Production of liquid core-polymer shell microcapsules

Polylactide (PLA) is a biodegradable polymer that has been used in particle form for drug release, due to its biocompatibility, tailorable degradation kinetics, and desirable mechanical properties. Active pharmaceutical ingredients (APIs) may be either dissolved or encapsulated within these biomaterials to create micro- or nanoparticles. Delivery of an AIP within fine particles may overcome solubility or stability issues that can result in early elimination or degradation of the AIP in a hostile biological environment. Furthermore, it is a promising method for controlling the rate of drug delivery and dosage. The goal of this project is to develop a simple and cost-effective device that allows us to produce monodisperse micro- and nanocapsules with controllable size and adjustable sheath thickness on demand. To achieve this goal, we have studied the dual-capillary electrospray and pulsed electrospray. Dual-capillary electrospray has received considerable attention in recent years due to its ability to create core-shell structures in a single-step. However, it also increases the difficulty of controlling the inner and outer particle morphology, since two simultaneous flows are required. Conventional electrospraying has been mainly conducted using direct-current (DC) voltage with little control over anything but the electrical potential. In contrast, control over the input voltage waveform (i.e. pulsing) in electrospraying offers greater control over the process variables. Poly(L-lactic acid) (PLLA) microspheres and microcapsules were successfully fabricated via pulsed-DC electrospray and dual-capillary electrospray, respectively. Core shell combinations produced include: Water/PLLA, PLLA/polyethylene glycol (PEG), and oleic Acid/PLLA. In this study, we designed a novel high-voltage pulse forming network and a set of new designs for coaxial electrospray nozzles. We also investigated the effect of the pulsed voltage characteristics (e.g. pulse frequency, pulse amplitude and pulse width) on the particle’s size and uniformity. We found that pulse frequency, pulse amplitude, pulse width, and the combinations of these factors had a statistically significant effect on the particle’s size. In addition, factors such as polymer concentration, solvent type, feed flow rate, collection method, temperature, and humidity can significantly affect the size and shape of the particles formed.

Apelin signaling modulates splanchnic angiogenesis and portosystemic collateral vessel formation in rats with portal hypertension

BACKGROUND/AIMS: Angiogenesis is a pathological hallmark of portal hypertension. Although VEGF is considered to be the most important proangiogenic factor in neoangiogenesis, this process requires the coordinated action of a variety of factors. Identification of novel molecules involved in angiogenesis is highly relevant, since they may represent potential new targets to suppress pathological neovascularization in angiogenesis-related diseases like portal hypertension. The apelin/APJ signaling pathway plays a crucial role in angiogenesis. Therefore, we determined whether the apelin system modulates angiogenesis-driven processes in portal hypertension. METHODS: Partial portal vein-ligated rats were treated with the APJ antagonist F13A for seven days. Splanchnic neovascularization and expression of angiogenesis mediators (Western blotting) was determined. Portosystemic collateral formation (microspheres), and hemodynamic parameters (flowmetry) were also assessed. RESULTS: Apelin and its receptor APJ were overexpressed in the splanchnic vasculature of portal hypertensive rats. F13A effectively decreased, by 52%, splanchnic neovascularization and expression of proangiogenic factors VEGF, PDGF and angiopoietin-2 in portal hypertensive rats. F13A also reduced, by 35%, the formation of portosystemic collateral vessels. CONCLUSIONS: This study provides the first experimental evidence showing that the apelin/APJ system contributes to portosystemic collateralization and splanchnic neovascularization in portal hypertensive rats, presenting a potential novel therapeutic target for portal hypertension.

