Encapsulation of anticancer drug curcumin and co-loading with photosensitizer hypericin into lipoproteins investigated by fluorescence resonance energy transfer.

Low-density lipoproteins (LDL) and high-density lipoproteins (HDL) are natural occurring vehicles attractive for drug delivery and targeting tumor cells. Here we have investigated the encapsulation and interaction of a well-known anticancer agent curcumin with LDL and HDL. LDL particles have been found to accumulate more curcumin molecules inside their structure than HDL. The chemical stability of curcumin is enhanced and its photo-physical properties are altered due to encapsulation inside both lipoproteins. Combining photodynamic therapy with chemotherapy can improve anticancer treatment by overcoming drug resistance in cancer therapy. Therefore, we have also investigated a co-loading of curcumin with a natural potent photosensitizer hypericin into molecules of LDL using fluorescence resonance energy transfer. The loading patterns of curcumin and hypericin into LDL particles were found to be different as revealed by the fluorescence resonance energy transfer experiments. Present study illustrates the potential of LDL nanoparticles in combination therapy because of simultaneous loading of more than one type of drugs into these nanoparticles with high level of efficiency.

Soft Hydrogel Zwitterionic Coatings Minimize Fibroblast and Macrophage Adhesion on Polyimide Substrates.

Minimizing the foreign body reaction to polyimide-based implanted devices plays a pivotal role in several biomedical applications. In this work, we propose materials exhibiting nonbiofouling properties and a Young's modulus reflecting that of soft human tissues. We describe the synthesis, characterization, and in vitro validation of poly(carboxybetaine) hydrogel coatings covalently attached to polyimide substrates via a photolabile 4-azidophenyl group, incorporated in poly(carboxybetaine) chains at two concentrations of 1.6 and 3.1 mol %. The presence of coatings was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy. White light interferometry was used to evaluate the coating continuity and thickness (between 3 and 6 µm under dry conditions). Confocal laser scanning microscopy allowed us to quantify the thickness of the swollen hydrogel coatings that ranged between 13 and 32 µm. The different hydrogel formulations resulted in stiffness values ranging from 2 to 19 kPa and led to different fibroblast and macrophage responses in vitro. Both cell types showed a minimum adhesion on the softest hydrogel type. In addition, both the overall macrophage activation and cytotoxicity were observed to be negligible for all of the tested material formulations. These results are a promising starting point toward future advanced implantable systems. In particular, such technology paves the way for novel neural interfaces able to minimize the fibrotic reaction, once implanted in vivo, and to maximize their long-term stability and functionality.

Unravelling the Excellent Chemical Stability and Bioavailability of Solvent Responsive Curcumin-Loaded 2-Ethyl-2-oxazoline-grad-2-(4-dodecyloxyphenyl)-2-oxazoline Copolymer Nanoparticles for Drug Delivery.

A new gradient copolymer has been synthesized by the living cationic ring-opening polymerization of hydrophilic 2-ethyl-2-oxazoline with lipophilic 2-(4-dodecyloxyphenyl)-2-oxazoline (EtOx-grad-DPOx). The prepared copolymer is capable of assembling in water to yield polymeric nanoparticles that are successfully loaded with an anticancer agent, curcumin. Self-assembly of the copolymer was found to be tuned by the polarity as well as the hydrogen bonding ability of solvents. Solvent took distinctive role in the preparation of unloaded and curcumin-loaded nanoparticles. The stability of the nanoparticles was increased by curcumin loading promoted by curcumin-polymer interactions. Further, the chemical stability of curcumin in water is largely enhanced inside the polymeric nanoparticles. Curcumin-loaded (EtOx-grad-DPOx) copolymer nanoparticles showed excellent stability in the biological medium, low cytotoxicity, and concentration dependent uptake by U87 MG and HeLa cells, which indicate the possibility of their efficient application in drug delivery.

Ex Vivo and In Vitro Studies on the Cytotoxicity and Immunomodulative Properties of Poly(2-isopropenyl-2-oxazoline) as a New Type of Biomedical Polymer.

