Cationic Liposomes Research Articles

In Vitro Effects of Charged and Zwitterionic Liposomes on Human Spermatozoa and Supplementation with Liposomes and Chlorogenic Acid during Sperm Freezing.

Semen handling and cryopreservation induce oxidative stress that should be minimized. In this study, human semen was supplemented during cryopreservation with formulations of handmade liposomes and chlorogenic acid (CGA), an antioxidant compound. Zwitterionic (ZL), anionic (AL), and cationic (CL) liposomes were synthesized and characterized. Three aliquots of swim-up-selected sperm were incubated with ZL, AL, and CL (1:10,000), respectively. The percentages of sperm with progressive motility, high mitochondrial membrane potential (MMP; JC-1), double-stranded DNA (dsDNA acridine orange), and acrosome integrity (Pisum sativum agglutinin) were assessed. Then, human semen was frozen using both 1:10,000 ZL and CGA as follows: freezing medium/empty ZL (EL), freezing medium/empty ZL/CGA in the medium (CGA + EL), freezing medium/CGA loaded ZL (CGA), freezing medium (CTR). The same sperm endpoints were evaluated. ZL were the most tolerated and used for semen cryopreservation protocols. All the supplemented samples showed better endpoints versus CTR (p < 0.001). In particular, spermatozoa from the CGA and CGA + EL A samples showed increased motility, dsDNA, and acrosome integrity versus CTR and EL (p < 0.001; motility EL vs. CGA + EL p < 0.05). ZL and CGA can improve post-thaw sperm quality, acting on both cold shock effect management and oxidative stress. These findings open new perspectives on human and animal reproduction.

CLPs-miR-103a-2-5p inhibits proliferation and promotes cell apoptosis in AML cells by targeting LILRB3 and Nrf2/HO-1 axis, regulating CD8 + T cell response

BackgroundLILRB3, a member of the leukocyte immunoglobulin-like receptor B (LILRB) family, has immunosuppressive functions and directly regulates cancer development, which indicates that LILRB3 is an attractive target for cancer diagnosis and therapy. Novel therapeutic treatments for acute myeloid leukemia (AML) are urgent and important, and RNA therapeutics including microRNAs (miRNAs) could be an effective option. Here, we investigate the role of dysregulated miRNA targeting LILRB3 in the AML microenvironment.MethodsPotential miRNAs binding to the 3ʹ-untranslated region (3ʹ-UTR) of the LILRB3 mRNA were predicted by bioinformatics websites. Then, we screened miRNAs targeting LILRB3 by quantitative real-time PCR, and the dual luciferase reporter assay. The expression of LILRB3 and microRNA (miR)-103a-2-5p in AML were determined and then their interactions were also analyzed. In vitro, the effects of miR-103a-2-5p were determined by CCK8, colony formation assay, and transwell assay, while cell apoptosis and cell cycle were analyzed by flow cytometry. Cationic liposomes (CLPs) were used for the delivery of miR-103a-2-5p in the AML mouse model, which was to validate the potential roles of miR-103a-2-5p in vivo.ResultsLILRB3 was upregulated in AML cells while miR-103a-2-5p was dramatically downregulated. Thus, a negative correlation was found between them. MiR-103a-2-5p directly targeted LILRB3 in AML cells. Overexpressed miR-103a-2-5p significantly suppressed the mRNA and protein levels of LILRB3, thereby inhibiting AML cell growth and reducing CD8 + T cell apoptosis. In addition, overexpressed miR-103a-2-5p reduced both the relative expression of Nrf2/HO-1 pathway-related proteins and the ratio of GSH/ROS, leading to the excessive intracellular ROS that may promote AML cell apoptosis. In the mouse model, the delivery of miR-103a-2-5p through CLPs could inhibit tumor growth.ConclusionsMiR-103a-2-5p serves as a tumor suppressor that could inhibit AML cell proliferation and promote their apoptosis by downregulating LILRB3 expression, suppressing the Nrf2/HO-1 axis, and reducing the ratio of GSH/ROS. Besides, our findings indicate that miR-103a-2-5p may enhance the CD8 + T cell response by inhibiting LILRB3 expression. Therefore, the delivery of miR-103a-2-5p through CLPs could be useful for the treatment of AML.

