Structural Data Showing the Existence of LDI Bonds between the Rings of Dimeric Cofacial Siloxysilicon Phthalocyanines.

In a pair of earlier papers, the existence of long directional interaction bonds, LDI bonds, was postulated on the basis of data for cofacial oligomeric siloxysilicon phthalocyanines from this laboratory and data for other cofacial oligomeric phthalocyanines from the literature. However, the combined data are not fully suited to the purpose for which they were used. Here an alternative approach is taken in which a carefully chosen group of dimeric cofacial siloxysilicon phthalocyanines is used. Structural data derived from these phthalocyanines is examined in some detail to determine where it conforms to normal expectations and where it does not. To a high degree of certainty, consideration of the results obtained shows that long directional (LDI) bonds exist in dimeric cofacial siloxysilicon phthalocyanines. The new data also provide an opportunity for other research on chemical bonds.

Dual Receptor-Targeted Theranostic Nanoparticles for Localized Delivery and Activation of Photodynamic Therapy Drug in Glioblastomas.

Targeting gold nanoparticles (AuNPs) with two or more receptor binding peptides has been proposed to address intratumoral heterogeneity of glioblastomas that overexpress multiple cell surface receptors to ultimately improve therapeutic efficacy. AuNPs conjugated with peptides against both the epidermal growth factor and transferrin receptors and loaded with the photosensitizer phthalocyanine 4 (Pc 4) have been designed and compared with monotargeted AuNPs for in vitro and in vivo studies. The (EGFpep+Tfpep)-AuNPs-Pc 4 with a particle size of ∼41 nm improved both specificity and worked synergistically to decrease time of maximal accumulation in human glioma cells that overexpressed two cell surface receptors as compared to cells that overexpressed only one. Enhanced cellular association and increased cytotoxicity were achieved. In vivo studies show notable accumulation of these agents in the brain tumor regions.

Synthesis, properties and drug potential of the photosensitive alkyl- and alkylsiloxy-ligated silicon phthalocyanine Pc 227.

The photosensitive, alkyl- and alkylsiloxy-ligated silicon phthalocyanine, SiPc[(CH2)3SH][OSi(CH3)2(CH2)3N(CH3)2], Pc 227, has been prepared and characterized. This phthalocyanine yields the experimental photodynamic therapy (PDT) drug Pc 4, SiPc[OH][OSi(CH3)2(CH2)3N(CH3)2], when irradiated with red light. To provide an understanding of the process by which Pc 227 and other alkyl-alkylsiloxysilicon phthalocyanines such as Pc 227 are photolyzed, bond dissociation energy, natural bond orbital (NBO) charge distribution, spin density distribution, nucleus-independent chemical shift (NICS), and electron localization function (ELF) calculations have been carried out on two models related to it. These show that the lowest energy pathway for the photolysis of Pc 227 is a homolysis involving a phthalocyanine π radical having a low SiPc-C bond dissociation energy. The promise of the results of this study for synthetic chemistry and drug development is discussed.

The Synthesis and Characterization of a Group of Transition Metal Octabutoxynaphthalocyanines and the Absorption and Emission Properties of the Co, Rh, Ir, Ni, Pd and Pt Members of This Group.

The synthesis and photophysical properties of new metallo-octabutoxynaphthalocyanines with Rh(III), Ir(III), and Pt(II) are reported. Various metals were inserted into the metal-free octabutoxynaphthalocyanine and the resultant metal complexes were fully characterized by NMR, UV-vis spectroscopy, and mass spectrometry. The absorption and emission properties of these new complexes were also examined and compared to those of Co(II), Ni(II), and Pd(II) octabutoxynaphthalocyanines. The results provide useful information to understand the effect of these transition metals on the properties of this macrocyclic ring.

Long directional interactions (LDIs) in oligomeric cofacial silicon phthalocyanines and other oligomeric and polymeric cofacial phthalocyanines.

