Commercial Transfection Agent Research Articles

Persistent Targeting DNA Nanocarrier Made of 3D Structural Unit Assembled from Only One Basic Multi-Palindromic Oligonucleotide for Precise Gene Cancer Therapy.

Construction of a simple, reconfigurable, and stimuli-responsive DNA nanocarrier remains a technical challenge. In this contribution, by designing three palindromic fragments, a simplest four-sticky end-contained 3D structural unit (PS-unit) made of two same DNA components is proposed. Via regulating the rotation angle of central longitudinal axis of PS-unit, theorientedassembly of one-component spherical architecture is accomplished with high efficiency. Introduction of an aptamer and sticky tail warehouse into one component creates a size-change-reversible targeted siRNA delivery nanovehicle. Volume swelling of 20 nm allows one carrier to load 1987 siPLK1s. Once entering cancer cells and responding to glutathione (GSH) stimuli, siPLK1s are almost 100% released and original size of nanovehicle is restored, inhibiting the expression of PLK1 protein and substantially suppressing tumor growth (superiorto commercial transfection agents) in tumor-bearing mice without systemic toxicity.

Mitochondria-Targeted Gene Silencing Facilitated by Mito-CPDs.

Mitochondrial DNA (mtDNA) plays an essential role in maintaining normal cellular activities. Its heteroplasmic mutations are known to cause various genetic diseases. Current genetic engineering strategies, such as those based on RNA interference (RNAi) and antisense technology, are difficult to genetically alter mtDNA, however, due to the inability of highly negatively charged oligonucleotides to translocate across the double-membrane mitochondria. We report herein a universal mitochondria-targeted gene-delivery approach by using cell-penetrating poly(disulfide)s (CPDs). Novel CPD-based mitochondrial transporters, named Mito-CPDs, were synthesized by using triphenylphosphonium (TPP)-fused propagating monomers containing either disulfide or diselenide backbones. Upon spontaneous complex formation with an oligonucleotide (single- or double-stranded), the resulting nanoscale Mito-CPD@Oligo exhibited excellent properties in common biological media. While the intracellular gene-delivery efficiency of these Mito-CPDs was comparable to that of commercial transfection agents, their unique mitochondria-localized properties enabled effective release of the loaded cargo inside these organelles. Subsequent mitochondrial delivery of siRNA and antisense oligonucleotides against suitable mtDNA-encoded proteins showed successful down-regulation of target protein expression, leading to profound effects on mitochondrial functions. Mito-CPDs thus provide a useful tool for future investigations of mitochondrial biology and treatment of mitochondria-related diseases.

GSH-Activatable Aggregation-Induced Emission Cationic Lipid for Efficient Gene Delivery.

The key to gene therapy is the design of biocompatible and efficient delivery systems. In this work, a glutathione (GSH)-activated aggregation-induced-emission (AIE) cationic amphiphilic lipid, termed QM-SS-KK, was prepared for nonviral gene delivery. QM-SS-KK was composed of a hydrophilic biocompatible lysine tripeptide headgroup, a GSH-triggered disulfide linkage, and a hydrophobic AIE fluorophore QM-OH (QM: quinoline-malononitrile) tail. The peptide moiety could not only efficiently compact DNA but also well modulate the dispersion properties of QM-SS-KK, leading to the fluorescence-off state before GSH treatment. The cleavage of disulfide in QM-SS-KK by GSH generated AIE signals in situ with a tracking ability. The liposomes consisted of QM-SS-KK, and 1,2-dioleoylphosphatidylethanolamine (DOPE) (QM-SS-KK/DOPE) delivered plasmid DNAs (pDNAs) into cells with high efficiency. In particular, QM-SS-KK/DOPE had an enhanced transfection efficiency (TE) in the presence of 10% serum, which was two times higher than that of the commercial transfection agent PEI25K. These results highlighted the great potential of peptide and QM-based fluorescence AIE lipids for gene delivery applications.

