A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

Targeting OGG1 and PARG radiosensitises head and neck cancer cells to high-LET protons through complex DNA damage persistence.

Complex DNA damage (CDD), containing two or more DNA lesions within one or two DNA helical turns, is a signature of ionising radiation (IR) and contributes significantly to the therapeutic effect through cell killing. The levels and complexity of CDD increases with linear energy transfer (LET), however, the specific cellular response to this type of DNA damage and the critical proteins essential for repair of CDD is currently unclear. We performed an siRNA screen of ~240 DNA damage response proteins to identify those specifically involved in controlling cell survival in response to high-LET protons at the Bragg peak, compared to low-LET entrance dose protons which differ in the amount of CDD produced. From this, we subsequently validated that depletion of 8-oxoguanine DNA glycosylase (OGG1) and poly(ADP-ribose) glycohydrolase (PARG) in HeLa and head and neck cancer cells leads to significantly increased cellular radiosensitivity specifically following high-LET protons, whilst no effect was observed after low-LET protons and X-rays. We subsequently confirmed that OGG1 and PARG are both required for efficient CDD repair post-irradiation with high-LET protons. Importantly, these results were also recapitulated using specific inhibitors for OGG1 (TH5487) and PARG (PDD00017273). Our results suggest OGG1 and PARG play a fundamental role in the cellular response to CDD and indicate that targeting these enzymes could represent a promising therapeutic strategy for the treatment of head and neck cancers following high-LET radiation.

Phototoxicity of cyclometallated Ir(III) complexes bearing a thio-bis-benzimidazole ligand, and its monodentate analogue, as potential PDT photosensitisers in cancer cell killing.

Two novel cyclometallated iridium(III) complexes have been prepared with one bidentate or two monodentate imidazole-based ligands, 1 and 2, respectively. The complexes showed intense emission with long lifetimes of the excited state. Femtosecond transient absorption experiments established the nature of the lowest excited state as 3IL state. Singlet oxygen generation with good yields (40% for 1 and 82% for 2) was established by detecting 1O2 directly, through its emission at 1270 nm. Photostability studies were also performed to assess the viability of the complexes as photosensitizers (PS) for photodynamic therapy (PDT). Complex 1 was selected as a good candidate to investigate light-activated killing of cells, whilst complex 2 was found to be toxic in the dark and unstable under light. Complex 1 demonstrated high phototoxicity indexes (PI) in the visible region, PI > 250 after irradiation at 405 nm and PI > 150 at 455 nm, in EJ bladder cancer cells.

Gelatin-containing porous polycaprolactone PolyHIPEs as substrates for 3D breast cancer cell culture and vascular infiltration.

Tumour survival and growth are reliant on angiogenesis, the formation of new blood vessels, to facilitate nutrient and waste exchange and, importantly, provide a route for metastasis from a primary to a secondary site. Whilst current models can ensure the transport and exchange of nutrients and waste via diffusion over distances greater than 200 µm, many lack sufficient vasculature capable of recapitulating the tumour microenvironment and, thus, metastasis. In this study, we utilise gelatin-containing polymerised high internal phase emulsion (polyHIPE) templated polycaprolactone-methacrylate (PCL-M) scaffolds to fabricate a composite material to support the 3D culture of MDA-MB-231 breast cancer cells and vascular ingrowth. Firstly, we investigated the effect of gelatin within the scaffolds on the mechanical and chemical properties using compression testing and FTIR spectroscopy, respectively. Initial in vitro assessment of cell metabolic activity and vascular endothelial growth factor expression demonstrated that gelatin-containing PCL-M polyHIPEs are capable of supporting 3D breast cancer cell growth. We then utilised the chick chorioallantoic membrane (CAM) assay to assess the angiogenic potential of cell-seeded gelatin-containing PCL-M polyHIPEs, and vascular ingrowth within cell-seeded, surfactant and gelatin-containing scaffolds was investigated via histological staining. Overall, our study proposes a promising composite material to fabricate a substrate to support the 3D culture of cancer cells and vascular ingrowth.

Progesterone receptor expression contributes to gemcitabine resistance at higher ECM stiffness in breast cancer cell lines.

