Magnetic conjugated polymer nanoparticles doped with a europium complex for biomedical imaging.

Self-assembling conjugated polymer nanoparticles containing PVK and PLGA-PEG as a matrix polymer were doped with both a luminescent rare-earth complex and magnetic nanoparticles (SPIONs), giving rise to materials that are both luminescent and magnetic. Nanoparticle sizes ranged from 80-110 nm without SPIONs and showed an increase in size (200-1000 nm) with additional SPION content (11-54%). Quantum yields (QYs) of 24% and 18% were measured for systems without and with 11% SPIONs, respectively. Optical properties were stable and suitable for biological imaging applications.

Insights on animal models to investigate inhalation therapy: Relevance for biotherapeutics.

Acute and chronic respiratory diseases account for major causes of illness and deaths worldwide. Recent developments of biotherapeutics opened a new era in the treatment and management of patients with respiratory diseases. When considering the delivery of therapeutics, the inhaled route offers great promises with a direct, non-invasive access to the diseased organ and has already proven efficient for several molecules. To assist in the future development of inhaled biotherapeutics, experimental models are crucial to assess lung deposition, pharmacokinetics, pharmacodynamics and safety. This review describes the animal models used in pulmonary research for aerosol drug delivery, highlighting their advantages and limitations for inhaled biologics. Overall, non-clinical species must be selected with relevant scientific arguments while taking into account their complexities and interspecies differences, to help in the development of inhaled medicines and ensure their successful transposition in the clinics.

Bright conjugated polymer nanoparticles containing a biodegradable shell produced at high yields and with tuneable optical properties by a scalable microfluidic device.

This study compares the performance of a microfluidic technique and a conventional bulk method to manufacture conjugated polymer nanoparticles (CPNs) embedded within a biodegradable poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG5K-PLGA55K) matrix. The influence of PEG5K-PLGA55K and conjugated polymers cyano-substituted poly(p-phenylene vinylene) (CN-PPV) and poly(9,9-dioctylfluorene-2,1,3-benzothiadiazole) (F8BT) on the physicochemical properties of the CPNs was also evaluated. Both techniques enabled CPN production with high end product yields (∼70-95%). However, while the bulk technique (solvent displacement) under optimal conditions generated small nanoparticles (∼70-100 nm) with similar optical properties (quantum yields ∼35%), the microfluidic approach produced larger CPNs (140-260 nm) with significantly superior quantum yields (49-55%) and tailored emission spectra. CPNs containing CN-PPV showed smaller size distributions and tuneable emission spectra compared to F8BT systems prepared under the same conditions. The presence of PEG5K-PLGA55K did not affect the size or optical properties of the CPNs and provided a neutral net electric charge as is often required for biomedical applications. The microfluidics flow-based device was successfully used for the continuous preparation of CPNs over a 24 hour period. On the basis of the results presented here, it can be concluded that the microfluidic device used in this study can be used to optimize the production of bright CPNs with tailored properties with good reproducibility.

Red-emitting protein-coated conjugated polymer nanoparticles.

Red emitting materials are desirable in biology due to the transparency of certain biological tissues at these wavelengths. Here, we report the synthesis of aqueous dispersions of amphiphilic protein (hydrophobin) capped red-emitting cyano-substituted poly(p-phenylenevinylene) conjugated polymer nanoparticles (CPNs) and their use in labeling live mammalian (HeLa) cells.

The Biosynthesis of Infrared-Emitting Quantum Dots in Allium Fistulosum.

The development of simple routes to emissive solid-state materials is of paramount interest, and in this report we describe the biosynthesis of infrared emitting quantum dots in a living plant via a mutual antagonistic reaction. Exposure of common Allium fistulosum to mercury and tellurium salts under ambient conditions resulted in the expulsion of crystalline, non-passivated HgTe quantum dots that exhibited emissive characteristics in the near-infrared spectral region, a wavelength range that is important in telecommunications and solar energy conversion.

In vivo biocompatibility, clearance, and biodistribution of albumin vehicles for pulmonary drug delivery.