Synthesis, Characterization, and In vitro Evaluation of Short Cationic Peptides for Gene Delivery Vehicle Candidate

Background: Amongst gene delivery vehicles, peptide-based vectors have drawn the intensive attraction of experts globally due to their simplicity and many advantages due to ease in design, biocompatibility, and safety. Rationally designed peptides are capable of condensing DNA molecules efficiently and facilitating gene expression in the target cells. Objective: This study aims to design, synthesize and evaluate short cationic peptides composed of several positively charges amino acids of lysine (K) and arginine (R) for gene delivery vehicle candidates. Methods: The short cationic peptides of PKKKRKV (P1), CHSPKKKRKV (P2), and YGRKKRRQRRR (P3) were synthesized using a solid-phase method on 2-chlorotrityl chloride resin. The crude peptides were purified using RP-HPLC and characterized by HR-TOF-ESI-MS and 1H-NMR. The capability of the peptides to condense DNA was evaluated by ethidium bromide exclusion assay. Cytotoxicity study of the peptides was carried out in HEK-293T, CHO-K1, and HepG2 cells using MTT assay. Gene expression facilitated by the peptides was determined in the HEK-293T. Results: The peptides were successfully synthesized with high purity (&gt; 90%) and in a high consistency with the synthetic products, as shown by the spectroscopic data. Physicochemical and biological evaluation showed that the cationic peptides are capable of condensing DNA molecule and have low cytotoxicity to the cells of HEK-293T, CHO-K1, and HepG2. Moreover, the cationic peptides facilitated gene delivery of green fluorescence protein more efficiently compared to PLL. Conclusion: The short cationic peptides rich in lysine and arginine have been successfully synthesized using solid-phase peptide synthesis method. They were found to be capable of condensing DNA, have low cytotoxicity, and facilitate gene delivery. However, structure modification or formulation of cationic peptide with lipid components to form cationic liposome is still needed to enhance transgene expression by these peptides.

Sialic Acid Conjugate-Modified Cationic Liposomal Paclitaxel for Targeted Therapy of Lung Metastasis in Breast Cancer: What a Difference the Cation Content Makes.

Cationic lipids play a pivotal role in developing novel drug delivery systems for diverse biomedical applications, owing to the success of mRNA vaccines against COVID-19 and the Phase III antitumor agent EndoTAG-1. However, the therapeutic potential of these positively charged liposomes is limited by dose-dependent toxicity. While an increased content of cationic lipids in the formulation can enhance the uptake and cytotoxicity toward tumor-associated cells, it is crucial to balance these advantages with the associated toxic side effects. In this work, we synthesized the cationic lipid HC-Y-2 and incorporated it into sialic acid (SA)-modified cationic liposomes loaded with paclitaxel to target tumor-associated immune cells efficiently. The SA-modified cationic liposomes exhibited enhanced binding affinity toward both RAW264.7 cells and 4T1 tumor cells in vitro due to the increased ratios of cationic HC-Y-2 content while effectively inhibiting 4T1 cell lung metastasis in vivo. By leveraging electrostatic forces and ligand-receptor interactions, the SA-modified cationic liposomes specifically target malignant tumor-associated immune cells such as tumor-associated macrophages (TAMs), reduce the proportion of cationic lipids in the formulation, and achieve dual objectives: high cellular uptake and potent antitumor efficacy. These findings highlight the potential advantages of this innovative approach utilizing cationic liposomes.

