Search Results (95)

Background: The C1858T PTPN22 variant is strongly associated with type 1 diabetes and autoimmune thyroid disease. Current treatment is substitutive hormonal administration, which does not target the disease pathogenetic mechanism. We previously implemented a novel immunotherapy, employing siRNA directed against the C1858T variant of PTPN22 delivered via functionalized lipoplexes, in order to halt autoimmune disease progression. Objectives: The objective of this study was to optimize lipoplex formulations functionalized with F9-PEG (a Siglec-10’s ligand) to facilitate targeted delivery by investigating their physical and chemical properties to warrant the best performance in in vivo studies. Methods: The effectiveness of siRNA liposome binding was evaluated by varying the relative lipid/siRNA charge ratio and analyzing the stability of the different formulations with respect to the methods of F9-PEG addition and ATTO740 fluorescent labeling by electrophoresis, dynamic and dielectrophoretic light scattering (DLS and DELS), and high-performance liquid chromatography (HPLC). Results: The optimal charge ratio of +2/−1 (lipid/siRNA) ensured a greater stability of lipoplexes and complete complexation of siRNA. Stability was improved by selecting a protocol of preparation that envisages functionalization with F9-PEG and the addition of ATTO740 lipid in the lipid film preparation step. HPLC confirmed the integrity of siRNA after preparation. Conclusions: The results of this study lead to formulations of F9-PEG lipoplexes with optimal properties that could be used for biodistribution and safety/efficacy studies in mice. Lipoplexes functionalized with F9-PEG could therefore represent a promising personalized nanotherapeutic platform for targeted siRNA delivery in endocrine C1858T patients. Full article

Cutaneous lupus erythematosus (CLE) is a chronic autoimmune skin disorder with limited therapeutic options, particularly for refractory discoid lupus (DLE), which often results in scarring and atrophy. Recent studies have identified miR-31, miR-485-3p, and miR-885-5p as key regulators of inflammation, apoptosis, and fibrosis in CLE skin lesions. This research investigates a novel topical miRNA therapy using DDC642 elastic liposomes to target these pathways in CLE. DDC642 liposomes were complexed with miRNAs (anti-miR-31, anti-miR-485-3p, pre-miR-885-5p) and characterized through dynamic light scattering and Cryo-TEM. Cytotoxicity, cellular penetration, and therapeutic efficacy were evaluated in primary keratinocytes, PBMCs, and immune 3D-skin organoids. miRNA lipoplexes were successfully synthesized with optimized particle size, surface charge, and encapsulation efficiency. These lipoplexes exhibited effective cellular penetration and low cytotoxicity. Anti-miR-31 lipoplexes reduced miR-31 and NF-κB levels while increasing STK40 and PPP6C expression. Pre-miR-885-5p lipoplexes elevated miR-885-5p levels and downregulated PSMB5 and NF-κB in keratinocytes. While anti-miR-485-3p lipoplexes reduced T-cell activation markers. Anti-miR-31 and pre-miR-885-5p lipoplexes successfully modulated inflammatory pathways in 3D-skin CLE models. miRNA lipoplexes represent promising candidates for pioneering topical genetic therapies for CLE. Further studies, including animal models, are necessary to validate and optimize these findings. Full article

Background/Objectives: Bringing small interfering RNA (siRNA) into the cell cytosol to achieve specific gene silencing is an attractive but also very challenging option for improved therapies. The first step for successful siRNA delivery is the complexation with a permanent cationic or ionizable compound. This protects the negatively charged siRNA and enables transfection through the cell membrane. The current study explores the performance of the innovative, ionizable lipid 2-Tetradecylhexadecanoic acid-(2-bis{[2-(2,6-diamino-1-oxohexyl)amino]ethyl}aminoethyl)-amide (T14diLys), in combination with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), for siRNA delivery and the impact of the production method (sonication vs. extrusion) on the particle properties. Methods: Liposomes were produced either with sonication or extrusion and characterized. The extruded liposomes were combined with siRNA at different N/P ratios and investigated in terms of size zeta potential, encapsulation efficiency, lipoplex stability against RNase A, and knockdown efficiency using enhanced green fluorescent protein (eGFP)-marked colon adenocarcinoma cells. Results: The liposomes prepared by extrusion were smaller and had a narrower size distribution than the sonicated ones. The combination of siRNA and liposomes at a nitrogen-to-phosphate (N/P) ratio of 5 had optimal particle properties, high encapsulation efficiency, and lipoplex stability. Gene knockdown tests confirmed this assumption. Conclusions: Liposomes produced with extrusion were more reproducible and provided enhanced particle properties. The physicochemical characterization and in vitro experiments showed that an N/P ratio of 5 was the most promising ratio for siRNA delivery. Full article

