PEG-Coated Large Mesoporous Silicas as Smart Platform for Protein Delivery and Their Use in a Collagen-Based Formulation for 3D Printing.

Silica-based mesoporous systems have gained great interest in drug delivery applications due to their excellent biocompatibility and high loading capability. However, these materials face challenges in terms of pore-size limitations since they are characterized by nanopores ranging between 6-8 nm and thus unsuitable to host large molecular weight molecules such as proteins, enzymes and growth factors (GFs). In this work, for an application in the field of bone regeneration, large-pore mesoporous silicas (LPMSs) were developed to vehicle large biomolecules and release them under a pH stimulus. Considering bone remodeling, the proposed pH-triggered mechanism aims to mimic the release of GFs encased in the bone matrix due to bone resorption by osteoclasts (OCs) and the associated pH drop. To this aim, LPMSs were prepared by using 1,3,5-trimethyl benzene (TMB) as a swelling agent and the synthesis solution was hydrothermally treated and the influence of different process temperatures and durations on the resulting mesostructure was investigated. The synthesized particles exhibited a cage-like mesoporous structure with accessible pores of diameter up to 23 nm. LPMSs produced at 140 °C for 24 h showed the best compromise in terms of specific surface area, pores size and shape and hence, were selected for further experiments. Horseradish peroxidase (HRP) was used as model protein to evaluate the ability of the LPMSs to adsorb and release large biomolecules. After HRP-loading, LPMSs were coated with a pH-responsive polymer, poly(ethylene glycol) (PEG), allowing the release of the incorporated biomolecules in response to a pH decrease, in an attempt to mimic GFs release in bone under the acidic pH generated by the resorption activity of OCs. The reported results proved that PEG-coated carriers released HRP more quickly in an acidic environment, due to the protonation of PEG at low pH that catalyzes polymer hydrolysis reaction. Our findings indicate that LPMSs could be used as carriers to deliver large biomolecules and prove the effectiveness of PEG as pH-responsive coating. Finally, as proof of concept, a collagen-based suspension was obtained by incorporating PEG-coated LPMS carriers into a type I collagen matrix with the aim of designing a hybrid formulation for 3D-printing of bone scaffolds.

Oral self-emulsifying delivery systems for systemic administration of therapeutic proteins: science fiction?

Objective: The aim of this study was to develop self-emulsifying drug delivery systems (SEDDS) for oral delivery of therapeutic proteins through hydrophobic ion pairing. Method: Horseradish peroxidase (HRP), a model protein, was ion paired with sodium docusate to increase its hydrophobicity. The formed enzyme - surfactant complex was incorporated into SEDDS, followed by permeation studies across Caco-2 cell monolayer and freshly excised rat intestine. Results: Hydrophobic ion pairs (HIP) were formed between HRP and sodium docusate with the efficiency of 87.49 ± 1.35%. The formed complex maintained 60.97 ± 1.48% of the original enzyme activity. The ion pair was subsequently loaded into SEDDS with a payload of 0.1% (mass per cent, m/m). The obtained emulsion formed by SEDDS had a droplet size in the range from 20 to 200 nm with negative zeta potential. Permeation mechanism of the enzyme was energy-dependent and the encapsulation of the HIP complex in SEDDS enhanced the permeation of the enzyme through the Caco-2 cell monolayer and freshly excised rat intestine by 4 times and 2.5 times compared to the free enzyme, respectively. Conclusion: According to these findings, hydrophobic ion pairing followed by incorporation to SEDDS might be considered as a potential strategy for oral delivery of therapeutic proteins.

Photoinduced Cleavage and Hydrolysis of o-Nitrobenzyl Linker and Covalent Linker Immobilization in Gelatin Methacryloyl Hydrogels.

