Nanocarriers For siRNA Delivery Research Articles

Multifunctional magnetic nanocarriers for delivery of siRNA and shRNA plasmid to mammalian cells: Characterization, adsorption and release behaviors

Nucleic acids are promising candidates for treating various diseases. Nucleic acid is negatively charged and hydrophilic; therefore, it is not efficiently taken up by cells. Successful gene therapy requires the development of carriers for efficient delivery of gene-expressing DNA plasmid and small interfering RNA (siRNA) duplex. In this study, we developed MNP-CA-PEI, a citric acid (CA)-modified magnetic nanoparticle (MNP) cross-linked with polyethyleneimine (PEI), using carbonyldiimidazole as the crosslinker. The physical properties of MNP-CA-PEI (particle size, morphologies, surface coating, surface potentials, magnetic hystereses, superparamagnetic behaviors, and infrared spectra) were systematically characterized by transmission electron microscopy imaging, dynamic light scattering, thermogravimetric analysis, superconducting quantum interference device, and Fourier transform infrared spectroscopy. The adsorption isotherm and kinetics were determined by the Langmuir model, the Freundlich model, a pseudo-first-order equation, and a pseudo-second-order equation. MNP-CA-PEI could form polyelectrolyte complexes with negatively charged nucleic acids, enabling the efficient delivery of nucleic acids into cells. Using MNP-CA-PEI nanoparticles, we magnetically triggered the intracellular delivery of green fluorescence protein (GFP)-expressing DNA plasmid, plasmid-expressing short hairpin RNA (shRNA) against GFP, or siRNA targeting GFP into different cell lines. Nucleic acid/MNP-CA-PEI displayed the enhanced cellular uptake of GFP-expressing DNA plasmid, and it improved the silencing efficiency of shRNA and siRNA, determined by fluorescence imaging. Gene knockdowns mediated by shRNA and siRNA were also confirmed by a quantitative real-time polymerase chain reaction. MNP-CA-PEI delivered nucleic acids into cytosol through caveolae-mediated endocytosis. This study introduces a new MNP functionalization that can be used for the magnetically driven intracellular delivery of nucleic acids.

Nanocarriers for delivery of siRNA as gene silencing mediator.

The term nanocarrier refers to sub-micrometric particles of less than 100 nm, designed to transport, distribute, and release nanotechnology-based drug delivery systems. siRNA therapy is a novel strategy that has great utility for a variety of treatments, however naked siRNA delivery has not been an effective strategy, resulting in the necessary use of nanocarriers for delivery. This review aims to highlight the versatility of carriers based on smart drug delivery systems. The nanocarriers based on nanoparticles as siRNA DDS have provided a set of very attractive advantages related to improved physicochemical properties, such as high surface-to-volume ratio, versatility to package siRNA, provide a dual function to both protect extracellular barriers that lead to elimination and overcome intracellular barriers limiting cytosolic delivery, and possible chemical modifications on the nanoparticle surface to improve stability and targeting. Lipid and polymeric nanocarriers have proven to be stable, biocompatible, and effective in vitro, further exploration of the development of new nanocarriers is needed to obtain safe and biocompatible tools for effective therapy.

Nanoparticle‐Mediated siRNA Delivery and Multifunctional Modification Strategies for Effective Cancer Therapy

AbstractRNA interference (RNAi), which can inhibit the expression or transcription of specific genes, is considered to be a revolutionary tool for mediating gene therapy. Small interfering RNA (siRNA) is an important therapeutic agent for RNAi. However, the clinical application of siRNA‐based gene silencing is still limited due to the easy degradation of siRNA during the circulation in the body and the difficulty in delivering siRNA to desired tissues and cells. The rapid development of nanotechnology provides new ideas for siRNA delivery and a large variety of nanocarriers have been designed to solve the problems of siRNA delivery including easy degradation, lack of targeting ability, poor internalization ability, easy to cause induction of immune response and so on. Here, this review introduces the mechanism of RNAi in gene therapy, then summarizes the developments on nanocarriers for siRNA delivery, and specially focuses on the functional modification strategies of nanocarriers to construct multifunctional siRNA delivery vectors. Lastly, the future development and challenges of siRNA delivery vector are discussed, along with the current application of siRNA in tumor treatment clinical trials. It is hoped that this review can provide useful information to help transform siRNA‐mediated gene therapy into clinical application.

Exosomes as nanocarriers for siRNA delivery: paradigms and challenges.

