A new peptide vector for efficient delivery of oligonucleotides into mammalian cells.

The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.     

LHRH receptor-mediated delivery of siRNA using polyelectrolyte complex micelles self-assembled from siRNA-PEG-LHRH conjugate and PEI

Polyelectrolyte complex (PEC) micelles modified with cancer cell targeting moieties were prepared for intracellular delivery of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA). A luteinizing hormone-releasing hormone (LHRH) peptide analogue was coupled as a cancer targeting ligand to the distal end of the poly(ethylene glycol) (PEG)-siRNA conjugate. The siRNA-PEG-LHRH conjugate self-assembled to form nanosized PEC micelles upon mixing with poly(ethylenimine) (PEI) via ionic interactions. The PEC micelles showed spherical morphology with a hydrodynamic diameter of ca. 150 nm. For LHRH receptor overexpressing ovarian cancer cells (A2780), the PEC micelles with LHRH exhibited enhanced cellular uptake compared to those without LHRH, resulting in increased VEGF gene silencing efficiency via receptor-mediated endocytosis. This study showed that PEC micelles decorated with specific cell-recognizable targeting ligands could be used for targeted delivery of siRNA.     

Graphene oxide nanosheets in complex with cell penetrating peptides for oligonucleotides delivery

A new strategy for gene transfection using the nanocarrier of cell penetrating peptides (CPPs; PepFect14 (PF14) or PepFect14 (PF14) (PF221)) in complex with graphene oxide (GO) is reported. GO complexed with CPPs and plasmid (pGL3), splice correction oligonucleotides (SCO) or small interfering RNA (siRNA) are performed. Data show adsorption of CPPs and oligonucleotides on the top of the graphenic lamellar without any observed change of the particle size of GO. GO mitigates the cytotoxicity of CPPs and improves the material biocompatibility. Complexes of GO-pGL3-CPPs (CPPs; PF14 or PF221) offer 2.1–2.5 fold increase of the cell transfection compared to pGL3-CPPs (CPPs; PF14 or PF221). GO-SCO-PF14 assemblies effectively transfect the cells with an increase of > 10–25 fold compared to the transfection using PF14. The concentration of GO plays a significant role in the material nanotoxicity and the transfection efficiency. The results open a new horizon in the gene treatment using CPPs and offer a simple strategy for further investigations.     

Scavenger receptor-mediated uptake of cell-penetrating peptide nanocomplexes with oligonucleotides

Cell-penetrating peptides (CPPs) are short cationic peptides that penetrate cells by interacting with the negatively charged plasma membrane; however, the detailed uptake mechanism is not clear. In contrary to the conventional mode of action of CPPs, we show here that a CPP, PepFect14 (PF14), forms negatively charged nanocomplexes with oligonucleotides and their uptake is mediated by class-A scavenger receptors (SCARAs). Specific inhibitory ligands of SCARAs, such as fucoidin, polyinosinic acid, and dextran sulfate, totally inhibit the activity of PF14-oligonucleotide nanocomplexes in the HeLa pLuc705 splice-correction cell model, while nonspecific, chemically related molecules do not. Furthermore, RNA interference (RNAi) knockdown of SCARA subtypes (SCARA3 and SCARA5) that are expressed in this cell line led to a significant reduction of the activity to <50%. In line with this, immunostaining shows prevalent colocalization of the nanocomplexes with the receptors, and electron microscopy images show no binding or internalization of the nanocomplexes in the presence of the inhibitory ligands. Interestingly, naked oligonucleotides also colocalize with SCARAs when used at high concentrations. These results demonstrate the involvement of SCARA3 and SCARA5 in the uptake of PF14-oligonucleotide nanocomplexes and suggest for the first time that some CPP-based systems function through scavenger receptors, which could yield novel possibilities to understand and improve the transfection by CPPs.     