Light-induced release systems can be triggered remotely and are of interest for many controlled release applications due to the possibility for spatio-temporal release control. In this study a biotin-functionalized photocleavable macromer is incorporated with an o-nitrobenzyl moiety into gelatin methacryloyl(-acetyl) hydrogels via radical cross-linking. Stronger immobilization of streptavidin-coupled horseradish peroxidase occurs in linker-functionalized hydrogels compared to pure gelatin methacryloyl(-acetyl) hydrogels, and a controlled release of the streptavidin conjugate upon UV-irradiation is possible. Liquid chromatography coupled to mass spectrometry (LC-MS) analysis of aqueous linker solutions allows the identification of the main cleavage products and the cleavage kinetics. Thus, it is shown that a significant hydrolysis of the linker occurs at 37 °C. Nevertheless the system reported here is a promising controlled release scaffold for proteins and application in tissue engineering, if background releases of the immobilized drug are tolerable.

Hyperbaric oxygen therapy for five days increases blood-brain barrier permeability.

This study aimed to explore the effects of hyperbaric oxygen (HBO2) on blood-brain barrier (BBB) integrity in rats, when administered for one (at 2.5 ATA, 3 HBO2 sessions a day) and five days (at 2.5 ATA, 3 HBO2 sessions a day for the first two days, and twice a day for the last three days). Horseradish peroxidase (HRP) was used to evaluate the BBB permeability. Superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) levels were measured in the cerebral cortex and hippocampus regions. Frequent vesicles containing HRP reaction products were observed in capillary endothelial cells in the cerebral cortex and hippocampus of rats subjected to HBO2. The accumulation of HRP reaction products in these brain regions was significantly higher than that of control animals (P ⟨ 0.01). In animals that received HBO2, MDA levels (P ⟨ 0.01 for five days) and GSH (p ⟨ 0.05 for one day, and P ⟨ 0.01 for five days) were decreased in the cerebral cortex, whereas SOD activities slightly increased in this region. In animals that received HBO2 significant decreases in MDA (P ⟨ 0.05 for one day; P ⟨ 0.01 for five days) and GSH (P ⟨ 0.05 for five days) levels were observed in the hippocampus region, but SOD activities decreased in this region. We showed that HBO2 administered with the doses described above impaired BBB integrity in otherwise healthy rats. Therefore, we suggest that the results of this study should be taken into consideration when patients are exposed to HBO2 with the same doses.

Effect of Tinospora cordifolia (Guduchi) on the phagocytic and pinocytic activity of murine macrophages in vitro.

Tinospora cordifolia (Guduchi) is a widely used herb in Ayurvedic system of medicine known to possess immunomodulatory properties. The present study was aimed to study the activation of macrophages after in vitro guduchi treatment. The aqueous extract of T. cordifolia was found to enhance phagocytosis and pinocytosis in vitro. The rate of pinocytosis by macrophages when measured by uptake of horseradish peroxidase was significantly increased after guduchi treatment as compared to medium alone. The macrophages demonstrated an increased phagocytosis to non-infective microorganisms (heat killed yeast) and live infective microorganisms (E. coli) after guduchi treatment. The results demonstrate that Guduchi enhances macrophage activation as analyzed by cytochemical parameters.

Intra- and trans-cellular delivery of enzymes by direct conjugation with non-multivalent anti-ICAM molecules.

Intercellular adhesion molecule 1 (ICAM-1) is a cell-surface protein overexpressed in many diseases and explored for endocytosis and transcytosis of drug delivery systems. All previous evidence demonstrating ICAM-1-mediated transport of therapeutics into or across cells was obtained using nanocarriers or conjugates coupled to multiple copies of anti-ICAM antibodies or peptides. Yet, transport of therapeutics linked to non-multivalent anti-ICAM ligands has never been shown, since multivalency was believed to be necessary to induce transport. Our goal was to explore whether non-multivalent binding to ICAM-1 could drive endocytosis and/or transcytosis of model cargo in different cell types. We found that anti-ICAM was specifically internalized by all tested ICAM-1-expressing cells, including epithelial, fibroblast and neuroblastoma cells, primary or established cell lines. Uptake was inhibited at 4°C and in the presence of an inhibitor of the ICAM-1-associated pathway, rather than inhibitors of the clathrin or caveolar routes. We observed minimal transport of anti-ICAM to lysosomes, yet prominent and specific transcytosis across epithelial monolayers. Finally, we coupled a model cargo (the enzyme horseradish peroxidase (HRP)) to anti-ICAM and separated a 1:2 antibody:enzyme conjugate for non-multivalent ICAM-1 targeting. Similar to anti-ICAM, anti-ICAM-HRP was specifically internalized and transported across cells, which rendered intra- and trans-cellular enzyme activity. Therefore, non-multivalent ICAM-1 targeting also provides transport of cargoes into and across cells, representing a new alternative for future therapeutic applications via this route.

