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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

Superior tumor accumulation and retention of PEGylated aptamer than that of antibody. (a) PEGylated aptamer was developed by attaching a 20 kDa PEG-FITC to the 3'-end and a biotin or a DY647 dye to the 5'- end of the DNA strand. (b) Particle size of PEGylated aptamer and antibody as determined by dynamic light scattering. (c) Determination of the equilibrium dissociation constants (K'd) of PEGylated aptamer to HT29 cells using flow cytometry by incubating cells at varying concentrations (1-200 nmol/L). (d) Binding and internalization of PEGylated aptamer to HT29 cells which were incubated with 100 nM EpCAM aptamer at 37 °C for 30 min, followed by washing and confocal microscopy imaging. Scale bar = 10 μm. (e) Live animal imaging of antibodies and aptamers. NOD-SCID mice bearing HT29 tumor (150 mm3) received a single intravenous injection of 0.75 nmol of control PEGylated aptamer, PEGylated aptamer and antibody followed by live animal imaging at the indicated time points. (f) The fluorescence-time curve of PEGylated aptamer in tumors as in (e) was determined by Living Imaging Software v2.50 (Xenogen) with the units of photons/s/cm2/sr. Log-scale heat map (at the right) of photon flux applies to all panels. Data are means ± SEM, n=3. RFI: relative fluorescence intensity.
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Figure 4: Superior tumor accumulation and retention of PEGylated aptamer than that of antibody. (a) PEGylated aptamer was developed by attaching a 20 kDa PEG-FITC to the 3'-end and a biotin or a DY647 dye to the 5'- end of the DNA strand. (b) Particle size of PEGylated aptamer and antibody as determined by dynamic light scattering. (c) Determination of the equilibrium dissociation constants (K'd) of PEGylated aptamer to HT29 cells using flow cytometry by incubating cells at varying concentrations (1-200 nmol/L). (d) Binding and internalization of PEGylated aptamer to HT29 cells which were incubated with 100 nM EpCAM aptamer at 37 °C for 30 min, followed by washing and confocal microscopy imaging. Scale bar = 10 μm. (e) Live animal imaging of antibodies and aptamers. NOD-SCID mice bearing HT29 tumor (150 mm3) received a single intravenous injection of 0.75 nmol of control PEGylated aptamer, PEGylated aptamer and antibody followed by live animal imaging at the indicated time points. (f) The fluorescence-time curve of PEGylated aptamer in tumors as in (e) was determined by Living Imaging Software v2.50 (Xenogen) with the units of photons/s/cm2/sr. Log-scale heat map (at the right) of photon flux applies to all panels. Data are means ± SEM, n=3. RFI: relative fluorescence intensity.

Mentions: Next, the in vivo performance of aptamer and antibody was studied. In order to confer sufficient serum half-life, the EpCAM aptamer was further engineered by conjugating a terminal 20 kDa polyethylene glycol (PEG) (Fig. 4a). PEGylation of the aptamer increased the size of aptamer from 2.09 nm (non-PEGylated version) to approximately 12 nm, exceeding the 10 nm threshold of glomerular filtration (Fig. 4b) 46. The PEGylation of the aptamer did not introduce adverse effects to the binding affinity. As illustrated in Figure 4c, the K'd of the PEGylated aptamer corresponded well to the reported K'd of the parental EpCAM aptamer against target cell lines 38. In the case of HT29, the PEGylated EpCAM aptamer had a K'd of 51.79 nM to HT29, compared with that of 39.42 nM for the original EpCAM aptamer (Fig. 4c). In addition, the PEGylated aptamer maintained its binding ability to EpCAM-positive cancer cells (Fig. 4d). To study tumor delivery and retention, 0.75 nmole of DY647-labelled PEGylated aptamers, control DY647-labelled PEGylated aptamers or FITC-labeled EpCAM antibodies was injected i.v. into NOD/SCID mice-bearing HT29 xenograft tumors. The signal from the ROIs of HT29 tumors from mice receiving PEGylated aptamer gradually increased to a plateau at 5 h followed by a slow and prolonged decrease until 26 h or later. However, signals from either control PEGylated aptamer or antibody disappeared at around 6 h (Fig. 4e and f), suggesting that the prolonged tumor residence of PEGylated EpCAM aptamer was not a non-specific accumulation due to EPR effect but rather resulted from the combined EPR effect and specific ligand interaction. The duration of the antibody signal at the tumor, which is indicative of its tumor volume of distribution, was 4.3-fold shorter than the tumor residence time of the PEGylated aptamer. Taken together, the PEGylated aptamers achieved longer tumor retention than the antibody counterpart, paving the way for the development of aptamers as effective targeted drug delivery vehicles.


