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Donald J. Hnatowich - One of the best experts on this subject based on the ideXlab platform.
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Cell Culture and Xenograft-Bearing Animal Studies of Radiolabeled Antisense DNA–Carrier Nanoparticles with Streptavidin as a Linker
Journal of nuclear medicine : official publication Society of Nuclear Medicine, 2007Co-Authors: Kayoko Nakamura, Yi Wang, Xinrong Liu, Atsushi Kubo, Donald J. HnatowichAbstract:UNLABELLED Transmembrane transfectors (carriers) are increasingly being viewed as helpful or even necessary to improve cellular delivery in connection with Antisense tumor targeting and other applications requiring cell membrane transport of DNAs, RNAs, and other oligomers. We are investigating streptavidin as a convenient linker for biotinylated carriers and oligomers because it requires only simple mixing for preparation. The goal of this study was to evaluate Antisense DNA-streptavidin-carrier nanoparticles for accumulation in cell culture and in xenograft-bearing mice. METHODS The 3 carriers were cholesterol, a 10-mer Tat peptide, and a 10-mer polyarginine peptide. A 20-mer DNA targeting the mdr1 messenger RNA coding for Pgp expression was used as the phosphodiester (PO) DNA as well as the phosphorothioate (PS) DNA. In all cases, the (99m)Tc radiolabel was on the DNA. The 8 nanoparticles were first tested in mdr1(++) KB-G2 and TCO-1 cells and in mdr1(+/-) KB-31 cells in culture for evidence of improved accumulation and Antisense targeting. Thereafter, the PS DNA-streptavidin-Tat, PO DNA-streptavidin-Tat, and PS DNA-streptavidin-cholesterol nanoparticles were administered intravenously to KB-G2 xenograft-bearing mice, and tissue distributions were measured. RESULTS In culture, the PO nanoparticles showed increased accumulation compared with the corresponding nanoparticles without the carrier in all 3 cell types; in contrast, with the PS nanoparticles, any similar carrier-mediated increase may have been obscured by the much higher protein-binding affinity of PS DNA. As evidence of Antisense targeting, the Tat and cholesterol PS nanoparticles showed statistically significant accumulation at 23 h in cells in the descending order TCO-1, KB-G2, and KB-31, although there were no significant differences among the PO nanoparticles. In xenograft-bearing mice, the tissue accumulation of both forms of the PS nanoparticles greatly exceeded that of the PO nanoparticles and, including in the tumor, were similar to that obtained previously for naked PS DNA. CONCLUSION The presence of the streptavidin linker had no obvious detrimental effect on the functions of the carriers and Antisense DNAs. The higher protein-binding affinity of the PS nanoparticles than the PO nanoparticles was still apparent both in vitro and in vivo, the pharmacokinetics of the PS nanoparticles were similar to that of naked PS DNA, and the carriers improved cellular accumulation, at least for the PO nanoparticles. These observations, taken together with the higher accumulation of both forms of the Antisense PS nanoparticles in mdr1(++) KB-G2 and TCO-1 cells than in mdr1(+/-) KB-31 cells, suggest that further effort is justified to confirm that the Antisense properties of the DNAs were not compromised by the presence of streptavidin.
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Influence of Two Transfectors on Delivery of ^99mTc Antisense DNA in Tumor-Bearing Mice
Molecular Imaging and Biology, 2006Co-Authors: Kayoko Nakamura, Yi Wang, Xinrong Liu, Atsushi Kubo, Donald J. HnatowichAbstract:Purpose The aim of the study is to determine whether delivery into tumor cells in vivo of a ^99mTc-labeled Antisense phosphorothioate DNA targeting the mdr1 mRNA improves after electrostatic complexation with the transmembrane transfector (TF) carriers Neophectin™ or jetPEI™ as was observed by us in vitro . Methods The biodistribution of the labeled Antisense DNA before and after complexation with either TF was determined in nude mice bearing KB-G2 (Pgp++) tumors. Results Complexation with either TF resulted in significantly higher background radioactivity levels in almost all normal tissues and modest improvement in tumor accumulation at best. The tumor accumulation was lower compared to naked at six hours (0.34 and 0.23 vs. 0.40% ID/g) and modestly higher at 24–28 hours (0.15 and 0.15 vs. 0.12% ID/g) for Neophectin and jetPEI, respectively. That blood was less than 0.18% ID/g for both TFs even at six hours suggests that tumor accumulations may have suffered from rapid blood clearance. Conclusion The results of this investigation show that because of the unfavorable pharmacokinetics of radiolabeled phosphorothioate DNAs when electrostatically complexed to jetPEI or Neophectin, neither TF appears to be useful in vivo despite favorable results in vitro . Future studies will devote greater consideration to the relative rates of tumor accumulation and blood clearance.
