Lipid Bilayer Structure Research Articles

Engineered and Mimicked Extracellular Nanovesicles for Therapeutic Delivery.

Exosomes are spherical extracellular nanovesicles with an endosomal origin and unilamellar lipid-bilayer structure with sizes ranging from 30 to 100 nm. They contain a large range of proteins, lipids, and nucleic acid species, depending on the state and origin of the extracellular vesicle (EV)-secreting cell. EVs' function is to encapsulate part of the EV-producing cell content, to transport it through biological fluids to a targeted recipient, and to deliver their cargos specifically within the aimed recipient cells. Therefore, exosomes are considered to be potential biological drug-delivery systems that can stably deliver their cargo into targeted cells. Various cell-derived exosomes are produced for medical issues, but their use for therapeutic purposes still faces several problems. Some of these difficulties can be avoided by resorting to hemisynthetic approaches. We highlight here the uses of alternative exosome-mimes involving cell-membrane coatings on artificial nanocarriers or the hybridization between exosomes and liposomes. We also detail the drug-loading strategies deployed to make them drug-carrier systems and summarize the ongoing clinical trials involving exosomes or exosome-like structures. Finally, we summarize the open questions before considering exosome-like disposals for confident therapeutic delivery.

Eugenol-Loaded Transethosomal Gel for Improved Skin Delivery and Treatment of Atopic Dermatitis.

Atopic dermatitis is a skin condition characterized by lichenification (thickening and increased skin marking), eczematous lesions, dry skin, itching, and pruritus. Eugenol is an aromatic polyphenolic compound that has attracted the attention of researchers due to its anti-inflammatory, anti-oxidant, and anti-cancer properties. The primary goal of the present study was to develop and evaluate eugenol-loaded transethosomes for the treatment of AD. Eugenol-loaded transethosomes were formulated using the ethanol injection method and subsequently subjected to particle size analysis, zeta potential, entrapment efficiency, deformability index, and HRTEM analysis. Transethosomal gel was prepared by direct-dispersion method by using Carbopol 940®. Results showed transethosomes to be lipid bilayer structures with acceptable size, and high entrapment efficiency. Transethosomal formulation showed shear-thinning behavior. Eugenol-loaded transethosomal gel was significantly able to enhance the retention of the drug in the skin. Transethosomal gel was significantly able to reduce Ear thickness, DLC, TLC, and IL-6 levels in mice model of AD. These results indicate that the eugenol-loaded transethosomal gel could be a promising carrier for the topical administration of eugenol for the treatment of AD.

Ganglioside GM1 Drives Hemin and Protoporphyrin Adsorption in Phospholipid Membranes: A Structural Study.

Monosialoganglioside (GM1), a ubiquitous component of lipid rafts, and hemin, an integral part of heme proteins such as hemoglobin, are essential to the cell membranes of brain neurons and erythrocyte red blood cells for regulating cellular communication and oxygen transport. Protoporphyrin IX (PPIX) and its derivative hemin, on the contrary, show significant cytotoxic effects when in excess causing hematological diseases, such as thalassemia, anemia, malaria, and neurodegeneration. However, the in-depth molecular etiology of their interactions with the cell membrane has so far been poorly understood. Herein, the structure of the polymer cushion-supported lipid bilayer (SLB) of the binary mixture of phospholipid and GM1 in the presence of PPIX and its derivative hemin has been investigated to predict the molecular interactions in model phospholipid membranes. A high-resolution synchrotron-based X-ray scattering technique has been employed to explore the out-of-plane structure of the assembly at different compositions and concentrations. The structural changes have been complemented with the isobaric changes in the mean molecular area obtained from the Langmuir monolayer isotherm to predict the additive-induced membrane condensation and fluidization. PPIX-induced fluidization of phospholipid SLB without GM1 was witnessed, which was reversed to condensation with 2-fold higher structural changes in the presence of GM1. A hemin concentration-dependent linear condensing effect was observed in the pristine SLB. The effect was significantly reduced, and the linearity was observed to be lost in the mixed SLB containing GM1. Our study shows that GM1 alters the interaction of hemin and PPIX with the membrane, which could be explained with the aid of hydrophobic and electrostatic interactions. Our study indicates favorable and unfavorable interactions of GM1 with PPIX and hemin, respectively, in the membrane. The observed structural changes in both SLB and the underlying polymer cushion layer lead to the proposal of a molecule-specific interaction model that can benefit the pharmaceutical industries specialized for drug designing. Our study potentially enriches our fundamental biophysical understanding of neurodegenerative diseases and drug-membrane interactions.

