Key Process and Factors Controlling the Direct Translocation of Cell-Penetrating Peptide through Bio-Membrane

Cell-penetrating peptide (CPP) can directly penetrate the cytosol (cytolysis) and is expected to be a potent vector for a drug delivery system (DDS). Although there is general agreement that CPP cytolysis is related to dynamic membrane deformation, a distinctive process has yet to be established. Here, we report the key process and factors controlling CPP cytolysis. To elucidate the task, we have introduced trypsin digestion of adsorbed CPP onto giant unilamellar vesicle (GUV) to quantify the adsorption and internalization (cytolysis) separately. Also, the time-course analysis was introduced for the geometric calculation of adsorption and internalization amount per lipid molecule consisting of GUV. As a result, we found that adsorption and internalization assumed to occur successively by CPP molecule come into contact with membrane lipid. Adsorption is quick to saturate within 10 min, while cytolysis of each CPP on the membrane follows successively. After adsorption is saturated, cytolysis proceeds further linearly by time with a different rate constant that is dependent on the osmotic pressure. We also found that temperature and lipid composition influence cytolysis by modulating lipid mobility. The electrolyte in the outer media is also affected as a chemical mediator to control CPP cytolysis by following the Hoffmeister effect for membrane hydration. These results confirmed the mechanism of cytolysis as temporal and local phase transfer of membrane lipid from Lα to Mesh1, which has punctured bilayer morphologies

Physicochemical analysis of liposome membranes consisting of model lipids in the stratum corneum

International audienceLamellar lipid layers in the stratum corneum (SC), the outermost layer of the skin, act as a primary permeability barrier to protect the body. The roles of SC lipid composition and membrane structure in skin barrier function have been extensively investigated using ex-vivo SC samples and reconstructed SC lipids in the form of multi-lamellar lipids or liposomes. The primary lipids, especially ceramide, have been found to be highly important. Atopic dermatitis (AD) is a well-known chronic inflammatory skin disease with immunologic and epidermal abnormalities of the permeability barrier; therefore, a comparison of SC lipids in AD skin with those in normal skin is a promising method to explore the mechanisms of skin barrier function. Here, we focused on the effect of sphingoids (ceramide metabolites and a minor component of the SC lipids) and their content/species on skin barrier function. A significant difference in the leakage ratio was observed between model SC lipid liposomes with a different sphingolipid ratio (sphingosine/sphinganine), with a value of 5.43 for normal skin vs. 14.3 for AD skin. This result shows a good concordance with AD mouse experiments. Therefore, an alteration in the composition of minor SC lipids resulting from a ceramide metabolic abnormality can affect the membrane integrity (i.e., skin barrier function). Small angle X-ray scattering (SAXS) measurements revealed no distinct differences in the SAXS pattern between the 3 models, with all models forming a rigid membrane (i.e., a nearly hydrated solid). According to increasing the temperature, the peaks indicated that the lamellar structures decreased in all models and that the lateral packing of lipids decreased, which suggested annealing or melting of the gel to a liquid crystal, although no distinct phase transition was observed through fluorescence anisotropy measurements. Hence, we assume that the altered sphingoid composition triggers local membrane structural changes (i.e., formation of domains or clusters)

Surfactant-like Properties of an Amphiphilic α‑Helical Peptide Leading to Lipid Nanodisc Formation

Nanodiscs are self-assembled discoidal nanoparticles composed of amphiphilic α-helical scaffold proteins or peptides that wrap themselves around the circumference of a lipid bilayer in a beltlike manner. In this study, an amphiphilic helical peptide that mimics helix 10 of human apoA-I was newly synthesized by solid phase peptide synthesis using Fmoc chemistry, and its physicochemical properties, including surface tension, self-association, and solubilization abilities, were evaluated and related directly to nanodisc formation. The synthesized peptide having hydrophobic and hydrophilic faces behaves like a general surfactant, affording a critical association concentration (CAC) of 2.7 × 10^–5 M and a γ_CAC of 51.2 mN m^–1 in aqueous solution. Interestingly, only a peptide solution above its CAC was able to microsolubilize L-α-dimyristoylphosphatidylcholine (DMPC) vesicles, and lipid nanodiscs with an average diameter of 9.5 ± 2.7 nm were observed by dynamic light scattering and negative stain transmission electron microscopy. Moreover, the ζ potentials of the lipid nanodiscs were measured for the first time as a function of pH, and the values changed from positive (20 mV) to negative (−30 mV). In particular, nanodisc solutions at acidic pH 4 (20 mV) or basic pH 9 (−20 mV) were found to be stable for more than 6 months as a result of the electrostatic repulsion between the particles

Effect of Vesicle Size on the Cytolysis of Cell-Penetrating Peptides (CPPs)

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the mechanism of cytolysis as a temporal and local phase transfer of membrane lipid caused by positive membrane curvature generation. Moreover, we showed how to control the CPP cytolysis. Here, we investigate the phospholipid vesicle’s size effect on CPP cytolysis because this is the most straightforward way to control membrane curvature. Contrary to our expectation, we found that the smaller the vesicle diameter (meaning a higher membrane curvature), the more cytolysis was suppressed. Such controversial findings led us to seek the reason for the unexpected results, and we ended up finding out that the mobility of membrane lipids as a liquid crystal is the key to cytolysis. As a result, we could explain the cause of cytolysis suppression by reducing the vesicle size (because of the restriction of lipid mobility); osmotic pressure reduction to enhance positive curvature generation works as long as the membrane is mobile enough to modulate the local structure. Taking all the revealed vital factors and their effects as a tool, we will further explore how to control CPP cytolysis for developing a DDS system combined with appropriate cargo selection to be tagged with CPPs

Enhancement of Cellulose Degradation by Cattle Saliva.

