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Thermally cross-linked superparamagnetic iron oxide nanoparticle-A10 RNA aptamer-doxorubicin conjugate

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: October 8, 2008.

Chemical name:Thermally cross-linked superparamagnetic iron oxide nanoparticle-A10 RNA aptamer-doxorubicin conjugate
Abbreviated name:TCL-SPION-Apt(Dox)
Agent category:Nucleic acid (nanoparticle)
Target:Prostate-specific membrane antigen (PSMA)
Target category:Nucleic acid binding protein
Method of detection:Magnetic resonance imaging (MRI), optical imaging
Source of signal/contrast:Iron oxides, doxorubicin-RNA complex
  • Checkbox In vitro
  • Checkbox Rodents
No structure is currently available in PubChem.



Prostate-specific membrane antigen (PSMA) is a type II membrane glycoprotein with a molecular weight of ~100 kDa (1). PSMA is composed of several domains, including a potential phosphorylation site in the cytoplasmic tail (amino acids 1–18), a highly hydrophobic α-helix in the transmembrane region (amino acids 19–43), and catalytic sites in the extensive extracellular domain (amino acids 44–750). Two unique enzymatic functions are found in PSMA: N-acetylated, α-linked, dipeptidase (NAALADase) activity and folate hydrolase activity. As a prostate cancer cell (PCa) marker, PSMA expression is primarily prostate-specific (~100% in androgen-independent PCa cells), with very low levels (~1,000-fold less) in the brain, salivary glands, and small intestine. Thus, PSMA has become an excellent target for imaging and therapy.

Aptamers (from the Latin aptus, to fit, and the Greek meros, part or region) are single-stranded or double-stranded oligonucleotides (RNA or DNA, respectively) that are modified to bind a variety of targets with high binding affinity and specificity (2). Aptamers range in size from 20 to 80 base pairs (~6–26 kDa) with dissociation constants in the range of 10 pM to 10 nM (3). Unlike linear oligonucleotides, which contain genetic information or antisense oligonucleotides that interrupt the transcription of genetic information, aptamers are globular molecules with a shape similar to tRNA and bind to target proteins specifically (4). A10 RNA aptamer (Apt) is a nuclease-stabilized 2’-fluoropyrimidine RNA molecule of 57 base pairs with a molecular weight of 18.5 kDa (5). Its 2’-fluoro-modified ribose on all pyrimidines and 3’-inverted deoxythymidine cap provide significant resistance to nuclease in blood (6). Apt has a single 5’-CG-3’ sequence in its predicted double-stranded stem region, which is a preferred binding site for the anthracycline class of anticancer drugs such as doxorubicin (Dox) (7). Dox intercalates within the GC pair in Apt to form physical conjugate Apt(Dox) at molar ratio of 1.11:1 (dissociation constant = 600 nM) and emit fluorescence simultaneously. Because Dox possesses high efficacy against a range of neoplasms, including acute lymphoblastic and myeloblastic leukemias, malignant lymphomas, soft tissue and bone sarcomas, and breast, ovarian, prostate bladder, gastric, and bronchogenic carcinomas (8), this complex can be used as a PSMA-specific drug carrier to deliver Dox to PCa.

Superparamagnetic iron oxide nanoparticles (SPION) consist of magnetite (Fe3O4), which has a high magnetic susceptibility to induce a significant magnetization inside a magnetic field. The resulting microscopic field gradients diphase nearby protons and cause a reduction of T2 relaxation times (9). SPION readily forms aggregates as a result of nonspecific adsorption of plasma protein in blood, and SPION is cleared rapidly from the body by functions of the reticular endothelial system (RES) such as via macrophages (10). Coating the surface of SPION with a protein-repelling polymer layer such as poly(ethyleneglycol) (PEG) can prevent the absorption of plasma proteins or cells onto the surface of SPION (11). The coating can be conducted via surface trialkoxysilane as an anchor part (12). For example, poly(3-trimethoxysilyl)propyl methacrylate-r-PEG methyl ether methacrylate) (poly(TMSMA-r-PEGMA)) is a PEG-silane copolymer consisting of surface-reactive Si(OMe)3 groups and protein-repelling PEG chains (11). This coating becomes highly stabilized by simple heat treatment through condensation of hydrolyzed silane group (Si(OH)3) to form a thermally cross-linked (TCL) polymer.