Exosomes in Renal Cell Cancer: Diagnostic and Therapeutic Nanovehicles.

Early diagnosis is crucial for enhancing the survival rate of renal cell cancer patients, and exosomes present potential advantages in this area. Their small size, high mobility, and lipid bilayer structure enable exosomes to cross biological membranes easily, protecting the bioactive cargo within from degradation. Exosomes significantly influence the invasion and metastasis of RCC, and they also contribute to tumor drug resistance and immune evasion.

Harnessing genetically engineered cell membrane-derived vesicles as biotherapeutics

Cell membrane-derived vesicles (CMVs) are particles generated from living cells, including extracellular vesicles (EVs) and artificial extracellular vesicles (aEVs) prepared from cell membranes. CMVs possess considerable potential in drug delivery, regenerative medicine, immunomodulation, disease diagnosis, etc . owing to their stable lipid bilayer structure, favorable biocompatibility, and low toxicity. Although the majority of CMVs inherit certain attributes from the original cells, it is still difficult to execute distinct therapeutic functions, such as organ targeting, signal regulation, and exogenous biotherapeutic supplementation. Hence, engineering CMVs by genetic engineering, chemical modification, and hybridization is a promising way to endow CMVs with specific functions and open up novel vistas for applications. In particular, there is a growing interest in genetically engineered CMVs harnessed to exhibit biotherapeutics. Herein, we outline the preparation strategies and their characteristics for purifying CMVs. Additionally, we review the advances of genetically engineered CMVs utilized to target organs, regulate signal transduction, and deliver biomacromolecules and chemical drugs. Furthermore, we also summarize the emerging therapeutic applications of genetically engineered CMVs in addressing tumors, diabetes, systemic lupus erythematosus, and cardiovascular diseases.

Engineered bacterial outer membrane vesicles: a versatile bacteria-based weapon against gastrointestinal tumors.

Outer membrane vesicles (OMVs) are nanoscale lipid bilayer structures released by gram-negative bacteria. They share membrane composition and properties with their originating cells, making them adept at traversing cellular barriers. These OMVs have demonstrated exceptional membrane stability, immunogenicity, safety, penetration, and tumor-targeting properties, which have been leveraged in developing vaccines and drug delivery systems. Recent research efforts have focused on engineering OMVs to increase production yield, reduce cytotoxicity, and improve the safety and efficacy of treatment. Notably, gastrointestinal (GI) tumors have proven resistant to several traditional oncological treatment strategies, including chemotherapy, radiotherapy, and targeted therapy. Although immune checkpoint inhibitors have demonstrated efficacy in some patients, their usage as monotherapy remains limited by tumor heterogeneity and individual variability. The immunogenic and modifiable nature of OMVs makes them an ideal design platform for the individualized treatment of GI tumors. OMV-based therapy enables combination therapy and optimization of anti-tumor effects. This review comprehensively summarizes recent advances in OMV engineering for GI tumor therapy and discusses the challenges in the clinical translation of emerging OMV-based anti-tumor therapies.

Tumor-derived microvesicles for cancer therapy.

Extracellular vesicles (EVs) are vesicles with lipid bilayer structures shed from the plasma membrane of cells. Microvesicles (MVs) are a subset of EVs containing proteins, lipids, nucleic acids, and other metabolites. MVs can be produced under specific cell stimulation conditions and isolated by modern separation technology. Due to their tumor homing and large volume, tumor cell-derived microvesicles (TMVs) have attracted interest recently and become excellent delivery carriers for therapeutic vaccines, imaging agents or antitumor drugs. However, preparing sufficient and high-purity TMVs and conducting clinical transformation has become a challenge in this field. In this review, the recent research achievements in the generation, isolation, characterization, modification, and application of TMVs in cancer therapy are reviewed, and the challenges facing therapeutic applications are also highlighted.

Liver Extracellular Vesicles and Particles Enriched β-Sitosterol Effectively Promote Liver Regeneration in Mice.

