Liposome-based mucus-penetrating particles (MPP) for mucosal theranostics: demonstration of diamagnetic chemical exchange saturation transfer (diaCEST) magnetic resonance imaging (MRI).

Mucus barriers lining mucosal epithelia reduce the effectiveness of nanocarrier-based mucosal drug delivery and imaging ("theranostics"). Here, we describe liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, e.g., the diaCEST MRI contrast agent barbituric acid (BA). We observed that polyethylene glycol (PEG)-coated liposomes containing ≥7 mol% PEG diffused only ~10-fold slower in human cervicovaginal mucus (CVM) compared to their theoretical speeds in water. 7 mol%-PEG liposomes contained sufficient BA loading for diaCEST contrast, and provided improved vaginal distribution compared to 0 and 3mol%-PEG liposomes. However, increasing PEG content to ~12 mol% compromised BA loading and vaginal distribution, suggesting that PEG content must be optimized to maintain drug loading and stability. Non-invasive diaCEST MRI illustrated uniform vaginal coverage and longer retention of BA-loaded 7 mol%-PEG liposomes compared to unencapsulated BA. Liposomal MPP with optimized PEG content hold promise for drug delivery and imaging at mucosal surfaces. FROM THE CLINICAL EDITOR: This team of authors characterized liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, such as barbituric acid (a diaCEST MRI contrast agent) and concluded that liposomal MPP with optimized PEG coating enables drug delivery and imaging at mucosal surfaces.

Emerging paradigms for the initiation of mucin-type protein O-glycosylation by the polypeptide GalNAc transferase family of glycosyltransferases.

Mammalian mucin-type O-glycosylation is initiated by a large family of ∼20 UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer α-GalNAc from UDP-GalNAc to Ser and Thr residues of polypeptide acceptors. Characterizing the peptide substrate specificity of each isoform is critical to understanding their properties, biological roles, and significance. Presently, only the specificities of ppGalNAc T1, T2, and T10 and the fly orthologues of T1 and T2 have been systematically characterized utilizing random peptide substrates. We now extend these studies to ppGalNAc T3, T5, and T12, transferases variously associated with human disease. Our results reveal several common features; the most striking is the similar pattern of enhancements for the three residues C-terminal to the site of glycosylation for those transferases that contain a common conserved Trp. In contrast, residues N-terminal to the site of glycosylation show a wide range of isoform-specific enhancements, with elevated preferences for Pro, Val, and Tyr being the most common at the -1 position. Further analysis reveals that the ratio of positive (Arg, Lys, and His) to negative (Asp and Glu) charged residue enhancements varied among transferases, thus further modulating substrate preference in an isoform-specific manner. By utilizing the obtained transferase-specific preferences, the glycosylation patterns of the ppGalNAc Ts against a series of peptide substrates could roughly be reproduced, demonstrating the potential for predicting isoform-specific glycosylation. We conclude that each ppGalNAc T isoform may be uniquely sensitive to peptide sequence and overall charge, which together dictates the substrate sites that will be glycosylated.

Development of Absorbable, Antibiotic-Eluting Sutures for Ophthalmic Surgery.

PURPOSE: To develop and evaluate an antibiotic-eluting suture for ophthalmic surgery. METHODS: Wet electrospinning was used to manufacture sutures composed of poly(L-lactide), polyethylene glycol (PEG), and levofloxacin. Size, morphology, and mechanical strength were evaluated via scanning electron microscopy and tensile strength, respectively. In vitro drug release was quantified using high performance liquid chromatography. In vitro suture activity against Staphylococcus epidermidis was investigated through bacterial inhibition studies. Biocompatibility was determined via histological analysis of tissue sections surrounding sutures implanted into Sprague-Dawley rat corneas. RESULTS: Sutures manufactured via wet electrospinning were 45.1 ± 7.7 µm in diameter and 0.099 ± 0.007 newtons (N) in breaking strength. The antibiotic release profile demonstrated a burst followed by sustained release for greater than 60 days. Increasing PEG in the polymer formulation, from 1% to 4% by weight, improved drug release without negatively affecting tensile strength. Sutures maintained a bacterial zone of inhibition for at least 1 week in vitro and elicited an in vivo tissue reaction comparable to a nylon suture. CONCLUSIONS: There is a need for local, postoperative delivery of antibiotics following ophthalmic procedures. Wet electrospinning provides a suitable platform for the development of sutures that meet size requirements for ophthalmic surgery and are capable of sustained drug release; however, tensile strength must be improved prior to clinical use. TRANSLATIONAL RELEVANCE: No antibiotic-eluting suture exists for ophthalmic surgery. A biocompatible, high strength suture capable of sustained antibiotic release could prevent ocular infection and preclude compliance issues with topical eye drops.