Anti-infective therapy of peri-implantitis with adjunctive local drug delivery or photodynamic therapy: 12-month outcomes of a randomized controlled clinical trial

OBJECTIVE: The objective of the study is to compare the clinical, microbiological and host-derived effects in the non-surgical treatment of initial peri-implantitis with either adjunctive local drug delivery (LDD) or adjunctive photodynamic therapy (PDT) after 12 months. MATERIALS AND METHODS: Forty subjects with initial peri-implantitis, that is, pocket probing depths (PPD) 4-6 mm with bleeding on probing (BoP) and radiographic bone loss ≤2 mm, were randomly assigned to two treatment groups. All implants were mechanically debrided with titanium curettes and with a glycine-based powder airpolishing system. Implants in the test group (N = 20) received adjunctive PDT, whereas minocycline microspheres were locally delivered into the peri-implant pockets of control implants (N = 20). At sites with residual BoP, treatment was repeated after 3, 6, 9 and 12 months. The primary outcome variable was the change in the number of peri-implant sites with BoP. Secondary outcome variables included changes in PPD, clinical attachment level (CAL), mucosal recession (REC) and in bacterial counts and crevicular fluid (CF) levels of host-derived biomarkers. RESULTS: After 12 months, the number of BoP-positive sites decreased statistically significantly (P < 0.05) from baseline in both groups (PDT: 4.03 ± 1.66-1.74 ± 1.37, LDD: 4.41 ± 1.47-1.55 ± 1.26). A statistically significant (P < 0.05) decrease in PPD from baseline was observed at PDT-treated sites up to 9 months (4.19 ± 0.55 mm to 3.89 ± 0.68 mm) and up to 12 months at LDD-treated sites (4.39 ± 0.77 mm to 3.83 ± 0.85 mm). Counts of Porphyromonas gingivalis and Tannerella forsythia decreased statistically significantly (P < 0.05) from baseline to 6 months in the PDT and to 12 months in the LDD group, respectively. CF levels of IL-1β decreased statistically significantly (P < 0.05) from baseline to 12 months in both groups. No statistically significant differences (P > 0.05) were observed between groups after 12 months with respect to clinical, microbiological and host-derived parameters. CONCLUSIONS: Non-surgical mechanical debridement with adjunctive PDT was equally effective in the reduction of mucosal inflammation as with adjunctive delivery of minocycline microspheres up to 12 months. Adjunctive PDT may represent an alternative approach to LDD in the non-surgical treatment of initial peri-implantitis.

NANOPARTICLE AGGLOMERATES FOR PULMONARY DRUG DELIVERY

The Pulmonary route has been traditionally used to treat diseases of the respiratory tract. However, important research within the last two decades have shown that in addition to treating local diseases, a wide range of systemic diseases can be treated by delivering drugs to the lungs. The recent FDA approval to market Exubera, an inhalable form of insulin developed by Pfizer, to treat Diabetes, may just be the stepping stone that the pharmaceutical industry needs to market other drugs to treat systemic diseases via the lungs. However, this technology still needs repeated drug doses to control glucose levels, as the inhaled drug is cleared rapidly. Technologies have been developed where inhaled particles are capable of controlled release of drug from the lungs. An important feature of these technologies is the large geometric size of the particles that makes it difficult for the lung macrophages to clear these particles, which results in longer residence times for the particles in the lungs. Owing to the porosity, these particles have lower densities making them deliverable to the deep lungs. However, no modulation of drug release can be achieved with these technologies when more drug release may be required. This additional requirement can only be assuaged by additional dosing of the drug formulation, which can have undesirable effects due to excess loading of excipients in the lungs. In an attempt to bring about modulation of release from long residence time particles, a novel concept was developed in our laboratory that has been termed as the Agglomerated Vesicle Technology (AVT). Liposomes with encapsulated drug were agglomerated using well known cross linking chemistries to form agglomerates in the micron sized range. The large particles exhibited aerodynamic sizes in the respirable size range with minimal damage to the particles upon nebulization. By breaking the cross links between the liposomes with a cleaving agent, it was anticipated that triggered release of drug from the AVT particles could be achieved. In vivo studies done in healthy rabbits showed that post-administration modulation of drug release is possible from the AVT particles after the introduction of the cleaving agent. This study has important implications for the future development of this technology, where the AVT particles can be made “sensitive” to the product of disease. It is envisaged that a single dose of AVT containing the appropriate drug when administered to the lungs would maintain drug levels at a controlled rate over an extended period of time. When the need for more drug arises, the product of the disease would trigger the AVT particles to release more drug as needed to control the condition, thus eliminating the need for repeated drug doses and improved compliance amongst patients.

