Dalia Freeman

A method for the quantitation of hypericin, an antiviral agent, in biological fluids by high-performance liquid chromatography

Hypericin, a polycyclic aromatic dianthroquinone, is a natural plant product with antiviral properties. We report here the development of a methodology for the extraction and quantitation of hypericin from plasma and biological fluids and the adaptation of a sensitive and selective method for detection of the compound by high-performance liquid chromatography. The methodology offers a rapid and specific means of monitoring drug blood levels in clinical and pharmacokinetic studies. The chromatographic procedure utilizes the substantial retentive properties of hypericin on reverse-phase media and detection by the strong visible absorbance maximum at 590 nm. Verification by the fluorescence spectral properties of hypericin in organic media can also be utilized. The assay is linear over a 3 log concentration range and hypericin is consistently recovered from murine, simian, and human plasma. The methodology was applied to assess the pharmacokinetic properties of hypericin in mice receiving a single bolus injection of 350 micrograms. A distribution half-life of 2.0 h and an elimination half-life of 38.5 h were calculated. We also discuss the limitations of direct analysis of hypericin by absorbance or fluorescence measurements.

Electrochromic Behavior of a Self-Propagating Molecular-Based Assembly

A metallo-supramolecular network undergoes reversible redox chemistry on indium-tin oxide (ITO) coated glass substrates with concurrent color change. The switching time, long-term stability, and coloration efficiency are competitive with polymeric materials such as the industrially important PEDOT.

Liposome binding constants and singlet oxygen quantum yields of hypericin, tetrahydroxy helianthrone and their derivatives: studies in organic solutions and in liposomes

The spectroscopy and photophysics of several hypericin and helianthrone derivatives were studied in methanol and when bound to liposomes. The singlet oxygen quantum yields (phi(delta)) were measured indirectly relative to Rose Bengal and hematoporphyrin IX, employing 9,10-dimethylanthracene as a singlet oxygen trap. Hypericin was found to have a phi(delta) of 0.39+/-0.01 in methanol, and 0.35+/-0.05 in lecithin vesicles, in agreement with literature values. A heavy atom effect was evident upon bromination, resulting in phi(delta) for tetrabromohypericin of 0.72+/-0.02, presumably due to enhanced intersystem crossing. Elimination of the anionic hydroxyls by methylation also enhanced phi(delta) to 0.81+/-0.01. Conversely, addition of anionic sulfate groups drastically reduced phi(delta) resulting in phi(delta)s of 0.12+/-0.01, 0.052+/-0.003 and 0.40+/-0.01 for hypericin disulfonate, hypericin tetrasulfonate and hexamethyl hypericin tetrasulfonate, respectively. The non-sulfonated helianthrones exhibited low phi(delta)s in solution. The liposome binding constants, Kb, were measured using a spectroscopic assay. Except for hexamethyl hypericin, all non-sulfonated compounds bound well with Kbs ranging from 15.5+/-0.1 to 48.7+/-3.9 (mg/ml)(-1). None of the tetrasulfonated compounds bound, however the hypericin disulfonate had a Kb of 4.1+/-0.2 (mg/ml)(-1). The phi(delta)s of the compounds capable of binding were measured and, in the case of the hypericin derivatives, were found not to vary dramatically from those in the free state. Liposome-bound helianthrone and dimethyl tetrahydroxy helianthrone both exhibited high phi(delta)s, i.e. >0.5. The variations in binding constant and sensitization efficiencies are explained in conjunction with the molecular structure. The relevance of the above data to photodynamic therapy is briefly discussed.

Assembly of crystalline halogen-bonded materials by physical vapor deposition.

Polycrystalline halogen-bonded assemblies fabricated by physical vapor deposition (PVD) exhibit controllable morphologies and microstructures. Although the solid-state packing may vary going from a solution crystal growth process (used for chromophore single-crystal determination) to a vapor-phase deposition process (used for PVD film fabrication), the corresponding film microstructures are independent of the substrate surface chemistry.

The correlation between hydrophilicity of hypericins and helianthrone: internalization mechanisms, subcellular distribution and photodynamic action in colon carcinoma cells

The internalization mechanism and subcellular distribution of hypericin (Hyp), hypericin tetrasulfonic acid (HypS4) and 1,3,4,6-tetrahydroxyhelianthrone (Hel) were studied in murine colon carcinoma CT26 cells, in protein-free medium or in the presence of serum proteins. The correlation between the extent of uptake of the sensitizers by cells that were incubated in the presence of different serum components, and the internalization mechanisms, was studied. The results indicate that sensitizer internalization may be a result of three mechanisms: partitioning, pinocytosis and endocytosis, and as a direct consequence is targeted to specific subcellular sites. While Hyp and Hel, the two lipophilic sensitizers, were localized in the endoplasmic reticulum after protein-free internalization, the hydrophilic HypS4 was localized in the cytoplasmic membrane and in lysosomes. An endolysosomal internalization route was revealed for Hyp and Hel under serum-enriched conditions showing lysosomal localization, as for HypS4. The lysosomal accumulation of Hyp-serum and specifically Hyp-LDL points to an endocytotic mechanism which is supported by its higher uptake parameter in an LDL-enriched medium, compared to the medium with 10% serum. The different uptake parameters of Hyp to cells, with or without serum, reflect the different mechanisms. Smaller differences in the uptake parameter for HypS4 reflect the distinction between partitioning and endocytosis, which, in this case, are both targeted to the lysosomes. The same uptake parameter of Hel to cells incubated in media with or without serum indicates the absence of the endocytotic mechanism. The interrelationship between subcellular targeting and photodynamic treatment was shown for the three sensitizers Hyp was found to be the most efficient sensitizer for PDT under our illumination protocol and it was dependent on internalization and localization sites.

