Search Results (36)

Background: Transfection is vital for gene therapy, mRNA treatments, CAR-T cell therapy, and regenerative medicine. While viral vectors are effective, non-viral systems like lipid nanoparticles (LNPs) offer safer, more flexible alternatives. This work explores emerging non-viral transfection technologies to improve delivery efficiency and therapeutic outcomes. Methods: This review synthesizes the current literature and recent advancements in non-viral transfection technologies. It focuses on the mechanisms, advantages, and limitations of various delivery systems, including lipid nanoparticles, biodegradable polymers, electroporation, peptide-based carriers, and microfluidic platforms. Comparative analysis was conducted to evaluate their performance in terms of transfection efficiency, cellular uptake, biocompatibility, and potential for clinical translation. Several academic search engines and online resources were utilized for data collection, including Science Direct, PubMed, Google Scholar Scopus, the National Cancer Institute’s online portal, and other reputable online databases. Results: Non-viral systems demonstrated superior performance in delivering mRNA, siRNA, and antisense oligonucleotides, particularly in clinical applications. Biodegradable polymers and peptide-based systems showed promise in enhancing biocompatibility and targeted delivery. Electroporation and microfluidic systems offered precise control over transfection parameters, improving reproducibility and scalability. Collectively, these innovations address key challenges in gene delivery, such as stability, immune response, and cell-type specificity. Conclusions: The continuous evolution of transfection technologies is pivotal for advancing gene and cell-based therapies. Non-viral delivery systems, particularly LNPs and emerging platforms like microfluidics and biodegradable polymers, offer safer and more adaptable alternatives to viral vectors. These innovations are critical for optimizing therapeutic efficacy and enabling personalized medicine, immunotherapy, and regenerative treatments. Future research should focus on integrating these technologies to develop next-generation transfection platforms with enhanced precision and clinical applicability. Full article

Background: Lipid nanoparticles (LNPs) represent one of the most effective non-viral vectors for nucleic acid delivery and have demonstrated clinical success in siRNA therapies and mRNA vaccines. While considerable research has focused on optimizing ionizable lipids and helper lipids, the impact of PEGylated lipid content on LNP-mediated mRNA delivery, especially in terms of in vitro transfection efficiency and in vivo performance, remains insufficiently understood. Methods: In this study, LNPs were formulated using a self-synthesized ionizable lipid and varying molar ratios of DMG-PEG2000. Nanoparticles were prepared via nanoprecipitation, and their physicochemical properties, mRNA encapsulation efficiency, cellular uptake, and transfection efficiency were evaluated in HeLa and DC2.4 cells. In vivo delivery efficiency and organ distribution were assessed in mice following intravenous administration. Results: The PEGylated lipid content exerted a significant influence on both the in vitro and in vivo performance of LNPs. A bell-shaped relationship between PEG content and transfection efficiency was observed: 1.5% DMG-PEG2000 yielded optimal mRNA transfection in vitro, while 5% DMG-PEG2000 resulted in the highest transgene expression in vivo. This discrepancy in optimal PEG content may be attributed to the trade-off between cellular uptake and systemic circulation: lower PEG levels enhance cellular internalization, whereas higher PEG levels improve stability and in vivo bioavailability at the expense of cellular entry. Furthermore, varying the PEG-lipid content enabled the partial modulation of organ distribution, offering a formulation-based strategy to influence biodistribution without altering the ionizable lipid structure. Conclusions: This study highlights the critical role of PEGylated lipid content in balancing nanoparticle stability, cellular uptake, and in vivo delivery performance. Our findings provide valuable mechanistic insights and suggest a straightforward formulation-based strategy to optimize LNP/mRNA systems for therapeutic applications. Full article

