Enhanced anti-colon cancer efficacy of 5-fluorouracil by epigallocatechin-3- gallate co-loaded in wheat germ agglutinin-conjugated nanoparticles.

Colon adenocarcinoma is the third most common cause of cancer-related deaths worldwide owing to its aggressive nature. Here, we developed a novel oral drug delivery system (DDS) that comprised active targeted nanoparticles made from gelatin and chitosan (non-toxic polymers). The nanoparticles were fabricated using a complex coacervation method, which was accompanied by conjugation of wheat germ agglutinin (WGA) onto their surface by glutaraldehyde cross-linking. Specifically, we integrated 5-fluorouracil (5-FU), the first-line treatment agent against colon cancer, and (-)-epigallocatechin-3-gallate (EGCG), which inhibits tumor growth via anti-angiogenesis and apoptosis-inducing effects, into the nanoparticles, named WGA-EF-NP. The 5-FU and EGCG co-loaded nanoparticles showed sustained drug release, enhanced cellular uptake, and longer circulation time. WGA-EF-NP exhibited superior anti-tumor activity and pro-apoptotic efficacy compared to the drugs and nanoparticles without WGA decoration owing to better bioavailability and longer circulation time in vivo. Thus, WGA-EF-NP shows promise as a DDS for enhanced efficacy against colon cancer.

Lectin uptake and incorporation into the calcitic spicule of sea urchin embryos.

Primary mesenchyme cells (PMCs) are skeletogenenic cells that produce a calcareous endoskeleton in developing sea urchin larvae. The PMCs fuse to form a cavity in which spicule matrix proteins and calcium are secreted forming the mineralized spicule. In this study, living sea urchin embryos were stained with fluorescently conjugated wheat germ agglutinin, a lectin that preferentially binds to PMCs, and the redistribution of this fluorescent tag was examined during sea urchin development. Initially, fluorescence was associated primarily with the surface of PMCs. Subsequently, the fluorescent label redistributed to intracellular vesicles in the PMCs. As the larval skeleton developed, intracellular granular staining diminished and fluorescence appeared in the spicules. Spicules that were cleaned to remove membranous material associated with the surface exhibited bright fluorescence, which indicated that fluorescently labelled lectin had been incorporated into the spicule matrix. The results provide evidence for a cellular pathway in which material is taken up at the cell surface, sequestered in intracellular vesicles and then incorporated into the developing spicule.

[Modification by wheat germ agglutinin delays the ocular elimination of liposome].

The purpose of this study is to explore the feasibility of wheat germ agglutinin (WGA) modified liposome as a vehicle for ophthalmic administration. Liposome loaded with 5-carboxyfluorescein (FAM) was prepared by lipid film hydration method. WGA was thiolated and then conjugated to the surface of the liposome via polyethylene glycol linker to constitute the WGA-modified and FAM-loaded liposome (WGA-LS/FAM). The amount of thiol groups on each WGA molecule was determined, and the bioactivity of WGA was estimated after it was modified to the surface of liposome. The physical and chemical features of the WGA-modified liposome were characterized and the ocular bioadhesive performance was evaluated in rats. The result showed that each thiolated WGA molecule was conjugated with 1.32 thiol groups. WGA-LS/FAM had a mean size of (97.40 +/- 1.39) nm, with a polydispersity index of 0.23 +/- 0.01. The entrapment efficacy of FAM was about (2.95 +/- 0.21)%, and only 4% of FAM leaked out of the liposome in 24 h. Erythrocyte agglutination test indicated that after modification WGA preserved the binding activity to glycoprotein. The in vivo ocular elimination of WGA-LS/FAM fitted first-order kinetics, and the elimination rate was significantly slower than that of the unmodified liposome, demonstrating WGA-modified liposome is bioadhesive and suitable for ophthalmic administration.

Tri-partite complex for axonal transport drug delivery achieves pharmacological effect.