A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses.

Intercellular protein-protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection, and synaptic transmission, but they are challenging to visualize because existing techniques have insufficient sensitivity and/or specificity. Here we report a split horseradish peroxidase (sHRP) as a sensitive and specific tool for the detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable to studying mechanisms of communication between a variety of cell types.

The molecular mechanism and effect of cannabinoid-2 receptor agonist on the blood-spinal cord barrier permeability induced by ischemia-reperfusion injury.

Previous studies have shown that modulation of the receptor-mediated endocannabinoid system during ischemia injury can induce potent neuroprotective effects. However, little is known about whether cannabinoid-2 (CB2) receptor agonist would produce a protective effect on blood-spinal cord barrier (BSCB) during ischemia. Using an in vivo transient spinal cord ischemia model in rats, JWH-015 (1mg/kg, i.p.), a CB2 receptor selective agonist, or vehicles were injected 20 min before ischemia. The effects of JWH-015 on BSCB permeability, the major structural protein for the formation of caveolae, caveolin-1 (cav-1), tight junction (TJ) protein Occludin and zona occludens protein-1 (ZO-1) were examined at day 1, day 3 and day 7 of reperfusion after transient spinal cord ischemia in rats. Here we demonstrated that JWH-015 significantly down-regulated the expression of cav-1, up-regulated the expression of TJ proteins, and then decreased the permeability of BSCB compared with control group. In addition, using an in vitro BBB model, oxygen glucose deprivation (OGD) was applied to simulate spinal cord ischemia in vitro in Human brain microvascular endothelial cells (HBMECs). JWH-015 greatly increased the transepithelial electrical resistance (TEER) and changed the distribution of ZO-1 and Occludin. Moreover, JWH-015 induced the expression of p-PKB and p-FoxO1 protein and decreased the expression of cav-1, which were greatly reversed by ROS inhibitor or PI3K inhibitor. Taken together, all of these results suggested that JWH-015 might regulate the BSCB permeability and this effect could be related to paracellular and transcellular pathway. And pharmacological CB2R ligands offer a new strategy for BSCB protection during ischemic injury.

DT(270-326) , a Truncated Diphtheria Toxin, Increases Blood-Tumor Barrier Permeability by Upregulating the Expression of Caveolin-1.

AIM: The nontoxic mutant of diphtheria toxin (DT) has been demonstrated to act as a receptor-specific carrier protein to delivery drug into brain. Recent research showed that the truncated "receptorless" DT was still capable of being internalized into cells. This study investigated the effects and potential mechanisms of DT(270-326) , a truncated "receptorless" DT, on the permeability of the blood-tumor barrier (BTB). METHODS: BTB and GECs were subjected to DT(270-326) treatment. HRP flux assays, immunofluorescent, co-immunoprecipitation, Western blot, CCK-8, and Flow cytometry analysis were used to evaluate the effects of DT(270-326) administration. RESULTS: Our results revealed that 5 µM of DT(270-326) significantly increased the permeability of BTBin vitro, which reached its peak at 6 h. The permeability was reduced by pretreatment with filipinIII. DT(270-326) co-localized and interacted with caveolin-1 via its caveolin-binding motif. The mRNA and protein expression levels of caveolin-1 were identical with the changes of BTB permeability. The upregulated expression of caveolin-1 was associated with Src kinase-dependent tyrosine phosphorylation of caveolin-1, which subsequently induced phosphorylation and inactivation of the transcription factor Egr-1. The combination of DT(270-326) with doxorubicin significantly enhanced the loss of cell viability and apoptosis of U87 glioma cells in contrast to doxorubicin alone. CONCLUSIONS: DT(270-326) might provide a novel strategy to increase the delivery of macromolecular therapeutic agents across the BTB.

