[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. In this context, this review focuses on our research using atomic force microscopy (AFM) to study liposomes and lipid nanoparticles. Our work significantly contributes to the capability of AFM to measure various types of liposomes in an aqueous medium, providing valuable insights into the mechanical properties of these nanoparticles. We discuss the applications of this AFM technique in assessing the quality of nanoparticle-based pharmaceuticals and developing membrane-active peptides.

Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability.

Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide while focusing on structural flexibility. We designed conformationally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose α-helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher α-helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17, whereas StchA2-17 remained on the cell membrane without cell penetration. Although the tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core, StchA2-17 exhibited a higher membrane affinity than A2-17 or StpA2-17. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

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. The obtained AFM images showed spherical and bleb-like structures of mRNA-LNPs, consistent with previous observations made using cryo-transmission electron microscopy. The AFM method also revealed the predominant existence of nanoparticles with a diameter < 60 nm, which were not detectable by dynamic light scattering and nanoparticle tracking analysis. As mRNA-LNPs are usually not monodisperse, but rather polydisperse, the AFM method can provide useful complementary information about mRNA-LNPs in their development and quality assessment.

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. This explains the validity of the formation of doxorubicin sulfate crystals of various shapes, e.g., curved, U-shaped, or circular, in addition to cylinder and/or rod-like crystals reported in the literature. Liposomes that do not contain crystals have inner aqueous phases with high electron density, suggesting that the doxorubicin is remotely loaded and remains as a solute without precipitation.

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. However, the effect of the different dietary soybean oils on cardiovascular disease remains unknown. To test whether diets rich in CSO versus MSO would attenuate atherosclerosis development, LDL receptor knock-out (LDLR-KO) mice were fed a Western diet enriched in saturated fatty acids (control), or a Western diet supplemented with 5% (w/w) LA-rich CSO or high-oleic MSO for 12 weeks. Both soybean oils contained a similar amount of linolenic acid (C18:3 n-3). The CSO diet decreased plasma lipid levels and the cholesterol content of VLDL and LDL by approximately 18% (p < 0.05), likely from increased hepatic levels of PUFA, which favorably regulated genes involved in cholesterol metabolism. The MSO diet, but not the CSO diet, suppressed atherosclerotic plaque size compared to the Western control diet (Control Western diet: 6.5 ± 0.9%; CSO diet: 6.4 ± 0.7%; MSO diet: 4.0 ± 0.5%) (p < 0.05), independent of plasma lipid level changes. The MSO diet also decreased the ratio of n-6/n-3 PUFA in the liver (Control Western diet: 4.5 ± 0.2; CSO diet: 6.1 ± 0.2; MSO diet: 2.9 ± 0.2) (p < 0.05), which correlated with favorable hepatic gene expression changes in lipid metabolism and markers of systemic inflammation. In conclusion, supplementation of the Western diet with MSO, but not CSO, reduced atherosclerosis development in LDLR-KO mice independent of changes in plasma lipids.

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. The circular dichroism spectra showed that the secondary structures were similar between the four congeners even at high temperature. The molar ellipticity of the four congeners in the presence of liposomes, as a cell membrane model, differed, and cyclosporines D and A showed lower molar ellipticity, while cyclosporine C exhibited higher molar ellipticity. Fluorescent spectra analysis using Laurdan indicated that liposome hydration was decreased in the presence of the cyclosporines, especially cyclosporines D and A. HPLC analysis also quantitatively showed that the amount of cyclosporine molecules internalized in HpG2 cells was the largest for cyclosporine D. We determined, using spectroscopy and HPLC, that the intensity of the interaction of the congeners with cell membranes was overall correlated with the lipophilicity derived from the side chains of each congener. Our results will contribute to the design of new macrocyclic peptides with favorable drug properties.

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. The present review also examined the trends in standardization required to validate the size and surface measurements of NPs. It was determined that there is a lack of available reference materials and it is difficult to select appropriate ones for modified NP surface characterization. Research and development are in progress on innovative surface-modified NP-based cancer and gene therapies targeting cells, tissues, and organs. Next-generation nanomedicine should compile studies on the practice and standardization of the measurement methods for NPs to design surface modifications and ensure the quality of NPs.

