Huguette Savoie

Polyacrylamide Nanoparticles as a Delivery System in Photodynamic Therapy

Nanoparticles can be targeted towards, and accumulate in, tumor tissue by the enhanced permeability and retention effect, if sequestration by the reticuloendothelial system (RES) is avoided. The application of nanoparticles in the field of drug delivery is thus an area of great interest, due to their potential for delivering high payloads of drugs site selectively. One area which may prove to be particularly attractive is photodynamic therapy, as the reactive oxygen species (ROS) which cause damage to the tumor tissue are not generated until the drug is activated with light, minimizing generalized toxicity and giving a high degree of spatial control over the clinical effect. In the present study, we have synthesized two types of nanoparticles loaded with photodynamic sensitizers: polylysine bound tetrasulfonato-aluminum phthalocyanine entrapped nanoparticles (PCNP) and polylysine bound tetrasulfonato-aluminum phthalocyanine entrapped nanoparticles coated with a second, porphyrin based, photosensitizer (PCNP-P) to enhance the capacity for ROS generation, and hence therapeutic potential. The mean sizes of these particles were 45 ± 10 nm and 95 ± 10 nm respectively. Uptake of the nanoparticles by human Caucasian colon adenocarcinoma cells (HT29) was determined by flow cytometry and confocal microscopy. Cell viability assays using PCNP-P and PCNP corresponding to the minimum uptake time (<5 min) and maximum uptake time (25 h) demonstrated that these cancer cells can be damaged by light activation of these photodynamic nanoparticles both in the external media and after internalization. The results suggest that, in order to induce photodynamic damage, the nanoparticles need only to be associated with the tumor cell closely enough to deliver singlet oxygen: their internalization within target cells may not be necessary. Clinically, this could be of great importance as it may help to combat the known ability of many cancer cells to actively expel conventional anticancer drugs.

Development and characterization of novel photosensitizer : scFv conjugates for use in photodynamic therapy of cancer.

Photodynamic therapy (PDT) is becoming an evermore useful tool in oncology but is frequently limited by side‐effects caused by a lack of targeting of the photosensitizer. This problem can often be circumvented by the conjugation of photosensitizers to tumour‐specific monoclonal antibodies. An alternative is the use of single chain (sc) Fv fragments which, whilst retaining the same binding specificity, are more efficient at penetrating tumour masses because of their smaller size; and are more effectively cleared from the circulation because of the lack of an Fc domain. Here we describe the conjugation of two isothiocyanato porphyrins to colorectal tumour‐specific scFv, derived from an antibody phage display library. The conjugation procedure was successfully optimized and the resulting immunoconjugates showed no loss of cell binding. In vitro assays against colorectal cell lines showed these conjugates had a selective photocytotoxic effect on cells. Annexin V and propidium iodide staining of treated cells confirmed cell death was mediated principally via an apoptotic pathway. This work suggests that scFv : porphyrin conjugates prepared using isothiocyanato porphyrins show promise for use as targeted PDT agents.

Regioselective and stoichiometrically controlled conjugation of photodynamic sensitizers to a HER2 targeting antibody fragment.

The rapidly increasing interest in the synthesis of antibody-drug conjugates as powerful targeted anticancer agents demonstrates the growing appreciation of the power of antibodies and antibody fragments as highly selective targeting moieties. This targeting ability is of particular interest in the area of photodynamic therapy, as the applicability of current clinical photosensitizers is limited by their relatively poor accumulation in target tissue in comparison to healthy tissue. Although synthesis of porphyrin-antibody conjugates has been previously demonstrated, existing work in this area has been hindered by the limitations of conventional antibody conjugation methods. This work describes the attachment of azide-functionalized, water-soluble porphyrins to a tratuzumab Fab fragment via a novel conjugation methodology. This method allows for the synthesis of a homogeneous product without the loss of structural stability associated with conventional methods of disulfide modification. Biological evaluation of the synthesized conjugates demonstrates excellent selectivity for a HER2 positive cell line over the control, with no dark toxicity observed in either case.

