Theodossis A. Theodossiou

Interactive transport, subcellular relocation and enhanced phototoxicity of hypericin encapsulated in guanidinylated liposomes via molecular recognition.

Hypericin (HYP), a photocytotoxic phenanthroperylenquinone was encapsulated in liposomes outfitted with guanidinium‐bearing lipids to ensure efficient cell binding through molecular recognition with anionic groups resident on the plasma membrane. The uptake of HYP encapsulated in these liposomes by DU145 human prostate cancer cells, was studied employing fluorescence, versus nonguadinylated liposomes and free HYP. The subcellular localization was in all cases studied by confocal microscopy employing specific subcellular organelle probes. The photocytotoxicity of HYP was assessed, 24 h following irradiation with 15 mWcm−2 light through a GG 495 Schott filter, by a standard tetrazolium to formazan assay (XTT). HYP uptake by DU145 cells was found to be profoundly enhanced by using guanidinylated liposomes. Also the distance of the guanidinium group from the liposomal surface was found to significantly affect HYP loading, subcellular localization and phototoxicity. The two different modes of liposome cell internalization observed, i.e. plasma membrane fusion and endocytosis, were found to greatly affect the phototoxicity of HYP. Molecular recognition was overall appraised as a promising, novel route for photodynamic therapy, profoundly enhancing its efficacy. HYP encapsulated in liposomes‐bearing guanidinium groups was more efficiently taken up by cells, leading to enhanced phototoxicity, in contrast to HYP encapsulated in their nonguanidinylated counterparts.

Synthesis and characterization of multifunctional hyperbranched polyesters as prospective contrast agents for targeted MRI.

Based on a commercially available hyperbranched aliphatic polyester, novel multifunctional gadolinium complexes were prepared bearing protective PEG chains, a folate targeting ligand and EDTA or DTPA chelate moieties. Their relatively high water relaxivity values coupled with biodegradability of the hyperbranched scaffold, folate receptor specificity render these non-toxic dendritic polymers promising candidates for MRI applications.

Protoporphyrin IX–β-Cyclodextrin Bimodal Conjugate: Nanosized Drug Transporter and Potent Phototoxin

Topical or systemic administration of 5‐aminolevulinic acid (ALA) and its esters results in increased production and accumulation of protoporphyrin IX (PpIX) in cancerous lesions allowing effective application of photodynamic therapy (PDT). The large concentrations of exogenous ALA practically required to bypass the negative feedback control exerted by heme on enzymatic ALA synthesis and the strong dimerization propensity of ALA are shortcomings of the otherwise attractive PpIX biosynthesis. To circumvent these limitations and possibly enhance the phototoxicity of PpIX by adjuvant chemotherapy, covalent bonding of PpIX with a drug carrier, β‐cyclodextrin (βCD) was implemented. The resulting PpIX + βCD product had both carboxylic termini of PpIX connected to the CD. PpIX + βCD was water soluble, was found to preferentially localize in mitochondria rather than in lysosomes both in MCF7 and DU145 cell lines while its phototoxiciy was comparable to that of PpIX. Moreover, PpIX + βCD effectively solubilized the breast cancer drug tamoxifen metabolite N‐desmethyltamoxifen (NDMTAM) in water. The PpIX + βCD/NDMTAM complex was readily internalized by both cell lines employed. Furthermore, the multimodal action of PpIX + βCD was demonstrated in MCF7 cells: while it retains the phototoxic profile of PpIX and its fluorescence for imaging purposes, PpIX + βCD can efficiently transport tamoxifen citrate intracellularly and confer cell death through a synergy of photo‐ and chemotoxicity.

Tamoxifen subcellular localization; observation of cell-specific cytotoxicity enhancement by inhibition of mitochondrial ETC complexes I and III.