TCL-SPION-A10 RNA aptamer-doxorubicin conjugate (TCL-SPION-Apt(Dox)) is a PSMA-specific agent used for magnetic resonance imaging the delivery of the anticancer drug Dox (13). TCL-SPION-Apt(Dox) contains three components: a PSMA-specific RNA aptamer A10 (Apt) covalently attached to the core surface as a targeting molecule and drug carrier, an anthracycline class of anticancer drug Dox as chemotherapeutic agent and optical sensor, and a TCL-SPION coated with a TCL carboxylic acid-PEG-silane copolymer as a magnetic resonance contrast agent and as a carrier for Apt(Dox). The PEGylated surface prevents protein and cell adsorption, while the carboxyl groups allow for conjugation of targeting moieties such as Apt. TCL-SPION-Apt(Dox) can be used to image the drug delivery to PCas.



The preparation of TCL-SPION-Apt(Dox) was conducted in several steps (11, 13). First, N-acryloxysuccinimide (NAS), 3-(trimethoxysilyl)propyl methacrylate (TMSMA), and PEG-methyl ether methacrylate (PEGMA; molecular weight ~475) were mixed at a molar ratio of 1:1:0.86 (11). After degassing with N2 for 15 min, 2,2’-azo-bis-isobutyronitrile was added for 24 h at 70ºC to initiate a free radical polymerization. The produced poly(TMSMA-r-PEGMA-r-NAS) (26.795 kDa) possessed a polydispersity of 1.646. Poly(TMSMA-r-pPEGMA-r-NAS) was further hydrolyzed to convert its trimethoxysilane and N-hydroxysuccinimide (NHS) ester into trihydroxysilane and carboxylic acid, respectively. Second, freshly prepared magnetite (Fe3O4) was reacted with the hydrolyzed form of poly(TMSMA-r-PEGMA-r-NSA) followed by thermal cross-linking for 2 h at 80ºC. The produced TCL-SPION, with carboxyl groups as the surface functional groups (carboxyl-TCL-SPION), was then treated with N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide and NHS (13). Commercially available N-terminated A10 aptamer (57 base pairs) was added 15 min later and reacted for 4 h to produce TCL-SPION-Apt, in which the molar ratio of PEG, Si(OCH3)3, and NHS-activated carboxylic acid was determined with 1H nuclear magnetic resonance imaging to be 1:0.85:0.71. Finally, unreacted Apt was removed and Dox was loaded to TCL-SPION-Apt to form the intercalated conjugate TCL-SPION-Apt(Dox). The molecular sizes were 60.8 ± 1.9 nm for TCL-SPION and 66.4 ± 1.5 nm for TCL-SPION-Apt.

In Vitro Studies: Testing in Cells and Tissues


Wang et al. studied the in vitro cellular uptake of TCL-SPION-Apt (13). PSMA-expressing PCa cells (LNCaP) and non-PSMA-expressing PCa cells (PC3) were incubated with 0.1 mg/ml TCL-SPION-Apt at 37ºC at time intervals of 1, 3, 6, 12, 18, and 24 h and followed by Prussian blue staining. The uptake of TCL-SPION-Apt in LNCaP cells was found as early as 3 h and gradually increased in a time-dependent manner, whereas no apparent cellular uptake was observed in PC3 cells. This result demonstrated differential targeting of PSMA-expressing PCa cells by TCL-SPION-Apt. The alteration of relaxation times (T1, T2) caused by cellular uptake of TCL-SPION-Apt was examined with nuclear magnetic resonance measurement (13). LNCap cells and PC3 cells were incubated for 6 h with TCL-SPION-Apt. A dramatic decrease was found only in the LNCaP cells, in which T1 decreased from 1,939 ± 116 to 263 ± 23 ms and T2 decreased from 104 ± 1.4 to 26.6 ± 0.4 ms. As a control, LNCap cells and PC3 cells were also incubated with non-targeted TCL-SPION for 6 h. Only a small alteration was observed in the LNCaP cells in that T1 decreased slightly from 1,939 ± 116 to 1,521 ± 201 ms and T2 decreased 104 ± 1.4 to 89.8 ± 1.1 ms. These data exhibited a high sensitivity in detecting PSMA-expressing PCa cells. The effect of TCL-SPION-Apt(Dox) on cellular proliferation was examined in vitro (13). After incubation with TCL-SPION-Apt(Dox) (0.1 mg/ml intercalated with 5 μM Dox) for 3 h, the viability was evaluated with methylthiazole tetrazolium (MTT) assay, which was 47.3 ± 1.4% for LNCaP cells and 69.3 ± 1.7% for PC3 cells. The free Dox was shown to be equipotent against both LNCaP and PC3 cell lines.

Animal Studies



No publication is currently available.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

CA 119349, EB 03647


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