Food allergens are transferred intact across the rat blood-placental barrier in vivo.

We investigated the mechanism of transplacental macromolecular transport in rats on the nineteenth day of pregnancy using tracers, transmission electron microscopy and immunohistochemistry. The blood-placental barrier of full-term rat placentas was composed of a trilaminar layer of trophoblast cells that separates the fetal capillaries from the maternal blood spaces: a layer of cytotrophoblasts lining the maternal blood space and a bilayer of syncytiotrophoblast surrounding the fetal capillaries. Horseradish peroxidase, intravenously injected into the maternal circulation, was found in the maternal blood spaces, the interspaces between the cytotrophoblasts and the syncytiotrophoblast I, many pits and small vesicles in the syncytiotrophoblast I, vesicles of the syncytiotrophoblast II, fetal connective tissue and fetal capillaries. Intravenously injected ovalbumin was detected in the maternal blood spaces, a trilaminar layer and the fetal capillaries. Neonatal Fc receptor (FcRn), a receptor for IgG, was localized at the maternal side of the blood-placental barrier. These results show that the structure of the rat blood-placental barrier is quite similar to the human blood-placental barrier, and non-specific macromolecules and food allergens may penetrate through the blood-placental barrier of the full-term placenta from the maternal to fetal circulation mediated by FcRn.

Minoxidil sulfate induced the increase in blood-brain tumor barrier permeability through ROS/RhoA/PI3K/PKB signaling pathway.

Adenosine 5'-triphosphate-sensitive potassium channel (KATP channel) activator, minoxidil sulfate (MS), can selectively increase the permeability of the blood-tumor barrier (BTB); however, the mechanism by which this occurs is still under investigation. Using a rat brain glioma (C6) model, we first examined the expression levels of occludin and claudin-5 at different time points after intracarotid infusion of MS (30 µg/kg/min) by western blotting. Compared to MS treatment for 0 min group, the protein expression levels of occludin and claudin-5 in brain tumor tissue of rats showed no changes within 1 h and began to decrease significantly after 2 h of MS infusion. Based on these findings, we then used an in vitro BTB model and selective inhibitors of diverse signaling pathways to investigate whether reactive oxygen species (ROS)/RhoA/PI3K/PKB pathway play a key role in the process of the increase of BTB permeability induced by MS. The inhibitor of ROS or RhoA or PI3K or PKB significantly attenuated the expression of tight junction (TJ) protein and the increase of the BTB permeability after 2 h of MS treatment. In addition, the significant increases in RhoA activity and PKB phosphorylation after MS administration were observed, which were partly inhibited by N-2-mercaptopropionyl glycine (MPG) or C3 exoenzyme or LY294002 pretreatment. The present study indicates that the activation of signaling cascades involving ROS/RhoA/PI3K/PKB in BTB was required for the increase of BTB permeability induced by MS. Taken together, all of these results suggested that MS might increase BTB permeability in a time-dependent manner by down-regulating TJ protein expression and this effect could be related to ROS/RhoA/PI3K/PKB signal pathway.

Single molecule kinetics of horseradish peroxidase exposed in large arrays of femtoliter-sized fused silica chambers.

