Greg F. Walker

Amine-reactive pyridylhydrazone-based PEG reagents for pH-reversible PEI polyplex shielding

PEGylation which is reversed after the therapeutic agent reaches the target cell presents an attractive feature for drug, protein or nucleic acid delivery. Amine-reactive, endosomal pH cleavable polyethylene glycol aldehyde-carboxypyridylhydrazone, N-hydroxysuccinimide esters (PEG-HZN-NHS) were synthesized and applied for bioreversible surface shielding of DNA polyplexes. Monofunctional mPEG-HZN-NHS was synthesized by reacting succinimidyl hydraziniumnicotinate with mPEG-butyraldehyde (20 kDa). Bifunctional OPSS-PEG-HZN-NHS was synthesized analogously via a omega-2-pyridyldithio-PEG (10 kDa) propionaldehyde intermediate. Polyethylenimine (PEI) polyplexes were reacted with the pH-sensitive (mPEG-HZN-NHS) or the corresponding stable (mPEG-NHS) reagent. Both types of polyplexes remained shielded at pH 7.4 as demonstrated by particle size and zeta potential measurements after 4h of incubation at 37 degrees C. Polyplex deshielding at endosomal pH 5 was observed only with the mPEG-HZN-NHS shielded particles. This was confirmed by fluorescence correlation spectroscopy using the analogous Alexa-488 fluorescently labeled bifunctional PEGylation reagents. Luciferase gene transfections with epidermal growth factor (EGF) containing polyplexes using EGF-receptor overexpressing hepatoma HUH7 cells showed an up to 16-fold enhancement in gene expression with the reversibly shielded polyplexes as compared to stably shielded polyplexes. Consistently, the reversibly shielded polyplexes mediated also an enhanced tumor specific in vivo transgene expression after intravenous administration in a subcutaneous HUH7 tumor model in SCID mice.

Monomolecular Assembly of siRNA and Poly(ethylene glycol)-Peptide Copolymers

In this work, we design and investigate the complex formation of highly uniform monomolecular siRNA complexes utilizing block copolymers consisting of a cationic peptide moiety covalently bound to a poly(ethylene glycol) (PEG) moiety. The aim of the study was to design a shielded siRNA construct containing a single siRNA molecule to achieve a sterically stabilized complex with enhanced diffusive properties in macromolecular networks. Using a 14 lysine-PEG (K14-PEG) linear diblock copolymer, formation of monomolecular siRNA complexes with a stoichiometric 1:3 grafting density of siRNA to PEG is realized. Alternatively, similar PEGylated monomolecular siRNA particles are achieved through complexation with a graft copolymer consisting of six cationic peptide side chains bound to a PEG backbone. The hydrodynamic radii of the resulting complexes as measured by fluorescence correlation spectroscopy (FCS) were found to be in good agreement with theoretical predictions using polymer brush scaling theory of a PEG decorated rodlike molecule. It is furthermore demonstrated that the PEG coating of the siRNA-PEG complexes can be rendered biodegradable through the use of a pH-sensitive hydrazone or a reducible disulfide bond linker between the K14 and the PEG blocks. To model transport under in vivo conditions, diffusion of these PEGylated siRNA complexes is studied in various charged and uncharged matrix materials. In PEG solutions, the diffusion coefficient of the siRNA complex is observed to decrease with increasing polymer concentration, in agreement with theory of probe diffusion in semidilute solutions. In charged networks, the behavior is considerably more complex. FCS measurements in fibrin gels indicate complete dissociation of the diblock copolymer from the complex, while transport in collagen solutions results in particle aggregation.

Optimized lipopolyplex formulations for gene transfer to human colon carcinoma cells under in vitro conditions

Polycation (PC, polyplex), cationic lipid (CL, lipoplex), and a combination of PC/CL (lipopolyplex) formulations were investigated for gene transfer to slow‐proliferating human colon carcinoma cell lines (COGA).

