Escape Of siRNA Research Articles

Functional Ginger-Derived Extracellular Vesicles-Coated ZIF-8 Containing TNF-α siRNA for Ulcerative Colitis Therapy by Modulating Gut Microbiota.

Tumor necrosis factor-α (TNF-α) plays a causal role in the pathogenesis of ulcerative colitis (UC), and anti-TNF-α siRNA shows great promise in UC therapy. However, delivering siRNA with site-targeted stability and therapeutic efficacy is still challenging due to the complex and dynamic intestinal microenvironment. Here, based on the functional plant-derived ginger extracellular vesicles (EVs) and porous ZIF-8 nanoparticles, we propose a novel TNF-α siRNA delivery strategy (EVs@ZIF-8@siRNA) for UC targeted therapy. Ginger EVs show strong colon and macrophage targeting, as well as robust resistance to acidic degradation in the stomach. Moreover, 6-shogaol in ginger-derived EVs displays anti-inflammatory effects, which enhance the treatment efficiency by cooperation with TNF-α siRNA. In vitro experiments reveal that ZIF-8 nanoparticles have high TNF-α siRNA loading capacity and promote siRNA escape from cellular lysosomes. In vivo experiments show that the TNF-α level is reduced more significantly in colonic tissue than other nontargeted inflammation related factors, showing a good targeting of this composite nanoparticle. Furthermore, gut microbiota sequencing results demonstrate that the nanoparticles can promote intestinal barrier repair by regulating the intestinal microbial balance and restoring the intestinal health of UC mice. Therefore, the developed EVs@ZIF-8@siRNA nanoparticles may represent a novel colon-targeted oral drug, providing a promising therapeutic strategy for UC therapy.

Polymer-DNA Carriers Co-Deliver Photosensitizer and siRNA for Light-Promoted Gene Transfection and Hypoxia-Relieved Photodynamic Therapy.

Photochemical internalization is an efficient strategy relying on photodynamic reactions to promote siRNA endosomal escape for the success of RNA-interference gene regulation, which makes gene-photodynamic combined therapy highly synergistic and efficient. However, it is still desired to explore capable carriers to improve the delivery efficiency of the immiscible siRNA and organic photosensitizers simultaneously. Herein, we employ a micellar nanostructure (PSNA) self-assembled from polymer-DNA molecular chimeras to fulfill this task. PSNA can plentifully load photosensitizers in its hydrophobic core simply by the nanoprecipitation method. Moreover, it can organize siRNA self-assembly by the densely packed DNA shell, which leads to a higher loading capacity than the typical electrostatic condensation method. The experimental results prove that this PSNA carrier can greatly facilitate siRNA escape from the endosome/lysosome and enhance transfection. Accordingly, the PSNA-administrated therapy exhibits a significantly improved anti-tumor efficacy owing to the highly efficient co-delivery capability.

Polymer‐DNA Carriers Co‐Deliver Photosensitizer and siRNA for Light‐Promoted Gene Transfection and Hypoxia‐Relieved Photodynamic Therapy

AbstractPhotochemical internalization is an efficient strategy relying on photodynamic reactions to promote siRNA endosomal escape for the success of RNA‐interference gene regulation, which makes gene‐photodynamic combined therapy highly synergistic and efficient. However, it is still desired to explore capable carriers to improve the delivery efficiency of the immiscible siRNA and organic photosensitizers simultaneously. Herein, we employ a micellar nanostructure (PSNA) self‐assembled from polymer‐DNA molecular chimeras to fulfill this task. PSNA can plentifully load photosensitizers in its hydrophobic core simply by the nanoprecipitation method. Moreover, it can organize siRNA self‐assembly by the densely packed DNA shell, which leads to a higher loading capacity than the typical electrostatic condensation method. The experimental results prove that this PSNA carrier can greatly facilitate siRNA escape from the endosome/lysosome and enhance transfection. Accordingly, the PSNA‐administrated therapy exhibits a significantly improved anti‐tumor efficacy owing to the highly efficient co‐delivery capability.