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Antisense Targeting of P-Glycoprotein Expression in Tissue Culture
Journal of nuclear medicine : official publication Society of Nuclear Medicine, 2005Co-Authors: Kayoko Nakamura, Atsushi Kubo, Donald J. HnatowichAbstract:Radiolabeled DNA and other oligomers are now under investigation for Antisense targeting of a variety of messenger RNA (mRNA). Multidrug resistance (MDR) is detectable as P-glycoprotein (Pgp) expression in most cells and is often elevated in tumor cells, especially those exposed to chemotherapeutic drugs. Radiolabeled Antisense DNA has not previously been considered for the targeting of mdr1 mRNA, the product of the mdr1 gene controlling Pgp expression of MDR. Methods: A 20-mer uniform phosphorothioate DNA, described elsewhere as targeting the AUG start codon of mdr1 mRNA, was used naked along with the sense phosphorothioate DNA control. The 3 cell lines were KB-G2, an epidermal carcinoma cell line that had been transfected to overexpress mdr1 mRNA (i.e., Pgp++) compared with its parent (Pgp+) KB-31, and TCO-1, a thyroid carcinoma cell line also reported to be Pgp++. The relative expression of mdr1 mRNA in these 3 cell lines was confirmed elsewhere by reverse transcriptase polymerase chain reaction. As a marker of Pgp expression, the uptake of 99mTc-sestamibi was measured in the 3 cell lines after 20 h of incubation with different concentrations of both Antisense and sense DNA. Both DNAs were radiolabeled with 99mTc via mercaptoacetyltriglycine, and cellular uptake was measured after 24 h of incubation. Results: In the case of the sense DNA, the ratio of sestamibi uptake in cells incubated with the DNA to those not exposed to the DNA was unaffected regardless of cell line and regardless of DNA concentration. In contrast, this ratio was significantly higher in both the KB-G2 and TCO-1 cells when incubated with Antisense DNA at concentrations greater than about 25 nmol/L (i.e., 150 ng/mL). Only in the KB-31 cells was the sestamibi accumulation unaffected by incubation with the Antisense DNA. Thus, the Antisense DNA was interfering with Pgp expression to a measurable extent in both Pgp++ cells, but not the Pgp+ cells. This behavior is almost certainly due to Antisense targeting of mdr1 mRNA by the Antisense DNA since the sense control DNA had no effect. A significant increased accumulation of 99mTc-Antisense versus 99mTc-sense DNA was observed in all 3 cell lines. In all cases, this difference was greatest at the lowest DNA concentrations and decreased with increasing concentration as expected for specific binding. In the KB-G2 cells, cellular accumulation of 99mTc-Antisense DNA was strikingly high at the lowest concentration at 54%, compared with 22% for 99mTc-sense DNA. These accumulations therefore probably reflect the higher mRNA target concentration in the MDR++ cells than in the MDR+ cells and the higher specific binding of 99mTc-Antisense DNA than nonspecific binding of 99mTc-sense DNA. Conclusion: Further evidence was obtained suggesting that an Antisense mechanism is responsible for the accumulation of 99mTc-oligomers in cells in culture. Finally, whereas evidence of in vitro targeting is not necessarily evidence of in vivo targeting, our results do suggest that radiolabeled Antisense DNA against the mdr1 mRNA may potentially be useful for Antisense imaging of MDR in cancer.