Heteronuclear NMR studies of 13C-labeled yeast cell wall β-glucan oligosaccharides

SummaryThe structures of uniformly 13C-labeled β-glucan octa- and undeca-oligosaccharides enzymatically prepared by the yeast cell wall glucanosyl transferase of Candida albicans were characterized by using a combination of HCCH-COSY, HCCH-TOCSY, and HMBC experiments. The oligosaccharide structures indicate that the cell wall glucanosyl transferase cleaves two glucosyl units from the reducing end of the initial linear β(1→3) penta-oligosaccharide and subsequently transfers the remainder to another oligosaccharide at the nonreducing end via a β(1→6) linkage. These results indicate that the combined action of cell wall glucanase and glucanosyl transferase activities could not only introduce intrachain β(1→6) linkages within a single glucan strand, but also result in cross-linking of two initially separate glucan strands with concurrent introduction of intrachain β(1→6) linkages. Since isolated fungal membranes only synthesize linear β(1→3) glucan strands, wall-associated enzymes probably participate in the assembly of the final wall glucan structure during cell growth and division.

Carbomer Inhibits Tryptic Proteolysis of Luteinizing Hormone-Releasing Hormone and N-α-Benzoyl-L-Arginine Ethyl Ester by Binding the Enzyme

AbstractPurpose. To determine the mechanism by which Carbomer inhibits the enzymatic activity of trypsin in hydrolysis of N-α-benzoyl-L-arginine ethyl ester (BAEE) and luteinizing hormone-releasing hormone (LHRH). Methods. Inhibition of enzymatic activity was studied by measuring the formation of metabolites from LHRH and BAEE. Binding of trypsin and substrates to 0.35% (w/v) Carbomer at pH 7.0 was studied by centrifugal filtration. Gel filtration and reverse phase HPLC was used to determine the stability of trypsin. Results. Carbomer reduced the rate of hydrolysis of BAEE and LHRH by trypsin to 34% and 28% of the control activity, respectively. The rate of metabolite formation for both substrates followed pseudo-zero order kinetics in the presence and absence of carbomer. Binding studies showed that 68% of the trypsin protein and 10% of BAEE was bound to carbomer, but no LHRH was bound. No low molecular weight autolysis products of trypsin could be identified by gel filtration. Reverse phase HPLC analysis of the unbound carbomer-treated-trypsin suggests a number of conformational forms of trypsin. The equilibrium binding capacity was 30 μg of trypsin to 1000 μg of carbomer. Conclusions. Decreased hydrolysis of LHRH and BAEE by trypsin in the presence of carbomer is due to enzyme-polymer interaction.

The metabolic barrier of the lower intestinal tract of salmon to the oral delivery of protein and peptide drugs

Oral delivery of peptide and protein drugs has potential advantages for the aquaculture industry. The bioavailability of proteins and peptides from the intestinal tract is very low. This can be attributed in part to the proteolytic activities of the intestine. Bovine serum albumin (BSA), human (hLHRH) and salmon (sLHRH) luteinizing-hormone releasing hormones were used to evaluate the proteolytic activity of anterior, middle and posterior sections of the Quinnat salmon (Oncorhynchus tshawytscha) intestinal tract. The lumenal proteolytic activities of the posterior intestinal section towards BSA were approximately half that of the anterior and middle sections. The half-lives of the LHRH analogues in the posterior were twofold longer than for the anterior and middle sections. Proteolytic activity of the posterior mucosal homogenates towards BSA was fourfold higher than the middle mucosal homogenates. LHRH analogues were hydrolysed by the posterior mucosal homogenate, whereas in the middle mucosal homogenate they were stable. Soybean trypsin inhibitor was shown to be the most effective inhibitor of lumenal proteolytic activity towards LHRH analogues. Sodium deoxycholate, EDTA and bestatin significantly inhibited the posterior mucosal hydrolytic activity towards the LHRH analogues. The posterior intestine of salmon is the most favourable site for the delivery of BSA and LHRH analogues with respect to the lumen, however the higher proteolytic activity of the posterior mucosa has to be overcome.