Crystal structures have been determined for the three-member set of cofacial silicon phthalocyanines, ((n-C(6)H(13))(3)SiO)[SiPcO](1-3)(Si(n-C(6)H(13))(3)). The staggering angles between adjacent rings in the dimer and trimer of this set are ∼16°. The interactions leading to these angles have been investigated by the atoms-in-molecules (AIM) and reduced-density-gradient (RDG) methods. The results show that long directional interactions (LDIs) are responsible for these angles. A survey of the staggering angles in various cofacial phthalocyanines described in the literature has revealed the existence of significant LDIs in a number of them. It is apparent that in many cases the ability of LDIs to dominate the forces giving rise to the staggering angles observed in cofacial phthalocyanines depends on their inter-ring separations.

Deep penetration of a PDT drug into tumors by noncovalent drug-gold nanoparticle conjugates.

Efficient drug delivery to tumors is of ever-increasing importance. Single-visit diagnosis and treatment sessions are the goal of future theranostics. In this work, a noncovalent PDT cancer drug-gold nanoparticle (Au NP) conjugate system performed a rapid drug release and deep penetration of the drug into tumors within hours. The drug delivery mechanism of the PDT drug through Au NPs into tumors by passive accumulation was investigated via fluorescence imaging, elemental analysis, and histological staining. The pharmacokinetics of the conjugates over a 7-day test period showed rapid drug excretion, as monitored via the fluorescence of the drug in urine. Moreover, the biodistribution of Au NPs in this study period indicated clearance of the NPs from the mice. This study suggests that noncovalent delivery via Au NPs provides an attractive approach for cancer drugs to penetrate deep into the center of tumors.

Addressing brain tumors with targeted gold nanoparticles: a new gold standard for hydrophobic drug delivery?

EGF-modified Au NP-Pc 4 conjugates showed 10-fold improved selectivity to the brain tumor compared to untargeted conjugates. The hydrophobic photodynamic therapy drug Pc 4 can be delivered efficiently into glioma brain tumors by EGF peptide-targeted Au NPs. Compared to the untargeted conjugates, EGF-Au NP-Pc 4 conjugates showed 10-fold improved selectivity to the brain tumor. This delivery system holds promise for future delivery of a wider range of hydrophobic therapeutic drugs for the treatment of hard-to-reach cancers.

Near-infrared-emitting phthalocyanines. A combined experimental and density functional theory study of the structural, optical, and photophysical properties of Pd(II) and Pt(II) α-butoxyphthalocyanines.

The structural, optical, and photophysical properties of 1,4,8,11,15,18,22,25-octabutoxyphthalocyaninato-palladium(II), PdPc(OBu)(8), and the newly synthesized platinum analogue PtPc(OBu)(8) are investigated combining X-ray crystallography, static and transient absorption spectroscopy, and relativistic zeroth-order regular approximation (ZORA) Density Functional Theory (DFT)/Time Dependent DFT (TDDFT) calculations where spin-orbit coupling (SOC) effects are explicitly considered. The results are compared to those previously reported for NiPc(OBu)(8) (J. Phys. Chem. A 2005, 109, 2078) in an effort to highlight the effect of the central metal on the structural and photophysical properties of the group 10 transition metal octabutoxyphthalocyanines. Different from the nickel analogue, PdPc(OBu)(8) and PtPc(OBu)(8) show a modest and irregular saddling distortion of the macrocycle, but share with the first member of the group similar UV-vis spectra, with the deep red and intense Q-band absorption experiencing a blue shift down the group, as observed in virtually all tetrapyrrolic complexes of this triad. The blue shift of the Q-band along the MPc(OBu)(8) (M = Ni, Pd, Pt) series is interpreted on the basis of the metal-induced electronic structure changes. Besides the intense deep red absorption, the title complexes exhibit a distinct near-infrared (NIR) absorption due to a transition to the double-group 1E (π,π*) state, which is dominated by the lowest single-group (3)E (π,π*) state. Unlike NiPc(OBu)(8), which is nonluminescent, PdPc(OBu)(8) and PtPc(OBu)(8) show both deep red fluorescence emission and NIR phosphorescence emission. Transient absorption experiments and relativistic spin-orbit TDDFT calculations consistently indicate that fluorescence and phosphorescence emissions occur from the S(1)(π,π*) and T(1)(π,π*) states, respectively, the latter being directly populated from the former, and the triplet state decays directly to the S(0) surface (the triplet lifetime in deaerated benzene solution was 3.04 µs for Pd and 0.55 µs for Pt). Owing to their triplet properties, PdPc(OBu)(8) and PtPc(OBu)(8) have potential for use in photodynamic therapy (PDT) and are potential candidates for NIR light emitting diodes or NIR emitting probes.