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)

Superior tumor accumulation and retention of PEGylated aptamer than that of antibody. (a) PEGylated aptamer was developed by attaching a 20 kDa PEG-FITC to the 3'-end and a biotin or a DY647 dye to the 5'- end of the DNA strand. (b) Particle size of PEGylated aptamer and antibody as determined by dynamic light scattering. (c) Determination of the equilibrium dissociation constants (K'd) of PEGylated aptamer to HT29 cells using flow cytometry by incubating cells at varying concentrations (1-200 nmol/L). (d) Binding and internalization of PEGylated aptamer to HT29 cells which were incubated with 100 nM EpCAM aptamer at 37 °C for 30 min, followed by washing and confocal microscopy imaging. Scale bar = 10 μm. (e) Live animal imaging of antibodies and aptamers. NOD-SCID mice bearing HT29 tumor (150 mm3) received a single intravenous injection of 0.75 nmol of control PEGylated aptamer, PEGylated aptamer and antibody followed by live animal imaging at the indicated time points. (f) The fluorescence-time curve of PEGylated aptamer in tumors as in (e) was determined by Living Imaging Software v2.50 (Xenogen) with the units of photons/s/cm2/sr. Log-scale heat map (at the right) of photon flux applies to all panels. Data are means ± SEM, n=3. RFI: relative fluorescence intensity.
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Related In: Results  -  Collection

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Figure 4: Superior tumor accumulation and retention of PEGylated aptamer than that of antibody. (a) PEGylated aptamer was developed by attaching a 20 kDa PEG-FITC to the 3'-end and a biotin or a DY647 dye to the 5'- end of the DNA strand. (b) Particle size of PEGylated aptamer and antibody as determined by dynamic light scattering. (c) Determination of the equilibrium dissociation constants (K'd) of PEGylated aptamer to HT29 cells using flow cytometry by incubating cells at varying concentrations (1-200 nmol/L). (d) Binding and internalization of PEGylated aptamer to HT29 cells which were incubated with 100 nM EpCAM aptamer at 37 °C for 30 min, followed by washing and confocal microscopy imaging. Scale bar = 10 μm. (e) Live animal imaging of antibodies and aptamers. NOD-SCID mice bearing HT29 tumor (150 mm3) received a single intravenous injection of 0.75 nmol of control PEGylated aptamer, PEGylated aptamer and antibody followed by live animal imaging at the indicated time points. (f) The fluorescence-time curve of PEGylated aptamer in tumors as in (e) was determined by Living Imaging Software v2.50 (Xenogen) with the units of photons/s/cm2/sr. Log-scale heat map (at the right) of photon flux applies to all panels. Data are means ± SEM, n=3. RFI: relative fluorescence intensity.
Mentions: Next, the in vivo performance of aptamer and antibody was studied. In order to confer sufficient serum half-life, the EpCAM aptamer was further engineered by conjugating a terminal 20 kDa polyethylene glycol (PEG) (Fig. 4a). PEGylation of the aptamer increased the size of aptamer from 2.09 nm (non-PEGylated version) to approximately 12 nm, exceeding the 10 nm threshold of glomerular filtration (Fig. 4b) 46. The PEGylation of the aptamer did not introduce adverse effects to the binding affinity. As illustrated in Figure 4c, the K'd of the PEGylated aptamer corresponded well to the reported K'd of the parental EpCAM aptamer against target cell lines 38. In the case of HT29, the PEGylated EpCAM aptamer had a K'd of 51.79 nM to HT29, compared with that of 39.42 nM for the original EpCAM aptamer (Fig. 4c). In addition, the PEGylated aptamer maintained its binding ability to EpCAM-positive cancer cells (Fig. 4d). To study tumor delivery and retention, 0.75 nmole of DY647-labelled PEGylated aptamers, control DY647-labelled PEGylated aptamers or FITC-labeled EpCAM antibodies was injected i.v. into NOD/SCID mice-bearing HT29 xenograft tumors. The signal from the ROIs of HT29 tumors from mice receiving PEGylated aptamer gradually increased to a plateau at 5 h followed by a slow and prolonged decrease until 26 h or later. However, signals from either control PEGylated aptamer or antibody disappeared at around 6 h (Fig. 4e and f), suggesting that the prolonged tumor residence of PEGylated EpCAM aptamer was not a non-specific accumulation due to EPR effect but rather resulted from the combined EPR effect and specific ligand interaction. The duration of the antibody signal at the tumor, which is indicative of its tumor volume of distribution, was 4.3-fold shorter than the tumor residence time of the PEGylated aptamer. Taken together, the PEGylated aptamers achieved longer tumor retention than the antibody counterpart, paving the way for the development of aptamers as effective targeted drug delivery vehicles.

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