Reply to the editor

We appreciate the comments and concerns expressed by Arakawa and colleagues regarding our article, titled “Pulsatile control of rotary blood pumps: Does the modulation waveform matter?”1 Unfortunately, we have to disagree with Arakawa and colleagues. As is obvious from the title of our article, it investigates the effect of different waveforms on the heart–device interaction. In contrast to the authors' claim, this is the first article in the literature that uses basic waveforms (sine, triangle, saw tooth, and rectangular) with different phase shifts to examines their impact on left ventricular unloading. The previous publications2, 3 and 4 just varied the pump speed during systole and diastole, which was first reported by Bearnson and associates5 in 1996, and studied its effect on aortic pressure, coronary flow, and end-diastolic volume. We should mention that dp/dtmax is a load-sensitive parameter of contractility and not representative for the degree of unloading. Moreover, none of the aforementioned reports has studied mechanical unloading and in particular the stroke work of the left ventricle. Our method is unique because we do not just alternate between high and low speed but have accurate control of the waveform because of the direct drive system of Levitronix Technologies LLC (Waltham, Mass) and a custom-developed pump controller. Without referring, Arakawa and associates state “several previous studies have already reported the coronary flow diminishes as the left ventricular assist device support increases.” It should be noted that all the waveforms used in our study have 2000 rpm average value with 1000 rpm amplitude, which is not an excessive speed for the CentriMag rotary pump (Levitronix) to collapse the ventricle and diminish the coronary flow. We agree with Arakawa and coworkers that there is a need for a heart failure model to come to more relevant results with respect to clinical expectations. However, we have explored many existing models, including species and breeds that have a native proneness to cardiomyopathy, but all of them differ from the genetic presentation in humans. We certainly do not believe that the use of microembolization, in which the coronary circulation is impaired by the injection of microspheres, would form a good model from which to draw conclusions about coronary flow change under different loading conditions. A model would be needed in which either an infarct is created to mimic ischemic heart failure or the coronary circulation remains untouched to simulate, for instance, dilated cardiomyopathy. Furthermore, in discussion we clearly mention that “lack of heart failure is a major limitation of our study.” We also believe that unloading is not the only factor of the cardiac functional recovery, and an excessive unloading of the left ventricle might lead to cardiac tissue atrophy. Therefore, in our article we mention that control of the level of cardiac unloading by assist devices has been suggested as a mechanical tool to promote recovery, and more studies are required to find better strategies for the speed modulation of rotary pumps and to achieve an optimal heart load control to enhance myocardial recovery. Finally, there are many publications about pulsing rotary blood pumps and it was impossible to include them all. We preferred to reference some of the earlier basic works such as an original research by Bearnson and coworkers5 and another article published by our group,6 which is more relevant.

Dielectrophoresis-based analyte separation and analysis

Dielectrophoresis—the tendency of a material of high dielectric permittivity to migrate in an electrical field gradient to a region of maximum field strength—provides an ideal motive force for manipulating small volumes of biological analytes in microfluidic microsystems. The work described in this thesis was based on the hypothesis that dielectrophoresis could be exploited to provide high-resolution cell separations in microsystems as well as a means for the electrically-controllable manipulation of solid supports for molecular analysis. To this end, a dielectrophoretic/gravitational field-flow-fractionation (DEP/G-FFF) system was developed and the separation performance evaluated using various types and sizes of polystyrene microspheres as model particles. It was shown that separation of the polystyrene beads was based on the differences in their effective dielectrophoretic properties. The ability of an improved DEP/G-FFF system to separate genetically identical, but phenotypically dissimilar cell types was demonstrated using mixtures of 6m2 mutant rat kidney cells grown under transforming and non-transforming culture conditions. Additionally, a panel of engineered dielectric microspheres was designed with specific, predetermined dielectrophoretic properties such that their dielectrophoretic behaviors would be controllable and predictable. The fabrication method involved the use of gold-coated polystyrene microsphere cores coated with a self-assembled monolayer of alkanethiol and, optionally, a self-assembled monolayer of phospholipid to form a thin-insulating-shell-over-conductive-interior structure. The successful development of the DEP/G-FFF separation system and the dielectrically engineered microspheres provides proof-of-principle demonstrations of enabling dielectrophoresis-based microsystem technology that should provide powerful new methods for the manipulation, separation and identification of analytes in many diverse fields. ^

