[Atomic Force Microscopy to Measure the Mechanical Property of Nanosized Lipid Vesicles and Its Applications].

Nanoparticles, including liposomes and lipid nanoparticles, have garnered global attention due to their potential applications in pharmaceuticals, vaccines, and gene therapies. These particles enable targeted delivery of new drug modalities such as highly active small molecules and nucleic acids. However, for widespread use of nanoparticle-based formulations, it is crucial to comprehensively analyze their characteristics to ensure both efficacy and safety, as well as enable consistent production.

Atomic Force Microscopic Imaging of mRNA-lipid Nanoparticles in Aqueous Medium.

The efficacy of mRNA-lipid nanoparticles (mRNA-LNPs) depends on several factors, including their size and morphology. This study presents a new technique to characterize mRNA-LNPs in an aqueous medium using atomic force microscopy (AFM). This method utilizes an anti-polyethylene glycol antibody to immobilize mRNA-LNPs onto a glass substrate without corruption, which cannot be avoided with conventional procedures using solid substrates such as mica and glass.

Folded, undulating, and fibrous doxorubicin sulfate crystals in liposomes.

High-resolution cryogenic transmission electron microscopy (cryo-TEM) evidenced that doxorubicin sulfate crystals in liposomes (prepared by remote loading with ammonium sulfate) form folded, undulating, and fibrous crystals with a diameter of approximately 2.4 nm. An undulating, fibrous crystal considered to be undergrowth, in addition to bundles of fibrous crystals, was also observed in doxorubicin-loaded liposomes.

Differential Effect of Dietary Supplementation with a Soybean Oil Enriched in Oleic Acid versus Linoleic Acid on Plasma Lipids and Atherosclerosis in LDLR-Deficient Mice.

Both monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) play important roles in lipid metabolism, and diets enriched with either of these two fatty acids are associated with decreased cardiovascular risk. Conventional soybean oil (CSO), a common food ingredient, predominantly contains linoleic acid (LA; C18:2), a n-6 PUFA. Recently, a modified soybean oil (MSO) enriched in oleic acid (C18:1), a n-9 MUFA, has been developed, because of its improved chemical stability to oxidation.

Analysis of the interaction of cyclosporine congeners with cell membrane models.

Owing to the relatively high molecular weight of macrocyclic peptides, investigation of the cellular uptake mechanism is required for the efficient design of macrocyclic peptides as potential drugs. We have previously reported, using HPLC, that cyclosporine A, a model macrocyclic peptide, and its congeners B, C, and D had different lipophilicity despite differing by only one amino acid. In the present study, we investigated how this difference in lipophilicity affected the interaction of the congeners with cell membranes.

Current Status and Challenges of Analytical Methods for Evaluation of Size and Surface Modification of Nanoparticle-Based Drug Formulations.

The present review discusses the current status and difficulties of the analytical methods used to evaluate size and surface modifications of nanoparticle-based pharmaceutical products (NPs) such as liposomal drugs and new SARS-CoV-2 vaccines. We identified the challenges in the development of methods for (1) measurement of a wide range of solid-state NPs, (2) evaluation of the sizes of polydisperse NPs, and (3) measurement of non-spherical NPs. Although a few methods have been established to analyze surface modifications of NPs, the feasibility of their application to NPs is unknown.

Detection of material-derived differences in the stiffness of egg yolk phosphatidylcholine-containing liposomes using atomic force microscopy.

Naturally sourced phospholipids are used in many liposomal pharmaceuticals. The present report describes a method to detect the effects of different egg yolk phosphatidylcholines (EPCs) on liposomal physicochemical properties. Five EPC-containing liposomes were prepared using five different EPCs obtained from different suppliers.

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

Exosomes mediate communication between cells in the body by the incorporation and transfer of biological materials. To design an artificial liposome, which would mimic the lipid composition and physicochemical characteristics of naturally occurring exosomes, we first studied the physicochemical properties of exosomes secreted from HepG2 cells. The exosome stiffness obtained by atomic force microscopy was moderate.

