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.

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.

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.

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.

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.

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.

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.

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.

Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

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. Liposomes modified with both apolipoproteins A-I and E were internalized in HepG2 cells in FBS-depleted culture medium at the same levels as unmodified liposomes in FBS-containing culture medium, which indicates that apolipoproteins A-I and E were the major serum components involved in liposomal binding to SR-B1 or LDLR (or both). These results increase our understanding of the disposition of liposomes, processes that can directly affect the efficacy and safety of drug products.

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. The prepared liposomes were composed of phosphatidylcholines and varying contents of cholesterol and/or a polyethylene glycol-modified phospholipid. We demonstrated that the efficiency of penetration into spheroids increased with the bending modulus (i.e., membrane rigidity) of the liposome, as determined by atomic force microscopy (correlation coefficient, 0.84). To clarify the mechanism by which membrane rigidity contributes to the penetration behavior of liposomes, we also analyzed the cellular uptake using monolayer cells. We showed that penetration efficiency was explained partially by cellular uptake efficiency, but that other factors such as liposome diffusion efficiency in the intercellular space of tumor spheroids contributed. Our results quantitatively demonstrate that the bending modulus of the liposomal membrane is a major determinant of liposomal penetration into three-dimensional spheroids. The present study will contribute to the understanding and control of tumor penetration of liposomal formulations.

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.

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

The effect of carrier morphology on the intestinal absorption of insulin was investigated using a morphology-tunable polymeric carrier, ß-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. The network structure in the acetate buffer was maintained even after dilution with a phosphate buffer, but the nanoparticles in the citrate buffer caused aggregation after dilution. Both complexes enhanced the intestinal absorption of insulin. Interestingly, their absorption profiles were totally different; prompt absorption was observed for the complex prepared in acetate buffer, whereas sustained absorption was observed for the complex prepared in citrate buffer. The difference in the absorption patterns was attributed to the difference in the complex morphology. Next, penetratin, a cell-penetrating peptide, was grafted to BCC to find further improvement in the absorption behavior. A simple mixture of penetratin and BCC was also effective. An oral administration study was also conducted in mice to observe effective suppression of glucose levels, which was further enhanced by coadministration of penetratin. Thus, BCC was proven to be an effective carrier for enhancing oral absorption of peptide drugs, and it is suggested that the carrier morphology is also an important factor that influences the absorption profile.

Atomic Force Microscopic Analysis of the Effect of Lipid Composition on Liposome Membrane Rigidity.

Mechanical rigidity of the liposome membrane is often defined by the membrane bending modulus and is one of the determinants of liposome stability, but the quantitative experimental data are still limited to a few kinds of liposomes. Here, we used atomic force microscopy to investigate the membrane bending moduli of liposomes by immobilizing them on bovine serum albumin-coated glass in aqueous medium. The following lipids were used for liposome preparation: egg yolk phosphatidylcholine, dioleoylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, cholesterol, and N-(carbonylmethoxypoly(ethylene glycol) 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine. By using liposomes of various compositions, we showed that the thermodynamic phase state of the membrane rather than the electric potential or liposome surface modification with poly(ethylene glycol) is the predominant determinant of the bending modulus, which decreased in the following order: solid ordered > liquid ordered > liquid disordered. By using the generalized polarization value of the Laurdan fluorescent probe, we investigated membrane rigidity in terms of membrane fluidity. Atomic force microscopic analysis was superior to the Laurdan method, especially in evaluating the membrane rigidity of liposomes containing hydrogenated soybean phosphatidylcholine and cholesterol. Positively charged liposomes with a large bending modulus were taken up by cells more efficiently than those with a small bending modulus. These findings offer a quantitative method of analyzing the membrane rigidity of nanosized liposomes with different lipid compositions and will contribute to the control of liposome stability and cellular uptake efficiency of liposomal formulations intended for clinical use.

Formation of stable nanodiscs by bihelical apolipoprotein A-I mimetic peptide.

Nanodiscs are composed of scaffold protein or peptide such as apolipoprotein A-I (apoA-I) and phospholipids. Although peptide-based nanodiscs have an advantage to modulate the size of nanodiscs by changing phospholipid/peptide ratios, they are usually less stable than apoA-I-based nanodiscs. In this study, we designed a novel nanodisc scaffold peptide (NSP) that has proline-punctuated bihelical amphipathic structure based on apoA-I mimetic peptides. NSP formed α-helical structure on 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) nanodiscs prepared by cholate dialysis method. Dynamic light scattering measurements demonstrated that diameters of NSP nanodiscs vary depending upon POPC/NSP ratios. Comparison of thermal unfolding of nanodiscs monitored by circular dichroism measurements demonstrated that NSP forms much more stable nanodiscs with POPC than monohelical peptide, 4F, exhibiting comparable stability to apoA-I-POPC nanodiscs. Intrinsic Trp fluorescence measurements showed that Trp residues of NSP exhibit more hydrophobic environment than that of 4 F on nanodiscs, suggesting the stronger interaction of NSP with phospholipids. Thus, the bihelical structure of NSP appears to increase the stability of nanodiscs because of the enhanced interaction of peptides with phospholipids. In addition, NSP as well as 4F spontaneously solubilized POPC vesicles into nanodiscs without using detergent. These results indicate that bihelical NSP forms nanodiscs with comparable stability to apoA-I and has an ability to control the size of nanodiscs simply by changing phospholipid/peptide ratios.

Molecular complex composed of ß-cyclodextrin-grafted Chitosan and pH-sensitive amphipathic peptide for enhancing cellular cholesterol efflux under acidic pH.

Excess of cholesterol in peripheral cells is known to lead to atherosclerosis. In this study, a molecular complex composed of ß-cyclodextrin-grafted chitosan (BCC) and cellular cholesterol efflux enhancing peptide (CEEP), synthesized by modifying pH sensitive amphipathic GALA peptide, is introduced with the eventual aim of treating atherosclerosis. BCC has a markedly enhanced ability to induce cholesterol efflux from cell membranes compared to ß-cyclodextrin, and the BCC-CEEP complex exhibited a 2-fold increase in cellular cholesterol efflux compared to BCC alone under weakly acidic conditions. Isothermal titration calorimetry and fluorescence spectroscopy measurements demonstrated that the random coil structure of CEEP at neutral pH converted to the α-helical structure at acidic pH, resulting in a three-order larger binding constant to BCC (K = 3.7 × 10(7) at pH 5.5) compared to that at pH 7.4 (K = 7.9 × 10(4)). Such high-affinity binding of CEEP to BCC at acidic pH leads to the formation of 100-nm-sized aggregate with positive surface charge, which would efficiently interact with cell membranes and induce cholesterol efflux. Since the cholesterol efflux ability of HDL is thought to be impaired under acidic environments in advanced atherosclerotic lesions, the BCC-CEEP complex might serve as a novel nanomaterial for treating atherosclerosis.

[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.

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.