Large arrays of femtoliter-sized chambers were etched into the surface of fused silica slides to enclose and observe hundreds of single horseradish peroxidase (HRP) molecules in parallel. Individual molecules of HRP oxidize the fluorogenic substrate Amplex Red to fluorescent resorufin in separate chambers, which was monitored by fluorescence microscopy. Photooxidation of Amplex Red and photobleaching of resorufin have previously limited the analysis of HRP in femtoliter arrays. We have strongly reduced these effects by optimizing the fluorescence excitation and detection scheme to yield accurate single molecule substrate turnover rates. We demonstrate the presence of long-lived kinetic states of single HRP molecules that are individually different for each molecule in the array. The large number of molecules investigated in parallel provides excellent statistics on the activity distribution in the enzyme population, which is similar to that reported for other enzymes such as ß-galactosidase. We have further confirmed that the product formation of HRP in femtoliter chambers is 10-fold lower than that in the bulk solution due to the particular two-step redox reaction mechanism of HRP.

Poly (dextrogyr-levogyr) lactide acid-triiodothyronine scaffold for peripheral nerve regeneration.

OBJECTIVES: To evaluate the poly(dextrogyr-levogyr) lactide acid-triiodothyronine (PDLLA-T3) seeded with Schwann cells conduit for repairing sciatic nerve defect. MATERIALS & METHODS: The rats were divided into three groups: autologous nerve transplantation (Group A), PDLLA-T3 + Schwann cells (Group B) and PDLLA + Schwann cells (Group C). RESULTS: Myelin sheath thickness was significantly greater in Group A compared with Group B and Group C. The regenerated nerves had nearly normal structure in Group A, and in Groups B and C nerve tissues filled in the anastomotic site and angiogenesis was noted. The mean number of myelinated nerve fibers and neurons in Group B was greater than in Group C. CONCLUSIONS: PDLLA-T3 is superior to PDLLA alone for repairing nerve defects.

Differential retinal origins of separate anatomical channels for pattern and motion vision in rabbit.

The most conspicuous feature of the rabbit retina is the visual streak that extends along the horizontal azimuth from the nasal margin to the temporal limit of the retina. We believe the streak processes movement vision and that the temporal region (area centralis) is responsible for pattern perception. Both anatomical and behavioural experiments were used to test this hypothesis. Behavioural measures of pattern vision in normal and chiasma-sectioned rabbits revealed both to have the same visual acuity. Using OKN as a measure of movement vision, normal rabbits showed both a directional and velocity-tuned response. The chiasma-sectioned rabbits, with only uncrossed fibre projections remaining, showed a total loss of movement detection. The injection of HRP into the vitreal chamber of one eye in normal rabbits revealed extensive uptake throughout the contralateral thalamus. In the ipsilateral thalamus, there was uptake solely from the ipsilateral retinal projection to a restricted wafer of the lateral geniculate nucleus (LGN). The chiasma cut rabbits showed a very different distribution of HRP in the thalamus. The uptake was restricted to a thin wafer of the LGN, with no contralateral uptake. Thus, the thalamic projections from the retinal area centralis were strictly segregated from the thalamic target areas for the visual streak without any overlap. These findings provide strong evidence for separate retinal origins with anatomically separate pathways for pattern and movement vision in the rabbit.

A system to enrich for primitive streak-derivatives, definitive endoderm and mesoderm, from pluripotent cells in culture.

Two lineages of endoderm develop during mammalian embryogenesis, the primitive endoderm in the pre-implantation blastocyst and the definitive endoderm at gastrulation. This complexity of endoderm cell populations is mirrored during pluripotent cell differentiation in vitro and has hindered the identification and purification of the definitive endoderm for use as a substrate for further differentiation. The aggregation and differentiation of early primitive ectoderm-like (EPL) cells, resulting in the formation of EPL-cell derived embryoid bodies (EPLEBs), is a model of gastrulation that progresses through the sequential formation of primitive streak-like intermediates to nascent mesoderm and more differentiated mesoderm populations. EPL cell-derived EBs have been further analysed for the formation of definitive endoderm by detailed morphological studies, gene expression and a protein uptake assay. In comparison to embryoid bodies derived from ES cells, which form primitive and definitive endoderm, the endoderm compartment of embryoid bodies formed from EPL cells was comprised almost exclusively of definitive endoderm. Definitive endoderm was defined as a population of squamous cells that expressed Sox17, CXCR4 and Trh, which formed without the prior formation of primitive endoderm and was unable to endocytose horseradish peroxidase from the medium. Definitive endoderm formed in EPLEBs provides a substrate for further differentiation into specific endoderm lineages; these lineages can be used as research tools for understanding the mechanisms controlling lineage establishment and the nature of the transient intermediates formed. The similarity between mouse EPL cells and human ES cells suggests EPLEBs can be used as a model system for the development of technologies to enrich for the formation of human ES cell-derived definitive endoderm in the future.