Gene transfection is a fundamental technique in the fields of biological research and therapeutic innovation. Due to their biocompatibility and membrane-mimetic properties, lipid vectors serve as essential tools in transfection. The successful delivery of genetic material into the cytoplasm is contingent upon the fusion of the vector and cellular membranes, which enables hydrophilic polynucleic acids to traverse the hydrophobic barriers of two intervening membranes. This review examines the critical role of membrane fusion in lipofection efficiency, with a particular focus on the molecular mechanisms that govern lipoplex–membrane interactions. This analysis will examine the key challenges inherent to the fusion process, from achieving initial membrane proximity to facilitating final content release through membrane remodeling. In contrast to viral vectors, which utilize specialized fusion proteins, lipid vectors necessitate a strategic formulation and environmental optimization to enhance their fusogenicity. This review discusses recent advances in vector design and fusion-promoting strategies, emphasizing their potential to improve gene delivery yield. It highlights the importance of understanding lipoplex–membrane fusion mechanisms for developing next-generation delivery systems and emphasizes the need for continued fundamental research to advance lipid-mediated transfection technology. Full article

This work aims to contribute to nucleic acid therapy advances by highlighting RNA interactions with novel compounds, namely lipoaminoacids (LAAs), which show great potential as non-viral vectors. LAAs not only retain the advantages associated with current non-viral vectors, namely fewer health risks, but also can form stable lipoplexes with genetic material, positioning them as promising candidates for advanced gene delivery systems. The biosynthesis of LAAs, using the enzymes, porcine pancreatic lipase (PPL), and a mixture of PPL and papain, enhanced the production, making them more environmentally friendly with reduced production costs, increasing their interest. Conductivity, absorbance at 260 nm, viscosity, and ethidium bromide fluorescence displacement assays were performed to study the characteristics of these lipoaminoacids and their interactions with nucleic acids (RNA and DNA) regarding a potential vector gene delivery system. The Gibbs energy of micellization of lipoaminoacid biosynthesized with PPL was −27.7 kJ/mol, showing great efficiency in micelle formation. All the obtained lipoaminoacids showed successful encapsulation of RNA, demonstrating the potential of these compounds for vector gene therapy. Full article

One of the major challenges in gene therapy is the efficient and safe introduction of nucleic acids into eukaryotic cells. This process requires overcoming various biological barriers and navigating complex pathways to reach target cells and achieve their biological function. To address this obstacle, numerous transfection methods have been developed, including physical techniques and the use of genetic vectors, both viral and non-viral. However, to date, no transfection method is 100% safe and efficient. Within the spectrum of non-viral genetic vectors, cationic liposomes formed by cationic lipids stand out for their ability to protect and deliver therapeutic NA. These liposomes offer greater biocompatibility and lower immunogenicity compared to viral vectors, although they still do not match the efficiency of viral delivery systems. Consequently, ongoing research focuses on synthesizing a wide variety of cationic lipids in the search for compounds that provide high transfection efficiency with minimal cytotoxicity. This study aimed to design and synthesize a novel cationic lipid (CholCadLys) derived from natural cellular molecules for transferring genetic material to eukaryotic cells. The lipid was synthesized using cholesteryl chloroformate for the hydrophobic region, cadaverine as a linker, and lysine for the polar region, connected by carbamate and amide bonds, respectively. Identification was confirmed through thin-layer chromatography, purification through preparative chromatography, and characterization via infrared spectroscopy and mass spectrometry. The synthesis yielded a 60% success rate, with stable nanoliposomes averaging 76 nm in diameter. Liposomes were formed using this CL and commercial neutral lipids, characterized by transmission electron microscopy and Nanoparticle Tracking Analysis. These liposomes, combined with plasmid DNA, formed lipoplexes used to transfect Hek-293 FT cells, achieving up to 40% transfection efficiency without cytotoxicity in the mixture of CholCadLys and CholCad. This novel CL demonstrates potential as an efficient, safe, and cost-effective gene transfer system, facilitating further development in gene therapy. Full article

