Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro

Background Increasing concern has been expressed regarding the potential adverse health effects that may be associated with human exposure to inhaled multi-walled carbon nanotubes (MWCNTs). Thus it is imperative that an understanding as to the underlying mechanisms and the identification of the key factors involved in adverse effects are gained. In the alveoli, MWCNTs first interact with the pulmonary surfactant. At this interface, proteins and lipids of the pulmonary surfactant bind to MWCNTs, affecting their surface characteristics. Aim of the present study was to investigate if the pre-coating of MWCNTs with pulmonary surfactant has an influence on potential adverse effects, upon both (i) human monocyte derived macrophages (MDM) monocultures, and (ii) a sophisticated in vitro model of the human epithelial airway barrier. Both in vitro systems were exposed to MWCNTs either pre-coated with a porcine pulmonary surfactant (Curosurf) or not. The effect of MWCNTs surface charge was also investigated in terms of amino (−NH2) and carboxyl (−COOH) surface modifications. Results Pre-coating of MWCNTs with Curosurf affects their oxidative potential by increasing the reactive oxygen species levels and decreasing intracellular glutathione depletion in MDM as well as decreases the release of Tumour necrosis factor alpha (TNF-α). In addition, an induction of apoptosis was observed after exposure to Curosurf pre-coated MWCNTs. In triple cell-co cultures the release of Interleukin-8 (IL-8) was increased after exposure to Curosurf pre-coated MWCNTs. Effects of the MWCNTs functionalizations were minor in both MDM and triple cell co-cultures. Conclusions The present study clearly indicates that the pre-coating of MWCNTs with pulmonary surfactant more than the functionalization of the tubes is a key factor in determining their ability to cause oxidative stress, cytokine/chemokine release and apoptosis. Thus the coating of nano-objects with pulmonary surfactant should be considered for future lung in vitro risk assessment studies. Keywords: Multi-walled carbon nanotubes (MWCNTs); Pulmonary surfactant (Curosurf); Macrophages; Epithelial cells; Dendritic cells; Triple cell co-culture; Pro-inflammatory and oxidative reactions

Scale Up of PLA Nanoparticle Synthesis by Solvent Displacement and Functionalization of the Particles with a Silica Shell for Targeted Drug Delivery Applications

Biodegradable polymer nanoparticles have the properties necessary to address many of the issues associated with current drug delivery techniques including targeted and controlled delivery. A novel drug delivery vehicle is proposed consisting of a poly(lactic acid) nanoparticle core, with a functionalized, mesoporous silica shell. In this study, the production of PLA nanoparticles is investigated using solvent displacement in both a batch and continuous manner, and the effects of various system parameters are examined. Using Pluronic F-127 as the stabilization agent throughout the study, PLA nanoparticles are produced through solvent displacement with diameters ranging from 200 to 250 nm using two different methods: dropwise addition and in an impinging jet mixer. The impinging jet mixer allows for easy scale-up of particle production. The concentration of surfactant and volume of quench solution is found to have minimal impact on particle diameter; however, the concentration of PLA is found to significantly impact the diameter mean and polydispersity. In addition, the stability of the PLA nanoparticles is observed to increase as residual THF is evaporated. Lastly, the isolated PLA nanoparticles are coated with a silica shell using the Stöber Process. It is found that functionalizing the silica with a phosphonic silane in the presence of excess Pluronic F-127 decreases coalescence of the particles during the coating process. Future work should be conducted to fine-tune the PLA nanoparticle synthesis process by understanding the effect of other system parameters and in synthesizing mesoporous silica shells.

Effect Of Confined Impinging Jet Mixing Processing Parameters On Poly (Lactic Acid) Nanoparticle Morphology

Biodegradable nanoparticles are at the forefront of drug delivery research as they provide numerous advantages over traditional drug delivery methods. An important factor affecting the ability of nanoparticles to circulate within the blood stream and interact with cells is their morphology. In this study a novel processing method, confined impinging jet mixing, was used to form poly (lactic acid) nanoparticles through a solvent-diffusion process with Pluronic F-127 being used as a stabilizing agent. This study focused on the effects of Reynolds number (flow rate), surfactant presence in mixing, and polymer concentration on the morphology of poly (lactic acid) nanoparticles. In addition to looking at the parameters affecting poly (lactic acid) morphology, this study attempted to improve nanoparticle isolation and purification methods to increase nanoparticle yield and ensure specific morphologies were not being excluded during isolation and purification. The isolation and purification methods used in this study were centrifugation and a stir cell. This study successfully produced particles having pyramidal and cubic morphologies. Despite successful production of these morphologies the yield of non-spherical particles was very low, additionally great variability existed between redundant trails. Surfactant was determined to be very important for the stabilization of nanoparticles in solution but appears to be unnecessary for the formation of nanoparticles. Isolation and purification methods that produce a high yield of surfactant free particles have still not been perfected and additional testing will be necessary for improvement.¿

