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Superior Performance of Aptamer in Tumor Penetration over Antibody: Implication of Aptamer-Based Theranostics in Solid Tumors.

Xiang D, Zheng C, Zhou SF, Qiao S, Tran PH, Pu C, Li Y, Kong L, Kouzani AZ, Lin J, Liu K, Li L, Shigdar S, Duan W - Theranostics (2015)

Bottom Line: Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy.To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications.We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation.

View Article: PubMed Central - PubMed

Affiliation: 1. School of Medicine, Deakin University, Pigdons Road, Waurn Ponds, Victoria 3216, Australia.

ABSTRACT
Insufficient penetration of therapeutic agents into tumor tissues results in inadequate drug distribution and lower intracellular concentration of drugs, leading to the increase of drug resistance and resultant failure of cancer treatment. Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy. Monoclonal antibodies have been commonly used in clinic for cancer treatment, but their limitation of penetrating into tumor tissues still remains because of their large size. Aptamers, as "chemical antibodies", are 15-20 times smaller than antibodies. To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications. Penetration and retention were studied in in vitro three-dimensional tumorspheres, in vivo live animal imaging and mouse colorectal cancer xenograft model. We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation. As observed from in vivo live animal imaging, EpCAM aptamers displayed a maximum tumor uptake at around 10 min followed by a rapid clearance after 80 min, while the signal of peak uptake and disappearance of antibody appeared at 3 h and 6 h after intravenous injection, respectively. The signal of PEGylated EpCAM aptamers in xenograft tumors was sustained for 26 h, which was 4.3-fold longer than that of the EpCAM antibody. Consistently, there were 1.67-fold and 6.6-fold higher accumulation of PEGylated aptamer in xenograft tumors than that of antibody, at 3 h and 24 h after intravenous administration, respectively. In addition, the aptamer achieved at least a 4-time better tumor penetration in xenograft tumors than that of the antibody at a 200 μm distances from the blood vessels 3 h after intravenous injection. Taken together, these data indicate that aptmers are superior to antibodies in cancer theranostics due to their better tumor penetration, more homogeneous distribution and longer retention in tumor sites. Thus, aptamers are promising agents for targeted tumor therapeutics and molecular imaging.

No MeSH data available.


Related in: MedlinePlus

Biodistribution of PEGylated aptamer and antibody in mice bearing xenograft colorectal tumors. NOD/SCID mice bearing HT29 xenograft tumors (~150 mm3) received a single i.v. injection of 1 nmol/mouse of PEGylated aptamer or antibody. The concentration of aptamer or antibody, expressed as % of injected dose (ID) per g of tissue, in tissues iundicated was determined at 3 h and 24 h after the agent administration using ELISA. Data are means ± SEM (n = 4). *, P < 0.05; **, P < 0.01 compared to antibody.
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Figure 5: Biodistribution of PEGylated aptamer and antibody in mice bearing xenograft colorectal tumors. NOD/SCID mice bearing HT29 xenograft tumors (~150 mm3) received a single i.v. injection of 1 nmol/mouse of PEGylated aptamer or antibody. The concentration of aptamer or antibody, expressed as % of injected dose (ID) per g of tissue, in tissues iundicated was determined at 3 h and 24 h after the agent administration using ELISA. Data are means ± SEM (n = 4). *, P < 0.05; **, P < 0.01 compared to antibody.

Mentions: In the above sequences, 2'-F represents 2'-fluoropyrimidine, 2'-O-Me indicates 2'-O-methyl modification. Lowercase letters indicate DNAs which are modified with 5'-methyl-deoxycytidine (5-Methyl-dC). The negative control aptamer is an aptamer of the same sequence as the EpCAM targeting aptamer but with a different side-chain modification that could affect the 3-dimensional structure of aptamer 29. As a result, this control aptamer is not able to bind to EpCAM and does not target the cancer cells overexpressing EpCAM. For engineering an effective DOX loading segment for future therapeutic applications (described in a separate manuscript) 5'-methyl-deoxycytidine (dC) was deployed in the newly engineered DNA stem as 5-Methyl dC when substituted for dC will increase the Tm by as much as 0.5°C per insertion. In addition, the presence of 5'-Methyl dC in CpG motifs can prevent or limit unwanted immune responses (Fig. 5-2a) 30-32. Prior to conducting all the experiments using aptamers, the aptamers are prepared in PBS containing 5 mM MgCl2, and then folded by denaturation at 85˚C for 5 min, followed by 10 min incubation at room temperature and refolding at 37˚C for at least 15 min.


