Milan Balaz

Imaging intracellular viscosity of a single cell during photoinduced cell death.

Diffusion-mediated cellular processes, such as metabolism, signalling and transport, depend on the hydrodynamic properties of the intracellular matrix. Photodynamic therapy, used in the treatment of cancer, relies on the generation of short-lived cytotoxic agents within a cell on irradiation of a drug. The efficacy of this treatment depends on the viscosity of the medium through which the cytotoxic agent must diffuse. Here, spectrally resolved fluorescence measurements of a porphyrin-dimer-based molecular rotor are used to quantify intracellular viscosity changes in single cells. We show that there is a dramatic increase in the viscosity of the immediate environment of the rotor on photoinduced cell death. The effect of this viscosity increase is observed directly in the diffusion-dependent kinetics of the photosensitized formation and decay of a key cytotoxic agent, singlet molecular oxygen. Using these tools, we provide insight into the dynamics of diffusion in cells, which is pertinent to drug delivery, cell signalling and intracellular mass transport.

Photophysical properties and intracellular imaging of water-soluble porphyrin dimers for two-photon excited photodynamic therapy

We have investigated the photophysical properties and intracellular behaviour of a series of hydrophilic conjugated porphyrin dimers. All the dimers exhibit intense linear absorption at 650-800 nm and high singlet oxygen quantum yields (0.5-0.9 in methanol), as required for an efficient sensitiser for photodynamic therapy (PDT). They also exhibit fluorescence at 700-800 nm, with fluorescence quantum yields of up to 0.13 in methanol, and show extremely large two-photon absorption maxima of 8,000-17,000 GM in the near-IR. The dimers aggregate in aqueous solution, but aggregation is reduced by binding to bovine serum albumin (BSA), as manifested by an increase in fluorescence intensity and a sharpening in the emission bands. This process can be regarded as a model for the interaction with proteins under physiological conditions. Confocal fluorescence microscopy of live cells was used to monitor the rate of cellular uptake, intracellular localisation and photostability. Porphyrin dimers with positively charged substituents partition into cells more efficiently than the negatively charged dimers. The photostability of these dimers, in living cells, is significantly better than that of the clinical photosensitiser verteporfin. Analysis of the photophysical parameters and intracellular imaging data indicates that these dimers are promising candidates for one-photon and two-photon excited PDT.

Synthesis of hydrophilic conjugated porphyrin dimers for one-photon and two-photon photodynamic therapy at NIR wavelengths

We report the synthesis of a series of hydrophilic butadiyne-linked conjugated zinc porphyrin dimers, designed as photodynamic therapy (PDT) agents. These porphyrin dimers exhibit exceptionally high two-photon absorption cross sections (delta(max) approximately 8,000-17,000 GM) and red-shifted linear absorption spectra (lambda(max) approximately 700-800 nm) making them ideal candidates for one-photon and two-photon excited photodynamic therapy. Four polar triethyleneglycol substituents are positioned along the sides of each dimer, but, on their own, these TEG chains do not confer sufficient solubility in aqueous physiological media for reproducible delivery into live cells. Charged cationic (methylpyridinium and trimethylammonium) and anionic (sulfonate and carboxylate) substituents have been appended to the meso-positions of porphyrin dimers using three synthetic strategies: 1) Suzuki coupling, 2) Sonogashira coupling, and 3) nucleophilic Senge arylation. Approaches 1 and 3 both allow attachment of aromatic substituents directly to the meso-positions of porphyrins. Approach 2 provides a route to hydrophilic porphyrin dimers with an ethyne link between the porphyrin and the polar aromatic substituent. The palladium-catalysed approaches 1 and 2 allow the synthesis of a broader range of meso-capped porphyrins, as many aryl halides are available. However the synthesis of the intermediate required for these routes necessitates a statistical reaction step, which decreases the overall yield. On the other hand, Senge-arylation provides highly regioselective nucleophilic aromatic substitution, and offers higher overall yield than the other routes. All these charged dimers exhibit good solubility in polar solvents (e.g. methanol) and aqueous solvent mixtures (aqueous DMSO or DMF).

Interactions of a Tetraanionic Porphyrin with DNA: from a Z-DNA Sensor to a Versatile Supramolecular Device

The anionic nickel(II) porphyrin NiTPPS is able to selectively sense the spermine induced left-handed Z-form of DNA while it is completely silent in the presence of right-handed B-DNA. Interactions between the DNA and the porphyrin can be easily modulated by pH and temperature. The resulting Z-DNA-porphyrin-spermine complex behaves as a supramolecular reversible information storage system and as a reversible AND logic gate.

Ligand Induced Circular Dichroism and Circularly Polarized Luminescence in CdSe Quantum Dots

Chiral thiol capping ligands L- and D-cysteines induced modular chiroptical properties in achiral cadmium selenide quantum dots (CdSe QDs). Cys-CdSe prepared from achiral oleic acid capped CdSe by postsynthetic ligand exchange displayed size-dependent electronic circular dichroism (CD) and circularly polarized luminescence (CPL). Opposite CPL signals were measured for the CdSe QDs capped with D- and L-cysteine. The CD profile and CD anisotropy varied with size of CdSe nanocrystals with largest anisotropy observed for CdSe nanoparticles of 4.4 nm. Magic angle spinning solid state NMR (MAS ssNMR) experiments suggested bidentate interaction between cysteine and the surface of CdSe. Time Dependent Density Functional Theory (TDDFT) calculations verified that attachment of L- and D-cysteine to the surface of model (CdSe)13 nanoclusters induces measurable opposite CD signals for the exitonic band of the nanocluster. The origin of the induced chirality is consistent with the hybridization of highest occupied CdSe molecular orbitals with those of the chiral ligand.

