Injectable biodegradable starch/chitosan delivery system for the sustained release of gentamicin to treat bone infections

Starch-conjugated chitosan microparticles were produced aimed to be used as a carrier for the long term sustained/controlled release of antibiotic drugs to control bone infection. The microparticles were prepared by a reductive alkylation crosslinking method. The obtained microparticles showed a spherical shape, with a slightly rough and porous surface, and a size range of 80-150 μm. Gentamicin was entrapped into the starch-conjugated chitosan microparticles and its release profile was studied in vitro. Increasing concentrations of gentamicin (from 50 to 150 mg/mL) led to a decrease in the encapsulation efficiency (from 67 to 55%), while drug loading increased from 4 to 27%. A sustained release of gentamicin was observed over a period of 30 days. The release kinetics could be controlled using an ionic crosslinker agent. In addition, a bacterial inhibition test on Staphylococcus aureus shows a diameter of the sample inhibition zone ranging from 12 to 17 mm (70-100% of relative activity).     

Swellable Ciprofloxacin-Loaded Nano-in-Micro Hydrogel Particles for Local Lung Drug Delivery

Incorporation of drug-loaded nanoparticles into swellable and respirable microparticles is a promising strategy to avoid rapid clearance from the lung and achieve sustained drug release. In this investigation, a copolymer of polyethylene glycol grafted onto phthaloyl chitosan (PEG-g-PHCs) was synthesized and then self-assembled with ciprofloxacin to form drug-loaded nanoparticles. The nanoparticles and free drug were encapsulated into respirable and swellable alginate micro hydrogel particles and assessed as a novel system for sustained pulmonary drug delivery. Particle size, morphology, dynamic swelling profile, and in vitro drug release were investigated. Results showed that drug-loaded nanoparticles with size of 218 nm were entrapped into 3.9-μm micro hydrogel particles. The dry nano-in-micro hydrogel particles exhibited a rapid initial swelling within 2 min and showed sustained drug release. Preliminary in vivo pharmacokinetic studies were performed with formulations delivered to rats by intratracheal insufflation. Ciprofloxacin concentrations in plasma and in lung tissue and lavage were measured up to 7 h. The swellable particles showed lower ciprofloxacin levels in plasma than the controlled group (a mixture of lactose with micronized ciprofloxacin), while swellable particles achieved higher concentrations in lung tissue and lavage, indicating the swellable particles could be used for controlling drug release and prolonging lung drug concentrations.KEY WORDS: alveolar macrophage, antibiotics, cross-linking, hydrogel swelling, intratracheal insufflation     

Spray-dried poly(D,L-lactide) microspheres containing carboplatin for veterinary use: In vitro and in vivo studies

The aim of this study was the development of a veterinary dosage form constituted by injectable biodegradable microspheres designed for the subcutaneous release of carboplatin, a chemotherapeutic drug. Poly(D,L-lactide) (PDLLA) microspheres were prepared by an emulsification/spray-drying method, using the drug-to-polymer weight ratios 1∶9 and 1∶5; blank microspheres (1% w/v) were prepared as a comparison. Microparticles were characterized in terms of morphology, encapsulation efficiency, and in vitro drug release behavior. In vivo tests were conducted on rats by subcutaneous injection of microsphere aqueous suspensions. Levels of carboplatin were evaluated both in the skin and in serum. The microparticles obtained had a spherical shape; particle size ranged from 5 to 7 μm, dependent on drug loading. Microspheres were able to control the in vitro release of the drug: approximately 90% to 100% of the carboplatin was released over 30 days. In vivo results showed that the microspheres were able to release high drug amounts locally, and sustained serum levels of drug were also achieved. Based on these results, carboplatin-loaded PDLLA microspheres may be useful for local delivery of the antineoplastic drug to the tumor, avoiding tumor recurrence in small animals, and may decrease the formation of distant metastases. Published: September 20, 2005     