Nebulization of siRNA for inhalation therapy based on a microfluidic surface acoustic wave platform

The local delivery of therapeutic small interfering RNA or siRNA to the lungs has the potential to improve the prognosis for patients suffering debilitating lung diseases. Recent advances in materials science have been aimed at addressing delivery challenges including biodistribution, bioavailability and cell internalization, but an equally important challenge to overcome is the development of an inhalation device that can deliver the siRNA effectively to the lung, without degrading the therapeutic itself. Here, we report the nebulization of siRNA, either naked siRNA or complexed with polyethyleneimine (PEI) or a commercial transfection agent, using a miniaturizable acoustomicrofluidic nebulization device. The siRNA solution could be nebulised without significant degradation into an aerosol mist with tunable mean aerodynamic diameters of approximately 3 µm, which is appropriate for deep lung deposition via inhalation. The nebulized siRNA was tested for its stability, as well as its toxicity and gene silencing properties using the mammalian lung carcinoma cell line A549, which demonstrated that the gene silencing capability of siRNA is retained after nebulization. This highlights the potential application of the acoustomicrofluidic device for the delivery of efficacious siRNA via inhalation, either for systemic delivery via the alveolar epithelium or local therapeutic delivery to the lung.

Dual Inhibitions on Glucose/Glutamine Metabolisms for Nontoxic Pancreatic Cancer Therapy.

Glucose and glutamine are two principal nutrients in mammalian cells that provide energy and biomass for cell growth and proliferation. Especially in cancer cells, glutamine could be a main alternative for energy and biomass supply once glucose metabolism is suppressed. Therefore, single inhibition of enzymes in either glucose metabolism or glutaminolysis, though maybe efficient in vitro, is far from being satisfactory for efficient in vivo cancer therapy. Here, we proposed a new strategy for dual inhibitions on both glucose and glutamine metabolisms concurrently by silencing mutated gene Kras and glutaminase 1 (GLS1) via nanomaterial-based siKras and siGLS1 delivery, rather than conventional highly toxic chemodrugs. Such a combination therapy could overcome the challenge that glucose and glutamine are alternatives to each other in the biosynthesis and energy production for cancer cells, resulting in much elevated treatment efficacy. In addition, layered double hydroxide (LDH), the siRNA carrier, enables an enhanced gene delivery efficiency compared to the commercial transfection agent Lipofectamine 2000. Briefly, Mg-Al LDH nanosheets, loaded with siKras and siGLS1 onto their surfaces by electrostatic adsorption, could release siRNA from lysosomes into the cytoplasm via the proton sponge effect of LDH, favoring the siRNA stability and gene silencing efficiency enhancements. The thus released siRNA could downregulate the expressions of Kras, GLS1, and other enzymes involved in glucose metabolism, resulting in the downregulations of ATP and other metabolites. Such a biosafe LDH/siRNA nanomedicine is able to efficiently suppress the growth of xenografts through cancer cell proliferation suppression, displaying its great potential as a simultaneous glucose/glutamine metabolism coinhibitor for treating pancreatic cancer.

A NIR Aggregation-Induced Emission Fluoroamphiphile as Visually Trackable and Serum-Tolerant Nonviral Gene Carrier

Functional fluorescence (FL) nonviral gene vectors with high serum tolerance bear broad application prospects in gene delivery. Fluorination has been widely utilized as an effective strategy to enhance serum tolerance. Herein, we show the combination of fluorination and aggregation-induced emission (AIE) for the construction of a nonviral gene vector with low cytotoxicity, visual tracking ability, and high serum tolerance. Large π-conjugation triphenylamine (TPA) derivative with a characteristic D-π-A structure was modified with two polar [12]aneN3 heads and a long fluorocarbon tail, giving the vector molecule FluoTPA. FluoTPA features near-infrared (NIR) emission, large Stokes shift, and strong binding affinity toward nucleic acids. Liposomes consisting of FluoTPA and dioleoylphosphatidylethanolamine (DOPE) (FluoTPA/DOPE) can effectively deliver both plasmid DNAs (pDNAs) and siRNAs into cells. Impressively, FluoTPA/DOPE showed comparable transfection efficiency (TE) in the presence of serum content up to 30% with that in the serum-free condition and achieved 7.4 times higher TE than the commercial transfection agent lipofectamine 2000 at the same condition. Finally, spatiotemporal tracking of the delivery process in cells was demonstrated. The results in this work suggest that FluoTPA could be a reliable theranostic platform for the nonviral delivery of nucleic acid therapeutics in serum condition.

Chitosan-Crosslinked Low Molecular Weight PEI-Conjugated Iron Oxide Nanoparticle for Safe and Effective DNA Delivery to Breast Cancer Cells.

