Effective siRNA Research Articles

Single-cell encoded gene silencing for high-throughput combinatorial siRNA screening.

The use of combinatorial siRNAs shows great promise for drug discovery, but the identification of safe and effective siRNA combinations remains challenging. Here, we develop a massively multiplexed technology for systematic screening of siRNA-based cocktail therapeutics. We employ composite micro-carriers that are responsive to near infrared light and magnetic field to achieve photoporation-facilitated siRNA transfection to individual cells. Thus, randomized gene silencing by different siRNA formulations can be performed with high-throughput single-cell-based analyses. For screening anti-cancer siRNA cocktails, we test more than 1300 siRNA combinations for knocking down multiple genes related to tumor growth, discovering effective 3-siRNA formulations with an emphasis on the critical role of inhibiting Cyclin D1 and survivin, along with their complementary targets for synergic efficacy. This approach enables orders of magnitude reduction in time and cost associated with largescale siRNA screening, and resolves key insights to siRNA pharmacology that are not permissive to existing methods.

Design, synthesis, molecular dynamics and gene silencing studies of novel therapeutic HIF-1α siRNAs in hypoxic cancer cells

Hypoxia inducible factors (HIFs) are heterodimeric proteins that belong to a small group of transcription factors, which mainly regulates transcription of genes under hypoxic conditions. Particularly, oxygen sensing subunit of HIF-1α is a predominant subtype that heterodimerizes with oxygen-independent HIF-1β subunit, to trigger the transcription of hypoxia responsive genes. Due to poor supply of blood and rapid division of cancerous cells, tumor microenvironment exhibits low oxygen condition and therefore increased levels of HIF-1α. One of the promising therapeutic strategies to cancer is modulation of HIF-1α signaling pathway. Small interfering RNA (siRNA) mediated downregulation of HIF-1α has been reported to prevent growth and progression of various types of cancer and holds great promise in the cancer treatment. In this study, computational approaches were used to design potential siRNAs targeting HIF-1α and investigate their interaction with the human argonaute-2 (hAgo2). Molecular dynamic simulation of HIF-1α siRNAs-hAgo2 complexes revealed key interactions required for the efficient binding of guide strand to hAgo2 protein. Two siRNAs (S2 and S5) exhibiting strong binding with hAgo2 were further considered. Subsequently, we transfected the MCF-7 cell line with both standard HIF-1α and our designed siRNAs (S2 and S5). Following transfection, translation changes in the MCF-7 cells were assessed through western blotting. S2 and S5 efficiently reduced the expression of HIF-1α in hypoxic conditions. The aim of the present study is to understand the siRNA-hAgo2 interaction. It is also focused on the desiging of effective siRNA against HIF-1α.

Combinatorial Synthesis of Alkyl Chain-Capped Poly(β-Amino Ester)s for Effective siRNA Delivery.

Poly (β-amino ester) (PBAE) is a class of biodegradable polymers containing ester bonds in their main chain, extensively investigated as cationic polymer carriers for siRNA. Most current PBAE carriers rely on termination with hydrophilic or charged amines. In this study, a polymer platform consisting of 168 PBAE polymers with hydrophobic alkyl chain terminals is constructed through sequential aza-Michael addition. A large number of effective carriers are identified through in vitro screening of the PBAE platform for siLuc delivery to HeLa-Luc cells. Specifically, PA8-C6 and PA8-C8 achieve remarkable gene knockdown efficacies of up to 80% with low cytotoxicity. Certain materials from the PA2 and PA5 series demonstrate potent siRNA delivery capabilities associated with elevated cytotoxicity. The pKa value of PBAE is predominantly determined by the hydrophilic amine side chains rather than the end-capping groups. A pKa range of ≈6.2-6.5 may contribute to the excellent delivery capability for PA8 series carriers. The co-formulation of PBAE carriers with helper lipids leads to the reduced size and surface charges of the polyplex NPs with siRNA, consequently decreasing the cytotoxicity and enhancing siRNA delivery efficacy. These findings hold significant implications for the development of novel degradable polymer carriers for siRNA delivery.