A diaCEST MRI approach for monitoring liposomal accumulation in tumors.

Nanocarrier-based chemotherapy allows preferential delivery of therapeutics to tumors and has been found to improve the efficacy of cancer treatment. However, difficulties in tracking nanocarriers and evaluating their pharmacological fates in patients have limited judicious selection of patients to those who might most benefit from nanotherapeutics. To enable the monitoring of nanocarriers in vivo, we developed MRI-traceable diamagnetic Chemical Exchange Saturation Transfer (diaCEST) liposomes. The diaCEST liposomes were based on the clinical formulation of liposomal doxorubicin (i.e. DOXIL®) and were loaded with barbituric acid (BA), a small, organic, biocompatible diaCEST contrast agent. The optimized diaCEST liposomal formulation with a BA-to-lipid ratio of 25% exhibited 30% contrast enhancement at B1=4.7µT in vitro. The contrast was stable, with ~80% of the initial CEST signal sustained over 8h in vitro. We used the diaCEST liposomes to monitor the response to tumor necrosis factor-alpha (TNF-α), an agent in clinical trials that increases vascular permeability and uptake of nanocarriers into tumors. After systemic administration of diaCEST liposomes to mice bearing CT26 tumors, we found an average diaCEST contrast at the BA frequency (5ppm) of 0.4% at B1=4.7µT while if TNF-α was co-administered the contrast increased to 1.5%. This novel approach provides a non-radioactive, non-metallic, biocompatible, semi-quantitative, and clinically translatable approach to evaluate the tumor targeting of stealth liposomes in vivo, which may enable personalized nanomedicine.

Scalable method to produce biodegradable nanoparticles that rapidly penetrate human mucus.

Mucus typically traps and rapidly removes foreign particles from the airways, gastrointestinal tract, nasopharynx, female reproductive tract and the surface of the eye. Nanoparticles capable of rapid penetration through mucus can potentially avoid rapid clearance, and open significant opportunities for controlled drug delivery at mucosal surfaces. Here, we report an industrially scalable emulsification method to produce biodegradable mucus-penetrating particles (MPP). The emulsification of diblock copolymers of poly(lactic-co-glycolic acid) and polyethylene glycol (PLGA-PEG) using low molecular weight (MW) emulsifiers forms dense brush PEG coatings on nanoparticles that allow rapid nanoparticle penetration through fresh undiluted human mucus. In comparison, conventional high MW emulsifiers, such as polyvinyl alcohol (PVA), interrupts the PEG coating on nanoparticles, resulting in their immobilization in mucus owing to adhesive interactions with mucus mesh elements. PLGA-PEG nanoparticles with a wide range of PEG MW (1, 2, 5, and 10 kDa), prepared by the emulsification method using low MW emulsifiers, all rapidly penetrated mucus. A range of drugs, from hydrophobic small molecules to hydrophilic large biologics, can be efficiently loaded into biodegradable MPP using the method described. This readily scalable method should facilitate the production of MPP products for mucosal drug delivery, as well as potentially longer-circulating particles following intravenous administration.

Mechanism of FGF23 processing in fibrous dysplasia.

Fibroblast growth factor-23 (FGF23) is a phosphate- and vitamin D-regulating hormone derived from osteoblasts/osteocytes that circulates in both active (intact, iFGF23) and inactive (C-terminal, cFGF23) forms. O-glycosylation by O-glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3) and differential cleavage by furin have been shown to be involved in regulating the ratio of active to inactive FGF23. Elevated iFGF23 levels are observed in a number of hypophosphatemic disorders, such as X-linked, autosomal recessive, and autosomal dominant hypophosphatemic rickets, whereas low iFGF23 levels are found in the hyperphosphatemic disorder familial tumoral calcinosis/hyperphosphatemic hyperostosis syndrome. Fibrous dysplasia of bone (FD) is associated with increased total FGF23 levels (cFGF23 + iFGF23); however, classic hypophosphatemic rickets is uncommon. Our results suggest that it can be explained by increased FGF23 cleavage leading to an increase in inactive cFGF23 relative to active iFGF23. Given the fact that FD is caused by activating mutations in the small G-protein G(s) α that results in increased cyclic adenosine monophosphate (cAMP) levels, we postulated that there may be altered FGF23 cleavage in FD and that the mechanism may involve alterations in cAMP levels and ppGalNacT3 and furin activities. Analysis of blood specimens from patients with FD confirmed that the elevated total FGF23 levels are the result of proportionally increased cFGF23 levels, consistent with less glycosylation and enhanced cleavage by furin. Analysis of primary cell lines of normal and mutation-harboring bone marrow stromal cells (BMSCs) from patients with FD demonstrated that BMSCs harboring the causative G(s) α mutation had higher cAMP levels, lower ppGalNAcT3, and higher furin activity. These data support the model wherein glycosylation by ppGalNAcT3 inhibits FGF23 cleavage by furin and suggest that FGF23 processing is a regulated process that controls overall FGF23 activity in FD patients.