Recently, a nongenomic cytotoxic component of the chemotherapeutic agent tamoxifen (TAM) has been identified that predominantly triggers mitochondrial events. The present study delineates the intracellular fate of TAM and studies its interaction with a spectrum of cell homeostasis modulators primarily relevant to mitochondria. The subcellular localization of TAM was assessed by confocal fluorescence microscopy. The effect of the modulators on TAM cytotoxicity was assessed by standard MTT assays. Our findings show that in estrogen receptor positive MCF7 breast adenocarcinoma cells and DU145 human prostate cancer cells, TAM largely accumulates in the mitochondria and endoplasmic reticulum, but not lysosomes. Our results further demonstrate that in MCF7, but not in DU145 cells, mitochondrial electron transport chain complex I and III inhibitors exacerbate TAM toxicity with an order of potency of myxothiazol ≥ stigmatellin > rotenone > antimycin A, suggesting a cell‐specific cytotoxic interplay between mitochondrial complex I and III function and TAM action.

A novel mitotropic oligolysine nanocarrier: Targeted delivery of covalently bound D-Luciferin to cell mitochondria

New and emerging therapeutic approaches focus on the targeted delivery of therapeutic agents to cell mitochondria with high specificity. Herein we present a novel mitotropic nanocarrier based on an oligolysine scaffold by addition of two triphenylphosphonium cations per oligomer. Although the parent oligolysine failed to enter healthy cells, the triphenylphosphonium modified carrier, with or without D-Luciferin, attached as cargo molecule, demonstrated striking mitochondrial specificity. Furthermore, the oligolysine bound d-Luciferin exhibited chemiluminescence, of lower intensity than free d-Luciferin, yet of remarkably longer steady-state temporal profile.

Carboxylated Hydroxyethyl Starch: A novel Polysaccharide for the Delivery of Doxorubicin

Hydroxyethyl starch (HES) was interacted with succinic anhydride affording a carboxylated derivative which has proved to be a promising polymeric drug delivery system. Specifically, this polymer is conveniently prepared, is biodegradable, non‐immunogenic, and can encapsulate doxorubicin due to the protonation of the primary amino group of doxorubicin by the carboxylic group located on the branched scaffold of the polysaccharide. In addition, due to the polyhydroxylated character of the polysaccharide, the latter can act as a protective coating in an analogous manner to the PEG‐chains ensuring prolonged circulation in vivo. In vitro experiments showed controlled release of doxorubicin to the nuclei of DU145 prostate cancer cells when the anticancer drug is incorporated in the carboxylated hydroxyethyl starch.

Hypericin hydroquinone: potential as a red-far red photosensitizer?

Hypericin hydroquinone is the product of two‐electron reduction of hypericin (quinone), a potent phenanthroperylenequinone photosensitizer. In contrast to the quinone, the hydroquinone exhibits strong absorbance in the far‐red spectral region. Herein we provide initial evidence on the potential of hypericin hydroquinone as a far‐red photosensitizer.

A versatile δ-aminolevulinic acid (ΑLA)-cyclodextrin bimodal conjugate-prodrug for PDT applications with the help of intracellular chemistry.

Summary Grafting of δ-aminolevulinic acid (1) moieties on the narrow periphery of a β-cyclodextrin (β-CD) derivative through hydrolysable bonds was implemented, in order to generate a water-soluble, molecular/drug carrier with the capacity to undergo intracellular transformation into protoporphyrin IX (PpIX), an endogenous powerful photosensitizer for photodynamic therapy (PDT). The water-soluble derivative 2 was prepared by esterifying δ-azidolevulinic acid with heptakis(6-hydroxyethylamino-6-deoxy)-β-cyclodextrin, with an average degree of substitution, DS = 3. Delivery of water-soluble, colorless 2 to cells resulted in intense red fluorescence registered by confocal microscopy, evidently due to the engagement of the intracellular machinery towards formation of PpIX. Conjugate 2 was further complexed with a fluorescein-labeled model guest molecule which was successfully transported into the cells, thereby demonstrating the bimodal action of the derivative. The present work shows the versatility of CDs in smart applications and constitutes advancement to our previously shown PpIX-β-CD conjugation both in terms of water solubility and lack of aggregation.

