Anionically Functionalized Glycogen Encapsulates Melittin by Multivalent Interaction.

We developed acid-functionalized glycogen conjugates as supramolecular carriers for efficient encapsulation and inhibition of a model cationic peptide melittin─the main component of honeybee venom. For this purpose, we synthesized and characterized a set of glycogens, functionalized to various degrees by several different acid groups. These conjugates encapsulate melittin up to a certain threshold amount, beyond which they precipitate. Computer simulations showed that sufficiently functionalized conjugates electrostatically attract melittin, resulting in its efficient encapsulation in a broad pH range around the physiological pH. Hemolytic assays confirmed in vitro that the effective inhibition of melittin's hemolytic activity occurs for highly functionalized samples, whereas no inhibition is observed when using low-functionalized conjugates. It can be concluded that functional glycogens are promising carriers for cationic molecular cargos or antidotes against animal venoms under conditions, in which suitable properties such as biodegradability and biocompatibility are crucial.

Fluorinated diselenide nanoparticles for radiosensitizing therapy of cancer.

Radiation resistance of cancer cells represents one of the major challenges in cancer treatment. The novel self-assembled fluoralkylated diselenide nanoparticles (fluorosomes) based on seleno-l-cystine (17FSe2) possess redox-active properties that autocatalytically decompose hydrogen peroxide (H2O2) and oxidize the intracellular glutathione (GSH) that results in regulation of cellular oxidative stress. Alkylfluorinated diselenide nanoparticles showed a significant cytotoxic and radiosensitizing effect on cancer cells. The EL-4 tumor-bearing C56BL/6 mice treated with 17FSe2 followed by fractionated radiation treatment (4 × 2Gy) completely suppressed tumor growth. Our results suggest that described diselenide system behaves as a potent radiosensitizer agent targeting tumor growth and preventing tumor recurrence.

Thiolated poly(2-hydroxyethyl methacrylate) hydrogels as a degradable biocompatible scaffold for tissue engineering.

Research of degradable hydrogel polymeric materials exhibiting high water content and mechanical properties resembling tissues is crucial not only in drug delivery systems but also in tissue engineering, medical devices, and biomedical-healthcare sensors. Therefore, we newly offer development of hydrogels based on poly(2-hydroxyethyl methacrylate-co-2-(acetylthio) ethyl methacrylate-co-2-methacryloyloxyethyl phosphorylcholine) [P(HEMA-ATEMA-MPC)] and optimization of their mechanical and in vitro and in vivo degradability. P(HEMA-ATEMA-MPC) hydrogels differed in chemical composition, degree of crosslinking, and starting molar mass of polymers (15, 19, and 30 kDa). Polymer precursors were synthesized by a reversible addition fragmentation chain transfer (RAFT) polymerization using 2-(acetylthio)ethyl methacrylate containing protected thiol groups, which enabled crosslinking and gel formation. Elastic modulus of hydrogels increased with the degree of crosslinking (Slaughter et al., 2009) [1]. In vitro and in vivo controlled degradation was confirmed using glutathione and subcutaneous implantation of hydrogels in rats, respectively. We proved that the hydrogels with higher degree of crosslinking retarded the degradation. Also, albumin, γ-globulin, and fibrinogen adsorption on P(HEMA-ATEMA-MPC) hydrogel surface was tested, to simulate adsorption in living organism. Rat mesenchymal stromal cell adhesion on hydrogels was improved by the presence of RGDS peptide and laminin on the hydrogels. We found that rat mesenchymal stromal cells proliferated better on laminin-coated hydrogels than on RGDS-modified ones.

Microscopic Structure of Swollen Hydrogels by Scanning Electron and Light Microscopies: Artifacts and Reality.