Monoglycoconjugated phthalocyanines: Effect of sugar and linkage on photodynamic activity

BACKGROUND Click chemistry can be advantageously used to graft carbohydrates on phthalocyanines which are potent photosensitisers, but the effect of the presence of triazole moieties on photodynamic efficiency was not investigated systematically to date. The nature and linkage of the sugar were investigated in order to define structure-activity relationships. METHOD Two sets of monoglycoconjugated water-soluble phthalocyanines have been designed and their photodynamic activity and uptake investigated in HT-29 human colon adenocarcinoma cells. Carbohydrates: galactose, mannose or lactose were grafted onto Zn(II) phthalocyanines either by glycosylation or by click reaction. RESULTS The triazole linkage formed by click conjugation lowered the biological efficiency for mannose and galactose, compared to classical glycosylation grafting. The mannose conjugate formed by glycosylation was the most photodynamically active, without correlation with the photosensitiser cell uptake.

Generation of Triplet Excited States via Photoinduced Electron Transfer in meso-anthra-BODIPY: Fluorogenic Response toward Singlet Oxygen in Solution and in Vitro

Heavy atom-free BODIPY-anthracene dyads (BADs) generate locally excited triplet states by way of photoinduced electron transfer (PeT), followed by recombination of the resulting charge-separated states (CSS). Subsequent quenching of the triplet states by molecular oxygen produces singlet oxygen (1O2), which reacts with the anthracene moiety yielding highly fluorescent species. The steric demand of the alkyl substituents in the BODIPY subunit defines the site of 1O2 addition. Novel bis- and tetraepoxides and bicyclic acetal products, arising from rearrangements of anthracene endoperoxides were isolated and characterized. 1O2 generation by BADs in living cells enables visualization of the dyads distribution, promising new imaging applications.

Near IR emitting isothiocyanato-substituted fluorophores: their synthesis and bioconjugation to monoclonal antibodies

Two near IR emitting fluorophores, based on the phthalocyanine and naphthalocyanine chromophores, which also bear a single isothiocyanato group suitable for conjugation to proteins are reported; their utility as luminescent probes is demonstrated by conjugation to monoclonal antibodies and the ability of these conjugates to selectively bind cells bearing the relevant antigen.

Synthesis and phototoxicity of polyethylene glycol (PEG) substituted metal-free and metallo-porphyrins: effect of PEG chain length, coordinated metal, and axial ligand.

Treatment of 5,10,15,20-tetra(pentafluorophenyl) porphyrins with polyethylene glycol (PEG) halides and sodium sulfide is a mild and efficient route to water-soluble PEG substituted porphyrins. The method has been used to access a series of free-base and metallated PEG porphyrins, which have been investigated for photodynamic activity against Human Caucasian colon adenocarcinoma (Caco2) cells. Toxicity, in the presence and absence of light, is shown to be affected by the length of the PEG chain, nature of coordinated metal and axial ligand.

NanoSOSG: a Nanostructured Fluorescent Probe for the Detection of Intracellular Singlet Oxygen

A biocompatible fluorescent nanoprobe for singlet oxygen (1 O2 ) detection in biological systems was designed, synthesized, and characterized, that circumvents many of the limitations of the molecular probe Singlet Oxygen Sensor Green® (SOSG). This widely used commercial singlet oxygen probe was covalently linked to a polyacrylamide nanoparticle core using different architectures to optimize the response to 1 O2 . In contrast to its molecular counterpart, the optimum SOSG-based nanoprobe, which we call NanoSOSG, is readily internalized by E. coli cells and does not interact with bovine serum albumin. Furthermore, the spectral characteristics do not change inside cells, and the probe responds to intracellularly generated 1 O2 with an increase in fluorescence.

Lipophilic cationic porphyrins as photodynamic sensitisers—Synthesis and structure–activity relationships

A convenient synthesis of a range of phosphorous and nitrogen centred lipophilic cationic porphyrins is described. In vitro assays for photodynamic activity against human colorectal adenocarcinoma cells (HT-29) reveal significant differences based on substituents around the cation centres.

Isothiocyanato Boron Dipyrromethenes—The First BODIPY Analogues of Fluorescein Isothiocyanate

Abstract Two boron complexes of 5-phenyldipyrromethenes bearing isothiocyanate groups on the phenyl ring have been synthesized for the first time. The utility of these new fluorescence probes for labeling biologically relevant proteins is demonstrated on two monoclonal antibodies that bind to antigens overexpressed on cancer cells. Spectral comparison of the two structures reveals significant photophysical differences, including bathochromically shifted excitation and emission bands, increased molar absorptivity and a large increase in fluorescence quantum yield of approximately 10 times. Differences in photophysical parameters are linked to hindered rotation of the phenyl ring in one of the probes.