The development of clinically acceptable albumin-based nanoparticle formulations for use in pulmonary drug delivery has been hindered by concerns about the toxicity of nanomaterials in the lungs combined with a lack of information on albumin nanoparticle clearance kinetics and biodistribution. In this study, the in vivo biocompatibility of albumin nanoparticles was investigated following a single administration of 2, 20, and 390µg/mouse, showing no inflammatory response (TNF-α and IL-6, cellular infiltration and protein concentration) compared to vehicle controls at the two lower doses, but elevated mononucleocytes and a mild inflammatory effect at the highest dose tested. The biodistribution and clearance of (111)In labelled albumin solution and nanoparticles over 48h following a single pulmonary administration to mice was investigated by single photon emission computed tomography and X-ray computed tomography imaging and terminal biodistribution studies. (111)In labelled albumin nanoparticles were cleared more slowly from the mouse lung than (111)In albumin solution (64.1±8.5% vs 40.6±3.3% at t=48h, respectively), with significantly higher (P<0.001) levels of albumin nanoparticle-associated radioactivity located within the lung tissue (23.3±4.7%) compared to the lung fluid (16.1±4.4%). Low amounts of (111)In activity were detected in the liver, kidneys, and intestine at time points >24h indicating that small amounts of activity were cleared from the lungs both by translocation across the lung mucosal barrier, as well as mucociliary clearance. This study provides important information on the fate of albumin vehicles in the lungs, which may be used to direct future formulation design of inhaled nanomedicines.

Biosynthesis of luminescent quantum dots in an earthworm.

The synthesis of designer solid-state materials by living organisms is an emerging field in bio-nanotechnology. Key examples include the use of engineered viruses as templates for cobalt oxide (Co(3)O(4)) particles, superparamagnetic cobalt-platinum alloy nanowires and gold-cobalt oxide nanowires for photovoltaic and battery-related applications. Here, we show that the earthworm's metal detoxification pathway can be exploited to produce luminescent, water-soluble semiconductor cadmium telluride (CdTe) quantum dots that emit in the green region of the visible spectrum when excited in the ultraviolet region. Standard wild-type Lumbricus rubellus earthworms were exposed to soil spiked with CdCl(2) and Na(2)TeO(3) salts for 11 days. Luminescent quantum dots were isolated from chloragogenous tissues surrounding the gut of the worm, and were successfully used in live-cell imaging. The addition of polyethylene glycol on the surface of the quantum dots allowed for non-targeted, fluid-phase uptake by macrophage cells.

Sparing methylation of beta-cyclodextrin mitigates cytotoxicity and permeability induction in respiratory epithelial cell layers in vitro.

Cyclodextrins (CDs) are promising solubility enhancers for inhaled drug delivery. However, they have dose-dependent effects on the respiratory epithelium, which may have advantages for permeability enhancement but also gives rise to safety concerns. In this study, the methyl thiazol tetrazolium (MTT) assay was used to compare a new sparingly methylated beta-CD, Kleptose Crysmebeta (Crysmeb) with the more established CD derivatives hydroxypropyl-gamma-cyclodextrin (HPgammaCD), randomly methylated beta-cyclodextrin (Rameb) and hydroxypropyl-beta-cyclodextrin (HPbetaCD). The betaCD derivatives affected cell metabolism in A549 cells in a concentration dependent manner with LD(50) of 56, 31 and 11 mM obtained for HPbetaCD, Crysmeb and Rameb, respectively. Calu-3 cells were less susceptible to betaCD with an LD(50) of 25 mM being obtained for Rameb only. Permeability increases in Calu-3 cell layers were observed with betaCD derivatives and a concentration dependency shown. The mechanism of permeability enhancement and its reversibility was investigated. Rameb produced an irreversible loss of cell layer barrier function at > or = 25 mM, but perturbations of epithelial integrity were moderate and reversible in the case of HPbetaCD and Crysmeb (25-50 mM). Given its high solubilisation capacity, the low toxicity and transient absorption promoting properties, this study identifies Crysmeb as a promising adjuvant in formulations for inhalation.

Investigation of the proinflammatory potential of biodegradable nanoparticle drug delivery systems in the lung.