Solid-State Compatibilization of Immiscible Polymer Blends: Cryogenic Milling and Solid-State Shear Pulverization

The blending of common polymers allows for the rapid and facile synthesis of new materials with highly tunable properties at a fraction of the costs of new monomer development and synthesis. Most blends of polymers, however, are completely immiscible and separate into distinct phases with minimal phase interaction, severelydegrading the performance of the material. Cross-phase interactions and property enhancement can be achieved with these blends through reactive processing or compatibilizer addition. A new class of blend compatibilization relies on the mechanochemical reactions between polymer chains via solid-state, high energy processing. Two contrasting mechanochemical processing techniques are explored in this thesis: cryogenic milling and solid-state shear pulverization (SSSP). Cryogenic milling is a batch process where a milling rod rapidly impacts the blend sample while submerged within a bath of liquid nitrogen. In contrast, SSSP is a continuous process where blend components are subjected to high shear and compressive forces while progressing down a chilled twin-screw barrel. In the cryogenic milling study, through the application of a synthesized labeledpolymer, in situ formation of copolymers was observed for the first time. The microstructures of polystyrene/high-density polyethylene (PS/HDPE) blends fabricated via cryomilling followed by intimate melt-state mixing and static annealing were found to be morphologically stable over time. PS/HDPE blends fabricated via SSSP also showed compatibilization by way of ideal blend morphology through growth mechanisms with slightly different behavior compared to the cryomilled blends. The new Bucknell University SSSP instrument was carefully analyzed and optimized to produce compatibilized polymer blends through a full-factorial experiment. Finally, blends of varying levels of compatibilization were subjected to common material tests to determine alternative means of measuring and quantifying compatibilization,

Characterization of Biodegradable Polymer Nanocomposites Fabricated via Solid-State Processing

Biodegradable polymer/clay nanocomposites were prepared withpristine and organically modified montmorillonite in polylactic acid (PLA) and polycaprolactone (PCL) polymer matrices. Nanocomposites were fabricated using extrusion and SSSP to compare the effects of melt-state and solid-state processing on the morphology of the final nanocomposite. Characterization of various material properties was performed on prepared biodegradable polymer/clay nanocomposites to evaluate property enhancements from different clays and/or processing methods.

A fluorescently labeled dendronized polymer-enzyme conjugate carrying multiple copies of two different types of active enzymes

A hybrid structure of a synthetic dendronized polymer, two different types of enzymes (superoxide dismutase and horseradish peroxidase), and a fluorescent dye (fluorescein) was synthesized. Thereby, a single polymer chain carried multiple copies of the two enzymes and the fluorescein. The entire attachment chemistry is based on UV/vis-quantifiable bis-aryl hydrazone bond formation that allows direct quantification of bound molecules: 60 superoxide dismutase, 120 horseradish peroxidase, and 20 fluorescein molecules on an average polymer chain of 2000 repeating units. To obtain other enzyme ratios the experimental conditions were altered accordingly. Moreover, it could be shown that both enzymes remained fully active and catalyzed a two-step cascade reaction.

Wetting and spreading of a surfactant film on solid particles: influence of sharp edges and surface irregularities

In addition to particle size and surface chemistry, the shape of particles plays an important role in their wetting and displacement by the surfactant film in the lung. The role of particle shape was the subject of our investigations using a model system consisting of a modified Langmuir-Wilhelmy surface balance. We measured the influence of sharp edges (lines) and other highly curved surfaces, including sharp corners or spikes, of different particles on the spreading of a dipalmitoylphosphatidyl (DPPC) film. The edges of cylindrical sapphire plates (circular curved edges, 1.65 mm radius) were wetted at a surface tension of 10.7 mJ/m2 (standard error (SE) = 0.45, n = 20) compared with that of 13.8 mJ/m2 (SE = 0.20, n = 20) for cubic sapphire plates (straight linear edges, edge length 3 mm) (p < 0.05). The top surfaces of the sapphire plates (cubic and cylindrical) were wetted at 8.4 mJ/m2 (SE = 0.54, n = 20) and 9.1 mJ/m2 (SE = 0.50, n = 20), respectively, but the difference was not significant (p > 0.05). The surfaces of the plates showed significantly higher resistance to spreading compared to that of the edges, as substantially lower surface tensions were required to initiate wetting (p < 0.05). Similar results were found for talc particles, were the edges of macro- and microcrystalline particles were wetted at 7.2 mJ/m2 (SE = 0.52, n = 20) and 8.2 mJ/m2 (SE = 0.30, n = 20) (p > 0.05), respectively, whereas the surfaces were wetted at 3.8 mJ/m2 (SE = 0.89, n = 20) and 5.8 mJ/m2 (SE = 0.52, n = 20) (p < 0.05), respectively. Further experiments with pollen of malvaceae and maize (spiky and fine knobbly surfaces) were wetted at 10.0 mJ/m2 (SE = 0.52, n = 10) and 22.75 mJ/m2 (SE = 0.81, n = 10), respectively (p < 0.05). These results show that resistance to spreading of a DPPC film on various surfaces is dependent on the extent these surfaces are curved. This is seen with cubic sapphire plates which have at their corners a radius of curvature of about 0.75 microm, spiky malvaceae pollen with an even smaller radius on top of their spikes, or talc with various highly curved surfaces. These highly curved surfaces resisted wetting by the DPPC film to a higher degree than more moderately curved surfaces such as those of cylindrical sapphire plates, maize pollens, or polystyrene spheres, which have a surface free energy similar to that of talc but a smooth surface. The macroscopic plane surfaces of the particles demonstrated the greatest resistance to spreading. This was explained by the extremely fine grooves in the nanometer range, as revealed by electron microscopy. In summary, to understand the effects of airborne particles retained on the surfaces of the respiratory tract, and ultimately their pathological potential, not only the particle size and surface chemistry but also the particle shape should be taken in consideration.