The monomer/dimer transition of enzyme I of the Escherichia coli phosphotransferase system.

Enzyme I (EI) is the first protein in the phosphotransfer sequence of the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This system catalyzes sugar phosphorylation/transport and is stringently regulated. Since EI homodimer accepts the phosphoryl group from phosphoenolpyruvate (PEP), whereas the monomer does not, EI may be a major factor in controlling sugar uptake. Previous work from this and other laboratories (e.g. Dimitrova, M. N., Szczepanowski, R. H., Ruvinov, S. B., Peterkofsky, A., and Ginsburg A. (2002) Biochem. 41, 906-913), indicate that K(a) is sensitive to several parameters. We report here a systematic study of K(a) determined by sedimentation equilibrium, which showed that it varied by 1000-fold, responding to virtually every parameter tested, including temperature, phosphorylation, pH (6.5 versus 7.5), ionic strength, and especially the ligands Mg(2+) and PEP. This variability may be required for a regulatory protein. Further insight was gained by analyzing EI by sedimentation velocity, by near UV CD spectroscopy, and with a nonphosphorylatable active site mutant, EI-H189Q, which behaved virtually identically to EI. The singular properties of EI are explained by a model consistent with the results reported here and in the accompanying paper (Patel, H. V., Vyas, K. A., Mattoo, R. L., Southworth, M., Perler, F. B., Comb, D., and Roseman, S. (2006) J. Biol. Chem. 281, 17579-17587). We suggest that EI and EI-H189Q each comprise a multiplicity of conformers and progressively fewer conformers as they dimerize and bind Mg(2+) and finally PEP. Mg(2+) alone induces small or no detectable changes in structure, but large conformational changes ensue with Mg(2+)/PEP. This effect is explained by a "swiveling mechanism" (similar to that suggested for pyruvate phosphate dikinase (Herzberg, O., Chen, C. C., Kapadia, G., McGuire, M., Carroll, L. J., Noh, S. J., and Dunaway-Mariano, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2652-2657)), which brings the C-terminal domain with the two bound ligands close to the active site His(189).

Properties of the C-terminal domain of enzyme I of the Escherichia coli phosphotransferase system.

The bacterial phosphoenolpyruvate (PEP):glycose phosphotransferase system (PTS) mediates uptake/phosphorylation of sugars. The transport of all PTS sugars requires Enzyme I (EI) and a phosphocarrier histidine protein of the PTS (HPr). The PTS is stringently regulated, and a potential mechanism is the monomer/dimer transition of EI, because only the dimer accepts the phosphoryl group from PEP. EI monomer consists of two major domains, at the N and C termini (EI-N and EI-C, respectively). EI-N accepts the phosphoryl group from phospho-HPr but not PEP. However, it is phosphorylated by PEP(Mg(2+)) when complemented with EI-C. Here we report that the phosphotransfer rate increases approximately 25-fold when HPr is added to a mixture of EI-N, EI-C, and PEP(Mg(2+)). A model to explain this effect is offered. Sedimentation equilibrium results show that the association constant for dimerization of EI-C monomers is 260-fold greater than the K(a) for native EI. The ligands have no detectable effect on the secondary structure of the dimer (far UV CD) but have profound effects on the tertiary structure as determined by near UV CD spectroscopy, thermal denaturation, sedimentation equilibrium and velocity, and intrinsic fluorescence of the 2 Trp residues. The binding of PEP requires Mg(2+). For example, there is no effect of PEP on the T(m), an increase of 7 degrees C in the presence of Mg(2+), and approximately 14 degrees C when both are present. Interestingly, the dissociation constants for each of the ligands from EI-C are approximately the same as the kinetic (K(m)) constants for the ligands in the complete PTS sugar phosphorylation assays.