The liver's regenerative capacity allows it to repair itself after injury. Extracellular vesicles and particles (EVPs) in the liver's interstitial space are crucial for signal transduction, metabolism, and immune regulation. Understanding the role and mechanism of liver-derived EVPs in regeneration is significant, particularly after partial hepatectomy, where the mechanisms remain unclear. A 70% hepatectomy model was established in mice, and EVPs were isolated and characterized using electron microscopy, nanocharacterization, and Western blot analysis. Combined metabolomic and transcriptomic analyses revealed β-sitosterol enrichment in EVPs and activation of the Hedgehog signaling pathway during regeneration. The role of β-sitosterol in EVPs on the Hedgehog pathway and its targets were identified using qRT-PCR, Western blot analysis. The regulation of carnitine synthesis by this pathway was determined using a dual luciferase assay. The effect of a β-sitosterol diet on liver regeneration was verified in mice. After 70% hepatectomy, the liver successfully regenerated without liver failure or death. At 24 hours post-surgery, tissue staining showed transient regeneration-associated steatosis (TRAS), with increased Ki67 positivity at 48 hours. EVPs displayed a spherical lipid bilayer structure with particle sizes of 70-130 nm. CD9, CD63, and CD81 in liver-derived EVPs were confirmed. Transcriptomic and metabolomic analyses showed EVPs supplementation significantly promoted carnitine synthesis and fatty acid oxidation. Tissue staining confirmed accelerated TRAS resolution and enhanced liver regeneration with EVP supplementation. Mass spectrometry identified β-sitosterol in EVPs, which binds to Smo protein, activating the Hedgehog pathway. This led to the nuclear transport of Gli3, stimulating Setd5 transcription and inducing carnitine synthesis, thereby accelerating fatty acid oxidation. Mice with increased β-sitosterol intake showed faster TRAS resolution and liver regeneration compared to controls. Liver-derived EVPs promote regeneration after partial hepatectomy. β-sitosterol from EVPs accelerates fatty acid oxidation and promotes liver regeneration by activating Hedgehog signaling pathway.

Imaging of Light-Enhanced Extracellular Vesicle-Mediated Delivery of Oxaliplatin to Colorectal Cancer Cells via Laser Ablation, Inductively Coupled Plasma Mass Spectrometry.

Extracellular vesicles (EVs) are lipid bilayer structures released by all cells that mediate cell-to-cell communication via the transfer of bioactive cargo. Because of the natural origin of EVs, their efficient uptake by recipient cells, capacity to stabilize and transport biomolecules and their potential for cell/tissue targeting and preferential uptake by cancer cells, they have enormous potential for bioengineering into improved and targeted drug delivery systems. In this work, we investigated the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) as a tool to measure the loading of platinum-based chemotherapeutic agents. The EV loading of oxaliplatin via co-incubation was demonstrated, and LA-ICP-MS imaging showed greater efficiency of delivery to colorectal cancer cells compared to free oxaliplatin, leading to enhanced cytotoxic effect. Further, the impact of EV co-loading with a porphyrin (C5SHU, known as 'C5') photosensitizer on oxaliplatin delivery was assessed. Fluorescence analysis using nano-flow cytometry showed dose-dependent EV loading as well as a trend towards the loading of larger particles. Exposure of OXA-C5-EV-treated colorectal cancer cells to light indicated that delivery was enhanced by both light exposure and porphyrins, with a synergistic effect on cell viability observed between oxaliplatin, EVs and light exposure after the delivery of the co-loaded EVs. In summary, this work demonstrates the utility of LA-ICP-MS and mass spectrometry imaging in assessing the loading efficiency and cellular delivery of platinum-based therapeutics, which would also be suitable for agents containing other elements, confirms that EVs are more efficient at delivery compared to free drugs, and describes the use of light exposure in optimizing delivery and therapeutic effects of EV-mediated drug delivery both in combination and independently of porphyrin-based photosensitizers.

Solidification of Aerial CO2‐Captured Aralkylamines in Water Triggered by Self‐Assembly Against Affinity for Formation of Reverse Lipid Bilayer

AbstractIt is found that aralkylamines solidified even in water by absorption of aerial CO2. Despite being amphiphilic, the aralkylamines absorbed CO2 and self‐assembled against the amphiphilicity to form a reverse lipid bilayer structure and solidified. Once the solid‐liquid separation of aerial CO2‐captured amines in water is achieved, it becomes possible to select the optimum conditions for both absorption and desorption.