Self-assembled redox-responsive BRD4 siRNA nanoparticles: fomulation and its in vitro delivery in gastric cancer cells

With the development of newer biomarkers in the diagnosis of gastric cancer (GC), therapeutic targets are emerging and molecular-targeted therapy is in progress RNA interference has emerged as a promising method of gene targeting therapy. However, naked small interfering RNA (siRNA) is unstable and susceptible to degradation, so employing vectors for siRNA delivery is the focus of our research. Therefore, we developed LMWP modified PEG-SS-PEI to deliver siRNA targeting BRD4 (L-NPs/siBRD4) for GC therapy. L-NPs/siBRD4 were prepared by electrostatic interaction and characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The release characteristics, cellular uptake and intracellular localization were also investigated. The in vitro anticancer activity of the prepared nanoparticles was analysed by MTT, Transwell invasion and wound healing assay. Quantitative real time-polymerase chain reaction (qRT-PCR) and Western blot were used to detect the effect of gene silencing. The results showed that the optimal N/P was 30 and the prepared L-NPs/siBRD4 uniformly distributed in the system with a spherical and regular shape. L-NPs/siBRD4 exhibited an accelerated release in GSH-containing media from 12h to 24h. The uptake of L-NPs/siBRD4 was enhanced and mainly co-localized in the lysosomes. After 6h incubation, LMWP modified PEG-SS-PEI helped siRNA escape from the lysosomes and diffused into the cytoplasm. L-NPs/siBRD4 significantly inhibited the proliferation, migration and invasion of cells. This might be related with the silence of BRD4, then inhibition of PI3K/Akt and c-Myc. Our results demonstrate that L-NPs/siBRD4 are a novel delivery system with anticancer, which may provide a more effective strategy for GC treatment.

Development of magnetic nanoparticles with double silica shells of different porosities for efficient siRNA delivery to breast cancer cells

Silica-based magnetic nanoparticles are designed to deliver siRNA to triple negative breast cancer cells. The cells are killed due to siRNA-induced apoptosis upon the magnetic field-enhanced efficient cellular uptake and endosome escape of siRNA.

The alpha-adrenergic antagonist prazosin promotes cytosolic siRNA delivery from lysosomal compartments

The widespread use of small interfering RNA (siRNA) is limited by the multiple extra- and intracellular barriers upon in vivo administration. Hence, suitable delivery systems, based on siRNA encapsulation in nanoparticles or its conjugation to targeting ligands, have been developed. Nevertheless, at the intracellular level, these state-of-the-art delivery systems still suffer from a low endosomal escape efficiency. Consequently, the bulk of the endocytosed siRNA drug rapidly accumulates in the lysosomal compartment. We recently reported that a wide variety of cationic amphiphilic drugs (CADs) can promote small nucleic acid delivery from the endolysosomal compartment into the cytosol via transient induction of lysosomal membrane permeabilization. Here, we describe the identification of alternate siRNA delivery enhancers from the NIH Clinical Compound Collection that do not have the typical physicochemical properties of CADs. Additionally, we demonstrate improved endolysosomal escape of siRNA via a cholesterol conjugate and polymeric carriers with the α1-adrenergic antagonist prazosin, which was identified as the best performing delivery enhancer from the compound screen. A more detailed assessment of the mode-of-action of prazosin suggests that a different cellular phenotype compared to typical CAD adjuvants drives cytosolic siRNA delivery. As it has been described in the literature that prazosin also induces cancer cell apoptosis and promotes antigen cross-presentation in dendritic cells, the proof-of-concept data in this work provides opportunities for the repurposing of prazosin in an anti-cancer combination strategy with siRNA.

Electrostatic Attractive Self-Delivery of siRNA and Light-Induced Self-Escape for Synergistic Gene Therapy.

Small interfering RNA (siRNA) holds immense promise for suppressing gene expression and treating various life-threatening diseases, including cancer. However, efficient delivery and lysosomal escape remain critical challenges that hinder the therapeutic effectiveness of siRNA. Herein, cationic photosensitizer (NB-Br) is grafted onto polo-like kinase 1 (PLK1) siRNA to form an amphiphilic siRNA-photosensitizer conjugate (siPLK1-NB), which can self-assemble into nanoparticles (siPLK1-NB NPs) via electrostatic attraction. Notably, siPLK1-NB NPs exhibit rapid and efficient cell endocytosis, as well as outstanding tumor-targeting property in multiple tumor-bearing mice models. When siPLK1-NB NPs are located inside tumor cell lysosomes, the generated reactive oxygen species (ROS) after photoactivation can disrupt the lysosome membrane structure and facilitate siRNA escape from lysosomes. Under light irradiation, siPLK1-NB NPs can downregulate PLK1 expression and induce photodynamic killing, effectively inhibiting tumor cell growth both in vitro and in vivo. Consequently, this study provides a novel design strategy for carrier-free siRNA delivery systems. As far as it is known, this is the first report of a carrier-free siRNA delivery system based on electrostatic attraction.