Poly(2-alkenyl-2-oxazoline)s are promising functional polymers for a variety of biomedical applications, such as drug delivery systems, peptide conjugates, or gene delivery. In this study, poly(2-isopropenyl-2-oxazoline) (PIPOx) is prepared through free-radical polymerization initiated with azobisisobutyronitrile. Reactive 2-oxazoline units in the side chain support an addition reaction with different compounds containing a carboxylic group, which facilitates the preparation of polymers labeled with two different fluorescent dyes. The cytotoxicities of 2-oxazoline monomers, PIPOx, and fluorescently labeled PIPOx are evaluated in vitro using an 3-(4,5-Dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and ex vivo using a cell proliferation assay with adenosine triphosphate bioluminescence. The cell uptake of labeled PIPOx is used to determine the colocalization of PIPOx with cell organelles that are part of the endocytic pathway. For the first time, it is shown that poly(2-isopropenyl-2-oxazoline) is a biocompatible material and is suitable for biomedical applications; further, its immunomodulative properties are evaluated.

Bacterial inclusion bodies as potential synthetic devices for pathogen recognition and a therapeutic substance release.

BACKGROUND: Adhesins of pathogens recognise the glycans on the host cell and mediate adherence. They are also crucial for determining the tissue preferences of pathogens. Currently, glyco-nanomaterials provide potential tool for antimicrobial therapy. We demonstrate that properly glyco-tailored inclusion bodies can specifically bind pathogen adhesins and release therapeutic substances. RESULTS: In this paper, we describe the preparation of tailored inclusion bodies via the conjugation of indicator protein aggregated to form inclusion bodies with soluble proteins. Whereas the indicator protein represents a remedy, the soluble proteins play a role in pathogen recognition. For conjugation, glutaraldehyde was used as linker. The treatment of conjugates with polar lysine, which was used to inactivate the residual glutaraldehyde, inhibited unwanted hydrophobic interactions between inclusion bodies. The tailored inclusion bodies specifically interacted with the SabA adhesin from Helicobacter pylori aggregated to form inclusion bodies that were bound to the sialic acids decorating the surface of human erythrocytes. We also tested the release of indicator proteins from the inclusion bodies using sortase A and Ssp DNAB intein self-cleaving modules, respectively. Sortase A released proteins in a relatively short period of time, whereas the intein cleavage took several weeks. CONCLUSIONS: The tailored inclusion bodies are promising "nanopills" for biomedical applications. They are able to specifically target the pathogen, while a self-cleaving module releases a soluble remedy. Various self-cleaving modules can be enabled to achieve the diverse pace of remedy release.

Light-mimicking cockroaches indicate Tertiary origin of recent terrestrial luminescence.

Bioluminescence is a common feature of the communication and defence of marine organisms, but this phenomenon is highly restricted in the terrestrial biota. Here, we present a geographical distribution of only the third order of luminescent insects--luminescent cockroaches, with all 13 known and/or herein reported new living species (based on deposited specimens). We show that, for the first time, photo-characteristics of three examined species are nearly identical with those of toxic luminescent click beetles, which they mimic. These observations are the evidence for the mimicry by light--a new type of defensive, batesian and interordinal mimicry. Our analysis surprisingly reveals an evolutionary novelty of all living luminescent insects, while in the sea (and possibly in the soil) luminescence is present also phylogenetically in very primitive organisms.

Purinergic P2X7 receptors participate in disturbed intracellular calcium homeostasis in peripheral blood mononuclear cells of patients with chronic kidney disease.