Development of Novel Chitosan Microcapsules for Pulmonary Delivery of Dapsone: Characterization,Aerosol Performance,and In Vivo Toxicity Evaluation

Pneumocystis carinii pneumonia (PCP) is a major opportunistic infection that affects patients with human immunodeficiency virus. Although orally administered dapsone leads to high hepatic metabolism, decreasing the therapeutic index and causing severe side effects, this drug is an effective alternative for the treatment of PCP. In this context, microencapsulation for pulmonary administration can offer an alternative to increase the bioavailability of dapsone, reducing its adverse effects. The aim of this work was to develop novel dapsone-loaded chitosan microcapsules intended for deep-lung aerosolized drug delivery. The geometric particle size (D_4,3) was approximately 7 μm, the calculated aerodynamic diameter (d_aero) was approximately 4.5 μm, and the mass median aerodynamic diameter from an Andersen cascade impactor was 4.7 μm. The in vitro dissolution profile showed an efficient dapsone encapsulation, demonstrating the sustained release of the drug. The in vitro deposition (measured by the Andersen cascade impactor) showed an adequate distribution and a high fine particles fraction (FPF = 50%). Scanning electron microscopy of the pulmonary tissues demonstrated an adequate deposition of these particles in the deepest part of the lung. An in vivo toxicity experiment showed the low toxicity of the drug-loaded microcapsules, indicating a protective effect of the microencapsulation process when the particles are microencapsulated. In conclusion, the pulmonary administration of the novel dapsone-loaded microcapsules could be a promising alternative for PCP treatment.KEY WORDS: dapsone, dry powders inhalers, in vivo toxicity, microparticles, pulmonary drug delivery     

Poly (DL-lactide-co-glycolide) microparticles as carriers for antimycobacterial drug rifampicin

Poly (DL-lactide-co-glycolide) polymers were investigated as carriers for the first line antitubercular drug rifampicin. Different formulations of PLG microparticles viz. porous, non porous and hardened exhibited sustained release of rifampicin up to 7 weeks in vitro. However, hardened PLG microparticles exhibited the most sustained release in vivo in different organs up to 6 weeks. In case of free rifampicin, release was detected in vivo only up to 48 hr. In addition, no hepatotoxicity was observed on a biochemical basis (levels of SGPT, ALP and total bilirubin) in comparison to control animals. Taken together, these results suggest that polymer encapsulated antitubercular drug rifampicin may serve as an ideal therapeutic approach for treatment of tuberculous infections.     

IKK2 Inhibition Using TPCA-1-Loaded PLGA Microparticles Attenuates Laser-Induced Choroidal Neovascularization and Macrophage Recruitment

The inhibition of NF-κB by genetic deletion or pharmacological inhibition of IKK2 significantly reduces laser-induced choroid neovascularization (CNV). To achieve a sustained and controlled intraocular release of a selective and potent IKK2 inhibitor, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) (MW: 279.29), we developed a biodegradable poly-lactide-co-glycolide (PLGA) polymer-delivery system to further investigate the anti-neovascularization effects of IKK2 inhibition and in vivo biosafety using laser-induced CNV mouse model. The solvent-evaporation method produced spherical TPCA-1-loaded PLGA microparticles characterized with a mean diameter of 2.4 ¼m and loading efficiency of 80%. Retrobulbar administration of the TPCA-1-loaded PLGA microparticles maintained a sustained drug level in the retina during the study period. No detectable TPCA-1 level was observed in the untreated contralateral eye. The anti-CNV effect of retrobulbarly administrated TPCA-1-loaded PLGA microparticles was assessed by retinal fluorescein leakage and isolectin staining methods, showing significantly reduced CNV development on day 7 after laser injury. Macrophage infiltration into the laser lesion was attenuated as assayed by choroid/RPE flat-mount staining with anti-F4/80 antibody. Consistently, laser induced expressions of Vegfa and Ccl2 were inhibited by the TPCA-1-loaded PLGA treatment. This TPCA-1 delivery system did not cause any noticeable cellular or functional toxicity to the treated eyes as evaluated by histology and optokinetic reflex (OKR) tests; and no systemic toxicity was observed. We conclude that retrobulbar injection of the small-molecule IKK2 inhibitor TPCA-1, delivered by biodegradable PLGA microparticles, can achieve a sustained and controllable drug release into choroid/retina and attenuate laser-induced CNV development without causing apparent systemic toxicity. Our results suggest a potential clinical application of TPCA-1 delivered by microparticles in treatment of CNV in the patients with age-related macular degeneration and other retinal neovascularization diseases.     