Ionic liquid combined with cationic liposome co-delivers microphthalmia-associated transcription factor small interfering RNA to regulate melanogenesis

Suppressing allele-specific genes using small interfering RNAs (siRNAs) can effectively whiten skin by influencing cellular gene and protein expression. Topical delivery of siRNA is a promising alternative to injections for RNA interference. However, the barrier function of the skin hinders the effective penetration of siRNA. Here, we report, a novel approach to achieve the transdermal delivery of effective siRNA doses using a complementary synergistic strategy of an ionic liquid (IL) and cationic liposome (CL). Microphthalmia-associated transcription factor (MITF) siRNA molecules were formed through electrostatic adsorption of the IL and CL to form positively charged nanocomposites, which were named IL-CL/p-siM. IL-CL/p-siM has a particle size of 171.47 nm, ζ-potential of 29.94 mV, high encapsulation rate of 92.11 %, and pH-sensitive release properties. In vitro studies on porcine skin confirmed the additive/synergistic effect of this strategy in enhancing epidermal and dermal penetration. This combination enabled superior transfection efficiency and cell viability while inhibiting melanin synthesis in skin melanocytes by downregulating the expression of genes downstream of MITF, namely tyrosinase-related protein-1, tyrosinase, and tyrosinase-related protein-2, which are associated with the melanocortin 1 receptor. We also conducted clinical studies that demonstrated its potential in treating melasma and its anti-melanotic efficacy. To summarize, IL-CL/p-siM represents a simple, personalized, and scalable platform for effective local delivery of siRNA to treat skin complications.

Exosome-mediated delivery of siRNA molecules in cancer therapy: triumphs and challenges.

The discovery of novel and innovative therapeutic strategies for cancer treatment and management remains a major global challenge. Exosomes are endogenous nanoscale extracellular vesicles that have garnered increasing attention as innovative vehicles for advanced drug delivery and targeted therapy. The attractive physicochemical and biological properties of exosomes, including increased permeability, biocompatibility, extended half-life in circulation, reduced toxicity and immunogenicity, and multiple functionalization strategies, have made them preferred drug delivery vehicles in cancer and other diseases. Small interfering RNAs (siRNAs) are remarkably able to target any known gene: an attribute harnessed to knock down cancer-associated genes as a viable strategy in cancer management. Extensive research on exosome-mediated delivery of siRNAs for targeting diverse types of cancer has yielded promising results for anticancer therapy, with some formulations progressing through clinical trials. This review catalogs recent advances in exosome-mediated siRNA delivery in several types of cancer, including the manifold benefits and minimal drawbacks of such innovative delivery systems. Additionally, we have highlighted the potential of plant-derived exosomes as innovative drug delivery systems for cancer treatment, offering numerous advantages such as biocompatibility, scalability, and reduced toxicity compared to traditional methods. These exosomes, with their unique characteristics and potential for effective siRNA delivery, represent a significant advancement in nanomedicine and cancer therapeutics. Further exploration of their manufacturing processes and biological mechanisms could significantly advance natural medicine and enhance the efficacy of exosome-based therapies.

The impact of heparin and direct thrombin inhibitors on cell-penetrating polymer siRNA transfection.

Gene therapy using siRNA has become a promising strategy to achieve targeted gene knockdown for treatment of cardiovascular pathologies. However, efficient siRNA transfection often relies on cationic delivery vectors such as synthetic cell-penetrating polymers which are susceptible to interference by negatively charged molecules. Anticoagulants such as heparin, which is negatively charged and widely used in cardiovascular applications, may pose a significant barrier to effective siRNA delivery. We therefore conducted in vitro studies utilizing human smooth muscle and endothelial cells transfected with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β2-microglobulin (B2M) siRNA in the presence of heparin, argatroban, and bivalirudin in order to determine which anticoagulant therapy is most compatible for siRNA delivery. We observed that while heparin, at clinical doses, decreases the efficiency of siRNA targeted mRNA knockdown, mRNA knockdown is not inhibited in the presence of either argatroban or bivalirudin. Our data suggests that heparin should be avoided during siRNA therapy with cationic transfection agents, and argatroban and bivalirudin should be used in its stead.