BACKGROUND: Targeted delivery of pharmaceutical agents into selected populations of CNS (Central Nervous System) neurons is an extremely compelling goal. Currently, systemic methods are generally used for delivery of pain medications, anti-virals for treatment of dermatomal infections, anti-spasmodics, and neuroprotectants. Systemic side effects or undesirable effects on parts of the CNS that are not involved in the pathology limit efficacy and limit clinical utility for many classes of pharmaceuticals. Axonal transport from the periphery offers a possible selective route, but there has been little progress towards design of agents that can accomplish targeted delivery via this intraneural route. To achieve this goal, we developed a tripartite molecular construction concept involving an axonal transport facilitator molecule, a polymer linker, and a large number of drug molecules conjugated to the linker, then sought to evaluate its neurobiology and pharmacological behavior. RESULTS: We developed chemical synthesis methodologies for assembling these tripartite complexes using a variety of axonal transport facilitators including nerve growth factor, wheat germ agglutinin, and synthetic facilitators derived from phage display work. Loading of up to 100 drug molecules per complex was achieved. Conjugation methods were used that allowed the drugs to be released in active form inside the cell body after transport. Intramuscular and intradermal injection proved effective for introducing pharmacologically effective doses into selected populations of CNS neurons. Pharmacological efficacy with gabapentin in a paw withdrawal latency model revealed a ten fold increase in half life and a 300 fold decrease in necessary dose relative to systemic administration for gabapentin when the drug was delivered by axonal transport using the tripartite vehicle. CONCLUSION: Specific targeting of selected subpopulations of CNS neurons for drug delivery by axonal transport holds great promise. The data shown here provide a basic framework for the intraneural pharmacology of this tripartite complex. The pharmacologically efficacious drug delivery demonstrated here verify the fundamental feasibility of using axonal transport for targeted drug delivery.

Quantum dots bearing lectin-functionalized nanoparticles as a platform for in vivo brain imaging.

Delivery of imaging agents to the brain is highly important for the diagnosis and treatment of central nervous system (CNS) diseases, as well as the elucidation of their pathophysiology. Quantum dots (QDs) provide a novel probe with unique physical, chemical, and optical properties, and become a promising tool for in vivo molecular and cellular imaging. However, their poor stability and low blood-brain barrier permeability severely limit their ability to enter into and act on their target sites in the CNS following parenteral administration. Here, we developed a QDs-based imaging platform for brain imaging by incorporating QDs into the core of poly(ethylene glycol)-poly(lactic acid) nanoparticles, which was then functionalized with wheat germ agglutinin and delivered into the brain via nasal application. The resulting nanoparticles, with high payload capacity, are water-soluble, stable, and showed excellent and safe brain targeting and imaging properties. With PEG functional terminal groups available on the nanoparticles surface, this nanoprobe allows for conjugation of various biological ligands, holding considerable potential for the development of specific imaging agents for various CNS diseases.

Vinorelbine cationic liposomes modified with wheat germ agglutinin for inhibiting tumor metastasis in treatment of brain glioma.

Glioma is the most common primary malignant brain tumor with a poor prognosis. The application of chemotherapeutic drugs is limited due to the existence of blood-brain barrier and serious side effects. Liposomes have been proven to be a stable and useful drug delivery system for tumors. In this paper, WGA (wheat germ agglutinin) modified vinorelbine cationic liposomes had been successfully constructed for treating glioma. In the liposomes, WGA was modified on the liposomal surface for crossing the blood-brain barrier and increasing the targeting effects, 3-(N-(N', N'-dimethylaminoethane) carbamoyl) cholesterol (DC-Chol) was used as cationic material and vinorelbine was encapsulated in the aqueous core of liposomes to inhibit tumor metastasis and kill tumor cells. Studies were performed on C6 cells in vitro and were verified in brain glioma-bearing mice in vivo. Results in vitro demonstrated that the targeting liposomes could induce C6 cells apoptosis, promote drugs across the blood-brain barrier, inhibit the metastasis of tumor cells and increase targeting effects to tumor cells. Meanwhile, action mechanism studies showed that the targeting liposomes could down-regulate PI3K, MMP-2, MMP-9 and FAK to inhibit tumor metastasis. Results in vivo exhibited that the targeting liposomes displayed an obvious antitumor efficacy by accumulating selectively in tumor site and exhibited low toxicity to blood system and major organs. Hence, WGA modified vinorelbine cationic liposomes might provide a safe and efficient therapy strategy for glioma.

