Improved Atomic Force Microscopy Stiffness Measurements of Nanoscale Liposomes by Cantilever Tip Shape Evaluation

Abstract

The stiffness of nanoscale liposomes, as measured by atomic force microscopy (AFM), was investigated as a function of temperature, immobilization on solid substrates, and cantilever tip shape. The liposomes were composed of saturated lipids and cholesterol, and the stiffness values did not change over the temperature range of 25−37 °C and were independent of immobilization methods. However, the stiffness varied with the tip shape of the cantilever. Therefore, 24 cantilevers were evaluated in terms of tip shape and aspect ratio (length/width) via a nonblind tip reconstruction (NBTR) method that used a tip characterizer with isolated line structures having specified dimensions. A standard for screening the tip geometry was established. A 24-fold improvement in stiffness precision in terms of relative standard deviation was demonstrated by using at least three cantilevers that meet the criteria of having a tip aspect ratio greater than 2.5 and a quadratic tip shape function. A significant difference in stiffness was subsequently revealed between dipalmitoylphosphatidylcholine−cholesterol (1:1 molar ratio) and egg yolk phosphatidylcholine−cholesterol (1:1 molar ratio) liposomes. Tip analysis using NBTR improved the precision of AFM stiffness measurements, which will enable the control of mechanical properties of nanoscale liposomes for various applications.

Effect of hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic α-helical peptides

“…AFM at 25 ± 1 °C in 10 mM Tris buffer containing 150 mM NaCl (pH 7.4) using a BioLever mini cantilever (BL-AC40TS, Olympus Co., Tokyo, Japan) via the QI mode of a JPK Nanowizard Ultra Speed microscope equipped with the Data Processing JPK software v.6.0 (JPK Instruments AG, Berlin, Germany) was performed by a slight modification of our previous procedure 54 . This method can directly measure the stiffness of a lipid vesicle immobilized on a solid substrate in an aqueous environment while simultaneously imaging the vesicle.…”

“…Briefly, 200 μL of EPC/EPG/cholesterol-LUVs or DSPC/DSPG-LUVs (50 μM of total lipid) in Tris buffer solution was incubated on an aminopropyl-modified mica substrate for 20 min, and an additional 1.4 mL of Tris buffer solution was added, followed by AFM measurement. We chose these lipid compositions for lipid vesicle immobilization on the substrate, which is the main limitation of this method as discussed previously 34 , 51 , 54 . For the AFM measurement of peptide samples, an additional 1.4 mL of the Tris buffer solution containing peptide was added so that the final peptide/lipid molar ratio was 1.…”

Physicochemical Characterization of Liposomes That Mimic the Lipid Composition of Exosomes for Effective Intracellular Trafficking

“…Sample Preparation for Analysis by Atomic Force Microscopy (AFM). For AFM measurements in an aqueous medium, the liposomes were immobilized intact on mica treated with APTES (APmica) as follows: 23 a mica disk was glued onto a slide glass with silicon adhesive, and then a plastic ring that surrounded the mica disk was glued to the slide glass with biocompatible glue (referred to as "container A"). For AFM measurements at 37 and 50 °C, a mica disk was glued onto a 35 mm sterile polystyrene Petri dish (AGC Techno Glass Co., Ltd., Shizuoka, Japan) with silicon adhesive (referred to as "container B").…”

Robust Nanoparticle Morphology and Size Analysis by Atomic Force Microscopy for Standardization

“…All reagents were of analytical grade. Immobilization on mica treated with 3-aminopropyl triethoxysilane (APTES) was performed as follows 19) : Immediately after mica cleavage, the new surface was incubated with 200 µL of 0.3% (v/v) APTES aqueous solution for 30 min, followed by rinsing with MilliQwater and air drying. The APTES was purchased from Tokyo Chemical Ind.…”

“…[13][14][15] There have been reports of AFM applied to observe nanomedicines. 8,[16][17][18] To use AFM as a technique for characterizing nanomedicines for quality control, robust analytical methods should be established. However, to our knowledge, there have been no such reports, which have comprehensively studied the analytical conditions for measuring the size and images of nanoparticles by the use of AFM, including validation of instrumentdependent repeatability nor any listings in pharmacopoeia.…”