Studies on uptake, sub-cellular trafficking and efflux of antisense oligodeoxynucleotides in glioma cells using self-assembling cationic lipoplexes as delivery systems

The cellular uptake of antisense oligodeoxynucleotides (ODNs) may be enhanced by the use of carriers such as cationic liposomes or lipoplexes, but little is known about the intracellular fate and subcellular trafficking of these systems in target cells. In this study, we report on the cellular uptake and biodistribution of ODNs in the presence and absence of optimised self-assembled cationic lipoplexes using the C6 glioma cell line as an in vitro model. Biotin or radiolabelled 15-mer phosphorothioate (PS) ODNs were synthesised and their cellular uptake and subcellular biodistribution characterised in the presence and absence of an optimised cationic lipoplex delivery system using studies ranging from cellular association, cellular efflux and transmission electron microscopy (TEM). Ultrastructural studies clearly showed PS ODNs in the absence of liposomal delivery to be sequestered within endosomal and lysosomal vesicular bodies indicative of endocytic uptake. ODNs were also visible, to a lesser extent, in the nucleus and cytoplasm. By employing DOSPA (2'-(1",2"-dioleoyloxypropyldimethyl-ammonium bromide)-N-ethyl-6-amidospermine tetra trifluoroacetic acid) and DOPE (dioleoylphosphatidylethanolamine) complex in a 3 : 1 ratio, as a delivery system for ODNs at a optimal lipid/DNA charge ratio of 1 : 1, the level of ODN cellular association was significantly increased by approximately 10-12 fold with a concomitant change in subcellular distribution of PS ODN. TEM studies indicated enhanced penetration of ODN within the cytosol and the cell nucleus with reduced presence in vesicular compartments. Efflux studies confirmed that cationic lipoplexes promoted entry of ODNs into 'deeper' cellular compartments, consistent with endosomal release. Optimised cationic lipoplexes improved cellular delivery of ODNs by enhancing cell association, uptake and by favourably modulating the intracellular trafficking and distribution of ODNs into non-vesicular compartments including the cytosol and nucleus.     

丝裂素活化的蛋白激酶反义寡脱氧核苷酸防治血管内膜增殖

目的：探讨Ca^2 -钙调蛋白依赖性蛋白激酶（丝裂素活化的蛋白激酶）（CCDPK）在生长因子诱导体外培养大鼠血管平滑肌细胞增殖中的作用及反义CCDPK寡脱氧核苷酸（ODN）对球囊损伤后大白鼠血管内膜增生的抑制作用。方法：利用脂质体转染17－mer CCDPK反义ODN进入培养的血管平滑肌细胞以抑制CCDPK活性,设正义及随机ODN作对照。用蛋白质印迹法测定CCDPK表达。[^3H]胸腺嘧啶核苷酸掺入测定平滑肌细胞DNA合成。用2F球囊导管造成大白鼠颈动脉再狭窄模型,利用多聚胶F127－ODN系统由血管外膜部位给药。于损伤后2周取样,固定及HE染色观察内膜增生情况。FITC标记的ODN观察体内外给药方法的分布及吸收情况。结果：CCDPK反义ODN能明显抑制PDGF及ET诱导的CCDPK蛋白表达及[^3H]胸腺嘧啶核苷酸掺入。在大鼠颈动脉再狭窄模型,能明显抑制血管内膜增生。结论：CCDPK介导了PDGF及ET诱导的血管平滑肌细胞增殖。针对p42-和p44-CCDPK起始部位设计的17－mer反义ODN能有效抑制生长因子诱导的血管平滑肌细胞的增殖及球囊损伤大鼠血管内膜增生。     

Serotonin-induced proliferation of pulmonary arterysmooth muscle cells is serotonin transporter and ERK pathway dependent

AIM: To investigate the effect of serotonin transporter (5-HTT)inhibitor fluoxetine and antisense oligodeoxynucleotide (ODN)to extracelluar signal-regulated kinases (ERKs) on pulmonary arterial smooth muscle cells (PASMCs) proliferation induced by 5-HT. METHODS: Liposomal transfection was used to introduce ODNs to ERK1/2 into cultured rat PASMCs and the transfection efficiency was measured by observing the uptake of the     

Folate-Mediated Targeting of Antisense Oligodeoxynucleotides to Ovarian Cancer Cells

