Zinc-Substituted Pheophorbide A Is a Safe and Efficient Antivascular Photodynamic Agent.

The present study focuses on the photodynamic activity of zinc-substituted pheophorbide a against human endothelial cells. Previously, zinc pheophorbide a has been shown to be a very potent photosensitizer but also a strong albumin binder. Binding to albumin significantly reduces its availability to cancer cells, which may necessitate the use of relatively high doses. Here we show that zinc pheophorbide a is very effective against vascular endothelial cells, even in its albumin-complexed form. Albumin complexation increases the lysosomal accumulation of the drug, thus enhancing its efficiency. Zinc pheophorbide a at nanomolar concentrations induces endothelial cell death via apoptosis, which in many cases is considered a desirable cell death mode because of its anti-inflammatory effect. Additionally, we demonstrate that in comparison to tumor cells, endothelial cells are much more susceptible to photodynamic treatment with the use of the investigated compound. Our findings demonstrate that zinc pheophorbide a is a very promising photosensitizer for use in vascular-targeted photodynamic therapy against solid tumors, acting as a vascular shutdown inducer. It can also possibly find application in the treatment of a range of vascular disorders. Numerous properties of zinc pheophorbide a are comparable or even more favorable than those of the well-known photosensitizer of a similar structure, palladium bacteriopheophorbide (TOOKAD®).

The Co-Culture of Staphylococcal Biofilm and Fibroblast Cell Line: The Correlation of Biological Phenomena with Metabolic NMR1 Footprint.

Staphylococcus aureus is one of the most prevalent pathogens associated with several types of biofilm-based infections, including infections of chronic wounds. Mature staphylococcal biofilm is extremely hard to eradicate from a wound and displays a high tendency to induce recurring infections. Therefore, in the present study, we aimed to investigate in vitro the interaction between S. aureus biofilm and fibroblast cells searching for metabolites that could be considered as potential biomarkers of critical colonization and infection. Utilizing advanced microscopy and microbiological methods to examine biofilm formation and the staphylococcal infection process, we were able to distinguish 4 phases of biofilm development. The analysis of staphylococcal biofilm influence on the viability of fibroblasts allowed us to pinpoint the moment of critical colonization-12 h post contamination. Based on the obtained model we performed a metabolomics analysis by 1H NMR spectroscopy to provide new insights into the pathophysiology of infection. We identified a set of metabolites related to the switch to anaerobic metabolism that was characteristic for staphylococcal biofilm co-cultured with fibroblast cells. The data presented in this study may be thus considered a noteworthy but preliminary step in the direction of developing a new, NMR-based tool for rapid diagnosing of infection in a chronic wound.

Polymeric Nanocarriers with Luminescent Colloidal Nanoplatelets as Hydrophilic and Non-Toxic Two-Photon Bioimaging Agents.

INTRODUCTION: Semiconductor nanoplatelets (NPLs) are promising materials for nonlinear optical microscopy since they feature good two-photon absorption (TPA) properties, narrow photoluminescence spectra and high quantum yields of luminescence. Nevertheless, the use of semiconductor NPLs is inevitably connected with concerns about heavy metal ion toxicity and their intrinsically hydrophobic character. METHODS: Our contribution focuses on the design and engineering of coloidal bionanomaterial consisting of two-dimensional highly luminescent CdSe semiconductor NPLs loaded into spherical and homogeneous polymeric nanocarriers (NCs) based on poly(ethylene oxide) and poly(propylene oxide) block co-polymer. The biocompatibility and usefulness of the NPLs-loaded polymeric NCs in two-photon induced bioimaging was demonstrated in vitroby cytotoxicity and two-photon microscopic studies using eukaryotic (normal fibroblasts and cancer ovarian) cells. RESULTS: The encapsulated NPLs maintain their intensive and spectrally narrow photoluminescence, as well as preserve good TPA properties, while the surrounding polymer shell imparts hydrophilic character and non-toxicity towards eukaryotic cells. Specifically, TPA cross-sections of the colloidal NCs loaded with NPLs show large values reaching up to 2.0 × 108 GM, with simultaneously two-photon brightness reaching 2.2 × 107 GM at 870 nm. MTT proliferation assay performed on cell lines treated with encapsulated NPLs revealed at least 70% viability of normal human gingival fibroblast (HGF) and cancer ovarian (MDAH-2774) cells, while the results of multiphoton imaging of murine (L-929) fibroblasts suggest that the encapsulated NPLs are capable of labelling the target cells enabling their visualization. CONCLUSION: As a result, we obtained water dispersible and temporally stable hydrophilic NPLs-loaded NCs that offer excellent, both one- and two-photon excited fluorescence preserving optical properties of the raw hydrophobic and colloidal NPLs. The biological responses upon eukaryotic cells indicate that the encapsulation process protects cells from the toxic influence of cadmium simultaneously preserving the unique multiphoton properties of the active cargo which opens a promising perspective for its application in multiphoton cancer bioimaging excited at the "optical transmission window" of biological tissues in near-infrared range.

