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

Excerpt

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.