The exact knowledge of hydrogel microstructure, mainly its pore topology, is a key issue in hydrogel engineering. For visualization of the swollen hydrogels, the cryogenic or high vacuum scanning electron microscopies (cryo-SEM or HVSEM) are frequently used while the possibility of artifact-biased images is frequently underestimated. The major cause of artifacts is the formation of ice crystals upon freezing of the hydrated gel. Some porous hydrogels can be visualized with SEM without the danger of artifacts because the growing crystals are accommodated within already existing primary pores of the gel. In some non-porous hydrogels the secondary pores will also not be formed due to rigid network structure of gels that counteracts the crystal nucleation and growth. We have tested the limits of true reproduction of the hydrogel morphology imposed by the swelling degree and mechanical strength of gels by investigating a series of methacrylate hydrogels made by crosslinking polymerization of glycerol monomethacrylate and 2-hydroxyethyl methacrylate including their interpenetrating networks. The hydrogel morphology was studied using cryo-SEM, HVSEM, environmental scanning electron microscopy (ESEM), laser scanning confocal microscopy (LSCM) and classical wide-field light microscopy (LM). The cryo-SEM and HVSEM yielded artifact-free micrographs for limited range of non-porous hydrogels and for macroporous gels. A true non-porous structure was observed free of artifacts only for hydrogels exhibiting relatively low swelling and high elastic modulus above 0.5 MPa, whereas for highly swollen and/or mechanically weak hydrogels the cryo-SEM/HVSEM experiments resulted in secondary porosity. In this contribution we present several cases of severe artifact formation in PHEMA and PGMA hydrogels during their visualization by cryo-SEM and HVSEM. We also put forward empirical correlation between hydrogel morphological and mechanical parameters and the occurrence and intensity of artifacts.

Biomimetic modification of dual porosity poly(2-hydroxyethyl methacrylate) hydrogel scaffolds-porosity and stem cell growth evaluation.

The macroporous synthetic poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels as 3D cellular scaffolds with specific internal morphology, so called dual pore size, were designed and studied. The morphological microstructure of hydrogels was characterized in the gel swollen state and the susceptibility of gels for stem cells was evaluated. The effect of specific chemical groups covalently bound in the hydrogel network by copolymerization on cell adhesion and growth, followed by effect of laminin coating were investigated. The evaluated gels contained either carboxyl groups of the methacrylic acid or quaternary ammonium groups brought by polymerizable ammonium salt or their combinations. The morphology of swollen gel was visualized using the laser scanning confocal microscopy. All hydrogels had very similar porous structures - their matrices contained large pores (up to 102 µm) surrounded with gel walls with small pores (100 µm). The total pore volume in hydrogels swollen in buffer solution ranged between 69 and 86 vol%. Prior to the seeding of the mouse embryonal stem cells, the gels were coated with laminin. The hydrogel with quaternary ammonium groups (with or without laminin) stimulated the cell growth the most. The laminin coating lead to a significant and quaternary ammonium groups. The gel chemical modification influenced also the topology of cell coverage that ranged from individual cell clusters to well dispersed multi cellular structures. Findings in this study point out the laser scanning confocal microscopy as an irreplaceable method for a precise and quick assessment of the hydrogel morphology. In addition, these findings help to optimize the chemical composition of the hydrogel scaffold through the combination of chemical and biological factors leading to intensive cell attachment and proliferation.

Biopolymer strategy for the treatment of Wilson's disease.

Wilson's disease is a genetic disorder that causes excessive accumulation of copper in the body, leading to toxic damage, especially in the liver and nervous system. The current treatment cause burdensome side effects. We describe the use of chemically modified biopolymer carriers based on microcrystalline cellulose and chitosan containing the highly specific copper chelator 8-hydroxyquinoline as a new type of therapy for Wilson's disease. The chelators can scavenges copper ions released from food during digestion and copper ions present in secretions in the gastrointestinal tract. Because the chelator is covalently bound to indigestible biopolymer carriers (crosslinked chitosan or modified cellulose), it is not taken up by the gastrointestinal tract and it can be eliminated through the feces, avoiding unwanted side effects. This concept was tested on Wistar rats, which received a radioactive 64CuCl2 solution together with the polymers with covalently bound 8-hydroxyquinoline through a gastric probe. 64Copper complex uptake from the gastrointestinal tract was significantly inhibited by both chelating polymers. With the modified polymers, the presence of 64Cu was detected mostly in the gastrointestinal tract, not in the internal organs. These findings indicate modified cellulose and crosslinked chitosan, with covalently bound 8-hydroxyquinoline exhibited the potential to be excellent therapeutics for treating Wilson's disease.