Lectin-conjugated PEG-PLA nanoparticles: preparation and brain delivery after intranasal administration.

In order to improve the absorption of nanoparticles in the brain following nasal administration, a novel protocol to conjugate biorecognitive ligands-lectins to the surface of poly (ethylene glycol)-poly (lactic acid) (PEG-PLA) nanoparticles was established in the study. Wheat germ agglutinin (WGA), specifically binding to N-acetyl-D-glucosamine and sialic acid, both of which were abundantly observed in the nasal cavity, was selected as a model lectin. The WGA-conjugated nanoparticles were prepared by incorporating maleimide in the PLA-PEG molecular and taking advantage of its thiol group binding reactivity to conjugate with 2-iminothialane thiolated WGA. Coupling of WGA with the PEG-PLA nanoparticles was confirmed by the existence of gold-labeled WGA-NP under TEM. The retention of biorecognitive activity of WGA after the covalent coupling procedure was confirmed by haemagglutination test. The resulting nanoparticles presented negligible nasal ciliatoxicity and the brain uptake of a fluorescent marker-coumarin carried by WGA functionized nanoparticles was about 2 folds in different brain tissues compared with that of coumarin incorporated in the unmodified ones. Thus, the technique offered a novel effective noninvasive system for brain drug delivery, especially for brain protein and gene delivery.

Differences in the connectivity of rat pudendal motor nuclei as revealed by retrograde transneuronal transport of wheat germ agglutinin.

Bilateral coordinated activation of pudendal motoneurons is an essential component of penile reflexes in male rats. However, little is known about the intraspinal organization of these reflexes. In the present study, retrograde transneuronal transport of wheat germ agglutinin (WGA) was used to examine the organization of spinal motoneurons and putative interneurons mediating penile reflexes in adult male rats. Injection of WGA into the ventral bulbospongiosus muscle resulted in direct retrograde labeling of motoneurons in the ipsilateral dorsomedial (DM) nucleus and transneuronal labeling of ipsilateral and contralateral DM motoneurons. Motoneurons in the ipsilateral and contralateral dorsolateral (DL) nuclei were not labeled. WGA-labeled putative interneurons were observed bilaterally, primarily in the ventromedial spinal gray matter extending dorsally to the central canal and the dorsal gray commissure. The number of transneuronally labeled putative interneurons increased with longer survival times. Injection of WGA into the ischiocavernosus muscle resulted in direct retrograde labeling of motoneurons in the medial subdivision of the ipsilateral DL nucleus. However, no WGA labeling was detected in motoneurons in the lateral subdivision of the ipsilateral DL nucleus, the contralateral DL nucleus, or the DM nuclei at any of the survival times studied (1-7 days). Only a small number of transneuronally labeled putative interneurons was observed in the ventrolateral gray matter at longer survival times (3-7 days). Thus, marked differences were observed between the DM and DL nuclei with respect to the transneuronal transport of WGA. These results are discussed with respect to the organization of the spinal circuits that mediate pudendal motor reflexes.

Motoneurons of twitch and nontwitch extraocular muscle fibers in the abducens, trochlear, and oculomotor nuclei of monkeys.

Eye muscle fibers can be divided into two categories: nontwitch, multiply innervated muscle fibers (MIFs), and twitch, singly innervated muscle fibers (SIFs). We investigated the location of motoneurons supplying SIFs and MIFs in the six extraocular muscles of monkeys. Injections of retrograde tracers into eye muscles were placed either centrally, within the central SIF endplate zone; in an intermediate zone, outside the SIF endplate zone, targeting MIF endplates along the length of muscle fiber; or distally, into the myotendinous junction containing palisade endings. Central injections labeled large motoneurons within the abducens, trochlear or oculomotor nucleus, and smaller motoneurons lying mainly around the periphery of the motor nuclei. Intermediate injections labeled some large motoneurons within the motor nuclei but also labeled many peripheral motoneurons. Distal injections labeled small and medium-large peripheral neurons strongly and almost exclusively. The peripheral neurons labeled from the lateral rectus muscle surround the medial half of the abducens nucleus: from superior oblique, they form a cap over the dorsal trochlear nucleus; from inferior oblique and superior rectus, they are scattered bilaterally around the midline, between the oculomotor nucleus; from both medial and inferior rectus, they lie mainly in the C-group, on the dorsomedial border of oculomotor nucleus. In the medial rectus distal injections, a "C-group extension" extended up to the Edinger-Westphal nucleus and labeled dendrites within the supraoculomotor area. We conclude that large motoneurons within the motor nuclei innervate twitch fibers, whereas smaller motoneurons around the periphery innervate nontwitch, MIF fibers. The peripheral subgroups also contain medium-large neurons which may be associated with the palisade endings of global MIFs. The role of MIFs in eye movements is unclear, but the concept of a final common pathway must now be reconsidered.