BACKGROUND: P2X(7) receptors intervene with lymphocyte activation and are responsible for multiple processes, including calcium influx. Here, we studied the participation of P2X(7) receptors in disturbed intracellular calcium homeostasis regulation in early-stage chronic kidney disease (CKD). METHODS: The study involved 20 healthy volunteers and 20 CKD stage 2-3 patients. The free cytosolic calcium concentration ([Ca(2+)](i)) was measured using fluorimetry. The P2X(7) pore function was evaluated by the fluorescent dye ethidium bromide. RESULTS: In peripheral blood mononuclear cells (PBMCs) of patients, [Ca(2+)](i), intracellular calcium stores and the capacitative calcium entry were increased when compared with healthy subjects. The agonist of P2X(7) receptor BzATP caused a sustained increase in [Ca(2+)](i) in both groups, but the effect was smaller in patients. The antagonist at the P2X(7) receptor KN-62 reduced [Ca(2+)](i) in patients, but had no effect in healthy subjects. In patients, the permeability of ethidium bromide through P2X(7) pores, as well as through BzATP-activated and KN-62-inhibited pores, was distinct from permeability in healthy volunteers. CONCLUSIONS: These results demonstrate that the calcium signaling pathway in PBMCs of CKD patients is defective already in CKD stage 2-3, and the pore-forming P2X(7) receptors are involved in these pathophysiological processes.

Antibody response to the 45 kDa Candida albicans antigen in an animal model and potential role of the antigen in adherence.

The Candida antigen CR3-RP (complement receptor 3-related protein) is supposed to be a 'mimicry' protein because of its ability to bind antibody directed against the alpha subunit of the mammalian CR3 (CD11b/CD18). This study aimed to (i) investigate the specific humoral isotypic response to immunization with CR3-RP in vivo in a rabbit animal model, and (ii) determine the role of CR3-RP in the adherence of Candida albicans in vitro using the model systems of buccal epithelial cells (BECs) and biofilm formation. The synthetic C. albicans peptide DINGGGATLPQ corresponding to 11 amino-acids of the CR3-RP sequence DINGGGATLPQALXQITGVIT, determined by N-terminal sequencing, was used for immunization of rabbits to obtain polyclonal anti-CR3-PR serum and for subsequent characterization of the humoral isotypic response of rabbits. A significant increase of IgG, IgA and IgM anti-CR3-RP specific antibodies was observed after the third (P<0.01) and the fourth (P<0.001) immunization doses. The elevation of IgA levels suggested peptide immunomodulation of the IgA1 subclass, presumably in coincidence with Candida epithelial adherence. Blocking CR3-RP with polyclonal anti-CR3-RP serum reduced the ability of Candida to adhere to BECs, in comparison with the control, by up to 35 % (P<0.001), and reduced biofilm formation by 28 % (P<0.001), including changes in biofilm thickness and integrity detected by confocal laser scanning microscopy. These properties of CR3-RP suggest that it has potential for future vaccine development.

Rapid effects of 1alpha,25(OH)2D3 in resting human peripheral blood mononuclear cells.

The steroid hormone 1alpha,25(OH)2D3 produces biological responses via both genomic and nongenomic mechanisms. Stimulation of rapid, nongenomic responses by 1alpha,25(OH)2D3 has been postulated to result from interaction of the ligand with cell membrane 1alpha,25(OH)2D3 receptors and to involve membrane receptors. We examined the rapid effects of 1alpha,25(OH)2D3 on calcium mobilization and calcium entry into resting human peripheral blood mononuclear cells isolated from healthy volunteers. We also investigated the possible involvement of purinergic receptors in this action. 1alpha,25(OH)2D3 induced a time-dependent increase in intracellular calcium concentration ([Ca2+]i). The initial 1alpha,25(OH)2D3-stimulated calcium increment was sensitive to thapsigargin (Tg), indicating its origins in calcium release from intracellular stores. 2-Aminoethyldiphenyl borate (2APB), an inhibitor of capacitative calcium entry, caused a significant [Ca2+]i decrease in human cells treated with 1alpha,25(OH)2D3. Furthermore, in contrast to observations in osteoblasts and skeletal muscle cells, nifedipine had no effect on 1alpha,25(OH)2D3-induced calcium entry, suggesting that L-type calcium channels were not implicated in this action. Besides, 1alpha,25(OH)(2)D3 prevented the calcium entry induced by 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a specific agonist of purinergic P2X7 receptors. This finding was further confirmed by 1alpha,25(OH)2D3-induced reduction of BzATP- and 4-aminopyridine (4AP)-stimulated ethidium bromide fluorescence. The presented results demonstrate, for the first time in healthy, resting human peripheral blood mononuclear cells that 1alpha,25(OH)2D3 is capable of exerting a rapid, nongenomic effect on [Ca2+]i, while inhibiting of the P2X7 channel permeability.

