Antisense delivery using protamine-oligonucleotide particles

Protamine, a polycationic peptide (mol. wt 4000-4500), was evaluated as a potential penetration enhancer for phosphodiester antisense oligonucleotides (ODNs). Unique complexes in the form of nanoparticles were spontaneously formed, which we call 'proticles'. The stability of the particles and the ODNs bound into the proticles was examined in foetal calf serum and cell culture medium. FITC-labelled ODNs bound to protamine showed an increased cellular uptake into human histiocytic lymphoma U 937 cells compared to free ODNs. Proticles significantly decreased cellular growth in a cell proliferation assay using ODNs against the c- myc proto-oncogene.     

Antisense delivery using protamine–oligonucleotide particles

Protamine, a polycationic peptide (mol. wt 4000–4500), was evaluated as a potential penetration enhancer for phosphodiester antisense oligonucleotides (ODNs). Unique complexes in the form of nanoparticles were spontaneously formed, which we call ‘proticles’. The stability of the particles and the ODNs bound into the proticles was examined in foetal calf serum and cell culture medium. FITC-labelled ODNs bound to protamine showed an increased cellular uptake into human histiocytic lymphoma U 937 cells compared to free ODNs. Proticles significantly decreased cellular growth in a cell proliferation assay using ODNs against the c-myc proto-oncogene.     

3'-methylphosphonate-modified oligo-2'-O-methylribonucleotides and their Tat peptide conjugates: uptake and stability in mouse fibroblasts in culture

Antisense oligo-2'-O-methylribonucleotides and their methylphosphonate derivatives show high binding affinities for their complementary targets under essentially physiological conditions. Additionally, the methylphosphonate linkage is resistant to nuclease hydrolysis. Here we show that a single methylphosphonate internucleotide linkage at the 3'-end of an oligo-2'-O-methylribonucleotide is sufficient to prevent degradation by the 3'-exonuclease activity found in mammalian serum. Complexes formed between a cationic lipid, Oligofectamine, and 5'-[(32)P]-labeled methylphosphonate modified oligo-2'-O-methylribonucleotides are taken up by mouse L(929) fibroblasts in culture. The extent of uptake appears to be dependent upon the sequence of the oligonucleotide. Examination of lysates of oligonucleotide treated cells by polyacrylamide gel electrophoresis showed that no degradation of the oligonucleotide occurred, even after incubation for 24 h. A fluorescein-derivatized oligomer was shown to localize mainly in the cell nucleus as monitored by fluorescence microscopy. Covalent conjugates of fluorescein-derivatized 3'-methylphosphonate modified oligo-2'-O-methylribonucleotides with Tat peptide, a cell permeating peptide, were also prepared. The Tat peptide was coupled to the 5'-end of the oligonucleotide using either disulfide coupling chemistry or conjugation of a keto derivative of the Tat peptide via a 4-(2-aminooxyethoxy-2-(ethylureido)quinoline group at the 5'-end of the oligonucleotide. Although formation of the Tat peptide conjugates was confirmed by mass spectrometry, the propensity of these oligonucleotides to form aggregates and their apparent high affinity for plastic and glass made the conjugates unsuitable for studies of uptake by cells in culture.     

Required structure of cationic peptide for oligonucleotide-binding and -delivering into cells.

Improvement of the methods for oligonucleotide delivery into cells is necessary for the development of antisense therapy. In the present work, a new strategy for oligonucleotide delivery into cells was tested using cationic peptides as a vector. At first, to understand what structure of the peptide is required for binding with an oligonucleotide, several kinds of alpha-helical and non-alpha-helical peptides containing cationic amino acids were employed. As a result, the amphiphilic alpha-helix peptides were best for binding with the oligonucleotide, and the long chain length and large hydrophobic region in the amphiphilic structure of the peptide were necessary for the binding and forming of aggregates with the oligonucleotide. In the case of non-alpha-helical peptides, no significant binding ability was observed even if their chain lengths and number of cationic amino acid residues were equal to those of the alpha-helical peptides. The remarkable ability of oligonucleotide delivery into COS-7 cells was observed in the alpha-helical peptides with a long chain length and large hydrophobic region in the amphiphilic structure, but was not observed in the non-alpha-helical peptides. It is considered that such alpha-helical peptides could form optimum aggregates with the ODN for uptake into cells. Based on these results, the alpha-helical peptide with a long chain length and large hydrophobic region is applicable as a vector for the delivery of oligonucleotides into cells.     

