Polymeric micelles: Theranostic co-delivery system for poorly water-soluble drugs and contrast agents

Abstract

Interest in theranostic agents has continued to grow because of their promise for simultaneous cancer detection and therapy. A platform-based nanosized combination agent suitable for the enhanced diagnosis and treatment of cancer was prepared using polymeric polyethylene glycol-phosphatidylethanolamine-based micelles loaded with both, poorly soluble chemotherapeutic agent paclitaxel and hydrophobic superparamagnetic iron oxide nanoparticles (SPION), a Magnetic Resonance Imaging contrast agent. The co-loaded paclitaxel and SPION did not affect each other's functional properties in vitro. In vivo, the resulting paclitaxel-SPION-co-loaded PEG-PE micelles retained their Magnetic Resonance contrast properties and apoptotic activity in breast and melanoma tumor mouse models. Such theranostic systems are likely to play a significant role in the combined diagnosis and therapy that leads to a more personalized and effective form of treatment.

Keywords: Cancer therapy; Diagnostics; Drug delivery; Micelles; Nanoparticles; Theranostics.

Figure 1. Characterization of micellar theranostic agent

Scheme (A) illustrates a PEG-PE micellar theranostic agent co-incorporated with paclitaxel and MRI contrast agents along with the chemical structure of PEG-PE showing the hydrophilic PEG which depicts the micelle corona and core-hydrophobic PE group, responsible for the solubilization of PTX and entrapment of SPIONs. (B) indicates micelle size and size distribution of empty PEG-PE/Plain micelles, PTX-loaded PEG-PE micelles, SPION-loaded PEG-PE micelles and co-loaded PTX/SPION PEG-PE theranostic micelles with their respective electron micrographs (C) at 75,000x magnification, scale bar repesents 100nm. (n=3)

Figure 2. T2-weighted MR image and relaxation times

MR imaging of contrast agent in the absence (A) and presence (B) of 1.5% w/w PTX during formulation optimization and their T2 relaxation times (D). T2-weighted MR images of 3% SPION-containing micelles and controls (C) along with their respective T2 relaxation times (E). MR images were obtained using a 7T Bruker magnet. T2 relaxation times were calculated using Paravision 5.0 software. (mean ± S.D., n=3).

Figure 3. In vitro cytotoxicity of micellar formulations

Percent viability of 4T1 and B16F10 cells after treatment with plain, SPION, PTX, theranostic micelles or free drug (PTX) at +24 hours (A, B) and + 48 hours (C, D) incubation. Data represent mean ± S.D. (n=3). (*) p < 0.05 between paclitaxel micelles and free paclitaxel; (#) p < 0.05 between theranostic micelles and free paclitaxel (Student’s t-test).

Figure 4. Whole body in vivo images

T-2 weighted MR image and color-coded composite maps acquired with breast (A) and melanoma (B) tumor-bearing mice, pre- and post-injection of various formulations containing SPIONs as MR contrast agent. Images were obtained using a 7 Tesla Bruker scanner. Similar images (C) were acquired 72 hours post-injection from both tumor models. Non-color coded images show regions of the tumor bounded by a dotted line. The arrow points the accumulation of contrast agent at the tumor site. Theranostic micelles retained their MR contrast properties equivalent to that of SPION-only micelles.

Figure 5. Prussian blue staining of tumor cryosections

Breast (4T1) and melanoma (B16F10) tumor sections stained for SPIONs localized within the tumor interstitium at +48 (A) and +72 hours (B). Blue stain (as indicated by the arrow) from the sections indicates the presence of SPION micelles against a light-red to pink background counterstained with nuclear red that stains cytoplasm and nucleus. The melanoma cells are characterized by their natural dark brown pigments. (Scale bar = 1cm)

Figure 6. TUNEL assay for apoptotic activity of paclitaxel

In vivo tumor cell apoptosis was induced by intra-tumoral administration of PTX-containing formulations in breast (4T1) and melanoma (B16F10) tumor-bearing mice. At +48 hours (A, B) and +72 hours (C, D) tumor apoptosis was observed after treatment with theranostic and PTX-only-containing formulations as determined by TUNEL assay. Panel (E) shows bar graph of average fluorescence intensity in arbitrary units across all formulations, (n=5) and data is represented as mean±S.D. Cells undergoing apoptosis were observed in the green/FITC channel (see arrow). Sections observed with a blue filter indicate DAPI stain for nuclei. In both cases, tumors treated with plain and SPION micelles lacked DNA fragmenting cells. (Scale bar = 1cm).