Monoolein-based nanocarriers for enhanced folate receptor-mediated RNA delivery to cancer cells

We report the development and characterization of a novel nanometric system for specific delivery of therapeutic siRNA for cancer treatment. This vector is based on a binary mixture of the cationic surfactant dioctadecyldimethylammonium chloride (DODAC) and the helper lipid monoolein (MO). These liposomes were previously validated by our research group as promising non-viral vectors for nucleic acid delivery. In this work, the DODAC:MO vesicles were for the first time functionalized with polyethylene glycol and PEG-folate conjugates to achieve both maximal stability in biological fluids and increase selectivity toward folate receptor α expressing cells. The produced DODAC:MO:PEG liposomes were highly effective in RNA complexation (close to 100%), and the resulting lipoplexes also demonstrated high stability in conditions simulating their administration by intravenous injection (physiological pH, high NaCl, heparin and fetal bovine serum concentrations). In addition, cell uptake of the PEG-folate-coated lipoplexes was significantly greater in folate receptor α positive breast cancer cells (39% for 25?µg/mL of lipid and 31% for 40?µg/mL) when compared with folate receptor α negative cells (31% for 25?µg/mL of lipid and 23% for 40?µg/mL) and to systems without PEG-folate (≈13% to 16% for all tested conditions), supporting their selectivity towards the receptor. Overall, the results support these systems as appealing vectors for selective delivery of siRNA to cancer cells by folate receptor α-mediated internalization, aiming at future therapeutic applications of interest.     

Effective cancer therapy based on selective drug delivery into cells across their membrane using receptor-mediated endocytosis

Cancer is one of the major causes of death globally. The current treatment options are insufficient, leading to unmet medical needs in cancer treatment. Off-target side effects, multidrug resistance, selective distribution to cancerous tissues, and cell membrane permeation of anti-cancer agents are critical problems to overcome. There is a method to solve these problems by using receptor-mediated endocytosis (RME). It is well known that proteins such as integrin, HER2, EGFR, or other cancer biomarkers are specifically overexpressed on the surface of target cancer cells. By taking advantage of such specific receptors, payloads can be transported into cells through endocytosis using a conjugate composed of the corresponding ligands connected to the payloads by an appropriate linker. After RME, the payloads released by endosomal escape into the cytoplasm can exhibit the cytotoxic activity against cancer cells. Cell-penetrating peptides (CPPs), tumor-homing peptides (THPs), and monoclonal antibodies (mAbs) are utilized as ligands in this system. Antibody drug conjugates (ADCs) based on RME have already been used to cure cancer. In addition to the canonical conjugate method, nanocarriers for spontaneous accumulation in cancer tissue due to enhanced permeability and retention (EPR) effect are extensively used. In this review, I introduce the possibilities and advantages of drug design and development based on RME for the treatment of cancer.     

Efficient cationic lipid-mediated delivery of antisense oligonucleotides into eukaryotic cells: down-regulation of the corticotropin-releasing factor receptor

Oligonucleotides (ODNs) can be employed as effective gene-specific regulators. However, before ODNs can reach their targets, several physical barriers have to be overcome, as although ODNs may pass cell membranes, most become sequestered in endocytic compartments. Accordingly, sophisticated strategies are required for efficient delivery. Here we have employed a pyridinium-based synthetic amphiphile, called SAINT-2, which carries ODNs into cells in a highly efficient, essentially non-toxic and serum-insensitive manner. Intracellular delivery was examined by monitoring the trafficking of fluorescent ODNs and lipid, and by measuring the effect of specific antisense ODNs on target mRNA and protein levels of the receptor for the neuropeptide corticotropin-releasing factor (CRF-R), expressed in Chinese hamster ovary cells. ODN delivery is independent of lipoplex size, and fluorescently tagged ODNs readily acquire access to the nucleus, whereas the carrier itself remains sequestered in the endosomal–lysosomal pathway. While the release is independent of the presence of serum, it is not observed when serum proteins gain access within the lipoplex, and which likely stabilizes the lipoplex membrane. We propose that the amphiphile-dependent aggregate structure governs complex dissociation, and hence, the biological efficiency of ODNs. We demonstrate an essentially non-toxic and effective antisense-specific down-regulation of the CRF-R, both at the mRNA and protein level.     

