Cellular Uptake Of Oligonucleotides Research Articles

Nano and Microtechnologies for the Delivery of Oligonucleotides with Gene Silencing Properties

Oligonucleotides (ONs) are synthetic fragments of nucleic acid designed to modulate the expression of target proteins. DNA-based ONs (antisense, antigene, aptamer or decoy) and more recently a new class of RNA-based ONs, the small interfering RNAs (siRNAs), have gained great attention for the treatment of different disease states, such as viral infections, inflammation, diabetes, and cancer. However, the development of therapeutic strategies based on ONs is hampered by their low bioavailability, poor intracellular uptake and rapid degradation in biological fluids. The use of a non-viral carrier can be a powerful tool to overcome these drawbacks. Lipid or polymer-based nanotechnologies can improve biological stability and cellular uptake of ONs, with possibility of tissue and/or cellular targeting. The use of polymeric devices can also produce a prolonged release of the ON, thus reducing the need of frequent administrations. This review summarizes advantages and issues related to the main non-viral vectors used for ON delivery.

Time‐resolved Microspectrofluorometry and Fluorescence Imaging Techniques: Study of Porphyrin‐mediated Cellular Uptake of Oligonucleotides

Time-resolved confocal microspectrofluorometry and fluorescence microscopy imaging were applied to monitor the cellular uptake of fluorescent-labeled oligonucleotides (ONs) delivered by a porphyrin molecule. The fate of porphyrin-ON complexes inside living cells has also been monitored. Due to intrinsic fluorescence of the porphyrin and sensitivity of its characteristics to microenvironment, multicomponent analysis of time-resolved fluorescence provides unique information about stability of the porphyrin-ON complexes, ON interactions with their target sequences, and ON and porphyrin distributions after delivery inside the cells. Time-resolved confocal microspectrofluorometry indeed delivers additional information compared with fluorescence confocal microscopy imaging widely employed to study ON uptake.

Hybridization and cellular uptake properties of lipophilic oligonucleotide-dendrimer conjugates

Dendrimeric compounds were conjugated to oligonucleotides in order to improve their cellular uptake. Second and third generation lipophilic dendrimers were covalently attached either to the 5'- or 3'- end of oligonucleotides. Thermal denaturation experiments showed that the attachment of a third generation dendrimer leads to a substantial decrease in binding affinity. The considerably smaller second-generation dendrimers, however, are well tolerated. Fluorescence measurements revealed that the presence of a second-generation dendrimer leads to a marked increase in the cellular uptake of oligonucleotides.

Liposomal delivery of antisense oligonucleotides for efficient downregulation of Bcl-2 and induction of apoptosis.

The aim of this study was to enhance the delivery and thus anti-tumoral efficiency of antisense bcl-2 oligonucleotides (ODN's). Bcl-2 overexpressing DoHH2 lymphoma and HeLa-cells were transfected with ODN's using a polycationic liposome preparation. Specific hybridization of antisense ODN's was demonstrated by gel-shift assays and in vitro transcription/translation studies. Cellular uptake of oligonucleotides was evaluated by fluorescence microscopy. Inhibition of bcl-2 translation was demonstrated by quantitative RT-PCR and Western Blot. TUNEL assay, ANNEXIN V-binding and Apo-2.7 expression were performed to evaluate induction of apoptosis. Using polycationic liposomes, a ODN transfection rate of 95% in HeLa and 45% in DoHH2 cells were demonstrated by fluorescence microscopy. 24 hours after transfection quantitative RT-PCR detected a 56% decrease of bcl-2 mRNA in antisense and a 7% decrease in sense transfected DoHH2 cells (p < 0.05). In HeLa-cells, bcl-2 expression was almost completely inhibited 72 hours after antisense ODN transfection. Antisense treated cells also showed significant induction of apoptosis. Polycationic liposome-mediated transfection of bcl-2 antisense ODN's causes enhanced cellular uptake and efficient bcl-2 downregulation in bcl-2 overexpressing cell lines. This delivery strategy may explain why significant induction of apoptosis was achieved at low oligonucleotide concentrations (approximately 200 pmol/5 x 10(5) tumor cells).