Particulate nanocarriers have been praised for their advantageous drug delivery properties in the lung, such as avoidance of macrophage clearance mechanisms and long residence times. However, instilled non-biodegradable polystyrene nanospheres with small diameters and thus large surface areas have been shown to induce pulmonary inflammation. This study examines the potential of biodegradable polymeric nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) and the novel PLGA derivative, diethylaminopropylamine polyvinyl alcohol-grafted-poly(lactic-co-glycolic acid) (DEAPA-PVAL-g-PLGA), to provoke inflammatory responses in the murine lung after intratracheal instillation. Lactate dehydrogenase (LDH) release, protein concentration, MIP-2 mRNA induction, and polymorphonucleocyte (PMN) recruitment in the bronchial alveolar lavage fluid (BALF) were used to evaluate an inflammatory response in Balb-C mice. Two sizes of polystyrene (PS) nanospheres (diameters: 75 nm and 220 nm) were included in the study for comparison. All nanoparticle suspensions were instilled at concentrations of 1 microg/microl and 2.5 microg/microl, representative of an estimated "therapeutic dose" and a concentrated "dose" of particles. In all experiments, the 75 nm PS particles exhibited elevated responses for the inflammatory markers investigated. In contrast, biodegradable particles of comparable hydrodynamic diameter showed a significantly lower inflammatory response. The most marked differences were observed in the extent of PMN recruitment. While the 75 nm and 220 nm PS nanospheres exhibited 41 and 74% PMN within the total BALF cell population after 24 h, respectively, PMN recruiting in lungs instilled with both types of biodegradable particles did not exceed values of the negative isotonic glucose control. In conclusion, evidence suggests that biodegradable polymeric nanoparticles designed for pulmonary drug delivery may not induce the same inflammatory response as non-biodegradable polystyrene particles of comparable size.

Modified polyethylenimines as non viral gene delivery systems for aerosol therapy: effects of nebulization on cellular uptake and transfection efficiency.

This study examined the effect of nebulization on the cellular uptake and transfection efficiency of polyplexes from four polyethylenimine (PEI) modifications: branched 25 kDa PEI (bPEI), linear 22 kDa PEI (linPEI), pegylated PEI (pegPEI) and biodegradable PEI (bioPEI). Polyplexes were aerosolized with air-jet and ultrasonic nebulizers. The aerosol was collected and used to determine complex size and zeta potential. Fluorescence-assisted cell sorting (FACS) was used to quantify the cellular association of polyplexes in primary alveolar cells (AEC), A549 cells and primary bronchial cells (BEC). Confocal laser scanning microscopic images provided information about the internalization of polyplexes. Transfection efficiencies of polyplexes were quantified via measurement of luciferase expression. All polymers were stable during nebulization, although size increases were observed after air-jet nebulization. FACS studies showed a two- to three-fold increase in polyplex association with BEC compared to A549 cells, while polyplex association with AEC was negligible. BPEI, linPEI and bioPEI polyplexes were internalized, while pegPEI polyplexes remained predominately attached to the cellular membrane. Luciferase expression was detected only in BEC and A549 cells with transfection efficiencies approximately one order of magnitude higher in BEC. All PEI modifications investigated were suitable for aerosol therapy, although cell type and polymer structure significantly influenced the uptake and transfection efficiency of the polyplexes.

Modified polyethylenimines as non-viral gene delivery systems for aerosol gene therapy: investigations of the complex structure and stability during air-jet and ultrasonic nebulization.

Polyelectrolyte complexes between DNA and polyethylenimine (PEI) are promising non-viral delivery systems for pulmonary inhalation gene therapy and thus require sufficient stability during nebulization. The structure and stability of four different PEI-DNA polyplexes, namely branched (bPEI), linear (linPEI), poly(ethylene glycol)-grafted PEI (PEGPEI), biodegradable (bioPEI) PEI with DNA, were investigated. Using atomic force microscopy, the morphology of DNA and polyplexes before and after both air-jet and ultrasonic nebulization was characterized. The influence of nebulization on physico-chemical properties, particle size and zeta potential, was studied. Efficient DNA condensation to spherical particles was achieved with bPEI (90 nm) and PEGPEI (110 nm). By contrast, incomplete DNA condensations, seen as flower structures, were observed with linPEI (110 nm) and bioPEI (105 nm). Air-jet nebulization altered the polyplex structure to a greater extent than ultrasonic nebulization and resulted mainly in smaller and non-spherical particles (30-200 nm). Ultrasonic nebulization did not change the spherical structure or particle size of the polyplexes. In particular, the shape and size of the PEGPEI polyplexes did not change. We conclude that ultrasonic nebulization is a milder aerosolization method for gene delivery systems based on PEI. Additionally, PEGPEI-DNA polyplexes seem to be more stable than their counterparts, which may be advantageous in pulmonary inhalation gene therapy.

A classification of drug substances according to their mechanism of action.