Novel cationic amphiphiles as delivery agents for antisense oligonucleotides.

There has been great interest recently in therapeutic use of nucleic acids including genes, ribozymes and antisense oligonucleotides. Despite recent improvements in delivering antisense oligonucleotides to cells in culture, nucleic acid-based therapy is still often limited by the poor penetration of the nucleic acid into the cytoplasm and nucleus of cells. In this report we describe nucleic acid delivery to cells using a series of novel cationic amphiphiles containing cholic acid moieties linked via alkylamino side chains. We term these agents 'molecular umbrellas' since the cationic alkylamino chains provide a 'handle' for binding of nucleic acids, while the cholic acid moieties are likely to interact with the lipid bilayer allowing the highly charged nucleic acid backbone to traverse across the cell membrane. Optimal gene and oligonucleotide delivery to cells was afforded by a derivative (amphiphile 5) containing four cholic acid moieties. With this amphiphile used as a constituent in cationic liposomes, a 4-5 log increase in reporter gene delivery was measured. This amphiphile used alone provided a 250-fold enhancement of oligo-nucleotide association with cells as observed by flow cytometry. A substantial fraction of cells exposed to complexes of amphiphile 5 and fluorescent oligo-nucleotide showed nuclear accumulation of the fluorophore. Enhanced pharmacological effectiveness of antisense oligonucleotides complexed with amphiphile 5 was observed using an antisense splicing correction assay that activates a Luciferase reporter. Intracellular delivery, nuclear localization and pharmacological effectiveness of oligonucleotides using amphiphile 5 were similar to those afforded by commercial cytofectins. However, in contrast to most commercial cytofectins, the umbrella amphiphile showed substantial delivery activity even in the presence of high concentrations of serum.     

DNA complexes with polycations useful for delivery of DNA into cells

Generation and physicochemical properties of complexes formed by high-molecular thymus DNA and plasmid DNA with synthetic polymers of (dimethyl amino)ethyl methacrylate, (diethyl amino)ethyl methacrylate, and poly(vinyl amine) were studied in solutions of different ionic strength using low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The complexes were tested for toxicity with T98G cell cultures. Condensation of DNA was shown to occur when the ratio of charged groups in the polycations and DNA exceeded unity. This condensation manifested itself as an increase in the optical density of DNA solutions. Condensation-associated changes in the dimensions of DNA molecules were determined, and phase diagrams of DNA-polycation systems were analyzed in the presence of NaCl. MTT analysis revealed no toxicity of these complexes.     

Lentiviral vectors for gene delivery into cells

Human immunodeficiency virus type I (HIV) is the etiologic agent of acquired immunodeficiency syndrome or AIDS. Vectors based upon HIV have been in use for over a decade. Beginning in 1996, with the demonstration of improved pseudotyping using vesicular stomatitis virus (VSV) G protein along with transduction of resting mammalian cells, a series of improvements have been made in these vectors, making them both safer and more efficacious. Taking a cue from vector development of murine leukemia virus (MLV), split coding and self-inactivating HIV vectors now appear quite suitable for phase I clinical trials. In parallel, a number of pre-clinical efficacy studies in animals have demonstrated the utility of these vectors for various diseases processes, especially neurodegenerative and hematopoietic illnesses. These vectors are also appropriate for the study of other viruses (specifically of viral entry) and investigation of the HIV replicative cycle, along with straightforward transgene delivery to target cells of interest. Vectors based upon other lentiviruses have shown similar abilities and promise. Although concerns remain, particularly with regards to detection and propagation of replication-competent lentivirus, it is almost certain that these vectors will be introduced into the clinic within the next 3-5 years.     