Molecular mechanism of calcitriol enhances membrane water permeability

Helicobacter pylori (H. pylori) exhibits a unique membrane lipid composition, including dimyristoyl phosphatidylethanolamine (DMPE) and cholesterol, unlike other Gram-negative bacteria. Calcitriol has antimicrobial activity against H. pylori, but cholesterol enhances antibiotics resistance in H. pylori. This study explored the changes in membrane structure and the molecular mechanisms of cholesterol/calcitriol translocation using well-tempered metadynamics (WT-MetaD) simulations and microsecond conventional molecular dynamics (CMD) simulations. Calcitriol facilitated water transport across the membrane, while cholesterol had the opposite effect. The differing effects might result from the tail 25-hydroxyl group and a wider range of orientations of calcitriol in the DMPE/dimyristoyl phosphatidylglycerol (DMPG) (3:1) membrane. Calcitriol moves across the bilayer center without changing its orientation along the membrane Z-axis, becomes parallel to the membrane surface at the membrane-water interface, and then rotates approximately 90° in this interface. The translocation mechanism of calcitriol is quite different from the flip-flop of cholesterol. Moreover, calcitriol crossed from one layer to another more easily than cholesterol, causing successive perturbations to the hydrophobic core and increasing water permeation. These results improve our understanding of the relationship between cholesterol/calcitriol concentrations and the lipid bilayer structure and the role of lipid composition in water permeation.

The Emerging Roles of Exosomal miRNAs in Breast Cancer Progression and Potential Clinical Applications.

Breast cancer remains the leading malignancy in terms of morbidity and mortality today. The tumor microenvironment of breast cancer includes multiple cell types, secreted proteins, and signaling components such as exosomes. Among these, exosomes have a lipid bilayer structure. Exosomes can reflect the biological traits of the parent cell and carry a variety of biologically active components, including proteins, lipids, small molecules, and non-coding RNAs, which include miRNA, lncRNA, and circRNA. MiRNAs are a group of non-coding RNAs of approximately 20-23 nucleotides in length encoded by the genome, triggering silencing and functional repression of target genes. MiRNAs have been shown to play a significant role in the development of cancer owing to their role in the prognosis, pathogenesis, diagnosis, and treatment of cancer. MiRNAs in exosomes can serve as effective mediators of information transfer from parental cells to recipient cells and trigger changes in biological traits such as proliferation, invasion, migration, and drug resistance. These changes can profoundly alter the progression of breast cancer. Therefore, here, we systematically summarize the association of exosomal miRNAs on breast cancer progression, diagnosis, and treatment in the hope of providing novel strategies and directions for subsequent breast cancer treatment.

Absolute scattering length density profile of liposome bilayers obtained by SAXS combined with GIXOS: a tool to determine model biomembrane structure

Lipid membranes play an essential role in biology, acting as host matrices for biomolecules like proteins and facilitating their functions. Their structures and structural responses to physiologically relevant interactions (i.e. with membrane proteins) provide key information for understanding biophysical mechanisms. Hence, there is a crucial need of methods to understand the effects of membrane host molecules on the lipid bilayer structure. Here, a purely experimental method is presented for obtaining the absolute scattering length density profile and the area per lipid of liposomal bilayers, by aiding the analysis of small-angle X-ray scattering (SAXS) data with the volume of bare headgroups obtained from grazing-incidence X-ray off-specular scattering (GIXOS) data of monolayers of the same model membrane lipid composition. The GIXOS data experimentally demonstrate that the variation of the bare headgroup volume upon change in lipid packing density is small enough to allow its usage as a reference value without knowing the lipid packing stage in a bilayer. This approach also has the advantage that the reference volume is obtained in the same aqueous environment as used for the model membrane bilayers. The validity of this method is demonstrated using several typical membrane compositions, as well as one example of a phospholipid membrane with an incorporated transmembrane peptide. This methodology allows us to obtain absolute scale rather than relative scale values using solely X-ray-based instrumentation, retaining a similar resolution to SAXS experiments. The method presented has high potential for understanding the structural effects of membrane proteins on the biomembrane structure.