Iron‐siRNA Nanohybrids for Enhanced Chemodynamic Therapy via Ferritin Heavy Chain Downregulation

AbstractFerrous iron (Fe2+) has more potent hydroxyl radical (⋅OH)‐generating ability than other Fenton‐type metal ions, making Fe‐based nanomaterials attractive for chemodynamic therapy (CDT). However, because Fe2+ can be converted by ferritin heavy chain (FHC) to nontoxic ferric form and then sequestered in ferritin, therapeutic outcomes of Fe‐mediated CDT agents are still far from satisfactory. Here we report the synthesis of siRNA‐embedded Fe0 nanoparticles (Fe0‐siRNA NPs) for self‐reinforcing CDT via FHC downregulation. Upon internalization by cancer cells, pH‐responsive Fe0‐siRNA NPs are degraded to release Fe2+ and FHC siRNA in acidic endo/lysosomes with the aid of oxygen (O2). The accompanied O2 depletion causes an intracellular pH decrease, which further promotes the degradation of Fe0‐siRNA NPs. In addition to initiating chemodynamic process, Fe2+‐catalyzed ⋅OH generation facilitates endo/lysosomal escape of siRNA by disrupting the membranes, enabling FHC downregulation‐enhanced CDT.

Iron-siRNA Nanohybrids for Enhanced Chemodynamic Therapy via Ferritin Heavy Chain Downregulation.

Ferrous iron (Fe2+) has more potent hydroxyl radical (•OH)-generating ability than other Fenton-type metal ions, making Fe-based nanomaterials attractive for chemodynamic therapy (CDT). However, because Fe2+ can be converted by ferritin heavy chain (FHC) to nontoxic ferric form and then sequestered in ferritin, therapeutic outcomes of Fe-mediated CDT agents are still far from satisfactory. Here we report the synthesis of siRNA-embedded Fe0 nanoparticles (Fe0-siRNA NPs) for self-reinforcing CDT via FHC downregulation. Upon internalization by cancer cells, pH-responsive Fe0-siRNA NPs are degraded to release Fe2+ and FHC siRNA in acidic endo/lysosomes with the aid of oxygen (O2). The accompanied O2 depletion causes an intracellular pH decrease, which further promotes the degradation of Fe0-siRNA NPs. In addition to initiating chemodynamic process, Fe2+-catalyzed •OH generation facilitates endo/lysosomal escape of siRNA by disrupting the membranes, enabling FHC downregulation-enhanced CDT.

Light-Activated siRNA Endosomal Release (LASER) by Porphyrin Lipid Nanoparticles.

Lipid nanoparticles (LNPs) have achieved clinical success in delivering small interfering RNAs (siRNAs) for targeted gene therapy. However, endosomal escape of siRNA into the cytosol remains a fundamental challenge for LNPs. Herein, we report a strategy termed light-activated siRNA endosomal release (LASER) to address this challenge. We established a porphyrin-LNP by incorporating porphyrin-lipids into the clinically approved Onpattro formulation. The porphyrin-LNP maintained the physical properties of an LNP and generated reactive oxygen species (ROS) when irradiated with near-infrared (NIR) light. Using confocal microscopy, we revealed that porphyrin-lipids within the LNP translocate to endosomal membranes during endocytosis. The translocated porphyrin-lipids generated ROS under light irradiation and enabled LASER through endosomal membranes disruption as observed through GAL-9 recruitment and transmission electron microscopy (TEM). By establishing a quantitative confocal imaging method, we confirmed that porphyrin-LNPs can increase siRNA endosomal escape efficiency by up to 2-fold via LASER and further enhance luciferase target knockdown by 4-fold more in luciferase-transfected prostate cancer cells. Finally, we formulated porphyrin-LNPs encapsulated with gold nanoparticles (GNP) and visualized the LASER effect within prostate tumors via TEM, confirming the light-activated endosomal membrane disruption and subsequent GNP release into cytosols in vivo. Overall, porphyrin-LNPs and the LASER approach enhanced siRNA endosomal escape and significantly improved knockdown efficacy. We believe the versatility of this technology could be applied to various LNP-based RNA therapeutics.