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

Because of the complexity of nanomedicines, analysis of their morphology and size has attracted considerable attention both from researchers and regulatory agencies. The atomic force microscope (AFM) has emerged as a powerful tool because it can provide detailed morphological characteristics of nanoparticles both in the air and in aqueous medium. However, to our knowledge, AFM methods for nanomedicines have yet to be standardized or be listed in any pharmacopeias.

Enhancement of direct membrane penetration of arginine-rich peptides by polyproline II helix structure.

The left-handed, extended polyproline II (PPII) helix is a unique secondary structure which potently modulates peptide/protein functions through its constraint conformation. To investigate the effect of PPII helix on the direct cell membrane penetration of arginine-rich peptides, we designed a polyproline-containing arginine-rich peptide P9R7W (PPPPPPPPPRRRRRRRW) by introducing nine proline residues into a linear R7W (RRRRRRRW) peptide. Circular dichroism spectroscopy showed that P9R7W has the PPII helix structure in solution whereas R7W is predominantly in random coil structure.

Instrument-Dependent Factors Affecting the Precision in the Atomic Force Microscopy Stiffness Measurement of Nanoscale Liposomes.

The mechanical strength (stiffness) of liposomes affects their cellular uptake efficiency and drug release in drug delivery processes. We recently developed a tip shape evaluation method for improving the precision of liposome stiffness measurement by quantitative imaging (QI)-mode atomic force microscopy (AFM). The present study applied our method to the widely-used AFM instruments equipped for intermittent contact (IC)-mode force curve measurements, and examined instrument-dependent factors that affect the liposome stiffness measurements.

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

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.

Refining Calibration Procedures of Circular Dichroism Spectrometer to Improve Usability.

Circular dichroism (CD) is a technique used for conformational studies of peptides and proteins. We studied the specific calibration procedures of CD spectrometers based on procedures specified in the European Pharmacopoeia. We aimed to develop procedures to improve the usability of CD, in addition to reducing adverse effects on users' health.

Detailed Morphological Characterization of Nanocrystalline Active Ingredients in Solid Oral Dosage Forms Using Atomic Force Microscopy.

The characterization of nanocrystalline active ingredients in multicomponent formulations for the design and manufacture of products with increased bioavailability is often challenging. The purpose of this study is to develop an atomic force microscopy (AFM) imaging method for the detailed morphological characterization of nanocrystalline active ingredients in multicomponent oral formulations. The AFM images of aprepitant and sirolimus nanoparticles in aqueous suspension show that their sizes are comparable with those measured using dynamic light scattering (DLS) analysis.

A novel amphipathic cell-penetrating peptide based on the N-terminal glycosaminoglycan binding region of human apolipoprotein E.

In the direct cell membrane penetration, arginine-rich cell-penetrating peptides are thought to penetrate into cells across the hydrophobic lipid membranes. To investigate the effect of the amphipathic property of arginine-rich peptide on the cell-penetrating ability, we designed a novel amphipathic cell-penetrating peptide, A2-17, and its derivative, A2-17KR, in which all lysine residues are substituted with arginine residues, based on the glycosaminoglycan binding region in the N-terminal α-helix bundle of human apolipoprotein E. Isothermal titration calorimetry showed that A2-17 variants have a strong ability to bind to heparin with high affinity.

Atomic Force Microscopy Study on the Stiffness of Nanosized Liposomes Containing Charged Lipids.

It has recently been recognized that the mechanical properties of lipid nanoparticles play an important role during in vitro and in vivo behaviors such as cellular uptake, blood circulation, and biodistribution. However, there have been no quantitative investigations of the effect of commonly used charged lipids on the stiffness of nanosized liposomes. In this study, by means of atomic force microscopy (AFM), we quantified the stiffness of nanosized liposomes composed of neutrally charged lipids combined with positively or negatively charged lipids while simultaneously imaging the liposomes in aqueous medium.

Current Understanding of Physicochemical Mechanisms for Cell Membrane Penetration of Arginine-rich Cell Penetrating Peptides: Role of Glycosaminoglycan Interactions.