Paracellular versus transcellular intestinal permeability to gliadin peptides in active celiac disease.

The intestinal permeability of undegraded α9-gliadin peptide 31-49 (p31-49) and 33-mer gliadin peptides is increased in active celiac disease. Two distinct transport pathways have been proposed: paracellular leakage through epithelial tight junctions and protected transcellular transport. To analyze the relative contribution of these pathways, we compared mucosa-to-serosa permeability of small and large permeability markers [ionic conductance (G), mannitol, 182 Da; horseradish peroxidase, 40 kDa] and gliadin peptides [33-mer (p56-88, 3900 Da), 19-mer (p31-49, 2245 Da; and p202-220, 2127 Da), and 12-mer (p57-68, 1453 Da)] in duodenal biopsy specimens mounted in Ussing chambers. The permeability of intact peptides was much higher for p31-49 or 33-mer than for horseradish peroxidase, p202-220, and p57-68. A positive correlation was observed between G, an index of paracellular diffusion of ions, and mannitol permeability. The absence of correlation between G and permeability to intact 33-mer or p31-49 did not favor paracellular diffusion of the peptides. Immunofluorescence studies indicated that 33-mer enters the early endosome antigen 1-positive compartment but escapes the lysosomal-associated protein 2-positive compartment. The results underline that mannitol and ionic conductance G cannot be considered markers of permeability to gliadin peptides. In active celiac disease, increases in transcellular permeability to intact gliadin peptides might be considered in treatment strategies aimed at controlling epithelial permeability to gluten.

Cortical layer V neurons in the auditory and visual cortices of normal, reeler, and yotari mice.

Both in the Reelin-deficient reeler and Dab1-deficient yotari mice, layer V corticospinal tract neurons in the sensory-motor cortex are radially spread instead of being confined to a single cortical layer. In the present study, we examined distribution pattern of cortical layer V neurons in the visual and auditory cortices of reeler and yotari mice with the injection of HRP into the superior and inferior colliculi of the adult animals, respectively. After the injection of HRP into the superior colliculus of the normal mouse, retrogradely labeled cells were distributed in layer V of the visual cortex, while the similar injection of HRP in the reeler and yotari mice produced radial dispersion of retrograde labeling through all of the depths of the visual cortex of these mutant mice. Next, we injected HRP into the inferior colliculus of the normal, reeler and yotari mice. Retrogradely labeled neurons were distributed in layer V of the normal auditory cortex, whereas they were again radially scattered in the auditory cortex of the reeler and yotari mice. Taken together with the previous and present findings, layer V cortical efferent neurons are radially scattered in the sensory-motor, visual and auditory cortices of the reeler and yotari mice.

Delivering horseradish peroxidase as a respirable powder to the isolated, perfused, and ventilated lung of the rat: the pulmonary disposition of an inhaled model biopharmaceutical.

