the desired size, while the presence of imogolites in the hydrogel promotes hydroxyapatites formation. These results demonstrate that imogolites can be effectively employed as functional fillers in polymer-based scaffolds.

Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation

Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide “stapling” to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR.

Mechanistic Insight into Sonoporation with Ultrasound-Stimulated Polymer Microbubbles

Sonoporation is emerging as a feasible, non-viral gene delivery platform for the treatment of cardiovascular disease and cancer. Despite promising results, this approach remains less efficient than viral methods. The objective of this work is to help substantiate the merit of polymeric microbubble sonoporation as a non-viral, localized cell permeation and payload delivery strategy by taking a ground-up approach to elucidating the fundamental mechanisms at play. In this study, we apply simultaneous microscopy of polymeric microbubble sonoporation over its intrinsic biophysical timescales–with sub-microsecond resolution to examine microbubble cavitation and millisecond resolution over several minutes to examine local macromolecule uptake through enhanced endothelial cell membrane permeability–bridging over six orders of magnitude in time. We quantified microbubble behavior and resulting sonoporation thresholds at transmit frequencies of 0.5, 1 and 2 MHz, and determined that sonic cracking is a necessary but insufficient condition to induce sonoporation. Further, sonoporation propensity increases with the extent of sonic cracking, namely, from partial to complete gas escape from the polymeric encapsulation. For the subset that exhibited complete gas escape from sonic cracking, a proportional relationship between the maximum projected gas area and resulting macromolecule uptake was observed. These results have revealed one aspect of polymeric bubble activity on the microsecond time scale that is associated with eliciting sonoporation in adjacent endothelial cells, and contributes toward an understanding of the physical rationale for sonoporation with polymer-encapsulated microbubble contrast agents.

Polyphenols from guaraná after in vitro digestion: Evaluation of bioacessibility and inhibition of activity of carbohydrate-hydrolyzing enzymes

Guaraná (Paullinia cupana) is a typical product from Amazon biota that exerts antioxidant capacity due to the presence of phenolic compounds, such as catechin, epicatechin and proanthocyanidins. The objective of this study was to evaluate the potential inhibitory activity of guaraná extracts, after digestion in vitro, on carbohydrates-metabolism enzymes and to assess the bioacessibility of guaraná polyphenols. The guaraná samples before and after enzymatic digestion, were compared for total phenolic content and phenolic profile. Furthermore, we investigated the uptake of polyphenols from guarana, using Caco-2 cells, and the effect of digested guaraná on carbohydrate metabolism enzymes. The amount of total phenolic compounds extracted from guaraná decreased after digestion in vitro, and native phenolics were not identified after cell permeation. On the other hand, polyphenols from guaraná were able to inhibit α-glucosidase and α-amylase activities. In conclusion, guaraná can be considered as a dietary source with anti-hyperglycemic potential.

Cell Imaging: Spatiotemporally Controllable Peptide-Based Nanoassembly in Single Living Cells for a Biological Self-Portrait (Adv. Mater. 32/2017)

A spatiotemporally-controllable peptide-based nanoassembly in a single living cell, for a biological self-portrait is reported by Xing-Jie Liang and co-workers in article number 1601128. The suitability of the synthesized probe is indicated by rapid cell permeation, sitespecific in situ self-assembly, responsive fluorescence, superior biocompatibility and sensitivity, and robustness, which allows longterm recording and simultaneously evaluating the real-time location and bioactivity. This noninvasive investigation can be further expanded to other bioactive molecules, thus facilitating quantitative imaging in vivo.

Structural Determinant and Its Underlying Molecular Mechanism of STPC2 Related to Anti-Angiogenic Activity.

