Crosslinker concentration controls TGFß-3 release and annulus fibrosus cell apoptosis in genipin-crosslinked fibrin hydrogels.

Back pain is a leading cause of global disability associated with intervertebral disc (IVD) pathologies. Discectomy alleviates disabling pain caused by IVD herniation without repairing annulus fibrosus (AF) defects, which can cause accelerated degeneration and recurrent pain. Biological therapies show promise for IVD repair but developing high-modulus biomaterials capable of providing biomechanical stabilisation and delivering biologics remains an unmet challenge. The present study identified critical factors and developed an optimal formulation to enhance the delivery of AF cells and transforming growth beta-3 (TGFß-3) in genipin-crosslinked fibrin (FibGen) hydrogels. Part 1 showed that AF cells encapsulated in TGFß-3-supplemented high-modulus FibGen synthesised little extracellular matrix (ECM) but could release TGFß-3 at physiologically relevant levels. Part 2 showed that AF cells underwent apoptosis when encapsulated in FibGen, even after reducing fibrin concentration from 70 to 5 mg/mL. Mechanistic experiments, modifying genipin concentration and integrin binding site presence demonstrated that genipin crosslinking caused AF cell apoptosis by inhibiting cell-biomaterial binding. Adding integrin binding sites with fibronectin partially rescued apoptosis, indicating genipin also caused acute cytotoxicity. Part 3 showed that FibGen formulations with 1 mg/mL genipin had enhanced ECM synthesis when supplemented with fibronectin and TGFß-3. In conclusion, FibGen could be used for delivering biologically active compounds and AF cells, provided that formulations supplied additional sites for cell-biomaterial binding and genipin concentrations were low. Results also highlighted a need for developing strategies that protect cells against acute crosslinker cytotoxicity to overcome challenges of engineering high-modulus cell carriers for musculoskeletal tissues that experience high mechanical demands.

Hepatotoxic potential of asarones: in vitro evaluation of hepatotoxicity and quantitative determination in herbal products.

α and ß asarones are natural constituents of some aromatic plants, especially species of the genus Acorus (Araceae). In addition to reports of beneficial properties of asarones, genotoxicity and carcinogenicity are also reported. Due to potential toxic effects of ß-asarone, a limit of exposure from herbal products of ~2 µg/kg body weight/day has been set temporarily until a full benefit/risk assessment has been carried out by the European Medicines Agency. Therefore, it is important to monitor levels of ß-asarone in herbal products. In this study, we developed a simple, rapid and validated GC-MS method for quantitative determination of asarones and applied it in 20 pediatric herbal products after detecting high concentrations of ß-asarone in a product suspected to be implicated in hepatotoxicity in a 3 month old infant. Furthermore, targeted toxicological effects were further investigated in human hepatocytes (THLE-2 cells) by employing various in vitro assays, with the goal of elucidating possible mechanisms for the observed toxicity. Results showed that some of the products contained as much as 4-25 times greater amounts of ß-asarone than the recommended levels. In 4 of 10 samples found to contain asarones, the presence of asarones could not be linked to the labeled ingredients, possibly due to poor quality control. Cell-based investigations in THLE-2 cells confirmed the cytotoxicity of ß-asarone (IC50 = 40.0 ± 2.0 µg/mL) which was associated with significant lipid peroxidation and glutathione depletion. This observed cytotoxic effect is likely due to induction of oxidative stress by asarones. Overall, the results of this study ascertained the usability of this GC-MS method for the quantitative determination of asarones from herbal products, and shed light on the importance of controlling the concentration of potentially toxic asarones in herbal products to safeguard consumer safety, especially when the target consumers are young children. Further investigations of the toxicity of asarones are warranted.

Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes.

Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes.

Immune stimulation by a CpG-containing oligodeoxynucleotide is enhanced when encapsulated and delivered in lipid particles.