A quantitative study of anterograde and retrograde axonal transport of exogenous proteins in olfactory nerve C-fibers.

The pike olfactory nerve which consists of a homogeneous population of C-fibers of 0.25 micron diameter or less was used to study quantitatively both anterograde and retrograde axoplasmic transport of wheat germ agglutinin and horseradish peroxidase. It was found that even in these extremely thin axons anterograde and retrograde transport takes place. Activity distribution profiles (transport profiles) for retrograde transport were established and found to be similar to the typical profiles of anterograde transport as they consisted of a small rapidly moving peak and a saddle region followed by the bulk of the material which moved more slowly. Horseradish peroxidase activity profiles were obtained both after injection into the synaptic region and after injection into the perikaryal region. From these transport profiles a maximal velocity of 25 mm/d (19 degrees C) for the leading peak and of about 7 mm/d for the slower component could be determined. There is no significant difference between the velocities for anterograde and retrograde transport. In the case of wheat germ agglutinin, only injection into the synaptic region resulted in typical transport profiles (retrograde transport) with a peak and saddle region. The maximum velocities of retrograde transport were about the same as for horseradish peroxidase [26 mm/d and 7 mm/d (19 degrees C)]. The electron microscopic analysis of horseradish peroxidase revealed that after injection into the olfactory bulb it was taken up into the neurons where it was found mainly in multivesicular bodies (0.5 micron diameter). In longitudinal sections of the nerve similar but slightly more elongated organelles (diameter 0.25 micron, length 0.4 micron) were found in those segments in which the slowly moving bulk of the peroxidase activity was located. The number of these organelles decreased with distance from the site of injection. The horseradish peroxidase transported within the leading peak could not be assigned to specific structures although several electron microscopic-histochemical methods were applied. It was concluded that anterograde and retrograde transport occur simultaneously in these axons, and that, therefore, even the large organelles, each of which almost fills the axon, must be able to pass each other. This would necessitate that the axons are able to transiently enlarge their diameter considerably.

Medullary neurones regulate hypothalamic corticotropin-releasing factor cell responses to an emotional stressor.

Hypothalamic-pituitary-adrenal axis activation is a hallmark of the stress response. In the case of physical stressors, there is considerable evidence that medullary catecholamine neurones are critical to the activation of the paraventricular nucleus corticotropin-releasing factor cells that constitute the apex of the hypothalamic-pituitary-adrenal axis. In contrast, it has been thought that hypothalamic-pituitary-adrenal axis responses to emotional stressors do not involve brainstem neurones. To investigate this issue we have mapped patterns of restraint-induced neuronal c-fos expression in intact animals and in animals prepared with either paraventricular nucleus-directed injections of a retrograde tracer, lesions of paraventricular nucleus catecholamine terminals, or lesions of the medulla corresponding to the A1 or A2 noradrenergic cell groups. Restraint-induced patterns of neuronal activation within the medulla of intact animals were very similar to those previously reported in response to physical stressors, including the fact that most stressor-responsive, paraventricular nucleus-projecting cells were certainly catecholaminergic and probably noradrenergic. Despite this, the destruction of paraventricular nucleus catecholamine terminals with 6-hydroxydopamine did not alter corticotropin-releasing factor cell responses to restraint. However, animals with ibotenic acid lesions encompassing either the A1 or A2 noradrenergic cell groups displayed significantly suppressed corticotropin-releasing factor cell responses to restraint. Notably, these medullary lesions also suppressed neuronal responses in the medial amygdala, an area that is now considered critical to hypothalamic-pituitary-adrenal axis responses to emotional stressors and that is also known to display a significant increase in noradrenaline turnover during restraint. We conclude that medullary neurones influence corticotropin-releasing factor cell responses to emotional stressors via a multisynaptic pathway that may involve a noradrenergic input to the medial amygdala. These results overturn the idea that hypothalamic-pituitary-adrenal axis response to emotional stressors can occur independently of the brainstem.