In this study, we aimed to use different strategies to further uncover the anti-angiogenic molecular mechanism of a fucoidan-like polysaccharide STPC2, isolated from brown alga Sargassum thunbergii. A desulfated derivative, STPC2-DeS, was successfully prepared and identified. The native polysaccharide and desulfated product were subjected to evaluate their anti-angiogenic effects. In the tube formation assay, STPC2 showed dose-dependent inhibition. In addition, STPC2 could distinctly inhibit the permeation of HUVEC cells into the lower chamber. Moreover, a significant reduction of microvessel density was observed in chick chorioallantoic membrane assay treated with STPC2. Meanwhile, STPC2 was found to repress the VEGF-induced neovessel formation in the matrigel plug assay in vivo. However, STPC2-DeS failed to suppress the anti-angiogenic activity via these in vitro and in vivo strategies. In addition, we demonstrated that STPC2 could significantly downregulate the phosphorylation of VEGFR2 and its related downstream Src family kinase, focal adhesion kinase, and AKT kinase. Furthermore, surface plasmon resonance assay revealed that STPC2 bound strongly to VEGF to interfere with VEGF–VEGFR2 interaction. Taken together, these results evidently demonstrated that STPC2 exhibited a potent anti-angiogenic activity through binding to VEGF via sulfated groups to impede VEGF–VEGFR2 interaction, thus affected the downstream signaling molecules.

Identification of the hydrogen diffusion parameters in bearing steel by Evolutionary Algorithm

Abstract A novel mathematical method for the identification of hydrogen diffusion parameters in metals has been implemented. This method is based on a clustering-based Evolutionary Algorithm which provides robust and efficient optimization of the diffusion parameters. It is assumed that hydrogen diffusion obeys the McNabb–Foster equations, and by using the Evolutionary Algorithm the 1D time-dependent solution is calibrated to the experimental curve measured by the two cells permeation test. The original McNabb–Foster diffusion model, including reversible and irreversible traps, was simplified to the models with only reversible or irreversible traps and the calibration quality was tested. Due to the random nature of Evolutionary Algorithms, multiple calibration sets were identified, which corresponds to the non-uniqueness of calibration by the McNabb–Foster diffusion model. The definition of the invariant diffusion parameters describing uniquely the material and the test is the main novelty of the proposed study.

Modular sub-monomeric cell-penetrating guanidine-rich peptoids – synthesis, assembly and biological evaluation

Development of a guanidinium-rich transporters toolset to study GAG-mediated cell permeation.

PUB097 Carbon Nanotubes for Efficient Mitochondrial Tumor Targeting

Cancer is uncontrollable growth of cells which are devoid of apoptosis. We developed a novel strategy ie Ligand mediated tumor targeting via carrier systems. Multiwalled Carbon nanotubes (MWCNTs) were used as it directly enters into the cell without passing through endo-lysosomes, large inner volume, distinct inner and outer surfaces & have ability to enter the cell by spontaneous mechanism. Thus, proposed work envisages Rhodamine-123 conjugated Paclitaxel loaded functionalized-CNTs to provide enhanced cell permeation in order to enhance mitochondrial availability of Paclitaxel. The raw MWCNT were procured and purified, oxidized & then conjugated with rhodamine-123 by carbodimide method. The MWCNT’s were characterized in-vitro for shape & size by Scanning(SEM) & Transmission Electron Microscopy(TEM), FTIR analysis, X-ray diffraction and zeta potential determined. Stability studies were performed at exaggerated conditions along with Hemolytic Toxicity Study. The Cell Cytotoxicity Study-MTT Assay was done using Hela cell lines. Mitochondrial localization was determined by CLSM study. The in-vivo part of the study comprised of determining the distribution of drug in various organs by fluorescence microscopy. The Rhodamine-123 conjugated MWCNTs were prepared and characterized. The CNTs showed high paclitaxel loading, sustained release, and excellent biocompatibility as evident by in-vitro drug release and low hemolytic toxicity. MTT assay against HeLa cell lines suggested the potential anticancer activity of the developed system. CLSM study suggested that mitochondrial specific localization of Rhodamine-123 conjugated MWCNTs in HeLa cells. Thus, Rhodamine-123 conjugated Paclitaxel loaded f-CNTs system have potential to provide an enhanced cell permeation and mitochondrial localization for effective tumour chemotherapy.