Evaluation of residential bioaerosol concentrations and their related health effects

Limited research has been conducted on the collection of bioaerosols and their health effects on individuals in the El Paso area. A year long study was conducted in the region to evaluate indoor bioaerosol concentrations (Mota et al., unpublished data). As part of the study, air samples were collected during each season for a year from 38 homes from the El Paso area. The main objective of the study was to assess seasonality differences in bioaerosol concentrations. The air samples were then cultured and analyzed for bacterial and fungal concentrations. As a supplement to that study, a health questionnaire was given during each seasonal air sampling to the participating resident to complete regarding their health status. The aim of this study was to evaluate the health questionnaire and assess any associations between the collected bioaerosol concentrations and the self-reported respiratory symptoms of the participating home residents. Symptom frequencies were tabulated and basic descriptive statistics, along with logistic regressions, were conducted on the relationship between “High” reporters of symptoms and bioaerosol concentrations and environmental factors. The most commonly reported symptoms by homeowners were nasal symptoms and allergies. In addition, there was evidence to support an association between indoor respirable bacteria concentrations and homeowners that report greater than or equal to 8 respiratory symptoms (OR=1.10, p=0.045). Smoking status, indoor humidity and season also displayed associations with homeowners that report greater than or equal to 8 respiratory symptoms (OR=3.3, p=0.045; OR=71.0, p=0.030; OR=7.2, 3.2, p=0.001, 0.008). With such a strong association, future assessment of symptoms, bioaerosol concentrations and environmental factors is needed to further establish their relationship. ^

ASSESSMENT OF SKELETAL MUSCLE BLOOD FLOW AND GLUCOSE METABOLISM WITH POSITRON EMITTING RADIONUCLIDES

In order to evaluate factors regulating substrate metabolism in vivo positron emitting radionuclides were used for the assessment of skeletal muscle blood flow and glucose utilization. The potassium analog, Rb-82 was used to measure skeletal muscle blood flow and the glucose analog, 18-F-2-deoxy-2-fluoro-D-glucose (FDG) was used to examine the kinetics of skeletal muscle transport and phosphorylation.^ New Zealand white rabbits' blood flow ranged from 1.0-70 ml/min/100g with the lowest flows occurring under baseline conditions and the highest flows were measured immediately after exercise. Elevated plasma glucose had no effect on increasing blood flow, whereas high physiologic to pharmacologic levels of insulin doubled flow as measured by the radiolabeled microspheres, but a proportionate increase was not detected by Rb-82. The data suggest that skeletal muscle blood flow can be measured using the positron emitting K+ analog Rb-82 under low flow and high flow conditions but not when insulin levels in the plasma are elevated. This may be due to the fact that insulin induces an increase in the Na+/K+-ATPase activity of the cell indirectly through a direct increase in the Na+/H+pump activity. This suggests that the increased cation pump activity counteracts the normal decrease in extraction seen at higher flows resulting in an underestimation of flow as measured by rubidium-82.^ Glucose uptake as measured by FDG employed a three compartment mathematical model describing the rates of transport, countertransport and phosphorylation of hexose. The absolute values for the metabolic rate of FDG were found to be an order of magnitude higher than those reported by other investigators. Changes noted in the rate constant for transport (k1) were found to disagree with the a priori information on the effects of insulin on skeletal muscle hexose transport. Glucose metabolism was however, found to increase above control levels with administration of insulin and electrical stimulation. The data indicate that valid measurements of skeletal muscle glucose transport and phosphorylation using the positron emitting glucose analog FDG requires further model application and biochemical validation. (Abstract shortened with permission of author.) ^

A cross-sectional study of human symptomatology and residential formaldehyde concentrations