ROS-Responsive Ferrocenyl Amphiphilic PAMAM Dendrimers for On-Demand Delivery of siRNA Therapeutics to Cancer Cells.

Small interfering RNA (siRNA) therapeutics, characterized by high specificity, potency, and durability, hold great promise in the treatment of cancer and other diseases. However, the clinic implementation of siRNA therapeutics critically depends on the safe and on-demand delivery of siRNA to the target cells. Here, we reported a family of ferrocenyl amphiphilic dendrimers (Fc-AmDs) for on-demand delivery of siRNA in response to the high ROS content in cancer cells. These dendrimers bear ROS-sensitive ferrocene moieties in the hydrophobic components and positively chargeable poly(amidoamine) dendrons as the hydrophilic entities, possessing favorable safety profiles and ROS responsive properties. One of these ferrocenyl amphiphilic dendrimers, Fc-C8-AmD 8A, outperforms in siRNA delivery, benefiting from its optimal balance of hydrophobicity and hydrophilicity. Its ROS feature facilitates specific and efficient disassembly of its complex with siRNA in ROS-rich cancer cells for effective siRNA delivery and gene silencing. Moreover, Fc-C8-AmD 8A also integrates the features and beneficial properties of both lipid and dendrimer vectors. Therefore, it represents a novel on-demand delivery system for cancer cell-specific siRNA delivery. This work opens new perspectives for designing self-assembly nanosystems for on-demand drug delivery.

Inhibition of SARS-CoV-2 Replication by Self-Assembled siRNA Nanoparticles Targeting Multiple Highly Conserved Viral Sequences.

Coronavirus infectious disease 2019 (COVID-19), caused by severe acute respiratory virus type 2 (SARS-CoV-2), has caused a global public health crisis. As an RNA virus, the high gene mutability of SARS-CoV-2 poses significant challenges to the development of broad-spectrum vaccines and antiviral therapeutics. There remains a lack of specific therapeutics directly targeting SARS-CoV-2. With the ability to efficiently inhibit the expression of target genes in a sequence-specific way, small interfering RNA (siRNA) therapy has exhibited significant potential in antiviral and other disease treatments. In this work, we presented a highly effective self-assembled siRNA nanoparticle targeting multiple highly conserved regions of SARS-CoV-2. The siRNA sequences targeting viral conserved regions were first screened and evaluated by their thermodynamic features, off-target effects, and secondary structure toxicities. RNA motifs including siRNA sequences were then designed and self-assembled into siRNA nanoparticles. These siRNA nanoparticles demonstrated remarkable uniformity and stability and efficiently entered cells directly through cellular endocytic pathways. Moreover, these nanoparticles effectively inhibited the replication of SARS-CoV-2, exhibiting a superior inhibitory effect compared to free siRNA. These results demonstrated that these self-assembled siRNA nanoparticles targeting highly conserved regions of SARS-CoV-2 represent highly effective antiviral candidates for the treatment of infections, and are promisingly effective against current and future viral variants.