Acid sphingomyelinase activity triggers microparticle release from glial cells

We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1β, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X₇, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1β release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1β release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1β release, thus, opening new strategies for the treatment of neuroinflammatory diseases.     

Design and <Emphasis Type="Italic">In Vitro</Emphasis> Performance Evaluation of Purified Microparticles of Pravastatin Sodium for Intestinal Delivery

The purpose of research was to develop a mucoadhesive multiparticulate sustained drug delivery system of pravastatin sodium, a highly water-soluble and poorly bioavailable drug, unstable at gastric pH. Mucoadhesive microparticles were formulated using eudragit S100 and ethyl cellulose as mucoadhesive polymers. End-step modification of w/o/o double emulsion solvent diffusion method was attempted to improve the purity of the product, that can affect the dose calculations of sustained release formulations and hence bioavailability. Microparticles formed were discrete, free flowing, and exhibited good mucoadhesive properties. DSC and DRS showed stable character of drug in microparticles and absence of drug polymer interaction. The drug to polymer ratio and surfactant concentration had significant effect on mean particle size, drug release, and entrapment efficiency. Microparticles made with drug: eudragit S100 ratio of 1:3 (F6) exhibited maximum entrapment efficiency of 72.7% and ex vivo mucoadhesion time of 4.15 h. In vitro permeation studies on goat intestinal mucosa demonstrated a flux rate (1,243 μg/cm²/h) that was 169 times higher than the flux of pure drug. The gastric instability problem was overcome by formulating the optimized microparticles as enteric-coated capsules that provided a sustained delivery of the highly water-soluble drug for 12 h beyond the gastric region. The release mechanism was identified as fickian diffusion (n = 0.4137) for the optimized formulation F6. Conclusively, a drug delivery system was successfully developed that showed delayed and sustained release up to 12 h and could be potentially useful to overcome poor bioavailability problems associated with pravastatin sodium.     

Association of Veterinary Third-Generation Cephalosporin Use with the Risk of Emergence of Extended-Spectrum-Cephalosporin Resistance in Escherichia coli from Dairy Cattle in Japan

The use of extended-spectrum cephalosporins in food animals has been suggested to increase the risk of spread of Enterobacteriaceae carrying extended-spectrum β-lactamases to humans. However, evidence that selection of extended-spectrum cephalosporin–resistant bacteria owing to the actual veterinary use of these drugs according to criteria established in cattle has not been demonstrated. In this study, we investigated the natural occurrence of cephalosporin-resistant Escherichia coli in dairy cattle following clinical application of ceftiofur. E. coli isolates were obtained from rectal samples of treated and untreated cattle (n = 20/group) cultured on deoxycholate-hydrogen sulfide-lactose agar in the presence or absence of ceftiofur. Eleven cefazoline-resistant isolates were obtained from two of the ceftiofur-treated cattle; no cefazoline-resistant isolates were found in untreated cattle. The cefazoline-resistant isolates had mutations in the chromosomal ampC promoter region and remained susceptible to ceftiofur. Eighteen extended-spectrum cephalosporin–resistant isolates from two ceftiofur-treated cows were obtained on ceftiofur-supplemented agar; no extended-spectrum cephalosporin–resistant isolates were obtained from untreated cattle. These extended-spectrum cephalosporin–resistant isolates possessed plasmid-mediated β-lactamase genes, including bla _CTX-M-2 (9 isolates), bla _CTX-M-14 (8 isolates), or bla _CMY-2 (1 isolate); isolates possessing bla _CTX-M-2 and bla _CTX-M-14 were clonally related. These genes were located on self-transmissible plasmids. Our results suggest that appropriate veterinary use of ceftiofur did not trigger growth extended-spectrum cephalosporin–resistant E. coli in the bovine rectal flora; however, ceftiofur selection in vitro suggested that additional ceftiofur exposure enhanced selection for specific extended-spectrum cephalosporin–resistant β-lactamase-expressing E. coli clones     