Selective degradation of targeted mRNAs using partially modified oligonucleotides

At a theoretical level, the utilization of an oligonucleotide complementary to a target message to prevent translation sounds extraordinarily simple. There are, however, significant hurdles to the use of antisense oligonucleotides in vivo. Issues of nuclease stability and RNase H activity, which are presented in this chapter, must be considered in any discussion involving antisense oligonucleotides. A third important issue, cellular uptake of oligonucleotides, has not been discussed. This chapter has circumvented this complex and poorly understood area by directly microinjecting chimeric oligonucleotides into single cells. The ability to microinject oligonucleotides into Xenopus oocytes and embryos has made this an ideal system to ask important mechanistic questions involving oligonucleotide metabolism and mode of action. It is not possible, for example, to determine the rate of intracellular oligonucleotide degradation in cell or tissue culture because the rate of internalization is generally unknown. Additionally, it is not possible to compare the efficacy of two oligonucleotides adequately if they have different rates of internalization.

Peptide Conjugates of Oligonucleotides As Enhanced Antisense Agents

The use of synthetic oligonucleotides and their analogs to block gene expression by binding the complementary RNA sequences in cells, the antisense principle, has been limited by poor uptake of the agents by cells in culture. This review describes attempts to harness by chemical conjugation the ability of certain peptides that may cross membranes to enhance the cellular uptake of oligonucleotides. These include fusogenic and hydrophobic peptides, nuclear localization signals, receptor targeting and translocating peptides, and various combinations. We also outline briefly some popular methods of peptide–oligonucleotide conjugation. Finally, we review the use of noncovalent peptide additives and the recent studies of conjugates of peptide nucleic acid (PNA) with peptides.

Evaluation of aminoalkylmethacrylate nanoparticles as colloidal drug carrier systems. Part II: characterization of antisense oligonucleotides loaded copolymer nanoparticles

Aminoalkylmethacrylate methylmethacrylate copolymer nanoparticles were evaluated for their use as potential drug carrier systems. Their cytotoxicity, as well as the loading of antisense oligonucleotides that were employed as anionic model drugs depended on the substitution of the basic aminoalkyl copolymer. Toxic influences on the integrity of cell membranes depended on aminoalkyl groups located on the particle surfaces. Toxicity was observed either by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays using African green monkey kidney (AGMK) cells or by a hemolysis test, where the efflux of haemoglobin from disrupted erythrocytes was measured. The cytotoxic effects were increased by the elongation of the N-alkyl chain by four additional methylene groups. Lipophilic polymethylmethacrylate (PMMA) homopolymer nanoparticles showed a negative surface charge and, therefore, were not suitable for the adsorption of anionic drugs. The surface charge was changed to positive values by the incorporation of basic monomers. Consequently, the loading efficacy was increased by raising the basic copolymer portion. Additionally, a pH-dependent loading behaviour of oligonucleotides was observed. Substitution of the amino nitrogen protons by methyl groups led to a decreased oligonucleotide loading and to a reduced cytotoxicity. Nanoparticles with permanent positively charged quarternary ammonium groups showed a high pH-independent loading efficacy, but also possessed a high cytotoxic potential. In this study, cationic copolymer nanoparticles containing 30% (w/w) methylaminoethyl-methacrylate (MMAEMC) were found to be optimal with regard to biocompatibility and carrier properties for hydrophilic anionic antisense oligonucleotides. A significant portion of adsorbed oligonucleotides were protected from enzymatic degradation. The cellular uptake of oligonucleotides into Vero cells was significantly enhanced by this methylaminoethyl-methacrylate derivative.

Advanced glycation end product (AGE)-mediated induction of tissue factor in cultured endothelial cells is dependent on RAGE.

