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Restoration of Membrane Environments for Membrane Proteins for Structural and Functional Studies
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
The WT HCN2 and the human BK proteins were expressed and purified as previously described [19,25]. Briefly, cells expressing FLAG-tagged HCN2 and BK proteins were broken by sonication for 1 minute; and sonication was repeated twice. Cell debris and nuclei were removed by spinning down at 1,000 g for 15 minutes at 4°C. Supernatants were spun down at 40,000 g for 1 hour at 4°C. Subsequently, pellets were mixed with the extraction buffer (16 mM n-Dodecyl β-D-maltoside (DDM), 200 mM KCl, 50 mM Tris-HCl, pH7.4, 5 mM EDTA, 1x Protease Inhibitor Cocktail (P8340, Sigma)), and rotated for 2 hours at 4°C. Then detergent-resistant membranes were spun down at 17,000 g for 40 minutes at 4°C. Supernatants were collected and mixed with FLAG-beads (anti-FLAG M2 affinity gel A2220, Sigma) and rotated at 4°C for 2 hours for binding. Proteins were eluted with 0.5 mg/mL FLAG peptide (F3290, Sigma) and concentrated.
Crystallizing Membrane Proteins: Experiments on Different Systems
Published in Hartmut Michel, Crystallization of Membrane Proteins, 1991
The obvious solutions to the Tween-20 problem would be to use either a more effective detergent exchange procedure (e.g., affinity chromatography) or another more easily exchangeable detergent. Because of technical difficulties with the former solution, we explored the use of decyl maltoside (C10-M) as the solubilization and purification detergent. C10-M solubilizes PGS well and retains the enzyme’s activity at all concentrations above the CMC. Furthermore, the CMC is high enough (see Table 1) to allow facile exchange with most of the other detergents, yet low enough to minimize the cost of its use. PGS purified in C10-M had a minute lipid content and crystallized readily as large, orthorhombic prisms (lower panel in Figure 5; see Reference 44). With a preparation free of unwanted detergent we have examined the crystallization behavior of PGS in the presence of other detergents and detergent mixtures. PGS is much less sensitive to the detergent environment than OmpF porin, though detergent effects can be seen, primarily in nucleation rates, crystal size and crystal habit.
Fundamentals in nasal drug delivery
Published in Anthony J. Hickey, Heidi M. Mansour, Inhalation Aerosols, 2019
Zachary Warnken, Yu Jin Kim, Heidi M. Mansour, Robert O. Williams, Hugh D.C. Smyth
The cells of the nasal epithelium are connected by tight junctions, creating a limiting barrier for drug absorption across the membrane. Drug absorption across the nasal epithelium favors small lipophilic molecules that can passively diffuse across the membrane for uptake into the blood and lymphatic system. Larger, more hydrophilic molecules such as proteins and peptides are limited in their absorption by their poor permeability across the nasal epithelium. The use of absorption enhancers is a frequent option to improve the nasal absorption of polar small molecules and macromolecules (99). Several compounds have been evaluated as absorption enhancers for the nasal route, including surfactants such as laureth-9, bile salts and derivatives such as sodium glycocholate and sodium tauro-24,25-dihydrofusidate, phospholipids such as didecanoyl-L-α-phosphatidylcholine, cyclodextrins, and cationic polymers such as chitosan (82). Absorption enhancers have been commercially developed by companies for nasal drug delivery (100,101). For example, CriticalSorb™ (Critical Pharmaceuticals Ltd.) is hydroxy fatty acid ester of polyethylene glycol that promotes the nasal absorption of hydrophilic small molecules, proteins, and peptides across mucosal epithelial cells (101). Preclinical studies have demonstrated the effectiveness of CriticalSorb in the nasal absorption of insulin and human growth hormone (101). It is used in marketed oral and intravenous products, and it is available in liquid and powder drug formulations. ChiSys® (Archimedes Pharma Ltd.) is a chitosan-based absorption enhancer that was developed for the nasal delivery of various drugs. Chitosan is bioadhesive due to its positive charge that extends drug residence time in the nasal cavity (101). There are studies demonstrating that chitosan transiently opens tight junctions in a mucosal membrane with its bioadhesive property, which enhances the nasal absorption of drugs (100–102). Intravail® (Aegis Therapeutics Inc.), a class of alkylsaccharides, is a nontoxic and nonirritating agent that can be employed as a nasal delivery system for small polar molecules, proteins, and peptides (100,101). Tetradecyl maltoside (TDM) is a class of alkylsaccharides and its effectiveness has been evaluated in a preclinical model. Previous studies have shown that TDM improved the nasal absorption of insulin and leptin, and increased their bioavailability (101,103). However, it should be noted that absorption enhancers may cause irritation and possibly irreversible damage of nasal tissues (1,101). Hence, it may need to be cautiously applied, and it especially may not be suitable for multiple dosing and/or long-term treatments. Formulation development is another critical approach to improve the nasal delivery of proteins and peptides. Different formulations, including microparticles, nanoparticles, and liposomes, have been developed and evaluated in a preclinical stage for their utilization in a nasal drug delivery system (104–113).
Transient Permeation Enhancer® (TPE®) technology for oral delivery of octreotide: a technological evaluation
Published in Expert Opinion on Drug Delivery, 2021
Other attempts to deliver octreotide orally involve the use of other PEs. Maggio and Grasso [71] achieved 4% oral bioavailability in mice using 0.5% w/v tetradecyl maltoside (Intravail®, Neurelis Inc., CA, USA) in a mixture. However, oral BA was only 0.7% and 0.5% when 1.5 to 3% w/v concentrations of tetradecyl maltoside were tested. In extrapolating from rodents to large animals, a substantial reduction in oral bioavailability can be expected, and perhaps the murine data were not enough to encourage such studies in the 10 years since. Older studies tested super-porous hydrogel and composite polymers in combination with the PE, trimethylated chitosan, to achieve an impressive 16% oral bioavailability for octreotide in pigs [72]. Again, this concept does not appear to have been advanced in the 20 years since, perhaps illustrating the difficulties in reducing promising technology concepts to highly-loaded oral solid dosage forms that can be synthesized and scaled. Table 2 summarizes a selection of alternative approaches to orally deliver octreotide.
Overview of intranasally delivered peptides: key considerations for pharmaceutical development
Published in Expert Opinion on Drug Delivery, 2018
Wisam Al Bakri, Maureen D. Donovan, Maria Cueto, Yunhui Wu, Chinedu Orekie, Zhen Yang
Dodecyl maltoside is a nonionic polysaccharide-containing surfactant. Dodecyl maltoside has been used to solubilize membrane-associated proteins [89,90] and has been used as an absorption enhancer to increase the bioavailability of large MW peptides and proteins delivered across the nasal mucosa. Intravail™ was reported to be effective in increasing the transmucosal permeability of peptides and proteins with MWs up to 20,000 Da [91]. Increases in transmucosal permeability are attributed to the ability of Intravail™ to open the tight junctions as evidenced by the reduction in the TEER measured across intestinal epithelial cells. This reduction was associated with an increase in insulin permeability across the rat intestinal membrane [92]. Dodecyl maltoside has also been shown to produce a drastic decrease in the TEER values of EpiAirway®, a human tracheal/bronchial cell culture model [93].