Purpose. Receptors for vitamin folic acid are frequently overexpressed on epithelial cancer cells, especially ovarian cancer cells. In this study, we examined whether this expression might be exploited to specifically deliver antisense oligodeoxynucleotides (ODN) to tumor cells. Methods. A conjugate was prepared by directly coupling folic acid to the 3 terminus of an anti-c-fos ODN and its cellular uptake and tumor inhibitory effect were evaluated using FD2008 cells that overexpress folate receptors. Results. When a phosphorothioate (PS)/phosphodiester (PO) chimeric ODN was conjugated with folic acid, its uptake by FD2008 cells was increased by about 8-fold (P < 0.01). In contrast, conjugation of folate to the ODN did not increase its uptake by CHO cells that lack the expression of FBP (P > 0.05). Furthermore, the increase in the uptake of conjugated ODN by FD2008 cells could be blocked by adding an excess amount of folic acid. The PS/PO antisense ODN had some inhibitory effect on the growth of FD2008 cells. However, its activity was significantly increased following conjugation with folic acid (P < 0.01). ODN of scrambled sequences with and without conjugation with folic acid failed to inhibit the growth of FD2008 cells. Finally, the antisense effect of the conjugated ODN on FD2008 cells was inhibited by an excess amount of free folic acid, suggesting that the sequence-dependent effect of folate-antisense ODN conjugate was mediated by folate binding protein. Conclusions. Direct derivatization of ODN with folate significantly improves their targeting efficiency to tumor cells in vitro. The folate-conjugated ODN, due to their small size and possibly efficient extravasation at tumor site, has the potential for treating solid tumors that overexpress folate receptors.     

丝裂素活化的蛋白激酶反义寡核苷酸抑制表皮生长因子诱导体外 …

目的：探讨丝裂素活化的蛋白激酶（ＮＡＰＫ）反义寡核苷（ＯＤＮ）对表皮生长因子（ＥＧＦ）诱导的培养大鼠血管平滑肌细胞增生的抑制作用。方法：用脂质体将Ｐ４２－和Ｐ４４－ＭＡＰＫ ＯＤＮ０．２μｍｏｌˉＬ＾－１转染入大鼠血管平滑肌细胞，设正义及随机ＯＤＮ为对照，用Ｗｅｓｔｅｒｎ Ｂｌｏｔ法结合Ｐ－８１滤纸法以髓磷脂碱性蛋白为底物测定ＭＡＰＫ活性。［＾３Ｈ］胸腺嘧啶核苷酸掺入测定平滑肌细胞ＤＮＡ合成。结果     

Target selectivity of MAPK phosphorothioate antisense ODN on p42/p44, p38 MAPK, and JNK protein expression and its inhibitory effect on VSMC DNA synthesis.

AIM: To analyze the target selective and sequence-specific inhibitory effect of mitogen-activated protein kinase (MAPK) phosphorothioate antisense oligodeoxynucleotides (ODN) on p42/p44, p38 MAPK, c-jun NH2-terminal protein kinases (JNK) protein expression, and DNA synthesis in vascular smooth muscle cell (VSMC). METHODS: Using a phosphorothioate-protected 17-mer antisense MAPK ODN directed against the initiation of translation sites of the p42/p44 MAPK isoforms by liposomal transfection to deplete cultured rat, rabbit, and fetal calf VSMC MAP kinases. The 17-mer sense and random sequence MAPK ODN were used as controls. After liposomal transfection, cells were exposed to 20% serum for 24 h, and then harvested in lysis buffer. P42/p44, p38 MAPK, and p46/p58 JNK protein expression were measured by Western blot. DNA synthesis was measured by [3H]thymidine incorporation. RESULTS: Treatment with MAPK antisense ODN (0.1-0.8 mumol.L-1) for 48 h reduced phosphored p42/p44 MAPK protein expression but without effect on p38 MAPK and JNK expression, and inhibited cultured rat, rabbit, and fetal calf VSMC [3H]thymidine incorporation stimulated by 20% serum in a concentration-dependent manner. CONCLUSION: The MAPK antisense ODN target-selectively and sequence-specifically reduces the p42/p44 MAPK protein expression and concentration-dependently inhibits proliferation of rat, rabbit and fetal calf VSMC.     

