Dicer-labile PEG conjugates for siRNA delivery

Poly(ethylene glycol) (PEG) conjugates of Dicer-substrate small interfering RNA (DsiRNA) have been prepared to investigate a new siRNA release strategy. 3'-sense or 5'-antisense thiol-modified, blunt-ended DsiRNAs, inhibiting enhanced green fluorescent protein (eGFP) expression, were covalently conjugated to PEG with varying molecular weights (2, 10, and 20 kg/mol) through a stable thioether bond using a Michael addition reaction. The DsiRNA conjugates with 2 kg/mol PEG (both 3'-sense or 5'-antisense strand conjugated) and the 10 kg/mol PEG conjugated to the 3'-sense strand of DsiRNA were efficiently cleaved by recombinant human Dicer to 21-mer siRNA, as determined by gel electrophoresis. Importantly, 2 and 10 kg/mol PEG conjugated to the 3'-sense strand of DsiRNA showed potent gene silencing activity in human neuroblastoma (SH-EP) cells, stably expressing eGFP, at both the mRNA and protein levels. Moreover, the 10 kg/mol PEG conjugates of the 3'-sense strand of DsiRNA were less immunogenic when compared with the unmodified DsiRNA, determined via an immune stimulation assay on human peripheral blood mononuclear cells.     

siRNA delivery technologies for mammalian systems

Inhibition of gene expression using the RNA interference (RNAi) pathway is rapidly becoming the method of choice for studying gene function in mammalian cells. However, successful knockdown of the target gene requires efficient delivery of short interfering RNAs (siRNAs). Several technologies have been developed that enable effective delivery of siRNAs to both cells in culture and whole animals. These technologies will allow the use of RNAi to study gene function in mammalian model systems in which classical methods are often limited and costly.     

Intrathecal polymer-based interleukin-10 gene delivery for neuropathic pain

Research on communication between glia and neurons has increased in the past decade. The onset of neuropathic pain, a major clinical problem that is not resolved by available therapeutics, involves activation of spinal cord glia through the release of proinflammatory cytokines in acute animal models of neuropathic pain. Here, we demonstrate for the first time that the spinal action of the proinflammatory cytokine, interleukin 1 (IL-1) is involved in maintaining persistent (2 months) allodynia induced by chronic-constriction injury (CCI). The anti-inflammatory cytokine IL-10 can suppress proinflammatory cytokines and spinal cord glial amplification of pain. Given that IL-1 is a key mediator of neuropathic pain, developing a clinically viable means of long-term delivery of IL-10 to the spinal cord is desirable. High doses of intrathecal IL-10-gene therapy using naked plasmid DNA (free pDNA-IL-10) is effective, but the dose required limits its potential clinical utility. Here we show that intrathecal gene therapy for neuropathic pain is improved sufficiently using two, distinct synthetic polymers, poly(lactic-co-glycolic) and polyethylenimine, that substantially lower doses of pDNA-IL-10 are effective. In conclusion, synthetic polymers used as i.t. gene-delivery systems are well-tolerated and improve the long-duration efficacy of pDNA-IL-10 gene therapy.     

Transferrin-containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery

Transferrin is a well-studied ligand for tumor targeting due to upregulation of transferrin receptors in numerous cancer cell types. Here, we report the development of a transferrin-modified, cyclodextrin polymer-based gene delivery system. The delivery system is comprised of a nanoparticle (formed by condensation of a cyclodextrin polycation with nucleic acid) that is surface-modified to display poly(ethylene glycol) (PEG) for increasing stability in biological fluids and transferrin for targeting of cancer cells that express transferrin receptor. A transferrin-PEG-adamantane conjugate is synthesized for nanoparticle modification. The transferrin conjugate retains high receptor binding and self-assembles with the nanoparticles by adamantane (host) and particle surface cyclodextrin (guest) inclusion complex formation. At low transferrin modification, the particles remain stable in physiologic salt concentrations and transfect K562 leukemia cells with increased efficiency over untargeted particles. The increase in transfection is eliminated when transfections are conducted in the presence of excess free transferrin. The transferrin-modified nanoparticles are appropriate for use in the systemic delivery of nucleic acid therapeutics for metastatic cancer applications.     