Assessment of cell viability and permeation enhancement in presence of lipid-based self-emulsifying drug delivery systems using Caco-2 cell model: Polysorbate 80 as the surfactant

PurposeLipid-based self-emulsifying drug delivery systems (SEDDS) are commonly used for solubilizing and enhancing oral bioavailability of poorly water-soluble drugs. However, their effects on viability of intestine epithelial cells and influence on membrane permeation are poorly understood. The present study was undertaken for safety assessment of lipid-based formulations containing medium-chain fatty acid esters as lipids and polysorbate 80 as the surfactant using the Caco-2 in vitro model. Any possible paracellular permeation enhancement through Caco-2 monolayers by the nontoxic formulations was also investigated. MethodsMixtures of monoglyceride (Capmul MCM EP or 708G) or propylene glycol monoester (Capmul PG-8 NF) of medium chain fatty acids with polysorbate 80, with and without the incorporation of a medium-chain triglyceride (Captex 355), were prepared. After suitable dilution with aqueous culture medium, the formulations were incubated with a series of Caco-2 cultures of different maturity. Cell viability and membrane integrity were assessed. Any effects of nontoxic formulations on the transport of the fluorescent dye, Lucifer yellow, through Caco-2 monolayers were also determined. ResultsFormulations containing 1:1 ratios of monoglyceride or propylene glycol monoester to triglyceride (30% polysorbate 80, 35% monoglyceride or monoester and 35% triglyceride) were best tolerated by Caco-2 cells. Increased maturity obtained through longer culture durations rendered Caco-2 cells greater tolerance towards lipid-based formulations, and maximum tolerance to lipid-based formulations was observed with Caco-2 monolayers after being cultured for 21–23days. Furthermore, extent of cell membrane rupture caused by lipid-surfactant mixtures correlated positively with levels of cytotoxicity, suggesting a potential underlying mechanism. Permeation studies using Caco-2 monolayer model revealed that certain formulations significantly enhanced paracellular transport activities. ConclusionsLipid-based SEDDS containing mixtures of monoglyceride (or monoester) and triglyceride of medium chain fatty acids formed fine microemulsions and were significantly less toxic than other formulations. Fully differentiated Caco-2 monolayer was more resistant to lipid-surfactant mixtures than less mature cultures. Certain formulations were also capable of enhancing paracellular permeation.

In vitro studies of dermally absorbed Cu(II) tripeptide complexes as potential anti-inflammatory drugs

Copper complexes have been reported to alleviation the inflammation associated with rheumatoid arthritis (RA). The aim of this research was to design and test new ligands that are able to promote the percutaneous absorption of copper and/or mobilise endogenous copper reserves, with the ultimate aim of testing these as anti-inflammatory agents. Formation constants of H+ and Cu(II) with the tripeptides sarcosyl-l-histidyl-l-histidine (Sar-HH) and sarcosyl-l-glycyl-l-histidine (Sar-GH) were measured at 25±0.01°C and at an ionic strength of 0.15M (NaCl) using glass electrode potentiometry. In the pH range 2–11, four species; CuLH, CuL, CuLH-1, and CuLH-2 were observed. The two tripeptides showed significantly different coordination behaviour at physiological pH, Sar-HH forming a CuLH-1 species while Sar-GH formed a CuLH-2 species. The solution structures of the different Cu(II) species formed with these ligands were investigated using visible spectroscopy (UV–Vis), electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy. The CuLH-2 species involved coordination of two deprotonated amide groups to give a neutral complex. 1H NMR identified the binding sites to be the imidazole nitrogen, the two-amide nitrogens and the terminal amino group. UV–Vis and EPR spectroscopy confirmed this mode of coordination. Speciation calculations using a blood plasma model were used to estimate the complexing ability of the ligands in silico. Octanol/water partition coefficients and Franz cell permeation studies were used to evaluate percutaneous skin absorption. The results showed that the Cu(II) complexes are hydrophilic but that Sar-GH caused a 2 fold increase in membrane permeability of Cu(II).