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

We previously developed an amphipathic arginine-rich peptide, A2-17, which has high ability to directly penetrate across cell membranes. To understand the mechanism of the efficient cell-penetrating ability of the A2-17 peptide, we designed three structural isomers of A2-17 having different values of the hydrophobic moment and compared their membrane interaction and direct cell penetration. Confocal fluorescence microscopy revealed that cell penetration efficiency of peptides tends to increase with their hydrophobic moment, in which A2-17 L14R/R15L, an A2-17 isomer with the highest hydrophobic moment, predominantly remains on plasma cell membranes. Consistently, Trp fluorescence analysis indicated the deepest insertion of A2-17 L14R/R15L into lipid membranes among all A2-17 isomers. Electrophysiological analysis showed that the duration and charge flux of peptide-induced pores in lipid membranes were prominent for A2-17 L14R/R15L, indicating the formation of stable membrane pores. Indeed, the A2-17 L14R/R15L peptide exhibited the strongest membrane damage to CHO-K1 cells. Atomic force microscopy quantitatively defined the peptide-induced membrane perturbation as the decrease in the stiffness of lipid vesicles, which was correlated with the hydrophobic moment of all A2-17 isomers. These results indicate that optimal membrane perturbation by amphipathic A2-17 peptide is critical for its efficient penetration into cells without inducing stabilized membrane pores.

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. Some liposomes were then fabricated to mimic the representative reported lipid composition of exosomes. Their physicochemical properties and cellular internalization efficiencies were investigated to optimize the cellular internalization efficiency of the liposomes. A favorable internalization efficiency was obtained by incubating HeLa cells with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol)/1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) (40/40/20 mol %) liposomes, which have a similar stiffness and zeta potential to exosomes. A dramatic increase in internalization efficiency was demonstrated by adding DOPS to simple DSPC/Chol liposomes. We found that DOPS had a more desirable effect on cellular internalization than its saturated lipid counterpart, 1,2-distearoyl-sn-glycero-3-phospho-l-serine. Furthermore, it was shown that the phosphatidylserine-binding protein, T-cell immunoglobulin mucin protein 4, was largely involved in the intracellular transfer of DSPC/Chol/DOPS liposomes. Thus, DOPS was a key lipid to provide the appropriate stiffness, zeta potential, and membrane surface affinity of the resulting liposome. Our results may help develop efficient drug carriers aiming to internalize active substances into cells.

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. There was no significant difference in purity between each EPC. The stiffness of the liposomes was examined via atomic force microscopy (AFM) in relation to the liposomal membrane permeability coefficient of encapsulated calcein after gel filtration, which is indicative of liposomal stability including the release of a hydrophilic drug from a liposome. Although the size of the liposome and the encapsulation efficiency of calcein did not significantly change with the type of EPC used, the liposome stiffness was found to vary depending on the EPC used, and liposomes with a similar stiffness were found to show a similar membrane permeability to calcein. Our results indicate the usefulness of stiffness measurement, using AFM as the analytical method, to detect material-derived differences in EPC-containing liposomes that affect drug release from the liposomes. Because drug release is one of the most important liposomal functions, combining this method with other analytical methods could be useful in selecting material for the development and quality control of EPC-containing liposomes.

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. To assess the applicability of standardization of AFM, in this study, we aimed to identify robust conditions for assessing the morphology and size of nanoparticles based on a polystyrene nanoparticle certified reference material standard. The spring constant of the cantilever did not affect the size of the nanoparticles but needed to be optimized depending on the measurement conditions. The size analysis method of the obtained images affected the results of the analyzed size values. The results analyzed by cross-sectional line profiling were independent of the measurement conditions and gave similar results to those from dynamic light scattering. It was indicated that approximately 100 particles are required for a representative measurement. Under the optimized conditions, there were no significant inter-instrument differences in the analyzed size values of polystyrene nanoparticles both in air and under aqueous conditions.

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. Tryptophan fluorescence measurements demonstrated that P9R7W binds to negatively charged lipid vesicles with similar affinity to R7W, in which there was no significant change in the PPII helix structure. Flow cytometry and confocal laser scanning microscopy analyses showed that P9R7W has an ability to penetrate into CHO-K1 cells more efficiently compared to R7W with no cytotoxicity. Consistently, a channel current analysis unveiled that P9R7W penetrates planar lipid bilayer membranes more efficiently than R7W without significant membrane perturbation. Our results indicate that the PPII helix structure can enhance the membrane penetration efficiency of arginine-rich peptides without lipid membrane perturbation.

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. We demonstrated that the evaluation of the tip shape for cantilever selection can be applicable to the IC mode as well as the QI mode. With the cantilever selection, the improved precision of the liposome stiffness was obtained when the stiffness of each liposome was determined from the slope in the force-deformation curve by the IC-mode force curve measurement. Further, the stiffness values were found to be similar to that measured by QI-mode measurements. These results indicate that our developed method can be widely used via IC-mode force curve measurements as well as via QI mode. It was also revealed that spatial drift of the cantilever position was instrument-dependent factors which could affect the precision of liposome stiffness measurements in the case of IC-mode force curve measurement. Therefore, in case of stiffness measurement by IC-mode force curve measurement, it is vital to obtain force-deformation curves immediately after imaging a liposome for the precise stiffness measurement of liposomes. These findings will promote the usage of the AFM stiffness measurement method for the characterization of lipid nanoparticle-based drug delivery systems.

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. 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.