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Electrostatic binding with tat and other cationic peptides increases cell accumulation of 99mTc-Antisense DNAs without entrapment
Molecular imaging and biology, 2003Co-Authors: Yu-min Zhang, Ning Liu, Mary Rusckowski, Changbin Liu, Guillermina Ferro Flores, Donald J. HnatowichAbstract:Abstract An important limitation restricting Antisense nuclear medicine imaging is low radioactivity accumulations in target cells. The Tat peptide (Tat), a basic domain of the HIV Tat protein, has been shown to enhance cell accumulation of various biomolecules. PURPOSE: The influence of Tat, as a cationic carrier, on the accumulation in cell culture of anionic Antisense DNAs bound electrostatically rather than covalently was investigated. To establish specificity of the accumulation, Antisense DNA and control sequence were studied along with four different peptides. The technique of in situ reverse transcription was used to assay the in vivo hybridization of Antisense DNA to the target mRNA in cultured live cells when transducted with the Tat peptide. METHODS: Uniform phosphorothioated DNAs were radiolabeled with 99mTc via Hynic/tricine. This 18 mer Antisense DNA against RIα mRNA along with its sense and random control was studied in ACHN cells with the four peptides as carriers. RESULTS: The addition of Tat significantly increased cell accumulations. At 12 hours accumulations went from 14% to 45% for the Antisense DNA and from 4% to 12% for control. Furthermore, an Antisense effect was again suggested, now with the Tat carrier, by the significantly higher accumulation of 99mTc on both Antisense DNAs vs. controls. Moreover, the accumulated Antisense DNA enhanced with the Tat carrier was capable of priming reverse transcription as determined by an in situ assay suggesting that the DNA could escape from entrapment in endosome or lysosome vesicles for hybridization. However, differences in cellular accumulation with either Tat compared to either scrambled peptide were not significant, showing that the Tat in this study was functioning merely as a cationic carrier. CONCLUSIONS: Although electrostatic binding of Antisense DNA to Tat is convenient, the association may mask the unique transduction properties of the peptide. Nevertheless, a promising improvement in cellular accumulation of Antisense DNA was observed through the use of these carriers. In addition, at least a fraction of the transducted DNA appears to be free of entrapment to hybridize to its mRNA target in live cells.
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in vitro investigations of tumor targeting with 99m tc labeled Antisense DNA
The Journal of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with γ- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an Antisense mechanism. Methods: An 18mer uniform phosphorothioate DNA Antisense to the messenger RNA (mRNA) of the type I regulatory subunit α of cyclic adenosine monophosphate-dependent protein kinase A (RIα) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. Results: By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of 99mTc-MAG3-DNA was lower than that of 35S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an Antisense effect was suggested by 3 findings: an increased accumulation of 35S- or 99mTc-labeled Antisense versus sense DNA, an increased accumulation of 99mTc-Antisense DNA in another RIα-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of 99mTc-Antisense DNA with increasing dosage of Antisense DNA. Higher than expected cellular accumulations of about 105 Antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the Antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5′-triphosphate into RNA in cells exposed to the Antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the Antisense DNA but not to the control DNA. Conclusion: This evidence suggests tumor cell accumulation by an Antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.
Yu-min Zhang - One of the best experts on this subject based on the ideXlab platform.
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Electrostatic binding with tat and other cationic peptides increases cell accumulation of 99mTc-Antisense DNAs without entrapment
Molecular imaging and biology, 2003Co-Authors: Yu-min Zhang, Ning Liu, Mary Rusckowski, Changbin Liu, Guillermina Ferro Flores, Donald J. HnatowichAbstract:Abstract An important limitation restricting Antisense nuclear medicine imaging is low radioactivity accumulations in target cells. The Tat peptide (Tat), a basic domain of the HIV Tat protein, has been shown to enhance cell accumulation of various biomolecules. PURPOSE: The influence of Tat, as a cationic carrier, on the accumulation in cell culture of anionic Antisense DNAs bound electrostatically rather than covalently was investigated. To establish specificity of the accumulation, Antisense DNA and control sequence were studied along with four different peptides. The technique of in situ reverse transcription was used to assay the in vivo hybridization of Antisense DNA to the target mRNA in cultured live cells when transducted with the Tat peptide. METHODS: Uniform phosphorothioated DNAs were radiolabeled with 99mTc via Hynic/tricine. This 18 mer Antisense DNA against RIα mRNA along with its sense and random control was studied in ACHN cells with the four peptides as carriers. RESULTS: The addition of Tat significantly increased cell accumulations. At 12 hours accumulations went from 14% to 45% for the Antisense DNA and from 4% to 12% for control. Furthermore, an Antisense effect was again suggested, now with the Tat carrier, by the significantly higher accumulation of 99mTc on both Antisense DNAs vs. controls. Moreover, the accumulated Antisense DNA enhanced with the Tat carrier was capable of priming reverse transcription as determined by an in situ assay suggesting that the DNA could escape from entrapment in endosome or lysosome vesicles for hybridization. However, differences in cellular accumulation with either Tat compared to either scrambled peptide were not significant, showing that the Tat in this study was functioning merely as a cationic carrier. CONCLUSIONS: Although electrostatic binding of Antisense DNA to Tat is convenient, the association may mask the unique transduction properties of the peptide. Nevertheless, a promising improvement in cellular accumulation of Antisense DNA was observed through the use of these carriers. In addition, at least a fraction of the transducted DNA appears to be free of entrapment to hybridize to its mRNA target in live cells.