The therapeutic benefit from phosphorothioate oligodeoxynucleotides (PS ODN) containing immune stimulatory sequences (ISS) has been demonstrated in animal models of cancer and infection. In particular, when CpG-containing PS ODN are administered to mice, activation of macrophages and dendritic, NK, T, and B cells occurs, resulting in the release of an array of cytokines, including interleukin-12 (IL-12), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). We have previously described stabilized antisense-lipid particles (SALP) for the i.v. administration of antisense ODN [Biochim Biophys Acta (2001) 1510:152--166]. Given the propensity for SALP to target macrophages in vivo it was of interest to determine whether they could enhance the potency of CpG ODN to induce an immune response. In this report we show that when CpG-containing SALP are administered intravenously to ICR mice the plasma concentrations of IL-12, IFN-gamma, IL-6, monocyte chemoattractant protein-1, and TNF-alpha are greatly increased compared with the same dose of free ODN. The pattern of cytokine induction indicates that the immune response is T helper cell type 1-biased, similar to that observed for PS CpG ODN ISS in general. Furthermore, when phosphodiester (PO) ODN is substituted for PS ODN in the SALP formulation cytokine induction is even greater at the early time points, in marked contrast to free PO ODN, which is inactive. These results demonstrate that the immunogenicity of ISS is not only enhanced by encapsulation in lipid particles, which more closely mimic the way ISS DNA would normally be presented to antigen presenting cells by pathogens in vivo, but also SALP enable unmodified PO CpG ODN to be used as immune stimulants.

Synthesis and characterization of phosphorescent cyclometalated iridium complexes.

The preparation, photophysics, and solid state structures of octahedral organometallic Ir complexes with several different cyclometalated ligands are reported. IrCl3.nH2O cleanly cyclometalates a number of different compounds (i.e., 2-phenylpyridine, 2-(p-tolyl)pyridine, benzoquinoline, 2-phenylbenzothiazole, 2-(1-naphthyl)benzothiazole, and 2-phenylquinoline), forming the corresponding chloride-bridged dimers, CwedgeN2Ir(mu-Cl)2IrCwedgeN2 (CwedgeNis a cyclometalated ligand) in good yield. These chloride-bridged dimers react with acetyl acetone (acacH) and other bidentate, monoanionic ligands such as picolinic acid (picH) and N-methylsalicylimine (salH), to give monomeric CwedgeN2Ir(LX) complexes (LX = acac, pic, sal). The emission spectra of these complexes are largely governed by the nature of the cyclometalating ligand, leading to lambda(max) values from 510 to 606 nm for the complexes reported here. The strong spin-orbit coupling of iridium mixes the formally forbidden 3MLCT and 3pi-pi* transitions with the allowed 1MLCT, leading to a strong phosphorescence with good quantum efficiencies (0.1-0.4) and room temperature lifetimes in the microsecond regime. The emission spectra of the CwedgeN2Ir(LX) complexes are surprisingly similar to the fac-IrCwedgeN3 complex of the same ligand, even though the structures of the two complexes are markedly different. The crystal structures of two of the CwedgeN2Ir(acac) complexes (i.e., CwedgeN = ppy and tpy) have been determined. Both complexes show cis-C,C', trans-N,N' disposition of the two cyclometalated ligands, similar to the structures reported for other complexes with a "CwedgeN2Ir" fragment. NMR data (1H and 13C) support a similar structure for all of the CwedgeN2Ir(LX) complexes. Close intermolecular contacts in both (ppy)2Ir(acac) and (tpy)2Ir(acac) lead to significantly red shifted emission spectra for crystalline samples of the ppy and tpy complexes relative to their solution spectra.

Membrane perturbation and the mechanism of lipid-mediated transfer of DNA into cells.

Mixtures of cationic lipids and unsaturated phosphatidylethanolamine are used extensively for the intracellular delivery of plasmids and antisense oligodeoxynucleotides (ODN) in vitro. However, the mechanism by which cytoplasmic delivery of these large molecules is achieved remains unclear. The common hypothesis is that phosphatidylethanolamine promotes fusion of lipid/DNA particles with endosomal membranes, but this is inconsistent with several reports that have failed to correlate the fusogenic activity of a wide variety of lipid/DNA particles, measured by lipid mixing techniques, with their transfection activity. To address this issue further we have conducted a detailed analysis of the lipid mixing and DNA transfer activity of two, physically similar but functionally different, lipid/DNA particles composed of equimolar dioleyldimethylammonium chloride (DODAC) and dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC). In combination with DODAC both phospholipids form almost identical lipid/DNA particles, they are endocytosed by cells to the same extent and each undergoes equivalent lipid mixing with cell membranes after uptake. Despite this, DNA transfer is 10- to 100-fold more extensive for lipid/DNA particles containing DOPE. We conclude that lipid mixing between lipid-based delivery systems and endosomal membranes must occur for DNA transfer to occur. However, the potency of different lipid/DNA particles correlates better with the ability of the exogenous lipid to disrupt membrane integrity.