One- and two-photon activated phototoxicity of conjugated porphyrin dimers with high two-photon absorption cross sections

Two-photon excited photodynamic therapy (PDT) has the potential to provide a highly targeted treatment for neoplastic diseases, as excitation can be pin-pointed to small volumes at the laser focus. In addition, two-photon PDT offers deeper penetration into mammalian tissue due to the longer wavelength of irradiation. Here we report the one-photon and two-photon excited PDT results for a collection of conjugated porphyrin dimers with high two-photon absorption cross sections. These dimers demonstrate high one-photon PDT efficacy against a human ovarian adenocarcinoma cell line (SK-OV-3) and exhibit no significant dark-toxicity at concentrations of up to 20 microM. Their one-photon excited PDT efficiencies, following irradiation at 657 nm, approach that of Visudyne, a drug used clinically for PDT. We investigated and optimised the effect of the photosensitizer concentration, incubation time and the light dose on the PDT efficacy of these dimers. These studies led to the selection of P2C2-NMeI as the most effective porphyrin dimer. We have demonstrated that P2C2-NMeI undergoes a two-photon activated process following excitation at 920 nm (3.6-6.8 mW, 300 fs, 90 MHz) and compared it to Visudyne. We conclude that the in vitro two-photon PDT efficacy of P2C2-NMeI is about twice that of Visudyne. This result highlights the potential of this series of porphyrin dimers for two-photon PDT.

Porphyrins as spectroscopic sensors for conformational studies of DNA

Molecular systems containing two or more interacting porphyrins show remarkable spectroscopic features that allow for a very sensitive detection of conformational changes on the microscale level by different methods, such as fluorescence and electronic circular dichroism (ECD). Covalent porphyrin-DNA assemblies can provide a CD profile (exciton couplet) within the porphyrin Soret band region which is very diagnostic for DNA conformational changes. Additionally, covalently linked porphyrins have been shown to function as DNA molecular caps and to stabilize the non-self-complementary non-Watson-Crick guanine-adenine DNA sequence via their strong π-π stacking.

Intramolecular rotation in a porphyrin dimer controls singlet oxygen production.

The efficiency with which a conjugated porphyrin dimer photosensitizes singlet oxygen production is shown to depend on the excitation wavelength, particularly in a viscous medium. This unprecedented behavior reflects viscosity-dependent dynamics that serve to interconvert two excited singlet state conformations of the porphyrin dimer. The efficiency of intersystem crossing from the two singlet state conformations to a common triplet state is shown to be different. In a viscous medium, each excited state conformation can be prepared selectively. Hence, wavelength-specific irradiation of the porphyrin allows fine control over the concentration of the triplet state produced which, in turn, is reflected in the photosensitized yield of singlet oxygen. This property may be beneficial for many applications requiring the controlled release of an oxidizing species, e.g., microfabrication and singlet oxygen-mediated cell death.

Achiral CdSe quantum dots exhibit optical activity in the visible region upon post-synthetic ligand exchange with D- or L-cysteine

Semiconductor cadmium selenide (CdSe) quantum dots (QDs) exhibited mirror-image circular dichroism (CD) spectra in the visible region (350-570 nm) after replacing the trioctylphosphine oxide/oleic acid ligands on achiral nanocrystals with D- and L-cysteines. Chiroptical properties of cysteine-capped CdSe QDs depend on their size and can be fine-tuned by changing the radius of QDs.

Chiroptical detection of condensed nickel(II)-Z-DNA in the presence of the B-DNA via porphyrin exciton coupled circular dichroism.

Here, we report a highly sensitive and specific chiroptical detection method of condensed left-handed Z-DNA in the presence of canonical right-handed B-DNA. The selective formation of a left-handed cytosine-guanine oligonucleotide (CG ODN) in the presence of a right-handed adenine-thymine oligonucleotide (AT ODN) was induced by millimolar concentrations of NiCl(2) and confirmed by electronic circular dichroism. The nickel(II) induced B- to Z-DNA transition of the CG ODN was accompanied by the concurrent condensation of the Ni(II)-Z-DNA, as confirmed by resonance light scattering, transmission spectroscopy, and centrifugation. The selective condensation of the CG ODN allowed its separation from the AT ODN using centrifugation. No structural changes were observed for the AT ODN upon addition of Ni(II). Anionic nickel(II) meso-tetra(4-sulfonatophenyl) porphyrin (NiTPPS) spectroscopically detected the left-handed Z-DNA in the Z-DNA/B-DNA mixture via a strong exciton coupled circular dichroism (ECCD) signal induced in the porphyrin Soret band absorption region. The bisignate ECCD signal originates from the assembly of achiral porphyrins into helical arrays by intermolecular interactions with the condensed Z-DNA scaffold. No induced CD signal was observed for the Ni(II)-B-DNA-NiTPPS complex. Hence, an unambiguous spectroscopic recognition of Ni(II) induced condensed Z-DNA in the presence of B-DNA is possible. The sensitivity of this chiroptical method was as low as 5% of the Z-DNA (4.4 μmol base pair concentration) in the presence of 95% B-DNA (80 μmol). Thus, NiTPPS is a highly sensitive probe for applications in biosensing via the CD signal amplification.