The cross-sectional study was performed to quantify the prevalence of symtomatology in residents of mobile homes as a function of indoor formaldehyde concentration. Formaldehyde concentrations were monitored for a seven hour period with an automated wet-chemical colorimetric analyzer. The health status of family members was ascertained by administration of questionnaires and physical exams. This is the first investigation to perform clinical assessments on residents undergoing concurrent exposure assessment in the home.^ Only 22.8% of households eligible for participation chose to cooperate. Monitoring data and health evaluations were obtained from 155 households in four Texas counties. A total of 428 residents (86.1%) were available for examination during the sampling hours. The study population included 45 infants, 126 children, and 257 adults.^ Formaldehyde concentration was not found to be significantly associated with increased risks for symptoms and signs of ocular irritation, dermal anomalies, or malaise. Three associations were identified that warrant further investigation. The relative odds associated with a doubling of formaldehyde concentration was significantly associated with parenchymal rales in adults and children. However, risk was modified by log respirable suspended particulate concentrations. Due to the presence of modification by a continuous variable, prevalence odds ratios (POR) and 95% confidence intervals (95% CI) for these associations are presented in tables. A doubling of formaldehyde concentration was also associated with an increased risk of perceived tightness in the chest in adults. Prevalence odds ratios are presented in a table due to effect modification by the average number of hours spent indoors on weekdays. Furthermore, a doubling of formaldehyde concentration was associated with an increased risk of drowsiness in children (POR = 2.60; 95% CI 1.04-6.51) and adults (POR = 1.94; 95% CI 1.20-3.14). ^

DEVELOPMENT AND APPLICATION OF SHORT-TERM MUTAGENICITY AND CYTOTOXICITY BIOASSAYS FOR INTEGRATED HEALTH ASSESSMENT STUDIES OF COAL FLY ASH EMISSIONS

Two respirable coal fly ash samples ((LESSTHEQ) 3(mu)m), one from a pressurized fluidized-bed combustion miniplant and one from a conventional combustion power plant, were investigated for physical properties, chemical composition and biological activity. Electron microscopy illustrated irregularity in fluidized-bed combustion fly ash and sphericity in conventional combustion fly ash. Elemental analysis of these samples showed differences in trace elements. Both fly ash samples were toxic in rabbit alveolar macrophage and Chinese hamster ovary cell systems in vitro. The macrophages were more sensitive to toxicity of fly ash than the ovary cells. For measuring the cytotoxicity of fly ash, the most sensitive parameters were adenosine triphosphate in the alveolar macrophage system and viability index in the hamster ovary system. Intact fluidized-bed combustion fly-ash particles showed mutagenicity only in strains TA98 and TA1538 without metabolic activation in the Ames Salmonella assay. No mutagenicity was detected in bioassay of conventional combustion fly ash particles. Solvent extraction yielded more mass from fluidized-bed combustion fly ash than from conventional combustion fly ash. The extracts of fluidized-bed combustion fly ash showed higher mutagenic activity than conventional combustion fly ash. These samples contained direct-acting, frameshift mutagens.^ Fly ash samples collected from the same fluidized-bed source by cyclones, a fabric filter, and a electrostatic precipitator at various temperatures were compared for particle size, toxicity, and mutagenicity. Results demonstrated that the biological activity of coal fly ash were affected by the collection site, device, and temperature.^ Coal fly ash vapor-coated with 1-nitropyrene was developed as a model system to study the bioavailability and recovery of nitroaromatic compounds in fly ash. The effects of vapor deposition on toxicity and mutagenicity of fly ash were examined. The nitropyrene coating did not significantly alter the ash's cytotoxicity. Nitropyrene was bioavailable in the biological media, and a significant percentage was not recovered after the coated fly ash was cultured with alveolar macrophages. 1-Nitropyrene loss increased as the number of macrophages was increased, suggesting that the macrophages are capable of metabolizing or binding 1-nitropyrene present in coal fly ash. ^

Profiles of diatoms, pollen, XRD, XRF and organic composition of two sediment cores (PG2022 and PG2023) from Lake Kyutyunda in Yakutia, NE Siberia, Russia