The effect of Toll-like receptor stimulation on the motility of regulatory T cells.

Regulatory T cells (Tregs) have suppressive functions and play an important role in controlling inflammation and autoimmunity. The migratory capacity of Tregs determines their location and their location determines whether they inhibit the priming of naïve lymphocytes in lymphoid tissues or the effector phase of immune responses at inflamed sites. Tregs generated or expanded in vitro are currently being tested in clinics for the treatment of autoimmune disorders, however, little is known about the factors controlling their migration towards therapeutically relevant locations. In this study, we have modulated Treg dynamics using Toll-like receptor (TLR) agonists. Dynamic imaging with confocal and two-photon microscopy revealed that Tregs generated in vitro and stimulated with P3C (a TLR2 agonist) but not with R848 (a TLR7 agonist) or LPS (a TLR4 agonist) showed enhanced cell migration within splenic white pulp or draining lymph node when transferred into mice intravenously or into the footpad, respectively. In summary, our data demonstrate that Tregs are more motile in response to direct TLR stimulation in particular towards TLR2 signals. This may have implications for efficient clinical Treg induction protocols.

Superabsorbent crosslinked bacterial cellulose biomaterials for chronic wound dressings.

In this work, we present a novel ex situ modification of bacterial cellulose (BC) polymer, that significantly improves its ability to absorb water after drying. The method involves a single inexpensive and easy-to-perform process of BC crosslinking, using citric acid along with catalysts, such as disodium phosphate, sodium bicarbonate, ammonium bicarbonate or their mixtures. In particular, the mixture of disodium phosphate and sodium bicarbonate was the most promising, yielding significantly greater water capacity (over 5 times higher as compared to the unmodified BC) and slower water release (over 6 times as compared to the unmodified BC). Further, our optimized crosslinked BC had over 1.5x higher water capacity than modern commercial dressings dedicated to highly exuding wounds, while exhibiting no cytotoxic effects against fibroblast cell line L929 in vitro. Therefore, our novel BC biomaterial may find application in super-absorbent dressings, designed for chronic wounds with imbalanced moisture level.

Development and biological evaluation of Ti6Al7Nb scaffold implants coated with gentamycin-saturated bacterial cellulose biomaterial.

Herein we present an innovative method of coating the surface of Titanium-Aluminium-Niobium bone scaffold implants with bacterial cellulose (BC) polymer saturated with antibiotic. Customized Ti6Al7Nb scaffolds manufactured using Selective Laser Melting were immersed in a suspension of Komagataeibacter xylinus bacteria which displays an ability to produce a 3-dimensional structure of bio-cellulose polymer. The process of complete implant coating with BC took on average 7 days. Subsequently, the BC matrix was cleansed by means of alkaline lysis and saturated with gentamycin. Scanning electron microscopy revealed that BC adheres and penetrates into the implant scaffold structure. The viability and development of the cellular layer on BC micro-structure were visualized by means of confocal microscopy. The BC-coated implants displayed a significantly lower cytotoxicity against osteoblast and fibroblast cell cultures in vitro in comparison to non-coated implants. It was also noted that gentamycin released from BC-coated implants inhibited the growth of Staphylococcus aureus cultures in vitro, confirming the suitability of such implant modification for preventing hostile microbial colonization. As demonstrated using digital microscopy, the procedure used for implant coating and BC chemical cleansing did not flaw the biomaterial structure. The results presented herein are of high translational value with regard to future use of customized, BC-coated and antibiotic-saturated implants designed for use in orthopedic applications to speed up recovery and to reduce the risk of musculoskeletal infections.