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in vitro investigations of tumor targeting with 99m tc labeled Antisense DNA
The Journal of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with γ- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an Antisense mechanism. Methods: An 18mer uniform phosphorothioate DNA Antisense to the messenger RNA (mRNA) of the type I regulatory subunit α of cyclic adenosine monophosphate-dependent protein kinase A (RIα) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. Results: By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of 99mTc-MAG3-DNA was lower than that of 35S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an Antisense effect was suggested by 3 findings: an increased accumulation of 35S- or 99mTc-labeled Antisense versus sense DNA, an increased accumulation of 99mTc-Antisense DNA in another RIα-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of 99mTc-Antisense DNA with increasing dosage of Antisense DNA. Higher than expected cellular accumulations of about 105 Antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the Antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5′-triphosphate into RNA in cells exposed to the Antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the Antisense DNA but not to the control DNA. Conclusion: This evidence suggests tumor cell accumulation by an Antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.
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In vitro investigations of tumor targeting with (99m)Tc-labeled Antisense DNA.
Journal of nuclear medicine : official publication Society of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:UNLABELLED One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with gamma- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an Antisense mechanism. METHODS An 18mer uniform phosphorothioate DNA Antisense to the messenger RNA (mRNA) of the type I regulatory subunit alpha of cyclic adenosine monophosphate-dependent protein kinase A (RI alpha) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. RESULTS By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of (99m)Tc-MAG3-DNA was lower than that of (35)S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an Antisense effect was suggested by 3 findings: an increased accumulation of (35)S- or (99m)Tc-labeled Antisense versus sense DNA, an increased accumulation of (99m)Tc-Antisense DNA in another RI alpha-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of (99m)Tc-Antisense DNA with increasing dosage of Antisense DNA. Higher than expected cellular accumulations of about 10(5) Antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the Antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5'-triphosphate into RNA in cells exposed to the Antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the Antisense DNA but not to the control DNA. CONCLUSION This evidence suggests tumor cell accumulation by an Antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.
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Influence of different chelators (HYNIC, MAG3 and DTPA) on tumor cell accumulation and mouse biodistribution of technetium-99m labeled to Antisense DNA.
European journal of nuclear medicine, 2000Co-Authors: Yu-min Zhang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:We have shown recently that cell accumulation in culture of Antisense DNA is strongly influenced by the presence of a 99mTc-MAG3 group for radiolabeling. We have now compared the in vitro and mouse in vivo behavior of 99mTc when radiolabeled to one Antisense phosphorothioate DNA by three different methods. The 18-mer Antisense DNA against the RIα subunit of PKA was conjugated via a primary amine on the 5'-end with the NHS esters of HYNIC and MAG3 and by the cyclic anhydride of DTPA. Surface plasmon resonance measurements revealed that the association rate constant for hybridization was unchanged for all three chelators as compared with that of the native DNA. Size exclusion HPLC showed rapid and quantitative protein binding for all three chelators upon incubation of labeled DNAs in 37°C serum and cell culture medium. However, in each case, radiolabeled and intact oligonucleotide was still detectable after 24 h. Cellular uptake was tested in an RIα mRNA-positive cancer cell line. The order of cellular accumulation of 99mTc was DTPA>HYNIC(tricine)>MAG3, with the differences increasing with time between 4 and 24 h. The rate of 99mTc egress from cells was found to be MAG3>HYNIC>DTPA, which may explain the order of cellular accumulation. The biodistribution in normal mice was heavily influenced by the labeling method and followed a pattern similar to that seen previously by us for peptides labeled with the same chelators. In conclusion, although these studies concerned only one Antisense DNA in one cell line, the results suggest that the success of Antisense imaging may depend, in part, on the method of radiolabeling.