Optimization of a procedure for rebonding dislodged orthodontic brackets.

The purpose of this study was to compare shear bond strength (SBS) of bonded and rebonded orthodontic brackets following a variety of commonly used conditioning treatments and using both light-cured and self-cured composite resin systems. Brackets debonded during the initial determination of SBS were rebonded after the removal of residual resin from enamel surfaces using five different treatments: (1) Remove residual resin using a tungsten carbide bur, re-etch enamel surface, then bond a new bracket; (2) Remove resin from the base mesh with micro-etching then rebond the same bracket, (3) Remove residual resin from the enamel surface using resin-removing pliers, recondition the enamel with an air-powder polisher, then bond a new bracket; (4) Remove residual resin using a rubber cup and pumice, then bond a new bracket; (5) Remove residual resin using pliers alone, then bond a new bracket. The results revealed that the light-cured system produced higher shear bond strength in the initial bond than the self-cured system (p<0.005). Reconditioning the enamel surfaces using a tungsten carbide bur and acid-etching gave the highest SBS (difference 5.8 MPa; p<0.01) and clinically favorable fracture characteristics. The data suggest that the optimal procedure for rebonding dislodged orthodontic brackets is to resurface the enamel using a tungsten carbide bur, acid-etch the enamel, and use a new or re-use an old bracket after microetching.

Lipid-based systems for the intracellular delivery of genetic drugs.

Currently available delivery systems for genetic drugs have limited utility for systemic applications. Cationic liposome/plasmid DNA or oligonucleotide complexes are rapidly cleared from circulation, and the highest levels of activity are observed in 'first pass' organs, such as the lungs, spleen and liver. Engineered viruses can generate an immune response, which compromises transfection resulting from subsequent injections and lack target specificity. A carrier, which can accumulate at sites of diseases such as infections, inflammations and tumours, has to be a small, neutral and highly serum-stable particle, which is not readily recognized by the fixed and free macrophages of the reticuloendothelial system (RES). This review summarizes lipid-based technologies for the delivery of nucleic acid-based drugs and introduces a new class of carrier systems, which solve, at least in part, the conflicting demands of circulation longevity and intracellular delivery. Plasmid DNA and oligonucleotides are entrapped into lipid particles that contain small amounts of a positively charged lipid and are stabilized by the presence of a polythylene glycol (PEG) coating. These carriers protect nucleic acid-based drugs from degradation by nucleases, are on average 70 nm in diameter, achieve long circulation lifetimes and are capable of transfecting cells.

Cationic lipids, phosphatidylethanolamine and the intracellular delivery of polymeric, nucleic acid-based drugs (review).

Polymeric, nucleic acid drugs must be protected from endogenous nucleases and delivered to target cell nuclei in order to maximize their activity. Constructs expressing therapeutic genes, antisense oligonucleotides and ribozymes can be delivered into cells by viral vectors, but concerns over safety and clinical utility have led to research into the development of alternative, non-viral delivery systems. Antisense and ribozyme drug development has focused upon modifications to the natural oligonucleotide chemistry which make the molecules resistant to nuclease degradation. These novel oligonucleotides cannot be generated by transgenes and must be administered in similar fashion to conventional drugs. However, oligonucleotides cannot cross membranes by passive diffusion and intracellular delivery for these drugs is very inefficient. Here we review the recent advances in forming lipid-DNA particles designed to mimic viral delivery of DNA. Most evidence now supports the hypothesis that lipid-DNA drugs enter target cells by endocytosis and disrupt the endosomal membrane, releasing nucleic acid into the cytoplasm. The mechanisms of particle formation and endosome disruption are not well understood. Cationic lipids are employed to provide an electrostatic interaction between the lipid carrier and polyanionic nucleic acids, and they are critical for efficient packaging of the drugs into a form suitable for systemic administration. However, their role in endosome disruption and other aspects of successful delivery leading to gene expression or inhibition of mRNA translation are less clear. We discuss the propensity of lipid-nucleic acid particles to undergo lipid mixing and fusion with adjacent membranes, and how phosphatidylethanolamine and other lipids may act as factors capable of disrupting bilayer structure and the endosomal pathway. Finally, we consider the challenges that remain in bringing nucleic acid based drugs into the realm of clinical reality.