Epidermal T Cell Dendrites Serve as Conduits for Bidirectional Trafficking of Granular Cargo.

Dendritic epidermal T cells (DETCs) represent a prototypical lineage of intraepithelial γδ T cells that participate in the maintenance of body barrier homeostasis. Unlike classical T cells, DETCs do not recirculate and they remain persistently activated through their T cell receptors (TCR) at steady state, i.e., in absence of infection or tissue wounding. The steady state TCR signals sustain the formation of immunological synapse-like phosphotyrosine-rich aggregates located on projections (PALPs) which act to anchor and polarize DETC's long cellular projections toward the apical epidermis while the cell bodies reside in the basal layers. The PALPs are known to contain pre-synaptic accumulations of TCR-containing and lysosomal granules, but how this cargo accumulates there remains unclear. Here, we combined anti-Vγ5 TCR, cholera toxin subunit B (CTB), and LysoTracker (LT)-based intravital labeling of intracellular granules, with high resolution dynamic microscopy and fluorescence recovery after photobleaching (FRAP) to characterize the steady state composition and transport of DETC granules in steady state epidermis. Intradermal fluorescent Vγ5 antibody decorated DETCs without causing cellular depletion, dendrite mobilization or rounding up and became slowly internalized over 48 h into intracellular granules that, after 6 days, colocalized with LAMP-1 and less so with LT or early endosomal antigen-1. Intradermal CTB was likewise internalized predominantly by DETCs in epidermis, labeling a partly overlapping set of largely LAMP-1+ intracellular granules. These as well as LT-labeled granules readily moved into newly forming dendrites and accumulated at the apical endings. FRAP and spatiotemporal tracking showed that the inside tubular lengths of DETC cellular projections supported dynamic trafficking of lysosomal cargo toward and away from the PALPs, including internalized TCR and lipid raft component ganglioside GM1 (labeled with CTB). By contrast, the rate of GM1 granules transport through comparable dendrites of non-DETCs was twice slower. Our observations suggest that DETCs use chronic TCR activation to establish a polarized conduit system for long-range trans-epithelial transport aimed to accumulate mature lysosomes at the barrier-forming apical epidermis. The biological strategy behind the steady state lysosome polarization by DETCs remains to be uncovered.

[Liposomes as non-viral carriers for genetic drugs]. / Liposomy jako niewirusowe systemy dostarczania leków genetycznych.

Methods in cancer therapy particularly in recent years, are rapidly changing, due to the need of design of new, more effective therapeutic strategies. Very promising approach to treatment of the neoplastic diseases is antisense gene therapy. Due to the low toxicity of treatment and eliminating not only the symptoms but also the molecular causes of the disease it may represent a breakthrough in cancer therapies. Delivery of a therapeutic DNA or RNA oligonucleotides to the target cells in vivo requires suitable carrier system. Non-viral drug carriers are increasingly used in new systems of targeted gene therapy. This review presents new generation of non-viral carriers, and is focused on immunoliposomes finding potential application in targeted gene therapy.

PKC-θ is a negative regulator of TRAIL-induced and FADD-mediated apoptotic spectrin aggregation.

INTRODUCTION: During studies on chemotherapy-induced apoptosis in lymphoid cells, we noted that aggregation of spectrin occurred early in apoptosis, i.e. before activation of initiator caspase(s) and prior to exposure of phosphatidylserine (PS). We also found that protein kinase C theta (PKC-θ) co-localized with spectrin in these aggregates. Our previously published studies indicated that in formation of early apoptotic spectrin aggregates, either PKC-θ or other apoptosis-related proteins are involved. Taking into consideration above data, we decided to test the effect of PKC-θ and Fas-associated death domain protein (FADD) on spectrin aggregation in these cells during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. MATERIAL AND METHODS: For PKC-θ gene (PRKCQ) or FADD gene expression silencing in Jurkat T cells we used lentiviral particles containing shRNA and scrambled shRNA, respectively. Spectrin aggregates were detected by Western blotting after Triton-X 100 extraction in pellet and soluble fractions or by confocal imaging. RESULTS: TRAIL-induced apoptosis results in spectrin aggregation and leads to translocation and aggregation of PKC-θ. We found that phorbol-myristate acetate, a PKC activator and translocation inducer, has only a small effect on spectrin aggregation. To further confirm this, we have also shown that knock down ofPRKCQin Jurkat T cells accelerates the formation of TRAIL-induced spectrin aggregates. Transient overexpression of theß-spectrin C-terminal fragment, containing multiple S/T phosphorylation sites, potential substrate sites for PKC-θ, accelerated the formation of spectrin aggregates. Silencing of downstream TRAIL receptor effector gene,FADD, delayed aggregation of spectrin, but did not reduce PKC-θ localization to the plasma membrane. CONCLUSIONS: In summary, our results show for the first time involvement of spectrin aggregation in TRAIL receptor-FADD apoptotic pathway and indicate that TRAIL-induced spectrin aggregate formation is mediated by FADD and negatively regulated by PKC-θ.