Application of therapeutic nucleic acids and RNA interference to create products for personalised medicine

INTRODUCTION. Small interfering RNAs (siRNAs) are among the most promising types of therapeutic nucleic acids aiming at the inhibition of pathogenetically relevant gene expression through the RNA interference mechanism. However, the limited bioavailability and immunogenicity of siRNAs and imperfect delivery systems hinder the clinical potential and applicability of siRNA medicinal products.AIM. This study aimed to summarise recent advances in the development of siRNA medicinal products and the corresponding delivery systems, review clinical trial results, and outline future development prospects for these medicinal products.DISCUSSION. This article covers the molecular mechanisms underlying RNA interference, the considerations for siRNA development, and the techniques for effective siRNA delivery. The article dwells upon various systems for nucleic acid delivery to targeted cells. The most promising delivery systems are non-viral systems, including liposomes, exosomes, nanoparticles, polymers, cell-penetrating peptides, and GalNAc ligands. Their main advantages include their ease of complexation with nucleic acids, modification and functionalization potential, favourable safety profile, ability to cross biological barriers, and tropism to target tissues. The article summarises the information that has accumulated over the past few years in clinical trials of siRNA medicinal products for a range of conditions, including metabolic disorders, infections, and cancers, as well as hereditary, ophthalmic, renal, and hepatic diseases. Special attention is paid to siRNA medicinal products undergoing clinical trials (over 10 products) and approved for clinical use (6 products, including MIR 19, the first authorised Russian siRNA medicinal product).CONCLUSION. Ultimately, siRNA medicinal products are a promising tool for personalised medicine, exhibiting therapeutic potential for a wide range of pathological conditions. Further studies of siRNA medicinal products should aim at improving siRNA production technology to increase their bioavailability and half-life period. In addition, these studies should aim at enhancing delivery systems for these products to mitigate toxicity risks and maximise efficacy.

Dual Effect by Chemical Electron Transfer Enhanced siRNA Lipid Nanoparticles: Reactive Oxygen Species-Triggered Tumor Cell Killing Aggravated by Nrf2 Gene Silencing.

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.

Effect of a novel siRNA delivery system, siRNA ternary complex, on melanoma lung metastasis

In this study, we determined effects of an anionic siRNA delivery vector, siRNA ternary complex, which is constructed with biodegradable dendrigraft poly-L-lysine (DGL) and γ-polyglutamic acid (γ-PGA) on the melanoma cells and melanoma lung metastasis. The siRNA ternary complex showed high cellular uptake and silencing effect in melanoma cell line B16-F10/Luc cells. After intravenous administration of the siRNA ternary complex, high silencing effect was also observed in the lung of B16-F10/Luc melanoma metastasis model mice. Therefore, we applied vascular endothelial growth factor (VEGF)-siRNA on the siRNA ternary complex and determined the effect on the melanoma lung metastasis. The siRNA ternary complex containing VEGF-siRNA reduced VEGF protein levels significantly in in vitro and in vivo, and the complex successfully inhibited melanoma lung metastasis. This biodegradable and effective siRNA delivery vector, siRNA ternary complex, could be available for clinical trials.

NSP4 promotes replication of porcine reproductive and respiratory syndrome virus-2

Porcine reproductive and respiratory syndrome (PRRS) is one of the most detrimental contagious swine ailments worldwide. Currently, no effective drugs are available for its treatment. Targeting the structural and non-structural proteins (NSP) of the type 2 PRRS virus (PRRSV-2) with small interfering RNA (siRNA) is an effective approach to inhibit PRRSV replication. NSP4, which is highly conserved and possesses 3 C-like serine protease activity (3CLSP), can cleave PRRSV self-proteins, thereby contributing to viral replication. To investigate the mechanism by which NSP4 regulates PRRSV-2 replication and screen for effective siRNA inhibitors of PRRSV-2 replication, the recombinant plasmid pEGFP-C1-NSP4 was constructed, and a control siRNA pair and two siRNA pairs targeting the PRRSV-2 NSP4 gene (shRNA-ctr, shRNA-150, and shRNA-536) were synthesized and cloned into the pSilencer4.1-CMV vector. After 24 h of incubation, Marc-145 cells were transfected with recombinant plasmids, and subsequently infected with different PRRSV-2 (XH-GD, ZQ-GD, GDr180, and JXA1-R). Subsequently, the effects of NSP4 overexpression, shRNA on PRRSV-2 replication were evaluated by assessing cytopathic effects (CPE), TCID50, quantitative real-time PCR (qPCR), immunofluorescence assays (IFA), and Western blotting. The data from these CPE, TCID50, qPCR, and IFA experiments revealed that NSP4 overexpression significantly enhanced PRRSV-2 replication and shRNA targeting NSP4 can inhibit PRRSV-2 replication in Marc-145 cells, indicating that shRNA could serve as candidate molecules for fundamental research on PRRSV-2.