Anionic liposomes prepared without organic solvents for effective siRNA delivery.

Currently, organic solvents are necessary for the preparation of anionic liposomes for siRNA delivery. The removal of organic solvent is time-consuming and the residual organic solvent is not only a hidden danger, but also affects the stability of anionic liposomes. Glycerol, which is physiologically compatible and does not need to be removed, is used to promote the dispersion of lipids and the formation of anionic liposomes. Additionally, the preparation process is simple and not time-consuming. The results showed that anionic liposomes, which were typically spherical with a particle size of 188.9nm were successfully prepared with glycerol. And with the help of Ca2+ , siRNA was encapsulated in anionic liposomes. The highest encapsulation efficiency at 2.4mM Ca2+ reached 91%. And the formation of calcium phosphate could promote the endosomal escape of siRNA effectively. The results from cell viability showed that the anionic liposomes had no obvious cytotoxicity. It was also verified that anionic liposomes could improve the resistance of siRNA against degradation. Additionally, siRNA delivered by anionic liposomes could play an effective role in knockout. Therefore, anionic liposomes prepared with glycerol will be a safe and effective delivery platform for siRNA and even other nucleic acid drugs.

A hybrid nanoassembly for ultrasound-inducible cytosolic siRNA delivery and cancer sono-gene therapy.

Cancer gene therapy by small-interfering RNAs (siRNAs) holds great promise but is impeded by a low cytoplasmic delivery efficiency. The past two decades have witnessed many efforts that are dedicated to discover biomaterials in order to increase cellular uptake efficiency of siRNAs. However, less attention has been paid to the lysosomal trapping dilemma that greatly restricts gene silencing outcomes. Herein, to address this challenge, we developed a sono-controllable strategy for ultrasound-promoted cytosolic siRNA delivery. A hybrid nanoassembly (HNA) was prepared via electrostatic self-assembly of a siRNA and a nona-arginine modified with protoporphyrin IX that is a sonosensitizer. After cellular uptake and exposure to sono-irradiation, HNA generated singlet oxygen to facilitate the lysosomal escape of siRNA to knock down anti-apoptotic Bcl-2 in the cytoplasm. We showed that the colocalization ratios between siRNA and the lysosome decreased from 91% to 33% post sono-irradiation; meanwhile, the gene silencing efficacy increased from 46% to 68% at 300nM of HNA. Furthermore, sonodynamic therapy was achieved by the sonosensitizer under ultrasound irradiation, which combined gene therapy to eradicate cancer cells, resulting in a cell death rate of 82%. This study thus presents a novel ultrasonic approach for effective cytoplasmic delivery of siRNAs and combinational sono-gene therapy of cancer.

Peptidyl Virus-Like Nanovesicles as Reconfigurable "Trojan Horse" for Targeted siRNA Delivery and Synergistic Inhibition of Cancer Cells.

The self-assembly of peptidyl virus-like nanovesicles (pVLNs) composed of highly ordered peptide bilayer membranes that encapsulate the small interfering RNA (siRNA) is reported. The targeting and enzyme-responsive sequences on the bilayer's surface allow the pVLNs to enter cancer cells with high efficiency and control the release of genetic drugs in response to the subcellular environment. By transforming its structure in response to the highly expressed enzyme matrix metalloproteinase 7 (MMP-7)in cancer cells, it helps the siRNA escape from the lysosomes, resulting in a final silencing efficiency of 92%. Moreover, the pVLNs can serve as reconfigurable "Trojan horse" by transforming into membranes triggered by the MMP-7 and disrupting the cytoplasmic structure, thereby achieving synergistic anticancer effects and 96% cancer cell mortality with little damage to normal cells. The pVLNs benefit from their biocompatibility, targeting, and enzyme responsiveness, making them a promising platform for gene therapy and anticancer therapy.