Cationic gemini surfactants have emerged as potential gene delivery agents as they can co-assemble with DNA due to a strong electrostatic association. Commonly, DNA complexation is enhanced by the inclusion of a helper lipid (HL), which also plays a key role in transfection efficiency. The formation of lipoplexes, used as non-viral vectors for transfection, through electrostatic and hydrophobic interactions is affected by various physicochemical parameters, such as cationic surfactant:HL molar ratio, (+/−) charge ratio, and the morphological structure of the lipoplexes. Herein, we investigated the DNA complexation ability of mixtures of serine-based gemini surfactants, (nSer)₂N5, and monoolein (MO) as a helper lipid. The micelle-forming serine surfactants contain long lipophilic chains (12 to 18 C atoms) and a five CH₂ spacer, both linked to the nitrogen atoms of the serine residues by amine linkages. The (nSer)₂N5:MO aggregates are non-cytotoxic up to 35–90 µM, depending on surfactant and surfactant/MO mixing ratio, and in general, higher MO content and longer surfactant chain length tend to promote higher cell viability. All systems efficaciously complex DNA, but the (18Ser)₂N5:MO one clearly stands as the best-performing one. Incorporating MO into the serine surfactant system affects the morphology and size distribution of the formed mixed aggregates. In the low concentration regime, gemini–MO systems aggregate in the form of vesicles, while at high concentrations the formation of a lamellar liquid crystalline phase is observed. This suggests that lipoplexes might share a similar bilayer-based structure. Full article

We propose a nucleic acids dilution-induced assembly (NADIA) method for the preparation of lipid nanoparticles. In the conventional method, water-soluble polymers such as nucleic acids and proteins are mixed in the aqueous phase. In contrast, the NADIA method, in which self-assembly is triggered upon dilution, requires dispersion in an alcohol phase without precipitation. We then investigated several alcohols and discovered that propylene glycol combined with sodium chloride enabled the dispersion of plasmid DNA and protamine sulfate in the alcohol phase. The streamlined characteristics of the NADIA method enable the preparation of extracellular vesicles-mimicking lipid nanoparticles (ELNPs). Among the mixing methods using a micropipette, a syringe pump, and a microfluidic device, the lattermost was the best for decreasing batch-to-batch differences in size, polydispersity index, and transfection efficiency in HepG2 cells. Although ELNPs possessed negative ζ-potentials and did not have surface antigens, their transfection efficiency was comparable to that of cationic lipoplexes. We observed that lipid raft-mediated endocytosis and macropinocytosis contributed to the transfection of ELNPs. Our strategy may overcome the hurdles linked to supply and quality owing to the low abundance and heterogeneity in cell-based extracellular vesicles production, making it a reliable and scalable method for the pharmaceutical manufacture of such complex formulations. Full article

The E6 and E7 oncoproteins of high-risk types of human papillomavirus (HR-HPV) are crucial for the development of cervical cancer (CC). Small interfering RNAs (siRNAs) are explored as novel therapies that silence these oncogenes, but their clinical use is hampered by inefficient delivery systems. Modification (pegylation) with polyethylene glycol (PEG) of liposomal siRNA complexes (siRNA lipoplexes) may improve systemic stability. We studied the effect of siRNA targeting HPV16 E6, delivered via cationic liposomes (lipoplexes), on cellular processes in a cervical carcinoma cell line (CaSki) and its potential therapeutic use. Lipoplexes-PEG-HPV16 E6, composed of DOTAP, Chol, DOPE, and DSPE-PEG2000 were prepared. The results showed that pegylation (5% DSPE-PEG2000) provided stable siRNA protection, with a particle size of 86.42 ± 3.19 nm and a complexation efficiency of over 80%; the siRNA remained stable for 30 days. These lipoplexes significantly reduced HPV16 E6 protein levels and restored p53 protein expression, inhibiting carcinogenic processes such as proliferation by 25.74%, migration (95.7%), and cell invasion (97.8%) at concentrations of 20 nM, 200 nM, and 80 nM, respectively. In conclusion, cationic lipoplexes-PEG-HPV16 E6 show promise as siRNA carriers for silencing HPV16 E6 in CC. Full article