Highly efficient cellular uptake of c-myb antisense oligonucleotides through specifically designed polymeric nanospheres.

c-myb antisense oligonucleotides (AS ODNs) were reversibly immobilized to a novel polymeric core shell nanosphere and their cellular uptake and inhibitory effect on HL60 leukemia cell proliferation studied. The nanosphere surface was so designed as to directly bind ODNs via ionic interactions and reversibly release them inside the cells. Compared with the cellular uptake of free oligonucleotide, the use of AS ODN (immobilized to the nanospheres) produced a 50-fold increase in the intracellular concentration. Specifically, a single dose of 320 nM of AS ODN immobilized to the nanospheres was capable of inhibiting HL60 cell proliferation with the same degree of efficiency obtained using a 50-fold higher dose of free AS ODN. Flow cytometric experiments with fluoresceinated ODNs showed a temperature-dependent uptake, which was detectable as early as 2 h after the beginning of treatment. The inhibitory effect on cell proliferation was maintained for up to 8 days of culture. Moreover, the level of c-Myb protein decreased by 24% after 2 days and by 60% after 4 days of treatment, thus indicating a continuous and sustained release of non-degraded AS ODN from the nanospheres inside the cells.     

Stepwise synthesis of oligonucleotide–peptide conjugates containing guanidinium and lipophilic groups in their 3′-termini

Two different series of oligonucleotide–peptide conjugates have been efficiently synthesized by stepwise solid-phase synthesis. First, oligonucleotides and oligonucleotide phosphorothioates containing polar groups at the 3′-termini, such as amine and guanidinium groups were prepared. ODNs conjugates carrying several lysine residues were obtained directly from Fmoc deprotection whereas ODN conjugates with guanidinium groups were obtained by post-synthetic guanidinylation. The second family contains different urea moieties that were achieved by standard protocols. All products were fully characterized by reversed phase HPLC and MALDI-TOF mass spectrometry yielding satisfactory results. Oligonucleotide–phosphorothioate conjugates were evaluated as potential antisense oligonucleotides in the inhibition of the luciferase gene.     

A novel 11-residual peptaibol-derived carrier peptide for in vitro oligodeoxynucleotide delivery into cell

Using a pore- and channel-forming peptide, TV-XIIa, which is an 11-residual peptaibol isolated from the fungus Trichoderma viride, we developed a vehicle for the cellular delivery of such polar biologically active agents as antisense oligodeoxynucleotides (ODNs). To function as an ODN carrier, basic amino acids, 10-mer of lysine, were conjugated to the C-terminus of TV-XIIa and the designed carrier peptide, Ac-U-N-I-I-U-P-L-L-U-P-I-K-K-K-K-K-K-K-K-K-K-OH (U: alpha-aminoisobutyric acid), was synthesized by the Fmoc-based solid-phase method. The complex between the carrier peptide and ODNs, which was electrostatically formed, was capable of crossing the membranes of NIH3T3 cells and the ODNs were accumulated in the cytoplasm and the nucleus. However, the complex was not taken up by A549 cells. The translocation of the complex occurred at both 4 and 37 degrees C in NIH3T3 cells and did not seem to involve an energy-dependent endocytic process.     

Uptake of minute virus of mice into cultured rodent cells.

The uptake of minute virus of mice into cells in tissue culture was examined biochemically and by electron microscopy. Cell-virus complexes were formed at 4 degrees C, and uptake of virus was followed after the cells were shifted to 37 degrees C. The infectious particles appeared to enter cells at 37 degrees C by a two-step process. The first and rapid phase was measured by the resistance of cell-bound virus to elution by EDTA. The bulk of the bound virus particles became refractory to elution with EDTA within 30 min of incubation at 37 degrees C. The infectious particles became resistant to EDTA elution at the same rate. The second, slower phase of the uptake process was measured by the resistance of infectious particles to neutralization by antiserum. This process was complete within 2 h of incubation at 37 degrees C. During this 2-h period, labeled viral DNA became progressively associated with the nuclear fraction of disrupted cells. The uptake of infectious virus could occur during the G1 phase of the cell cycle and was not an S phase-specific event. The uptake process was not the cause of the S phase dependence of minute virus of mice replication. In electron micrographs, virus absorbed to any area of the cell surface appeared to be taken into the cell by pinocytosis.     