Chemoresistance poses a great barrier to breast cancer treatment and is thought to correlate with increased matrix stiffness. We developed two-dimensional (2D) polyacrylamide (PAA) and three-dimensional (3D) alginate in vitro models of tissue stiffness that mimic the stiffness of normal breast and breast cancer. We then used these to compare cell viability in response to chemotherapeutic treatment. In both 2D and 3D we observed that breast cancer cell growth and size was increased at a higher stiffness corresponding to tumours compared to normal tissue. When chemotherapeutic response was measured, a specific differential response in cell viability was observed for gemcitabine in 2 of the 7 breast cancer cell lines investigated. MCF7 and T-47D cell lines showed gemcitabine resistance at 4 kPa compared to 500 Pa. These cell lines share a common phenotype of progesterone receptor (PGR) expression and, indeed, pre-treatment with the selective progesterone receptor modulator (SPRM) mifepristone abolished resistance to gemcitabine at high stiffness. Our data reveals that combined treatment with SPRMs may therefore help in reducing resistance to gemcitabine in stiffer breast tumours which are PGR positive.

Morphological Response in Cancer Spheroids for Screening Photodynamic Therapy Parameters.

Photodynamic therapy (PDT) is a treatment which uses light-activated compounds to produce reactive oxygen species, leading to membrane damage and cell death. Multicellular cancer spheroids are a preferable alternative for PDT evaluation in comparison to monolayer cell cultures due to their ability to better mimic in vivo avascular tumour characteristics such as hypoxia and cell-cell interactions, low cost, and ease of production. However, inconsistent growth kinetics and drug responsiveness causes poor experimental reproducibility and limits their usefulness. Herein, we used image analysis to establish a link between human melanoma C8161 spheroid morphology and drug responsiveness. Spheroids were pre-selected based on sphericity, area, and diameter, reducing variation in experimental groups before treatment. Spheroid morphology after PDT was analyzed using AnaSP and ReViSP, MATLAB-based open-source software, obtaining nine different parameters. Spheroids displayed a linear response between biological assays and morphology, with area (R2 = 0.7219) and volume (R2 = 0.6138) showing the best fit. Sphericity, convexity, and solidity were confirmed as poor standalone indicators of spheroid viability. Our results indicate spheroid morphometric parameters can be used to accurately screen inefficient treatment combinations of novel compounds.

Increased Replication Stress Determines ATR Inhibitor Sensitivity in Neuroblastoma Cells.

Despite intensive high-dose multimodal therapy, high-risk neuroblastoma (NB) confers a less than 50% survival rate. This study investigates the role of replication stress in sensitivity to inhibition of Ataxia telangiectasia and Rad3-related (ATR) in pre-clinical models of high-risk NB. Amplification of the oncogene MYCN always imparts high-risk disease and occurs in 25% of all NB. Here, we show that MYCN-induced replication stress directly increases sensitivity to the ATR inhibitors VE-821 and AZD6738. PARP inhibition with Olaparib also results in replication stress and ATR activation, and sensitises NB cells to ATR inhibition independently of MYCN status, with synergistic levels of cell death seen in MYCN expressing ATR- and PARP-inhibited cells. Mechanistically, we demonstrate that ATR inhibition increases the number of persistent stalled and collapsed replication forks, exacerbating replication stress. It also abrogates S and G2 cell cycle checkpoints leading to death during mitosis in cells treated with an ATR inhibitor combined with PARP inhibition. In summary, increased replication stress through high MYCN expression, PARP inhibition or chemotherapeutic agents results in sensitivity to ATR inhibition. Our findings provide a mechanistic rationale for the inclusion of ATR and PARP inhibitors as a potential treatment strategy for high-risk NB.

In Vitro Low-Fluence Photodynamic Therapy Parameter Screening Using 3D Tumor Spheroids Shows that Fractionated Light Treatments Enhance Phototoxicity.