Targeted siRNA Therapy for Psoriasis: Translating Preclinical Potential into Clinical Treatments.

Psoriasis is a chronic inflammatory skin disease characterized by the excessive proliferation of keratinocytes and heightened immune activation. Targeting pathogenic genes through small interfering RNA (siRNA) therapy represents a promising strategy for the treatment of psoriasis. This mini-review provides a comprehensive summary of siRNA research targeting the pathogenesis of psoriasis, covering aspects such as keratinocyte function, inflammatory cell roles, preclinical animal studies, and siRNA delivery mechanisms. It details recent advancements in RNA interference that modulate key factors including keratinocyte proliferation (Fibroblast Growth Factor Receptor 2, FGFR2), apoptosis (Interferon Alpha Inducible Protein 6, G1P3), differentiation (Grainyhead Like Transcription Factor 2, GRHL2), and angiogenesis (Vascular Endothelial Growth Factor, VEGF); immune cell infiltration and inflammation (Tumor Necrosis Factor-Alpha, TNF-α; Interleukin-17, IL-17); and signaling pathways (JAK-STAT, Nuclear Factor Kappa B, NF-κB) that govern immunopathology. Despite significant advances in siRNA-targeted treatments for psoriasis, several challenges persist. Continued scientific developments promise the creation of more effective and safer siRNA medications, potentially enhancing the quality of life for psoriasis patients and revolutionizing treatments for other diseases. This article focuses on the most recent research advancements in targeting the pathogenesis of psoriasis with siRNA and explores its future therapeutic prospects.

Poly(β-aminoester) Physicochemical Properties Govern the Delivery of siRNA from Electrostatically Assembled Coatings.

Localized short interfering RNA (siRNA) therapy has the potential to drive high-specificity molecular-level treatment of a variety of disease states. Unfortunately, effective siRNA therapy suffers from several barriers to its intracellular delivery. Thus, drug delivery systems that package and control the release of therapeutic siRNAs are necessary to overcome these obstacles to clinical translation. Layer-by-layer (LbL) electrostatic assembly of thin film coatings containing siRNA and protonatable, hydrolyzable poly(β-aminoester) (PBAE) polymers is one such drug delivery strategy. However, the impact of PBAE physicochemical properties on the transfection efficacy of siRNA released from LbL thin film coatings has not been systematically characterized. In this study, we investigate the siRNA transfection efficacy of four structurally similar PBAEs in vitro. We demonstrate that small changes in structure yield large changes in physicochemical properties, such as hydrophobicity, pKa, and amine chemical structure, driving differences in the interactions between PBAEs and siRNA in polyplexes and in LbL thin film coatings for wound dressings. In our polymer set, Poly3 forms the most stable interactions with siRNA (Keff,w/w = 0.298) to slow release kinetics and enhance transfection of reporter cells in both colloidal and thin film coating approaches. This is due to its unique physiochemical properties: high hydrophobicity (clog P = 7.86), effective pKa closest to endosomal pH (pKa = 6.21), and high cooperativity in buffering (nhill = 7.2). These properties bestow Poly3 with enhanced endosomal buffering and escape properties. Taken together, this work elucidates the connections between small changes in polymer structure, emergent properties, and polyelectrolyte theory to better understand PBAE transfection efficacy.