MiRNA‐21 Inhibitor‐Loaded Cationic Liposomes Development using the Quality by Design Approach

AbstractThe success of efficiently delivering genetic material to target cells depends on developing an appropriate vector. Among the variety of nanocarriers used for gene therapy, cationic liposomes are the most investigated for delivering genetic material in different forms of cancer. Cationic liposomes contain lipids with positively charged head groups that form electrostatic bonds with the negatively charged phosphate group of nucleic acids, ensuring efficient loading of the genetic material. The aim of this study was the development and in vitro characterization of liposomal formulations for genetic material delivery using the Quality by Design (QbD) approach. We have employed the Design of Experiments (DoE) methodology to study the impact of selected formulation factors on the quality of the proposed non‐viral vectors. Among the varied factors, the ones that had an impact on the responses were the total lipid concentration and cationic lipid concentration. Two liposomal formulations with adequate critical quality attributes (CQAs) were selected for miRNA‐21 inhibitor encapsulation. We continued with the in vitro evaluation on lung cancer cell line. Fluorescence microscopy helped visualize the internalization of all liposomal formulations and apoptotic effects. Finally, we evaluated the cellular viability and migration events after treatment with the cationic liposomes.

Intrinsic immunogenicity of liposomes for tuberculosis vaccines: Effect of cationic lipid and cholesterol

Tuberculosis (TB) is still among the deadliest infectious diseases, hence there is a pressing need for more effective TB vaccines. Cationic liposome subunit vaccines are excellent vaccine candidates offering effective protection with a better safety profile than live vaccines. In this study, we aim to explore intrinsic adjuvant properties of cationic liposomes to maximize immune activation while minimizing aspecific cytotoxicity. To achieve this, we developed a rational strategy to select liposomal formulation compositions and assessed their physicochemical and immunological properties in vitro models using human monocyte-derived dendritic cells (MDDCs). A broad selection of commercially available cationic compounds was tested to prepare liposomes containing Ag85B-ESAT6-Rv2034 (AER) fusion protein antigen. 1,2-Dioleoyl-sn‑glycero-3-ethylphosphocholine (EPC)-based liposomes exhibited the most advantageous activation profile in MDDCs as assessed by cell surface activation markers, cellular uptake, antigen-specific T-cell activation, cytokine production, and cellular viability. The addition of cholesterol to 20 mol% improved the performance of the tested formulations compared to those without it; however, when its concentration was doubled there was no further benefit, resulting in reduced cell viability. This study provides new insights into the role of cationic lipids and cholesterol in liposomal subunit vaccines.

Enhanced anti-malarial efficacy of mefloquine delivered via cationic liposome in a murine model of experimental cerebral malaria

Malaria is a longstanding global health challenge that continues to afflict over 90 countries located in tropical and subtropical regions of the globe. The rise of drug-resistant malarial parasites has curtailed the therapeutic efficacy of a number of once-effective anti-malarials, including mefloquine. In the present study, we have taken advantage of drug encapsulation approach to elevate the anti-malarial potential of mefloquine. Encouragingly, our findings unveil that liposomal formulations of mefloquine outperform equivalent doses of free mefloquine, both in laboratory cultures and in a murine model of malaria. Intriguingly, a cationic liposomal mefloquine formulation, administered at four successive doses of 3 mg/kg body weight, achieves complete resolution of cerebral malaria in the murine model while avoiding noticeable toxic repercussions. Altogether, our study furnishes pre-clinical validation for a therapeutic strategy that can remarkably enhance the drug efficacy, offering a revitalizing solution for failing anti-malarials.