Due to the proliferation-induced high demand of cancer cells for folic acid (FA), significant overexpression of folate receptors 1 (FR1) is detected in most cancers. To our knowledge, a detailed characterization of FR1 expression and regulation regarding therapeutic and diagnostic feasibilities in prostate cancer (PCa) has not been described. In the present study, cell cultures, as well as tissue sections, were analyzed using Western blot, qRT-PCR and immunofluorescence. In addition, we utilized FA-functionalized lipoplexes to characterize the potential of FR1-targeted delivery into PCa cells. Interestingly, we detected a high level of FR1-mRNA in healthy prostate epithelial cells and healthy prostate tissue. However, we were able to show that PCa cells in vitro and PCa tissue showed a massively enhanced FR1 membrane localization where the receptor can finally gain its function. We were able to link these changes to the overexpression of GPI–transamidase (GPI-T) by image analysis. PCa cells in vitro and PCa tissue show the strongest overexpression of GPI-T and thereby induce FR1 membrane localization. Finally, we utilized FA-functionalized lipoplexes to selectively transfer pDNA into PCa cells and demonstrate the therapeutic potential of FR1. Thus, FR1 represents a very promising candidate for targeted therapeutic transfer pathways in PCa and in combination with GPI-T, may provide predictive imaging in addition to established diagnostics. Full article

Over the past decade, mRNA-based therapy has displayed significant promise in a wide range of clinical applications. The most striking example of the leap in the development of mRNA technologies was the mass vaccination against COVID-19 during the pandemic. The emergence of large-scale technology and positive experience of mRNA immunization sparked the development of antiviral and anti-cancer mRNA vaccines as well as therapeutic mRNA agents for genetic and other diseases. To facilitate mRNA delivery, lipid nanoparticles (LNPs) have been successfully employed. However, the diverse use of mRNA therapeutic approaches requires the development of adaptable LNP delivery systems that can control the kinetics of mRNA uptake and expression in target cells. Here, we report effective mRNA delivery into cultured mammalian cells (HEK293T, HeLa, DC2.4) and living mouse muscle tissues by liposomes containing either 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) or the newly applied 1,30-bis(cholest-5-en-3β-yloxycarbonylamino)-9,13,18,22-tetraaza-3,6,25,28-tetraoxatriacontane tetrahydrochloride (2X7) cationic lipids. Using end-point and real-time monitoring of Fluc mRNA expression, we showed that these LNPs exhibited an unusually delayed (of over 10 h in the case of the 2X7-based system) but had highly efficient and prolonged reporter activity in cells. Accordingly, both LNP formulations decorated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀) provided efficient luciferase production in mice, peaking on day 3 after intramuscular injection. Notably, the bioluminescence was observed only at the site of injection in caudal thigh muscles, thereby demonstrating local expression of the model gene of interest. The developed mRNA delivery systems hold promise for prophylactic applications, where sustained synthesis of defensive proteins is required, and open doors to new possibilities in mRNA-based therapies. Full article

Rhabdomyosarcoma is a pediatric cancer associated with aggressiveness and a tendency to develop metastases. Fusion-negative rhabdomyosarcoma (FN-RMS) is the most commonly occurring subtype of RMS, where metastatic disease can hinder treatment success and decrease survival rates. RMS-derived exosomes were previously demonstrated to be enriched with miRNAs, including miR-1246, possibly contributing to disease aggressiveness. We aimed to decipher the functional impact of exosomal miR-1246 on recipient cells and its role in promoting aggressiveness. Treatment of normal fibroblasts with FN-RMS-derived exosomes resulted in a significant uptake of miR-1246 paired with an increase in cell proliferation, migration, and invasion. In turn, delivery of miR-1246-mimic lipoplexes promoted fibroblast proliferation, migration, and invasion in a similar manner. Conversely, when silencing miR-1246 in FN-RMS cells, the resulting derived exosomes demonstrated reversed effects on recipient cells’ phenotype. Delivery of exosomal miR-1246 targets GSK3β and promotes β-catenin nuclear accumulation, suggesting a deregulation of the Wnt pathway, known to be important in tumor progression. Finally, a pilot clinical study highlighted, for the first time, the presence of high exosomal miR-1246 levels in RMS patients’ sera. Altogether, our results demonstrate that exosomal miR-1246 has the potential to alter the tumor microenvironment of FN-RMS cells, suggesting its potential role in promoting oncogenesis. Full article