Background: RNA interference (RNAi) is a powerful tool that can target many proteins without the expensive and time-consuming drug development studies. However, due to the challenges in delivering RNA molecules, the potential impact of RNAi approaches is yet to be fully realized in clinical settings. Lipid nanoparticles (LNPs) have been the most successful delivery system for nucleic acids, but targeted delivery to a solid tumor still eludes the developed LNPs. We hypothesized that specially designed low-molecular-weight PEIs can partially or completely replace the ionizable lipids for more accommodating vehicles due to the structural flexibility offered by polymers, which could lead to safer and more efficient nucleic acid delivery. Methods: To achieve this, we first optimized the LNP formulations as a point of reference for three outcomes: cellular uptake, cytotoxicity, and silencing efficiency. Using a response surface methodology (Design Expert), we optimized siRNA delivery by varying mole fractions of lipid components. Leveraging the optimal LNP formulation, we integrated specifically designed cationic polymers as partial or complete replacements for the ionizable lipid. This methodological approach, incorporating optimal combined designs and response surface methodologies, refined the LPNPs to an optimal efficiency. Results: Our data revealed that DOPE and Dlin-MC3-DMA contributed to higher efficiency in selected breast cancer cells over DSPC and ALC-0315 as neutral and ionizable lipids, respectively, based on the software analysis and direct comparative experiments. Incorporation of selected polymers enhanced the cellular internalization significantly, which in some formulations resulted in higher efficiency. Conclusions: These findings offer a framework for the rational design of LPNPs, that could enhance the passive targeting and silencing efficiency in cancer treatment and broader applications for RNAi-based strategies. Full article

The overdose of acetaminophen (APAP) has become the leading cause of acute liver failure in the United States and some Western countries. As a principal member of the cytochrome P450 enzymes (CYPs), CYP2E1 is a vital enzyme in regard to the production of toxic APAP metabolites and in the development of APAP-induced liver injury (AILI). In this study, we investigated the therapeutic effects and mechanisms of lipid nanoparticle (LNP)-based delivery of small interfering RNA targeting Cyp2e1 (si-Cyp2e1 LNPs) on AILI in mice. C57BL/6J male mice were injected with 300 mg/kg APAP to establish an AILI model, and si-Cyp2e1 LNPs were administered via the tail vein. The results showed that the levels of serum alanine aminotransferase and aspartate aminotransferase were lower than those in APAP mice after treatment with si-Cyp2e1 LNPs immediately. Moreover, si-Cyp2e1 LNPs significantly inhibited liver necrosis and oxidative stress in APAP mice. RNA sequencing revealed that si-Cyp2e1 LNPs exerted regulatory effects on pathways and genes related to peroxisome proliferator-activated receptor (PPAR). Consistent with this finding, we also proved that si-Cyp2e1 LNPs markedly regulated the expressions of genes involved in the PPAR signaling pathway (CYP4A, PPARα, FABP 1, and CD36) in APAP mice, as well as inflammatory factors (Il-6, Il-1β, and Tnf-α). These findings suggested that si-Cyp2e1 LNPs may alleviate APAP-induced oxidative stress and inflammation by regulating lipid metabolism via PPAR-related pathways. Full article

Background/Objectives: Transforming Growth Factor-beta (TGFβ1) plays a core role in the process of pulmonary fibrosis (PF). The progression of pulmonary fibrosis can be alleviated by siRNA-based inhibiting TGF-β1. However, the limitations of naked siRNA lead to the failure of achieving therapeutic effect. This study aimed to design lipid nanoparticles (LNPs) that can deliver siTGF-β1 to the lungs for therapeutic purposes. Methods: The cytotoxicity and transfection assay in vitro were used to screen ionizable lipids (ILs). Design of Experiments (DOE) was used to obtain novel LNPs that can enhance resistance to atomization shear forces. Meanwhile, the impact of LNPs encapsulating siTGF-β1 (siTGFβ1-LNPs) on PF was investigated. Results: When DLin-DMA-MC3 (MC3) was used as the ILs, the lipid phase ratio was MC3:DSPC:DMG-PEG2000:cholesterol = 50:10:3:37, and N/P = 3.25; the siTGFβ1-LNPs could be stably delivered to the lungs via converting the siTGFβ1-LNPs solution into an aerosol (atomization). In vitro experiments have confirmed that siTGFβ1-LNPs have high safety, high encapsulation, and can promote cellular uptake and endosomal escape. In addition, siTGFβ1-LNPs significantly reduced inflammatory infiltration and attenuated deposition of extracellular matrix (ECM) and protected the lung tissue from the toxicity of bleomycin (BLM) without causing systemic toxicity. Conclusions: The siTGFβ1-LNPs can be effectively delivered to the lungs, resulting in the silencing of TGF-β1 mRNA and the inhibition of the epithelial–mesenchymal transition pathway, thereby delaying the process of PF, which provides a new method for the treatment and intervention of PF. Full article