The evaluation of novel photosensitizers (PSs) for photodynamic therapy (PDT) is difficult due to the limitations of two-dimensional cell culture and multiple parameters (dose, light intensity, uptake time), which complicate progression to in vivo experiments and clinical translation. Three-dimensional (3D) cell culture models like multicellular cancer tumor spheroids (MCTS) show great similarities to in vivo avascular tumor conditions, improving the speed and accuracy of screening novel compounds with various treatment combinations. In this study, we utilize C8161 human melanoma spheroids to screen PDT treatment combinations using protoporphyrin IX (PpIX) and drug-loaded carbon dot (CD) conjugates PpIX-CD and PpIX@CD at ultralow fluence values (<10 J/cm2). Conjugates show equivalent light-induced damage to PpIX from 1 µg/mL with significantly less dark cytotoxicity up to 72 h after exposure, shown by LDH release and dsDNA content. Fractionated treatments, carried out by dividing light exposure with 24 h intervals, demonstrate an enhanced PDT effect compared to single exposure at equal concentrations. Light sheet fluorescence microscopy combined with live/dead staining demonstrates that spheroids sustain extensive damage after PDT, with PpIX and PpIX-CD showing improved uptake compared to PpIX@CD. We show that PDT parameter screening can be carried out using a low-cost and convenient combination of assays to improve the efficiency of evaluating novel compounds.

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts.

Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.

Poly(ADP-Ribose) Glycohydrolase (PARG) vs. Poly(ADP-Ribose) Polymerase (PARP) - Function in Genome Maintenance and Relevance of Inhibitors for Anti-cancer Therapy.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that catalyze the addition of poly(ADP-ribose) (PAR) subunits onto themselves and other acceptor proteins. PARPs are known to function in a large range of cellular processes including DNA repair, DNA replication, transcription and modulation of chromatin structure. Inhibition of PARP holds great potential for therapy, especially in cancer. Several PARP1/2/3 inhibitors (PARPi) have had success in treating ovarian, breast and prostate tumors harboring defects in the homologous recombination (HR) DNA repair pathway, especially BRCA1/2 mutated tumors. However, treatment is limited to specific sub-groups of patients and resistance can occur, limiting the use of PARPi. Poly(ADP-ribose) glycohydrolase (PARG) reverses the action of PARP enzymes, hydrolysing the ribose-ribose bonds present in poly(ADP-ribose). Like PARPs, PARG is involved in DNA replication and repair and PARG depleted/inhibited cells show increased sensitivity to DNA damaging agents. They also display an accumulation of perturbed replication intermediates which can lead to synthetic lethality in certain contexts. In addition, PARG is thought to play an important role in preventing the accumulation of cytoplasmic PAR and therefore parthanatos, a caspase-independent PAR-mediated type of cell death. In contrast to PARP, the therapeutic potential of PARG has been largely ignored. However, several recent papers have demonstrated the exciting possibilities that inhibitors of this enzyme may have for cancer treatment, both as single agents and in combination with cytotoxic drugs and radiotherapy. This article discusses what is known about the functions of PARP and PARG and the potential future implications of pharmacological inhibition in anti-cancer therapy.

Therapeutic vulnerabilities in the DNA damage response for the treatment of ATRX mutant neuroblastoma.

BACKGROUND: In neuroblastoma, genetic alterations in ATRX, define a distinct poor outcome patient subgroup. Despite the need for new therapies, there is a lack of available models and a dearth of pre-clinical research. METHODS: To evaluate the impact of ATRX loss of function (LoF) in neuroblastoma, we utilized CRISPR-Cas9 gene editing to generate neuroblastoma cell lines isogenic for ATRX. We used these and other models to identify therapeutically exploitable synthetic lethal vulnerabilities associated with ATRX LoF. FINDINGS: In isogenic cell lines, we found that ATRX inactivation results in increased DNA damage, homologous recombination repair (HRR) defects and impaired replication fork processivity. In keeping with this, high-throughput compound screening showed selective sensitivity in ATRX mutant cells to multiple PARP inhibitors and the ATM inhibitor KU60019. ATRX mutant cells also showed selective sensitivity to the DNA damaging agents, sapacitabine and irinotecan. HRR deficiency was also seen in the ATRX deleted CHLA-90 cell line, and significant sensitivity demonstrated to olaparib/irinotecan combination therapy in all ATRX LoF models. In-vivo sensitivity to olaparib/irinotecan was seen in ATRX mutant but not wild-type xenografts. Finally, sustained responses to olaparib/irinotecan therapy were seen in an ATRX deleted neuroblastoma patient derived xenograft. INTERPRETATION: ATRX LoF results in specific DNA damage repair defects that can be therapeutically exploited. In ATRX LoF models, preclinical sensitivity is demonstrated to olaparib and irinotecan, a combination that can be rapidly translated into the clinic. FUNDING: This work was supported by Christopher's Smile, Neuroblastoma UK, Cancer Research UK, and the Royal Marsden Hospital NIHR BRC.