S‐Nitroso‐β‐Cyclodextrins as New Bimodal Carriers: Preparation, Detailed Characterization, Nitric‐Oxide Release, and Molecular Encapsulation

6-Monoand 6-multi-S-nitroso-b-cyclodextrins (SNObCDs) were prepared from their corresponding thiols (SHbCDs) and characterized in detail for the first time in terms of their conformational preferences and SNO content, thermal and photochemical stability, their ability to encapsulate guest molecules, and their cell toxicity and permeation. The prevalence of gauche–trans (gt) over gauche–gauche (gg) conformations (with respect to rotation about the C5 C6SH bond and, hence, to the bCD cavity) and the presence of syn-to-anti equilibria (with respect to the C6S-NO configuration) in SNO-CDs and in a reference compound, S-nitrosoglutathione (GSNO), were suggested by H and N NMR spectroscopy. Quantum mechanical calculations indicated that the gt conformations indeed prevail in mono-SH-bCD and mono-SNO-bCD, whereas a blend of gt/gg conformations prevail in per-SH-bCD and per-SNO-bCD. This reflected the presence of two potentially dissimilar SNO groups with diverse stabilities toward NO release and propensities for forming interglucose S S bonds. Moreover, syn conformers were energetically preferred in all cases. MonoSNO-bCD is water soluble, thermally more stable than GSNO, and undergoes photocontrolled NO release. Furthermore, the CD cavity is available for guest encapsulation without noticeable perturbation of the SNO functionality whilst hosting, for example, the chemotherapeutic tamoxifen. Nitrosation of per-SH-bCD to form per-SNO-bCD was found to compete with SNO decomposition and disulfidebridge formation, thereby resulting in an average of 5.2 SNO groups instead of 7. Upon isolation, SNO-CDs, as well as GSNO, suffer a small additional loss of SNO groups, mostly to afford disulfides. Multi-SNO-bCD is soluble in DMSO and displays better thermal stability than GSNO and cell permeability. Both SNO-CDs were found to be chemically non-toxic to cells at high incubation concentrations (>200 mm); thus, they represent a potentially new family of bimodal drug-delivery systems.

Artesunate shows potent anti-tumor activity in B-cell lymphoma

BackgroundAlthough chemo-immunotherapy has led to an improved overall survival for most B-cell lymphoma types, relapsed and refractory disease remains a challenge. The malaria drug artesunate has previously been identified as a growth suppressor in some cancer types and was tested as a new treatment option in B-cell lymphoma.MethodsWe included artesunate in a cancer sensitivity drug screen in B lymphoma cell lines. The preclinical properties of artesunate was tested as single agent in vitro in 18 B-cell lymphoma cell lines representing different histologies and in vivo in an aggressive B-cell lymphoma xenograft model, using NSG mice. Artesunate-treated B lymphoma cell lines were analyzed by functional assays, gene expression profiling, and protein expression to identify the mechanism of action.ResultsDrug screening identified artesunate as a highly potent anti-lymphoma drug. Artesunate induced potent growth suppression in most B lymphoma cells with an IC50 comparable to concentrations measured in serum from artesunate-treated malaria patients, while leaving normal B-cells unaffected. Artesunate markedly inhibited highly aggressive tumor growth in a xenograft model. Gene expression analysis identified endoplasmic reticulum (ER) stress and the unfolded protein response as the most affected pathways and artesunate-induced expression of the ER stress markers ATF-4 and DDIT3 was specifically upregulated in malignant B-cells, but not in normal B-cells. In addition, artesunate significantly suppressed the overall cell metabolism, affecting both respiration and glycolysis.ConclusionsArtesunate demonstrated potent apoptosis-inducing effects across a broad range of B-cell lymphoma cell lines in vitro, and a prominent anti-lymphoma activity in vivo, suggesting it to be a relevant drug for treatment of B-cell lymphoma.