In this study, novel solid lipid particles containing the adjuvant lipid monophosphoryl lipid A (termed ‘SLN-A’) were synthesised. The SLN-A particles were able to efficiently bind and form complexes with a DNA vaccine encoding the urease alpha subunit of Helicobacter pylori. The resultant nanoparticles were termed lipoplex-A. In a mouse model of H. pylori infection, the lipoplex-A nanoparticles were used to immunise mice, and the resultant immune responses were analysed. It was found that the lipoplex-A vaccine was able to induce high levels of antigen-specific antibodies and an influx of gastric CD4⁺ T cells in vaccinated mice. In particular, a prime with lipoplex-A and a boost with soluble UreA protein induced significantly high levels of the IgG1 antibody, whereas two doses of lipoplex-A induced high levels of the IgG2c antibody. In this study, lipoplex-A vaccination did not lead to a significant reduction in H. pylori colonisation in a challenge model; however, these results point to the utility of the system for delivering DNA vaccine-encoded antigens to induce immune responses and suggest the ability to tailor those responses. Full article

In this study, the impact of different delivery systems on the cytokine-inducing, antiproliferative, and antitumor activities of short immunostimulatory double-stranded RNA (isRNA) was investigated. The delivery systems, consisting of the polycationic amphiphile 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20 tetraazahexacosan tetrahydrochloride (2X3), and the lipid-helper dioleoylphosphatidylethanolamine (DOPE), were equipped with polyethylene glycol lipoconjugates differing in molecular weight and structure. The main findings of this work are as follows: (i) significant activation of MCP-1 and INF-α, β, and γ production in CBA mice occurs under the action of isRNA complexes with liposomes containing lipoconjugates with long PEG chains, while activation of MCP-1 and INF-γ, but not INF-α or β, was observed under the action of isRNA lipoplexes containing lipoconjugates with short PEG chains; (ii) a pronounced antiproliferative effect on B16 melanoma cells in vitro, as well as an antitumor and hepatoprotective effect in vivo, was induced by isRNA pre-complexes with non-pegylated liposomes, while complexes containing lipoconjugates with long-chain liposomes were inactive; (iii) the antitumor activity of isRNA correlated with the efficiency of its accumulation in the cells and did not explicitly depend on the activation of cytokine and interferon production. Thus, the structure of the delivery system plays a vital role in determining the response to isRNA and allows for the choice of a delivery system depending on the desired effect. Full article

Liposomes and other nanoparticles have been widely studied as innovative nanomaterials because of their unique properties. Pyridinium salts, on the basis of 1,4-dihydropyridine (1,4-DHP) core, have gained significant attention due to their self-assembling properties and DNA delivery activity. This study aimed to synthesize and characterize original N-benzyl substituted 1,4-dihydropyridines and evaluate the influence on structure modifications on compound physicochemical and self-assembling properties. Studies of monolayers composed of 1,4-DHP amphiphiles revealed that the mean molecular areas values were dependent on the compound structure. Therefore, the introduction of N-benzyl substituent to the 1,4-DHP ring enlarged the mean molecular area by almost half. All nanoparticle samples obtained by ethanol injection method possessed positive surface charge and average diameter of 395–2570 nm. The structure of the cationic head-group affects the size of the formed nanoparticles. The diameter of lipoplexes formed by 1,4-DHP amphiphiles and mRNA at nitrogen/phosphate (N/P) charge ratios of 1, 2, and 5 were in the range of 139–2959 nm and were related to the structure of compound and N/P charge ratio. The preliminary results indicated that more prospective combination are the lipoplexes formed by pyridinium moieties containing N-unsubstituted 1,4-DHP amphiphile 1 and pyridinium or substituted pyridinium moieties containing N-benzyl 1,4-DHP amphiphiles 5a–c at N/P charge ratio of 5, which would be good candidates for potential application in gene therapy. Full article