Saccharification of cellulose is a promising technique for producing alternative source of energy. However, the efficiency of conversion of cellulose into soluble sugar using any currently available methodology is too low for industrial application. Many additives, such as surfactants, have been shown to enhance the efficiency of cellulose-to-sugar conversion. In this study, we have examined first whether cattle saliva, as an additive, would enhance the cellulase-catalyzed hydrolysis of cellulose, and subsequently elucidated the mechanism by which cattle saliva enhanced this conversion. Although cattle saliva, by itself, did not degrade cellulose, it enhanced the cellulase-catalyzed degradation of cellulose. Thus, the amount of reducing sugar produced increased approximately 2.9-fold by the addition of cattle saliva. We also found that non-enzymatic proteins, which were present in cattle saliva, were responsible for causing the enhancement effect. Third, the mechanism of cattle saliva mediated enhancement of cellulase activity was probably similar to that of the canonical surfactants. Cattle saliva is available in large amounts easily and cheaply, and it can be used without further purification. Thus, cattle saliva could be a promising additive for efficient saccharification of cellulose on an industrial scale

Removal of protein in cattle saliva.

<p>(a) Protein concentration in cattle saliva treated with methanol or acetone. Protein concentration was measured using Bradford protein assay. (b) Enhancement effect of cattle saliva treated with methanol or acetone. Cattle saliva treated with methanol or acetone used in the cellulose degradation assay. The reaction condition follows the basic experimental protocol. All experiments were performed in triplicate and average mean values were plotted. Error bars indicate ± standard deviations. Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).</p

Addition order assay.

<p>(a) Schematic representation of the experimental design. (b) Effect on the production of reducing sugar. The amount of reducing sugar produced at each addition order experimental condition, shown schematically in (a), was measured. Simultaneous: A mixture in which cellulose, cellulase and cattle saliva were added simultaneously. Added with cellulase: Cellulase was added to a mixture containing cellulose and cattle saliva. Added with cellulose: Cellulose was added to a mixture containing cellulase and cattle saliva. Added with saliva: Cattle saliva was added to a mixture containing cellulose and cellulase. Simultaneous (25 hours): A mixture in which cellulose, cellulase and cattle saliva were added simultaneously and incubated for 25 h. Error bars indicate ± standard deviations (n = 9). Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).</p

Properties of cattle saliva on real biomass degradation.

<p>Effects of (a) cellulase concentration and (b) incubation time on cellulose conversion. (a) Cellulase concentrations were 0, 10, 50, 100 and 250 μg/mL, while concentration of cattle saliva was constantly 10%. The reaction mixtures were incubated at 50°C for 24 h. (b) Different incubation times were tested (0, 12, 24, 48 and 72 h), while concentrations of cellulase and cattle saliva were constantly 50 μg/mL and 10%, respectively. The reaction mixtures were incubated at 50°C. All experiments were performed in triplicate and average mean values were plotted. Error bars indicate ± standard deviations. Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).</p

Effects of cattle saliva on cellulose degradation.

<p>(a) Enhancement effect of cattle saliva. Effect of cattle saliva addition on the production of reducing sugar from micro-crystalline cellulose. Reaction mixtures containing 10 μg/mL cellulase and 0.8% (wt%) cellulose were incubated in the presence or absence of 10% cattle saliva at 50°C for 24 h. Effects of (b) cellulase concentration, (c) incubation time and (d) cattle saliva concentration on reducing sugar production. In (b), concentrations of cellulase used were 0, 1, 5, 10, 50, 100, 500 and 1000 μg/mL, while the concentration of cellulose was kept same as in (a) above and the reaction mixtures were incubated at 50°C for 24 h. In <b>(c),</b> different incubation times were used (0, 1, 3, 6, 12, 24, 48 and 72 h) while keeping the composition of the reaction mixture same as in (a) above. In (d), different concentrations of cattle saliva were used here: 0, 0.5, 1, 2, 3, 4, 7 and 10%; concentrations of cellulase and cellulose and reaction conditions were same as in (a) above. All experiments were performed in triplicate and results are expressed as average means. Error bars indicate ± standard deviations. Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).</p

Effect of various treatments on the enhancement effect of cattle saliva.

<p>(a) Denatured and dialyzed cattle saliva. Denaturation of cattle saliva: Cattle saliva was autoclaved for 13 minites at 121°C to denature proteins. After that, the saliva was centrifuged at 20,400 x <i>g</i> for 10 min. The supernatant (called ‘Autoclaved saliva’) was collected and subsequently used in experiments. Dialysis of cattle saliva: Cattle saliva was dialyzed against distilled water for 72 h at room temperture. The water was exchanged every other day. (b) Proteinase K treatment. Twenty microliters cattle saliva was mixed with 20 μL proteinase K (20 mg/mL) and the mixture was incubated at 50°C for 12 h. After the incubation, the mixture was incubated at 96°C for 10 min to denature proteinase K. This mixture was called ‘Proteinase K Saliva’ and used in the cellulose degradation assay. The concentration of cattle saliva in the reaction mixture was 5%. All experiments were performed in triplicate and average mean values were plotted. Error bars indicate ± standard deviations. Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).</p