Metal-organic framework-mediated siRNA delivery and sonodynamic therapy for precisely triggering ferroptosis and augmenting ICD in osteosarcoma

The complex genomics, immunosuppressive tumor microenvironment (TME), and chemotherapeutic resistance of osteosarcoma (OS) have resulted in limited therapeutic effects in the clinic. Ferroptosis is involved in tumor progression and is regulated mainly by glutathione peroxidase 4 (GPX4). Small interfering RNA (siRNA)-based RNA interference (RNAi) can precisely target any gene. However, achieving effective siRNA delivery is highly challenging. Here, we fabricated a TME-responsive metal-organic framework (MOF)-based biomimetic nanosystem (mFeP@si) with siGPX4 delivery and sonodynamic therapy (SDT) to treat OS by targeting ferroptosis. Under ultrasound (US) irradiation, mFeP@si achieves lysosomal escape via singlet oxygen (1O2)-mediated lysosomal membrane disruption and then accelerates ROS generation and glutathione (GSH) depletion. Meanwhile, siGPX4 silences GPX4 expression by binding to GPX4 mRNA and leads to the accumulation of toxic phospholipid hydroperoxides (PL-OOH), further magnifying the ROS storm and triggering ferroptosis. Notably, synergistic therapy remarkably enhances antitumor effects, improves the immunosuppressive TME by inducing potent immunogenic cell death (ICD), and increases the sensitivity of chemotherapy-resistant OS cells to cisplatin. Overall, this novel nanosystem, which targets ferroptosis by integrating RNAi and SDT, exhibits strong antitumor effects both in vitro and in vivo, providing new insights for treating OS.

Abstract 7238: Exploring the potential of targeted DNA Nanobot delivery systems for gene delivery

Abstract Gene therapy is a cornerstone of modern medicine's quest for precision treatment to cure human disease including cancer. The current approach to gene delivery technology employs viral vector delivery systems. While effective, these vectors present significant challenges including immunogenicity, safety concerns due to off-target non-tissue specific delivery, payload limitations, and significant cost ultimately diluting their therapeutic potential. Thus, novel approaches to gene delivery are necessary. One such approach to address these critical limitations involves DNA Nanobots, a DNA-based nanotechnology that allows for a tissue-/target-cell specific gene delivery system. Utilizing DNA origami molecular assembly techniques, our DNA Nanobots offer a customizable and highly precise delivery platform, free from viral vector constraints. This innovative approach has the potential to transform the landscape of gene therapy by providing a safer, more efficient, and targeted alternative. Key technical advancements with our DNA Nanobots Gene Delivery Platform include: •CRISPR Cas-9 Integration: By incorporating CRISPR Cas-9 functional proteins, our nanobots were shown to enhance gene editing precision in primary human T cells, broadening therapeutic applications across various genetic conditions. •Targeted mRNA Delivery: Our nanobots revealed promising results in the targeted delivery of mRNA, vital for vaccine development and personalized treatments. •Efficient antisense/siRNA Delivery: Prior studies have overcome traditional challenges including endosomal escape and cytoplasmic entry, demonstrating effective siRNA delivery critical for gene silencing therapeutic development. In summary, DNA Nanobots mark a paradigm shift in gene therapy. Their ability to deliver various nucleic acids with high specificity and efficiency addresses many of the current methodological limitations especially off-target safety concerns and high cost. This advancement not only enhances the therapeutic index of existing treatments but also opens avenues for novel therapeutic strategies, potentially reshaping the future of molecular medicine and gene therapy. Citation Format: Melika Shahhoessini, Jeffrey R. Spitzner, Christopher R. Lucas, Carlos E. Castro, Patrick D. Halley. Exploring the potential of targeted DNA Nanobot delivery systems for gene delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7238.

Development and Characterization of Modified Chitosan Lipopolyplex for an Effective siRNA Delivery.

