Self-Organization of Lipid-Porphyrin Conjugates at the Air/Water Interface.

Lipid-porphyrin conjugates are versatile compounds which can self-assemble into liposome-like structures with multifunctional properties. Most of the conjugates that have been described so far, consisted in grafting pyropheophorbide-a (Pyro-a) or other porphyrin derivatives through the esterification of the hydroxyl group in the sn-2 position of a lysophosphatidylcholine. However, despite the versatility of these conjugates, less is known about the impact of the lipid backbone structure on their 2D phase behavior at the air/water interface and more precisely on their fine structures normal to the interface as well as on their in-plane organization. Herein, we synthesized a new lipid-porphyrin conjugate (PyroLSM) based on the amide coupling of Pyro-a to a lysosphingomyelin backbone (LSM) and we compared its interfacial behavior to that of Pyro-a and Pyro-a conjugated lysophosphatidylcholine (PyroLPC) using Langmuir balance combined to a variety of other physical techniques. Our results provided evidence on the significant impact of the lipid backbone on the lateral packing of the conjugates as well as on the shape and size of the formed domains. Compared to Pyro-a and PyroLPC monolayers, PyroLSM exhibited the highest lateral packing which highlights the role of the lipid backbone in controlling their 2D organization which in turn may impact the photophysical properties of their assemblies.

Design and Synthesis of New PEGylated Polydopamine-Based Nanoconstructs Bearing ROS-Responsive Linkers and a Photosensitizer for Bimodal Photothermal and Photodynamic Therapies against Cancer.

Polydopamine (PDA) nanoparticles (NPs) have recently acquired considerable attention for the development of nanoplatforms with multifunctional properties including photothermal (PTT) and photodynamic (PDT) activities. In addition to their high PTT performance, they can be easily conjugated to different types of photosensitizers (PSs) to acquire PDT activity. However, because of PDA free-radical scavenging properties, grafting the PSs directly to PDA surfaces may lead to an inefficient PDT outcome. Thus, the present work aims at synthesizing and characterizing a new PEGylated PDA-based nanoplatform with bifunctional PTT and PDT properties, which allows bimodal cancer therapy with the possibility to release the PS on demand in a spatiotemporal fashion. To do so, PDA NPs with a well-defined size and shape were prepared by the auto-oxidative self-polymerization process of dopamine hydrochloride in mild alkaline solution. The impact of the size on the PTT conversion efficiency was then determined. This allowed us to choose the optimal PDA NP size for PTT applications. Next, PDA NPs were decorated with SH-PEG polymers that bear at their extremity a thioketal reactive oxygen species-cleavable linker coupled to trisulfonated-tetraphenylporphyrin (TPPS3) chosen as a hydrophilic PS. The grafting efficiency of PS-conjugated PEG on PDA was demonstrated in situ using a quartz crystal microbalance with dissipation monitoring. In addition, the photoinduced release of the PS was demonstrated by 1H NMR. Finally, PTT/PDT bimodal therapy was assessed in vitro on human squamous esophageal cells by illuminating the PDA NPs at two different wavelengths, which showed the strong synergistic effect of combining PTT and PDT within this nanoplatform.

Cancer Stem Cell and Bulk Cancer Cell Active Copper(II) Complexes with Vanillin Schiff Base Derivatives and Naproxen.

Four copper(II) complexes, 1-4 containing regioisomeric vanillin Schiff base derivatives and the nonsteroidal anti-inflammatory drug (NSAID), naproxen, were synthesised and characterised. All complexes effectively cleave DNA in cell-free systems, with 4 displaying the highest nuclease activity. DNA binding studies suggest that 4 binds to DNA via the grooves prior to inducing oxidative DNA cleavage. Three of the complexes (1, 3, and 4) indiscriminately kill cancer stem cell (CSC)-enriched cells (HMLER-shEcad) and bulk cancer cells (HMLER) at micromolar concentrations. The most effective complex, 4 also reduced the formation and size of mammospheres to a similar extent as salinomycin, a well-established CSC-potent agent. Mechanistic studies show that 4 is readily taken up by CSCs, elevates intracellular reactive oxygen species (ROS) levels, causes DNA damage, and induces caspase-dependent apoptosis. Furthermore, 4 inhibits cyclooxygenase-2 (COX-2) expression and causes COX-2-dependent CSC death. The advantage of 4 over bulk cancer cell- or CSC-selective agents is that it has the potential to remove whole tumor populations (bulk cancer cells and CSCs) with a single dose.