Transscleral Sustained Vasohibin-1 Delivery by a Novel Device Suppressed Experimentally-Induced Choroidal Neovascularization

We established a sustained vasohibin-1 (a 42-kDa protein), delivery device by a novel method using photopolymerization of a mixture of polyethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and collagen microparticles. We evaluated its effects in a model of rat laser-induced choroidal neovascularization (CNV) using a transscleral approach. We used variable concentrations of vasohibin-1 in the devices, and used an enzyme-linked immunosorbent assay and Western blotting to measure the released vasohibin-1 (0.31 nM/day when using the 10 μM vasohibin-1 delivery device [10VDD]). The released vasohibin-1 showed suppression activity comparable to native effects when evaluated using endothelial tube formation. We also used pelletized vasohibin-1 and fluorescein isothiocyanate-labeled 40 kDa dextran as controls. Strong fluorescein staining was observed on the sclera when the device was used for drug delivery, whereas pellet use produced strong staining in the conjunctiva and surrounding tissue, but not on the sclera. Vasohibin-1 was found in the sclera, choroid, retinal pigment epithelium (RPE), and neural retina after device implantation. Stronger immunoreactivity at the RPE and ganglion cell layers was observed than in other retinal regions. Significantly lower fluorescein angiography (FA) scores and smaller CNV areas in the flat mounts of RPE-choroid-sclera were observed for the 10VDD, VDD (1 μM vasohibin-1 delivery device), and vasohibin-1 intravitreal direct injection (0.24 μM) groups when compared to the pellet, non-vasohibin-1 delivery device, and intravitreal vehicle injection groups. Choroidal neovascularization can be treated with transscleral sustained protein delivery using our novel device. We offer a safer sustained protein release for treatment of retinal disease using the transscleral approach.     

β-Cyclodextrin-Based Inclusion Complexation Bridged Biodegradable Self-Assembly Macromolecular Micelle for the Delivery of Paclitaxel

In this study, a novel adamantanamine-paclitaxel (AD-PTX) incorporated oligochitosan- carboxymethyl-β-cyclodextrin (CSO-g-CM-β-CD) self-assembly macromolecular (CSO-g-CM-β-CD@AD-PTX) micelle was successfully prepared in water through sonication. The formed molecules were characterized by Fourier transform infrared spectroscopy, proton nuclear magnetic resonance (NMR) spectroscopy, two-dimensional NMR, elemental analysis, and liquid chromatography-mass spectrometry, while the correspondent micelles were characterized by dynamic light scattering and transmission electron microscopy. We showed that the macromolecular micelle contained a spherical core-shell structure with a diameter of 197.1 ± 3.3 nm and zeta potential of −19.1 ± 4.3 mV. The CSO-g-CM-β-CD@AD-PTX micelle exhibited a high drug-loading efficacy up to 31.3%, as well as a critical micelle concentration of 3.4 × 10^-7 M, which indicated good stability. Additionally, the in vitro release profile of the CSO-g-CM-β-CD@AD-PTX micelle demonstrated a long-term release pattern, 63.1% of AD-PTX was released from the micelle during a 30-day period. Moreover, the CSO-g-CM-β-CD@AD-PTX micelle displayed cytotoxicity at a sub-μM scale similar to PTX in U87 MG cells, and CSO-g-CM-β-CD exhibited a good safety profile by not manifesting significant toxicity at concentrations up to 100 μM. These results indicated that β-CD-based inclusion complexation resulting in biodegradable self-assembled macromolecular micelles can be utilized as nanocarrier, and may provide a promising platform for drug delivery in the future medical applications.     