Breast cancer has attracted tremendous research interest in treatment development as one of the major threats to public health. The use of non-viral carriers for therapeutic DNA delivery has shown promise in treating various cancer types, including breast cancer, due to their high DNA loading capacity, high cell transfection efficiency, and design versatility. However, cytotoxicity and large sizes of non-viral DNA carriers often raise safety concerns and hinder their applications in the clinic. Here we report the development of a novel nanoparticle formulation (termed NP-Chi-xPEI) that can safely and effectively deliver DNA into breast cancer cells for successful transfection. The nanoparticle is composed of an iron oxide core coated with low molecular weight (800 Da) polyethyleneimine crosslinked with chitosan via biodegradable disulfide bonds. The NP-Chi-xPEI can condense DNA into a small nanoparticle with the overall size of less than 100 nm and offer full DNA protection. Its biodegradable coating of small-molecular weight xPEI and mildly positive surface charge confer extra biocompatibility. NP-Chi-xPEI-mediated DNA delivery was shown to achieve high transfection efficiency across multiple breast cancer cell lines with significantly lower cytotoxicity as compared to the commercial transfection agent Lipofectamine 3000. With demonstrated favorable physicochemical properties and functionality, NP-Chi-xPEI may serve as a reliable vehicle to deliver DNA to breast cancer cells.

Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells.

Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX—the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1208/s12248-021-00653-2.

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.

Naphthalimide‐modified dendrimers as efficient and low cytotoxic nucleic acid delivery vectors

AbstractPoly(amidoamine) (PAMAM) dendrimers have been widely used as nucleic acid delivery vectors. To improve the transfection efficacy, ligands such as amino acid, peptide and lipid are conjugated to the periphery of dendrimers. However, the structures of these ligands are generally flexible, and few reports about rigid‐ligand‐modified dendrimers are reported. In this work, three novel PAMAM dendrimers (G5‐1, G5‐2 and G5‐3) modified with rigid naphthalimide were synthesized as nucleic acid delivery vectors. Magnetic resonance spectroscopy showed that the average numbers of naphthalimide molecules conjugated on the PAMAM periphery in compounds G5‐1, G5‐2 and G5‐3 are 8, 30 and 50, respectively. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay revealed good cytocompatibility of these vectors in multiple cell lines, including MG63, HeLa, HEK293 and MC3T3‐E1 cells, with a cell survival rate of more than 90% in most cases. Among the three dendrimers, G5‐1 with fewer naphthalimide molecules showed stronger RNA binding ability than G5‐2 and G5‐3. G5‐1 also exhibited the best gene silencing efficacy at a dendrimer to miRNA weight ratio of 1:2 in MC3T3‐E1 cells, which is much higher than the commercial transfection agent Lipofectamine 2000 tested under the same conditions. In addition, G5‐1 showed high transfection efficacy in plasmid DNA‐mediated luciferase expression experiments. These results suggest that rigid ligand modification can be developed as a new strategy to design safe and highly efficient nucleic acid delivery vectors. © 2021 Society of Industrial Chemistry.

Two-Photon Near-Infrared AIE Luminogens as Multifunctional Gene Carriers for Cancer Theranostics.

Construction of multifunctional nonviral gene vectors to execute defined tasks holds great potential for the precise and effective treatment of gene-associated diseases. Herein, we have developed four large π-conjugation triphenylamine derivatives bearing two polar [12]aneN3 heads and a lipophilic tail for applications in gene delivery, one/two-photon-triggered near-infrared (NIR) fluorescence bioimaging, and combined photodynamic therapy (PDT) and gene therapy of cancer. These compounds possess typical NIR aggregation-induced emission characteristics, mega Stokes shifts, strong two-photon excitation fluorescence, and excellent DNA condensation abilities. Among them, vector 4 with a tail of n-hexadecane realized a transfection efficiency as high as 6.7 times that of the commercial transfection agent Lipofectamine 2000 in HEK293T cell lines. Using vector 4 as an example, transfection process tracking and ex vivo/in vivo tumoral imaging and retention with high resolution, high brightness, deep tissue penetration, and good biosafety were demonstrated. In addition, efficient singlet oxygen (1O2) generation by the DNA complex formed by vector 4 (4/DNA) resulted in effective PDT. Combined with anticancer gene therapy, collaborative cancer treatment with a dramatically enhanced cancer cell-killing effect was achieved. The development of this "three birds, one stone" approach suggests a new and promising strategy for better cancer treatment and real-time tracking of gene delivery.