Uptake and intracellular fate of multifunctional nanoparticles: a comparison between lipoplexes and polyplexes via quantum dot mediated Förster resonance energy transfer

Lipoplexes and polyplexes represent the two major nanocarrier systems for nucleic acid delivery. Previous studies examining their uptake and intracellular unpacking rely on organic fluorophores fraught with low signal intensity and photobleaching. In this work quantum dot mediated F?rster resonance energy transfer (QD-FRET) was first used to study and compare the cellular uptake and the intracellular fate of oligodeoxynucelotide (ODN)-based lipoplexes and polyplexes. QD605-amine and Cy5-labeled ODN (Cy5-GTI2040) were chosen as the FRET pair. By adjusting the lipid/ODN ratio of lipoplexes and the nitrogen/phosphate (N/P) ratio of polyplexes, lipoplexes and polyplexes with comparable physical properties were produced. The biological activities of dual-labeled lipoplexes and polyplexes remained unaltered compared to their unlabeled counterparts as evidenced by their comparable antisense activities against protein R2 in KB cells. Flow cytometry and confocal microscopy revealed similar pattern of uptake for these two types of nanoparticles, although polyplexes had a higher dissociation rate than lipoplexes in KB cells. We demonstrate that QD-FRET is a sensitive tool to study the uptake and intracellular unpacking of lipoplexes and polyplexes, which may help optimize their formulations for various theranostics applications.     

Histidine-rich peptides and polymers for nucleic acids delivery.

Nucleic acids transfer into mammalian cells requires devices to improve their escape from endocytic vesicles where they are mainly confined following cellular uptake. In this review, we describe histidine-rich molecules that enable the transfer of plasmid and oligonucleotides (ODN) in human and non-human cultured cells. An histidine-rich peptide which permeabilizes biological membrane at pH 6.4, favored the transfection mediated by lactosylated polylysine/pDNA complexes. Histidylated polylysine forms cationic particles of 100 nm with a plasmid and yielded a transfection of 3-4.5 orders of magnitude higher than polylysine. The biological activity of antisense ODN was increased more than 20-fold when it was complexed with highly histidylated oligolysine into small cationic spherical particles of 35 nm. Evidence that imidazole protonation mediates the effect of these molecules in endosomes are provided. We also describe a disulfide-containing polylysine conjugate capable of mediating DNA unpackaging in a reductive medium and to increase the transfection efficiency. Overall, these molecules constitute interesting devices for developing non-viral gene delivery systems.     

On the role of methacrylic acid copolymers in the intracellular delivery of antisense oligonucleotides.

The delivery of active biomacromolecules to the cytoplasm is a major challenge as it is generally hindered by the endosomal/lysosomal barrier. Synthetic titratable polyanions can overcome this barrier by destabilizing membrane bilayers at pH values typically found in endosomes. This study investigates how anionic polyelectrolytes can enhance the cytoplasmic delivery of an antisense oligonucleotide (ODN). Novel methacrylic acid (MAA) copolymers were examined for their pH-sensitive properties and ability to destabilize cell membranes in a pH-dependent manner. Ternary complex formulations prepared with the ODN, a cationic lipid and a MAA copolymer were systematically characterized with respect to their size, zeta potential, antisense activity, cytotoxicity and cellular uptake using the A549 human lung carcinoma cell line. The MAA copolymer substantially increased the activity of the antisense ODN in inhibiting the expression of protein kinase C-alpha. Uptake, cytotoxicity and antisense activity were strongly dependent on copolymer concentration. Metabolic inhibitors demonstrated that endocytosis was the major internalization pathway of the complexes, and that endosomal acidification was essential for ODN activity. Confocal microscopy analysis of cells incubated with fluorescently-labeled complexes revealed selective delivery of the ODN, but not of the copolymer, to the cytoplasm/nucleus. This study provides new insight into the mechanisms of intracellular delivery of macromolecular drugs, using synthetic anionic polyelectrolytes.     

Antisense-induced down-regulation of thymidylate synthase and enhanced cytotoxicity of 5-FUdR in 5-FUdR-resistant HeLa cells.

1. Thymidylate synthase (TS) is a target for several anticancer drugs. We previously showed that an antisense oligodeoxynucleotide (ODN) directed against TS mRNA down-regulated TS protein and enhanced cytotoxicity of TS-targeting drugs [including 5-fluorodeoxyuridine (5-FUdR)] in HeLa cells. Patient tumours with increased TS expression are resistant to TS-targeting drugs. It was hypothesized that TS mRNA and consequently TS protein could be down-regulated in 5-FUdR-resistant cells that overexpress TS, sensitizing them to 5-FUdR cytotoxicity. In this study we assessed the capacity of an anti-TS antisense ODN to circumvent resistance dependent on TS overexpression. 2. Variant HeLa clones exhibiting 2 - 20 fold resistance to 5-FUdR were selected by exposing cultured cells to drug. Clones FUdR-5a, -25b, and -50a expressed TS protein levels 10 fold, 10 fold, and 17 fold higher (respectively) than parental cells. Cells were treated with antisense ODN 83 (a 2'-methoxy-ethoxylated, phosphorothioated 20-mer, complementary to a portion of the 3'-untranslated region of TS mRNA), or ODN 32 (a control ODN with the same base composition as ODN 83, but in randomized order). Twenty-four and 48 h following transfection (50-100 nM ODN, plus polycationic liposome), TS mRNA levels (by RT-PCR) and protein levels (by radiolabelled 5-FUdR-monophosphate binding) were decreased by at least 60% in ODN 83-treated cells compared with control ODN 32-treated cells. ODN 83 enhanced the cytotoxicity of 5-FUdR by up to 85% in both parental and 5-FUdR-resistant cell lines. 3. Antisense ODN can be used to down-regulate TS and attenuate drug resistance in TS-overexpressing cells.     