Lipid nanoparticles (LNPs) have shown promise as a delivery system for nucleic acid-based therapeutics, including DNA, siRNA, and mRNA vaccines. The immune system plays a critical role in the response to these nanocarriers, with innate immune cells initiating an early response and adaptive immune cells mediating a more specific reaction, sometimes leading to potential adverse effects. Recent studies have shown that the innate immune response to LNPs is mediated by Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs), which recognize the lipid components of the nanoparticles. This recognition can trigger the activation of inflammatory pathways and the production of cytokines and chemokines, leading to potential adverse effects such as fever, inflammation, and pain at the injection site. On the other hand, the adaptive immune response to LNPs appears to be primarily directed against the protein encoded by the mRNA cargo, with little evidence of an ongoing adaptive immune response to the components of the LNP itself. Understanding the relationship between LNPs and the immune system is critical for the development of safe and effective nucleic acid-based delivery systems. In fact, targeting the immune system is essential to develop effective vaccines, as well as therapies against cancer or infections. There is a lack of research in the literature that has systematically studied the factors that influence the interaction between LNPs and the immune system and further research is needed to better elucidate the mechanisms underlying the immune response to LNPs. In this review, we discuss LNPs’ composition, physico-chemical properties, such as size, shape, and surface charge, and the protein corona formation which can affect the reactivity of the immune system, thus providing a guide for the research on new formulations that could gain a favorable efficacy/safety profile. Full article

Nucleic acid therapeutics are promising alternatives to conventional anti-cancer therapy, such as chemotherapy and radiation therapy. While conventional therapies have limitations, such as high side effects, low specificity, and drug resistance, nucleic acid therapeutics work at the gene level to eliminate the cause of the disease. Nucleic acid therapeutics treat diseases in various forms and using different mechanisms, including plasmid DNA (pDNA), small interfering RNA (siRNA), anti-microRNA (anti-miR), microRNA mimics (miRNA mimic), messenger RNA (mRNA), aptamer, catalytic nucleic acid (CNA), and CRISPR cas9 guide RNA (gRNA). In addition, nucleic acids have many advantages as nanomaterials, such as high biocompatibility, design flexibility, low immunogenicity, small size, relatively low price, and easy functionalization. Nucleic acid therapeutics can have a high therapeutic effect by being used in combination with various nucleic acid nanostructures, inorganic nanoparticles, lipid nanoparticles (LNPs), etc. to overcome low physiological stability and cell internalization efficiency. The field of nucleic acid therapeutics has advanced remarkably in recent decades, and as more and more nucleic acid therapeutics have been approved, they have already demonstrated their potential to treat diseases, including cancer. This review paper introduces the current status and recent advances in nucleic acid therapy for anti-cancer treatment and discusses the tasks and prospects ahead. Full article

Drug development costs can be significantly reduced if proven “platform” technologies are allowed to be used without having to validate their use. The most recent US Food and Drug Administration (FDA) guideline brings more clarity, as well as a greater focus on the most complex technologies that can now be used for faster drug development. The FDA has highlights the use of lipid nanoparticles (LNPs) to package and deliver mRNA vaccines, gene therapy, and short (2–20 length) synthetic nucleotides (siRNA). Additionally, monoclonal antibody cell development is targeted. The FDA provides a systematic process of requesting platform status to benefit from its advantages. It brings advanced science and rationality into regulatory steps for the FDA’s approval of drugs and biologicals. Full article