Development of an activated microglia cell line model for screening lipid nanoparticle formulations of BDNF siRNA for neuropathic pain therapy

Purpose Brain-derived neurotrophic factor (BDNF) released from activated spinal microglia via the ATP-P2 × 4R-BDNF signaling pathway plays a central role in initiating pain hypersensitivity in neuropathic pain. Therefore, knockdown of BDNF expression in microglia using molecules like siRNA can be a promising strategy for neuropathic pain therapy. Our lab is developing lipidoid nanoparticles (LNP) for the delivery of BDNF siRNA (siBDNF) for treating neuropathic pain. We characterized the Spontaneously Immortalized Microglia-A9 (SIM-A9) cell line and confirmed the expression of Iba1, P2 × 4R, and BDNF under resting (non-activated) and ATP-stimulated conditions. SIM-A9 cell line was cultured with and without external stimuli, adenosine triphosphate (ATP), and/or lipopolysaccharide (LPS) by varying parameters such as the concentration of stimuli, incubation time, and cell regeneration time post-activation. The cytocompatibility of SIM-A9 cells with ATP and LPS was determined using an ATP assay. Intracellular Iba1, P2 × 4R, and BDNF protein expression in the resting and stimulated cells were characterized using western blotting, in-cell western, and immunocytochemistry. In a pilot study, SIM-A9 cells were transfected with RNAiMAX (a commercial transfection agent)/siBDNF complexes and changes in BDNF expression were evaluated using western blotting. We present two novel findings: first, SIM-A9 cells expressed P2 × 4R and BDNF proteins. Second, ATP at a safe dose showed a time-dependent increase in Iba1 and BDNF expression without intracellular toxicity. At 40 nM siRNA concentration, RNAiMAX/siBDNF complexes showed about a 15% reduction in SIM-A9 BDNF expression. In preliminary studies, LNP/siBDNF at 40 nM were well-tolerated by SIM-A9 cells, however, they only showed negligible BDNF knockdown in activated SIM-A9 cells. Currently, we are optimizing LNP formulations and transfection conditions to increase the extent of BDNF gene silencing. We have developed an ATP-activated SIM-A9 cell line model system that can be utilized for screening of neuropathic pain therapeutics.

Fluorescent Nanoparticles for Targeted Tumor Imaging and DNA Tracking Gene Delivery In Vitro/In Vivo.

Fluorescence detection is desirable to track the gene transfer process in order to explain the mechanism. Here, a fluorescent nanoparticle, diketopyrrolopyrrole-based liposome (DPL), was prepared for DNA delivery and tumor imaging in vitro and in vivo. The process to deliver DNA into cells was detected in real time by DPL according to the fluorescent property. The transfection efficacies (TEs) for luciferase and enhanced green fluorescent protein (EGFP) analysis of DPL were 1.5 times those of the commercial transfection agent Lipo 2000. Importantly, the DPL/DNA system has high EGFP TE in vivo with tumor targeting ability. This work provided an effective strategy for monitoring transfection processes.

Cationic Polymer Nanoparticles-Mediated Delivery of miR-124 Impairs Tumorigenicity of Prostate Cancer Cells.

MicroRNAs (miRNAs) play a pivotal role in regulating the expression of genes involved in tumor development, invasion, and metastasis. In particular, microRNA-124 (miR-124) modulates the expression of carnitine palmitoyltransferase 1A (CPT1A) at the post-transcriptional level, impairing the ability of androgen-independent prostate cancer (PC3) cells to completely metabolize lipid substrates. However, the clinical translation of miRNAs requires the development of effective and safe delivery systems able to protect nucleic acids from degradation. Herein, biodegradable polyethyleneimine-functionalized polyhydroxybutyrate nanoparticles (PHB-PEI NPs) were prepared by aminolysis and used as cationic non-viral vectors to complex and deliver miR-124 in PC3 cells. Notably, the PHB-PEI NPs/miRNA complex effectively protected miR-124 from RNAse degradation, resulting in a 30% increase in delivery efficiency in PC3 cells compared to a commercial transfection agent (Lipofectamine RNAiMAX). Furthermore, the NPs-delivered miR-124 successfully impaired hallmarks of tumorigenicity, such as cell proliferation, motility, and colony formation, through CPT1A modulation. These results demonstrate that the use of PHB-PEI NPs represents a suitable and convenient strategy to develop novel nanomaterials with excellent biocompatibility and high transfection efficiency for cancer therapy.