Yoshihiro Ito - One of the best experts on this subject based on the ideXlab platform.
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Intracellular Delivery of Antisense DNA and siRNA with Amino Groups Masked with Disulfide Units.
Chemical & pharmaceutical bulletin, 2020Co-Authors: Zhaoma Shu, Azumi Ota, Naoko Abe, Kosuke Nakamoto, Fumiaki Tomoike, Seiichi Tada, Yukiya Takayama, Yuri Katsurada, Kosuke Kusamori, Yoshihiro ItoAbstract:Efficient methods for delivery of Antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.
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disulfide unit conjugation enables ultrafast cytosolic internalization of Antisense DNA and sirna
Angewandte Chemie, 2019Co-Authors: Zhaoma Shu, Iku Tanaka, Azumi Ota, Daichi Fushihara, Naoko Abe, Saki Kawaguchi, Kosuke Nakamoto, Fumiaki Tomoike, Seiichi Tada, Yoshihiro ItoAbstract:Development of intracellular delivery methods for Antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor-ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10 minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide-modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of Antisense DNA and siRNA.
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Disulfide‐Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA
Angewandte Chemie (International ed. in English), 2019Co-Authors: Zhaoma Shu, Iku Tanaka, Azumi Ota, Daichi Fushihara, Naoko Abe, Saki Kawaguchi, Kosuke Nakamoto, Fumiaki Tomoike, Seiichi Tada, Yoshihiro ItoAbstract:Development of intracellular delivery methods for Antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor-ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10 minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide-modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of Antisense DNA and siRNA.
Mary Rusckowski - One of the best experts on this subject based on the ideXlab platform.
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Initial Mechanistic Studies of Antisense Targeting in Cells
Journal of nuclear medicine : official publication Society of Nuclear Medicine, 2006Co-Authors: Xinrong Liu, Yi Wang, Mary Rusckowski, Kayoko Nakamura, Yee Wang, Guozheng Liu, Hongliu Ding, Atsushi KuboAbstract:The continued development of Antisense targeting will require a better understanding of the mechanism. Methods: We performed initial studies of the mechanism of intracellular Antisense targeting through measurements of in situ transcription, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), 32P-labeled uridine-5′-triphosphate (α-32P-UTP) incorporation, nuclear accumulations of 99mTc-labeled DNAs, and messenger RNA (mRNA) transcription rate. As reported earlier, an Antisense DNA against the mdr1 mRNA coding for P-glycoprotein (Pgp) and its sense DNA control were used in KB-G2 (Pgp++) cells. Results: Definitive evidence for Antisense targeting was obtained by in situ transcription showing complementary DNA elongation in cells exposed to Antisense DNA, acting therefore as an intracellular PCR primer of mdr1 mRNA, but not in cells exposed to sense DNA. Immunofluorescence staining showed higher accumulations of Antisense versus sense DNAs in KB-G2 cells. Transnuclear migration was confirmed by higher accumulations in the nucleus compared with the cytoplasm in cells incubated with 99mTc-labeled Antisense DNA. However, the observed specific accumulations of Antisense DNAs of about 106 per cell over 10 h could not be explained by a feedback mechanism upregulating transcription in cells exposed to Antisense DNA as no increase in mRNA levels was detected by both RT-PCR and 32P-UTP in these cells. To explore an alternative hypothesis, a novel approach using 99mTc-labeled Antisense DNA as a probe of total mRNA from cells previously saturated with unlabeled Antisense DNA was used to estimate the transcription rate. Compared with controls, mdr1 mRNA levels were found to be initially low after saturation and to recover at about 2,000 copies per minute per cell. If persistent, this transcription rate would provide 106 mRNAs in 10 h. Conclusion: The results of all studies are consistent with Antisense as the mechanism of targeting. Though a feedback mechanism leading to upregulation of mRNA transcription is an unlikely explanation for the high specific accumulations, our results may be explained if Antisense DNAs are targeting mdr1 mRNAs produced at high transcription rates. If the target is primarily pre-mRNA in the nucleus rather than mature mRNA in the cytoplasm, this would provide as well an explanation for the observed migration of 99mTc-labeled Antisense DNA into the nucleus.