Induction of Necroptosis in Cancer Stem Cells using a Nickel(II)-Dithiocarbamate Phenanthroline Complex.

The cytotoxic properties of a series of nickel(II)-dithiocarbamate phenanthroline complexes is reported. The complexes 1-6 kill bulk cancer cells and cancer stem cells (CSCs) with micromolar potency. Two of the complexes, 2 and 6, kill twice as many breast cancer stem cell (CSC)-enriched HMLER-shEcad cells as compared to breast CSC-depleted HMLER cells. Complex 2 inhibits mammosphere formation to a similar extent as salinomycin (a CSC-specific toxin). Detailed mechanistic studies suggest that 2 induces CSC death by necroptosis, a programmed form of necrosis. Specifically, 2 triggers MLKL phosphorylation, oligomerization, and translocation to the cell membrane. Additionally, 2 induces necrosome-mediated propidium iodide (PI) uptake and mitochondrial membrane depolarisation, as well as morphological changes consistent with necroptotosis. Strikingly, 2 does not evoke necroptosis by intracellular reactive oxygen species (ROS) production or poly(ADP) ribose polymerase (PARP-1) activation.

The Potent Inhibitory Effect of a Naproxen-Appended Cobalt(III)-Cyclam Complex on Cancer Stem Cells.

We report the potency against cancer stem cells (CSCs) of a new cobalt(III)-cyclam complex (1) that bears the nonsteroidal anti-inflammatory drug, naproxen. The complex displays selective potency for breast CSC-enriched HMLER-shEcad cells over breast CSC-depleted HMLER cells. Additionally, it inhibited the formation of three-dimensional tumour-like mammospheres, and reduced their viability to a greater extent than clinically used breast cancer drugs (vinorelbine, cisplatin and paclitaxel). The anti-mammosphere potency of 1 was enhanced under hypoxia-mimicking conditions. Detailed mechanistic studies revealed that DNA damage and cyclooxygenase-2 (COX-2) inhibition contribute to the cytotoxic mechanism of 1. To the best of our knowledge, 1 is the first cobalt-containing compound to show selective potency for CSCs over bulk cancer cells.

Breast Cancer Stem Cell Potent Copper(II)-Non-Steroidal Anti-Inflammatory Drug Complexes.

The breast cancer stem cell (CSC) potency of a series of copper(II)-phenanthroline complexes containing the nonsteroidal anti-inflammatory drug (NSAID), indomethacin, is reported. The most effective copper(II) complex in this series, 4, selectivity kills breast CSC-enriched HMLER-shEcad cells over breast CSC-depleted HMLER cells. Furthermore, 4 reduces the formation, size, and viability of mammospheres, to a greater extent than salinomycin, a potassium ionophore known to selectively inhibit CSCs. Mechanistic studies revealed that the CSC-specificity observed for 4 arises from its ability to generate intracellular reactive oxygen species (ROS) and inhibit cyclooxygenase-2 (COX-2), an enzyme that is overexpressed in breast CSCs. The former induces DNA damage, activates JNK and p38 pathways, and leads to apoptosis.

Effect of Phase-Change Nanodroplets and Ultrasound on Blood-Brain Barrier Permeability In Vitro.