Subcellular distribution of enzyme I of the Escherichia coli phosphoenolpyruvate:glycose phosphotransferase system depends on growth conditions.

The phosphoenolpyruvate:glycose phosphotransferase system (PTS) participates in important functions in the bacterial cell, including the phosphorylation/uptake of PTS sugars. Enzyme I (EI), the first protein of the PTS complex, accepts the phosphoryl group from phosphoenolpyruvate, which is then transferred through a chain of proteins to the sugar. In these studies, a mutant GFP, enhanced yellow fluorescent protein (YFP), was linked to the N terminus of EI, giving Y-EI. Y-EI was active both in vitro (>/=90% compared with EI) and in vivo. Unexpectedly, the subcellular distribution of Y-EI varied significantly. Three types of fluorescence were observed: (i) diffuse (dispersed throughout the cell), (ii) punctate (concentrated in numerous discrete spots throughout the cell), and (iii) polar (at one or both ends of the cell). Cells from dense colonies grown on agar plates with LB broth or synthetic (Neidhardt) medium showed primarily bipolar or punctate fluorescence. In liquid culture, under carefully defined carbon-limiting growth conditions [ribose (non-PTS), mannitol (PTS sugar), or dl-lactate], cellular levels of enzymatically active Y-EI remain essentially constant for each carbon source, but fluorescence distribution depends on C source, cell density, growth phase, and apparently on "conditioned medium." Fluorescence was diffuse during exponential growth on LB or ribose/Neidhardt medium. On ribose they became punctate in the stationary phase, reverting to diffuse when more ribose was added. In LB, both Y-EI and a nonphosphorylatable mutant, H189Q-Y-EI, showed a diffuse fluorescence during growth, but, shortly after the addition of isopropyl beta-d-thiogalactopyranoside, Y-EI became bipolar; H189Q-Y-EI did not. The functions of EI sequestration remain to be determined.

Molecular cloning and characterization of a unique beta-glucosidase from Vibrio cholerae.

We have recently reported the molecular cloning of a gene, gspK, in Vibrio cholerae that encodes a specific glucosamine kinase. We describe here the identification of bglA, a gene contiguous to gspK in a presumptive large chitin catabolic operon. BglA was molecularly cloned into Escherichia coli, and the protein BglA was overexpressed and purified to apparent homogeneity. BglA is 65 kDa (574 amino acids) with an N-terminal amino acid sequence predicted by the gene sequence, suggesting that the enzyme is cytoplasmic. The purified enzyme exhibited optimal activity with p-nitrophenyl beta-glucoside, cellobiose, and higher oligosaccharides of cellulose. No other glucosides or glycosides tested were hydrolyzed, including Glc-Glc disaccharides where the linkage is beta 1-->2, beta 1-->3, and beta 1-->6, respectively. The predicted BglA sequence bears little similarity to other proteins in the data banks. The Henrissat algorithm places BglA sequence in Family 9 of the glycosidases, suggesting it is an endoglucanase. However, the results summarized above suggested that BglA is an exoenzyme yielding Glc at each cleavage step. To resolve this apparent discrepancy, detailed kinetic studies were conducted with cellotetraose. Only exoglucanase activity was detected. The function of this enzyme in V. cholerae remains to be determined, especially because our strain of this organism does not utilize cellobiose.

Gangliosides are functional nerve cell ligands for myelin-associated glycoprotein (MAG), an inhibitor of nerve regeneration.

Myelin-associated glycoprotein (MAG) binds to the nerve cell surface and inhibits nerve regeneration. The nerve cell surface ligand(s) for MAG are not established, although sialic acid-bearing glycans have been implicated. We identify the nerve cell surface gangliosides GD1a and GT1b as specific functional ligands for MAG-mediated inhibition of neurite outgrowth from primary rat cerebellar granule neurons. MAG-mediated neurite outgrowth inhibition is attenuated by (i) neuraminidase treatment of the neurons; (ii) blocking neuronal ganglioside biosynthesis; (iii) genetically modifying the terminal structures of nerve cell surface gangliosides; and (iv) adding highly specific IgG-class antiganglioside mAbs. Furthermore, neurite outgrowth inhibition is mimicked by highly multivalent clustering of GD1a or GT1b by using precomplexed antiganglioside Abs. These data implicate the nerve cell surface gangliosides GD1a and GT1b as functional MAG ligands and suggest that the first step in MAG inhibition is multivalent ganglioside clustering.