Highly efficient gene silencing and bioimaging based on fluorescent carbon dots <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Small interfering RNA (siRNA) is an attractive therapeutic candidate for sequencespecific gene silencing to treat incurable diseases using small molecule drugs.However,its efficient intracellular delivery has remained a challenge.Here,we have developed a highly biocompatible fluorescent carbon dot (CD),and demonstrate a functional siRNA delivery system that induces efficient gene knockdown in vitro and in vivo.We found that CD nanoparticles (NPs) enhance the cellular uptake of siRNA,via endocytosis in tumor cells,with low cytotoxicity and unexpected immune responses.Real-time study of fluorescence imaging in live cells shows that CD NPs favorably localize in cytoplasm and successfully release siRNA within 12 h.Moreover,we demonstrate that CD NP-mediated siRNA delivery significantly silences green fluorescence protein (GFP) expression and inhibits tumor growth in a breast cancer cell xenograft mouse model of tumor-specific therapy.We have developed a multi functional siRNA delivery vehicle enabling simultaneous bioimaging and efficient downregulation of gene expression,that shows excellent potential for gene therapy.     

Delivery of multiple siRNAs using lipid-coated PLGA nanoparticles for treatment of prostate cancer

Nanotechnology can provide a critical advantage in developing strategies for cancer management and treatment by helping to improve the safety and efficacy of novel therapeutic delivery vehicles. This paper reports the fabrication of poly(lactic acid-co-glycolic acid)/siRNA nanoparticles coated with lipids for use as prostate cancer therapeutics made via a unique soft lithography particle molding process called Particle Replication In Nonwetting Templates (PRINT). The PRINT process enables high encapsulation efficiency of siRNA into neutral and monodisperse PLGA particles (32-46% encapsulation efficiency). Lipid-coated PLGA/siRNA PRINT particles were used to deliver therapeutic siRNA in vitro to knockdown genes relevant to prostate cancer.     

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.     

Targeted Delivery of siRNA‐Generating DNA Nanocassettes Using Multifunctional Nanoparticles

Molecular therapy using a small interfering RNA (siRNA) has shown promise in the development of novel therapeutics. Various formulations have been used for in vivo delivery of siRNAs. However, the stability of short double‐stranded RNA molecules in the blood and efficiency of siRNA delivery into target organs or tissues following systemic administration have been the major issues that limit applications of siRNA in human patients. In this study, multifunctional siRNA delivery nanoparticles are developed that combine imaging capability of nanoparticles with urokinase plasminogen activator receptor‐targeted delivery of siRNA expressing DNA nanocassettes. This theranostic nanoparticle platform consists of a nanoparticle conjugated with targeting ligands and double‐stranded DNA nanocassettes containing a U6 promoter and a shRNA gene for in vivo siRNA expression. Targeted delivery and gene silencing efficiency of firefly luciferase siRNA nanogenerators are demonstrated in tumor cells and in animal tumor models. Delivery of survivin siRNA expressing nanocassettes into tumor cells induces apoptotic cell death and sensitizes cells to chemotherapy drugs. The ability of expression of siRNAs from multiple nanocassettes conjugated to a single nanoparticle following receptor‐mediated internalization should enhance the therapeutic effect of the siRNA‐mediated cancer therapy.     

SiRNA drug delivery by biodegradable polymeric nanoparticles

RNA interference (RNAi) is an emerging technology in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. It offers a broad spectrum of applications in both biological and medical research. Small interference RNA (siRNA) was recently explored for its therapeutical potential. However, the drug delivery of siRNA oligos is very novel and is in great need of future research. To this end, a biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticle drug carrier system was prepared to load siRNA oligos with desired physicochemical properties. The nanoparticles were characterized by scanning electron microscopy and laser diffraction particle sizer. The delivery of siRNA into the targeted 293T cells was observed using fluorescent-labeled double-stranded Cy3-oligos. The model siRNA oligos, si-GFP-RNA, were also successfully loaded into PLGA nanoparticles and delivered in 293T cells. The gene silencing effect and the inhibition of GFP expression were investigated using fluorescent microscopy. Both positive and negative controls were used to compare with the new siRNA nanoparticle delivery system. It was found that nanoparticles offered both effective delivery of siRNA and prominent GFP gene silencing effect. Compared to conventional carrier systems, the new biodegradable polymeric nanoparticle system may also offer improved formulation stability, which is practically beneficial and may be used in the future clinical studies of siRNA therapeutics.     