Porous Heat-Treated Polyacrylonitrile Scaffolds for Bone Tissue Engineering.

Heat-treated polyacrylonitrile (HT-PAN), also referred to as black orlon (BO), is a promising carbon-based material used for applications in tissue engineering and regenerative medicine. To the best of our knowledge, no such complex bone morphology-mimicking three-dimensional (3D) BO structure has been reported to date. We report that BO can be easily made into 3D cryogel scaffolds with porous structures, using succinonitrile as a porogen. The cryogels possess a porous morphology, similar to bone tissue. The prepared scaffolds showed strong osteoconductive activity, providing excellent support for the adhesion, proliferation, and mitochondrial activity of human bone-derived cells. This effect was more apparent in scaffolds prepared from a matrix with a higher content of PAN (i.e., 10% rather than 5%). The scaffolds with 10% of PAN also showed enhanced mechanical properties, as revealed by higher compressive modulus and higher compressive strength. Therefore, these scaffolds have a robust potential for use in bone tissue engineering.

Tungsten (VI) based "molecular puzzle" photoluminescent nanoparticles easily covered with biocompatible natural polysaccharides via direct chelation.

Multimodal probes, which can be simultaneously visualized by multiple imaging modalities, enable the cellular uptake, intracellular fate, biodistribution and elimination to be tracked in organisms. In this study, we report the synthesis of crystalline WO3 and CaWO4 doped with Eu3+ or Tb3+ nanoparticles (size range of 10-160 nm) coated with polysaccharides, and these nanoparticles constitute a versatile easy-to-construct modular toolbox for multimodal imaging. The particles adsorb significant amounts of polysaccharides from the solution, providing biocompatibility and may serve as a platform for labeling. For WO3, the sorption is reversible. However, on CaWO4, stable coating is formed. CaWO4/Tb3+ coated with chemisorbed dextrin, mannan, guar gum and sodium alginate successfully underwent endocytosis with HepG2 cells and was visualized using confocal microscopy.

Modified glycogen as construction material for functional biomimetic microfibers.

We describe a conceptually new, microfibrous, biodegradable functional material prepared from a modified storage polysaccharide also present in humans (glycogen) showing strong potential as direct-contact dressing/interface material for wound healing. Double bonds were introduced into glycogen via allylation and were further exploited for crosslinking of the microfibers. Triple bonds were introduced by propargylation and served for further click functionalization of the microfibers with bioactive peptide. A simple solvent-free method allowing the preparation of thick layers was used to produce microfibers (diameter ca 2µm) from allylated and/or propargylated glycogen. Crosslinking of the samples was performed by microtron beta-irradiation, and the irradiation dose was optimized to 2kGy. The results from biological testing showed that these highly porous, hydrophilic, readily functionalizable materials were completely nontoxic to cells growing in their presence. The fibers were gradually degraded in the presence of cells.

Thermoresponsive Polymers for Nuclear Medicine: Which Polymer Is the Best?

Thermoresponsive polymers showing cloud point temperatures (CPT) in aqueous solutions are very promising for the construction of various systems in biomedical field. In many of these applications these polymers get in contact with ionizing radiation, e.g., if they are used as carriers for radiopharmaceuticals or during radiation sterilization. Despite this fact, radiosensitivity of these polymers is largely overlooked to date. In this work, we describe the effect of electron beam ionizing radiation on the physicochemical and phase separation properties of selected thermoresponsive polymers with CPT between room and body temperature. Stability of the polymers to radiation (doses 0-20 kGy) in aqueous solutions increased in the order poly(N-vinylcaprolactam) (PVCL, the least stable) ≪ poly[N-(2,2-difluoroethyl)acrylamide] (DFP) < poly(N-isopropylacrylamide) (PNIPAM) ≪ poly(2-isopropyl-2-oxazoline-co-2-n-butyl-2-oxazoline) (POX). Even low doses of ß radiation (1 kGy), which are highly relevant to the storage of polymer radiotherapeutics and sterilization of biomedical systems, cause significant increase in molecular weight due to cross-linking (except for POX, where this effect is weak). In the case of PVCL irradiated with low doses, the increase in molecular weight induced an increase in the CPT of the polymer. For PNIPAM and DFP, there is strong chain hydrophilization leading to an increase in CPT. From this perspective, POX is the most suitable polymer for the construction of delivery systems that experience exposure to radiation, while PVCL is the least suitable and PNIPAM and DFP are suitable only for low radiation demands.