Functional Analysis of Genomic DNA, cDNA, and Nucleotide Sequence of the Mature C-Type Natriuretic Peptide Gene in Vascular Cells

Objective—The aim of this study was to investigate the effect of various C-type natriuretic peptide (CNP) sequences (genomic DNA [CNPDNA], cDNA derived from mRNA [CNPcDNA], and sequence coding for 22 amino acids of the mature CNP [CNP22aa]) on the growth of primary porcine vascular cells. Methods and Results—Gene transfer was performed with cationic lipid DOCSPER or linear polyethylenimine. All 3 CNP sequences led to significant inhibition of smooth muscle cell (SMC) proliferation. In contrast, significant stimulation of cell growth was observed in endothelial cells (ECs) using CNPDNA or CNPcDNA but not CNP22aa. In a porcine restenosis model, a significant reduction in neointima hyperplasia was found 3 months after application of the CNPcDNA vector compared with the control transfection. Conclusions—The results demonstrate that the first intron in the CNP sequence does not contain any additional enhancer-binding sites. However, the signal sequence is indispensable for secretion of CNP and its appropriate physiological function. Furthermore, the results show for the first time the therapeutic effect of CNP using liposome-mediated gene transfer and local adventitial delivery. Advantages of the CNP gene are its dual effects with inhibition of SMC proliferation and simultaneous promotion of EC growth.

Quantitative capillary electrophoresis assay for the proteolytic stability of luteinizing hormone-releasing hormones.

A rapid and simple capillary electrophoresis (CE) assay for measuring the stability of luteinizing hormone-releasing hormone (LHRH) analogues in the presence of intestinal enzymes has been developed and validated. Buffer pH and sample stacking were important factors in controlling resolution and reproducibility. The CE assay for human (h) and salmon LHRH analogues between 0.05 and 0.25 mM was linear for peak height versus concentration (r2>0.99). Analysis of hLHRH at 0.1 mM had an intra-day relative standard deviation of 1.25% and an inter-day relative standard deviation of 5.0%. The method was applied to the stability of LHRH analogues in salmon intestinal digests.

Activity of pancreatic endopeptidases towards luteinizing hormone-releasing hormones

LHRH and its analogues have low oral bioavailability; this is in part due to their degradation by peptidases present in the intestinal lumen. To determine the appropriate inhibitors to co-administer with LHRH oral formulations, the peptidases involved in their digestion have to be identified. Human (hLHRH) and salmon (sLHRH) LHRH analogues contain a number of potential cleavage sites for the lumenal pancreatic secreted serine endopeptidases: chymotrypsin, trypsin and elastase. The rate of LHRH degradation by equimolar concentrations of chymotrypsin, trypsin and elastase were examined separately in vitro, at pH 8.0, 15 degrees C. At a molar ratio of 1:1000 (enzyme:LHRH), both LHRH analogues were rapidly hydrolysed by alpha-chymotrypsin with half-lives of 2.5+/-0.3 and 2.7+/-0.4 min (mean+/-S.D., n=3), respectively, whereas in the presence of elastase both LHRH analogues were slowly hydrolysed with half-lives of 90+/-15 and 114+/-21 min (mean+/-S.D., n=3), respectively. Trypsin had no activity towards either LHRH analogues after 2 h incubation. The degradation of the LHRH analogues by elastase is likely to be a property of the chymotrypsin impurity. It is concluded that protection of the LHRH analogues from alpha-chymotrypsin is a requirement for the development of oral absorbable product.

Formulation of olfactory-targeted microparticles with tamarind seed polysaccharide to improve nose-to-brain transport of drugs

Targeted delivery and retention of drug formulations in the olfactory mucosa, the target site for nose-to-brain drug absorption is a major challenge due to the geometrical complexity of the nose and nasal clearance. Recent modelling data indicates that 10μm-sized microparticles show maximum deposition in the olfactory mucosa. In the present study we tested the hypothesis that 10μm-sized mucoadhesive microparticles would preferentially deposit on, and increase retention of drug on, the olfactory mucosa in a novel 3D-printed human nasal-replica cast under simulated breathing. The naturally occurring mucoadhesive polymer, tamarind seed polysaccharide (TSP) was used to formulate the microparticles using a spray drying technique. Physicochemical properties of microparticles such as size, morphology and mucoadhesiveness was investigated using a combination of laser diffraction, electron microscopy and texture-analysis. Furthermore, FITC-dextrans (5-40kDa) were incorporated in TSP-microparticles as model drugs. Size-dependent permeability of the FITC-dextrans was observed ex vivo using porcine nasal mucosa. Using the human nasal-replica cast, greater deposition of 10μm TSP-microparticles in the olfactory region was observed compared to TSP-microparticles 2μm in size. Collectively, these findings support our hypothesis that 10μm-sized mucoadhesive microparticles can achieve selective deposition and retention of drug in the olfactory mucosa.