Influence of transbilayer area asymmetry on the morphology of large unilamellar vesicles.

The morphological consequences of differences in the monolayer surface areas of large unilamellar vesicles (LUVs) have been examined employing cryoelectron microscopy techniques. Surface area was varied by inducing net transbilayer transport of dioleoylphosphatidylglycerol (DOPG) in dioleoylphosphatidylcholine (DOPC):DOPG (9:1, mol:mol) LUVs in response to transmembrane pH gradients. It is shown that when DOPG is transported from the inner to the outer monolayer, initially invaginated LUVs are transformed to long narrow tubular structures, or spherical structures with one or more protrusions. Tubular structures are also seen in response to outward DOPG transport in DOPC:DOPG:Chol (6:1:3, mol:mol:mol) LUV systems, and when lyso-PC is allowed to partition into the exterior monolayer of DOPC:DOPG (9:1, mol:mol) LUVs in the absence of DOPG transport. Conversely, when the inner monolayer area is expanded by the transport of DOPG from the outer monolayer to the inner monolayer of non-invaginated LUVs, a reversion to invaginated structures is observed. The morphological changes are well described by an elastic bending theory of the bilayer. Identification of the difference in relaxed monolayer areas and of the volume-to-area ratio of the LUVs as the shape-determining factors allows a quantitative classification of the observed morphologies. The morphology seen in LUVs supports the possibility that factors leading to differences in monolayer surface areas could play important roles in intracellular membrane transport processes.

Influence of plasma on the osmotic sensitivity of large unilamellar vesicles prepared by extrusion.

In the presence of plasma, the osmotic differential required to trigger lysis of large unilamellar vesicles is significantly decreased with the membrane tension at rupture being reduced from about 36 to about 12 dynes/cm for vesicles composed of palmitoyloleoylphosphatidylcholine: cholesterol (55:45). Despite increasing vesicle sensitivity, however, plasma does not alter the characteristics of osmotically induced lysis. As in the absence of plasma, lysis is not an all-or-nothing event but instead results in only partial loss of intravesicular solute, so that following membrane resealing the vesicle interior remains hyperosmotic with respect to the external medium. To identify the component responsible for the observed increase in vesicle osmotic sensitivity, plasma was fractionated by density centrifugation. Albumin and other soluble plasma proteins, including those associated with the complement system, were found to exert only a modest influence on vesicle osmotic behavior. In contrast all of the lipoprotein fractions lowered vesicle tolerance to osmotic pressure, with high density lipoprotein exerting an effect comparable to whole plasma.

Osmotic properties of large unilamellar vesicles prepared by extrusion.

We have examined the morphology and osmotic properties of large unilamellar vesicles (LUVs) prepared by extrusion. Contrary to expectations, we observe by cryo-electron microscopy that such vesicles, under isoosmotic conditions, are non-spherical. This morphology appears to be a consequence of vesicle passage through the filter pores during preparation. As a result when such LUVs are placed in a hypoosmotic medium they are able to compensate, at least partially, for the resulting influx of water by "rounding up" and thereby increasing their volume with no change in surface area. The increase in vesicle trapped volume associated with these morphological changes was determined using the slowly membrane-permeable solute [3H]-glucose. This allowed calculation of the actual osmotic gradient experienced by the vesicle membrane for a given applied differential. When LUVs were exposed to osmotic differentials of sufficient magnitude lysis occurred with the extent of solute release being dependent on the size of the osmotic gradient. Surprisingly, lysis was not an all-or-nothing event, but instead a residual osmotic differential remained after lysis. This differential value was comparable in magnitude to the minimum osmotic differential required to trigger lysis. Further, by comparing the release of solutes of differing molecular weights (glucose and dextran) a lower limit of about 12 nm diameter can be set for the bilayer defect created during lysis. Finally, the maximum residual osmotic differentials were compared for LUVs varying in mean diameter from 90 to 340 nm. This comparison confirmed that these systems obey Laplace's Law relating vesicle diameter and lysis pressure. This analysis also yielded a value for the membrane tension at lysis of 40 dyn cm-1 at 23 degrees C, which is in reasonable agreement with previously published values for giant unilamellar vesicles.