Different classification systems for therapeutic agents exist. The most commonly used one is the ATC Code (ATC: Anatomy, Therapeutic properties, Chemical, pharmacological properties). Here, an alternative classification system (TCAT: Target-Chemistry-Anatomy-Therapy) is proposed which refers to the molecular mechanism of action or rather, target. The main subgroups of targets are: enzymes; substrates, metabolies, proteins; receptors; ion channels; transporter molecules and systems; nucleic acids, ribosomes; physicochemical mechanisms; antigen-antibody reactions; unknown targets. This target-oriented approach may be particularly useful in teaching advanced medicinal chemistry.

Surfactant-free, biodegradable nanoparticles for aerosol therapy based on the branched polyesters, DEAPA-PVAL-g-PLGA.

PURPOSE: This study describes the development of surfactant-free, biodegradable nanoparticle systems with varying physicochemical properties and their suitability for pulmonary application via nebulization. METHODS: Nanoparticle suspensions were formulated from the branched polyester, diethylaminopropyl amine-poly(vinyl alcohol)-grafted-poly(lactide-co-glycolide) (DEAPA-PVAL-g-PLGA) alone, as well as with increasing amounts of carboxymethyl cellulose (CMC). Particle size, zeta potential, turbidity, and morphology (atomic force microscopy) were characterized. Three formulations were chosen for further study: Cationic nanoparticles without CMC, cationic nanoparticles with CMC, and anionic nanoparticles with an excess of CMC. Nanoparticle degradation was characterized, as well as stability during nebulization. Nanoparticle-cell interactions were investigated and quantified using confocal laser scanning microscopy and fluorescence spectrometry. RESULTS: Nanoparticles ranged in size from 70-250 nm and displayed zeta potentials of +58.9 to -46.6 mV. Anionic nanoparticles showed the highest stability during nebulization. The degradation rate of each nanoparticle formulation decreased with increasing amounts of CMC. Cell association was highest among cationic nanoparticles (57% and 30%, respectively), although these were not internalized. Despite a lower rate of cell association (3%), anionic nanoparticles were internalized by A549 cells. CONCLUSIONS: Surfactant-free nanoparticles from DEAPA-PVAL-g-PLGA are versatile drug delivery systems; however, only the anionic formulations investigated were proven suitable for aerosol therapy.

Air pollution particles mediated oxidative DNA base damage in a cell free system and in human airway epithelial cells in relation to particulate metal content and bioreactivity.

Epidemiological studies demonstrate an association between increased human morbidity and mortality with exposure to air pollution particulate matter. We hypothesized that such effects may be associated with the ability of the particles to mediate generation of reactive oxygen species (ROS), either directly, via interaction with ambient oxygen or indirectly through initiation of an oxidative burst in phagocytes. To test this hypothesis, we determined 8-oxo-dG formation as a measure of direct generation of ROS, in response to particulate exposures to 2'-deoxyguanosine (dG), free and in calf thymus DNA in aerated solutions as the target molecule and cell culture, to assess the relationship between induction of oxidative damage, particulate metal content and metal bioreactivity. The HPLC-ECD technique was employed for separation and quantification of 8-oxo-dG, the most widely recognized marker of DNA oxidation. Particles used in this study include: Arizona desert dust (AZDD), coal fly ash (CFA and ECFA), oil fly ash (OFA and ROFA), and ambient air [SRM 1649 and Dusseldorf (DUSS), Germany]. The major difference between these particles is the concentration of water-soluble metals. The fly ash particulates OFA and ROFA showed a significant dose-dependent increase in dG hydroxylation to 8-oxo-dG formation over the control dG (p < 0.05), with yields 0.03 and 1.25% at the highest particulate concentration (1 mg/mL). Metal ion chelators and DMSO, a hydroxyl radical scavenger, inhibited this hydroxylation. In contrast, desert dust, coal fly ash and urban air particles induced 8-oxo-dG with yields ranging from 0.003 to 0.006%, respectively, with levels unaffected by pretreatment of the particles with metal ion chelators or addition of DMSO to the incubation mixture. When calf thymus DNA was used as a substrate, all the particles induced 8-oxo-dG in a pattern similar to that observed for dG hydroxylation, but with relatively less yield. Treatment of the particles with metal ion chelator before reacting with DNA or addition of catalase in the incubation mixture, suppressed 8-oxo-dG formation significantly (p < 0.05) in oil-derived fly ash particles only. To determine whether the oxidative responses of these particulates as shown in cell-free systems were consistent with responses using a more biologically relevant environment, human airway epithelial cells were treated with the particulates and induction of 8-oxo-dG was determined. All particles induced 8-oxo-dG in the DNA of cells above culture control, except CFA. Cells exposed to 10-400 mg/mL of ROFA for 2 h induced a dose-dependent increase in 8-oxo-dG formation. Treatment of ROFA with metal ion chelator attenuated these effects. Overall, damage enhancement by particulates in dG, calf thymus, and cellular DNA as determined by 8-oxo-dG formation under aerobic conditions is consistent with the concentration of water-soluble, not the total metal content of the particle.