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Electrostatic binding with tat and other cationic peptides increases cell accumulation of 99mTc-Antisense DNAs without entrapment
Molecular imaging and biology, 2003Co-Authors: Yu-min Zhang, Ning Liu, Mary Rusckowski, Changbin Liu, Guillermina Ferro Flores, Donald J. HnatowichAbstract:Abstract An important limitation restricting Antisense nuclear medicine imaging is low radioactivity accumulations in target cells. The Tat peptide (Tat), a basic domain of the HIV Tat protein, has been shown to enhance cell accumulation of various biomolecules. PURPOSE: The influence of Tat, as a cationic carrier, on the accumulation in cell culture of anionic Antisense DNAs bound electrostatically rather than covalently was investigated. To establish specificity of the accumulation, Antisense DNA and control sequence were studied along with four different peptides. The technique of in situ reverse transcription was used to assay the in vivo hybridization of Antisense DNA to the target mRNA in cultured live cells when transducted with the Tat peptide. METHODS: Uniform phosphorothioated DNAs were radiolabeled with 99mTc via Hynic/tricine. This 18 mer Antisense DNA against RIα mRNA along with its sense and random control was studied in ACHN cells with the four peptides as carriers. RESULTS: The addition of Tat significantly increased cell accumulations. At 12 hours accumulations went from 14% to 45% for the Antisense DNA and from 4% to 12% for control. Furthermore, an Antisense effect was again suggested, now with the Tat carrier, by the significantly higher accumulation of 99mTc on both Antisense DNAs vs. controls. Moreover, the accumulated Antisense DNA enhanced with the Tat carrier was capable of priming reverse transcription as determined by an in situ assay suggesting that the DNA could escape from entrapment in endosome or lysosome vesicles for hybridization. However, differences in cellular accumulation with either Tat compared to either scrambled peptide were not significant, showing that the Tat in this study was functioning merely as a cationic carrier. CONCLUSIONS: Although electrostatic binding of Antisense DNA to Tat is convenient, the association may mask the unique transduction properties of the peptide. Nevertheless, a promising improvement in cellular accumulation of Antisense DNA was observed through the use of these carriers. In addition, at least a fraction of the transducted DNA appears to be free of entrapment to hybridize to its mRNA target in live cells.
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in vitro investigations of tumor targeting with 99m tc labeled Antisense DNA
The Journal of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with γ- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an Antisense mechanism. Methods: An 18mer uniform phosphorothioate DNA Antisense to the messenger RNA (mRNA) of the type I regulatory subunit α of cyclic adenosine monophosphate-dependent protein kinase A (RIα) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. Results: By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of 99mTc-MAG3-DNA was lower than that of 35S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an Antisense effect was suggested by 3 findings: an increased accumulation of 35S- or 99mTc-labeled Antisense versus sense DNA, an increased accumulation of 99mTc-Antisense DNA in another RIα-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of 99mTc-Antisense DNA with increasing dosage of Antisense DNA. Higher than expected cellular accumulations of about 105 Antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the Antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5′-triphosphate into RNA in cells exposed to the Antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the Antisense DNA but not to the control DNA. Conclusion: This evidence suggests tumor cell accumulation by an Antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.
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In vitro investigations of tumor targeting with (99m)Tc-labeled Antisense DNA.
Journal of nuclear medicine : official publication Society of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:UNLABELLED One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with gamma- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an Antisense mechanism. METHODS An 18mer uniform phosphorothioate DNA Antisense to the messenger RNA (mRNA) of the type I regulatory subunit alpha of cyclic adenosine monophosphate-dependent protein kinase A (RI alpha) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. RESULTS By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of (99m)Tc-MAG3-DNA was lower than that of (35)S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an Antisense effect was suggested by 3 findings: an increased accumulation of (35)S- or (99m)Tc-labeled Antisense versus sense DNA, an increased accumulation of (99m)Tc-Antisense DNA in another RI alpha-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of (99m)Tc-Antisense DNA with increasing dosage of Antisense DNA. Higher than expected cellular accumulations of about 10(5) Antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the Antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5'-triphosphate into RNA in cells exposed to the Antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the Antisense DNA but not to the control DNA. CONCLUSION This evidence suggests tumor cell accumulation by an Antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.