Nicotine Elicits Prolonged Calcium Signaling along Ventral Hippocampal Axons

Presynaptic nicotinic acetylcholine receptors (nAChRs) have long been implicated in the modulation of CNS circuits. We previously reported that brief exposure to low concentrations of nicotine induced sustained potentiation of glutamatergic transmission at ventral hippocampal (vHipp)-striatal synapses. Here, we exploited nAChR subtype-selective antagonists and agonists and α7*nAChR knockout mutant mice (α7-/-) to elucidate the signaling mechanisms underlying nAChR-mediated modulation of synaptic transmission. Using a combination of micro-slices culture from WT and α7-/-mice, calcium imaging, and immuno-histochemical techniques, we found that nicotine elicits localized and oscillatory increases in intracellular Ca²⁺ along vHipp axons that persists for up to 30 minutes. The sustained phase of the nicotine-induced Ca²⁺ response was blocked by α-BgTx but not by DHβE and was mimicked by α7*nAChR agonists but not by non-α7*nAChR agonists. In vHipp slices from α7-/- mice, nicotine elicited only transient increases of axonal Ca²⁺ signals and did not activate CaMKII. The sustained phase of the nicotine-induced Ca²⁺ response required localized activation of CaMKII, phospholipase C, and IP₃ receptor mediated Ca²⁺-induced Ca²⁺ release (CICR). In conclusion, activation of presynaptic nAChRs by nicotine elicits Ca²⁺ influx into the presynaptic axons, the sustained phase of the nicotine-induced Ca²⁺ response requires that axonal α7*nAChR activate a downstream signaling network in the vHipp axons.     

Neutron Activated Samarium-153 Microparticles for Transarterial Radioembolization of Liver Tumour with Post-Procedure Imaging Capabilities

Introduction

Samarium-153 (¹⁵³Sm) styrene divinylbenzene microparticles were developed as a surrogate for Yttrium-90 (⁹⁰Y) microspheres in liver radioembolization therapy. Unlike the pure beta emitter ⁹⁰Y, ¹⁵³Sm possess both therapeutic beta and diagnostic gamma radiations, making it possible for post-procedure imaging following therapy.

Methods

The microparticles were prepared using commercially available cation exchange resin, Amberlite IR-120 H⁺ (620–830 μm), which were reduced to 20–40 μm via ball mill grinding and sieve separation. The microparticles were labelled with ¹⁵²Sm via ion exchange process with ¹⁵²SmCl₃, prior to neutron activation to produce radioactive ¹⁵³Sm through ¹⁵²Sm(n,γ)¹⁵³Sm reaction. Therapeutic activity of 3 GBq was referred based on the recommended activity used in ⁹⁰Y-microspheres therapy. The samples were irradiated in 1.494 x 10¹² n.cm^-2.s^-1 neutron flux for 6 h to achieve the nominal activity of 3.1 GBq.g^-1. Physicochemical characterisation of the microparticles, gamma spectrometry, and in vitro radiolabelling studies were carried out to study the performance and stability of the microparticles.

Results

Fourier Transform Infrared (FTIR) spectroscopy of the Amberlite IR-120 resins showed unaffected functional groups, following size reduction of the beads. However, as shown by the electron microscope, the microparticles were irregular in shape. The radioactivity achieved after 6 h neutron activation was 3.104 ± 0.029 GBq. The specific activity per microparticle was 53.855 ± 0.503 Bq. Gamma spectrometry and elemental analysis showed no radioactive impurities in the samples. Radiolabelling efficiencies of ¹⁵³Sm-Amberlite in distilled water and blood plasma over 48 h were excellent and higher than 95%.