BACKGROUND: Our aim was to investigate the potential of the DustGun aerosol technology integrated with the isolated, perfused, and ventilated lung of the rat (IPL) to study the pulmonary disposition of an inhaled model biopharmaceutical, the 40-kDa protein horseradish peroxidase (HRP). METHOD: The DustGun aerosol technology was used to deliver respirable powder aerosols of HRP (the mass median aerodynamic diameter: 1.7 µm) as an 80-sec bolus to the IPL perfused in a single-pass mode. Lung perfusate was repeatedly sampled for 125 min after the HRP exposure. The amount of active HRP clearing with the perfusate or being retained in the lung was measured enzymatically. RESULTS AND CONCLUSIONS: The total amount of HRP deposited in the lungs was 335 ± 100 µg and 568 ± 47 µg for a low- and high-dose exposure, respectively. After inhalation, the initial appearance of HRP in the perfusate was rapid. However, the total amount of HRP that cleared with the perfusate remained below 0.5% of the deposited dose. The effect of opening the tight junctions between the alveolar epithelial cells on HRP absorption was studied by exposing the IPL to nebulized aerosols of either 0.02, 0.2, or 2% poly-L-Arginine (PLA) (MW 42.5 kDa) in phosphate-buffered saline (PBS) for 5 min, at 40 min after the HRP exposure. Subsequent exposure to 0.02% PLA did not affect HRP absorption. However, exposure to 0.2% PLA increased the absorption rate ninefold, and the total amount of HRP clearing with the perfusate increased to approximately 4% of the deposited dose. No further increase was obtained with 2% PLA, indicating a steep dose-response for the enhancer. It was concluded that the pulmonary absorption of HRP is quite slow, and absorption enhancers affecting tight junctions have a distinctive, yet limited efficiency. The presented inhalation technology can be very useful in studying the pulmonary absorption of biopharmaceuticals.

Stabilization and release of enzymes from silk films.

A significant challenge remains to protect protein drugs from inactivation during production, storage, and use. In the present study, the stabilization and release of horseradish peroxidase (HRP) in silk films was investigated. Water-insoluble silk films were prepared under mild aqueous conditions, maintaining the activity of the entrapped enzyme. Depending on film processing and post-processing conditions, HRP retained more than 90% of the initial activity at 4 degrees C, room temperature and 37 degrees C over two months. The stability of protein drugs in silk films is attributed to intermolecular interactions between the silk and the enzymes, based on Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The unique structural feature of silk molecules, periodic hydrophobic-hydrophilic domains, enabled strong interactions with proteins. The entrapped protein was present in two states, untrapped active and trapped inactive forms. The ratio between the two forms varied according to processing conditions. Proteolytic degradation and dissolution of the silk films resulted in the release of the bound enzyme which was otherwise not released by diffusion; enzyme recovered full activity upon release. There was a linear relationship between silk degradation/dissolution and the release of entrapped enzyme. Modifying the secondary structure of the silk matrix and the interactions with the non-crystalline domains resulted in control of the film degradation or dissolution rate, and therefore the release rate of the entrapped enzyme. Based on the above results, silk materials are an intriguing carrier for proteins in terms of both retention of activity and controllable release kinetics from the films.

Descending brain-spinal cord projections in a primitive vertebrate, the lamprey: cerebrospinal fluid-contacting and dopaminergic neurons.

We used Neurobiotin as a retrograde tract tracer in both larval and adult sea lampreys and observed a number of neuronal brainstem populations (mainly reticular and octaval populations and some diencephalic nuclei) that project to the spinal cord, in agreement with the results of previous tracer studies. We also observed small labeled neurons in the ventral hypothalamus, the mammillary region, and the paratubercular nucleus, nuclei that were not reported as spinal projecting. Notably, most of the labeled cells of the mammillary region and some of the ventral hypothalamus were cerebrospinal fluid-contacting (CSF-c) neurons. Combined tract tracing and immunocytochemistry showed that some of the labeled neurons of the mammillary and paratubercular nuclei were dopamine immunoreactive. In addition, some CSF-c cells were labeled in the caudal rhombencephalon and rostral spinal cord, and many were also dopamine immunoreactive. Results with other tracers (biotinylated dextran amines, horseradish peroxidase, and the carbocyanine dye DiI) also demonstrated that the molecular weight or the molecular nature of the tracer was determinant in revealing diencephalic cells with very thin axons. The results show that descending systems afferent to the spinal cord in lampreys are more varied than previously reported, and reveal a descending projection from CSF-c cells, which is unknown in vertebrates. The present results also reveal the existence of large differences between agnathans and gnathostomes in the organization of the dopaminergic cells that project to the spinal cord.