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Influence of different chelators (HYNIC, MAG3 and DTPA) on tumor cell accumulation and mouse biodistribution of technetium-99m labeled to Antisense DNA.
European journal of nuclear medicine, 2000Co-Authors: Yu-min Zhang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:We have shown recently that cell accumulation in culture of Antisense DNA is strongly influenced by the presence of a 99mTc-MAG3 group for radiolabeling. We have now compared the in vitro and mouse in vivo behavior of 99mTc when radiolabeled to one Antisense phosphorothioate DNA by three different methods. The 18-mer Antisense DNA against the RIα subunit of PKA was conjugated via a primary amine on the 5'-end with the NHS esters of HYNIC and MAG3 and by the cyclic anhydride of DTPA. Surface plasmon resonance measurements revealed that the association rate constant for hybridization was unchanged for all three chelators as compared with that of the native DNA. Size exclusion HPLC showed rapid and quantitative protein binding for all three chelators upon incubation of labeled DNAs in 37°C serum and cell culture medium. However, in each case, radiolabeled and intact oligonucleotide was still detectable after 24 h. Cellular uptake was tested in an RIα mRNA-positive cancer cell line. The order of cellular accumulation of 99mTc was DTPA>HYNIC(tricine)>MAG3, with the differences increasing with time between 4 and 24 h. The rate of 99mTc egress from cells was found to be MAG3>HYNIC>DTPA, which may explain the order of cellular accumulation. The biodistribution in normal mice was heavily influenced by the labeling method and followed a pattern similar to that seen previously by us for peptides labeled with the same chelators. In conclusion, although these studies concerned only one Antisense DNA in one cell line, the results suggest that the success of Antisense imaging may depend, in part, on the method of radiolabeling.
Ning Liu - One of the best experts on this subject based on the ideXlab platform.
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Electrostatic binding with tat and other cationic peptides increases cell accumulation of 99mTc-Antisense DNAs without entrapment
Molecular imaging and biology, 2003Co-Authors: Yu-min Zhang, Ning Liu, Mary Rusckowski, Changbin Liu, Guillermina Ferro Flores, Donald J. HnatowichAbstract:Abstract An important limitation restricting Antisense nuclear medicine imaging is low radioactivity accumulations in target cells. The Tat peptide (Tat), a basic domain of the HIV Tat protein, has been shown to enhance cell accumulation of various biomolecules. PURPOSE: The influence of Tat, as a cationic carrier, on the accumulation in cell culture of anionic Antisense DNAs bound electrostatically rather than covalently was investigated. To establish specificity of the accumulation, Antisense DNA and control sequence were studied along with four different peptides. The technique of in situ reverse transcription was used to assay the in vivo hybridization of Antisense DNA to the target mRNA in cultured live cells when transducted with the Tat peptide. METHODS: Uniform phosphorothioated DNAs were radiolabeled with 99mTc via Hynic/tricine. This 18 mer Antisense DNA against RIα mRNA along with its sense and random control was studied in ACHN cells with the four peptides as carriers. RESULTS: The addition of Tat significantly increased cell accumulations. At 12 hours accumulations went from 14% to 45% for the Antisense DNA and from 4% to 12% for control. Furthermore, an Antisense effect was again suggested, now with the Tat carrier, by the significantly higher accumulation of 99mTc on both Antisense DNAs vs. controls. Moreover, the accumulated Antisense DNA enhanced with the Tat carrier was capable of priming reverse transcription as determined by an in situ assay suggesting that the DNA could escape from entrapment in endosome or lysosome vesicles for hybridization. However, differences in cellular accumulation with either Tat compared to either scrambled peptide were not significant, showing that the Tat in this study was functioning merely as a cationic carrier. CONCLUSIONS: Although electrostatic binding of Antisense DNA to Tat is convenient, the association may mask the unique transduction properties of the peptide. Nevertheless, a promising improvement in cellular accumulation of Antisense DNA was observed through the use of these carriers. In addition, at least a fraction of the transducted DNA appears to be free of entrapment to hybridize to its mRNA target in live cells.
