A Combinatorial Library of Lipid Nanoparticles for RNA Delivery to Leukocytes

Lipid nanoparticles (LNPs) are the most advanced nonviral platforms for small interfering RNA (siRNA) delivery that are clinically approved. These LNPs, based on ionizable lipids, are found in the liver and are now gaining much attention in the field of RNA therapeutics. The previous generation of ionizable lipids varies in linker moieties, which greatly influences in vivo gene silencing efficiency. Here novel ionizable amino lipids based on the linker moieties such as hydrazine, hydroxylamine, and ethanolamine are designed and synthesized. These lipids are formulated into LNPs and screened for their efficiency to deliver siRNAs into leukocytes, which are among the hardest to transfect cell types. Two potent lipids based on their in vitro gene silencing efficiencies are also identified. These lipids are further evaluated for their biodistribution profile, efficient gene silencing, liver toxicity, and potential immune activation in mice. A robust gene silencing is also found in primary lymphocytes when one of these lipids is formulated into LNPs with a pan leukocyte selective targeting agent (β₇ integrin). Taken together, these lipids have the potential to open new avenues in delivering RNAs into leukocytes.     

Tripartite siRNA micelles as controlled delivery systems for primary dendritic cells

Dendritic cells (DCs) are key cells in immunology that are able to stimulate or inhibit the immune response. RNA interference has appeared of great interest to modulate the expression of immunogenic or tolerogenic molecules. In our study, pH-sensitive polyion complex micelles based on a double-hydrophilic block copolymer and poly-l-lysine were formulated to entrap a small interfering RNA (siRNA). We show that siRNA-loaded micelles were cytotolerant and efficiently endocytosed by DCs. siRNA targeting eGFP, used as model siRNA, was released into the cytosol following endocytosis of the micelles and the silencing of eGFP expression was observed in DC isolated from transgenic mice.

Our results underscore the potential of pH-sensitive polyion complex micelles to formulate therapeutic siRNA for DC engineering in order to maintain the homeostasis of the immune response.     

Peptide‐like Polymers Exerting Effective Glioma‐Targeted siRNA Delivery and Release for Therapeutic Application

Lipopolymer 49, a solid‐phase synthesized T‐shaped peptide‐like oligoamide containing two central oleic acids, 20 aminoethane, and two terminal cysteine units, is identified as very potent and biocompatible small interfering RNA (siRNA) carrier for gene silencing in glioma cells. This carrier is combined with a novel targeting polymer 727, containing a precise sequence of Angiopep 2 targeting peptide, linked with 28 monomer units of ethylene glycol, 40 aminoethane, and two terminal cysteines in siRNA complex formation. Angiopep‐polyethylene glycol (PEG)/siRNA polyplexes exhibit good nanoparticle features, effective glioma‐targeting siRNA delivery, and intracellular siRNA release, resulting in an outstanding gene downregulation both in glioma cells and upon intravenous delivery in glioma model nude mice without significant biotoxicity. Therefore, this novel siRNA delivery system is expected to be a promising strategy for targeted and safe glioma therapy.     

A Highly Entangled Polymeric Nanoconstruct Assembled by siRNA and its Reduction‐Triggered siRNA Release for Gene Silencing

A nanoconstruct (NC) is developed from a biocompatible natural polymer and siRNA conjugates to deliver small interfering RNA (siRNA) target‐specifically without cationic condensation reagents. This study reports a novel siRNA‐mediated cross‐linked NC produced by hybridizing two complementary single‐stranded siRNAs that are conjugated to the polymer dextran via a disulfide linkage. The reducible disulfide bond between the siRNA and polymer allow siRNA release from the NC in the reducible cytoplasmic region after the NC enters the cell. In addition, when the NC contains the prostate‐carcinoma‐binding peptide aptamer DUP‐1, it can selectively deliver siRNA into prostate cancer cells of the PC‐3 lines; thus, the newly formulated NC has reduced the cytotoxicity and improved the efficacy of target‐specific siRNA delivery. Moreover, this new concept of NCs using biocompatible siRNA and a neutral polymer may provide insightful knowledge for future directions for designing NCs for stimuli‐responsive and advanced target‐specific siRNA delivery.     

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.     

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.     

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.     

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.     

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.     

Anionic liposomes for small interfering ribonucleic acid (siRNA) delivery to primary neuronal cells: Evaluation of alpha-synuclein knockdown efficacy

Alpha-synuclein (α-syn) deposition in Lewy bodies (LB) is one of the main neuropathological hallmarks of Parkinson's disease (PD).LB accumulation is considered a causative factor of PD,which suggests that strategies aimed at reducing α-syn levels could be relevant for its treatment.In the present study,we developed novel nanocarriers suitable for systemic delivery of small interfering ribonucleic acid (siRNA) that were specifically designed to reduce neuronal α-syn by RNA interference.Anionic liposomes loaded with an siRNA-protamine complex for α-syn gene silencing and decorated with a rabies virus glycoprotein (RVG)-derived peptide as a targeting agent were prepared.The nanoparticles were characterized for their ability to load,protect,and deliver the functional siRNA to mouse primary hippocampal and cortical neurons as well as their efficiency to induce gene silencing in these cells.Moreover,the nanocarriers were evaluated for their stability in serum.The RVG-decorated liposomes displayed suitable characteristics for future in vivo applications and successfully induced α-syn gene silencing in primary neurons without altering cell viability.Collectively,our results indicate that RVG-decorated liposomes may be an ideal tool for further studies aimed at achieving efficient in vivo α-syn gene silencing in mouse models of PD.     