The prevalence of excessive drinking-related alcoholic liver disease (ALD) is rising, yet therapeutic options remain limited. High alcohol consumption and consequent oxidative metabolism by cytochrome P450 (CYP) can lead to extremely high levels of reactive oxygen species, which overwhelm cellular defenses and harm hepatocytes. Our previous investigations showed that inhibiting Cyp2e1 using RNA interference reduced the incidence of ALD. However, compensatory mechanisms other than CYP2E1 contribute to oxidative stress in the liver. Therefore, we coupled triple siRNA lipid nanoparticles (LNPs) targeting Cyp2e1 with two isoenzymes Cyp4a10 and Cyp4a14 to treat ALD mouse models fed with Lieber–Decarli ethanol liquid diet for 12 weeks at the early (1st week), middle (5th week), and late (9th week) stages. The administration of triple siRNA LNPs significantly ameliorated chronic alcoholic liver injury in mice, and early treatment achieved the most profound effects. These effects can be attributed to a reduction in oxidative stress and increased expression of antioxidant genes, including Gsh-Px, Gsh-Rd, and Sod1. Moreover, we observed the alleviation of inflammation, evidenced by the downregulation of Il-1β, Il-6, Tnf-α, and Tgf-β, and the prevention of excessive lipid synthesis, evidenced by the restoration of the expression of Srebp1c, Acc, and Fas. Finally, triple siRNA treatment maintained normal metabolism in lipid oxidation. In brief, our research examined the possible targets for clinical intervention in ALD by examining the therapeutic effects of triple siRNA LNPs targeting Cyp2e1, Cyp4a10, and Cyp4a14. The in vivo knockdown of the three genes in this study is suggested as a promising siRNA therapeutic approach for ALD. Full article

Therapeutic HPV vaccines that induce potent HPV-specific cellular immunity and eliminate pre-existing infections remain elusive. Among various candidates under development, those based on DNA constructs are considered promising because of their safety profile, stability, and efficacy. However, the use of electroporation (EP) as a main delivery method for such vaccines is notorious for adverse effects like pain and potentially irreversible muscle damage. Moreover, the requirement for specialized equipment adds to the complexity and cost of clinical applications. As an alternative to EP, lipid nanoparticles (LNPs) that are already commercially available for delivering mRNA and siRNA vaccines are likely to be feasible. Here, we have compared three intramuscular delivery systems in a preclinical setting. In terms of HPV-specific cellular immune responses, mice receiving therapeutic HPV DNA vaccines encapsulated with LNP demonstrated superior outcomes when compared to EP administration, while the naked plasmid vaccine showed negligible responses, as expected. In addition, SM-102 LNP M exhibited the most promising results in delivering candidate DNA vaccines. Thus, LNP proves to be a feasible delivery method in vivo, offering improved immunogenicity over traditional approaches. Full article

Insufficient endosomal escape presents a major hurdle for successful nucleic acid therapy. Here, for the first time, a chemical electron transfer (CET) system was integrated into small interfering RNA (siRNA) lipid nanoparticles (LNPs). The CET acceptor can be chemically excited using the generated energy between the donor and hydrogen peroxide, which triggers the generation of reactive oxygen species (ROS), promoting endosomal lipid membrane destabilization. Tetra-oleoyl tri-lysino succinoyl tetraethylene pentamine was included as an ionizable lipopeptide with a U-shaped topology for effective siRNA encapsulation and pH-induced endosomal escape. LNPs loaded with siRNA and CET components demonstrated a more efficient endosomal escape, as evidenced by a galectin-8-mRuby reporter; ROS significantly augmented galectin-8 recruitment by at least threefold compared with the control groups, with a p value of 0.03. Moreover, CET-enhanced LNPs achieved a 24% improvement in apoptosis level by knocking down the tumor-protective gene nuclear factor erythroid 2-related factor 2, boosting the CET-mediated ROS cell killing. Full article

The aim of this study was to explore the efficacy and safety of TGFβ1 siRNA lipid nanoparticles (LNPs) modified with different PEG derivatives (PEG5000 cholesterol, abbreviated as CE; tocopherol polyethylene glycol 1000 succinate, abbreviated as TPGS) in the treatment of paclitaxel-resistant non-small-cell lung cancer. Three kinds of TGFβ1 siRNA LNPs were prepared via microfluidics technology, using different PEG derivatives and dosages (CE1.5, CE2.5, TPGS2.5) as variables. Their particle size, zeta potential, contents, and encapsulation efficiencies were determined. The inhibition of TGFβ1 mRNA and protein expression and the effects of the three kinds of LNPs on the proliferation of paclitaxel-resistant non-small-cell lung cancer cells (A549/T cell) were characterized. The distributions of the three siRNA LNPs in nude mice bearing A549/T tumors, especially at the tumor site, were observed using in vivo mouse imaging technology, and their corresponding efficacies were evaluated. The average particle size of the three kinds of TGFβ1 siRNA LNPs was about 70–80 nm, and they were capable of charge flipping. All three siRNA LNPs could effectively inhibit the expression of TGFβ1 mRNA and protein in A549/T cells and inhibit the proliferation of A549/T cells in vitro. The results of in vivo mice imaging showed that the three kinds of siRNA LNPs, when labeled with cypate, retain strong fluorescence in the tumor at 24 h. The pharmacodynamic results, such as for relative tumor volumes and tumor inhibition rates, reveal that TGFβ1 siRNA LNPs modified with CE1.5, CE2.5, or TPGS2.5 can be used to effectively treat paclitaxel-resistant lung adenocarcinoma. The histopathological results showed that the three kinds of LNPs have a certain toxicity but are relatively safe compared to common forms of chemotherapy such as cabazitaxel. TGFβ1 siRNA LNPs modified with CE1.5, CE2.5, and TPGS2.5 can inhibit TGFβ1 mRNA and protein expression in A549/T cells in vitro and can accumulate and play a role in the tumor tissue of nude mice, features that can be exploited for treating paclitaxel-resistant lung adenocarcinoma. Full article