Exosomes are nano-sized vesicles that facilitate intercellular communications through carrying genetic materials and functional biomolecules. Owing to their unique size and structure, exosomes have emerged as a useful tool to overcome the limitations of siRNA delivery. The use of exosomes as siRNA delivery vehicles lacks certain disadvantages of the existing foreign delivery systems such as viruses, polycationic polymers and liposomes, and introduces several advantages including inherent capacity to pass through biological barriers and escape from phagocytosis by the reticuloendothelial system, as well as being biocompatible, non-toxic, and immunologically inert. Different strategies have been employed to harness exosome-based delivery systems, including surface modification with targeting ligands, and using exosome-display technology, virus-modified exosomes, and exosome-mimetic vesicles. The present review provides a capsule summary of the recent advances and current challenges in the field of exosome-mediated siRNA delivery.

Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells

While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical conditions, siRNA-nanocarriers’ stability after intravenous administration is still a major obstacle for the development of RNA-interference based therapies. But, although the need for stability is well recognized, the notion that strong stabilization can decrease nanocarriers’ efficiency is sometimes neglected. In this work we evaluated two stealth functionalization strategies to stabilize the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. The nanocarriers were pre- and post-pegylated, forming vectors with different stabilities in biological fluids. The stealth nanocarriers’ behavior was tested under biological mimetic conditions, as the production of stable siRNA-lipoplexes is determinant to achieve efficient intravenous siRNA delivery to cancer cells. Upon incubation in human serum for 2h, by fluorescence Single Particle Tracking microscopy, PEG-coated lipoplexes were found to have better colloidal stability as they could maintain a relatively stable size. In addition, using fluorescence fluctuation spectroscopy, post-pegylation also proved to avoid siRNA dissociation from the nanocarriers in human serum. Concomitantly it was found that PEG-coated lipoplexes improved cellular uptake and transfection efficiency in H1299 cells, and had the ability to silence BCR-ABL, affecting the survival of K562 cells.Based on an efficient cellular internalization, good silencing effect, good siRNA retention and good colloidal stability in human serum, DODAB:MO (2:1) siRNA-lipoplexes coated with PEG-Cer are considered promising nanocarriers for further in vivo validation. Statement of SignificanceThis work describes two stealth functionalization strategies for the stabilization of the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. These nanocarriers are capable of efficiently incorporating and delivering siRNA molecules to cells in order to silence genes whose expression is implicated in a pathological condition. The main objective was to functionalize these nanocarriers with a coating conferring protection to siRNA in blood without compromising its efficient delivery to cancer cells, validating the potential of DODAB:MO (2:1) siRNA-lipoplexes as therapeutic vectors. We show that the stealth strategy is determinant to achieve a stable and efficient nanocarrier, and that DODAB:MO mixtures have a very promising potential for systemic siRNA delivery to leukemic cells.

Nanocarriers for delivery of siRNA and co-delivery of siRNA and other therapeutic agents.

A major problem in cancer treatment is the multidrug resistance. siRNA inhibitors have great advantages to solve the problem, if the bottleneck of their delivery could be well addressed by the various nanocarriers. Moreover, co-delivery of siRNA together with the various anticancer agents in one nanocarrier may maximize their additive or synergistic effect. This review provides a comprehensive summary on the state-of-the-art of the nanocarriers, which may include prodrugs, micelles, liposomes, dendrimers, nanohydrogels, solid lipid nanoparticles, nanoparticles of biodegradable polymers and nucleic acid nanocarriers for delivery of siRNA and co-delivery of siRNA together with anticancer agents with focus on synthesis of the nanocarrier materials, design and characterization, in vitro and in vivo evaluation, and prospect and challenges of nanocarriers.

Polymeric nanoparticles for the delivery of siRNA and the partnership between WSU and UNIT