Nanodiamond as a vector for siRNA delivery to Ewing sarcoma cells

The ability of diamond nanoparticles (nanodiamonds, NDs) to deliver small interfering RNA (siRNA) into Ewing sarcoma cells is investigated with a view to the possibility of in-vivo anticancer nucleic-acid drug delivery. siRNA is adsorbed onto NDs that are coated with cationic polymer. Cell uptake of NDs is demonstrated by taking advantage of the NDs' intrinsic fluorescence from embedded color-center defects. Cell toxicity of these coated NDs is shown to be low. Consistent with the internalization efficacy, a specific inhibition of EWS/Fli-1 gene expression is shown at the mRNA and protein level by the ND-vectorized siRNA in a serum-containing medium.     

Rekindling RNAi Therapy: Materials Design Requirements for In Vivo siRNA Delivery

With the recent FDA approval of the first siRNA‐derived therapeutic, RNA interference (RNAi)‐mediated gene therapy is undergoing a transition from research to the clinical space. The primary obstacle to realization of RNAi therapy has been the delivery of oligonucleotide payloads. Therefore, the main aims is to identify and describe key design features needed for nanoscale vehicles to achieve effective delivery of siRNA‐mediated gene silencing agents in vivo. The problem is broken into three elements: 1) protection of siRNA from degradation and clearance; 2) selective homing to target cell types; and 3) cytoplasmic release of the siRNA payload by escaping or bypassing endocytic uptake. The in vitro and in vivo gene silencing efficiency values that have been reported in publications over the past decade are quantitatively summarized by material type (lipid, polymer, metal, mesoporous silica, and porous silicon), and the overall trends in research publication and in clinical translation are discussed to reflect on the direction of the RNAi therapeutics field.     

Ionizable Lipid with Supramolecular Chemistry Features for RNA Delivery In Vivo

Lipid nanoparticles (LNPs) and ribonucleic acid (RNA) technology are highly versatile tools that can be deployed for diagnostic, prophylactic, and therapeutic applications. In this report, supramolecular chemistry concepts are incorporated into the rational design of a new ionizable lipid, C3-K2-E14, for systemic administration. This lipid incorporates a cone-shaped structure intended to facilitate cell bilayer disruption, and three tertiary amines to improve RNA binding. Additionally, hydroxyl and amide motifs are incorporated to further enhance RNA binding and improve LNP stability. Optimization of messenger RNA (mRNA) and small interfering RNA (siRNA) formulation conditions and lipid ratios produce LNPs with favorable diameter (<150 nm), polydispersity index (<0.15), and RNA encapsulation efficiency (>90%), all of which are preserved after 2 months at 4 or 37 °C storage in ready-to-use liquid form. The lipid and formulated LNPs are well-tolerated in animals and show no deleterious material-induced effects. Furthermore, 1 week after intravenous LNP administration, fluorescent signal from tagged RNA payloads are not detected. To demonstrate the long-term treatment potential for chronic diseases, repeated dosing of C3-K2-E14 LNPs containing siRNA that silences the colony stimulating factor-1 (CSF-1) gene can modulate leukocyte populations in vivo, further highlighting utility.     