Novel antisense therapeutics delivery systems: In vitro and in vivo studies of liposomes targeted with anti-CD20 antibody.

Antisense gene therapy using molecules such as antisense oligodeoxynucleotides, siRNA or miRNA is a very promising strategy for the treatment of neoplastic diseases. It can be combined with other treatment strategies to enhance therapeutic effect. In acute leukemias, overexpression of the antiapoptotic gene BCL2 is observed in more than 70% of cases. Therefore, reduction of the Bcl-2 protein level could, in itself, prevent the development of cancer or could possibly help sensitize cancer cells to apoptosis inducers. The main objective of our work is to develop therapeutic liposome formulations characterized by high transfection efficiency, stability in the presence of serum, as well as specificity and toxicity for target (leukemic) cells. Each of our liposomal formulations consists of a core composed of antisense oligonucleotides complexed by either cationic lipid, DOTAP, or a synthetic polycation, polyethyleneimine, encapsulated within liposomes modified with polyethylenoglycol. In addition, the liposomal shells are enriched with covalently-bound antibodies recognizing a well characterized bio-marker, CD20, exposed on the surface of leukemia cells. The resulting immunoliposomes selectively and effectively reduced the expression of BCL2 in target cells. Model animal experiments carried out on mice-engrafted tumors expressing the specific marker showed high efficiency of the liposome formulations against specific tumor development. In conclusion, we show that lipid formulations based on a polyplex or lipoplex backbone additionally equipped with antibodies are promising non-viral vectors for specific oligonucleotide transfer into human tumor cells.

The effect of statins on cancer cells--review.

Statins [3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, abbreviated HMGCR) inhibitors], are well-known cholesterol-depleting agents. Since the early 1990 s, it has been known that statins could be successfully used in cancer therapy, but the exact mechanism(s) of statin activity remains unclear and is now an extensive focus of investigation. So far, it was proven that there are several mechanisms that are activated by statins in cancer cells; some of them are leading to cell death. Statins exert different effects depending on cell line, statin concentration, duration of exposure of cells to statins, and the type of statin being used. It was shown that statins may inhibit the cell cycle by influence on both expression and activity of proteins involved in cell-cycle progression such as cyclins, cyclin-dependent kinases (CDK), and/or inhibitors of CDK. Also, statins may induce apoptosis by both intrinsic and extrinsic pathways. Statin treatment may lead to changes in molecular pathways dependent on the EGF receptor, mainly via inhibition of isoprenoid synthesis. By inhibition of the synthesis of cholesterol, statins may destabilize the cell membrane. Moreover, statins may change the arrangement of transporter OATP1, the localization of HMGCR, and could induce conformational changes in GLUT proteins. In this review, we have tried to gather and compare most of the recent outcomes of the research in this field. We have also attempted to explain why hydrophilic statins are less effective than hydrophobic statins. Finally, we have gathered results from in vivo experiments, presenting the use of statins in combined therapies and discussed a number of molecular targets that could serve as biomarkers predisposing to statin therapy.

Toward a magic or imaginary bullet? Ligands for drug targeting to cancer cells: principles, hopes, and challenges.