A light-triggerable formulation to control the stability of pro-angiogenic transcription factor hypoxia inducible factor-1α (HIF-1α).

The control of vascular remodeling mediated by transcription factor HIF-1α is critical in the treatment of several diseases including cancer, retinopathies, chronic wounds, and ischemic heart disease, among others. Gene silencing using a small interfering RNA (siRNA) is a promising therapeutic strategy to regulate HIF-1α; however, the delivery systems developed so far have limited endothelial targeting and efficiency. Herein, we have synthesized a light-triggerable polymeric nanoparticle (NP) library composed of 110 formulations which showed variable morphology, charge and disassembly rates after UV exposure. More than 35% of the formulations of the library were more efficient in gene knockdown than the siRNA delivered by a commercial transfection agent (lipofectamine RNAiMAX). The most efficient siRNA delivery formulations were tested against different cell types to identify one with preferential targeting to endothelial cells. Using a two-step methodology, we have identified a formulation that shows exquisite targeting to endothelial cells and is able to deliver more efficiently the siRNA that modulates HIF-1α than commercial transfection agents. Overall, the strategy reported here increases the specificity for tissue regulation and the efficiency for the intracellular delivery of siRNAs.

Engineering multifunctional bioactive citric acid-based nanovectors for intrinsical targeted tumor imaging and specific siRNA gene delivery in vitro/in vivo

Targeted tumor imaging and efficient specific gene delivery in vivo has been one of the main challenges in gene-based cancer diagnosis and therapy. Herein, we engineered a citric acid-based polymer with intrinsical photoluminescence and gene loading capacity to achieve targeted delivery of siRNA and tumor imaging in vitro and in vivo. The multifunctional platform was formed from the self-assembling of poly (citric acid)-polymine conjugated with folic acid and rhodamine B (PPFR). PPFR showed stable photoluminescent ability and could effectively bind and protect the siRNA against RNase degradation. PPFR also exhibited good blood compatibility and cell compatibility against C2C12, MCF-7 and A549. Compared with commercial transfection agent Lipofectamine™ 2000, PPFR had a high cellular uptake, equivalent transfection efficiency and effectively down-regulated intracellular p65 expression in A549 cancer cells. Importantly, PPFR could efficiently accumulate and label the tumor tissue through the fluorescent imaging, selectively deliver siRNA into tumor tissue in vivo based on the tumor-bearing nude mice model. This work may provide a facile strategy to synthesize multifunctional biocompatible biomaterials for targeted tumor imaging and gene therapy.

Functional DNA Delivery Enabled by Lipid-Modified Charge-Altering Releasable Transporters (CARTs).

Safe and effective DNA delivery systems are required to enable or enhance clinical strategies and research involving gene therapy and DNA vaccinations. To address this delivery problem, a series of charge-altering releasable transporters (CARTs) with varied lipid content were prepared and evaluated for plasmid DNA (pDNA) delivery into cultured cells. These lipid-modified CART co-oligomers were synthesized in only two steps via sequential organocatalytic ring-opening polymerization of lipid-containing cyclic carbonate monomers and morpholinone monomers. Lipid variations of the CARTs substantially impacted the delivery efficiency of pDNA, with oleyl- and linoleyl-based CARTs showing enhanced performance relative to the commercial transfection agent Lipofectamine 2000 (L2000). The best-performing oleyl CART was carried forward to study stable luciferase transfection with a Sleeping Beauty ( SB) transposon system. The oleyl CART outperformed the L2000 positive control with respect to stable transfection efficiency. CART-pDNA complexes represent a new DNA delivery system for research and clinical applications.

Evaluating the Mechanisms of Light-Triggered siRNA Release from Nanoshells for Temporal Control Over Gene Regulation.