Halogen-Bonding Mediated Stepwise Assembly of Gold Nanoparticles onto Planar Surfaces

In this study halogen bonding (XB) is used as the driving force for the noncovalent assembly of gold nanoparticles (AuNPs) on silicon and quartz substrates functionalized with organic monolayers. The AuNPs are functionalized with XB-donor ligands, whereas the monolayers have pyridine groups as XB-acceptors. The surface-confined systems are formed by iteratively exposing the monolayers to solutions of organic cross-linkers having 2-4 pyridine groups and functionalized AuNPs. UV-vis spectroscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM) reveal how the structure of the resulting surface-bound assemblies are controlled by (i) the properties of the monolayers, (ii) the molecular structure and the number of XB binding sites of the organic cross-linker, and (iii) the number of functionalized AuNP and cross-linker deposition steps. Moreover, these structures exhibit surface-enhanced Raman scattering and significant changes are observed in the morphology of some of the surface-bound assemblies upon aging.

Solvatochromic Effects in the Electronic Absorption and Nuclear Magnetic Resonance Spectra of Hypericin in Organic Solvents and in Lipid Bilayers

Abstract The natural product hypericin was tested in recent years as a biological photosensitizer with a potential for viral and cellular photodamage. We thus studied extensively its spectroscopy and membrane partitioning. Absorption, fluorescence excitation and emission spectra of the sodium salt (HyNa) were measured in 36 protic and aprotic, polar and apolar, solvents. Electronic transition bands as well as vibrational progressions were identified. Aggregation in some nonpolar solvents and protonation in organic acids were demonstrated. Modeling solvatochromism was done by Lippert equation, by the ET(30) parameter and by the Taft multiparameter approach. In all cases, separation into protic and aprotic solvents gave much better fits to the models. 13C chemical shift data could also be correlated with solvent polarity. They correlated best with Lipperts Δf polarity measure, but tended to fall into two distinct solvent groups—each along different lines—corresponding to protic and aprotic media, respectively. This interesting phenomenon suggests that in the case of the charged and slightly water soluble HyNa, two mechanisms of solvation are involved, each resulting in its own line equation. In aprotic media, dipole–dipole interaction is the predominant solvation mechanism. In protic solvents, the most effective means of solvation is likely to be hydrogen bonding. When intercalated into the liposomal phospholipid bilayer, HyNa is oriented at an angle to the interface, thus experiencing a gradient of solvent polarities: a highly polar environment (similar to methanol) for C-2/5, suggesting that they lie not far from the interface; a moderately polar environment (similar to that of n-propanol) for C-6a/14a, which are somewhat deeper within the bilayer; and a more lipophilic environment (akin to n-hexanol) for C-10/11. The fluorescence excitation peak in liposomes also correlates with an aprotic medium of relatively high polarity, as might be excepted from a molecule in a shallow position in the bilayer.

Acidic properties of hypericin and its octahydroxy analogue in the ground and excited states

pH dependent absorption and fluorescence spectra of hypericin 1 and its octahydroxy analogue 3 demonstrate their acidic properties, their deprotonation occuring in the ground and excited states from OH in the bay region and in the peri position to the carbonyl, respectively; the presence of the anion of 1 in the crystalline state is established by X-ray diffraction.

Conformation equilibria in vitamins D. The synthesis of 1α-hydroxy-3-epivitamin D3(1α-hydroxy-3α-cholecalciferol)

The ration of the two conformers of 1α-hydroxy-3-epivitamin D3, which has been synthesized from 1α,3β-dihydroxycholest-Δ6-ene, has been established.

Hypericin Derivatives: Substituent Effects on Radical-anion Formation

The electron‐transfer properties of the hypericin derivatives, dibromo‐, hexaacetyl‐, hexamethyl‐ and desmethylhypericin, were studied. Cyclovoltammetric measurements revealed that dibromo‐ and desmethylhypericin have almost the same redox potentials as the parent hypericin. Substitution of the hydroxyl groups by acetoxy leads to less negative E½ values, whereas methoxy substitution induces more negative values. Electron paramagnetic resonance (EPR)/electron nuclear double resonance/general TRIPLE spectroscopy and quantum mechanical calculations were used to establish the structure of the one‐electron reduced stages of hypericin derivatives. Proton loss in the bay region, already demonstrated for hypericin, was also found for dibromo‐ and desmethylhypericin. The spin and charge of the radical ions are predominately confined to the central biphenoquinone moiety of the hypericin skeleton. Generation of the radical ions by in situ electrolysis indicates that the redox potentials of hypericin, dibromo‐ and desmethylhypericin, containing hydroxyls at the 1, 3, 4, 6, 8 and 13 positions, largely depend on the solvent. With phosphate‐buffered saline (pH 7.4)/dimethylsulfoxide (DMSO) as the solvent the EPR spectra of the corresponding radical ions appear at markedly lower potentials than in pure DMSO and N,N′‐dimethylformamide. However, this effect is not observable for hexaacetyl‐ and hexamethylhypericin‐lacking hydroxyl groups. In all cases the EPR data and calculations revealed the presence of 7,14 tautomers.