Conscription of Immune Cells by Light-Activatable Silencing NK-Derived Exosome (LASNEO) for Synergetic Tumor Eradication.

Exosomes derived from natural killer (NK) cells (NEO) constitute promising antineoplastic nano‐biologics because of their versatile functions in immune regulation. However, a significant augment of their immunomodulatory capability is an essential need to achieve clinically meaningful treatment outcomes. Light‐activatable silencing NK‐derived exosomes (LASNEO) are orchestrated by engineering the NEO with hydrophilic small interfering RNA (siRNA) and hydrophobic photosensitizer Ce6. Profiling of genes involved in apoptosis pathway with Western blot and RNA‐seq in cells receiving NEO treatment reveals that NEO elicits effective NK cell‐like cytotoxicity toward tumor cells. Meanwhile, reactive oxygen species (ROS) generation upon laser irradiation not only triggers substantial photodynamic therapy effect but also boosts M1 tumor‐associated macrophages polarization and DC maturation in the tumor microenvironment (TME). In addition, ROS also accelerates the cellular entry and endosomal escape of siRNA in TME. Finally, siRNAs targeting PLK1 or PD‐L1 induce robust gene silencing in cancer cells, and downregulation of PD‐L1 restores the immunological surveillance of T cells in TME. Therefore, the proposed LASNEO exhibit excellent antitumor effects by conscripting multiple types of immune cells. Considering that its manufacture is quite simple and controllable, LASNEO show compelling potential for clinical translational application.

Radicals Scavenging MOFs Enabling Targeting Delivery of siRNA for Rheumatoid Arthritis Therapy.

Macrophages play essential roles in the progression of rheumatoid arthritis (RA), which are polarized into the pro-inflammatory M1 phenotype with significant oxidative stress and cytokines excretion. Herein, an active targeting nanomedicine based on metal-organic frameworks (MOFs) to re-educate the diseased macrophages for RA therapy is reported. The MOFs are prepared via coordination between tannic acid (TA) and Fe3+ , and anti-TNF-α siRNA is loaded via a simple sonication process, achieving high loading capacity comparable to cationic vectors. The MOFs show excellent biocompatibility, and enable rapid endo/lysosome escape of siRNA via the proton-sponge effect for effective cytokines down-regulation. Importantly, such nanomedicine displays intrinsic radicals scavenging capability to eliminate a broad spectrum of reactive oxygen and nitrogen species (RONS), which in turn repolarizes the M1 macrophages into anti-inflammatory M2 phenotypes for enhanced RA therapy in combination with siRNA. The MOFs are further modified with bovine serum albumin (BSA) to allow cascade RA joint and diseased macrophages targeted delivery. As a result, an excellent anti-RA efficacy is achieved in a collagen-induced arthritis mice model. This work provides a robust gene vector with great translational potential, and offers a vivid example of rationally designing MOF structure with multifunctionalities to synergize with its payload for enhanced disease treatment.

Carbon Dioxide-Derived Biodegradable and Cationic Polycarbonates as a New siRNA Carrier for Gene Therapy in Pancreatic Cancer.

Pancreatic cancer is an aggressive malignancy associated with poor prognosis and a high tendency in developing infiltration and metastasis. K-ras mutation is a major genetic disorder in pancreatic cancer patient. RNAi-based therapies can be employed for combating pancreatic cancer by silencing K-ras gene expression. However, the clinical application of RNAi technology is appreciably limited by the lack of a proper siRNA delivery system. To tackle this hurdle, cationic poly (cyclohexene carbonate) s (CPCHCs) using widely sourced CO2 as the monomer are subtly synthesized via ring-opening copolymerization (ROCOP) and thiol-ene functionalization. The developed CPCHCs could effectively encapsulate therapeutic siRNA to form CPCHC/siRNA nanoplexes (NPs). Serving as a siRNA carrier, CPCHC possesses biodegradability, negligible cytotoxicity, and high transfection efficiency. In vitro study shows that CPCHCs are capable of effectively protecting siRNA from being degraded by RNase and promoting a sustained endosomal escape of siRNA. After treatment with CPCHC/siRNA NPs, the K-ras gene expression in both pancreatic cancer cell line (PANC-1 and MiaPaCa-2) are significantly down-regulated. Subsequently, the cell growth and migration are considerably inhibited, and the treated cells are induced into cell apoptotic program. These results demonstrate the promising potential of CPCHC-mediated siRNA therapies in pancreatic cancer treatment.