Chitosan based hydrogel microspheres as drug carriers.

Chitosan/tripolyphosphate (CHIT/TPP) and chitosan/tripolyphosphate/chondroitin sulfate (CHIT/TPP/CHS) core-shell type microspheres were prepared by polyelectrolyte complexation in order to develop a biocompatible matrix for drug delivery. The continual method using a multi-loop reactor under sterile conditions was applied for microsphere preparation. All the types of microspheres produced were spherical in shape and had a porous structure. The mechanical resistance of the microspheres increased in the presence of CHS as the second polyanion, which toughened the microsphere shell structure. For a drug release application, the process of microsphere preparation was modified by dissolving ofloxacin (OFL), the fluoroquinolone antibiotic, in CHIT solution before complex formation. This study shows the difference in OFL release comparing the microspheres CHIT/TPP and CHIT/TPP/CHS and implies the potential to control this process.

Characterization of the interaction of hypericin with protein kinase C in U-87 MG human glioma cells.

A fluorescence imaging technique was used to monitor intracellular localization of protein kinase C (PKC) in U-87 MG human glioma cells in the presence of hypericin (Hyp) and phorbol 12-myristate-13-acetate (PMA). It is shown that PKC localization, which reflects its activity, is influenced by Hyp and this influence is different from that observed for PMA which acts as PKC activator. Fluorescence binding experiments were used to determine the binding constants of Hyp to several isoforms of PKC. The obtained values of K(d)s (approximately 100 nM) suggest that Hyp binds with high affinity to PKC. Finally, molecular modeling was used to compare structural models of the interaction of C1B domain of PKC (PKC isoforms alpha, delta, gamma) with Hyp and our previously published model of the (C1B domain PKCgamma)/PMA complex. The influence of Hyp on PKC translocation in U-87 MG cells in comparison with PMA, colocalization fluorescence pattern of Hyp and PKC, the higher binding affinity of Hyp to PKC in comparison with known binding constants of phorbol esters, as well as the binding mode of Hyp and PMA to the C1B domain of PKC suggested by molecular modeling, support the idea that Hyp and PMA might competitively bind to the regulatory domain of PKC.

4-Aminopyridine activates calcium influx through modulation of the pore-forming purinergic receptor in human peripheral blood mononuclear cells.

We investigated whether 4-aminopyridine (4AP), a drug recently linked to calcium influx and apoptosis, also affected purinergic receptor channels that are known to play an important role in the activation of T lymphocytes. The application of 4AP induced a rise in [Ca2+]i that was sensitive to nickel. This action was also observed in cells in which calcium reserves were emptied using thapsigargin (Tg). However, it was not present in the absence of extracellular Ca2+, despite full internal reserves. Adenosine trisphosphate (ATP), a partial agonist and a physiological activator of purinergic receptors, also stimulated Ca2+ entry independently of the calcium release from internal compartments. The effects of 4AP and ATP were not additive when studied on the same population of cells. KN-62 inhibited an increase in calcium entry induced by 4AP, while brilliant blue G (BBG) prevented it, supporting the hypothesis that purinergic P2X7 receptors are involved in this action. Furthermore, 4AP allowed entry of ethidium bromide (314 Da) but not propidium iodide (415 Da) into the cell, also corroborating the involvement of P2X7 pores. The presented results demonstrate, for the first time in human mononuclear cells isolated from healthy volunteers, that the P2X7 channel pore is involved in the action of 4AP and intervenes in the sustained calcium entry induced in response to 4AP.