Although the climate development over the Holocene in the Northern Hemisphere is well known, palaeolimnological climate reconstructions reveal spatiotemporal variability in northern Eurasia. Here we present a multi-proxy study from north-eastern Siberia combining sediment geochemistry, and diatom and pollen data from lake-sediment cores covering the last 38,000 cal. years. Our results show major changes in pyrite content and fragilarioid diatom species distributions, indicating prolonged seasonal lake-ice cover between ~13,500 and ~8,900 cal. years BP and possibly during the 8,200 cal. years BP cold event. A pollen-based climate reconstruction generated a mean July temperature of 17.8°C during the Holocene Thermal Maximum (HTM) between ~8,900 and ~4,500 cal. years BP. Naviculoid diatoms appear in the late Holocene indicating a shortening of the seasonal ice cover that continues today. Our results reveal a strong correlation between the applied terrestrial and aquatic indicators and natural seasonal climate dynamics in the Holocene. Planktonic diatoms show a strong response to changes in the lake ecosystem due to recent climate warming in the Anthropocene. We assess other palaeolimnological studies to infer the spatiotemporal pattern of the HTM and affirm that the timing of its onset, a difference of up to 3,000 years from north to south, can be well explained by climatic teleconnections. The westerlies brought cold air to this part of Siberia until the Laurentide ice-sheet vanished 7,000 years ago. The apparent delayed ending of the HTM in the central Siberian record can be ascribed to the exceedance of ecological thresholds trailing behind increases in winter temperatures and decreases in contrast in insolation between seasons during the mid to late Holocene as well as lacking differentiation between summer and winter trends in paleolimnological reconstructions.

Prokaryotic growth and presence of metabolic products and contamination tracers in rocks samples from ODP Leg 187 sites

The microbial population in samples of basalt drilled from the north of the Australian Antarctic Discordance (AAD) during Ocean Drilling Program Leg 187 were studied using deoxyribonucleic acid (DNA)-based methods and culturing techniques. The results showed the presence of a microbial population characteristic for the basalt environment. DNA sequence analysis revealed that microbes grouping within the Actinobacteria, green nonsulfur bacteria, the Cytophaga/Flavobacterium/Bacteroides (CFB) group, the Bacillus/Clostridium group, and the beta and gamma subclasses of the Proteobacteria were present in the basalt samples collected. The most dominant phylogenetic group, both in terms of the number of sequences retrieved and the intensities of the DNA bands obtained with the denaturing gradient gel electrophoresis analysis, was the gamma Proteobacteria. Enrichment cultures showed phylogenetic affiliation with the Actinobacteria, the CFB group, the Bacillus/Clostridium group, and the alpha, beta, gamma, and epsilon subclasses of the Proteobacteria. Comparison of native and enriched samples showed that few of the microbes found in native basalt samples grew in the enrichment cultures. Only seven clusters, two clusters within each of the CFB and Bacillus/Clostridium groups and five clusters within the gamma Proteobacteria, contained sequences from both native and enriched basalt samples with significant similarity. Results from cultivation experiments showed the presence of the physiological groups of iron reducers and methane producers. The presence of the iron/manganese-reducing bacterium Shewanella was confirmed with DNA analysis. The results indicate that iron reducers and lithotrophic methanogenic Archaea are indigenous to the ocean crust basalt and that the methanogenic Archaea may be important primary producers in this basaltic environment.

Technical developments for computed tomography on the CENBG nanobeam line

The use of ion microbeams as probes for computedtomography has proven to be a powerful tool for the three-dimensional characterization of specimens a few tens of micrometers in size. Compared to other types of probes, the main advantage is that quantitative information about mass density and composition can be obtained directly, using specific reconstruction codes. At the Centre d’Etudes Nucléaires de Bordeaux Gradignan (CENBG), this technique was initially developed for applications in cellular biology. However, the observation of the cell ultrastructure requires a sub-micron resolution. The construction of the nanobeamline at the Applications Interdisciplinaires des Faisceaux d’Ions en Region Aquitaine (AIFIRA) irradiation facility has opened new perspectives for such applications. The implementation of computedtomography on the nanobeamline of CENBG has required a careful design of the analysis chamber, especially microscopes for precise sample visualization, and detectors for scanning transmission ion microscopy (STIM) and for particle induced X-ray emission (PIXE). The sample can be precisely positioned in the three directions X, Y, Z and a stepper motor coupled to a goniometer ensures the rotational motion. First images of 3D tomography were obtained on a reference sample containing microspheres of certified diameter, showing the good stability of the beam and the sample stage, and the precision of the motion.