Carrier PNA for shRNA delivery into cells

A peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as ‘bridge-builder’ to connect a CPP with an shRNA. The carrier PNA successfully formed a hybrid with an shRNA bearing complementary dangling bases and the shRNA was introduced into cells by the carrier PNA, and RNAi was induced by the shRNA.     

DNA complexes with polycations used for delivery of DNA into cells

The formation and physicochemical properties of high-molecular thymus and plasmid DNA complexes with synthetic polymers based on (dimethyl-amino)ethyl methacrylate (DMAEM), (diethyl-amino)ethyl methacrylate (DEAEM), and polyvinyl amine (PVA) were investigated in solutions of different ionic strength by low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The toxicity of complexes in T98G cells was studied. It was shown that, when the ratio of polycations to DNA charged groups concentration (N+/P) reaches values > 1, DNA condensation occurs. It is accompanied by increasing optical density of solutions. Changes in DNA size after condensation were estimated. Phase diagrams of systems DNA/polycation in the presence of NaCl were obtained. It was shown by MTT-analysis that DNA complexes with polycations in the range of concentrations used have low toxicity.     

Mannose polyethylenimine conjugates for targeted DNA delivery into dendritic cells.

Cell surface-bound receptors represent suitable entry sites for gene delivery into cells by receptor-mediated endocytosis. Here we have taken advantage of the mannose receptor that is highly expressed on antigen-presenting dendritic cells for targeted gene transfer by employing mannosylpolyethylenimine (ManPEI) conjugates. Several ManPEI conjugates were synthesized and used for formation of ManPEI/DNA transfection complexes. Conjugates differed in the linker between mannose and polyethylenimine (PEI) and in the size of the PEI moiety. We demonstrate that ManPEI transfection is effective in delivering DNA into mannose receptor-expressing cells. Uptake of ManPEI/DNA complexes is receptor-specific, since DNA delivery can be competed with mannosylated albumin. Additionally, incorporation of adenovirus particles into transfection complexes effectively enhances transgene expression. This is particularly important for primary immunocompetent dendritic cells. It is demonstrated here that dendritic cells transfected with ManPEI/DNA complexes containing adenovirus particles are effective in activating T cells of T cell receptor transgenic mice in an antigen-specific fashion.     

Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

The condensation of nucleic acids into well-defined particles is an integral part of several approaches to artificial cellular delivery. Improvements in the efficiency of nucleic acid delivery in vivo are important for the development of DNA- and RNA-based therapeutics. Presently, most efforts to improve the condensation and delivery of nucleic acids have focused on the synthesis of novel condensing agents. However, short oligonucleotides are not as easy to condense into well-defined particles as gene-length DNA polymers and present particular challenges for discrete particle formation. We describe a novel strategy for improving the condensation and packaging of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. 2 kb). These non-covalent assemblies possess single-stranded nicks or single-stranded gaps at regular intervals along the duplex backbones. The condensation behavior of nicked- and gapped-DNA duplexes was investigated using several cationic condensing agents. Transmission electron microscopy and light-scattering studies reveal that these DNA duplexes condense much more readily than short duplex oligonucleotides (i.e. 21 bp), and more easily than a 3 kb plasmid DNA. The polymeric condensing agents, poly-l-lysine and polyethylenimine, form condensates with nicked- and gapped-DNA that are significantly smaller than condensates formed by the 3 kb plasmid DNA. These results demonstrate the ability for DNA structure and topology to alter nucleic acid condensation and suggest the potential for the use of this form of DNA in the design of vectors for oligonucleotide and gene delivery. The results presented here also provide new insights into the role of DNA flexibility in condensate formation.     