Long, directional interactions in cofacial silicon phthalocyanine oligomers.

Single crystal structures have been determined for the three cofacial, oxygen-bridged, silicon phthalocyanine oligomers, [((CH(3))(3)SiO)(2)(CH(3))SiO](SiPcO)(2-4)[Si(CH(3))(OSi(CH(3))(3))(2)], and for the corresponding monomer. The data for the oligomers give structural parameters for a matching set of three cofacial, oxygen-bridged silicon phthalocyanine oligomers for the first time. The staggering angles between the six adjacent cofacial ring pairs in the three oligomers are not in a random distribution nor in a cluster at the intuitively expected angle of 45° but rather are in two clusters, one at an angle of 15° and the other at an angle of 41°. These two clusters lead to the conclusion that long, directional interactions (LDI) exist between the adjacent ring pairs. An understanding of these interactions is provided by atoms-in-molecules (AIM) and reduced-density-gradient (RDG) studies. A survey of the staggering angles in other single-atom-bridged, cofacial phthalocyanine oligomers provides further evidence for the existence of LDI between cofacial phthalocyanine ring pairs in single-atom-bridged phthalocyanine oligomers.

Delivery and efficacy of a cancer drug as a function of the bond to the gold nanoparticle surface.

In this feature article, gold nanoparticle conjugates loaded with phthalocyanine-based PDT drugs are prepared and tested for delivery efficiency and PDT efficacy on HeLa cancer cells. It could be shown that the delivery and PDT outcome are strongly affected by the bond that links the drug load to the nanoparticle surface. Whereas labile amino adsorption to the Au nanoparticle surface allows for efficient drug release into the cancer cells and for efficient PDT, a covalent thiol bond to the Au nanoparticle leads to the delivery of the drug into cell vesicles, and no PDT effect is observed. This work highlights the importance of carefully choosing the interaction between drug molecules and the nanoparticle surface.

Structural factors and mechanisms underlying the improved photodynamic cell killing with silicon phthalocyanine photosensitizers directed to lysosomes versus mitochondria.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose-response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

Oxidative modification of cytochrome c by singlet oxygen.

Singlet oxygen ((1)O(2)) is a reactive oxygen species that may be generated in biological systems. Photodynamic therapy generates (1)O(2) by photoexcitation of sensitizers resulting in intracellular oxidative stress and induction of apoptosis. (1)O(2) oxidizes amino acid side chains of proteins and inactivates enzymes when generated in vitro. Among proteogenic amino acids, His, Tyr, Met, Cys, and Trp are known to be oxidized by (1)O(2) at physiological pH. However, there is a lack of direct evidence of oxidation of proteins by (1)O(2). Because (1)O(2) is difficult to detect in cells, identifying oxidized cellular products uniquely derived from (1)O(2) could serve as a marker of its presence. In the present study, (1)O(2) reactions with model peptides analyzed by tandem mass spectrometry provide insight into the mass of prominent adducts formed with the reactive amino acids. Analysis by MALDI-TOF and tandem mass spectrometry of peptides of cytochrome c exposed to (1)O(2) generated by photoexcitation of the phthalocyanine Pc 4 showed unique oxidation products, which might be used as markers of the presence of (1)O(2) in the mitochondrial intermembrane space. Differences in the elemental composition of the oxidized amino acid residues observed with cytochrome c and the model peptides suggest that the protein environment can affect the oxidation pathway.

Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization.

10-N-Nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipin-deficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.

Effect of the functionalization of the axial phthalocyanine ligands on the energy transfer in QD-based donor-acceptor pairs.