Molecular Structure, Biochemical Functions, Genetics, and Emerging Clinical Relevance of Glucose Transporters

In the human body, glucose acts as a major energy-producing fuel and regulator of energy homeostasis, enzyme functions, and gene transcription. The selective permeability of the lipid bilayer structure of the cell membrane makes it mandatory for glucose to require transport proteins for its transit into the cells. These include solute carrier integral membrane proteins such as glucose transporters (GLUTs) and sodium-glucose transporters. GLUTs belong to the major facilitator superfamily with a 12 transmembrane spanner topology, with GLUT1–13 sharing the same transmembrane sequence but variable transmembrane loops and terminal cytoplasmic ends of carbon and nitrogen. Phylogenetic analysis classifies GLUTs into three classes, with each class showing an affinity for a specific substrate. The tightly coupled relationship between glucose homeostasis and the nearly ubiquitous GLUTs has led to the investigation of their diverse roles in embryonic development, adult physiology, and clinical disorders including but not limited to inborn errors, diabetes mellitus, metabolic syndrome, and cancers. The current review is pivoted around the studies focusing on the structure and functions of members of the GLUT family, their chromosomal and organ-specific distribution, as well as the current evidence of their clinical implications and prospective therapeutic roles, specifically in cancers and metabolic disorders. The literature for the present work was retrieved from databases including Google Scholar, Web of Science, and PubMed.

Photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes to promote PD-L1 multivalent binding for effective immune checkpoint blockade therapy

Immune checkpoint blockade (ICB) therapy targeting PD-L1 via monoclonal antibody (mAb) has shown extensive clinical benefits in the diverse types of advanced malignancies. However, most patients are completely refractory to ICB therapy owing to the PD-L1 recycling mechanism. Herein, we propose photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes (immune checkpoint blockade liposomes; ICB-LPs) to promote PD-L1 multivalent binding for inducing lysosomal degradation of PD-L1 in tumor cells. The ICB-LPs are prepared by formulation of DC8,9PC with photo-polymerized diacetylenic moiety, 1,2-dipalmitoylphosphatidylcholine (DPPC) and anti-PD-L1 peptide (D-form NYSKPTDRQYHF)-conjugated DSPE-PEG2k (anti-PD-L1-DSPE-PEG2k) in a molar ratio of 45:45:10, followed by cross-linking of liposomal bilayer upon UV irradiation. The 10 mol% anti-PD-L1-DSPE-PEG2k incorporated ICB-LPs have a nano-sized lipid bilayer structure with an average diameter of 137.7 ± 1.04 nm, showing a high stability in serum condition. Importantly, the ICB-LPs efficiently promote the multivalent binding with PD-L1 on the tumor cell membrane, which are endocytosed with aim to deliver PD-L1 to the lysosomes, wherein the durable PD-L1 degradation is observed for 72 h, in contrast to anti PD-L1 mAbs showing the rapid PD-L1 recycling within 9 h. The in vitro co-culture experiments with CD8+ T cells show that ICB-LPs effectively enhance the T cell-mediated antitumor immune responses against tumor cells by blocking the PD-L1/PD-1 axis. When ICB-LPs are intravenously injected into colon tumor-bearing mice, they efficiently accumulate within the targeted tumor tissues via both passive and active tumor targeting, inducing a potent T cell-mediated antitumor immune response by effective and durable PD-L1 degradation. Collectively, this study demonstrates the superior antitumor efficacy of crosslinked and anti-PD-L1 peptide incorporated liposome formulation that promotes PD-L1 multivalent binding for trafficking of PD-L1 toward the lysosomes instead of the recycling endosomes.

Chorionic villi-derived nanofibers enhanced mesenchymal stem cell extracellular vesicle secretion and bioactivity for endometrium regeneration toward intrauterine adhesion treatment

Intrauterine adhesion (IUA) is one of the main causes of female infertility. Extracellular vesicles (EVs) secreted by mesenchymal stem cells (MSCs), are consisted of a complete lipid bilayer structure, rich in multiple factors and miRNAs related to tissue regeneration, which are the main effective paracrine components of MSCs to treat IUA. However, the poor secretion and bioactivity of EVs leading to the unexpected therapeutic efficacy of MSC-EVs, have been obstacles to their widespread use in clinical practice. Here, we developed human chorionic villi (CV)-derived nanofibers that mimic the tissue-specific ECM microenvironment of CV-derived MSCs, maintain the stemness of MSCs for a long time, and are rich in apolipoproteins, HSP90, ANXA1-A6, etc., which can enhance EV secretion and bioactivity. Then, the CV-nanofiber bionic culture system for mesenchymal stem cells was constructed to achieve large-scale cultivation of MSCs for continuous collection of high-concentration EVs enrichment supernatant. Additionally, EVs obtained by this system were encapsulated with CV-nanofibers to form EVs-ECM aggregate, which can effectively promote endometrium regeneration toward supporting fetal development and live births. Our study provides a new idea for enhancing EV secretion and bioactivity, and a cell-free therapy for treating IUA by EVs, thus promoting the potential of clinical application.