Bifunctional cyclam-based ligands with phosphorus acid pendant moieties for radiocopper separation: thermodynamic and kinetic studies.

Two macrocyclic ligands based on cyclam with trans-disposed N-methyl and N-(4-aminobenzyl) substituents as well as two methylphosphinic (H2L1) or methylphosphonic (H4L2) acid pendant arms were synthesised and investigated in solution. The ligands form stable complexes with transition metal ions. Both ligands show high thermodynamic selectivity for divalent copper over nickel(II) and zinc(II)-K(CuL) is larger than K(Ni/ZnL) by about seven orders of magnitude. Complexation is significantly faster for the phosphonate ligand H4L2, probably due to the stronger coordination ability of the more basic phosphonate groups, which efficiently bind the metal ion in an "out-of-cage" complex and thus accelerate its "in-cage" binding. The rate of Cu(II) complexation by the phosphinate ligand H2L1 is comparable to that of cyclam itself and its derivatives with non-coordinating substituents. Acid-assisted decomplexation of the copper(II) complexes is relatively fast (τ1/2 = 44 and 42 s in 1 M aq. HClO4 at 25 °C for H2L1 and H4L2, respectively). This combination of properties is convenient for selective copper removal/purification. Thus, the title ligands were employed in the preparation of ion-selective resins for radiocopper(II) separation. Glycidyl methacrylate copolymer beads were modified with the ligands through a diazotisation reaction. The separation ability of the modified polymers was tested with cold copper(II) and non-carrier-added (64)Cu in the presence of a large excess of both nickel(II) and zinc(II). The experiments exhibited high overall separation efficiency leading to 60-70% recovery of radiocopper with high selectivity over the other metal ions, which were originally present in 900-fold molar excess. The results showed that chelating resins with properly tuned selectivity of their complexing moieties can be employed for radiocopper separation.

Chelating polymeric beads as potential therapeutics for Wilson's disease.

Wilson's disease is a genetic disorder caused by a malfunction of ATPase 7B that leads to high accumulation of copper in the organism and consequent toxic effects. We propose a gentle therapy to eliminate the excessive copper content with oral administration of insoluble non-resorbable polymer sorbents containing selective chelating groups for copper(II). Polymeric beads with the chelating agents triethylenetetramine, N,N-di(2-pyridylmethyl)amine, and 8-hydroxyquinoline (8HQB) were investigated. In a preliminary copper uptake experiment, we found that 8HQB significantly reduced copper uptake (using copper-64 as a radiotracer) after oral administration in Wistar rats. Furthermore, we measured organ radioactivity in rats to demonstrate that 8HQB radiolabelled with iodine-125 is not absorbed from the gastrointestinal tract after oral administration. Non-resorbability and the blockade of copper uptake were also confirmed with small animal imaging (PET/CT) in mice. In a long-term experiment with Wistar rats fed a diet containing the polymers, we have found that there were no signs of polymer toxicity and the addition of polymers to the diet led to a significant reduction in the copper contents in the kidneys, brains, and livers of the rats. We have shown that polymers containing specific ligands could potentially be novel therapeutics for Wilson's disease.

Treating spinal cord injury in rats with a combination of human fetal neural stem cells and hydrogels modified with serotonin.