This study examines the electronic coupling between quantum dots (QDs) and molecules on their surfaces as a function of the modality of their interaction. As a probe, the energy transfer (ET) between CdSe QDs and phthalocyanines (Pcs) was monitored and evaluated with regard to the functionalization of the axial phthalocyanine ligand, bulkiness of the functional group bridging the QD donor and Pc acceptor, and the number of the functionalized axial ligands. New silicon PCs and their conjugates with CdSe QDs were synthesized. The ET efficiency and kinetics were studied by steady state and femtosecond time-resolved absorption spectroscopy. We observed a decrease in ET efficiency with the increase in functional group bulkiness, which could be explained by increasing steric hindrance between the ET pair. In addition, a higher ET efficiency was observed for amino and thiol functionalized Pcs compared to Pcs without functional group on the axial alkyl chain.

Irradiation-induced enhancement of Pc 4 fluorescence and changes in light scattering are potential dosimeters for Pc 4-PDT.

Phthalocyanine 4 (Pc 4) is a promising photosensitizer currently in clinical trials. Photobiological responses to Pc 4 photodynamic therapy (Pc 4-PDT) have been characterized extensively, but relatively little has been done to evaluate dose metrics for this sensitizer. We describe an irradiation-induced increase in fluorescence in tumor cell monolayers. This increase is due solely to enhanced fluorescence from Pc 4, as confirmed by confocal spectroscopy. In EMT6 cells incubated with 250 nM Pc 4 for 24 h, the maximum increase in fluorescence is approximately 3.7-fold above baseline levels. This increase occurs over a range of fluences, 0.05-0.6 J cm(-2), where clonogenic survival decreases by 3 orders of magnitude. Light scattering measurements performed on similarly treated EMT6 cells in suspension suggested a Pc 4-PDT-mediated mitochondrial swelling of approximately 13% at 0.6 J cm(-2), where fluorescence enhancement saturates under these treatment conditions. Fluorescence imaging and light scattering experiments performed at a five-fold lower Pc 4 incubation concentration revealed a reduced fluorescence enhancement at a five-fold higher fluence, which produced comparable mitochondrial swelling. Taken together, these data suggest that Pc 4 is initially aggregated at high local concentration in mitochondria and that irradiation relaxes the quenching of Pc 4 fluorescence through a mechanism that may involve mitochondrial swelling.

Fluorescence resonance energy transfer reveals a binding site of a photosensitizer for photodynamic therapy.

Phthalocyanine (Pc) 4, like many photosensitizers for photodynamic therapy (PDT), localizes to intracellular membranes, especially mitochondria. Pc 4-PDT photodamages Bcl-2 and Bcl-xL, antiapoptotic proteins interacting with the permeability transition pore complex that forms at contact sites between the inner and outer mitochondrial membranes. These complexes and the inner membrane are unique in containing the phospholipid cardiolipin. Nonyl-acridine orange (NAO) is a specific probe of cardiolipin. Here we show evidence for fluorescence resonance energy transfer from NAO to Pc 4, defining a binding site for the photosensitizer. This observation establishes an innovative tool for exploring the localization of other photosensitizers and additional fluorescent, mitochondrion-localizing drugs having appropriate spectral properties.

Theranostic Agents for Photodynamic Therapy of Prostate Cancer by Targeting Prostate-Specific Membrane Antigen.

Prostatectomy has been the mainstay treatment for men with localized prostate cancer. Surgery, however, often can result in major side effects, which are caused from damage and removal of nerves and muscles surrounding the prostate. A technology that can help surgeons more precisely identify and remove prostate cancer resulting in a more complete prostatectomy is needed. Prostate-specific membrane antigen (PSMA), a type II membrane antigen highly expressed in prostate cancer, has been an attractive target for imaging and therapy. The objective of this study is to develop low molecular weight PSMA-targeted photodynamic therapy (PDT) agents, which would provide image guidance for prostate tumor resection and allow for subsequent PDT to eliminate unresectable or remaining cancer cells. On the basis of our highly negatively charged, urea-based PSMA ligand PSMA-1, we synthesized two PSMA-targeting PDT conjugates named PSMA-1-Pc413 and PSMA-1-IR700. In in vitro cellular uptake experiments and in vivo animal imaging experiments, the two conjugates demonstrated selective and specific uptake in PSMA-positive PC3pip cells/tumors, but not in PSMA-negative PC3flu cells/tumors. Further in vivo photodynamic treatment proved that the two PSMA-1-PDT conjugates can effectively inhibit PC3pip tumor progression. The two PSMA-1-PDT conjugates reported here may have the potential to aid in the detection and resection of prostate cancers. It may also allow for the identification of unresectable cancer tissue and PDT ablation of such tissue after surgical resection with potentially less damage to surrounding tissues. Mol Cancer Ther; 15(8); 1834-44. ©2016 AACR.

