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A Synthetic Aptamer-Drug Adduct for Targeted Liver Cancer Therapy.

Trinh TL, Zhu G, Xiao X, Puszyk W, Sefah K, Wu Q, Tan W, Liu C - PLoS ONE (2015)

Bottom Line: AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure.In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox.We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure. AS1411 has shown promising utility as a treatment for cancers in Phase I and Phase II clinical trials without causing major side-effects. AS1411 inhibits tumor cell growth by binding to nucleolin which is aberrantly expressed on the cell membrane of many tumors. In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox. We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

No MeSH data available.


Related in: MedlinePlus

Preparation and affinity of the AS1411-Dox adduct.(a) Schematic description of the preparation of AS1411-Dox adduct by incubating Dox (500 μM), AS1411 (20 μM), and formaldehyde (0.37%) at 10°C overnight, followed by purification by reverse phase HPLC. (b) AS1411-Dox adduct remained its binding affinity towards Huh7 established by the equilibrium dissociation constant (Kd). (c) UV-Vis spectrum displaying the absorbance of purified AS1411-Dox adduct, with the characteristic absorbance peak of Dox at 490 nm. (d) Flow cytometry results indicating specific recognition ability of AS1411 and AS1411-Dox adduct to Huh7 HCC cells, note the non-specific DNA library does not detect Huh7 cells (e) Random library and control DNA aptamers and the control DNA-Dox adduct in contrast were not able to specifically bind to Huh7 cells.
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pone.0136673.g003: Preparation and affinity of the AS1411-Dox adduct.(a) Schematic description of the preparation of AS1411-Dox adduct by incubating Dox (500 μM), AS1411 (20 μM), and formaldehyde (0.37%) at 10°C overnight, followed by purification by reverse phase HPLC. (b) AS1411-Dox adduct remained its binding affinity towards Huh7 established by the equilibrium dissociation constant (Kd). (c) UV-Vis spectrum displaying the absorbance of purified AS1411-Dox adduct, with the characteristic absorbance peak of Dox at 490 nm. (d) Flow cytometry results indicating specific recognition ability of AS1411 and AS1411-Dox adduct to Huh7 HCC cells, note the non-specific DNA library does not detect Huh7 cells (e) Random library and control DNA aptamers and the control DNA-Dox adduct in contrast were not able to specifically bind to Huh7 cells.

Mentions: Dox is a widely prescribed chemotherapeutic used in cancer therapy. Dox is capable of forming adducts with DNA [30,31]. The Dox adduct was prepared by incubating Dox with formaldehyde, which was used as a reducing and crosslinking agent during adduct formation. Specifically, AS1411-Dox adduct was prepared by incubating aptamers with Dox and formaldehyde in reaction buffer at 10°C overnight (Fig 3A). Residual aptamer, drug, and formaldehyde were removed from the reaction using reverse phase high-performance liquid chromatography (HPLC). Results indicate that the DDA displayed strong absorbance at 260 nm (from drug and DNA) as well as some absorbance from 490 nm (exclusively from drug) (S3 Fig). Purified adduct was lyophilized and desalted. UV-Vis spectrometry results further verified the characteristic drug absorbance around 490 nm in purified DDA (Fig 3C). Based on previous reports, molar extinction coefficient of 7,677 M-1cm-1 at 506 nm for Dox in DDA was used for drug quantification in adduct [20]. Accordingly, AS1411-Dox adduct was determined to have 5.4 ± 0.1 (s.d., n = 3) copies of Dox moieties per AS1411 strand. Similarly, a non-binding control DNA (S1 Table) was also used to prepare a control adduct with Dox (control-Dox).


A Synthetic Aptamer-Drug Adduct for Targeted Liver Cancer Therapy.

Trinh TL, Zhu G, Xiao X, Puszyk W, Sefah K, Wu Q, Tan W, Liu C - PLoS ONE (2015)

Preparation and affinity of the AS1411-Dox adduct.(a) Schematic description of the preparation of AS1411-Dox adduct by incubating Dox (500 μM), AS1411 (20 μM), and formaldehyde (0.37%) at 10°C overnight, followed by purification by reverse phase HPLC. (b) AS1411-Dox adduct remained its binding affinity towards Huh7 established by the equilibrium dissociation constant (Kd). (c) UV-Vis spectrum displaying the absorbance of purified AS1411-Dox adduct, with the characteristic absorbance peak of Dox at 490 nm. (d) Flow cytometry results indicating specific recognition ability of AS1411 and AS1411-Dox adduct to Huh7 HCC cells, note the non-specific DNA library does not detect Huh7 cells (e) Random library and control DNA aptamers and the control DNA-Dox adduct in contrast were not able to specifically bind to Huh7 cells.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4629891&req=5

pone.0136673.g003: Preparation and affinity of the AS1411-Dox adduct.(a) Schematic description of the preparation of AS1411-Dox adduct by incubating Dox (500 μM), AS1411 (20 μM), and formaldehyde (0.37%) at 10°C overnight, followed by purification by reverse phase HPLC. (b) AS1411-Dox adduct remained its binding affinity towards Huh7 established by the equilibrium dissociation constant (Kd). (c) UV-Vis spectrum displaying the absorbance of purified AS1411-Dox adduct, with the characteristic absorbance peak of Dox at 490 nm. (d) Flow cytometry results indicating specific recognition ability of AS1411 and AS1411-Dox adduct to Huh7 HCC cells, note the non-specific DNA library does not detect Huh7 cells (e) Random library and control DNA aptamers and the control DNA-Dox adduct in contrast were not able to specifically bind to Huh7 cells.
Mentions: Dox is a widely prescribed chemotherapeutic used in cancer therapy. Dox is capable of forming adducts with DNA [30,31]. The Dox adduct was prepared by incubating Dox with formaldehyde, which was used as a reducing and crosslinking agent during adduct formation. Specifically, AS1411-Dox adduct was prepared by incubating aptamers with Dox and formaldehyde in reaction buffer at 10°C overnight (Fig 3A). Residual aptamer, drug, and formaldehyde were removed from the reaction using reverse phase high-performance liquid chromatography (HPLC). Results indicate that the DDA displayed strong absorbance at 260 nm (from drug and DNA) as well as some absorbance from 490 nm (exclusively from drug) (S3 Fig). Purified adduct was lyophilized and desalted. UV-Vis spectrometry results further verified the characteristic drug absorbance around 490 nm in purified DDA (Fig 3C). Based on previous reports, molar extinction coefficient of 7,677 M-1cm-1 at 506 nm for Dox in DDA was used for drug quantification in adduct [20]. Accordingly, AS1411-Dox adduct was determined to have 5.4 ± 0.1 (s.d., n = 3) copies of Dox moieties per AS1411 strand. Similarly, a non-binding control DNA (S1 Table) was also used to prepare a control adduct with Dox (control-Dox).

Bottom Line: AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure.In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox.We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure. AS1411 has shown promising utility as a treatment for cancers in Phase I and Phase II clinical trials without causing major side-effects. AS1411 inhibits tumor cell growth by binding to nucleolin which is aberrantly expressed on the cell membrane of many tumors. In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox. We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

No MeSH data available.


Related in: MedlinePlus