MYCN expression induces replication stress and sensitivity to PARP inhibition in neuroblastoma.

This study investigates the influence expression of the MYCN oncogene has on the DNA damage response, replication fork progression and sensitivity to PARP inhibition in neuroblastoma. In a panel of neuroblastoma cell lines, MYCN amplification or MYCN expression resulted in increased cell death in response to a range of PARP inhibitors (niraparib, veliparib, talazoparib and olaparib) compared to the response seen in non-expressing/amplified cells. MYCN expression slowed replication fork speed and increased replication fork stalling, an effect that was amplified by PARP inhibition or PARP1 depletion. Increased DNA damage seen was specifically induced in S-phase cells. Importantly, PARP inhibition caused a significant increase in the survival of mice bearing MYCN expressing tumours in a transgenic murine model of MYCN expressing neuroblastoma. Olaparib also sensitized MYCN expressing cells to camptothecin- and temozolomide-induced cell death to a greater degree than non-expressing cells. In summary, MYCN expression leads to increased replication stress in neuroblastoma cells. This effect is exaggerated by inhibition of PARP, resulting in S-phase specific DNA damage and ultimately increased tumour cell death. PARP inhibition alone or in combination with classical chemotherapeutics is therefore a potential therapeutic strategy for neuroblastoma and may be more effective in MYCN expressing tumours.

DNA Fiber Assay for the Analysis of DNA Replication Progression in Human Pluripotent Stem Cells.

Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabilities during prolonged in vitro culture that threaten to preclude their use in cell-based regenerative medicine. The rapid proliferation of pluripotent cells leads to constitutive replication stress, hindering the progression of DNA replication forks and in some cases leading to replication-fork collapse. Failure to overcome replication stress can result in incomplete genome duplication, which, if left to persist into the subsequent mitosis, can result in structural and numerical chromosomal instability. We have recently applied the DNA fiber assay to the study of replication stress in human PSC and found that, in comparison to somatic cells states, these cells display features of DNA replication stress that include slower replication fork speeds, evidence of stalled forks, and replication initiation from dormant replication origins. These findings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA fiber assay has enabled the development of an advanced nucleoside-enriched culture medium that increases replication fork progression and decreases DNA damage and mitotic errors in human PSC cultures. The DNA fiber assay allows for the study of replication fork dynamics at single-molecule resolution. The assay relies on cells incorporating nucleotide analogs into nascent DNA during replication, which are then measured to monitor several replication parameters. Here we provide an optimized protocol for the fiber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters. © 2020 Wiley Periodicals LLC. Basic Protocol 1: DNA fiber labeling Basic Protocol 2: DNA fiber spreading Basic Protocol 3: Immunostaining Support Protocol 1: Microscopy/data acquisition Support Protocol 2: Data analysis.

Investigation of the role of VHL-HIF signaling in DNA repair and apoptosis in zebrafish.

pVHL is a tumor suppressor. The lack of its function leads to various tumors, among which ccRCC (clear cell renal cell carcinoma) has the most serious outcome due to its resistance to chemotherapies and radiotherapies. Although HIF promotes the progression of ccRCC, the precise mechanism by which the loss of VHL leads to tumor initiation remains unclear. We exploited two zebrafish vhl mutants, vhl and vll, and Tg (phd3:: EGFP)i144 fish to identify crucial functions of Vhl in tumor initiation. Through the mutant analysis, we found that the role of pVHL in DNA repair is conserved in zebrafish Vll. Interestingly, we also discovered that Hif activation strongly suppressed genotoxic stress induced DNA repair defects and apoptosis in vll and brca2 mutants and in embryos lacking ATM activity. These results suggest the potential of HIF as a clinical modulator that can protect cells from accumulating DNA damage and apoptosis which can lead to cancers and neurodegenerative disorders.