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. The use of ethanol instead of 1,4-dioxane as the solvent for isoandrosterone was examined. Both solvents yielded the same maximum value of +3.3 for molar CD. We also studied a two-point calibration method using (1S)-(+)-ammonium 10-camphorsulfonate instead of (1S)-(+)-10-camphorsulfonic acid, which is a hygroscopic compound. Both compounds yielded similar results and the values for (1S)-(+)-ammonium 10-camphorsulfonate of 2.39 ± 0.04 and -4.92 ± 0.06 at 290.5 and 192.5 nm, respectively, were within the criteria defined in the European Pharmacopoeia. The inter-laboratory repeatability was also acceptable. These studies provide specific procedures for calibrating CD spectrometers for drug development.

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. The method also provides information on a wide-sized range of particles, including small particles that can often only be detected by DLS when larger particles are removed by additional filtration steps. An expected advantage of the AFM method is the ability to obtain a detailed information on particle morphology and stiffness, which allows the active pharmaceutical ingredient and excipient (titanium dioxide) particles to be distinguished. Selective imaging of particles can also be achieved by varying the surface properties of the AFM solid substrate, which allows to control the interactions between the substrate and the active pharmaceutical ingredient and excipient particles. AFM analysis in combination with other methods (e.g., DLS), should facilitate the rational development of formulations based on nanoparticles.

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. Circular dichroism and tryptophan fluorescence measurements demonstrated that A2-17 variants bind to lipid vesicles with a structural change from random coil to amphipathic α-helix, being inserted into the hydrophobic membrane interiors. Flow cytometric analysis and confocal laser scanning microscopy demonstrated the great cell penetration efficiency of A2-17 variants into CHO-K1 cells when incubated at low peptide concentrations (2 µM or less), suggesting that the increased amphipathicity with α-helix formation enhances the cell membrane penetration ability of arginine-rich peptides. Interestingly, A2-17KR exhibited lower efficiency of cell membrane penetration compared to A2-17 despite of their similar binding affinity to lipid membranes. Since high peptide concentrations (typically >10 µM) are usually prerequisite for efficient cell penetration of arginine-rich peptides, A2-17 is a unique amphipathic cell-penetrating peptide that exhibits an efficient cell penetration ability even at low peptide concentrations.

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. Our results showed that charged lipids, whether negatively or positively charged, have the effect of reducing the stiffness of nanosized liposomes, independently of the saturation degree of the lipid acyl chains; the measured stiffness values of liposomes containing charged lipids are 30-60% lower than those of their neutral counterpart liposomes. In addition, we demonstrated that the Laurdan generalized polarization values, which are related to the hydration degree of the liposomal membrane interface and often used as a qualitative indicator of liposomal membrane stiffness, do not directly correlate with the physical stiffness values of the liposomes prepared in this study. However, our results indicate that direct quantitative AFM measurement is a valuable method to gain molecular-scale information about how the hydration degree of liposomal interfaces reflects (or does not reflect) liposome stiffness as a macroscopic property. Our AFM method will contribute to the quantitative characterization of the nano-bio interaction of nanoparticles and to the optimization of the lipid composition of liposomes for clinical use.

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. Alternatively, CPPs favorably bind in a charge density- dependent manner to negatively charged, sulfated glycosaminoglycans (GAGs), such as heparan sulfate and chondroitin sulfate, which are abundant on the cell surface and are involved in many biological functions. We have recently demonstrated that the interaction of CPPs with sulfated GAGs plays a critical role in their direct cell membrane penetration: the favorable enthalpy contribution drives the high-affinity binding of arginine-rich CPPs to sulfated GAGs, initiating an efficient cell membrane penetration. The favorable enthalpy gain is presumably mainly derived from a unique property of the guanidino group of arginine residues forming multidentate hydrogen bonding with sulfate and carboxylate groups in GAGs. Such interactions can be accompanied with charge neutralization of arginine-rich CPPs, promoting their partition into cell membranes. This review summarizes the current understanding of the physicochemical mechanism for lipid membrane penetration of CPPs, and discusses the role of the GAG interactions on the cell membrane penetration of CPPs.

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). The lipid compositions of the studied liposomes were similar to those of commercial products. AFM proved to be a viable method for obtaining images of both polystyrene nanoparticles and liposomes in aqueous medium. For the polystyrene nanoparticles, the average height size observed by AFM was similar to the average number-weighted diameter obtained by DLS, indicating the usefulness of AFM for measuring the sizes of nanoparticles in aqueous medium. For the liposomes, the height sizes obtained by AFM differed depending upon the procedures of immobilizing the liposomes onto a solid substrate. In addition, the resultant average height sizes of the liposomes were smaller than those obtained by DLS. This knowledge will help the correct use of AFM as a powerful tool for imaging and size measurement of nanotechnology-based drug products for clinical use.