Lipid-based nanomedicines (LBNs), including liposomes and mRNA-lipid nanoparticles, have propelled modern drug delivery research; however, they possess significant challenges regarding structural characterization. Cryogenic transmission electron microscopy (cryo-TEM) preserves LBNs in a near-native, hydrated state, enabling high-resolution imaging of both external and internal features. This review discusses the key principles of cryo-TEM, highlights its advantages in characterizing LBNs, and addresses challenges such as precise sample preparation, beam-induced damage, and complexities in analyzing polydisperse specimens. Nevertheless, ongoing advances in instrumentation and workflow automation continue to expand the accessibility and data quality of cryo-TEM. Standardizing protocols and developing reference materials would further strengthen data reproducibility and facilitate regulatory considerations. By offering unparalleled insight into LBN morphology and functionality, cryo-TEM stands at the forefront of nanomedicine development, guiding both fundamental research and the optimization of next-generation therapeutic carriers.

Mitochondria are essential for cellular functions, including ATP production, calcium homeostasis, oxidative stress regulation, and apoptosis. Mitochondrial dysfunction is associated with a variety of diseases, including neurodegenerative disorders, skeletal muscle diseases, and mitochondrial diseases. This review explores the latest mitochondrial-targeted therapeutic approaches across the following key perspectives: (1) technological innovations in mitochondrial transplantation, focusing on tunnel nanotubes and extracellular vesicles; (2) the role of mitochondria in skeletal muscle diseases and therapeutic activation strategies; (3) advances in mitochondrial enhancement techniques within cell therapy, particularly in pediatric applications; and (4) the latest treatment modalities for mitochondrial diseases, such as gene and cell therapies. Taken together, these strategies demonstrate the transformative potential of mitochondrial targeting in cell- and organelle-specific medicine. Additionally, the MITO-Porter system is highlighted as an innovative drug delivery platform contributing to these advances.

Liposomes have a more complex structure than conventional low-molecular-weight pharmaceuticals, so there is concern that quality evaluation items will be diverse and evaluation methods will be complex. Raman spectroscopy has recently attracted attention as a Process Analytical Technology in the pharmaceutical manufacturing, and its application is expected to expand to biopharmaceuticals and other drugs with complex manufacturing in the future. We have demonstrated that the combination of probe-type Raman spectrometer and partial least squares analysis enables real-time quantification of drug inclusion rate and drug release rate from liposomes, and is useful as a new quality assessment method for liposomes. In this study, we evaluated the phase transition of drug-encapsulated liposomes and the accompanying drug release by using Raman spectroscopy. Drug-encapsulated liposomes were prepared by preparing liposomes with different cholesterol (CHOL) ratios. The phase transition and drug release of liposomes were evaluated by using Raman spectroscopy. Raman spectroscopic measurements showed that the peak intensity of the phase transition was large in systems with low CHOL ratios, while it was low in systems with high CHOL ratios. In the drug release test, a decrease in the peak intensity of the drug-derived spectra over time was observed significantly in the low CHOL ratio system compared to the high CHOL ratio system which is supposed to release drug lower due to liquid-ordered phase, suggesting that the drug release property increased in the low CHOL system. Thus, Raman spectroscopy can be used to evaluate the phase transition and the associated drug release properties of liposomes.

Liposomes are small vesicles composed of lipid bilayers, which have been widely studied and are used in drug delivery systems (DDSs). The lipid bilayers, as two-dimensional liquid crystalline structures, show different phase states, and temperature-dependent phase transitions occur as a result of the thermotropic alteration of the physicochemical properties of the lipid bilayers, resulting in drastic changes in the morphology and dynamics of the fluctuations of the lipid bilayers. Analysis of the thermotropic phase behavior of the liposomal lipid bilayer is crucial for the development and application of functional liposomes for DDSs. We constructed a differential scanning fluorimetry (DSF) method that enabled observation and analysis of the thermotropic phase transitions and temperatures of liposomal lipid bilayers using a real-time PCR device and solvatochromic dyes, which have fluorescence characteristics that reflect alterations in the polar environment. This DSF method using Nile Red and a tandem thermal sequence enabled analysis of the phase transition temperatures of three liposomal phosphatidylcholines, and not only the T_m and T_p, but also the T_sub values, except for the T_p value of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, were clearly determined. Other solvatochromic dyes could not be used to determine the T_sub values clearly. The measured phase transition temperatures of three liposomal phosphocholines correlated well with the reported values. Our DSF method has several practical advantages over the typical thermal analytical method, differential scanning calorimetry, including reduced sample volume and analytical time, which may contribute to expanding the opportunities for the physicochemical analysis of liposomal lipid bilayers.

Multiple sclerosis (MS) develops due to an abnormal T-cell immune response to autoantigens and control of T-cell activation is a mainstream approach for its treatment. In the present study, neogenin, a key molecule for T-cell activation, was used as a targeted molecular gene therapy for MS. Lipid nanoparticles (LNPs) loaded with small interfering RNA (siRNA) targeting neogenin (LNPsiNeo) were prepared, and their therapeutic effect on experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein, a model of MS was evaluated. Neogenin gene expression was reduced by LNPsiNeo in mouse EL-4 cells and splenocytes of LNPsiNeo-treated EAE mouse. Additionally, fluorescence-activated cell sorting (FACS) revealed that the number of CD4⁺ T cells in the splenocytes of EAE mouse decreased after intravenous injection of LNPsiNeo. Furthermore, the progression of encephalomyelitis symptoms was significantly suppressed by LNPsiNeo, whereas the lipid nanoparticle with control siRNA failed to show any effect. The present study suggests that neogenin is a target molecule for EAE gene therapy and LNPsiNeo may be suitable for the MS treatment.