Currently, there is no effective strategy for the treatment of spinal cord injury (SCI). A combination of biomaterials and stem cell therapy seems to be a promising approach to increase regenerative potential after SCI. We evaluated the use of a cellpolymer construct based on a combination of the conditionally immortalized spinal progenitor cell line SPC-01_GFP3, derived from human fetal spinal cord tissue, with a serotonin-modified poly(2-hydroxyethyl methacrylate) hydrogel (pHEMA-5HT). We compared the effect of treatment with a pHEMA-5HT hydrogel seeded with SPC-01_GFP3 cells, treatment with a pHEMA-5HT only and no treatment on functional outcome and tissue reconstruction in hemisected rats. Prior to transplantation the cell-polymer construct displayed a high potential to support the growth, proliferation and differentiation of SPC-01 cells in vitro. One month after surgery, combined hydrogel-cell treatment reduced astrogliosis and tissue atrophy and increased axonal and blood vessel ingrowth into the implant; however, two months later only the ingrowth of blood vessels remained increased. SPC-01_GFP3 cells survived well in vivo and expressed advanced markers of neuronal differentiation. However, a majority of the transplanted cells migrated out of the lesion and only rarely remained in the hydrogel. No differences among the groups in motor or sensory recovery were observed. Despite the support of the hydrogel as a cell carrier in vitro, and good results in vivo one month postsurgery, there was only a small effect on long term recovery, mainly due to the limited ability of the hydrogels to support the in vivo growth and differentiation of cells within the implant. Further modifications will be necessary to achieve stable long term improvement in functional outcome.

Glycogen as a biodegradable construction nanomaterial for in vivo use.

It is demonstrated that glycogen as a biodegradable and inexpensive material coming from renewable resources can be used as a carrier for the construction of in vivo imaging nanoagents. The model system considered is composed of glycogen modified with gadolinium and fluorescent labels. Systematic studies of properties of these nanocarriers by a variety of physical methods and results of in vivo tests of biodegradability are reported. This represents, to the authors' best knowledge, the first such use of glycogen.

New coupling strategy for radionuclide labeling of synthetic polymers.

We have developed a radiolabeling strategy for synthetic polymers based on the formation of azo dye usable for both covalent and chelating labeling modalities under mild conditions. Poly[N-(2-hydroxypropyl)methacrylamide] and poly(N-isopropyl acrylamide) were used as model polymers. N-methacryloyl tyrosinamide was introduced into the polymers and the phenolic moiety was then reacted with diazotized chelator precursors. The conjugates were radiolabeled with both the covalently bound (iodine-125) and chelated (indium-111) radionuclides in high yields and sufficient in vitro stability of the labels was proven.

Biological evaluation of polymeric micelles with covalently bound doxorubicin.

The main limitation of contemporary anticancer chemotherapy remains to be the insufficient specificity of the drugs for tumor tissue, which decreases the maximum tolerated dose due to severe side effects. Micellar drug delivery systems based on amphiphilic block copolymers with a very narrow size distribution (10 to 100 nm in diameter) is a novel innovative approach. Here, we report biological and pharmacological properties of polymeric micellar conjugate containing doxorubicin (DOX) covalently bound via hydrolytically cleavable hydrazone bonds to the micelle core. The system had a very low systemic toxicity (almost 20 times lower than free DOX) and long circulation in the bloodstream (with half of the dose after 24 h). Significant accumulation of tested micelles within the tumor was confirmed by fluorescent whole body imaging. Our new micellar system showed promising therapeutic activity against established murine EL-4 T-cell lymphoma; it was found that it is able to completely cure about 75% of tumor-bearing mice (with doses of either 1 x 150 mg DOX kg(-1) or 2 x 75 mg DOX kg(-1), administered i.v.). Moreover, treatment with micelles in cured mice induced tumor-specific resistance. Up to 80% of these mice survived rechallenge with original but not with distinct tumor cells.

Thermoresponsive, hydrolytically degradable polymer micelles intended for radionuclide delivery.

Novel polymer micelles, prepared by self-assembling thermoresponsive poly(N-isopropylacrylamide)-graft-poly[N-(2-hydroxypropyl)methacrylamide] copolymers with hydrolytically degradable N-glycosylamine groups between the polymer blocks are proposed for delivery of diagnostic and therapeutic radionuclides into solid tumors. The micelles are formed by fast heating of an aqueous solution of the copolymer to 37 degrees C. They have a hydrodynamic diameter of 128 nm (measured using dynamic light scattering) and slowly degrade during incubation in aqueous buffer at pH = 7.4. Labeling with both (131)I and (90)Y proceeds with high yields (>85%). The unlabeled polymers are not cytotoxic for any of the tested murine and human cell lines.