Conclusion

The laboratory work revealed that the ¹⁵³Sm-Amberlite microparticles demonstrated superior characteristics for potential use in hepatic radioembolization.     

Chitosan and Enteric Polymer Based Once Daily Sustained Release Tablets of Aceclofenac: In Vitro and In Vivo Studies

The purpose of this study was to develop a once daily sustained release tablet of aceclofenac using chitosan and an enteric coating polymer (hydroxypropyl methylcellulose phthalate or cellulose acetate phthalate). Overall sustained release for 24 h was achieved by preparing a double-layer tablet in which the immediate release layer was formulated for a prompt release of the drug and the sustained release layer was designed to achieve a prolonged release of drug. The preformulation studies like IR spectroscopic and differential scanning calorimetry showed the absence of drug–excipient interactions. The tablets were found within the permissible limits for various physicochemical parameters. Scanning electron microscopy was used to visualize the surface morphology of the tablets and to confirm drug release mechanisms. Good equivalence in the drug release profile was observed when drug release pattern of the tablet containing chitosan and hydroxypropyl methylcellulose phthalate (M-7) was compared with that of marketed tablet. The optimized tablets were stable at accelerated storage conditions for 6 months with respect to drug content and physical appearance. The results of pharmacokinetic studies in human volunteers showed that the optimized tablet (M-7) exhibited no difference in the in vivo drug release in comparison with marketed tablet. No significant difference between the values of pharmacokinetic parameters of M-7 and marketed tablets was observed (p > 0.05; 95% confidence intervals). However the clinical studies in large scale and, long term and extensive stability studies at different conditions are required to confirm these results.Key words: aceclofenac, chitosan, matrix tablet, pharmacokinetics, sustained release     

In vitro and in vivo evaluation of single-unit commercial conventional tablet and sustained-release capsules compared with multiple-unit polystyrene microparticle dosage forms of ibuprofen

The major aims of the present study were (1) to select a multiple-unit formulation that matched the in vitro dissolution profile of single-unit sustained-release commercial capsules, (2) to compare the sustaining/controlling efficacy of the selected multiple-unit formulation with that of the single-unit commercial conventional tablet and sustained-release capsules, and (3) to determine whether an in vitro-in vivo correlation exists for single- and multiple-unit formulations. Ibuprofen (20%–60% wt/wt)-loaded multiple-unit polystyrene microparticles were prepared by an emulsion-solvent evaporation method from an aqueous system. The in vitro release profiles obtained in phosphate buffer of pH 6.8 for drug-loaded polystyrene microparticles and for commercial sustained-release capsules (Fenlong-SR, 400 mg) were compared. Since the microparticles with 30% ibuprofen load showed a release profile comparable to that of the Fenlong-SR release profile, the microparticles with this drug load were considered to be the optimized/selected formulation and, therefore, were subjected to stability study and in vivo study in human volunteers. A single-dose oral bioavailability study revealed significant differences in C_max, T_max, t^1/2a, t^1/2e, K_a, K_e, and AUC between the conventional tablet and optimized or Fenlong-SR capsule dosage forms. However, all the parameters, with the exception of K_a along with relative bioavailability (F) and retard quotient (R_Δ), obtained from the optimized ibuprofenloaded microparticles were lower than that obtained from the commercial Fenlong-SR formulation. Furthermore, linear relationship obtained between the percentages dissolved and absorbed suggests a means to predict in vivo absorption by measuring in vitro dissolution. Published: September 1, 2006     

Urine from Treated Cattle Drives Selection for Cephalosporin Resistant Escherichia coli in Soil