Increased Non-Homologous End Joining Makes DNA-PK a Promising Target for Therapeutic Intervention in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular tumour in adults, with a mean survival of six months following metastasis. The survival rates have not improved in over 30 years. This study has shown that sister chromatid exchange (SCE) is low in UM which is likely due to a reduced expression of FANCD2. As FANCD2 can function to suppress non-homologous end joining (NHEJ), this study therefore investigated NHEJ in UM. The activation of the catalytic subunit of the NHEJ pathway protein DNA-dependent protein kinase (DNA-PK) was measured by analysing the foci formation and the ligation efficiency by NHEJ determined using a plasmid-based end-joining assay. Using small-interfering RNA (siRNA) knock-down, and chemical inhibitors of DNA-PK, the survival of primary UM cultures and two cell lines were determined. To assess the homologous recombination capacity in response to the inhibition of DNA-PK, a SCE analysis was performed. In addition, to support the findings, the messenger RNA (mRNA) expression of genes associated with NHEJ was analysed using the Cancer Genome Atlas (TCGA)-UM RNAseq data (n = 79). The NHEJ activity and DNA-PKcs activation was upregulated in UM and the inhibition of DNA-PK selectively induced apoptosis and sensitized to ionising radiation and inter-strand cross-linking agents. The inhibition of the NHEJ protein DNA-PK is lethal to UM, indicating a potentially effective therapeutic option, either alone or as a sensitizer for other treatments.

Carbon dot-protoporphyrin IX conjugates for improved drug delivery and bioimaging.

Photodynamic therapy (PDT) uses photosensitisers such as protoporphyrin IX (PpIX) to target tumours via the release of toxic singlet oxygen when irradiated. The effectivity of the treatment is limited by the innate properties of the photosensitizers; they typically exhibit inefficient accumulation in target tissue and high dark toxicity. Carbon dots (CDs) are biocompatible fluorescent nanoparticles which can improve PpIX cellular uptake and solubility. In this work, we present conjugates synthesised by host-guest encapsulation (PpIX@CD) and amide cross-linking (PpIX-CD). Characterization demonstrated conjugates have a loading efficiency of 34-48% and similar singlet oxygen production to PpIX. PpIX-containing CDs showed a 2.2 to 3.7-fold decrease in dark toxicity. PpIX-CD and PpIX@CD showed equivalent light-induced toxicity to PpIX in concentrations >1 µg/ml, leading to a 3.2 to 4.1-fold increase in photo-toxicity index (PI). The less soluble fraction of cross-linked conjugates (PpIX-CD)p did not show significant difference from PpIX. Confocal light scanning microscopy demonstrated rapid intracellular uptake and accumulation of conjugates. Our results demonstrate the variations between cross-linking strategies in CD-based conjugates, highlighting their potential as carriers in drug delivery and bioimaging applications.

ESCRT-III is necessary for the integrity of the nuclear envelope in micronuclei but is aberrant at ruptured micronuclear envelopes generating damage.

Micronuclei represent the cellular attempt to compartmentalize DNA to maintain genomic integrity threatened by mitotic errors and genotoxic events. Some micronuclei show aberrant nuclear envelopes (NEs) that collapse, generating damaged DNA that can promote complex genome alterations. However, ruptured micronuclei also provide a pool of cytosolic DNA that can stimulate antitumor immunity, revealing the complexity of micronuclear impact on tumor progression. The ESCRT-III (Endosomal Sorting Complex Required for Transport-III) complex ensures NE reseals during late mitosis and is repaired in interphase. Therefore, ESCRT-III activity maybe crucial for maintaining the integrity of other genomic structures enclosed by a NE. ESCRT-III activity at the NE is coordinated by the subunit CHMP7. We show that CHMP7 and ESCRT-III protect against the genomic instability associated with micronuclei formation. Loss of ESCRT-III activity increases the population of micronuclei with ruptured NEs, revealing that its NE repair activity is also necessary to maintain micronuclei integrity. Surprisingly, aberrant accumulation of ESCRT-III are found at the envelope of most acentric collapsed micronuclei, suggesting that ESCRT-III is not recycled efficiently from these structures. Moreover, CHMP7 depletion relieves micronuclei from the aberrant accumulations of ESCRT-III. CHMP7-depleted cells display a reduction in micronuclei containing the DNA damage marker RPA and a sensor of cytosolic DNA. Thus, ESCRT-III activity appears to protect from the consequence of genomic instability in a dichotomous fashion: ESCRT-III membrane repair activity prevents the occurrence of micronuclei with weak envelopes, but the aberrant accumulation of ESCRT-III on a subset of micronuclei appears to exacerbate DNA damage and sustain proinflammatory pathways.