Cytotoxicity, speedy degradation, and limited cellular absorption are the foremost features influencing the successful delivery of RNAs. Chitosan (Cs) is a polymer that offers an advantage due to its bio-compatibility and biodegradable nature, making it an ideal polycationic vector for delivering siRNA. In this study, chitosan has been modified with arginine in order to increase its encapsulation of siRNA and improve cellular absorption. It was discovered that arginine and guanidino moieties could transport through membranes of cells and play an important part in membrane permeability. FTIR and 13C NMR were used to characterize the complex. These chitosan-arginine (CsAr) siRNA complexes are further encapsulated in anionic DPPC/cholesterol liposomes to combine the effects of liposome-chitosan-arginine complexes called lipopolyplexes (LCAr). Formed LCAr were investigated for their lipid/CsAr-siRNA ratios, size, zeta-potential, heparin, and serum RNase stability by agarose gel retardation, and cell uptake efficiency compared to their "parent" polyplexes. Results revealed complete lipidation of CsAr-siRNA polyplexes at lipid mass ratio 10 resulting in lipopolyplexes in the 120 to 230nm range. Polyplex entrapped ~70% of siRNA, whereas lipidation increases siRNA encapsulationto ~95%. Developed LCAr showed ~4 times less hemolytic potential as compared to the parent polyplexes at the highest siRNA dose. The CsAr-siRNA and its lipid-coated form showed enhanced cellular association as compared to the marketed Lipofectamine 2000 proving its effectiveness in siRNA delivery. CsAr-liposome conjugation is simple and safe, and serves as a robust carrier for gene transport in physiological situations without compromising transfection efficacy.

Flash nanoprecipitation assisted self-assembly of ionizable lipid nanoparticles for nucleic acid delivery.

Ionizable lipid nanoparticles (LNPs) have emerged as a powerful tool for the intracellular delivery of nucleic acids. Following the recent success of LNP-based siRNA therapeutics and mRNA vaccines, the use of ionizable lipids for nucleic acid delivery has tremendously increased. Here, we introduce a flash nanoprecipitation (FNP) approach using the confined impingement (CIJ) mixer to stably self-assemble ionizable LNPs. To validate this approach, we employed three clinically relevant LNP formulations containing SM102, ALC0315, and DLin-MC3-DMA as ionizable lipids. FNP-assembled LNPs showed >95% encapsulation efficiency of mRNA and siRNA payloads and particle sizes below 150 nm. SM102 or ALC0315 LNPs demonstrated efficient delivery of mRNA into immune cells in vitro and to lymphoid organs in vivo, whereas Dlin-MC3-DMA LNPs allowed effective intracellular siRNA delivery with great functional ability. The FNP technique could economically produce LNPs in smaller volumes that are highly suitable for the discovery phase.

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.

Effect of inhibiting PDHα1 gene expression on the metabolism of fatty liver cells

PDHα1 gene encodes catalytic subunit PDHE1α of pyruvate dehydrogenase (PDH) complex. Based on previous studies, it is hypothesized that inhibition of PDH activity prevents the entry of glycolytic pyruvate into TCA cycle, while promotes fatty acid oxidation and reduces liver triglyceride (TG) level, thereby alleviating nonalcoholic fatty liver disease (NAFLD). In this study, an in vitro model for NAFLD was established with medical fat emulsion; the most effective siRNA for PDHα1 gene was screened out by qPCR technology; the alterations in metabolism of glucose and lipid, and structure & function of mitochondria in the NAFLD cells were primarily evaluated after transfecting PDHα1 siRNA. As the results showed, after inhibiting the expression of PDHα1 gene, glucose level in culture medium was time-dependently increased, and LDH activity in the cells was moderately elevated after 24 h of transfection and then returned to the normal level after 48 h; intracellular TG level was decreased while LPS activity was increased in a time-dependent manner; no significant change in mitochondrial structure was observed with or without siRNA transfection, and ATP content was obviously reduced after 24 h of transfection, followed by restoration after 48 h. It can be concluded that inhibiting PDHα1 gene in fatty liver cells enhances lipid degradation, and represses the utilization of glucose to an extent, thus reducing TG level without impacting energy generation required for cell survival.