RNA interference (RNAi) is an endogenous regulatory process whereby double-stranded RNA (dsRNA) present in the cell cytoplasm causes sequence-specific, post-transcriptional gene silencing [1]. dsRNA is first cleaved into short (19-21 nucleotides long) dsRNA fragments (siRNA) by Dicer, an endoribonuclease. The siRNA, which may be endogenous or exogenous (synthetically produced therapeutic) is subsequently loaded onto the RNA-induced silencing complex (RISC). The sense or passenger strand of the siRNA is then unwound and cleaved. The retained anti-sense or guide strand is highly specific to the target messenger RNA (mRNA) through complementary base paring. The activated RISC-siRNA complex scans, binds and degrades the complementary target mRNA present in the cell cytoplasm at a highly specific position relative to the 5' end of the anti-sense strand, thus causing degradation of the complementary mRNA, which then leads to gene silencing. This process is catalytic, allowing the same RISC-siRNA complex to cleave multiple copies of the target mRNAs, and will continue until the siRNA is degraded or diluted upon cell division. With the decoding of the human genome siRNA has emerged as a broadly applicable and versatile therapeutic platform. The drug discovery aspect is greatly optimized for therapeutic siRNA compared to small molecular weight drugs, as one only requires knowledge of the sequence of the target gene in the case of siRNA, while trial and error through more or less random modification of the drug is required during screening of small drug molecules [2]. The specificity of siRNA is particularly relevant in the treatment of cancer, as there is the potential to selectively act only on cancerous cells, even when the therapeutic reaches non-target cell populations. siRNA therapy also holds promise in targeting the so-called 'non-druggable' diseases - those not amenable to conventional therapeutics such as small molecules, or monoclonal antibodies [3]. Another advantage of therapeutic siRNA is that gene regulation happens in the cell cytosol, and not the nucleus as in DNA-based gene therapies. This is significant, as the passive transport through the nuclear membrane is highly regulated, much more so than that across the cellular membrane, and is thus particularly relevant to postmitotic cells such as those in the airways. The critical limiting factor in translating siRNA therapeutics to the clinic is the ability to efficiently deliver siRNA to the cell cytoplasm.[4]. Free siRNA is very unstable in the extracellular milieu, with plasma half-lives of only ca. 10 min [5] Moreover, due to its negative charge and large size, free siRNA is not readily internalized through passive diffusion, and is easily degraded in the lysosomes following cellular internalization through endocytosis [4,6]. Therefore, only a small fraction of the initial siRNA dose reaches the cell cytosol when administered as a free molecule. The use of nanocarriers for the efficient delivery of siRNA, and the ability to formulate such nanocarriers for regional delivery to the tissue of interest is thus of great relevance in developing siRNA therapeutic platforms. Because viral carriers are potentially associated with severe immunological responses, non-viral siRNA carriers are preferred [6]. Non-viral (nano)carriers may involve complexation of siRNA with a positively charged carrier, chemical conjugation, or encapsulation [5,7]. The most widely used methods for the delivery of nucleic acids are lipoplexes, and polyplexes, and complexes/self-assembled domains. Conjugation of siRNA to singly functionalizable molecules such as PEG and cholesterol has been also explored. More recently, conjugation of siRNA to multivalent nanocarriers has been proposed, and this strategy may provide unique opportunities that may help overcome the challenges associated with the delivery of siRNA with complexes and singly functionalizable molecules. In this work we will discuss recent advances on the use of nanocarriers for siRNA delivery, with a special focus on the targeting of lung diseases. We will also discuss strategies for the formulation of such siRNA carriers for the regional delivery to the lungs using oral inhalation devices.

Lyophilised ready-to-use formulations of PEG-PCL-PEI nano-carriers for siRNA delivery

The purpose of the present study was to transfer aqueous PEG-PCL-PEI nano-suspensions into dry ready-to-use formulations, suitable for delivery of siRNA. Therefore, freshly prepared nano-suspensions were lyophilised with glucose as lyoprotectant. Firstly, the required glucose concentration for sufficient stabilisation of unloaded carriers was determined via dynamic light scattering. Morphology of fresh and rehydrated carriers was visualised by cryogenic scanning electron microscopy. Subsequently, the feasibility of siRNA loading before and after lyophilisation was investigated. For both strategies complex diameter and in vitro transfection efficiency were determined and correlated to freshly prepared samples. Hydrodynamic diameter (95.2±1.4nm) and size distribution (0.132±0.019) of unloaded nano-suspension were restored after rehydration by addition of ≥1.5% of glucose before lyophilisation. Moreover, after loading of rehydrated carriers with siRNA, no significant difference in complex size was observed as compared to freshly prepared ones. Stabilisation of pre-formed carrier/siRNA complexes during lyophilisation is feasible at elevated N/P (e.g. 20) and glucose concentrations above 5%. As determined via real-time-PCR, lyophilised samples were as active as freshly prepared ones regarding transfection efficiency. In conclusion, lyophilisation is an effective technique to produce physically stable PEG-PCL-PEI formulations. These general findings may be applicable to further particulate gene delivery systems to shelf ready-to-use formulations.

PEGylated Calcium Phosphate Nanocomposites as Smart Environment‐Sensitive Carriers for siRNA Delivery

A novel inorganic–organic hybrid nanocomposite is formed in situ using a simple and straightforward method. Conjugate of short interfering RNA (siRNA) duplex with poly(ethylene glycol) via a disulfide linkage (PEG–SS–siRNA) is demonstrated to regulate the crystal growth of calcium phosphate (CaP), yielding a monodispersed nanocomposite. The resultant nanocomposite can be utilized as nanocarriers for siRNA delivery. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

165. Characterization of Low Molecular Weight Folate-Conjugated Chitosan as Polymeric Nanocarriers for siRNA Delivery

165. Characterization of Low Molecular Weight Folate-Conjugated Chitosan as Polymeric Nanocarriers for siRNA Delivery