There are many problems directly correlated with the systemic administration of drugs and how they reach their target site. Targeting promises to be a hopeful strategy as an improved means of drug delivery, with reduced toxicity and minimal adverse side effects. Targeting exploits the high affinity of cell-surface-targeted ligands, either directly or as carriers for a drug, for specific retention and uptake by the targeted diseased cells. One of the most important parameters which should be taken into consideration in the selection of an appropriate ligand for targeting is the binding affinity (K D). In this review we focus on the importance of binding affinities of monoclonal antibodies, antibody derivatives, peptides, aptamers, DARPins, and small targeting molecules in the process of selection of the most suitable ligand for targeting of nanoparticles. In order to provide a critical comparison between these various options, we have also assessed each technology format across a range of parameters such as molecular size, immunogenicity, costs of production, clinical profiles, and examples of the level of selectivity and toxicity of each. Wherever possible, we have also assessed how incorporating such a targeted approach compares with, or is superior to, original treatments.

Liposome-coated lipoplex-based carrier for antisense oligonucleotides.

The chemical nature of genetic drugs (e.g. antisense oligonucleotides, siRNA, vectors) requires a suitable carrier system to protect them from enzymatic degradation without changing their properties and enable efficient delivery into target cells. Lipid vectors for nucleic acid delivery that have been widely investigated for years can be very effective. As the majority of attempts made in the field of cancer gene therapy have focused on solid tumors, while blood cancer cells have attracted less attention, the latter became the subject of our investigation. The lipid carrier proposed here is based on liposomes constructed by others but the lipid composition is original. A liposome-coated lipoplex (L-cL) consists of a core arising from complexation of positively charged lipid and negatively charged oligodeoxynucleotide (ODN) or plasmid DNA coated by a neutral or anionic lipid bilayer. Moreover, our lipid vector demonstrates size stability and is able to retain a high content of enclosed plasmid DNA or antisense oligodeoxynucleotides (asODNs). Observed transfection efficacies of the tested preparation using a plasmid coding for fluorescent protein were up to 60-85% of examined leukemia cells (Jurkat T and HL-60 lines) in the absence or the presence of serum. When BCL­2 asODN was encapsulated in the L-cL, specific silencing of this gene product at both the mRNA and protein level and also a markedly decreased cell survival rate were observed in vitro. Moreover, biodistribution analysis in mice indicates prolonged circulation characteristic for PEG-modified liposomal carriers. Experiments on tumor-engrafted animals indicate substantial inhibition of tumor growth.

Release of an ~55kDa fragment containing the actin-binding domain of ß-spectrin by caspase-8 during FND-induced apoptosis depends on the presence of protein 4.1.

Analyses of the status of the membrane spectrin-based skeleton during fludarabine/mitoxantrone/dexamethasone-induced (FND-induced) apoptosis revealed proteolytic degradation of ß-spectrin, with the prevalent appearance of a specific fragment with a molecular weight of ~55kDa, containing the actin-binding domain (ABD). Appearance of this fragment was dependent on induction of apoptosis. In silico proteolysis of spectrin identified caspase-8 as a candidate protease responsible for the generation of this ~55kDa ABD-containing fragment. Analyses of spectrin and procaspase-8 localization during early apoptosis indicated temporary (<30-120min) submembranous colocalization of both proteins. Proteolytic release of the N-terminal ~55kDa fragment of purified spectrin by recombinant caspase-8 does not occur in normal cells, but does occur in isolated membrane, such as red blood cell ghosts, or in vitro in the presence of apoptotic cell extracts. Surprisingly, proteolysis of purified spectrin by recombinant caspase-8 resulted in the generation of the ~55kDa fragment only in the presence of purified protein 4.1. This suggests that only the appropriate spatial arrangement of the spectrin-based membrane skeleton or the appropriate conformational state of spectrin, which are both known to be induced by 4.1, can sensitize ß-spectrin to cleavage by caspase-8 at the N-terminal ABD-containing region.

Human DHHC proteins: a spotlight on the hidden player of palmitoylation.

Palmitoylation is one of the most common posttranslational lipid modifications of proteins and we now know quite a lot about it. However, the state of knowledge about the enzymes that catalyze this process is clearly insufficient. This review is focused on 23 human DHHC genes and their products - protein palmitoyltransferases. Here we describe mainly the structure and function of these proteins, but also, to a lesser degree, what the substrates of the enzymes are and whether they are related to various diseases. The main aim of this review was to catalogue existing information concerning the human DHHC family of genes/proteins, making them and their functions easier to understand.