Increasing Angiogenesis Factors in Hypoxic Diabetic Wound Conditions by siRNA Delivery: Additive Effect of LbL-Gold Nanocarriers and Desloratadine-Induced Lysosomal Escape.

Impaired wound healing in people with diabetes has multifactorial causes, with insufficient neovascularization being one of the most important. Hypoxia-inducible factor-1 (HIF-1) plays a central role in the hypoxia-induced response by activating angiogenesis factors. As its activity is under precise regulatory control of prolyl-hydroxylase domain 2 (PHD-2), downregulation of PHD-2 by small interfering RNA (siRNA) could stabilize HIF-1α and, therefore, upregulate the expression of pro-angiogenic factors as well. Intracellular delivery of siRNA can be achieved with nanocarriers that must fulfill several requirements, including high stability, low toxicity, and high transfection efficiency. Here, we designed and compared the performance of layer-by-layer self-assembled siRNA-loaded gold nanoparticles with two different outer layers—Chitosan (AuNP@CS) and Poly L-arginine (AuNP@PLA). Although both formulations have exactly the same core, we find that a PLA outer layer improves the endosomal escape of siRNA, and therefore, transfection efficiency, after endocytic uptake in NIH-3T3 cells. Furthermore, we found that endosomal escape of AuNP@PLA could be improved further when cells were additionally treated with desloratadine, thus outperforming commercial reagents such as Lipofectamine® and jetPRIME®. AuNP@PLA in combination with desloratadine was proven to induce PHD-2 silencing in fibroblasts, allowing upregulation of pro-angiogenic pathways. This finding in an in vitro context constitutes a first step towards improving diabetic wound healing with siRNA therapy.

Minimal Physiologically Based Pharmacokinetic-Pharmacodynamic (mPBPK-PD) Model of N-Acetylgalactosamine-Conjugated Small Interfering RNA Disposition and Gene Silencing in Preclinical Species and Humans.

Conjugation of small interfering RNA (siRNA) to tris N-acetylgalactosamine [(GalNAc)3] can enable highly selective, potent, and durable knockdown of targeted proteins in the liver. However, potential knowledge gaps between in vitro experiments, preclinical species, and clinical scenarios remain. A minimal physiologically based pharmacokinetic-pharmacodynamic model for GalNAc-conjugated siRNA (GalNAc-siRNA) was developed using published data for fitusiran (ALN-AT3), an investigational compound targeting liver antithrombin (AT), to delineate putative determinants governing the whole-body-to-cellular pharmacokinetic (PK) and pharmacodynamic (PD) properties of GalNAc-siRNA and facilitate preclinical-to-clinical translation. The model mathematically linked relevant mechanisms: 1) hepatic biodistribution, 2) tris-GalNAc binding to asialoglycoprotein receptors (ASGPRs) on hepatocytes, 3) ASGPR endocytosis and recycling, 4) endosomal transport and escape of siRNA, 5) cytoplasmic RNA-induced silencing complex (RISC) loading, 6) degradation of target mRNA by bound RISC, and 7) knockdown of protein. Physiologic values for 36 out of 48 model parameters were obtained from the literature. Kinetic parameters governing (GalNAc)3-ASGPR binding and internalization were derived from published studies of uptake in hepatocytes. The proposed model well characterized reported pharmacokinetics, RISC dynamics, and knockdown of AT mRNA and protein by ALN-AT3 in mice. The model bridged multiple PK-PD data sets in preclinical species (mice, rat, monkey) and successfully captured reported plasma pharmacokinetics and AT knockdown in a phase I ascending-dose study. Estimates of in vivo potency were similar (∼2-fold) across species. Subcutaneous absorption and serum AT degradation rate constants scaled across species by body weight with allometric exponents of -0.29 and -0.22. The proposed mechanistic modeling framework characterizes the unique PK-PD properties of GalNAc-siRNA. SIGNIFICANCE STATEMENT: Tris N-acetylgalactosamine (GalNAc)3-conjugated small interfering RNA (siRNA) therapeutics enable liver-targeted gene therapy and precision medicine. Using a translational and systems-based minimal physiologically based pharmacokinetic-pharmacodynamic (mPBPK-PD) modeling approach, putative determinants influencing GalNAc-conjugated siRNA (GalNAc-siRNA) functionality in three preclinical species and humans were investigated. The developed model successfully integrated and characterized relevant published in vitro-derived biomeasures, mechanistic PK-PD profiles in animals, and observed clinical PK-PD responses for an investigational GalNAc-siRNA (fitusiran). This modeling effort delineates the disposition and liver-targeted pharmacodynamics of GalNAc-siRNA.