Phase-change nanodroplets (PCND;NDs) are emulsions with a perfluorocarbon (PFC) core that undergo acoustic vaporisation as a response to ultrasound (US). Nanodroplets change to microbubbles and cavitate while under the effect of US. This cavitation can apply forces on cell connections in biological barrier membranes, such as the blood-brain barrier (BBB), and trigger a transient and reversible increased permeability to molecules and matter. This study aims to present the preparation of lipid-based NDs and investigate their effects on the brain endothelial cell barrier in vitro. The NDs were prepared using the thin-film hydration method, followed by the PFC addition. They were characterised for size, cavitation (using a high-speed camera), and PFC encapsulation (using FTIR). The bEnd.3 (mouse brain endothelial) cells were seeded onto transwell inserts. Fluorescein with NDs and/or microbubbles were applied on the bEND3 cells and the effect of US on fluorescein permeability was measured. The Live/Dead assay was used to assess the BBB integrity after the treatments. Size and PFC content analysis indicated that the NDs were stable while stored. High-speed camera imaging confirmed that the NDs cavitate after US exposure of 0.12 MPa. The BBB cell model experiments revealed a 4-fold increase in cell membrane permeation after the combined application of US and NDs. The Live/Dead assay results indicated damage to the BBB membrane integrity, but this damage was less when compared to the one caused by microbubbles. This in vitro study shows that nanodroplets have the potential to cause BBB opening in a similar manner to microbubbles. Both cavitation agents caused damage on the endothelial cells. It appears that NDs cause less cell damage compared to microbubbles.

Influence of the porphyrin structure and linker length on the interfacial behavior of phospholipid-porphyrin conjugates.

HYPOTHESIS: Phospholipid-porphyrin (Pl-Por) conjugates consist of porphyrin derivatives grafted to a lysophosphatidylcholine backbone. Owing to their structural similarities with phospholipids, Pl-Por conjugates can self-assemble into liposome-like assemblies. However, there is a significant lack of information concerning the impact of the porphyrin type and the length of the alkyl chain bearing the porphyrin on the interfacial behavior of the Pl-Por conjugates. We hypothesized that changing the chain length and the porphyrin type could impact their two-dimensional phase behavior and modulate the alignment between the two chains. EXPERIMENTS: 6 Pl-Por conjugates with different alkyl chain lengths in the sn2 position of C16 lysophosphatidylcholine and coupled to either pheophorbide-a or pyropheophorbide-a were synthesized. Their interfacial behavior at the air/water interface was assessed using Langmuir balance combined to a variety of other physical techniques including Brewster angle microscopy, atomic force microscopy and X-ray reflectometry. FINDINGS: Our results showed that all 6 Pl-Por form stable monolayers with the porphyrin moiety at the air/water interface. We also showed that changing the porphyrin moiety controlled the packing of the monolayer and thus the formation of organized domains. The chain length dictated the structure of the formed domains with no evidence of the alignment between the two chains.

Novel liposome-like assemblies composed of phospholipid-porphyrin conjugates with photothermal and photodynamic activities against bacterial biofilms.

Phospholipid-Porphyrin (PL-Por) conjugates are unique building blocks that can self assemble into liposome-like structures with improved photophysical properties compared to their monomeric counterparts. The high packing density of porphyrin moieties enables these assemblies to exhibit high photothermal conversion efficiency as well as photodynamic activity. Thus, PL-Por conjugates assemblies can be used for photodynamic therapy (PDT) and photothermal therapy (PTT) applications against resistant bacteria and biofilms. In order to tune the PD/PT properties of such nanosystems, we developed six different supramolecular assemblies composed of newly synthesized PL-Por conjugates bearing either pheophorbide-a (PhxLPC) or pyropheophorbide-a (PyrxLPC) photosensitizers (PSs) for combined PDT/PTT against planktonic bacteria and their biofilms. In this study, the influence of the chemical structure of the phospholipid backbone as well as that of the PS on the photothermal conversion efficiency, the photodynamic activity and the stability of these assemblies in biological medium were determined. Then their antimicrobial efficiency was assessed on S. aureus and P. aeruginosa planktonic cultures and biofilms. The two studied systems show almost the same photothermal effect against planktonic cultures and biofilms of S. aureus and P. aeruginosa. However, PhxLPC vesicles exhibit superior photodynamic activity, making them the best combination for PTT/PDT. Such results highlight the higher potential of the photodynamic activity of PL-Por nanoassemblies compared to their photothermal conversion in combating bacterial infections.