Clustered magnetite nanocrystals cross-linked with PEI for efficient siRNA delivery

Magnetofection has been utilized as a powerful tool to enhance gene transfection efficiency via magnetic field-enforced cellular transport processes. The accelerated accumulation of nucleic acid molecules by applying an external magnetic force enables the rapid and improved transduction efficiency. In this study, we developed magnetite nanocrystal clusters (PMNCs) cross-linked with polyethylenimine (PEI) to magnetically trigger intracellular delivery of small interfering RNA (siRNA). PMNCs were produced by cross-linked assembly of catechol-functionalized branched polyethylenimine (bPEI) around magnetite nanocrystals through an oil-in-water (O/W) emulsion and solvent evaporation method. The physical properties of PMNC were characterized by TEM, DLS, TSA, and FT-IR. Finely tuned formulation of clustered magnetite nanocrystals with controlled size and shape exhibited superior saturation of magnetization value. Magnetite nanocrystal clusters could form nanosized polyelectrolyte complexes with negatively charged siRNA molecules, enabling efficient delivery of siRNA into cells upon exposure to an external magnetic field within a short time. This study introduces a new class of magnetic nanomaterials that can be utilized for magnetically driven intracellular siRNA delivery.     

Dicetyl phosphate-tetraethylenepentamine-based liposomes for systemic siRNA delivery

Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.     

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.     

Targeted quantum dot conjugates for siRNA delivery

Treatment of human diseases such as cancer generally involves the sequential use of diagnostic tools and therapeutic modalities. Multifunctional platforms combining therapeutic and diagnostic imaging functions in a single vehicle promise to change this paradigm. in particular, nanoparticle-based multifunctional platforms offer the potential to improve the pharmacokinetics of drug formulations, while providing attachment sites for diagnostic imaging and disease targeting features. We have applied these principles to the delivery of small interfering RNA (siRNA) therapeutics, where systemic delivery is hampered by rapid excretion and nontargeted tissue distribution. Using a PEGlyated quantum dot (QD) core as a scaffold, siRNA and tumor-homing peptides (F3) were conjugated to functional groups on the particle's surface. We found that the homing peptide was required for targeted internalization by tumor cells, and that siRNA cargo could be coattached without affecting the function of the peptide. Using an EGFP model system, the role of conjugation chemistry was investigated, with siRNA attached to the particle by disulfide cross-linkers showing greater silencing efficiency than when attached by a nonreducible thioether linkage. Since each particle contains a limited number of attachment sites, we further explored the tradeoff between number of F3 peptides and the number of siRNA per particle, leading to an optimized formulation. Delivery of these F3/siRNA-QDs to EGFP-transfected HeLa cells and release from their endosomal entrapment led to significant knockdown of EGFP signal. By designing the siRNA sequence against a therapeutic target (e.g., oncogene) instead of EGFP, this technology may be ultimately adapted to simultaneously treat and image metastatic cancer.     

Strategies for enhancing antibody delivery to the brain

Antibodies and antibody conjugates have emerged as important tools for cancer therapy. However, a major therapeutic challenge for the use of antibodies is their inability to cross the blood–brain barrier (BBB) to reach tumors localized in the central nervous system (CNS). Multiple methods have been developed to enhance antibody delivery to the CNS, including direct injection, mechanical or biochemical disruption of the BBB, conjugation to a ‘molecular Trojan horse’, cationization, encapsulation in nanoparticles and liposomes, and more recently, stem cell-mediated antibody delivery. In this review, we discuss each of these approaches, highlighting their successes and the obstacles that remain to be overcome.     

Evaluation of two cationic delivery systems for siRNA

Small interference RNA (siRNA) is an important research tool, and also has the potential for clinical application. RNA interference (RNAi) approaches allow degradation of selective mRNA coding for pathogenic or disease-related proteins. RNAi pathway can be taken advantage of by the delivery of chemically synthesized siRNA. To fully attain its potential a sufficient siRNA must be delivered to the cell's cytoplasm. Cellular delivery of polyanions such as siRNA, while a challenging problem, may be addressed by the use of cationic macromolecules, the two major classes being lipids and polymers. In this study we compared two model cationic vectors liposomes (lipoplexes) and polyethelyenimine (PEI) (polyplexes). Complexes of the cationic macromolecules and siRNA did not differ in terms of their cellular uptake as determined by flow cytometry. However, it was demonstrated that the lipoplexes decomplexed more easily than the polyplexes. Differences in the biological activity of the siRNA were observed using commercially available siTOX siRNA. Lipoplexes resulted in dose-dependent siRNA activity; to 76.4 +/- 5.9% cell death was seen 48 hours posttransfection using 80 nmol siTOX. In summary, the selection of delivery vector can have a profound impact on biological activity of siRNA molecules. siRNA decomplexation from the cationic vector might be an important factor in the future development of new vectors.     