Morphological alterations of exogenous surfactant inhibited by meconium can be prevented by dextran

BACKGROUND: Surfactant dysfunction due to inhibition is involved in the pathophysiology of meconium aspiration syndrome. Dextran addition has been shown to reverse exogenous surfactant inactivation by meconium, but the precise mechanisms and the morphological correlate of this effect are yet unknown. Morphological surfactant analysis by transmission electron microscopy (TEM) and stereology allows the differentiation of active (large aggregates = LA) and inactive (small aggregates = SA) subtypes. METHODS: To determine the in vitro effects of meconium and dextran addition on the morphology of a modified porcine natural surfactant (Curosurf), Curosurf samples were either incubated alone or together with meconium or with meconium and dextran, fixed and processed for TEM. Volume fractions of surfactant subtypes [lamellar body-like forms (LBL), multilamellar vesicles (MV), unilamellar vesicles (UV)] were determined stereologically. RESULTS: All preparations contained LBL and MV (corresponding to LA) as well as UV (corresponding to SA). The volume fraction of UV increased with addition of meconium and decreased with further addition of dextran. Correspondingly, the UV/(LBL+MV) ratio (resembling the SA/LA ratio) increased when meconium was added and decreased when dextran was added to the surfactant-meconium mixture. CONCLUSION: Meconium causes alterations in the ultrastructural composition of Curosurf that can be visualized and analyzed by TEM and stereology. These alterations resemble an increase in the SA/LA ratio and are paralleled by an increase in minimum surface tension. Dextran prevents these effects and may therefore be a useful additive to exogenous surfactant preparations to preserve their structural and functional integrity, thereby improving their resistance to inactivation.

Alteration of the pulmonary surfactant system in full-term infants with hereditary ABCA3 deficiency

RATIONALE: ABCA3 mutations are known to cause fatal surfactant deficiency. OBJECTIVE: We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis. METHODS: Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting. RESULTS: ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro-SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro-SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro-SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively. CONCLUSION: Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis.

Polymer stent coating for prevention of neointimal hyperplasia

AIMS: Restenosis has been the principal limitation of bare metal stents. Based upon the presumption that platelet and inflammatory cell recruitment initiate neointimal proliferation, we explored a novel polymer coating that reduces cell-stent interactions. The purpose of the present study was to investigate the effect of poly(L-lysine)-graft-poly(ethyleneglycol) (PLL-g-PEG) adsorbed to stent surfaces to reduce neointimal hyperplasia in the porcine restenosis model. METHODS AND RESULTS: Seven animals were instrumented each with 2 stainless steel stents (15 mm length, 2.5-3.5 mm diameter), randomly implanted in 1 major epicardial coronary artery. One stent was dip-coated with PLL-g-PEG, whereas the other stent served as the uncoated control stent. All animals were sacrificed after 6 weeks for histological examination. Neointimal hyperplasia was significantly less (-51%) in the PLL-g-PEG-coated stents (1.15 +/- 0.59 mm2) than in the uncoated control stents (2.33 +/- 1.01 mm2; p < 0.001). Conversely, lumen size was larger in the PLL-g-PEG-coated stents (2.91 +/- 1.17 mm2) than in the uncoated stents (2.04 +/- 0.64 mm2; p < 0.001). High magnification histomorphologic examination revealed no signs of inflammation or thrombus formation in either stent group. CONCLUSIONS: Polymeric steric stabilization of stents with PLL-g-PEG significantly reduces neointimal hyperplasia in the porcine restenosis model. Reduction of cell-stent interactions mediated by PLL-g-PEG appear to improve biocompatibility of stainless steel stents without evidence of adverse inflammatory or prothrombotic effects.