Novel Ellipsoid Chitosan-Phthalate Lecithin Nanoparticles for siRNA Delivery.

Small interfering RNA (siRNA) has received increased interest as a gene therapeutic agent. However, instability and lack of safe, affordable, and effective carrier systems limit siRNA's widespread clinical use. To tackle this issue, synthetic vectors such as liposomes and polymeric nanoparticles have recently been extensively investigated. In this study, we exploited the advantages of reduced cytotoxicity and enhanced cellular penetration of chitosan-phthalate (CSP) together with the merits of lecithin (LC)-based nanoparticles (NPs) to create novel, ellipsoid, non-cytotoxic, tripolyphosphate (TPP)-crosslinked NPs capable of delivering siRNA efficiently. The resulting NPs were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and were found to be ellipsoid in the shape of ca. 180 nm in size, exhibiting novel double-layer shells, with excellent stability at physiological pH and in serum solutions. MTT assay and confocal fluorescence microscopy showed that CSP-LC-TPP NPs are non-cytotoxic and efficiently penetrate cancer cells in vitro. They achieved 44% silencing against SLUG protein in MDA-MB-453 cancer cells and were significantly superior to a commercial liposome-based transfection agent that achieved only 30% silencing under comparable conditions. Moreover, the NPs protected their siRNA cargos in 50% serum and from being displaced by variable concentrations of heparin. In fact, CSP-LC-TPP NPs achieved 26% transfection efficiency in serum containing cell culture media. Real-time wide-field fluorescence microscopy showed siRNA-loaded CSP-LC-TPP NPs to successfully release their cargo intracellularly. We found that the amphoteric nature of chitosan-phthalate polymer promotes the endosomal escape of siRNA and improves the silencing efficiency.

Regulating the immunosuppressive tumor microenvironment to enhance breast cancer immunotherapy using pH-responsive hybrid membrane-coated nanoparticles

The combination of an immuno-metabolic adjuvant and immune checkpoint inhibitors holds great promise for effective suppression of tumor growth and invasion. In this study, a pH-responsive co-delivery platform was developed for metformin (Met), a known immuno-metabolic modulator, and short interfering RNA (siRNA) targeting fibrinogen-like protein 1 mRNA (siFGL1), using a hybrid biomimetic membrane (from macrophages and cancer cells)-camouflaged poly (lactic-co-glycolic acid) nanoparticles. To improve the endo-lysosomal escape of siRNA for effective cytosolic siRNA delivery, a pH-triggered CO2 gas-generating nanoplatform was developed using the guanidine group of Met. It can react reversibly with CO2 to form Met-CO2 for the pH-dependent capture/release of CO2. The introduction of Met, a conventional anti-diabetic drug, promotes programmed death-ligand 1 (PD-L1) degradation by activating adenosine monophosphate-activated protein kinase, subsequently blocking the inhibitory signals of PD-L1. As a result, siFGL1 delivery by the camouflaged nanoparticles of the hybrid biomimetic membrane can effectively silence the FGL1 gene, promoting T-cell-mediated immune responses and enhancing antitumor immunity. We found that a combination of PD-L1/programmed death 1 signaling blockade and FGL1 gene silencing exhibited high synergistic therapeutic efficacy against breast cancer in vitro and in vivo. Additionally, Met alleviated tumor hypoxia by reducing oxygen consumption and inducing M1-type differentiation of tumor-related macrophages, which improved the tumor immunosuppressive microenvironment. Our results indicate the potential of hybrid biomimetic membrane-camouflaged nanoparticles and combined Met-FGL1 blockade in breast cancer immunotherapy.