Fluorescence Quenching of Carboxy-Substituted Phthalocyanines Conjugated with Nanoparticles under High Stoichiometric Ratios

Background: The search of the approaches towards a photosensitizer’s conjugation with multifunctional nanoparticles is an important step in the development of photodynamic therapy techniques. Association of photosensitizer molecules with nanoparticles that perform the delivery function can lead to a change in the functional state of the photosensitizer. Methods: We studied the effects observed upon incorporation of octa- and hexadeca-carboxyphthalocyanines of zinc(II) and aluminum(III) (Pcs) into the polymer shell of nanoparticles with a semiconductor CdSe/CdS/ZnS core with various spectral and optical methods. Results: First, the interaction of Pc with the polymer shell leads to a change in the spectral properties of Pc; the effect strongly depends on the structure of the Pc molecule (number of carboxyl groups as well as the nature of the central cation in the macrocycle). Secondly, upon incorporation of several Pc molecules, concentration effects become significant, leading to Pc aggregation and/or nonradiative energy transfer between neighboring Pc molecules within a single nanoparticle. Conclusions: These processes lead to the decrease of a number of the Pc molecules in an excited state. Such effects should be taken into account during the development of multifunctional platforms for the delivery of photosensitizers, including the use of nanoparticles as enhancers of photosensitizer activity by energy transfer

Molecular Mechanism of Uptake of Cationic Photoantimicrobial Phthalocyanine across Bacterial Membranes Revealed by Molecular Dynamics Simulations

Phthalocyanines are aromatic macrocyclic compounds, which are structurally related to porphyrins. In clinical practice, phthalocyanines are used in fluorescence imaging and photodynamic therapy of cancer and noncancer lesions. Certain forms of the substituted polycationic metallophthalocyanines have been previously shown to be active in photodynamic inactivation of both Gram-negative and Gram-positive bacteria; one of them is zinc octakis­(cholinyl)­phthalocyanine (ZnPcChol⁸⁺). However, the molecular details of how these compounds translocate across bacterial membranes still remain unclear. In the present work, we have developed a coarse-grained (CG) molecular model of ZnPcChol⁸⁺ within the framework of the popular MARTINI CG force field. The obtained model was used to probe the solvation behavior of phthalocyanine molecules, which agreed with experimental results. Subsequently, it was used to investigate the molecular details of interactions between phthalocyanines and membranes of various compositions. The results demonstrate that ZnPcChol⁸⁺ has high affinity to both the inner and the outer model membranes of Gram-negative bacteria, although this species does not show noticeable affinity to the 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphatidylcholine membrane. Furthermore, we found out that the process of ZnPcChol⁸⁺ penetration toward the center of the outer bacterial membrane is energetically favorable and leads to its overall disturbance and formation of the aqueous pore. Such intramembrane localization of ZnPcChol⁸⁺ suggests their twofold cytotoxic effect on bacterial cells: (1) via induction of lipid peroxidation by enhanced production of reactive oxygen species (i.e., photodynamic toxicity); (2) via rendering the bacterial membrane more permeable for additional Pc molecules as well as other compounds. We also found that the kinetics of penetration depends on the presence of phospholipid defects in the lipopolysaccharide leaflet of the outer membrane and the type of counterions, which stabilize it. Thus, the results of our simulations provide a detailed molecular view of ZnPcChol⁸⁺ “self-promoted uptake”, the pathway previously proposed for some small molecules crossing the outer bacterial membrane

Molecular Mechanism of Uptake of Cationic Photoantimicrobial Phthalocyanine across Bacterial Membranes Revealed by Molecular Dynamics Simulations

Phthalocyanines are aromatic macrocyclic compounds, which are structurally related to porphyrins. In clinical practice, phthalocyanines are used in fluorescence imaging and photodynamic therapy of cancer and noncancer lesions. Certain forms of the substituted polycationic metallophthalocyanines have been previously shown to be active in photodynamic inactivation of both Gram-negative and Gram-positive bacteria; one of them is zinc octakis­(cholinyl)­phthalocyanine (ZnPcChol⁸⁺). However, the molecular details of how these compounds translocate across bacterial membranes still remain unclear. In the present work, we have developed a coarse-grained (CG) molecular model of ZnPcChol⁸⁺ within the framework of the popular MARTINI CG force field. The obtained model was used to probe the solvation behavior of phthalocyanine molecules, which agreed with experimental results. Subsequently, it was used to investigate the molecular details of interactions between phthalocyanines and membranes of various compositions. The results demonstrate that ZnPcChol⁸⁺ has high affinity to both the inner and the outer model membranes of Gram-negative bacteria, although this species does not show noticeable affinity to the 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphatidylcholine membrane. Furthermore, we found out that the process of ZnPcChol⁸⁺ penetration toward the center of the outer bacterial membrane is energetically favorable and leads to its overall disturbance and formation of the aqueous pore. Such intramembrane localization of ZnPcChol⁸⁺ suggests their twofold cytotoxic effect on bacterial cells: (1) via induction of lipid peroxidation by enhanced production of reactive oxygen species (i.e., photodynamic toxicity); (2) via rendering the bacterial membrane more permeable for additional Pc molecules as well as other compounds. We also found that the kinetics of penetration depends on the presence of phospholipid defects in the lipopolysaccharide leaflet of the outer membrane and the type of counterions, which stabilize it. Thus, the results of our simulations provide a detailed molecular view of ZnPcChol⁸⁺ “self-promoted uptake”, the pathway previously proposed for some small molecules crossing the outer bacterial membrane