Heteronuclear d-d and d-f Ru(ii)/M complexes [M = Gd(iii), Yb(iii), Nd(iii), Zn(ii) or Mn(ii)] of ligands combining phenanthroline and aminocarboxylate binding sites: combined relaxivity, cell imaging and photophysical studies.

A ligand skeleton combining a 1,10-phenanthroline (phen) binding site and one or two heptadentate N3O4 aminocarboxylate binding sites, connected via alkyne spacers to the phen C3 or C3/C8 positions, has been used to prepare a range of heteronuclear Ru·M and Ru·M2 complexes which have been evaluated for their cell imaging, relaxivity, and photophysical properties. In all cases the phen unit is bound to a {Ru(bipy)2}2+ unit to give a phosphorescent {Ru(bipy)2(phen)}2+ luminophore, and the pendant aminocarboxylate sites are occupied by a secondary metal ion M which is either a lanthanide [Gd(iii), Nd(iii), Yb(iii)] or another d-block ion [Zn(ii), Mn(ii)]. When M = Gd(iii) or Mn(ii) these ions provide the complexes with a high relaxivity for water; in the case of Ru·Gd and Ru·Gd2 the combination of high water relaxivity and 3MLCT phosphorescence from the Ru(ii) unit provides the possibility of two different types of imaging modality in a single molecular probe. In the case of Ru·Mn and Ru·Mn2 the Ru(ii)-based phosphorescence is substantially reduced compared to the control complexes Ru·Zn and Ru·Zn2 due to the quenching effect of the Mn(ii) centres. Ultrafast transient absorption spectroscopy studies on Ru·Mn (and Ru·Zn as a non-quenched control) reveal the occurrence of fast (<1 ns) PET in Ru·Mn, from the Mn(ii) ion to the Ru(ii)-based 3MLCT state, i.e. MnII-(phen˙-)-RuIII → MnIII-(phen˙-)-RuII; the resulting MnIII-(phen˙-) state decays with τ ≈ 5 ns and is non-luminescent. This occurs in conformers when an ET pathway is facilitated by a planar, conjugated bridging ligand conformation connecting the two units across the alkyne bridge but does not occur in conformers where the two units are electronically decoupled by a twisted conformation of the bridging ligand. Computational studies (DFT) on Ru·Mn confirmed both the occurrence of the PET quenching pathway and its dependence on molecular conformation. In the complexes Ru·Ln and Ru·Ln2 (Ln = Nd, Yb), sensitised near-infrared luminescence from Nd(iii) or Yb(iii) is observed following photoinduced energy-transfer from the Ru(ii) core, with Ru → Nd energy-transfer being faster than Ru → Yb energy-transfer due to the higher density of energy-accepting states on Nd(iii).

Synthesis and Aggregation of a Porphyrin-Cored Hyperbranched Polyglycidol and Its Application as a Macromolecular Photosensitizer for Photodynamic Therapy.

Macromolecules are potentially useful delivery systems for cancer drugs, as their size allows them to utilize the enhanced permeability and retention effect (EPR), which facilitates selective delivery to (and retention within) tumors. In addition, macromolecular delivery systems can prolong circulation times as well as protect and solubilize toxic and hydrophobic drug moieties. Overall, these properties and abilities can result in an enhanced therapeutic effect. Photodynamic therapy (PDT) combines the use of oxygen and a photosensitizer (PS), which become toxic upon light irradiation. We proposed that a PS encapsulated within a water-soluble macromolecule could exploit the EPR effect and safely and selectively deliver the PS to a tumor. In this paper, we describe the synthesis of a porphyrin-cored hyperbranched polymer that aggregated into larger micellar structures. DLS and TEM indicated that these aggregated structures had diameters of 45 and 20 nm for the solvated and nonsolvated species, respectively. The porphyrin-cored HBP (PC-HBP), along with the nonencapsulated porphyrin (THPP), were screened against EJ bladder carcinoma cells in the dark and light. Both THPP and PC-HBP displayed good toxicity in the light, with LD50 concentrations of 0.5 and 1.7 µM, respectively. However, in the dark, the nonincorporated porphyrin (THPP) displayed significant toxicity, generating an LD50 of 4 µM. On the other hand, no dark toxicity was observed for the polymer system (PC-HBP) at concentrations of 100 µM or less. As such, incorporation within the large polymer aggregate serves to eliminate dark toxicity while maintaining excellent toxicity when irradiated.