Surfactant mediated adsorption of negatively charged latex particles to a cellulose surface

The adsorption of anionic, carboxyl functionalized latex particles, recharged by a cationic surfactant acting as fabric softener/conditioner, to a cellulose surface was investigated with evanescent wave video microscopy. This technique allows to monitor the deposition and release of individual particles in real-time with an excellent selectivity and sensitivity. Since the recharged particles and the conditioner compete for the free surface, the initial deposition rate and final surface coverage are found to be strongly dependent on the ratio of particle and conditioner concentrations.

The long-term mechanical integrity of non-reinforced PEEK-OPTIMA polymer for demanding spinal applications: experimental and finite-element analysis

Polyetheretherketone (PEEK) is a novel polymer with potential advantages for its use in demanding orthopaedic applications (e.g. intervertebral cages). However, the influence of a physiological environment on the mechanical stability of PEEK has not been reported. Furthermore, the suitability of the polymer for use in highly stressed spinal implants such as intervertebral cages has not been investigated. Therefore, a combined experimental and analytical study was performed to address these open questions. A quasi-static mechanical compression test was performed to compare the initial mechanical properties of PEEK-OPTIMA polymer in a dry, room-temperature and in an aqueous, 37 degrees C environment (n=10 per group). The creep behaviour of cylindrical PEEK polymer specimens (n=6) was measured in a simulated physiological environment at an applied stress level of 10 MPa for a loading duration of 2000 hours (12 weeks). To compare the biomechanical performance of different intervertebral cage types made from PEEK and titanium under complex loading conditions, a three-dimensional finite element model of a functional spinal unit was created. The elastic modulus of PEEK polymer specimens in a physiological environment was 1.8% lower than that of specimens tested at dry, room temperature conditions (P<0.001). The results from the creep test showed an average creep strain of less than 0.1% after 2000 hours of loading. The finite element analysis demonstrated high strain and stress concentrations at the bone/implant interface, emphasizing the importance of cage geometry for load distribution. The stress and strain maxima in the implants were well below the material strength limits of PEEK. In summary, the experimental results verified the mechanical stability of the PEEK-OPTIMA polymer in a simulated physiological environment, and over extended loading periods. Finite element analysis supported the use of PEEK-OPTIMA for load-bearing intervertebral implants.

Methylprednisolone Fails to Preserve Pulmonary Surfactant and Blood-Air Barrier Integrity in a Porcine Cardiopulmonary Bypass Model

BACKGROUND: Pulmonary inflammation after cardiac surgery with cardiopulmonary bypass (CPB) has been linked to respiratory dysfunction and ultrastructural injury. Whether pretreatment with methylprednisolone (MP) can preserve pulmonary surfactant and blood-air barrier, thereby improving pulmonary function, was tested in a porcine CPB-model. MATERIALS AND METHODS: After randomizing pigs to placebo (PLA; n = 5) or MP (30 mg/kg, MP; n = 5), animals were subjected to 3 h of CPB with 1 h of cardioplegic cardiac arrest. Hemodynamic data, plasma tumor necrosis factor-alpha (TNF-alpha, ELISA), and pulmonary function parameters were assessed before, 15 min after CPB, and 8 h after CPB. Lung biopsies were analyzed for TNF-alpha (Western blot) or blood-air barrier and surfactant morphology (electron microscopy, stereology). RESULTS: Systemic TNF-alpha increased and cardiac index decreased at 8 h after CPB in PLA (P < 0.05 versus pre-CPB), but not in MP (P < 0.05 versus PLA). In both groups, at 8 h after CPB, PaO(2) and PaO(2)/FiO(2) were decreased and arterio-alveolar oxygen difference and pulmonary vascular resistance were increased (P < 0.05 versus baseline). Postoperative pulmonary TNF-alpha remained unchanged in both groups, but tended to be higher in PLA (P = 0.06 versus MP). The volume fraction of inactivated intra-alveolar surfactant was increased in PLA (58 +/- 17% versus 83 +/- 6%) and MP (55 +/- 18% versus 80 +/- 17%) after CPB (P < 0.05 versus baseline for both groups). Profound blood-air barrier injury was present in both groups at 8 h as indicated by an increased blood-air barrier integrity score (PLA: 1.28 +/- 0.03 versus 1.70 +/- 0.1; MP: 1.27 +/- 0.08 versus 1.81 +/- 0.1; P < 0.05). CONCLUSION: Despite reduction of the systemic inflammatory response and pulmonary TNF-alpha generation, methylprednisolone fails to decrease pulmonary TNF-alpha and to preserve pulmonary surfactant morphology, blood-air barrier integrity, and pulmonary function after CPB.