Polyethylene glycol-stabilized cationic liposome encapsulating glucosamine sulfate: A promising nanoformulation for osteoarthritis therapy

Osteoarthritis (OA) is a common orthopedic chronic disease, and the use of nanomaterials as carriers is an effective way to realize slow and controlled release therapy for OA drugs. In this study, we synthesized a polyethylene glycol-stabilized bilayer-decorated cationic liposome (CLis) as a drug delivery system for delivering glucosamine sulfate (GS) drug to achieve the treatment of OA in rats. The CLis encapsulated with GS drug (GS-CLis) was prepared by a reverse evaporation method, and its physical properties, encapsulation rate, and drug release performance were evaluated. The biological properties of GS-CLis were evaluated in vitro. The therapeutic effect of GS-CLis on osteoarthritis was evaluated in vivo, and the in vivo biosafety of the drug was assessed by hematology, blood biochemistry, and hematoxylin-eosin staining. GS-CLis had a particle size of (236.28 ± 4.76) nm, a potential of (27.35 ± 4.86) mv, and a spherical shape. The encapsulation rate was (96.18 ± 5.77)% and the drug-loading capacity was (9.61 ± 0.28)%, which provided a good slow drug release. GS-CLis has low cytotoxicity, low hemolysis rate, and good biocompatibility. GS-CLis can more effectively alleviate joint surface damage and inhibit the expression of inflammatory factors, and has no significant effect on the body weight of rats, with good biological safety. In this study, we successfully synthesized novel GS-CLis for the treatment of OA, which improved the retention time and therapeutic effect of GS in OA and provided a research basis for its development as an OA nanoformulation.

Fusogenicity of cationic liposomes with phase-separating multicomponent lipid compositions

Fusogenicity of cationic liposomes with phase-separating multicomponent lipid compositions

Menthol-modified paclitaxel multifunctional cationic liposomes cross the blood-brain barrier and target glioma stem cells for treatment of glioblastoma

Glioblastoma is the most malignant glioma of the central nervous system, as the presence of cerebral glioma stem cells and the blood-brain barrier hamper its treatment. Menthol can improve blood-brain barrier permeability. Cationic liposomes can interact with negatively charged macromolecules on tumor neovascular endothelial cells to produce passive targeting. Ginsenoside Rh2 can replace cholesterol to make liposomes more stable. Paclitaxel has an excellent antitumor effect. Therefore, in this study, menthol-modified paclitaxel cationic liposomes were constructed to cross the blood-brain barrier and targeted the tumor, to achieve safe and effective treatment of glioblastoma. We discovered that the addition of DC-chol liposomes can enhance the uptake of C6 glioma stem cells, promote the apoptosis of C6 glioma stem cells and inhibit the proliferation of tumor cells in vitro. We found that targeted liposomes could cross the blood-brain barrier and target tumor sites to induce tumor cell apoptosis in vivo. In brief, menthol-modified paclitaxel cationic liposomes offer a safer approach to the treatment of glioblastoma.

Calreticulin P-domain-derived “Eat-me” peptides for enhancing liposomal uptake in dendritic cells

Discovering new ligands for enhanced drug uptake and delivery has been the core interest of the drug delivery field. This study capitalizes on the natural “eat-me” signal of calreticulin (CRT), proposing a novel strategy for functionalizing liposomes to improve cellular uptake. CRT is presented on the surfaces of apoptotic cells, and it plays a crucial role in immunogenic cell death (ICD). This is because it is essential for antigen uptake via low-density lipoprotein (LDL) receptor-mediated phagocytosis. Inspired by this mechanism, we interrogated CRT’s “eat-me” feature using CRT-derived peptides to functionalize liposomes. We studied liposomal formulation stability, properties, cellular uptake, toxicity, and intracellular trafficking in dendritic cells. We identified key peptide fragments of CRT, specifically from the hydrophilic P-domain, that are compatible with liposomal formulations. Contrary to the more hydrophobic N-domain peptides, the P-domain peptides induced significantly higher liposomal uptake in DC2.4 dendritic cells than cationic DOTAP and anionic DPPG liposomes without inducing toxicity. The P-domain-derived peptides led to enhanced liposomal uptake into DC2.4 dendritic cells compared to the standard DPPC liposomes. The uptake can be partially blocked by the receptor-associated protein (RAP). Upon internalization, P-domain-peptide-decorated liposomes showed higher co-localization with lysosomes compared to the standard DPPC liposomes. Our findings illuminate CRT's operational role and identify P-domain peptides as promising agents for developing biomimetic drug delivery systems that can potentially replicate CRT's “eat-me” function.

