Assessment of Aptamer as a Potential Drug Targeted Delivery for Retinal Angiogenesis Inhibition

Abstract

AT11-L0 is an aptamer derivative of AS1411 composed of G-rich sequences that can adopt a G-quadruplex (G4) structure and target nucleolin (NCL), a protein that acts as a co-receptor for several growth factors. Hence, this study aimed to characterize the AT11-L0 G4 structure and its interaction with several ligands for NCL targeting and to evaluate their capacity to inhibit angiogenesis using an in vitro model. The AT11-L0 aptamer was then used to functionalize drug-associated liposomes to increase the bioavailability of the aptamer-based drug in the formulation. Biophysical studies, such as nuclear magnetic resonance, circular dichroism, and fluorescence titrations, were performed to characterize the liposomes functionalized with the AT11-L0 aptamer. Finally, these liposome formulations with the encapsulated drugs were tested on the human umbilical vein endothelial cell (HUVEC) model to assess their antiangiogenic capacity. The results showed that the AT11-L0 aptamer-ligand complexes are highly stable, presenting melting temperatures from 45 °C to 60 °C, allowing for efficient targeting of NCL with a K_D in the order of nM. The aptamer-functionalized liposomes loaded with ligands C₈ and dexamethasone did not show cytotoxic effects in HUVEC cells compared with the free ligands and AT11-L0, as assessed by cell viability assays. AT11-L0 aptamer-functionalized liposomes encapsulating C₈ and dexamethasone did not present a significant reduction in the angiogenic process when compared with the free ligands. In addition, AT11-L0 did not show anti-angiogenic effects at the concentrations tested. However, C₈ shows potential as an angiogenesis inhibitor, which should be further developed and optimized in future experiments.

Keywords: G-quadruplex aptamers; angiogenesis; nanosystems; nucleolin; retinal diseases.

Figure 1

Evaluation of G4 formation using distinct spectroscopic techniques. (A) AS1411 derivatives. (B) CD spectra of AT11-L0 (10 μΜ) obtained with increasing concentrations of KCl in 20 mM of phosphate buffer in the range of 220–340 nm. (C) The effect of KCl salt on the structure formation of AT11-L0 G4 (50 μΜ) was monitored by ¹H NMR spectroscopy recorded in potassium buffer supplemented with 10% D₂O and increased concentrations of KCl (0 to 65 mM) (all regions of spectra in Figure S1). Imino protons of AT11-L0 are indicated with asterisks (∗). All measurements were performed at 20 °C.

Figure 2

Chemical structure of molecules tested: BRACO-19, C₈, dexamethasone, PDS, PhenDC3, and TMPyP4.

Figure 3

T_m radar plot of AT11-L0 in the presence of different molar equivalents of ligands obtained by FRET-melting experiments.

Figure 4

Fluorescence titrations of pre-folded (A) 5′-Cy5-AT11-L0 sequence, complexed with (B) C₈, (C) dexamethasone, and (D) PhenDC3 with increasing concentrations of NCL.

Figure 5

Reactional scheme of the conjugation of NH₂-AT11-L0 to DSPE-PEG-NHS used to obtain aptamer-functionalized liposomes.

Figure 6

HUVEC cell viability assay in the presence of (A) C₈-loaded liposomes and (B) dexamethasone-loaded liposomes.

Figure 7

HUVEC tube formation assay. (A) Tube formation was significantly inhibited by (1) dexamethasone (10 µM), (2) AT11-L0 dexamethasone-loaded liposomes (10 µM), (3) C₈ (0.05 µM), (4) AT11-L0 C₈-loaded liposomes (0.05 µM), and (5) suramin (10 µM) (positive control) in comparison to the control group. * p < 0.05 and ** p < 0.01. (B) Representative images of tube formation assay.