The ability to regulate intracellular gene expression with exogenous nucleic acids such as small interfering RNAs (siRNAs) has substantial potential to improve the study and treatment of disease. However, most transfection agents and nanoparticle-based carriers that are used for the intracellular delivery of nucleic acids cannot distinguish between diseased and healthy cells, which may cause them to yield unintended widespread gene regulation. An ideal delivery system would only silence targeted proteins in diseased tissue in response to an external stimulus. To enable spatiotemporal control over gene silencing, researchers have begun to develop nucleic acid-nanoparticle conjugates that keep their nucleic acid cargo inactive until it is released from the nanoparticle on-demand by externally applied near-infrared laser light. This strategy can overcome several limitations of other nucleic acid delivery systems, but the mechanisms by which these platforms operate remain ill understood. Here, we perform a detailed investigation of the mechanisms by which silica core/gold shell nanoshells (NSs) release conjugated siRNA upon excitation with either pulsed or continuous wave (CW) near-infrared (NIR) light, with the goal of providing insight into how these nanoconjugates can enable on-demand gene regulation. We demonstrate that siRNA release from NSs upon pulsed laser irradiation is a temperature-independent process that is substantially more efficient than siRNA release triggered by CW irradiation. Contrary to literature, which suggests that only pulsed irradiation releases siRNA duplexes, we found that both modes of irradiation release a mixture of siRNA duplexes and single-stranded oligonucleotides, but that pulsed irradiation results in a higher percentage of released duplexes. To demonstrate that the siRNA released from NSs upon pulsed irradiation remains functional, we evaluated the use of NSs coated with green fluorescent protein (GFP)-targeted siRNA (siGFP-NS) for on-demand knockdown of GFP in cells. We found that GFP-expressing cells treated with siGFP-NS and irradiated with a pulsed laser experienced a 33% decrease in GFP expression compared to cells treated with no laser. Further, we observed that light-triggered gene silencing mediated by siGFP-NS is more potent than using commercial transfection agents to deliver siRNA into cells. This work provides unprecedented insight into the mechanisms by which plasmonic NSs release siRNA upon light irradiation and demonstrates the importance of thoroughly characterizing photoresponsive nanosystems for applications in triggered gene regulation.

Branched Glycerol-Based Copolymer with Ultrahigh p65 siRNA Delivery Efficiency for Enhanced Cancer Therapy.

The small interfering RNA (siRNA) is emerging as a potential therapeutic for the treatment of various diseases because of the targeted gene silencing capability. The suppression of p65 expression has shown great potential in various cancer treatments. However, various substantial obstacles limit its clinical applications, including low cellular uptake, instability, and cytotoxicity of delivery vehicles. We show a highly branched and biocompatible glycerol-based copolymer (HBGC) to effectively deliver siRNA for excellent p65 gene silencing and safe lung cancer treatment in vitro and in vivo. HBGCs could be synthesized through a facile and modular one-spot Michael addition. HBGCs effectively protect siRNA in serum, enhance cellular uptake of siRNA, and show negligible cytotoxicity in various cells (A549, HeLa, HepG2, and C2C12). Additionally, the HBGC-siRNA complex potently downregulates the p65 expression in A549 cancer cells (almost the highest value of 96% in reported references) and enhances the cellular apoptosis compared to that of commercial transfection agents polyethyleneimine 25 kDa and Lipofectamine 2000. The HBGC-siRNA complex demonstrated significantly increased accumulation in the tumor sites and enhanced downregulation of p65 gene and cancer cell apoptosis. Furthermore, the tumor growth could be significantly inhibited in a subcutaneous lung tumor model with negligible adverse effects.

Plasmid DNA Delivery: Nanotopography Matters.

Plasmid DNA molecules with unique loop structures have widespread bioapplications, in many cases relying heavily on delivery vehicles to introduce them into cells and achieve their functions. Herein, we demonstrate that control over delicate nanotopography of silica nanoparticles as plasmid DNA vectors has significant impact on the transfection efficacy. For silica nanoparticles with rambutan-, raspberry-, and flower-like morphologies composed of spike-, hemisphere-, and bowl-type subunit nanotopographies, respectively, the rambutan-like nanoparticles with spiky surfaces demonstrate the highest plasmid DNA binding capability and transfection efficacy of 88%, higher than those reported for silica-based nanovectors. Moreover, it is shown that the surface spikes of rambutan nanoparticles provide a continuous open space to bind DNA chains via multivalent interactions and protect the gene molecules sheltered in the spiky layer against nuclease degradation, exhibiting no significant transfection decay. This unique protection feature is in great contrast to a commercial transfection agent with similar transfection performance but poor protection capability against enzymatic cleavage. Our study provides new understandings in the rational design of nonviral vectors for efficient gene delivery.