Binding of advanced glycation end products (AGEs) to the cellular surface receptor (RAGE) induces translocation of the transcription factor NF-kappaB into the nucleus and NF-kappaB-mediated gene expression. This study examines the role of RAGE in the AGE albumin-mediated induction of endothelial tissue factor, known to be partly controlled by NF-kappaB. Endothelial cells (ECs) were incubated in the presence of an 18-mer phosphorothioate oligodeoxynucleotide antisense to the 5'-coding sequence of the RAGE gene (antisense RAGE; 0.1 micromol/L). Sense oligonucleotides (sense RAGE, 0.1 micromol/L) of the same region served as control. The cellular uptake of oligonucleotides was controlled by immunofluorescence microscopy. RAGE transcription was suppressed by antisense RAGE, as demonstrated by RT-PCR reactions. AGE albumin-mediated activation of cultured ECs was studied after 48 hours of preincubation of ECs with antisense or sense RAGE. Electrophoretic mobility shift assays and Western blot analysis demonstrated that the AGE albumin-induced translocation of NF-kappaB from the cytoplasm into the nucleus was suppressed in the presence of antisense RAGE but not by sense RAGE. In parallel, AGE albumin-mediated tissue factor transcription, activity, and antigen were significantly reduced in ECs exposed to antisense RAGE, whereas sense RAGE (and nonspecific oligonucleotides) did not influence tissue factor expression. Activation of ECs and induction of tissue factor by AGE albumin in ECs is dependent on RAGE.

Cellular delivery of oligonucleotides by synthetic import peptide carrier.

Inefficient cellular uptake and endosomal entrapment are among the obstacles impeding the therapeutic use of oligonucleotides (ONs). The objectives of this study are to investigate the feasibility of utilizing a synthetic import peptide as a drug carrier for cytoplasmic delivery of ONs and to study its transport mechanisms. A molecular conjugate consisting of a signal import peptide (IP) derived from Kaposi fibroblast growth factor (K-FGF) and a polycationic ON linker, polylysine (PL), was synthesized and complexed with 5' fluorescently-labeled ON. Complex formation was verified by spectral shift assay and cellular uptake of the ON complex was studied fluorometrically. Microscopic studies were performed to visualize the intracellular distribution of the ON. Cells treated with the ON:IP-PL complex exhibited a dose-dependent increase in ON uptake over free ON-treated controls. The uptake of the complex was shown to occur via an energy-independent, non-endocytic, process since metabolic and endocytic inhibitors and low temperature did not prevent the uptake. Microscopic studies revealed a non-punctate fluorescence pattern, consistent with the non-endocytic transport process. Intense nuclear fluorescence was observed in cells treated with the complex but not with free ON, suggesting enhanced cytoplasmic delivery and nuclear accumulation of the ON by the conjugate. Efficient complex uptake was shown to require both the ON-binding moiety PL and the IP moiety. The delivery system was found to be non-toxic at the concentrations used. The peptide carrier was effective in promoting the cellular uptake of ON. The mechanism by which the peptide facilitates ON uptake appears to involve a direct translocation of ON via a non-endocytic process. The peptide carrier has the potential to overcome the problem of ON endosomal entrapment and degradation.

Oligonucleotide targeting to alveolar macrophages by mannose receptor-mediated endocytosis

Antisense oligonucleotides (ONs) have proven useful for selective inhibition of gene expression. However, their effective use is limited by inefficient cellular uptake and lack of cellular targeting. In this paper, we report a drug targeting system which utilizes mannose receptor-mediated endocytosis to enhance cellular uptake of ONs in alveolar macrophages (AMs). The system employs a molecular complex consisting of partially substituted mannosylated poly( l-lysine) (MPL), electrostatically linked to a 5′ fluorescently labeled ON. Upon recognition by the macrophage mannose receptors, the MPL was internalized by the receptor-mediated pathway, co-transporting the ON. Our results indicated that the AMs treated with the MPL:ON complex exhibited a significant increase in ON uptake (up to 17-fold) over free ON-treated controls. Effective ON uptake was shown to require the recognition of the mannose moiety since unmodified polylysine was much less effective in promoting ON uptake. Specific internalization of the ON:MPL complex by the mannose receptor pathway was verified by competitive inhibition using mannosylated albumin. Under this condition, the ON complex uptake was inhibited. The requirement of mannose receptors for complex uptake was further demonstrated using a macrophage cell line, J774.1, which expresses a low level of mannose receptors. When treated with the complex, these cells showed no susceptibility to ON uptake, thus suggesting the targeting ability of the carrier system to the AMs. Following cellular internalization, the ON complex appeared largely accumulated in endocytic vesicles. Enhanced endosomal exit of the ON was achieved using a fusogenic peptide derived from the amino terminal sequence of influenza virus hemagglutinin HA2. Cytotoxicity studies showed that at the concentrations effectively enhancing ON uptake, both MPL and the fusogenic peptide caused no toxic effects to the cells, thereby suggesting their potential safety and utilization in vivo.