Tritium labeling of antisense oligonucleotides by exchange with tritiated water.

We describe a simple, efficient, procedure for labeling oligonucleotides to high specific activity (< 1 x 10(8) cpm/mumol) by hydrogen exchange with tritiated water at the C8 positions of purines in the presence of beta-mercaptoethanol, an effective radical scavenger. Approximately 90% of the starting material is recovered as intact, labeled oligonucleotide. The radiolabeled compounds are stable in biological systems; greater than 90% of the specific activity is retained after 72 hr incubation at 37 degrees C in serum-containing media. Data obtained from in vitro cellular uptake experiments using oligonucleotides labeled by this method are similar to those obtained using 35S or 14C-labeled compounds. Because this protocol is solely dependent upon the existence of purine residues, it should be useful for radiolabeling modified as well as unmodified phosphodiester oligonucleotides.     

Cellular internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans

The exact mechanisms by which arginine-rich cell-penetrating peptides enter cells are still the subject of debate. Here, we have analyzed in detail the effects of serum and extracellular concentration on the internalization of oligoarginines (R n; n = 4, 8, 12, 16). The presence of serum in the incubation medium had a major influence on the uptake of R12 and R16 peptides but did not affect the uptake of R4 and R8 significantly. Incubation of cells at 37 degrees C with R12 and R16 peptides in serum-containing medium showed that the majority of labeling was confined to punctate endocytic structures. Performing the same experiments in serum-free media led to a dramatic increase in cytosolic labeling, and similarly diffuse R12 and R16 labeling was observed in cells treated with peptides at 4 degrees C. This suggests, in both cases, that the peptides were entering via a nonendocytic mechanism. Further studies on R12 peptide suggest that the initiation of nonendocytic uptake and cytosolic labeling is also dependent on serum concentration and extracellular peptide concentration. At relatively low concentrations, the peptide labels endocytic structures, but upon raising the peptide concentration, the fraction labeling the cytosol increases dramatically and this accompanies a nonlinear increase in total cellular fluorescence. Membrane-associated proteoglycans also contribute to increasing the peptide concentration at the cell surface by enhancing their recruitment via electrostatic interactions. These results demonstrate that uptake mechanisms of these compounds are highly dependent on both the presence of serum and the effective extracellular peptide concentration.     

Folate-liposome-mediated antisense oligodeoxynucleotide targeting to cancer cells: evaluation in vitro and in vivo

The objective of this study was to investigate the use of folate-targeted liposomes for the delivery of encapsulated oligonucleotides to folate receptor (FR)-positive tumor cells in vitro and in vivo. This project involved the synthesis and biological evaluation of many folate-PEG-lipid conjugates, where the chemical form of the folate moiety (pteroate) and the length of the PEG linker chain were varied widely. Folate-targeted oligonucleotide-containing liposomes were prepared using conventional methods, and the extent of cell uptake was evaluated using, among others, the FR positive KB cell line. Oligonucleotide-loaded folate-targeted liposomes were found to rapidly associate with the KB cells, and saturation was typically reached within the first hour of incubation at 37 degrees C. Nearly 100,000 liposomes per cell were bound or internalized at saturation. Importantly, cell association was blocked by a large excess folic acid, thus reflecting the FR-specific nature of the cell interaction. Full targeting potential was achieved with PEG linkers as low as 1000 in molecular weight, and pteroates bearing glycine or gamma-aminobutyryl residues juxtaposed to the pteroic acid moiety were also effective for targeting, provided that a terminal cysteine moiety was present at the distal end of the PEG chain for added hydrophilicity. When tested in vivo, folate-targeted liposomes were found to deliver approximately 1.8-fold more oligonucleotide to the livers of nude mice (relative to the nontargeted PEG-containing formulations); however, no improvement in KB tumor uptake was observed. We conclude from these results that folate liposomes can effectively deliver oligonucleotides into folate receptor-bearing cells in vitro, but additional barriers exist in vivo that prevent or decrease effective tumor uptake and retention.     

Mechanism of cellular uptake of modified oligodeoxynucleotides containing methylphosphonate linkages.