Preparation of siRNA encapsulated nanoliposomes suitable for siRNA delivery by simply discontinuous mixing

Previously a scalable and extrusion-free method has been developed for efficient liposomal encapsulation of DNA by twice stepwise mixing of lipids in ethanol and DNA solution using T-shape mixing chamber. In this study, we prepared nanoliposomes encapsulating siRNA by simply discontinuous mixing of lipids in ethanol/ether/water mixture and acidic siRNA solution without use of special equipment. The simple mixing siRNA/liposomal particles (siRNA/SMLs) prepared using ethanol/ether/water (3:1:1) mixture showed 120.4 ± 20.2 nm particle size, 0.174 ± 0.033 polydispersity and 86.5 ± 2.76% siRNA encapsulation rate. In addition, the SMLs almost completely protected the encapsulated siRNA from RNase A digestion. Coupling of anti-human epidermal growth factor receptor (EGFR) Fab′ to siRNA/SMLs enhanced EGFR-specific cell penetration of SMLs and induced siRNA dependent gene silencing. Unexpectedly, the Cy5.5-labeled Fab′ showed almost no in vivo targeting to the xenografted A549 tumors in SCID-NOD mice. However, multiple injection of the unmodified siRNA/SMLs accumulated in the tumors and induced siRNA-dependent in vivo gene silencing. These results demonstrate that the siRNA/SMLs can be used as a siRNA delivery tool for gene therapy.     

Acid-degradable cationic dextran particles for the delivery of siRNA therapeutics

We report a new acid-sensitive, biocompatible, and biodegradable microparticulate delivery system, spermine modified acetalated-dextran (Spermine-Ac-DEX), which can be used to efficiently encapsulate siRNA. These particles demonstrated efficient gene knockdown in HeLa-luc cells with minimal toxicity. This knockdown was comparable to that obtained using Lipofectamine, a commercially available transfection reagent generally limited to in vitro use due to its high toxicity.     

Biomimetic nanoparticles for siRNA delivery in the treatment of leukaemia

Leukaemia is a bone marrow cancer occurring in acute and chronic subtypes. Acute leukaemia is a rapidly fatal cancer potentially causing death within a few weeks, if untreated. Leukaemia arises as a result of disruption to haematopoietic precursors, caused either by acquired gene fusions, gene mutations or inappropriate expression of the relevant oncogenes. Current treatment options have made significant progress, but the 5 year survival for acute leukaemia remains under 10% in elderly patients, and less than 50% for some types of acute leukaemia in younger adults. For chronic leukaemias longer survival is generally expected and for chronic myeloid leukaemia patients on tyrosine kinase inhibitors the median survival is not yet reached and is expected to exceed 10 years. Chemotherapy and haematopoietic stem cell transplantation (HSCT) for acute leukaemia provide the mainstay of therapy for patients under 65 and both carry significant morbidity and mortality. Alternative and superior therapeutic strategies for acute leukaemias are urgently required. Recent molecular-based knowledge of recurring chromosome rearrangements, in particular translocations and inversions, has resulted in significant advances in understanding the molecular pathogenesis of leukaemia. Identification of a number of unique fusion genes has facilitated the development of highly specific small interfering RNAs (siRNA). Although delivery of siRNA using multifunctional nanoparticles has been investigated to treat solid cancers, the application of this approach to blood cancers is at an early stage. This review describes current treatments for leukaemia and highlights the potential of leukaemic fusion genes as therapeutic targets for RNA interference (RNAi). In addition, the design of biomimetic nanoparticles which are capable of responding to the physiological environment of leukaemia and their potential to advance RNAi therapeutics to the clinic will be critically evaluated.     

