Direct entry of cell-penetrating peptide can be controlled by maneuvering the membrane curvature.

A biomembrane's role is to be a barrier for interior cytosol from an exterior environment to execute the cell's normal biological functions. However, a water-soluble peptide called cell-penetrating peptide (CPP) has been known for its ability to directly penetrate through the biomembranes into cells (cytolysis) without perturbating cell viability and expected to be a promising drug delivery vector. Examples of CPP include peptides with multiple arginine units with strong cationic properties, which is the key to cytolysis. Here we show the conclusive evidence to support the mechanism of CPP's cytolysis and way to control it. The mechanism we proposed is attributed to biomembrane's physicochemical nature as lamellar liquid crystal (Lα). Cytolysis occurs as the temporal and local dynamic phase transitions from Lα to an undulated lamellar with pores called Mesh1. We have shown this phase transfer of Lα composed of dioleoyl-phosphatidylcholine (DOPC) with water by adding oligo-arginine (Rx) as CPP at the equilibrium. Using giant unilamellar vesicle composed of DOPC as a single cell model, we could control the level of cytolysis of CPP (FITC-R8) by changing the curvature of the membrane through osmotic pressure modulation. The cytolysis of CPP utilizes biomembrane's inherent topological and functional flexibility corresponding to the stimuli.

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.

Possibility of monitoring granulation by analyzing the amount of hydroxypropylcellulose, a binder on the surface of granules, using ToF-SIMS.

The purpose of this study was to clarify the mechanism responsible for high-shear wet granulation using time-of-flight secondary ion mass spectrometry (ToF-SIMS), which can be used for surface chemical mapping. A total of 15 kinds of granules, including hydroxypropylcellulose (HPC) as a binder, were obtained in a model formulation using different granulation conditions, such as the amount of sprayed water and the granulation time. Surface chemical mapping of these granules was then performed using a ToF-SIMS analysis, which distinguishes each component by detecting the specific mass-to-charge ratio (m/z). As a result, we found that HPC got to appear on the surface of granule with proceeding wet granulation. By considering this result, we concluded that the distributions of HPC might be closely related to the progress of granule consolidation and growth in wet granulation. Therefore, the progress of granulation can likely be understood by measuring the content of HPC on the granule surface.