Substrate reduction therapy (SRT) has been proposed as a new gene therapy for Fabry disease (FD) to prevent the formation of globotriaosylceramide (Gb3). Nanomedicines containing different siRNA targeted to Gb3 synthase (Gb3S) were designed. Formulation factors, such as the composition, solid lipid nanoparticles (SLNs) preparation method and the incorporation of different ligands, such as gold nanoparticles (GNs), protamine (P) and polysaccharides, were evaluated. The new siRNA–golden LNPs were efficiently internalized in an FD cell model (IMFE-1), with GNs detected in the cytoplasm and in the nucleus. Silencing efficacy (measured by RT-qPCR) depended on the final composition and method of preparation, with silencing rates up to 90% (expressed as the reduction in Gb3S-mRNA). GNs conferred a higher system efficacy and stability without compromising cell viability and hemocompatibility. Immunocytochemistry assays confirmed Gb3S silencing for at least 15 days with the most effective formulations. Overall, these results highlight the potential of the new siRNA–golden LNP system as a promising nanomedicine to address FD by specific SRT. Full article

Breast cancer is the most frequently diagnosed cancer among women. Breast cancer is also the key reason for worldwide cancer-related deaths among women. The application of small interfering RNA (siRNA)-based drugs to combat breast cancer requires effective gene silencing in tumor cells. To overcome the challenges of drug delivery to tumors, various nanosystems for siRNA delivery, including lipid-based nanoparticles that protect siRNA from degradation for delivery to cancer cells have been developed. These nanosystems have shown great potential for efficient and targeted siRNA delivery to breast cancer cells. Lipid-based nanosystems remain promising as siRNA drug delivery carriers for effective and safe cancer therapy including breast cancer. Lipid nanoparticles (LNPs) encapsulating siRNA enable efficient and specific silencing of oncogenes in breast tumors. This review discusses a variety of lipid-based nanosystems including cationic lipids, sterols, phospholipids, PEG-lipid conjugates, ionizable liposomes, exosomes for effective siRNA drug delivery to breast tumors, and the clinical translation of lipid-based siRNA nanosystems for solid tumors. Full article

The lipid nanoparticle (LNP) is one of the promising nanotechnologies for the delivery of RNA molecules, such as small interfering RNA (siRNA) and messenger RNA (mRNA). A series of LNPs that contain an mRNA encoding the antigen protein of SARS-CoV-2 were already approved as RNA vaccines against this infectious disease. Since LNP formulations are generally metastable, their physicochemical properties are expected to shift toward a more stable state during the long-time storage of suspensions. The current mRNA vaccines are supplied in the form of frozen formulations with a cryoprotectant for preventing deterioration. They must be stored in a freezer at temperatures from −80 °C to −15 °C. It is thought that therapeutic applications of this mRNA-LNP technology could be accelerated if a new formulation that permits mRNA-LNPs to be stored under milder conditions were available. We previously reported on a one-pot method for producing siRNA-encapsulated LNPs by combining freeze-drying technology with the conventional alcohol dilution method (referred to herein as the “alcohol dilution–lyophilization method”). In this study, this method was applied to the preparation of mRNA-LNPs to provide a freeze-dried formulation of mRNA LNPs. The resulting formulation can be stored at 4 °C for at least 4 months. Full article