Morphological evidence of the inhibitory effect of taxol on the fast axonal transport.

The short term effects of taxol, a stabilizing drug of microtubules, on the peripheral nerves in the rat was investigated using a new chamber system which can be applied to incubate a sciatic nerve with various solutions in vivo. A functional analysis of retrograde axonal transport using rhodamine-labeled wheat germ agglutinin (WGA-rhodamine) showed the inhibitory effect of the drug. An electron microscopic study also revealed that a variety of vesicles were observed to accumulate on both the proximal and the distal sides of the chamber, however, no significant increase in the number of microtubules in the axons, based on the pharmacological effect of the drug, was observed even though one had been expected. These findings support the inhibitory effect of taxol on the fast axonal transport of the neurons. Furthermore, the accumulated vesicles were morphologically different from those accumulated by ligation. These results suggest that a special component of the fast axonal transport was thus selectively blocked by the drug.

A hypothesis for the superior sensitivity of wheat germ agglutinin as a neuroanatomical probe.

We have shown that a conjugate of wheat germ agglutinin (WGA), with horseradish peroxidase (HRP), is more sensitive than native HRP as a probe of neuroanatomic connections involving the retrograde transport of the lectin. It has also been shown in our laboratory that WGA-HRP remains at the site of injection twice as long as HRP. The purpose of the present morphometric study was to investigate the basis for the higher sensitivity of WGA-HRP over HRP as a retrogradely transported tracer molecule. To do this, we modified the experiment of Heuser and Reese which utilized the tracing of HRP in the frog neuromuscular junction (Heuser, J.E. and Reese, T.S., J. Cell Biol., 57 (1973) 315-344). Instead of using HRP alone, we examined, in double labeling experiments, fluid and adsorptive endocytosis with free HRP and WGA coupled to ferritin (WGA-ferritin) respectively. Immediately after nerve stimulation, both markers are taken up simultaneously into cisternae, and in tubular structures strikingly similar to the described compartment of uncoupling of receptor from ligand (CURL). Frequently, cisternae were connected with putative CURL. This early double labeling of cisternae and putative CURL was followed by the appearance of synaptic vesicles labeled with WGA-ferritin only (72-79%), HRP only (6-11%), and both labels (13-16%). In contrast to the labeling pattern of synaptic vesicles, the majority of cisternae and putative CURL had both labels throughout the duration of the experiments (77-80%). The results of this study indicate that most of WGA-ferritin and HRP are co-localized in cisternae and putative CURL, compartments involved in endocytosis and surface receptor recycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Satellite cells as blood-ganglion cell barrier in autonomic ganglia.

In a preliminary study a difference in the uptake and transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) between the trigeminal ganglion and the superior cervical ganglion was observed. After injection of WGA-HRP and HRP into the trigeminal ganglion, peroxidase was found in the space between the satellite cell processes and the ganglion cells. The ganglion cells showed pinocytosis and uptake of WGA-HRP and HRP. In the superior cervical ganglion WGA-HRP and HRP were found alongside the satellite cells but were absent in the space between satellite cells and ganglion cells. Intravenous injection revealed the presence of HRP in the space between sensory ganglion cells and their satellite cells of the trigeminal and nodose ganglion whereas HRP was absent in the space between autonomic ganglion cells and their satellite cells of the superior cervical, medial cervical and pterygopalatine ganglion although HRP lined the satellite cell membranes. By means of electron microscopy, satellite cell processes in the superior cervical ganglion were found to enwrap ganglion cells very tightly with a marginal space between both cell types. Satellite cells and their processes were mutually anchored by numerous tight junctions. In the trigeminal ganglion the extracellular space between ganglion cells and satellite cells was larger and satellite cells were found to be more loosely arranged around the ganglion cells. Satellite cell processes were only occasionally linked by tight junctions. It is concluded that satellite cells in autonomic ganglia comprise an effective barrier for WGA-HRP and HRP and probably large molecules in general. This barrier is absent in sensory ganglia.