Phospholipid-porphyrin conjugates: deciphering the driving forces behind their supramolecular assemblies.

Phospholipid-porphyrin conjugates (PL-Por) are nowadays considered as a unique class of building blocks that can self-assemble into supramolecular structures that possess multifunctional properties and enhanced optoelectronics characteristics compared to their disassembled counterparts. However, despite their versatile properties, little is known about the impact of the packing parameter of PL-Por conjugates on their assembling mechanism and their molecular organization inside these assemblies. To gain a better understanding on their assembling properties, we synthesized two new series of PL-Por conjugates with different alkyl sn2-chain lengths linked via an amide bond to either pheophorbide-a (PhxLPC) or pyropheophorbide-a (PyrxLPC). By combining a variety of experimental techniques with molecular dynamics (MD) simulations, we investigated both the assembling and optical properties of the PL-Por either self-assembled or when incorporated into lipid bilayers. We demonstrated that independently of the linker length, PhxLPC assembled into closed ovoid structures, whereas PyrxLPC formed rigid open sheets. Interestingly, PyrxLPC assemblies displayed a significant red shift and narrowing of the Q-band indicating the formation of ordered J-aggregates. The MD simulations highlighted the central role of the interaction between porphyrin cores rather than the length difference between the two phospholipid chains in controlling the structure of the lipid bilayer membranes and thus their optical properties. Indeed, while PhxLPC have the tendency to form inter-leaflet π-stacked dimers, PyrxLPC conjugates formed dimers within the same leaflet. Altogether, this work could be used as guidelines for the design of new PL-Por conjugates that self-assemble into bilayer-like supramolecular structures with tunable morphology and optical properties.

Image-guided thermosensitive liposomes for focused ultrasound enhanced co-delivery of carboplatin and SN-38 against triple negative breast cancer in mice.

Triggerable nanocarriers have the potential to significantly improve the therapeutic index of existing anticancer agents. They allow for highly localised delivery and release of therapeutic cargos, reducing off-target toxicity and increasing anti-tumour activity. Liposomes may be engineered to respond to an externally applied stimulus such as focused ultrasound (FUS). Here, we report the first co-delivery of SN-38 (irinotecan's super-active metabolite) and carboplatin, using an MRI-visible thermosensitive liposome (iTSL). MR contrast enhancement was achieved by the incorporation of a gadolinium lipid conjugate in the liposome bilayer along with a dye-labelled lipid for near infrared fluorescence bioimaging. The resulting iTSL were successfully loaded with SN-38 in the lipid bilayer and carboplatin in the aqueous core - allowing co-delivery of both. The iTSL demonstrated both thermosensitivity and MR-imageability. In addition, they showed effective local targeted co-delivery of carboplatin and SN-38 after triggered release with brief FUS treatments. A single dosage induced significant improvement of anti-tumour activity (over either the free drugs or the iTSL without FUS-activation) in triple negative breast cancer xenografts tumours in mice.

MR-labelled liposomes and focused ultrasound for spatiotemporally controlled drug release in triple negative breast cancers in mice.