Cellular effects of antisense c-myc oligodeoxynucleotides are delivery dependent.

Among the transcription factors critical for cell cycle regulation is the proto-oncogene c-myc. The expression of c-myc upon vascular injury and its inhibition by antisense oligodeoxynucleotides have demonstrated the importance of this protein in the control of proliferation for many cell types, including vascular smooth muscle cells. Liposomes can enhance cellular incorporation of antisense oligodeoxynucleotides, but cellular uptake of oligonucleotides in this manner is still suboptimal, and the oligonucleotides are not protected from enzymatic degradation. Physico-chemical modifications of the oligomers must be developed before antisense oligodeoxynucleotides can be considered as a potential gene therapy for many of the human diseases. This study reports on the enhanced cellular incorporation of antisense phosphodiester oligonucleotides when conjugated to lipophilic linkers. Conjugated phosphodiesters of antisense c-myc oligodeoxynucleotides inhibited cultured human aortic smooth muscle cell growth by 47.5 ± 1.0% 4 days following only a 24-h exposure to the conjugated antisense phosphodiester oligonucleotides. Liposome-enhanced, but unconjugated, phosphodiester and phosphorothioate oligonucleotides were less effective (24.4 ± 1.9% and 29.5 ± 3.1% inhibition, respectively). Smooth muscle cell growth inhibition by antisense c-myc oligodeoxynucleotides correlated with the suppression of nuclear c-myc protein expression. Thus, antisense c-myc oligodeoxynucleotides conjugated to lipid-soluble linkers enhanced cellular incorporation as well as intracellular retention of oligodeoxynucleotides, resulting in rapid and sustained inhibition of c-myc expression of smooth muscle cells. This, in turn, caused a prolonged growth inhibition compared to unconjugated oligodeoxynucleotides.

The uptake and distribution of phosphorothioate oligonucleotides into vascular smooth muscle cells in vitro and in rabbit arteries.

Oligonucleotides are a class of compounds with potential as therapeutics for a variety of clinical applications. Local delivery of oligonucleotides to the arterial wall is a challenging aspect of the development of these therapeutics for restenosis, and herein we report experiments characterizing the uptake and distribution of phosphorothiate oligonucleotides into vascular smooth muscle cells in primary cultures and in rabbit arteries. Primary cultures of smooth muscle cells incubated with rhodamine-oligonucleotides showed uptake only into cytoplasmic vesicles. No nuclear or cytosolic localization was detected. In normal arteries there was no visible tissue or cellular uptake of oligonucleotides after intralumenal administration. However, in balloon-injured arteries there was significant oligonucleotide uptake into the tissue with apparent cytoplasmic delivery to the medial smooth muscle cells, as evinced by intense staining of their nuclei with labeled oligonucleotides. Measurement of FITC-oligonucleotide in artery extracts showed significantly greater uptake in injured, compared with normal arteries. Light and electron microscopic studies demonstrated a correlation between the degree of damage and the amount of uptake. These results demonstrate that oligonucleotides penetrate easily into the arterial wall of balloon-injured arteries and accumulate in the medial smooth muscle cells-the target cells for antirestenosis therapeutics following balloon angioplasty.