The cellular uptake and intracellular distribution of methylphosphonate oligonucleotides (15 mers) has been examined using both 32P labeled and fluorescent labeled oligonucleotides. The cellular uptake process for methylphosphonate oligonucleotides is highly temperature dependent, with a major increase in uptake occurring between 15 and 20 degrees C. Most of the label which becomes cell associated at 37 degrees C cannot be removed by acid washing or trypsinization and thus seems to be within the cell. Visualization of rhodamine labeled methylphosphonate oligonucleotides using digital imaging fluorescence microscopy reveals a vesicular subcellular distribution suggestive of an endosomal localization. There was extensive co-localization of rhodamine labeled methylphosphonate oligonucleotides with fluorescein-dextran, an endosomal/lysosomal marker substance. The apparent endocytotic uptake of labeled methylphosphonate oligonucleotides could not be blocked by competition with unlabeled methylphosphonate or phosphodiester oligonucleotides, nor by ATP. This contrasts with the situation for radiolabeled phosphodiester oligonucleotides whose uptake can be completely blocked with unlabeled competitor. Uptake of phosphodiester oligonucleotides, but not of methylphosphonate oligonucleotides, could be blocked by acidification of the cytosol. These observations suggest that the pathway of cellular uptake of methylphosphonate oligonucleotides involves fluid phase or adsorbtive endocytosis, and is distinct from the uptake pathway for phosphodiester oligonucleotides.     

Protamine enhances epidermal growth factor (EGF)-stimulated mitogenesis by increasing cell surface EGF receptor number. Implications for existence of cryptic EGF receptors

Treatment of Swiss mouse 3T3 cells and human epidermoid carcinoma A431 cells with protamine at 37 degrees C increased the 125I-epidermal growth factor (EGF) binding activity at 4 degrees C. The effect of protamine on the increase of 125I-EGF binding activity appeared to be time, temperature, and dose dependent. This up-modulation of 125I-EGF binding by protamine correlated with protamine enhancement of EGF-stimulated mitogenesis, with respect to the magnitude of the effect and the dose response curves. Scatchard plot analyses indicated that protamine induced an increase in numbers of both high and low affinity EGF receptors without affecting their affinities. Protamine also increased functionally active EGF receptors in plasma membranes and solubilized membranes. This was evidenced by Scatchard plot analyses and by a protamine-induced increase of 125I-EGF-EGF receptor complex and an increase in EGF-stimulated phosphorylation of the EGF receptor. Combined with column chromatography of the solubilized EGF receptor on protamine-agarose gel, these results suggest that protamine may increase the EGF receptor number by directly activating cryptic EGF receptors in the plasma membrane.     

Association of antisense oligonucleotides with lipoproteins prolongs the plasma half-life and modifies the tissue distribution.

In order to direct antisense oligonucleotides to specific tissues or cell types in vivo, we are exploring the possibility to utilize lipoproteins as transport vehicles. A 16-mer oligonucleotide (ODN) was derivatized at the 5' prime through a 32P phosphate spacer with cholesterol, yielding a 32P-labeled amphiphatic cholesteryl-oligonucleotide (cholODN). Incubation of cholODN with low-density lipoprotein (LDL) for 2 hr at 37 degrees C resulted in the formation of a cholODN-LDL complex that migrates as a single peak on agarose gel electrophoresis. The cholODN was found to bind quantitatively to both high-density lipoproteins (HDL) and LDL, but not to albumin. Stable oligonucleotide-LDL particles with up to 50 molecules of cholODN per LDL particle could be obtained. In contrast, the control ODN did not show affinity for plasma lipoproteins. Upon injection into rats, cholODN became rapidly associated with plasma lipoproteins while control ODNs were recovered in the lipoprotein deficient serum fraction. The plasma half-life of cholODN (9-11 min) is considerably prolonged as compared with the control ODN (t1/2 less than 1 min). The cholODN-LDL was at least 5 min stable against degradation by rat plasma nucleases. It is concluded that derivatization of antisense oligonucleotides with cholesterol profoundly modifies their in vivo fate and opens possibilities for efficient and specific receptor-dependent targeting, mediated by lipoproteins coupled with specific recognition markers to various hepatic cell types.     