The U.S. Food and Drug Administration recently issued new rules for using ceftiofur in food animals in part because of an increasing prevalence of enteric bacteria that are resistant to 3^rd-generation cephalosporins. Parenteral ceftiofur treatment, however, has limited effects on enteric bacteria so we tested the hypothesis that excreted ceftiofur metabolites exert significant selection pressure for ceftiofur-resistant Escherichia coli in soil. Test matrices were prepared by mixing soil with bovine feces and adding urine containing ceftiofur metabolites (CFM) (0 ppm, ∼50 ppm and ∼100 ppm). Matrices were incubated at 23°C or 4°C for variable periods of time after which residual CFM was quantified using a bioassay. Bla _CMY-2 plasmid-bearing ceftiofur resistant (cef^R) E. coli and one-month old calves were used to study the selection effects of CFM and transmission of cef^R bacteria from the environment back to animals. Our studies showed that urinary CFM (∼13 ppm final concentration) is biologically degraded in soil within 2.7 days at 23°C, but persists up to 23.3 days at 4°C. Even short-term persistence in soil provides a >1 log₁₀ advantage to resistant E. coli populations, resulting in significantly prolonged persistence of these bacteria in the soil (∼two months). We further show that resistant strains readily colonize calves by contact with contaminated bedding and without antibiotic selection pressure. Ceftiofur metabolites in urine amplify resistant E. coli populations and, if applicable to field conditions, this effect is far more compelling than reported selection in vivo after parenteral administration of ceftiofur. Because ceftiofur degradation is temperature dependent, these compounds may accumulate during colder months and this could further enhance selection as seasonal temperatures increase. If cost-effective engineered solutions can be developed to limit ex vivo selection, this may limit proliferation for ceftiofur resistant enteric bacteria while preserving the ability to use this important antibiotic in food animal production.     

Micro-CT Technique Is Well Suited for Documentation of Remodeling Processes in Murine Carotid Arteries

Background

The pathomechanisms of atherosclerosis and vascular remodelling are under intense research. Only a few in vivo tools to study these processes longitudinally in animal experiments are available. Here, we evaluated the potential of micro-CT technology.

Methods

Lumen areas of the common carotid arteries (CCA) in the ApoE^-/- partial carotid artery ligation mouse model were compared between in vivo and ex vivo micro-CT technique and serial histology in a total of 28 animals. AuroVist-15 nm nanoparticles were used as in vivo blood pool contrast agent in a Skyscan 1176 micro-CT at resolution of 18 μmeter voxel size and a mean x-ray dose of 0.5 Gy. For ex vivo imaging, animals were perfused with MicroFil and imaged at 9 μmeter voxel size. Lumen area was evaluated at postoperative days 7, 14, and 28 first by micro-CT followed by histology.

Results

In vivo micro-CT and histology revealed lumen loss starting at day 14. The lumen profile highly correlated (r = 0.79, P<0.0001) between this two methods but absolute lumen values obtained by histology were lower than those obtained by micro-CT. Comparison of in vivo and ex vivo micro-CT imaging revealed excellent correlation (r = 0.83, P<0.01). Post mortem micro-CT yielded a higher resolution than in vivo micro-CT but there was no statistical difference of lumen measurements in the partial carotid artery ligation model.

Conclusion

These data demonstrate that in vivo micro-CT is a feasible and accurate technique with low animal stress to image remodeling processes in the murine carotid artery.     

Acid- and Enzyme-Mediated Solubilization of Cell-Wall beta-1.3,beta-1.4-d-Glucan in Maize Coleoptiles : Implications for Auxin-Mediated Growth

The release of soluble carbohydrates from isolated cell wall of maize (Zea mays L.) was investigated in the range of pH 1 to 8.5. The pH profile demonstrated two peaks, a broad peak at pH 6 due to enzymatic breakdown of β-glucan to monosaccharides (wall autolysis) and a sharp peak at pH 2.5 due to acid-mediated, nonenzymatic liberation of macromolecular β-glucan from the wall. The pH dependence of acid-induced growth and cell-wall extensibility of coleoptile segments closely agrees with the pH dependence of acid-mediated β-glucan solubilization in the isolated wall. However, there is no evidence that enzymatic or nonenzymatic β-glucan solubilization is involved in the mechanism of auxin-mediated growth.     