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in vitro investigations of tumor targeting with 99m tc labeled Antisense DNA
The Journal of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with γ- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an Antisense mechanism. Methods: An 18mer uniform phosphorothioate DNA Antisense to the messenger RNA (mRNA) of the type I regulatory subunit α of cyclic adenosine monophosphate-dependent protein kinase A (RIα) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. Results: By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of 99mTc-MAG3-DNA was lower than that of 35S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an Antisense effect was suggested by 3 findings: an increased accumulation of 35S- or 99mTc-labeled Antisense versus sense DNA, an increased accumulation of 99mTc-Antisense DNA in another RIα-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of 99mTc-Antisense DNA with increasing dosage of Antisense DNA. Higher than expected cellular accumulations of about 105 Antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the Antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5′-triphosphate into RNA in cells exposed to the Antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the Antisense DNA but not to the control DNA. Conclusion: This evidence suggests tumor cell accumulation by an Antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.
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In vitro investigations of tumor targeting with (99m)Tc-labeled Antisense DNA.
Journal of nuclear medicine : official publication Society of Nuclear Medicine, 2001Co-Authors: Yu-min Zhang, Yi Wang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:UNLABELLED One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with gamma- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an Antisense mechanism. METHODS An 18mer uniform phosphorothioate DNA Antisense to the messenger RNA (mRNA) of the type I regulatory subunit alpha of cyclic adenosine monophosphate-dependent protein kinase A (RI alpha) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. RESULTS By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of (99m)Tc-MAG3-DNA was lower than that of (35)S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an Antisense effect was suggested by 3 findings: an increased accumulation of (35)S- or (99m)Tc-labeled Antisense versus sense DNA, an increased accumulation of (99m)Tc-Antisense DNA in another RI alpha-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of (99m)Tc-Antisense DNA with increasing dosage of Antisense DNA. Higher than expected cellular accumulations of about 10(5) Antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the Antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5'-triphosphate into RNA in cells exposed to the Antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the Antisense DNA but not to the control DNA. CONCLUSION This evidence suggests tumor cell accumulation by an Antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.
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Influence of different chelators (HYNIC, MAG3 and DTPA) on tumor cell accumulation and mouse biodistribution of technetium-99m labeled to Antisense DNA.
European journal of nuclear medicine, 2000Co-Authors: Yu-min Zhang, Ning Liu, Zhihong Zhu, Mary Rusckowski, Donald J. HnatowichAbstract:We have shown recently that cell accumulation in culture of Antisense DNA is strongly influenced by the presence of a 99mTc-MAG3 group for radiolabeling. We have now compared the in vitro and mouse in vivo behavior of 99mTc when radiolabeled to one Antisense phosphorothioate DNA by three different methods. The 18-mer Antisense DNA against the RIα subunit of PKA was conjugated via a primary amine on the 5'-end with the NHS esters of HYNIC and MAG3 and by the cyclic anhydride of DTPA. Surface plasmon resonance measurements revealed that the association rate constant for hybridization was unchanged for all three chelators as compared with that of the native DNA. Size exclusion HPLC showed rapid and quantitative protein binding for all three chelators upon incubation of labeled DNAs in 37°C serum and cell culture medium. However, in each case, radiolabeled and intact oligonucleotide was still detectable after 24 h. Cellular uptake was tested in an RIα mRNA-positive cancer cell line. The order of cellular accumulation of 99mTc was DTPA>HYNIC(tricine)>MAG3, with the differences increasing with time between 4 and 24 h. The rate of 99mTc egress from cells was found to be MAG3>HYNIC>DTPA, which may explain the order of cellular accumulation. The biodistribution in normal mice was heavily influenced by the labeling method and followed a pattern similar to that seen previously by us for peptides labeled with the same chelators. In conclusion, although these studies concerned only one Antisense DNA in one cell line, the results suggest that the success of Antisense imaging may depend, in part, on the method of radiolabeling.