Chiral cationic polyamines for chiral microcapsules and siRNA delivery

Reported herein is the use of chiral cationic polyamines for two intriguing applications: fabrication of chiral covalently-linked microcapsules, and enantiospecific delivery of siRNA to Huh 7 cells. The microcapsules are easily fabricated from homochiral polymers, and the resulting architectures can be used for supramolecular chiral catalysis and many other potential applications. Enantiospecific delivery of siRNA to Huh 7 cells is seen by one ‘enantiomer’ of the polymers delivering siRNA with significantly improved transfection efficiency and reduced toxicity compared to the ‘enantiomeric’ polymer and commercially available transfection reagents. Taken together, the use of these easily accessible polyamine structures for diverse applications is highlighted in this Letter herein and can lead to numerous future research efforts.     

Acetylation of PAMAM dendrimers for cellular delivery of siRNA

Background  

The advancement of gene silencing via RNA interference is limited by the lack of effective short interfering RNA (siRNA) delivery vectors. Rational design of polymeric carriers has been complicated by the fact that most chemical modifications affect multiple aspects of the delivery process. In this work, the extent of primary amine acetylation of generation 5 poly(amidoamine) (PAMAM) dendrimers was studied as a modification for the delivery of siRNA to U87 malignant glioma cells.     

Synthesis of folate-functionalized RAFT polymers for targeted siRNA delivery

Receptor-mediated, cell-specific delivery of siRNA enables silencing of target genes in specific tissues, opening the door to powerful therapeutic options for a multitude of diseases. However, the development of delivery systems capable of targeted and effective siRNA delivery typically requires multiple steps and the use of sophisticated, orthogonal chemistries. Previously, we developed diblock copolymers consisting of dimethaminoethyl methacrylate-b-dimethylaminoethyl methacrylate-co-butyl methacrylate-co-propylacrylic acid as potent siRNA delivery systems that protect siRNA from enzymatic degradation and enable its cytosolic delivery through pH-responsive, endosomolytic behavior. (1, 2) These architectures were polymerized using a living radical polymerization method, specifically reversible addition-fragmentation chain transfer (RAFT) polymerization, which employs a chain transfer agent (CTA) to modulate the rate of reaction, resulting in polymers with low polydispersity and telechelic chain ends reflecting the chemistry of the CTA. Here we describe the straightforward, facile synthesis of a folate receptor-targeted diblock copolymer siRNA delivery system because the folate receptor is an attractive target for tumor-selective therapies as a result of its overexpression in a number of cancers. Specifically, we detail the de novo synthesis of a folate-functionalized CTA, use the folate-CTA for controlled polymerizations of diblock copolymers, and demonstrate efficient, specific cellular folate receptor interaction and in vitro gene knockdown using the folate-functionalized polymer.     

Optimization of a synthetic siRNA delivery for the treatment of rhabdomyosarcoma

Silencing of a target-genes by small interfering RNA (siRNA) has emerged as a powerful new tool not only for basic research but also with potential therapeutic benefits. This paper demonstrates that optimal delivery strategy is crucial for effective target-gene silencing. Using lipofection, under defined conditions, we were able to markedly down-regulate expression of the selected genes involved in rhabdomyosarcoma metastasis: MET, CXCR4, LIFR and PAX3-FKHR.     

Functionalized silicon quantum dots tailored for targeted siRNA delivery

For RNA interference (RNAi) mediated silencing of the ABCB1 gene in Caco-2 cells biocompatible luminescent silicon quantum dots (SiQDs) were developed to serve as self-tracking transfection tool for ABCB1 siRNA. While the 2-3 nm sized SiQD core exhibits green luminescence, the QD surfaces are completely saturated with covalently linked 2-vinylpyridine that may electrostatically bind siRNA. For down-regulating P-glycoprotein (Pgp) expression of the ABCB1 gene the SiQDs were complexed with siRNA. The cellular uptake and allocation of SiQD-siRNA complexes in Caco-2 cells were monitored using confocal laser scanning microscopy and transmission electron microscopy. The release of siRNA to the cytoplasm was verified through real-time PCR quantification of the reduced ABCB1 mRNA level. Additional evidence was obtained from time-resolved in situ fluorescence spectroscopic monitoring of the Pgp efflux dynamics in transfected Caco-2 cells which yielded significantly reduced transporter efficiencies for the Pgp substrate Rhodamine 123.