Gelation under stress: impact of shear flow on the formation and mechanical properties of methylcellulose hydrogels.

We demonstrate that small unidirectional applied-stresses during temperature-induced gelation dramatically change the gel temperature and the resulting mechanical properties and structure of aqueous methylcellulose (MC), a material that forms a brittle gel with a fibrillar microstructure at elevated temperatures. Applied stress makes gelation more difficult, evidenced by an increased gelation temperature, and weakens mechanical properties of the hot gel, evidenced by a decreased elastic modulus and decreased apparent failure stress. In extreme cases, formation of a fully percolated polymer network is inhibited and a soft granular yield-stress fluid is formed. We quantify the effects of the applied stress using a filament-based mechanical model to relate the measured properties to the structural features of the fibril network. The dramatic changes in the gel temperature and hot gel properties give more design freedom to processing-dependent rheology, but could be detrimental to coating applications where gravitational stress during gelation is unavoidable.

High throughput research and evaporation rate modeling for solvent screening for ethylcellulose barrier membranes in pharmaceutical applications.

CONTEXT: Ethylcellulose is commonly dissolved in a solvent or formed into an aqueous dispersion and sprayed onto various dosage forms to form a barrier membrane to provide controlled release in pharmaceutical formulations. Due to the variety of solvents utilized in the pharmaceutical industry and the importance solvent can play on film formation and film strength it is critical to understand how solvent can influence these parameters. OBJECTIVE: To systematically study a variety of solvent blends and how these solvent blends influence ethylcellulose film formation, physical and mechanical film properties and solution properties such as clarity and viscosity. MATERIALS AND METHODS: Using high throughput capabilities and evaporation rate modeling, thirty-one different solvent blends composed of ethanol, isopropanol, acetone, methanol, and/or water were formulated, analyzed for viscosity and clarity, and narrowed down to four solvent blends. Brookfield viscosity, film casting, mechanical film testing and water permeation were also completed. RESULTS AND DISCUSSION: High throughput analysis identified isopropanol/water, ethanol, ethanol/water and methanol/acetone/water as solvent blends with unique clarity and viscosity values. Evaporation rate modeling further rank ordered these candidates from excellent to poor interaction with ethylcellulose. Isopropanol/water was identified as the most suitable solvent blend for ethylcellulose due to azeotrope formation during evaporation, which resulted in a solvent-rich phase allowing the ethylcellulose polymer chains to remain maximally extended during film formation. Consequently, the highest clarity and most ductile films were formed. CONCLUSION: Employing high throughput capabilities paired with evaporation rate modeling allowed strong predictions between solvent interaction with ethylcellulose and mechanical film properties.

Feasibility Investigation of Cellulose Polymers for Mucoadhesive Nasal Drug Delivery Applications.

The feasibility of various cellulose polymer derivatives, including methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), sodium-carboxymethylcellulose (sodium-CMC), and cationic-hydroxyethylcellulose (cationic-HEC), for use as an excipient to enhance drug delivery in nasal spray formulations was investigated. Three main parameters for evaluating the polymers in nasal drug delivery applications include rheology, ciliary beat frequency (CBF), and permeation across nasal tissue. Reversible thermally induced viscosity enhancement was observed at near nasal physiological temperature when cellulose derivatives were combined with an additional excipient, poly(vinyl caprolactam)-poly(vinyl acetate)-poly(ethylene glycol) graft copolymer (PVCL-PVA-PEG). Cationic-HEC was shown to enhance acyclovir permeation across the nasal mucosa. None of the tested cellulosic polymers caused any adverse effects on porcine nasal tissues and cells, as assessed by alterations in CBF. Upon an increase in polymer concentration, a reduction in CBF was observed when ciliated cells were immersed in the polymer solution, and this decrease returned to baseline when the polymer was removed. While each cellulose derivative exhibited unique advantages for nasal drug delivery applications, none stood out on their own to improve more than one of the performance characteristics examined. Hence, these data may be useful for the development of new cellulose derivatives in nasal drug formulations.

Identification of FAK substrate peptides via colorimetric screening of a one-bead one-peptide combinatorial library.

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is associated with regulating cellular functions such as cell adhesion and migration and has emerged as an important target for cancer research. Short peptide substrates that are selectively and efficiently phosphorylated by FAK have not been previously identified and tested. Here we report the synthesis and screening of a one-bead one-peptide combinatorial library to identify novel substrates for FAK. Using a solid-phase colorimetric antibody tagging detection platform, the peptide beads phosphorylated by FAK were sequenced via Edman degradation and then validated through radioisotope kinetic studies with [γ-(32)P] ATP to derive Michaelis-Menton constants. The combination of results gathered from both colorimetric and radioisotope kinase assays led to the rational design of a second generation of FAK peptide substrates. Out of all the potential peptide substrates evaluated, the most active was GDYVEFKKK with a K(M) = 92 µM and a Vmax = 1920 nmol/min/mg. Peptide substrates discovered within this study may be useful diagnostic tools for future kinase investigations and may lead to novel therapeutic agents.

Effect of formulation conditions on hypromellose performance properties in films used for capsules and tablet coatings.

This study investigated the effects of polymer dispersion and hydration conditions on hypromellose (HPMC) film properties, such as strength, oxygen permeability, water vapor transmission, clarity, and haze. The focus of the study was to build a better understanding of the impact that changes to HPMC dispersion and hydration conditions have on performance properties of the resulting films. This understanding could potentially lead to more flexible formulation guidelines for formulators. Films of HPMC 2906 (USP) were produced from aqueous solutions prepared using various formulation conditions. Results showed that tensile properties and oxygen permeability were not significantly affected by the variables used. The differences observed in water vapor transmission are unlikely to affect practical application of the material. However, the differences observed in clarity and haze at 50°C hydration temperature could affect the appearance of a capsule or coated tablet. Several methods were used to determine whether loss of optical properties was due to surface phenomena or bulk defects within a film. Results indicated that the cloudy appearance was primarily due to surface roughness. Based on this information, there is some flexibility in formulation conditions; however, hydration temperatures greater than 25°C are not recommended.

Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or ß-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41.

Native conformation at specific residues in recombinant inclusion body protein in whole cells determined with solid-state NMR spectroscopy.

Inclusion bodies are insoluble aggregates that are formed by bacteria to store excess recombinant protein produced during expression. The structure of the protein in inclusion bodies is poorly understood but it has been hypothesized that the protein may form misfolded beta sheet aggregates. This paper presents an isotopic labeling and solid-state nuclear magnetic resonance approach to determine the secondary structure of individual residues within a recombinant influenza virus "FHA2" protein in inclusion bodies. The inclusion bodies were studied either in the context of the unlysed hydrated E. coli cells or in the hydrated pellet formed from centrifugation of the material insoluble in the cell lysate. The native structure of FHA2 is predominantly helical and native helical structure was also observed for several specific residues in the inclusion body FHA2. This approach will be applicable to structural analysis of many inclusion body proteins and should provide useful information for optimizing solubilization and purification protocols of these proteins.

Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein.

This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of Escherichia coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A(600) approximately 8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the (13)CO chemical shifts measured using solid-state nuclear magnetic resonance spectroscopy.