Arginine-rich cell penetrating peptides (CPPs) are very promising drug carriers to deliver membrane-impermeable pharmaceuticals, such as siRNA, bioactive peptides and proteins. CPPs directly penetrate into cells across cell membranes via a spontaneous energy-independent process, in which CPPs appear to interact with acidic lipids in the outer leaflet of the cell membrane. However, acidic lipids represent only 10 to 20% of the total membrane lipid content and in mammalian cell membranes they are predominantly located in the inner leaflet.

Imaging and size measurement of nanoparticles in aqueous medium by use of atomic force microscopy.

Size control of nanoparticles in nanotechnology-based drug products is crucial for their successful development, since the in vivo pharmacokinetics of nanoparticles are size-dependent. In this study, we evaluated the use of atomic force microscopy (AFM) for imaging and size measurement of nanoparticles in aqueous medium. The height sizes of rigid polystyrene nanoparticles and soft liposomes were measured by AFM and were compared with the hydrodynamic sizes measured by dynamic light scattering (DLS).

Involvement of scavenger receptor class B type 1 and low-density lipoprotein receptor in the internalization of liposomes into HepG2 cells.

In this study, HepG2 cells, an in vitro model system for human hepatocytes, were used to evaluate the interaction of lipoprotein receptors with liposomes carrying fluorescently labeled cholesterol and their subsequent intracellular uptake. In these experiments, two lipoprotein receptors, scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR), accounted for approximately 20% and 10%, respectively, of the intracellular uptake of the labeled liposomes. These findings indicate that additional mechanisms contributed to liposomal internalization.

Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.

Glycosaminoglycans (GAGs), which are covalently-linked membrane proteins at the cell surface have recently been suggested to involve in not only endocytic cellular uptake but also non-endocytic direct cell membrane translocation of arginine-rich cell-penetrating peptides (CPPs). However, in-situ comprehensive observation and the quantitative analysis of the direct membrane translocation processes are challenging, and the mechanism therefore remains still unresolved. In this work, real-time in-cell NMR spectroscopy was applied to investigate the direct membrane translocation of octaarginine (R8) into living cells.

Control of Liposomal Penetration into Three-Dimensional Multicellular Tumor Spheroids by Modulating Liposomal Membrane Rigidity.

Effective penetration of drug-carrying nanoparticles into solid tumors is a major challenge in cancer therapy. Exploration of the physicochemical properties of nanoparticles that affect penetration efficiency is required to achieve maximum therapeutic effects. Here, we used confocal laser scanning microscopy to evaluate the efficiencies of penetration of fluorescently labeled liposomes into three-dimensional spheroids composed of HeLa cells.

Dependence of Intestinal Absorption Profile of Insulin on Carrier Morphology Composed of β-Cyclodextrin-Grafted Chitosan.

The effect of carrier morphology on the intestinal absorption of insulin was investigated using a morphology-tunable polymeric carrier, β-cyclodextrin-grafted chitosan (BCC). The insulin-BCC complexes were prepared in either acetate or citrate buffer solutions, followed by dilution with phosphate buffer for the administration. The complex had a molecular network structure in the acetate buffer, whereas nanoparticles formed in the citrate buffer.

Membrane Rigidity Determined by Atomic Force Microscopy Is a Parameter of the Permeability of Liposomal Membranes to the Hydrophilic Compound Calcein.

We determined the permeability coefficient of a model hydrophilic drug, calcein, encapsulated within saturated lipid-based nano-sized liposomes of various lipid profiles. We demonstrated that the addition of cholesterol to liposomes containing saturated lipids increased the permeability of the liposomal membrane to calcein via a decrease in the membrane bending modulus, as determined by means of atomic force microscopy. We found an inverse correlation between the membrane bending modulus of saturated lipid-based nano-sized liposomes and the permeability coefficient of encapsulated calcein, demonstrating that bending modulus, as determined by means of atomic force microscopy, is a quantitative parameter describing the permeability of liposomal membranes to calcein.