Role of Lipids and Divalent Cations in Membrane Fusion Mediated by the Heptad Repeat Domain 1 of Mitofusin.

Mitochondria are highly dynamic organelles that constantly undergo fusion and fission events to maintain their shape, distribution and cellular function. Mitofusin 1 and 2 proteins are two dynamin-like GTPases involved in the fusion of outer mitochondrial membranes (OMM). Mitofusins are anchored to the OMM through their transmembrane domain and possess two heptad repeat domains (HR1 and HR2) in addition to their N-terminal GTPase domain. The HR1 domain was found to induce fusion via its amphipathic helix, which interacts with the lipid bilayer structure. The lipid composition of mitochondrial membranes can also impact fusion. However, the precise mode of action of lipids in mitochondrial fusion is not fully understood. In this study, we examined the role of the mitochondrial lipids phosphatidylethanolamine (PE), cardiolipin (CL) and phosphatidic acid (PA) in membrane fusion induced by the HR1 domain, both in the presence and absence of divalent cations (Ca2+ or Mg2+). Our results showed that PE, as well as PA in the presence of Ca2+, effectively stimulated HR1-mediated fusion, while CL had a slight inhibitory effect. By considering the biophysical properties of these lipids in the absence or presence of divalent cations, we inferred that the interplay between divalent cations and specific cone-shaped lipids creates regions with packing defects in the membrane, which provides a favorable environment for the amphipathic helix of HR1 to bind to the membrane and initiate fusion.

EV-miRNA-Mediated Intercellular Communication in the Breast Tumor Microenvironment.

Cancer research has prioritized the study of the tumor microenvironment (TME) as a crucial area of investigation. Understanding the communication between tumor cells and the various cell types within the TME has become a focal point. Bidirectional communication processes between these cells support cellular transformation, as well as the survival, invasion, and metastatic dissemination of tumor cells. Extracellular vesicles are lipid bilayer structures secreted by cells that emerge as important mediators of this cell-to-cell communication. EVs transfer their molecular cargo, including proteins and nucleic acids, and particularly microRNAs, which play critical roles in intercellular communication. Tumor-derived EVs, for example, can promote angiogenesis and enhance endothelial permeability by delivering specific miRNAs. Moreover, adipocytes, a significant component of the breast stroma, exhibit high EV secretory activity, which can then modulate metabolic processes, promoting the growth, proliferation, and migration of tumor cells. Comprehensive studies investigating the involvement of EVs and their miRNA cargo in the TME, as well as their underlying mechanisms driving tumoral capacities, are necessary for a deeper understanding of these complex interactions. Such knowledge holds promise for the development of novel diagnostic and therapeutic strategies in cancer treatment.

Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by 1H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties.

1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.

Engineered Plant-Derived Nanovesicles Facilitate Tumor Therapy: Natural Bioactivity Plus Drug Controlled Release Platform.

Tumors are the second-most common disease in the world, killing people at an alarming rate. As issues with drug resistance, lack of targeting, and severe side effects are revealed, there is a growing demand for precision-targeted drug delivery systems. Plant-derived nanovesicles (PDNVs), which arecomposed of proteins, lipids, RNA, and metabolites, are widely distributed and readily accessible. The potential for anti-proliferative, pro-apoptotic, and drug-resistant-reversing effects on tumor cells, as well as the ability to alter the tumor microenvironment (TME) by modulating tumor-specific immune cells, make PDNVs promising anti-tumor therapeutics. With a lipid bilayer structure that allows drug loading and a transmembrane capacity readily endocytosed by cells, PDNVs are also expected to become a new drug delivery platform. Exogenous modifications of PDNVs enhance their circulating stability, tumor targeting ability, high cell endocytosis rate, and controlled-release capacity. In this review, we summarize PDNVs' natural antitumor activity, as well as engineered PDNVs as efficient precision-targeted drug delivery tools that enhance therapeutic effects. Additionally, we discuss critical considerations related to the issues raised in this area, which will encourage researchers to improve PDNVs as better anti-tumor therapeutics for clinic applications.