Superior Performance of Aptamer in Tumor Penetration over Antibody: Implication of Aptamer-Based Theranostics in Solid Tumors.

Xiang D, Zheng C, Zhou SF, Qiao S, Tran PH, Pu C, Li Y, Kong L, Kouzani AZ, Lin J, Liu K, Li L, Shigdar S, Duan W - Theranostics (2015)

Biodistribution of PEGylated aptamer and antibody in mice bearing xenograft colorectal tumors. NOD/SCID mice bearing HT29 xenograft tumors (~150 mm3) received a single i.v. injection of 1 nmol/mouse of PEGylated aptamer or antibody. The concentration of aptamer or antibody, expressed as % of injected dose (ID) per g of tissue, in tissues iundicated was determined at 3 h and 24 h after the agent administration using ELISA. Data are means ± SEM (n = 4). *, P < 0.05; **, P < 0.01 compared to antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4508498&req=5

Figure 5: Biodistribution of PEGylated aptamer and antibody in mice bearing xenograft colorectal tumors. NOD/SCID mice bearing HT29 xenograft tumors (~150 mm3) received a single i.v. injection of 1 nmol/mouse of PEGylated aptamer or antibody. The concentration of aptamer or antibody, expressed as % of injected dose (ID) per g of tissue, in tissues iundicated was determined at 3 h and 24 h after the agent administration using ELISA. Data are means ± SEM (n = 4). *, P < 0.05; **, P < 0.01 compared to antibody.
Mentions: In the above sequences, 2'-F represents 2'-fluoropyrimidine, 2'-O-Me indicates 2'-O-methyl modification. Lowercase letters indicate DNAs which are modified with 5'-methyl-deoxycytidine (5-Methyl-dC). The negative control aptamer is an aptamer of the same sequence as the EpCAM targeting aptamer but with a different side-chain modification that could affect the 3-dimensional structure of aptamer 29. As a result, this control aptamer is not able to bind to EpCAM and does not target the cancer cells overexpressing EpCAM. For engineering an effective DOX loading segment for future therapeutic applications (described in a separate manuscript) 5'-methyl-deoxycytidine (dC) was deployed in the newly engineered DNA stem as 5-Methyl dC when substituted for dC will increase the Tm by as much as 0.5°C per insertion. In addition, the presence of 5'-Methyl dC in CpG motifs can prevent or limit unwanted immune responses (Fig. 5-2a) 30-32. Prior to conducting all the experiments using aptamers, the aptamers are prepared in PBS containing 5 mM MgCl2, and then folded by denaturation at 85˚C for 5 min, followed by 10 min incubation at room temperature and refolding at 37˚C for at least 15 min.

Bottom Line: Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy.To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications.We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation.

View Article: PubMed Central - PubMed

Affiliation: 1. School of Medicine, Deakin University, Pigdons Road, Waurn Ponds, Victoria 3216, Australia.

ABSTRACT
Insufficient penetration of therapeutic agents into tumor tissues results in inadequate drug distribution and lower intracellular concentration of drugs, leading to the increase of drug resistance and resultant failure of cancer treatment. Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy. Monoclonal antibodies have been commonly used in clinic for cancer treatment, but their limitation of penetrating into tumor tissues still remains because of their large size. Aptamers, as "chemical antibodies", are 15-20 times smaller than antibodies. To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications. Penetration and retention were studied in in vitro three-dimensional tumorspheres, in vivo live animal imaging and mouse colorectal cancer xenograft model. We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation. As observed from in vivo live animal imaging, EpCAM aptamers displayed a maximum tumor uptake at around 10 min followed by a rapid clearance after 80 min, while the signal of peak uptake and disappearance of antibody appeared at 3 h and 6 h after intravenous injection, respectively. The signal of PEGylated EpCAM aptamers in xenograft tumors was sustained for 26 h, which was 4.3-fold longer than that of the EpCAM antibody. Consistently, there were 1.67-fold and 6.6-fold higher accumulation of PEGylated aptamer in xenograft tumors than that of antibody, at 3 h and 24 h after intravenous administration, respectively. In addition, the aptamer achieved at least a 4-time better tumor penetration in xenograft tumors than that of the antibody at a 200 μm distances from the blood vessels 3 h after intravenous injection. Taken together, these data indicate that aptmers are superior to antibodies in cancer theranostics due to their better tumor penetration, more homogeneous distribution and longer retention in tumor sites. Thus, aptamers are promising agents for targeted tumor therapeutics and molecular imaging.

No MeSH data available.


Related in: MedlinePlus