Morphological heterogeneity within the cingulate cortex in rat: a horseradish peroxidase transport study.

We compared the connections of two areas within rat cingulate cortex, the Cg1/Cg2 area vs the Cg3 area, by iontophoresing small quantities of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) into either of these two divisions and identifying afferent and efferent connections. Cortical projections were more widespread for the cingulate cortex (Cg3) area than for the Cg1/Cg2 area and included the dysgranular and agranular insular cortex, and perirhinal cortex. The Cg3 area received input from the CA1 layer of the hippocampus while the Cg1/Cg2 area was interconnected primarily with retrosplenial cortex. In the brainstem, both received input from Barrington's nucleus however, many of the subcortical connections of the two areas differed and supported the hypothesis that the Cg3 area is part of the limbic and visceral motor system while the Cg1/Cg2 area is more closely allied with somatic motor control. The Cg3 area received input from the basolateral nucleus of the amygdala, the supramammillary hypothalamic nucleus, the laterodorsal tegmental nucleus, and the lateral parabrachial nucleus. The Cg1/Cg2 area received input from the substantia nigra and targeted deep layers of the superior colliculus. Thus, rat cingulate cortex is a heterogeneous area that can be further subdivided into separate limbic/autonomic (Cg3) and somatic motor areas (Cg1/Cg2).

Preparation, characterization and application of artificial Caco-2 cell surfaces in the silver nanoparticle enhanced fluorescence technique.

To approach in vivo conditions during real time monitoring of biorecognitive interactions, biomimetic surfaces were prepared by fusion of purified plasma membrane fractions from Caco-2 cells with self-assembled monolayers attached to silver colloid coated standard microplates. Proper orientation and integrity of the membrane coating was confirmed by binding of fluorescein-labelled wheat germ agglutinin (F-WGA) which interacts with certain carbohydrates of the glycocalyx. Additionally, the competition with the complementary carbohydrate decreased the F-WGA binding to 15%. The assay setup offers information about the real time binding kinetics and the affinity of the interaction with the cell membrane excluding interfering events such as internalization and metabolism. As exemplified by F-WGA-binding, the mean velocity of the interaction is 627.07 mFU/s and the working range is 40-240 nM with a detection limit of 1.6 pmol F-WGA. The storage stability of the ready-to-use plates exceeded at least 1 month. The real time monitoring of lectin-prodrug binding to the biomimetic membranes revealed that high conjugation numbers reduces the affinity. The assays are simple and fast one-step reactions without any washing steps as this new technique discriminates between membrane bound and bulk fluorescence. Thus, biomimetic membranes on silver colloid layers represent a versatile tool for high throughput screening at early stages of drug discovery and development.

Lectin-mediated drug delivery: influence of mucin on cytoadhesion of plant lectins in vitro.

As the mucous layer represents the first barrier to peroral lectin-mediated drug delivery, the influence of mucin on the cytoadhesive properties of lectins was studied in vitro by establishing a rapid and simple microplate format assay using pig gastric mucin (PGM) for coating the wells. The lectin-binding capacity of mucin followed the order WGA>>UEA-I>>LCA=STL>PNA>DBA. The PGM-binding of wheat germ agglutinin (WGA) was strongly dependent on pH being highest at pH 5.0. In comparison, PGM-binding of WGA was about 15% at gastric pH and 60-70% at intestinal pH. This points to unimpeded gastric transit of WGA-grafted formulations and favorable conditions within the intestine for binding to mucus coated enterocytes. Moreover the WGA-PGM interaction was concentration-dependent, specific and fully reversible. According to a competitive assay in the presence of Caco-2 monolayers, the PGM-binding of WGA was saturated and influenced by the lectin-concentration yielding 28% Caco-2 bound WGA (125 ng WGA/0.29 cm(2) monolayer) and 68% Caco-2 bound WGA (4 microg WGA/0.29 cm(2) monolayer), respectively. Following on from these results, lectins are expected to suffer at least partially from premature inactivation by shed off mucus like bioadhesives of the first generation, however initial but reversible mucus-binding of lectins offers partititioning to the cell membrane followed by uptake into the enterocyte.