Mixed polymer micelles of amphiphilic and cationic copolymers for delivery of antisense oligonucleotides

Cationic copolymers were synthesized by conjugation of branched 2 kDa polyethylenimine (PEI) and Pluronic block copolymers (F38, P85, P123). Compositions of these copolymers mixed with corresponding free Pluronics at weight ratio 1:9 were used to complex phosphorothioate oligonucleotides (ODN). As a result stable suspensions of small micelle-like particles (<220 nm) were obtained. Incorporation of ODN in these formulations increased uptake of ODN in KBv cells and increased sequence specific activity of antisense ODN targeted against MDR gene in multidrug resistant cells resulting in inhibition of the functional activity of P-glycoprotein (P-gp) in these cells. Furthermore, these formulations increased transport of ODN across model intestinal barrier, Caco-2 cell monolayers, suggesting that they could be useful for oral delivery of biologically active ODN.     

Lipid-based delivery of combinations of antisense oligodeoxynucleotides for the in vitro inhibition of HIV-1 replication

We evaluated a new approach to AIDS therapy by using combinations of oligodeoxynucleotides (ODNs), delivered with a lipid-based carrier system, that target different HIV viral genome sites. We identified some of the factors that seem to influence the effectiveness of a combination strategy in cell cultures including ODN concentrations, type of infection (acute vs chronic), backbone modification of the ODN, and the number of sequences. When delivered by the DLS carrier system, some advantages of using a combination of ODNs over treatment with only one ODN could be observed in acute infection assays but not in the chronic infection model. These results suggest that in the acute infection model, the 3 different antisense ODNs in the “cocktail” might block an early step of virus replication by combined inhibitory effects. Various combinations of phosphorothioate-modified (PS) and unmodified oligonucleotides delivered by the DLS system were compared for their antiviral activity in a long-term acute assay using HIV-1 (IIIB strain)-infected MOLT-3 cells. The most effective combination had 3 phosphorothioate antisense ODNs: Srev, SDIS, and SPac (>99% inhibition at 100 pM). However, the additive effect determined when using ODN combinations was rather low, revealing the high level of nonsequence specificity in HIV-1 cell culture models. Data illustrated the high sequence nonspecific activity of ODNs, especially when comparing activity of antisense ODNs with activity of random control sequence ODNs. The latter exhibited an inhibitory effect similar to that of antisense ODNs under our experimental conditions. Nevertheless, we demonstrated that it is possible to achieve high anti-HIV activity by using, in combination, picomolar range concentrations of antisense oligonucleotides complexed to a lipid-based carrier system such as the DLS system, without increasing cell toxicity.     

Mixed Polymer Micelles of Amphiphilic and Cationic Copolymers for Delivery of Antisense Oligonucleotides

Cationic copolymers were synthesized by conjugation of branched 2 kDa polyethylenimine (PEI) and Pluronic® block copolymers (F38, P85, P123). Compositions of these copolymers mixed with corresponding free Pluronics® at weight ratio 1:9 were used to complex phosphorothioate oligonucleotides (ODN). As a result stable suspensions of small micelle-like particles (<220 nm) were obtained. Incorporation of ODN in these formulations increased uptake of ODN in KBv cells and increased sequence specific activity of antisense ODN targeted against MDR gene in multidrug resistant cells resulting in inhibition of the functional activity of P-glycoprotein (P-gp) in these cells. Furthermore, these formulations increased transport of ODN across model intestinal barrier, Caco-2 cell monolayers, suggesting that they could be useful for oral delivery of biologically active ODN.     