Aptamer-mediated delivery of splice-switching oligonucleotides to the nuclei of cancer cells

To reduce the adverse effects of cancer therapies and increase their efficacy, new delivery agents that specifically target cancer cells are needed. We and others have shown that aptamers can selectively deliver therapeutic oligonucleotides to the endosome and cytoplasm of cancer cells that express a particular cell surface receptor. Identifying a single aptamer that can internalize into many different cancer cell-types would increase the utility of aptamer-mediated delivery of therapeutic agents. We investigated the ability of the nucleolin aptamer (AS1411) to internalize into multiple cancer cell types and observed that it internalizes into a wide variety of cancer cells and migrates to the nucleus. To determine if the aptamer could be utilized to deliver therapeutic oligonucleotides to modulate events in the nucleus, we evaluated the ability of the aptamer to deliver splice-switching oligonucleotides. We observed that aptamer-splice-switching oligonucleotide chimeras can alter splicing in the nuclei of treated cells and are effective at lower doses than the splice switching oligonucleotides alone. Our results suggest that aptamers can be utilized to deliver oligonucleotides to the nucleus of a wide variety of cancer cells to modulate nuclear events such as RNA splicing.     

Incorporation of terminal phosphorothioates into oligonucleotides.

Considerable effort has been directed towards studying the structure and function of oligonucleotides and several approaches rely on the attachment of reporter groups to oligonucleotides. We report here the introduction of 3'- and 5'-terminal phosphorothioates into heptameric oligonucleotides and their post-synthetic modification with several reporter groups. The synthesis of terminal phosphorothioates is based on the coupling of a ribonucleoside phosphoramidite at the first or last nucleotide, respectively, which, after sulphurization, is removed by sequential oxidation of the vicinal hydroxyl groups and then beta-elimination. Product formation is of the order of 95%. The ratio of phosphorothioate- versus phosphate-terminated oligodeoxynucleotides as analysed by electrophoresis on a Hg2+gel is in general 85/15. Examples for the reactivity of the terminal phosphorothioates for conjugation with cholesterol, bimane and for sulphydryl exchange are described.     

Strategies for improved antigen delivery into dendritic cells

Efficacious vaccines against cancers and infectious diseases will, in general, need to elicit comprehensive immune responses, including cytotoxic T lymphocyte activity. Because of their unique T cell stimulatory capacities, dendritic cells (DC) have emerged as the most potent antigen-presenting cell. Vaccination strategies should therefore aim at the acquisition and display of the antigen(s) of choice by DC. Results from vaccination studies, in animal models and in humans, stress the need for optimized antigen delivery systems to DC, to increase vaccination efficacy as well as to improve control on the immunological outcome. Here, we discuss the advantages and limitations of several recently described methodologies for antigen delivery into DC.     

Polymeric micelles with water-insoluble drug as hydrophobic moiety for drug delivery

The hydrophobic block of polymeric micelles formed by amphiphilic copolymers has no direct therapeutical effect, and the metabolites of these hydrophobic segments might lead to some unexpected side effects. Here the hydrophobic core of polymeric micelles is replaced by highly water-insoluble drugs themselves, forming a new micellar drug delivery system. By grafting hydrophobic drugs of paclitaxel (PTX) onto the surface of hydrophilic hyperbranched poly(ether-ester) (HPEE), we constructed an amphiphilic copolymer (HPEE-PTX). HPEE-PTX could self-assemble into micellar nanoparticles in aqueous solution with tunable drug contents from 4.1 to 10.7%. Moreover, the hydrolysis of HPEE-PTX in serum resulted in the cumulative release of PTX. In vivo evaluation indicated that the dosage toleration of PTX in mice had been improved greatly and HPEE-PTX micellar nanoparticles could be used as an efficient prodrug with satisfactory therapeutical effect. We believe that most of the lipophilic drugs could improve their characters through this strategy.     

Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells.

To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (4(6), Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH( 2,) and Hel 11-7, KLLK-LLLK-LWKK-LLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 4(6) had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 4(6) and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide-DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.