Activation of RIG-I/MDA5 Signaling and Inhibition of CD47-SIRPα Checkpoint with a Dual siRNA-Assembled Nanoadjuvant for Robust Cancer Immunotherapy.

Antigen-presenting cells (APCs) play a crucial role in the anti-tumor immunity as they are responsible for capturing, processing, and presenting tumor antigens to T cells. However, their activation is often limited by the absence of adjuvants and the suppressive effects of immune checkpoints, such as CD47-SIRPα. Herein, we present a nanoadjuvant that is self-assembled from long RNA building blocks generated through rolling circle transcription (RCT) reaction and further modified with cationic liposomes. Owing to the high load of densely packed RNA, this nanoadjuvant could robustly activate RIG-I/MDA5 signaling in APCs, leading to the maturation of dendritic cells (DCs) and the polarization of tumor-associated macrophages (TAMs) toward an anti-tumor M1-like phenotype. In addition, with a well-designed template, the generated long RNA from RCT reaction includes two kinds of siRNA targeting both CD47 in tumor cells and SIRPα in APCs. This dual gene silencing results in efficient inhibition of the CD47-SIRPα checkpoint. Collectively, the robust activation of RIG-I/MDA5 signaling and efficient inhibition of CD47-SIRPα checkpoint enhance the phagocytic activity of APCs, which in turn promotes the cross-priming of effector T cells and the activation of anti-tumor immune responses. This study therefore provides a simple and robust RNA nanoadjuvant for cancer immunotherapy.

Activation of RIG‐I/MDA5 Signaling and Inhibition of CD47‐SIRPα Checkpoint with a Dual siRNA‐Assembled Nanoadjuvant for Robust Cancer Immunotherapy

AbstractAntigen‐presenting cells (APCs) play a crucial role in the anti‐tumor immunity as they are responsible for capturing, processing, and presenting tumor antigens to T cells. However, their activation is often limited by the absence of adjuvants and the suppressive effects of immune checkpoints, such as CD47‐SIRPα. Herein, we present a nanoadjuvant that is self‐assembled from long RNA building blocks generated through rolling circle transcription (RCT) reaction and further modified with cationic liposomes. Owing to the high load of densely packed RNA, this nanoadjuvant could robustly activate RIG‐I/MDA5 signaling in APCs, leading to the maturation of dendritic cells (DCs) and the polarization of tumor‐associated macrophages (TAMs) toward an anti‐tumor M1‐like phenotype. In addition, with a well‐designed template, the generated long RNA from RCT reaction includes two kinds of siRNA targeting both CD47 in tumor cells and SIRPα in APCs. This dual gene silencing results in efficient inhibition of the CD47‐SIRPα checkpoint. Collectively, the robust activation of RIG‐I/MDA5 signaling and efficient inhibition of CD47‐SIRPα checkpoint enhance the phagocytic activity of APCs, which in turn promotes the cross‐priming of effector T cells and the activation of anti‐tumor immune responses. This study therefore provides a simple and robust RNA nanoadjuvant for cancer immunotherapy.

Hybrid Milk Extracellular Vesicles as Potential Systems for Oral Delivery of siRNA

AbstractOral administration of RNA is currently not possible and current approaches for RNA delivery by injection are not suitable for the oral route. In this work, novel bioinspired nanoparticles for oral delivery of RNA therapies are reported. Specifically, the fabrication of hybrid nanovesicles (“hybridosomes”) based on bovine milk extracellular vesicles (mEVs) and liposomes is reported. Hybridosomes are fabricated using two related methods based on freeze‐thawing of mEVs and cationic liposomes. The systems are 180–230 nm and demonstrated efficient loading of small interfering (siRNA) cargo. Hybridosomes exhibit significantly lower cytotoxicity in intestinal Caco‐2 cells compared to cationic liposomes and superior stability in a fed‐state simulated intestinal fluid compared to liposomes. Furthermore, these systems significantly increase the transport of siRNA across in vitro intestinal model. Finally, hybridosomes loaded with siRNA successfully induce transfection in J774A.1 macrophages. Overall, the results show that the hybrid systems fabricated here offer potential for oral delivery of RNA therapeutics.