Coevolution of HMG domains and homeodomains and the generation of transcriptional regulation by Sox/POU complexes.

The highly conserved homeodomains and HMG domains are components of a large number of proteins that play a role in the transcriptional regulation of gene expression during embryogenesis. Both the HMG domain and the homeodomain serve as interfaces for factor interactions with DNA, as well as with other proteins, and it is likely that the high degree of structural and sequence conservation within these domains reflects the conservation of basic aspects of these interactions. Classical HMG domain proteins have an ancient origin, being found in all eukaryotes, and are thought to have given rise to the metazoan-specific class of HMG domain proteins called the Sox proteins. Similarly, the metazoan-specific POU domain proteins are thought to have arisen from genes encoding ancestral homeodomain proteins. In this review, we summarize several examples of different HMG-homeodomain interactions that illustrate not only the ancient origin of each of these protein families, but also their relationship to each other, and discuss how coevolution of HMG and homeodomains may have lead to creation of the specialized Sox/POU protein complexes. Using the FGF-4 gene as an example, we also speculate on how coevolution of regulatory Sox/POU target DNA sequences may have occurred, and how the summation of these changes may have lead to the emergence of new developmental pathways.

Effect of ozone on diesel exhaust particle toxicity in rat lung.

Ambient particulate matter (PM) concentrations have been associated with mortality and morbidity. Diesel exhaust particles (DEP) are present in ambient urban air PM. Coexisting with DEP (and PM) is ozone (O(3)), which has the potential to react with some components of DEP. Some reports have shown increased lung injury in rats coexposed to PM and O(3), but it is unclear whether this increased injury was due to direct interaction between the pollutants or via other mechanisms. To examine whether O(3) can directly react with and affect PM bioactivity, we exposed DEP to O(3) in a cell-free in vitro system and then examined the bioactivity of the resultant DEP in a rat model of lung injury. Standard Reference Material 2975 (diesel exhaust PM) was initially exposed to 0.1 ppm O(3) for 48 h and then instilled intratracheally in Sprague-Dawley rats. Rat lung inflammation and injury was examined 24 h after instillation by lung lavage. The DEP exposed to 0.1 ppm O(3) was more potent in increasing neutrophilia, lavage total protein, and LDH activity compared to unexposed DEP. The increased DEP activity induced by the O(3) exposure was not attributable to alteration by air that was also present during the O(3) exposure. Exposure of DEP to a higher O(3) concentration (1.0 ppm) led to a decreased bioactivity of the particles. In contrast, carbon black particles, low in organic content relative to DEP, did not exhibit an increase in any of the bioactivities examined after exposure to 0.1 ppm O(3). DEP incorporated O(3) (labeled with (18)O) in a linear fashion. These data suggest that ambient concentrations of O(3) can increase the biological potency of DEP. The ozonized DEP may play a role in the induction of lung responses by ambient PM.

Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer.