Photothermal sensitisation as a novel therapeutic approach for tumours: studies at the cellular and animal level.

Irradiation of B78H1 murine amelanotic melanoma cells with 850 nm light emitted from a Ti:sapphire laser, operated in a pulsed mode at high fluence rates and in the presence of Ni(II)-octabutoxy-naphthalocyanine (NiNc), promoted a photothermally sensitised process leading to fast and irreversible cell death. This resulted in the ejection of a consistent mass of cytoplasmic material from the irradiated cells that was detected by scanning electron microscopy. The extensive chemical and mechanical damage was probably caused by the photoinduced generation of an acoustic shock wave. The efficiency of the photoprocess was modulated by intracellular concentration of NiNc and maximally by the formation of aggregated naphthalocyanine clusters in specific subcellular areas. Very similar results were obtained upon irradiation of NiNc-loaded C32 human amelanotic melanoma cells and transformed murine HT-1080 and HaCaT fibroblasts. From these results, photothermal sensitisation appears to be a general phenomenon and preliminary studies with mice bearing subcutaneously transplanted amelanotic melanomas, irradiated with 850 nm light 24 h after intravenous injection of NiNc, suggest that this approach has potential for the therapy of some types of skin tumours.

Transferrin receptor-targeted theranostic gold nanoparticles for photosensitizer delivery in brain tumors.

Therapeutic drug delivery across the blood-brain barrier (BBB) is not only inefficient, but also nonspecific to brain stroma. These are major limitations in the effective treatment of brain cancer. Transferrin peptide (Tfpep) targeted gold nanoparticles (Tfpep-Au NPs) loaded with the photodynamic pro-drug, Pc 4, have been designed and compared with untargeted Au NPs for delivery of the photosensitizer to brain cancer cell lines. In vitro studies of human glioma cancer lines (LN229 and U87) overexpressing the transferrin receptor (TfR) show a significant increase in cellular uptake for targeted conjugates as compared to untargeted particles. Pc 4 delivered from Tfpep-Au NPs clusters within vesicles after targeting with the Tfpep. Pc 4 continues to accumulate over a 4 hour period. Our work suggests that TfR-targeted Au NPs may have important therapeutic implications for delivering brain tumor therapies and/or providing a platform for noninvasive imaging.

Peptide-Targeted Gold Nanoparticles for Photodynamic Therapy of Brain Cancer.

Targeted drug delivery using epidermal growth factor peptide-targeted gold nanoparticles (EGFpep-Au NPs) is investigated as a novel approach for delivery of photodynamic therapy (PDT) agents, specifically Pc 4, to cancer. In vitro studies of PDT show that EGFpep-Au NP-Pc 4 is twofold better at killing tumor cells than free Pc 4 after increasing localization in early endosomes. In vivo studies show that targeting with EGFpep-Au NP-Pc 4 improves accumulation of fluorescence of Pc 4 in subcutaneous tumors by greater than threefold compared with untargeted Au NPs. Targeted drug delivery and treatment success can be imaged via the intrinsic fluorescence of the PDT drug Pc 4. Using Pc 4 fluorescence, it is demonstrated in vivo that EGFpep-Au NP-Pc 4 impacts biodistribution of the NPs by decreasing the initial uptake by the reticuloendothelial system (RES) and by increasing the amount of Au NPs circulating in the blood 4 h after IV injection. Interestingly, in vivo PDT with EGFpep-Au NP-Pc 4 results in interrupted tumor growth when compared with EGFpep-Au NP control mice when selectively activated with light. These data demonstrate that EGFpep-Au NP-Pc 4 utilizes cancer-specific biomarkers to improve drug delivery and therapeutic efficacy over untargeted drug delivery.