Carboranyl-Chlorin e6 as a Potent Antimicrobial Photosensitizer.

Antimicrobial photodynamic inactivation is currently being widely considered as alternative to antibiotic chemotherapy of infective diseases, attracting much attention to design of novel effective photosensitizers. Carboranyl-chlorin-e6 (the conjugate of chlorin e6 with carborane), applied here for the first time for antimicrobial photodynamic inactivation, appeared to be much stronger than chlorin e6 against Gram-positive bacteria, such as Bacillus subtilis, Staphyllococcus aureus and Mycobacterium sp. Confocal fluorescence spectroscopy and membrane leakage experiments indicated that bacteria cell death upon photodynamic treatment with carboranyl-chlorin-e6 is caused by loss of cell membrane integrity. The enhanced photobactericidal activity was attributed to the increased accumulation of the conjugate by bacterial cells, as evaluated both by centrifugation and fluorescence correlation spectroscopy. Gram-negative bacteria were rather resistant to antimicrobial photodynamic inactivation mediated by carboranyl-chlorin-e6. Unlike chlorin e6, the conjugate showed higher (compared to the wild-type strain) dark toxicity with Escherichia coli ΔtolC mutant, deficient in TolC-requiring multidrug efflux transporters

Potassium permeability of BACE- and chlorin e₆-treated <i>B</i>. <i>subtilis</i> (A) and <i>E</i>. <i>coli</i> (B) cells after illumination measured by a potassium-selective electrode.

<p>Cells were incubated with photosensitizers for 10 min prior to illumination (4 J/cm²). Nigericin (1 μM) was added at the end of each recording to induce full potassium efflux.</p

Dark toxicity of BACE and chlorin e₆ towards WT <i>Escherichia coli</i> and the <i>E</i>. <i>coli</i> Δ<i>tolC</i> mutant.

<p>Chlorin e₆ or BACE (100 nM—50 μM) were added to bacterial cultures (1–5*10⁶ cells/ml), placed in 96-well plates. Cell density was determined by absorbance at 600 nm. After that bacteria were allowed to grow in the dark within 21 hours and cell density was measured again.</p

Accumulation of BACE and chlorin e₆ by <i>B</i>. <i>subtilis</i> (A) and <i>E</i>. <i>coli</i> (B) cells monitored by FCS.

<p>Fluorescence intensity traces of photosensitizers were recorded with the FCS set-up in the presence or absence of bacterial cells (10⁶ per ml). <i>Inserts</i>: Corresponding dependences of the number of peaks with the fluorescence intensity <i>F</i> exceeding the threshold <i>F</i>_<i>0</i>, <i>n(F>F</i>_<i>0</i><i>)</i>, on the value of <i>F</i>_<i>0</i>.</p

Uptake of BACE or chlorin e₆ by <i>B</i>. <i>subtilis</i> (A) and <i>E</i>. <i>coli</i> (B) cells.

<p>Cells were incubated for 10 min in the dark with the indicated amount of a photosensitizer. The pellet obtained after centrifugation was treated with 0.1 M NaOH / 1% SDS. The photosensitizer concentration was determined from its fluorescence by using a calibration curve for the solutions of different concentrations in 0.1 M NaOH / 1% SDS.</p

Photodynamic action on liposomes made from <i>E</i>. <i>coli</i> total lipids.

<p>Carboxyfluorescein leakage from liposomes induced by 1-min exposure to visible light (4 J/cm²) in the presence of BACE (red curve), chlorin e₆ (blue curve), and a control without illumination and photosensitizers (green curve). The solution was 100 mM KCl, 10 mM Tris, 10 mM MES, pH 7.0. Lipid concentration was 5 μg/ml.</p

Phototoxicity and dark toxicity of BACE and chlorin e₆ towards <i>Mycobacterium sp</i>. (A), <i>Staphyllococcus aureus</i> (B), and <i>Bacillus subtilis</i> (C) evaluated by CFU counts.

<p>Phototoxicity was determined upon illumination with red light at 4 J/cm². The data shown are mean values ± standard deviations.</p