Cell-penetrating peptide and cationic liposomes mediated siRNA delivery to arrest growth of chronic myeloid leukemia cells in vitro

Gene silencing through RNA interference (RNAi) is a promising therapeutic approach for a wide range of disorders, including cancer. Non-viral gene therapy, using specific siRNAs against BCR-ABL1, can be a supportive or alternative measure to traditional chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKIs) therapies, given the prevalence of clinical TKI resistance. The main challenge for such approaches remains the development of the effective delivery system for siRNA tailored to the specific disease model.The purpose of this study was to examine and compare the efficiency of endosomolytic cell penetrating peptide (CPP) EB1 and PEG2000-decorated cationic liposomes composed of polycationic lipid 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2Х3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for anti-bcr-abl siRNA delivery into the K562 human CML cell line. We show that both EB1 and 2Х3-DOPE-DSPE-PEG2000 (0.62 % mol.) liposomes effectively deliver siRNA into K562 cells by endocytic mechanisms, and the use of liposomes leads to more effective inhibition of expression of the targeted gene (BCR-ABL1) and cancer cell proliferation. Taken together, these findings suggest that PEG-decorated cationic liposomes mediated siRNA delivery allows an effective antisense suppression of certain oncogenes, and represents a promising new class of therapies for CML.

A PEGylated liposomal formulation of prochlorperazine that limits brain exposure but retains dynamin II activity: A potential adjuvant therapy for cancer patients receiving chemotherapeutic mAbs

Anti-cancer monoclonal antibodies often fail to provide therapeutic benefit in receptor-positive patients due to rapid endocytosis of antibody-bound cell surface receptors. High dose co-administration of prochlorperazine (PCZ) inhibits endocytosis and sensitises tumours to mAbs by inhibiting dynamin II but can also introduce neurological side effects. We examined the potential to use PEGylated liposomal formulations of PCZ (LPCZ) to retain the anti-cancer effects of PCZ, but limit brain uptake. Uncharged liposomes showed complete drug encapsulation and pH-dependent drug release, but cationic liposomes showed limited drug encapsulation and lacked pH-dependent drug release. Uncharged LPCZ showed comparable inhibition of EGFR internalisation to free PCZ in KJD cells. After IV administration to rats, LPCZ reduced the plasma clearance and brain uptake of PCZ compared to IV PCZ. The results suggest that LPCZ may offer some benefit over PCZ as an adjunct therapy in cancer patients receiving mAb treatment.

The interplay between trehalose and dextran as spray drying precursors for cationic liposomes

Successful oral delivery of liposomes requires formulations designed to withstand harsh gastrointestinal conditions, e.g., by converting to solid-state followed by loading into gastro-resistant delivery devices. The hypothesis was that the use of dextran-trehalose mixtures for spray drying would improve the rehydration kinetics of dried liposomes. The objectives were to determine the protective capacity of trehalose-dextran dehydration precursors and to increase the concentration of liposomes in the dry formulation volume. The study successfully demonstrated that 8.5% dextran combined with 76.5% trehalose protected CAF®04 liposomes during drying, with the liposome content maintained at 15% of the dry powder. Accordingly, the rehydration kinetics were slightly improved in formulations containing up to 8.5% dextran in the dry powder volume. Additionally, a 2.4-fold increase in lipid concentration (3 mM vs 7.245 mM) was achieved for spray dried CAF®04 liposomes. Ultimately, this study demonstrates the significance of trehalose as a primary carrier during spray drying of CAF®04 liposomes and highlights the advantage of incorporating small amounts of dextran to tune rehydration kinetics of spray-dried liposomes.

