"OA02" peptide facilitates the precise targeting of paclitaxel-loaded micellar nanoparticles to ovarian cancer in vivo

Abstract

Micellar nanoparticles based on linear polyethylene glycol (PEG) block dendritic cholic acids (CA) copolymers (telodendrimers), for the targeted delivery of chemotherapeutic drugs in the treatment of cancers, are reported. The micellar nanoparticles have been decorated with a high-affinity "OA02" peptide against α-3 integrin receptor to improve the tumor-targeting specificity which is overexpressed on the surface of ovarian cancer cells. "Click chemistry" was used to conjugate alkyne-containing OA02 peptide to the azide group at the distal terminus of the PEG chain in a representative PEG(5k)-CA(8) telodendrimer (micelle-forming unit). The conjugation of OA02 peptide had negligible influence on the physicochemical properties of PEG(5k)-CA(8) nanoparticles and as hypothesized, OA02 peptide dramatically enhanced the uptake efficiency of PEG(5k)-CA(8) nanoparticles (NP) in SKOV-3 and ES-2 ovarian cancer cells via receptor-mediated endocytosis, but not in α-3 integrin-negative K562 leukemia cells. When loaded with paclitaxel, OA02-NPs had significantly higher in vitro cytotoxicity against both SKOV-3 and ES-2 ovarian cancer cells as compared with nontargeted nanoparticles. Furthermore, the in vivo biodistribution study showed OA02 peptide greatly facilitated tumor localization and the intracellular uptake of PEG(5k)-CA(8) nanoparticles into ovarian cancer cells as validated in SKOV3-luc tumor-bearing mice. Finally, paclitaxel (PTX)-loaded OA02-NPs exhibited superior antitumor efficacy and lower systemic toxicity profile in nude mice bearing SKOV-3 tumor xenografts, when compared with equivalent doses of nontargeted PTX-NPs as well as clinical paclitaxel formulation (Taxol). Therefore, OA02-targeted telodendrimers loaded with paclitaxel have great potential as a new therapeutic approach for patients with ovarian cancer.

Figure 1

Chemical structure of OA02 peptide functionalized PEG^5K-CA₈ telodendrimer (A) and the preparation of stealth targeted NPs by the self-assembly of blank/OA02-functionalized telodendrimers (B). Alkyne-containing OA02 peptide was conjugated to the azide group on the distal terminus of PEG chain in PEG^5K-CA₈ telodendrimer via “click chemistry”. The Dde-protected amino group on the proximal lysine in OA02-PEG^5K-CA₈ telodendrimer can be removed and used for the conjugation of fluorescence dyes such as FITC or Cy5.5 for cell and animal imaging. The OA02 peptides presented on the surface of targeted NPs are able to specifically recognize and bind the alpha-3 integrin receptors, which are overexpressed on the cell membrane of ovarian cancer cells.

Figure 2

The morphology (A) and particle size distribution (B) of PTX-OA02-NPs (5 mg PTX in 20 mg/ml telodendrimer) measured by cryo-TEM and DLS, respectively. In Figure 2A, white arrows point to NPs, while Mosaic Virus (TMV) was used as calibration standard (18 nm in width). The ice crystals usually have clear edge contrast. C, In vitro PTX release kinetics from PTX-NPs or PTX-OA02-NPs in PBS at 37 °C. The concentration of PTX remained in the dialysis cartridge at various time points was measured by HPLC. Error bars were obtained from triplicate samples.

Figure 3

The uptake of FITC-labeled OA02-NPs in SKOV-3 ovarian cancer cells and K562 leukemia cells (alpha-3 integrin negative). A, Confocal microscopic images of SKOV-3 cell incubated with FITC-labeled NPs and OA02-NPs (green) for 2 h. To demonstrate the alpha-3 integrin dependent uptake, excess amount of alpha-3 integrin antibody, or free OA02 peptide was added prior to the incubation of OA02-NPs with SKOV-3 cells. The flow cytometric analysis of OA02-NPs uptake in K562 cells (B) and SKOV-3 cells (C). The mean fluorescence intensity (MFI) of cells incubated with NPs and OA02-NPs are shown as insets. * P < 0.05. D, the addition of free OA02 peptides at concentrations from 2 to 200 μM inhibited cellular uptake of OA02-NPs in SKOV-3 cells in a dose-dependent manner.

Figure 4

(A) Intracellular tracking of dual fluorescent-labeled OA02-NPs upon their uptake in live SKOV-3 cells. DiD dye (red) was encapsulated as drug surrogates into FITC (green) chemically labeled OA02-NPs. SKOV-3 cells were incubated with DiD/FITC dual labeled OA02-NPs for 2 h, and the cells were stained with lysosome tracker (red) before the live-cell imaging by confocal microscopy. (B) The differential cytotoxicities of 0.5 μg/ml PTX-loaded NPs (PTX-NPs), PTX-loaded OA02-NPs (PTX-OA02-NPs), and the equivalent dose of blank NPs, OA02-NPs against SKOV-3 ovarian cancer cells measured by MTT assay. NPs were incubated with cells for 2 h, and the cells were subsequently washed and incubated in fresh media for a total of 72 h before assessing cell viability in each group. * P < 0.05.

Figure 5

In vivo and ex vivo NIRF optical imaging of Cy5.5-labeled NPs or OA02-NPs biodistribution after i.v. injection in subcutaneous SKOV3-luc tumor bearing mice. (A) In vivo optical images of real-time tumor targeting characteristics of NPs or OA02-NPs. Tumor bearing mice were injected via tail vain with 4 nM Cy5.5-labeled NPs or OA02-NPs, and were scanned with Kodak multimodal imaging system IS2000MM at different time point. (B) Representative ex vivo optical images of tumors and organs of SKOV3-luc bearing mice sacrificed at 24 h. (C) quantitative fluorescence intensities of tumors and organs from ex vivo images (n = 3). (D) Histological analysis of NPs or OA02-NPs distribution (Cy5.5, red) in tumor cryosections. The nuclei were stained by DAPI (blue), alpha-3 integrin expression on SKOV-3 tumor cells and vascular endothelial cells were visualized by anti-alpha-3(green) and anti-CD31 (orange) staining, respectively.

Figure 6

In vivo tumor growth inhibition (A), Kaplan-Meier survival curves (B), and body weight changes (C) of SKOV-3 tumor bearing mice after the intravenous treatment of various PTX formulations. Tumor bearing mice were administered intravenously with PBS (control), Taxol^® (10 mg/kg), PTX-NPs (10 mg/kg and 30 mg/kg) and OA02-PTX-NPs (10 mg/kg and 30 mg/kg), respectively, every three day on days 0, 3, 6, 9, 12 and 15 for a total of 6 doses. Data represent mean ± SEM (n = 8–10).