Cationic liposomes mediated delivery of antisense oligonucleotides targeted to HPV 16 E7 mRNA in CaSki cells

The “high risk” types 16 and 18 of human papillomavirus (HPV) are involved in the etiology of genital squamous cell carcinoma. The early genes 6 and 7 (E6–E7) of these viruses code for the major transforming proteins, capable of inducing cell transformation alone or acting synergistically with other oncogenes. Antisense oligonucleotides, recently applied to inhibit the functions of a number of cellular and viral proteins, might provide the basis for a new therapeutic strategy against HPV-induced malignancies. We studied the proliferation of CaSki cells by the MTT assay after their exposure to HPV 16 E7 mRNA antisense oligonucleotides with and without cationic liposomes (containing dimethyldioctadecylammonium bromide DDAB, and dioleylphosphatidylethanolamine, DOPE). Unmodified oligonucleotides (either 12- or 23-mers) did not have nay effect on either CaSki cell proliferation or morphology when compared with the untreated cells. The cellular uptake of oligonucleotides was significantly enhanced by the cationic liposomes as assessed by confocal laser scanning microscopy (CLSM). The cationic liposomes were toxic to the cells as demonstrated by the reduced cell number and altered cell morphology. Only a slight reduction of the cell proliferation was seen when antisense 12-mer was protected from its 3′- and 5′-ends with thiolated and FITC, respectively. Both the 12- and the 23-mers with the cationic liposomes inhibited cell proliferation, the inhibitory effect being longer with the 23-mer. Overall, the MTT assay was less sensitive than light microscopy to reveal the toxic effects on CaSki cells. The results suggest that antisense oligonucleotides targeted to HPV 16 E7 mRNA can be introduced into CaSki cells with cationic liposomes.

Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake.

Oligonucleotides can be adsorbed on polyisohexylcyanoacrylate nanoparticles in the presence of hydrophobic quarternary ammonium salts. Oligonucleotides bound to nanoparticles are protected from nuclease attack both in buffer and in cell culture media. Cellular uptake of oligonucleotides is increased when they are adsorbed onto nanoparticles as a result of the capture of nanoparticles by an endocytic phagocytic pathway. Intracellular stability towards nucleolytic degradation is increased in the presence of nanoparticles. These results show that nanoparticles can be considered as convenient carriers for the protection and delivery of oligonucleotides to cells.

Interactions of antisense DNa oliginucletide analogs with phospholid membranes (liposomes)

Antisense oligonucleotides have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases. However, the mechanism by which many oligonucleotide analogs enter cells to exert the desired effects is unknown. In this study, we have used phospholipid model membranes (liposomes) to examine further the mechanisms by which oligonucleotide analogs cross biological membranes. Permeation characteristics of 32P or fluorescent labelled methylphosphonate (MP-oligo), phosphorothioate (S-oligo), alternating methylphosphonate-phosphodiester (Alt-MP) and unmodified phosphodiester (D-oligo) oligodeoxynucleotides were studied using liposomal membranes. Efflux rates (t1/2 values) at 37 degrees C for oligonucleotides entrapped within liposomes ranged from 7-10 days for D-, S- and Alt-MP-oligos to about 4 days for MP-oligos. This suggests that cellular uptake of oligonucleotides by passive diffusion may be an unlikely mechanism, even for the more hydrophobic MP-oligos, as biological effects are observed over much shorter time periods. We also present data that suggest oligonucleotides are unlikely to traverse phospholipid bilayers by membrane destabilization. We show further that MP-oligos exhibit saturable binding (adsorption) to liposomal membranes with a dissociation constant (Kd) of around 20nM. Binding appears to be a simple interaction in which one molecule of oligonucleotide attaches to a single lipid site. In addition, we present water-octanol partition coefficient data which shows that uncharged 12-15 mer MP-oligos are 20-40 times more soluble in water than octanol; the low organic solubility is consistent with the slow permeation of MP-oligos across liposome membranes. These results are thought to have important implications for both the cellular transport and liposomal delivery of modified oligonucleotides.