Pivotal Role of the α2A-Adrenoceptor in Producing Inflammation and Organ Injury in a Rat Model of Sepsis

Background

Norepinephrine (NE) modulates the responsiveness of macrophages to proinflammatory stimuli through the activation of adrenergic receptors (ARs). Being part of the stress response, early increases of NE in sepsis sustain adverse systemic inflammatory responses. The intestine is an important source of NE release in the early stage of cecal ligation and puncture (CLP)-induced sepsis in rats, which then stimulates TNF-α production in Kupffer cells (KCs) through the activation of the α₂-AR. It is important to know which of the three α₂-AR subtypes (i.e., α_2A, α_2B or α_2C) is responsible for the upregulation of TNF-α production. The aim of this study was to determine the contribution of α_2A-AR in this process.

Methodology/Principal Findings

Adult male rats underwent CLP and KCs were isolated 2 h later. Gene expression of α_2A-AR was determined. In additional experiments, cultured KCs were incubated with NE with or without BRL-44408 maleate, a specific α_2A-AR antagonist, and intraportal infusion of NE for 2 h with or without BRL-44408 maleate was carried out in normal animals. Finally, the impact of α_2A-AR activation by NE was investigated under inflammatory conditions (i.e., endotoxemia and CLP). Gene expression of the α_2A-AR subtype was significantly upregulated after CLP. NE increased the release of TNF-α in cultured KCs, which was specifically inhibited by the α_2A-AR antagonist BRL-44408. Equally, intraportal NE infusion increased TNF-α gene expression in KCs and plasma TNF-α which was also abrogated by co-administration of BRL-44408. NE also potentiated LPS-induced TNF-α release via the α_2A-AR in vitro and in vivo. This potentiation of TNF-α release by NE was mediated through the α_2A-AR coupled Gαi protein and the activation of the p38 MAP kinase. Treatment of septic animals with BRL-44408 suppressed TNF-α, prevented multiple organ injury and significantly improved survival from 45% to 75%.

Conclusions/Significance

Our novel finding is that hyperresponsiveness to α₂-AR stimulation observed in sepsis is primarily due to an increase in α_2A-AR expression in KCs. This appears to be in part responsible for the increased proinflammatory response and ensuing organ injury in sepsis. These findings provide important feasibility information for further developing the α_2A-AR antagonist as a new therapy for sepsis.     

Folic Acid Conjugated δ-Valerolactone-Poly(ethylene glycol) Based Triblock Copolymer as a Promising Carrier for Targeted Doxorubicin Delivery

The aim of this study is to test the hypothesis that the newly synthesized poly(δ-valerolactone)/poly(ethylene glycol)/poly(δ-valerolactone) (VEV) copolymer grafted with folic acid would impart targetability and further enhance the anti-tumor efficacy of doxorubicin (DOX). Here, folic acid conjugated VEV (VEV-FOL) was synthesized by a modified esterification method and characterized using IR and NMR. DOX loaded VEV-FOL micelles were synthesized using a novel solvent evaporation method and were obtained with a mean diameter of 97 nm with high encapsulation efficiency and sustained in vitro release profile. Comparative studies of polymer micelles with and without folate for cellular uptake and cytotoxicity were done on folate receptor-positive breast cancer cell line, MDAMB231. The intracellular uptake tests showed significant increase in folate micellar uptake when compared to non-folate-mediated micelles. MTT assay followed by apoptosis assays clearly indicated that folate decorated micelles showed significantly better cytotoxicity (IC₅₀ = 0.014 µM) and efficiency to induce apoptosis than other treated groups. Moreover, a significant G2/M arrest was induced by DOX loaded VEV-FOL micelles at a concentration where free drug failed to show any activity. Thus, our results show that the folic acid-labeled VEV copolymer is a promising biomaterial with controlled and sustainable tumor targeting ability for anticancer drugs which can open new frontiers in the area of targeted chemotherapy.