Gold Nanoparticles Capped with Polyethyleneimine for Enhanced siRNA Delivery

An efficient and safe delivery system for small interfering RNA (siRNA) is required for clinical application of RNA interfering therapeutics. Polyethyleneimine (PEI)‐capped gold nanoparticles (AuNPs) are successfully manufactured using PEI as the reductant and stabilizer, which bind siRNA at an appropriate weight ratio by electrostatic interaction and result in well‐dispersed nanoparticles with uniform structure and narrow size distribution. With siRNA binding, PEI‐capped AuNPs induce more significant and enhanced reduction in targeted green fluorescent protein expression in MDA‐MB‐435s cells, though more internalized PEI/siRNA complexes in cells are evidenced by confocal laser scanning microscopy observation and fluorescence‐activated cell sorting analyses. PEI‐capped AuNPs/siRNA targeting endogenous cell‐cycle kinase, an oncogene polo‐like kinase 1 (PLK1), display significant gene expression knockdown and induce enhanced cell apoptosis, whereas it is not obvious when the cells are treated with PLK1 siRNA using PEI as the carrier. Without exhibiting cellular toxicity, PEI‐capped AuNPs appear to be suitable as a potential carrier for intracellular siRNA delivery.     

N-Alkyl-PEI-functionalized iron oxide nanoclusters for efficient siRNA delivery

Small-interfering RNA (siRNA) is an emerging class of therapeutics, which works by regulating the expression of a specific gene involved in disease progression. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. In this study, a nonviral nanoparticle gene carrier is developed and its efficiency for siRNA delivery and transfection is validated at both in vitro and in vivo levels. Such a nanocarrier, abbreviated as Alkyl-PEI2k-IO, was constructed with a core of iron oxide nanoparticles (IOs) and a shell of alkylated polyethyleneimine of 2000 Da [corrected] molecualr weight (Alkyl-PEI2k). It is found to be able to bind with siRNA, resulting in well-dispersed nanoparticles with a controlled clustering structure and narrow size distribution. Electrophoresis studies show that the Alkyl-PEI2k-IOs could retard siRNA completely at N:P ratios (i.e., PEI nitrogen to nucleic acid phosphate) above 10, protect siRNA from enzymatic degradation in serum, and release complexed siRNA efficiently in the presence of polyanionic heparin. The knockdown efficiency of the siRNA-loaded nanocarriers is assessed with 4T1 cells stably expressing luciferase (fluc-4T1) and further, with a fluc-4T1 xenograft model. Significant down-regulation of luciferase is observed, and unlike high-molecular-weight analogues, the Alkyl-PEI2k-coated IOs show good biocompatibility. In conclusion, Alkyl-PEI2k-IOs demonstrate highly efficient delivery of siRNA and an innocuous toxic profile, making it a potential carrier for gene therapy.     

Functionalization of silica nanoparticles for nucleic acid delivery

Silica nanoparticles (SiNPs) have been widely engineered for biomedical applications, such as bioimaging and drug delivery, because of their high tunability, which allows them to perform specific functions. In this review, we discuss the functionalization and performance of SiNPs for nucleic acid delivery. Nucleic acids, including plasmid DNA (pDNA) and small interfering RNA (siRNA), constitute the next generation molecular drugs for the treatment of intractable diseases. However, their low bioavailability requires delivery systems that can circumvent nuclease attack and kidney filtration to ensure efficient access to the target cell cytoplasm or nucleus. First, we discussed the biological significance of nucleic acids and the parameters required for their successful delivery. Next, we reviewed SiNP designing for nucleic acid delivery with respect to nucleic acid loading and release, cellular uptake, endosomal escape, and biocompatibility. In addition, we discussed the co-delivery potential of SiNPs. Finally, we analyzed the current challenges and future directions of SiNPs for advanced nucleic acid delivery.     

Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands

T cells help regulate immunity, which makes them an important target for RNA therapies. While nanoparticles carrying RNA have been directed to T cells in vivo using protein‐ and aptamer‐based targeting ligands, systemic delivery to T cells without targeting ligands remains challenging. Given that T cells endocytose lipoprotein particles and enveloped viruses, two natural systems with structures that can be similar to lipid nanoparticles (LNPs), it is hypothesized that LNPs devoid of targeting ligands can deliver RNA to T cells in vivo. To test this hypothesis, the delivery of siRNA to 9 cell types in vivo by 168 nanoparticles using a novel siGFP‐based barcoding system and bioinformatics is quantified. It is found that nanomaterials containing conformationally constrained lipids form stable LNPs, herein named constrained lipid nanoparticles (cLNPs). cLNPs deliver siRNA and sgRNA to T cells at doses as low as 0.5 mg kg^?1 and, unlike previously reported LNPs, do not preferentially target hepatocytes. Delivery occurs via a chemical composition‐dependent, size‐independent mechanism. These data suggest that the degree to which lipids are constrained alters nanoparticle targeting, and also suggest that natural lipid trafficking pathways can promote T cell delivery, offering an alternative to active targeting approaches.     