反义碱性成纤维细胞生长因子寡核苷酸对血管紧张素Ⅱ诱导培养的 …

AIM: To study the effect of antisense basic fibroblast growth factor (bFGF) oligonucleotides (ODN) transfection on the growth of cultured aortic smooth muscle cells (SMC) in spontaneously hypertensive rats (SHR). METHODS: Using cationic liposome-mediated method, antisense bFGF ODN were introduced into SMC, bFGF gene expression was detected by Northern blotting, cell hyperplasia was evaluated by [3H] thymidine incorporation and cell counting. RESULTS: Transfection of antisense bFGF ODN (5 mumol.L-1) almost completely inhibited enhanced bFGF mRNA expression and inhibited cell proliferation induced by angiotensin II (Ang 1 mumol.L-1). In basal state and Ang-stimulated state, [3H]thymidine incorporation was inhibited by 26.5% (P < 0.01) and 42.0% (P < 0.01) and cell number was inhibited by 17.3% (P < 0.01) and by 22.2% (P < 0.01), respectively. CONCLUSION: The transfection of antisense bFGF ODN into cultured SMC effectively suppressed bFGF mRNA expression and inhibited the SMC proliferation induced by Ang.     

Antisense down-regulation of thymidylate synthase to suppress growth and enhance cytotoxicity of 5-FUdR, 5-FU and Tomudex in HeLa cells.

1. Thymidylate synthase (TS), the key enzyme in de novo synthesis of thymidine, is an important target for antitumour chemotherapy. It was hypothesized that antisense oligonucleotide down-regulation of TS mRNA would decrease TS levels and enhance the cytotoxicity of inhibitors of TS, including the pyrimidine analogues 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), and the folate analogue Tomudex (ICI D1694; N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theon yl-L-glutamic acid). 2. 2'-Methoxyethoxylated, phosphorothioated 20-mer oligodeoxynucleotides (ODNs), complementary to various sequences in TS mRNA, were synthesized, along with control oligomers consisting of the same, respective bases in randomized order, against which all the biological effects were compared. Following a 6-h transfection of HeLa cells using polycationic liposome at 3 microg ml(-1), ODN 83 (50 nM), complementary to a region in the 3'-untranslated region of the TS mRNA, decreased TS mRNA levels by approximately 70% within 24 h. ODN 83 also decreased TS enzyme activity, as measured by binding of TS to radiolabelled 5-fluorodeoxyuridine monophosphate. In addition to inhibiting proliferation by up to approximately 40%, ODN 83 enhanced the cytotoxicity of Tomudex or 5-FU, added 1 day following transfection, by 50 - 60%. ODN 83 also enhanced sensitivity to 5-FUdR by 70%, but did not affect the toxicity of cisplatin, chlorambucil, melphalan, doxorubicin, ionizing radiation, paclitaxel, or irinotecan. 3. These data indicate that antisense ODN down-regulation of TS can inhibit human tumour cell proliferation and enhance the efficacy of TS-targeted drugs.     

Stent-based delivery of antisense oligodeoxynucleotides targeted to the PDGF A-chain decreases in-stent restenosis of the coronary artery

BACKGROUND: Although the use of drug-eluting stents (DESs) has been shown to limit neointima hyperplasia, currently available DESs may adversely affect reendothelialization, possibly precipitating cardiac events. We evaluated the effect of an antisense oligodeoxynucleotide (ODN) targeted to the platelet-derived growth factor (PDGF) A-chain on in-stent restenosis in pig coronary artery. METHODS: A bare metal stent coated with phosphorothioate-linked antisense ODN or nonsense ODN, or a bare metal stent without ODN (control), was implanted in the mid segment of the left anterior descending artery (LAD). Twenty-eight days after implantation, angiography and intravascular ultrasound (IVUS) were performed, the LAD was removed, and stenosis was evaluated pathologically. RESULTS: Volumetric stenosis ratios were 64 +/- 11.9, 44 +/- 3.4, and 26 +/- 3.8% in coronary arteries implanted with control, nonsense ODN-coated, and antisense ODN-coated stents, respectively. In angioscopic findings, the lumen surface was smooth in the stented segments in all groups. Struts of antisense ODN-coated stents were observed embedded in the neointima, whereas embedding was not observed in nonsense ODN-coated stents or control stents, indicating a decrease in hyperplasia in response to antisense ODN treatment. Pathologic findings showed 77 +/- 5.8, 68 +/- 12.2, and 38 +/- 5.3% stenosis in coronary arteries implanted with control stents, nonsense ODN-coated stents, and antisense ODN-coated stents, respectively. A continuous lining of endothelial cells was observed along the lumen of coronary arteries implanted with antisense ODN-coated stents. CONCLUSIONS: Stent-based delivery of an antisense ODN targeted to the PDGF A-chain effectively inhibits neointima formation after stent implantation in pig coronary artery by suppressing VSMC hyperplasia and preserving endothelialization. Antisense-ODNs may provide a therapy for in-stent restenosis of the coronary artery.