Rationale: Image-guided, triggerable, drug delivery systems allow for precisely placed and highly localised anti-cancer treatment. They contain labels for spatial mapping and tissue uptake tracking, providing key location and timing information for the application of an external stimulus to trigger drug release. High Intensity Focused Ultrasound (HIFU or FUS) is a non-invasive approach for treating small tissue volumes and is particularly effective at inducing drug release from thermosensitive nanocarriers. Here, we present a novel MR-imageable thermosensitive liposome (iTSL) for drug delivery to triple-negative breast cancers (TNBC). Methods: A macrocyclic gadolinium-based Magnetic Resonance Imaging (MRI) contrast agent was covalently linked to a lipid. This was incorporated at 30 mol% into the lipid bilayer of a thermosensitive liposome that was also encapsulating doxorubicin. The resulting iTSL-DOX formulation was assessed for physical and chemical properties, storage stability, leakage of gadolinium or doxorubicin, and thermal- or FUS-induced drug release. Its effect on MRI relaxation time was tested in phantoms. Mice with tumours were used for studies to assess both tumour distribution and contrast enhancement over time. A lipid-conjugated near-infrared fluorescence (NIRF) probe was also included in the liposome to facilitate the real time monitoring of iTSL distribution and drug release in tumours by NIRF bioimaging. TNBC (MDA-MB-231) tumour-bearing mice were then used to demonstrate the efficacy at retarding tumour growth and increasing survival. Results: iTSL-DOX provided rapid FUS-induced drug release that was dependent on the acoustic power applied. It was otherwise found to be stable, with minimum leakage of drug and gadolinium into buffers or under challenging conditions. In contrast to the usually suggested longer FUS treatment we identified that brief (~3 min) FUS significantly enhanced iTSL-DOX uptake to a targeted tumour and triggered near-total release of encapsulated doxorubicin, causing significant growth inhibition in the TNBC mouse model. A distinct reduction in the tumours' average T1 relaxation times was attributed to the iTSL accumulation. Conclusions: We demonstrate that tracking iTSL in tumours using MRI assists the application of FUS for precise drug release and therapy.

The breast cancer stem cell potency of copper(ii) complexes bearing nonsteroidal anti-inflammatory drugs and their encapsulation using polymeric nanoparticles.

We report the cancer stem cell (CSC) potency of a novel series of copper(ii)-phenanthroline complexes bearing nonsteriodial anti-inflammatory drugs: naproxen, tolfenamic acid, and indomethacin (2a-3c). Two of the complexes, 2a and 3c, kill breast CSC-enriched HMLER-shEcad cells (grown in both monolayer and three-dimensional cell cultures) to a significantly better extent than salinomycin, a well-established CSC toxin. The most potent complex in the series, 3c induces its cytotoxic effect by generating intracellular reactive oxygen species (ROS) and inhibiting cyclooxgenase-2 (COX-2) activity. Encapsulation of 3c using biodegradable methoxy poly(ethylene glycol)-b-poly(d,l-lactic-co-glycolic) acid (PEG-PLGA) copolymers at the appropriate feed (5%, 3c NP5) enhances breast CSC uptake and reduces overall toxicity. The nanoparticle formulation, 3c NP5 selectively kills breast CSCs over bulk breast cancer cells, and evokes a similar cellular response to the payload, 3c. To the best of our knowledge, this is the first study to demonstrate that polymeric nanoparticles can be used to effectively deliver CSC-potent metal complexes into CSCs.

Primary options for acute care: general practitioners using their skills to manage "avoidable admission" patients in the community.

AIM: To enroll 600 primary care "avoidable admission" patients in a programme that utilised general practitioners to manage those patients in the community. METHODS: The Primary Options for Acute Care (POAC) programme ran from 26 February to 31 December 2001. Using networks already established, primary care teams were invited to manage patients using any resources they required, up to a cost of approximately $266 per patient. If needed, a Service Coordinator was available to arrange investigations, care, or treatment. RESULTS: From 26 February to 31 December 2001, 707 patients were enrolled in POAC by 100 GPs. 104 patients (15%) were eventually admitted to hospital. An average of $200.73 per patient per episode was spent (not including administrative costs). A wide variety of patients and diseases were managed. Patients and general practitioners reported high levels of satisfaction with the programme. CONCLUSION: POAC demonstrated the ability and willingness of primary care providers to successfully manage patients who traditionally would be sent to hospital, within a defined budget