Cationic Bovine Serum Albumin Based Self‐Assembled Nanoparticles as siRNA Delivery Vector for Treating Lung Metastatic Cancer

It is generally believed that intravenous application of cationic vectors is limited by the binding of abundant negatively charged serum components, which may cause rapid clearance of the therapeutic agent from the blood stream. However, previous studies show that systemic delivery of cationic gene vectors mediates specific and efficient transfection within the lung, mainly as a result of interaction of the vectors with serum proteins. Based on these findings, a novel and charge‐density‐controllable siRNA delivery system is developed to treat lung metastatic cancer by using cationic bovine serum albumin (CBSA) as the gene vector. By surface modification of BSA, CBSA with different isoelectric points (pI) is synthesized and the optimal cationization degree of CBSA is determined by considering the siRNA binding and delivery ability, as well as toxicity. The CBSA can form stable nanosized particles with siRNA and protect siRNA from degradation. CBSA also shows excellent abiliies to intracellularly deliver siRNA and mediate significant accumulation in the lung. When Bcl2‐specific siRNA is introduced to this system, CBSA/siRNA nanoparticles exhibit an efficient gene‐silencing effect that induces notable cancer cell apoptosis and subsequently inhibits the tumor growth in a B16 lung metastasis model. These results indicate that CBSA‐based self‐assembled nanoparticles can be a promising strategy for a siRNA delivery system for lung targeting and metastatic cancer therapy.     

Hyaluronidase-triggered anticancer drug and siRNA delivery from cascaded targeting nanoparticles for drug-resistant breast cancer therapy

Drug resistance renders standard chemotherapy ineffective in the treatment of connective tissue growth factor (CTGF)-overexpressing breast cancer.By co-embedding the breast tumor cell-penetrating peptide (PEGA-pVEC) and hyaluronic add (HA) as a targeting media,novel cascaded targeting nanoparticles (HACT NPs) were created on a rattle mesoporous silica (rmSiO2) scaffold for the pinpoint delivery of siRNAs along with an anticancer drug,aiming at overcoming the drug resistance of CTGF-overexpressing breast cancer in vivo.The targeting nanoparticles selectively accumulated in the vasculature under the guidance of the PEGA-pVEC peptide,cascaded by receptor-mediated endocytosis with the aid of another targeting agent,HA,presenting a greater in vivo tumor targeting ability than single targeting ligand vectors.In addition,an HA shell prevented the leakage of therapeutic drugs during the cargo transport process,until the hyaluronidase (HAase)-triggered degradation upon lysosomes entering,guaranteeing a controllable drug release inside the target cells.When the protective shell disintegrates,the released siRNA took charge to silence the gene associated with drug resistance,CTGF,thus facilitating doxorubicin-induced apoptosis.The cascaded targeting media (PEGA-pVEC and HA) advances precision-guided therapy in vivo,while the encapsulation of siRNAs into a chemotherapy drug delivery system provides an effident strategy for the treatment of drug resistance cancers.     

Rhodamine-loaded poly(lactic-co-glycolic acid) nanoparticles for investigation of in vitro interactions with breast cancer cells

Nanoparticle-based drug delivery systems are considered promising for the delivery of imaging agents and drugs for the detection and treatment of illnesses, including cancer. Investigation of nanoparticle interactions with the diseased cells can lead to better designs. In this work, poly(lactic-co-glycolic acid) nanoparticles loaded with rhodamine 6G were prepared by nanoprecipitation with high encapsulation efficiency. In vitro release studies demonstrated that rhodamine escaped from the nanoparticles at a very slow rate at physiological pH, thus making it ideal for imaging studies. At acidic pH this agent was released quickly, suggesting charge interactions between the polymer and rhodamine. Microscopy and flow cytometry studies show higher uptake in MDA-MB-231 breast cancer cells when exposed to rhodamine-loaded nanoparticles than to rhodamine in solution.     