Fibroblast growth factor (FGF)-4 gene expression in the inner cell mass of the blastocyst and in EC cells requires the combined activity of two transcriptional regulators, Sox2 and Oct-3, which bind to adjacent sites on the FGF-4 enhancer DNA and synergistically activate transcription. Sox2 and Oct-3 bind cooperatively to the enhancer DNA through their DNA-binding, high mobility group and POU domains, respectively. These two domains, however, are not sufficient to activate transcription. We have analyzed a number of Sox2 and Oct-3 deletion mutants to identify the domains within each protein that contribute to the activity of the Sox2 x Oct-3 complex. Within Oct-3, we have identified two activation domains, the N-terminal AD1 and the C-terminal AD2, that play a role in the activity of the Sox2 x Oct-3 complex. AD1 also displays transcriptional activation functions in the absence of Sox2 while AD2 function was only detected within the Sox2 x Oct-3 complex. In Sox2, we have identified three activation domains within its C terminus: R1, R2, and R3. R1 and R2 can potentiate weak activation by Sox2 in the absence of Oct-3 but their deletion has no effect on the Sox2 x Oct-3 complex. In contrast, R3 function is only observed when Sox2 is complexed with Oct-3. In addition, analysis of Oct-1/Oct-3 chimeras indicates that the Oct-3 homeodomain also plays a critical role in the formation of a functional Sox2 x Oct-3 complex. Our results are consistent with a model in which the synergistic action of Sox2 and Oct-3 results from two major processes. Cooperative binding of the factors to the enhancer DNA, mediated by their binding domains, stably tethers each factor to DNA and increases the activity of intrinsic activation domains within each protein. Protein-protein and protein-DNA interactions then may lead to reciprocal conformational changes that expose latent activation domains within each protein. These findings define a mechanism that may also be utilized by other Sox x POU protein complexes in gene activation.

The dhfr oribeta-binding protein RIP60 contains 15 zinc fingers: DNA binding and looping by the central three fingers and an associated proline-rich region.

Initiation of DNA replication occurs with high frequency within oribeta, a short region 3' to the Chinese hamster dhfr gene. Homodimers of RIP60 (replication initiation-region protein 60 kDA) purified from nuclear extract bind two ATT-rich sites in oribeta and foster the formation of a twisted 720 bp DNA loop in vitro. Using a one hybrid screen in yeast, we have cloned the cDNA for human RIP60. RIP60 contains 15 C(2)H(2)zinc finger (ZF) DNA binding motifs organized in three clusters, termed hand Z1 (ZFs 1-5), hand Z2 (ZFs 6-8) and hand Z3 (ZFs 9-15). A proline-rich region is located between hands Z2 and Z3. Gel mobility shift and DNase I footprinting experiments show hands Z1 and Z2 independently bind the oribeta RIP60 sites specifically, but with different affinities. Hand Z3 binds DNA, but displays no specificity for RIP60 sites. Ligation enhancement, DNase I footprinting, and atomic force microscopy assays show that hand Z2 and a portion of the associated proline-rich region is sufficient for protein multimerization on DNA and DNA looping in vitro. Polyomavirus origin-dependent plasmid replication assays show RIP60 has weak replication enhancer activity, suggesting that RIP60 does not harbor a transcriptional transactivation domain. Because vertebrate origins of replication have no known consensus sequence, we suggest that sequence-specific DNA binding proteins such as RIP60 may act as accessory factors in origin identification prior to the assembly of pre-initiation complexes.

Metals associated with both the water-soluble and insoluble fractions of an ambient air pollution particle catalyze an oxidative stress.

One potential mechanism of injury mediated by air pollution particles is through metal-catalyzed oxidant generation. In one emission source particle, soluble metals have been associated with biological effect and toxicity. However, a majority of metals in ambient air pollution particles can be associated with insoluble components. We tested the hypothesis that concentrations of catalytically active metal in ambient air pollution particles are not equivalent to the concentrations of water-soluble metal. Twelve filters collected from the North Provo, UT, monitoring station were agitated in deionized water. Both the aqueous extract and pellet were isolated, lyophilized, and defined as the water-soluble and insoluble fractions, respectively. The fractions were chemically characterized and ionizable concentrations of metals were measured using inductively coupled plasma emission spectroscopy. While the water-soluble fraction had significantly greater concentrations of ionizable metals per unit mass, the insoluble fraction also had measurable quantities. In vitro oxidant generation by the two fractions, measured as thiobarbituric acid-reactive-products of deoxyribose, corresponded to the concentrations of ionizable rather than total metals. The release of interleukin-8 by cultured respiratory epithelial cells after incubation with the two fractions also coincided with the ionizable metal concentrations. Finally, neutrophil influx and lavage protein levels 24 h after instillation of the two fractions in rats reflected the ionizable metal concentrations, in vitro oxidative stress, and mediator release. We conclude that catalytically active metals can be measured in both the soluble and insoluble fractions of an ambient air pollution particle. These metals corresponded to the biological activity of the two fractions. While in greater concentration in the water-soluble fraction, larger total quantities of catalytically and biologically active metals are likely to be associated with the insoluble fraction as a result of the abundance of the latter.