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.     

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.     

短发夹RNA介导RNA干涉的实验学控制研究

采用载体介导的RNA干涉(RNA interference,RNAi)技术抑制HEK293H细胞中外源报告基因的表达,并设置一系列的实验学控制以探讨其在RN加应用研究中的价值及意义。运用pAVU6 27,构建了针对增强型绿色荧光蛋白(EGFP)基因的一系列短发夹RNA表达载体,并用脂质体法转染于．HEK293H细胞中,检测EGFPmRNA及蛋白表达水平。瞬时转染48h后,RT-PCR及Western blot结果表明,针对EGFP三个靶点的干涉质粒均不同程度地抑制细胞内EGFP的表达,但仅转录正反义链、链内1—2个碱基错配的干涉载体不能介导特异的RNAi效应。此外,共转染实验结果表明干涉质粒两两间无相互增强或抑制的RNAi效应。在此基础上,通过G418筛选生成了HEK293H EGFP稳定表达株,在转染后不同时间点检测EGFPmRNA及蛋白表达变化,发现RNAi效应在一定时间范围内呈现明显的时间依赖性。这些结果表明,有效的实验控制在RNAi研究中十分必要,具有重要的参考价值。基于载体表达的RNAi作用呈现明显的时间依赖性效应,为RNAi应用研究提供了一定的参考及借鉴价值。     

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.     

Design of double-walled carbon nanotubes for biomedical applications

Double-walled carbon nanotubes (DWNTs) prepared by catalytic chemical vapour deposition were functionalized in such a way that they were optimally designed as a nano-vector for the delivery of small interfering RNA (siRNA), which is of great interest for biomedical research and drug development. DWNTs were initially oxidized and coated with a polypeptide (Poly(Lys:Phe)), which was then conjugated to thiol-modified siRNA using a heterobifunctional cross-linker. The obtained oxDWNT-siRNA was characterized by Raman spectroscopy inside and outside a biological environment (mammalian cells). Uptake of the custom-designed nanotubes was not associated with detectable biochemical perturbations in cultured cells, but transfection of cells with DWNTs loaded with siRNA targeting the green fluorescent protein (GFP) gene, serving as a model system, as well as with therapeutic siRNA targeting the survivin gene, led to a significant gene silencing effect, and in the latter case a resulting apoptotic effect in cancer cells.     

Biodegradable Nanocapsules as siRNA Carriers for Mutant K‐Ras Gene Silencing of Human Pancreatic Carcinoma Cells

The application of small interfering RNA (siRNA)‐based RNA interference (RNAi) for cancer gene therapy has attracted great attention. Gene therapy is a promising strategy for cancer treatment because it is relatively non‐invasive and has a higher therapeutic specificity than chemotherapy. However, without the use of safe and efficient carriers, siRNAs cannot effectively penetrate the cell membranes and RNAi is impeded. In this work, cationic poly(lactic acid) (CPLA)‐based degradable nanocapsules (NCs) are utilized as novel carriers of siRNA for effective gene silencing of pancreatic cancer cells. These CPLA‐NCs can readily form nanoplexes with K‐Ras siRNA and over 90% transfection efficiency is achieved using the nanoplexes. Cell viability studies show that the nanoparticles are highly biocompatible and non‐toxic, indicating that CPLA‐NC is a promising potential candidate for gene therapy in a clinical setting.     

Mussel Adhesion‐Inspired Reverse Transfection Platform Enhances Osteogenic Differentiation and Bone Formation of Human Adipose‐Derived Stem Cells

Using small interfering RNA (siRNA) to regulate gene expression is an emerging strategy for stem cell manipulation to improve stem cell therapy. However, conventional methods of siRNA delivery into stem cells based on solution‐mediated transfection are limited due to low transfection efficiency and insufficient duration of cell‐siRNA contact during lengthy culturing protocols. To overcome these limitations, a bio‐inspired polymer‐mediated reverse transfection system is developed consisting of implantable poly(lactic‐co‐glycolic acid) (PLGA) scaffolds functionalized with siRNA‐lipidoid nanoparticle (sLNP) complexes via polydopamine (pDA) coating. Immobilized sLNP complexes are stably maintained without any loss of siRNA on the pDA‐coated scaffolds for 2 weeks, likely due to the formation of strong covalent bonds between amine groups of sLNP and catechol group of pDA. siRNA reverse transfection with the pDA‐sLNP‐PLGA system does not exhibit cytotoxicity and induces efficient silencing of an osteogenesis inhibitor gene in human adipose‐derived stem cells (hADSCs), resulting in enhanced osteogenic differentiation of hADSCs. Finally, hADSCs osteogenically committed on the pDA‐sLNP‐PLGA scaffolds enhanced bone formation in a mouse model of critical‐sized bone defect. Therefore, the bio‐inspired reverse transfection system can provide an all‐in‐one platform for genetic modification, differentiation, and transplantation of stem cells, simultaneously enabling both stem cell manipulation and tissue engineering.     

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.