Orally delivered thioketal nanoparticles loaded with TNF-α-siRNA target inflammation and inhibit gene expression in the intestines

Small interfering RNAs (siRNAs) directed against proinflammatory cytokines have the potential to treat numerous diseases associated with intestinal inflammation; however, the side-effects caused by the systemic depletion of cytokines demands that the delivery of cytokine-targeted siRNAs be localized to diseased intestinal tissues. Although various delivery vehicles have been developed to orally deliver therapeutics to intestinal tissue, none of these strategies has demonstrated the ability to protect siRNA from the harsh environment of the gastrointestinal tract and target its delivery to inflamed intestinal tissue. Here, we present a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can localize orally delivered siRNA to sites of intestinal inflammation, and thus inhibit gene expression in inflamed intestinal tissue. TKNs are formulated from a polymer, poly-(1,4-phenyleneacetone dimethylene thioketal), that degrades selectively in response to reactive oxygen species (ROS). Therefore, when delivered orally, TKNs release siRNA in response to the abnormally high levels of ROS specific to sites of intestinal inflammation. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with siRNA against the proinflammatory cytokine tumour necrosis factor-alpha (TNF-α) diminish TNF-α messenger RNA levels in the colon and protect mice from ulcerative colitis.     

ROS‐Responsive Polymeric siRNA Nanomedicine Stabilized by Triple Interactions for the Robust Glioblastoma Combinational RNAi Therapy

Small interfering RNA (siRNA) holds inherent advantages and great potential for treating refractory diseases. However, lack of suitable siRNA delivery systems that demonstrate excellent circulation stability and effective at‐site delivery ability is currently impeding siRNA therapeutic performance. Here, a polymeric siRNA nanomedicine (3I‐NM@siRNA) stabilized by triple interactions (electrostatic, hydrogen bond, and hydrophobic) is constructed. Incorporating extra hydrogen and hydrophobic interactions significantly improves the physiological stability compared to an siRNA nanomedicine analog that solely relies on the electrostatic interaction for stability. The developed 3I‐NM@siRNA nanomedicine demonstrates effective at‐site siRNA release resulting from tumoral reactive oxygen species (ROS)‐triggered sequential destabilization. Furthermore, the utility of 3I‐NM@siRNA for treating glioblastoma (GBM) by functionalizing 3I‐NM@siRNA nanomedicine with angiopep‐2 peptide is enhanced. The targeted Ang‐3I‐NM@siRNA exhibits superb blood–brain barrier penetration and potent tumor accumulation. Moreover, by cotargeting polo‐like kinase 1 and vascular endothelial growth factor receptor‐2, Ang‐3I‐NM@siRNA shows effective suppression of tumor growth and significantly improved survival time of nude mice bearing orthotopic GBM brain tumors. New siRNA nanomedicines featuring triple‐interaction stabilization together with inbuilt self‐destruct delivery ability provide a robust and potent platform for targeted GBM siRNA therapy, which may have utility for RNA interference therapy of other tumors or brain diseases.     

Effective gene silencing by multilayered siRNA-coated gold nanoparticles

Small interfering RNA (siRNA) has been widely proposed to treat various diseases by silencing genes, but its delivery remains a challenge. A well controlled assembly approach is applied to prepare a protease-assisted nanodelivery system. Protease-degradable poly-L-lysine (PLL) and siRNA are fabricated onto gold nanoparticles (AuNPs), by alternating the charged polyelectrolytes. In this study, up to 4 layers of PLL and 3 layers of siRNA (sR3P) are coated. Due to the slow degradation of PLL, the incorporated siRNA is released gradually and shows extended gene-silencing effects. Importantly, the inhibition effect in cells is found to correlate with the number of siRNA layers.