Poly(propylacrylic acid) enhances cationic lipid-mediated delivery of antisense oligonucleotides

The use of antisense oligodeoxynucleotides (ODNs) to inhibit the expression of specific mRNA targets represents a powerful technology for control of gene expression. Cationic lipids and polymers are frequently used to improve the delivery of ODNs to cells, but the resulting complexes often aggregate, bind to serum components, and are trafficked poorly within cells. We show that the addition of a synthetic, pH-sensitive, membrane-disrupting polyanion, poly(propylacrylic acid) (PPAA), improves the in vitro efficiency of the cationic lipid, DOTAP, with regard to oligonucleotide delivery and antisense activity. In characterization studies, ODN complexation with DOTAP/ODN was maintained even when substantial amounts of PPAA were added. The formulation also exhibited partial protection of phosphodiester oligonucleotides against enzymatic digestion. In Chinese hamster ovary (CHO) cells, incorporation of PPAA in DOTAP/ODN complexes improved 2- to 3-fold the cellular uptake of fluorescently tagged oligonucleotides. DOTAP/ODN complexes containing PPAA also maintained high levels of uptake into cells upon exposure to serum. Addition of PPAA to DOTAP/ODN complexes enhanced the antisense activity (using GFP as the target) over a range of PPAA concentrations in both serum-free, and to a lesser extent, serum-containing media. Thus, PPAA is a useful adjunct that improves the lipid-mediated delivery of oligonucleotides.     

Uptake, cellular distribution and novel cellular binding proteins of immunostimulatory CpG oligodeoxynucleotides in glioblastoma cells

Glioblastomas are the most malignant and most frequent brain tumors and exciting targets of gene and immunotherapy. Despite rapid development of experimental therapy little is known about the cellular behaviour of therapeutic oligodeoxynucleotides (ODNs). Here we designed uptake, cellular distribution and cellular binding proteins of immunostimulatory CpG-ODNs in glioblastoma cells by flow cytometry, fluorescence microscopy and mass spectrometry. Our data show that the phosphorothioate (PS) CpG-ODNs uptake in T98G and C6 cells is dose-, time-, temperature-dependent and independent of the CpG dinucleotides. Uptake can be inhibited by sodium azide, polyanions but not by chloroquine. After internalisation FITC labelled CpG-ODNs showed a spotted distribution in cytoplasm. Dozens of cellular binding proteins were identified using mass spectrometry. The binding of ODNs to proteins is dependent on modification and sequence but independent on CpG motif. ODNs bind to cellular proteins that are important for RNA processing and transport. Furthermore, three novel membrane proteins were identified, which might contribute to uptake of ODNs. ODNs binding to these proteins might interfere with the physiological function and thus might cause unwanted effects. Such binding also might influence the uptake efficiency or cellular distribution of therapeutic ODNs.     

Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells

Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4-12 degrees C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 degrees C incubations. At temperatures between 12 and 30 degrees C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 degrees C. Small increases in the extracellular peptide concentration in 37 degrees C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-beta-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.     

Complex formation and vectorization of a phosphorothioate oligonucleotide with an amphipathic leucine- and lysine-rich peptide: study at molecular and cellular levels

Optical spectroscopic techniques such as CD, Raman scattering, and fluorescence imaging allowed us to analyze the complex formation and vectorization of a single-stranded 20-mer phosphorothioate oligodeoxynucleotide with a 15-mer amphipathic peptide at molecular and cellular levels. Different solvent mixtures (methanol and water) and molecular ratios of peptide/oligodeoxynucleotide complexes were tested in order to overcome the problems related to solubility. Optimal conditions for both spectroscopic and cellular experiments were obtained with the molecular ratio peptide/oligodeoxynucleotide equal to 21:4, corresponding to a 7:5 ratio for their respective +/- charge ratio. At the molecular level, CD and Raman spectra were consistent with a alpha-helix conformation of the peptide in water or in a methanol-water mixture. The presence of methanol increased considerably the solubility of the peptide without altering its alpha-helix conformation, as evidenced by CD and Raman spectroscopies. UV absorption melting profile of the oligodeoxynucleotide gave rise to a flat melting profile, corresponding to its random structure in solution. Raman spectra of oligodeoxynucleotide/peptide complexes could only be studied in methanol/water mixture solutions. Drastic changes observed in Raman spectra have undoubtedly shown: (a) the perturbation occurred in the peptide secondary structure, and (b) possible interaction between the lysine residues of the peptide and the oligodeoxynucleotide. At the cellular level, the complex was prepared in a mixture of 10% methanol and 90% cell medium. Cellular uptake in optimal conditions for the oligodeoxynucleotide delivery with low cytotoxicity was controlled by fluorescence imaging allowing to specifically locate the compacted oligonucleotide labeled with fluorescein at its 5'-terminus with the peptide into human glioma cells after 1 h of incubation at 37 degrees C.     

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.