Enucleation-induced transsynaptic labeling with WGA-HRP in the developing rat visual system.

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) is a neuroanatomical tracer which is transported transneuronally. In order to investigate whether transport of WGA-HRP is across synapses, labeling was studied in the developing retinotectal pathway where it is known that enucleation results in increased ipsilateral synaptic connections from the remaining eye. While little or no transneuronal labeling was evident in controls, after enucleation transneuronal labeling was consistently observed. Furthermore, the critical period for enucleation-induced transneuronal labeling coincides with the known critical period for enucleation-induced neuronal survival and synaptic formation. The results suggest that transneuronal exchange of WGA-HRP depends on the presence of synapses, and is therefore transsynaptic.

Lectin-mediated bioadhesion: preparation, stability and caco-2 binding of wheat germ agglutinin-functionalized Poly(D,L-lactic-co-glycolic acid)-microspheres.

To take advantage of the cytoadhesive characteristics of Wheat germ agglutinin (WGA) for improved particulate drug delivery, the interaction between WGA-grafted poly(D,L-lactic-co-glycolic acid)-microspheres and Caco-2 monolayers was investigated using bovine serum albumin (BSA) or glycine coated microspheres as a control. Covalent immobilization of WGA by the carbodiimide/N-hydroxysuccinimide-method on 4 microm microspheres yielded a surface density of 9.67+/-1.21x10(6) molecules/particle, whereas 0.22+/-0.04x10(6) WGA-molecules were bound by physical adsorption. After storage for 21 days in HEPES-buffer and treatment of the particles with 5 M urea, 86% of covalently linked lectin was still attached to the particles. At 4 degrees C the Caco-2 binding rate of both, WGA- and BSA-modified particles increased with addition of increasing numbers of particles until saturation was reached at 38150+/-1740 (WGA) or 12066+/-1195 (BSA) microspheres bound/mm(2) Caco-2 monolayer. Inhibition of Caco-2 binding of WGA-functionalized microspheres by chitotriose indicated for specificity of the interaction. As observed by confocal laser scanning microscopy, the fluorescein-loading of the particles was accumulated intracellularly after incubation of Caco-2 monolayers with WGA-modified microspheres contrary to glycine-grafted microspheres. Additionally, in case of WGA-functionalized microspheres the amount of cell associated fluorescein was 200-fold higher than that of the free solution. In conclusion, WGA-modified microspheres are expected to enhance intestinal transport of incorporated drugs due to cytoadhesion provided by the lectin coating.

Lectin-mediated drug delivery: discrimination between cytoadhesion and cytoinvasion and evidence for lysosomal accumulation of wheat germ agglutinin in the Caco-2 model.

Lectin-mediated drug delivery may become a promising strategy to improve the efficacy of poorly permeable drugs by utilising active high-capacity transport pathways of epithelial tissues. This requires the elucidation of the basic mechanisms of lectin uptake prior to their practical use. We studied the interaction between the dietary lectin wheat germ agglutinin (WGA) and Caco-2 cells (single cells and monolayers) by a newly established assay design that is able to discriminate between cellular binding and uptake as well as by confocal microscopy: (i) All binding sites available for WGA at the cell membrane were occupied within 10 min of incubation. (ii) Cytoadhesion was followed by immediate uptake. After 20 min, 60% (single cells) or 30% (monolayers) of the membrane bound lectin were internalised. However, regardless of cell arrangement, 80% of the surface bound lectin was taken up into the cells during the course of the experiment. (iii) About 50% of the internalised lectin accumulated within the lysosomes after 1 h. This was confirmed by assays in the presence of monensin, an inhibitor of endosomal acidification, and by colocalisation with lysosomal cathepsin followed by semiquantitative image analysis. Further analysis by immunocytochemistry suggested that the trans-Golgi complex and the caveoli were not involved. Due to cytoadhesion, cytoinvasion and partial lysosomal accumulation, WGA-mediated drug delivery may provide for improved intracellular availability of conjugated drugs or colloidal carrier systems.