Liposome-exosome hybrids for in situ detection of exosomal miR-1246 in breast cancer.

Several lines of evidence suggest that exosomal miRNAs are potential biomarkers for cancer monitoring. An urgent need remains for the in situ detection of exosomal miRNAs at low concentrations without destroying the exosome structure. In the present study, a novel sensitive exosomal miR-1246 in situ detection strategy has been developed by integrating the CRISPR/Cas13a system with the formation of hybrids between exosomes and cationic liposomes. The liposomes were loaded with CRISPR/Cas13a, CRISPR RNA (crRNA), and RNA reporter probes. In the presence of exosomes, the liposome-exosome hybrids were formed through electrostatic interactions, and CRISPR/Cas13a was activated to cleave the reporter probes by exosomal miR-1246. The acquired fluorescence signal showed a linear response to the logarithm of MCF-7 exosome concentrations, indicating a quantitative response to exosomal miR-1246. The regression equation is y = 5021 log C - 9976 (R2 = 0.9985) with a limit of detection of 3 × 102 particles per mL. This strategy could not only be used to detect serum exosomal miR-1246 in breast cancer patients but also to distinguish early form advanced disease. This strategy can be exploited in future exosomal miRNA analyses.

Efficient siRNA delivery to murine melanoma cells via a novel genipin-based nano-polymer.

Small-interfering RNAs (siRNAs) are therapeutic nucleic acids, often delivered via cationic polymers, liposomes, or extracellular vesicles, each method with its limitations. Genipin, a natural crosslinker for primary amines, was explored for siRNA delivery scaffolds. Spermine/genipin-based GxS5 polymers were synthesized, showing slightly positive ζ potential at neutral pH and intrinsic fluorescence. We then tuned their polymerization adding glycine to the reaction batch, from 1 to 10 molar ratio with genipin, therefore conferring them a "zwitterionic" character. GxS5 efficiently internalized into B16F10 murine melanoma cells, and exhibited strong siRNA-complexing ability and they were able to elicit up to 60% of gene knock-down without any toxicity. This highlights GxS5's potential as a safe, replicable, and tunable platform for therapeutic nucleic acid delivery, suggesting broader applications. This innovative approach not only sheds light on the intricate genipin reaction mechanism but also underscores the importance of fine-tuning nanoparticle properties for effective siRNA delivery. GxS5's success in mitigating cytotoxicity while maintaining delivery efficacy signifies a promising step towards safer and more efficient nucleic acid therapeutics.

Development of Liposomes That Target Axon Terminals Encapsulating Berberine in Cultured Primary Neurons.

Most of the energy in neurons is produced in mitochondria. Mitochondria generate the ATP that is essential for neuronal growth, function, and regeneration. Mitochondrial axonal transport plays a crucial role in maintaining neuronal homeostasis and biological activity. Decreased mitochondrial axonal transport at axon terminals, where the metabolism of substances is likely to be delayed, may contribute to neurological dysfunction. Therefore, regulation of mitochondrial dynamics at axon terminals has attracted considerable interest as a strategy to modulate neuronal function. Nanoparticles may be useful in controlling local mitochondrial dynamics. Nevertheless, there are few reports on the influence of drug delivery that nanoparticles impart on the mitochondrial dynamics in neurons. This paper reports the results of a study using liposomes (LPs) to examine local drug delivery and pharmacological actions on neurons. We tested berberine (BBR), which is an activator of AMP-activated protein kinase (AMPK), to examine the utility of this drug as a cellular energy sensor. Axon terminals targeting LPs were prepared. The amount of axon terminals targeting LPs was increased compared with treatment using cationic LPs. Moreover, axon terminal-targeting LPs increased anterograde transport by about 40% compared with that of either naked BBR or cationic LPs and suppressed axonal retraction. Our findings suggest that local drug delivery to neurons is important for enhancing pharmacological activity in axon terminals.