Paclitaxel-loaded iron platinum stealth immunomicelles are potent MRI imaging agents that prevent prostate cancer growth in a PSMA-dependent manner

Abstract

Background and methods: Problems with the clinical management of prostate cancer include the lack of both specific detection and efficient therapeutic intervention. We report the encapsulation of superparamagnetic iron platinum nanoparticles (SIPPs) and paclitaxel in a mixture of polyethyleneglycolated, fluorescent, and biotin-functionalized phospholipids to create multifunctional SIPP-PTX micelles (SPMs) that were conjugated to an antibody against prostate-specific membrane antigen (PSMA) for the specific targeting, magnetic resonance imaging (MRI), and treatment of human prostate cancer xenografts in mice.

Results: SPMs were 45.4 ± 24.9 nm in diameter and composed of 160.7 ± 22.9 μg/mL iron, 247.0 ± 33.4 μg/mL platinum, and 702.6 ± 206.0 μg/mL paclitaxel. Drug release measurements showed that, at 37°C, half of the paclitaxel was released in 30.2 hours in serum and two times faster in saline. Binding assays suggested that PSMA-targeted SPMs specifically bound to C4-2 human prostate cancer cells in vitro and released paclitaxel into the cells. In vitro, paclitaxel was 2.2 and 1.6 times more cytotoxic than SPMs to C4-2 cells at 24 and 48 hours of incubation, respectively. After 72 hours of incubation, paclitaxel and SPMs were equally cytotoxic. SPMs had MRI transverse relaxivities of 389 ± 15.5 Hz/mM iron, and SIPP micelles with and without drug caused MRI contrast enhancement in vivo.

Conclusion: Only PSMA-targeted SPMs and paclitaxel significantly prevented growth of C4-2 prostate cancer xenografts in nude mice. Furthermore, mice injected with PSMA-targeted SPMs showed significantly more paclitaxel and platinum in tumors, compared with nontargeted SPM-injected and paclitaxel-injected mice.

Keywords: MRI; iron platinum; micelle; paclitaxel; prostate cancer.

Figure 1

Transmission electron microscopic image of SPMs. Notes: SPMs were applied to a carbon-coated grid and allowed to dry. Adding a drop of 2% uranyl acetate negatively stained the grid. The samples were imaged on an Hitachi 7500 transmission electron microscope with an acceleration voltage of 80 kV. The scale bar is 50 nm. Abbreviation: SPMs, superparamagnetic iron platinum nanoparticles and paclitaxel in a mixture of PEGylated and biotin-functionalized phospholipids.

Figure 2

Temperature dependence of drug release rates for SPMs in serum and saline. Notes: A 100 μL aliquot of freshly prepared particles was collected as the zero-hour time point. At subsequent time points, the particles were magnetically retained on a column and the amount of paclitaxel in the nonmagnetic flow-through and in the magnetic particles was measured using an enzyme-linked immunosorbent assay. The amount of drug release is shown as the percentage of drug released compared with the initial amount of drug loaded into the particles, as measured with enzyme-linked immunosorbent assay immediately after encapsulation. SPMs were incubated in saline at 20°C (diamonds), saline at 37°C (squares), serum at 37°C (circles), or serum at 4°C (triangles). Abbreviation: SPMs, superparamagnetic iron platinum nanoparticles and paclitaxel in a mixture of PEgylated and biotin-functionalized phospholipids.

Figure 3

Specific binding of J591-SPMs to C4-2 prostate cancer cells. Notes: Confocal images of PSMA-targeted, rhodamine red-containing SPMs containing fluorescent paclitaxel (green, top row) and control IgG-SPMs (bottom row) incubated with C4-2 human prostate cancer cells and stained with DAPI. The last column on the right shows the summed images, which display all three colors for the J591-SPMs, and only shows DAPI staining for the Igg-SPMs. Abbreviations: PSMA, prostate-specific membrane antigen; SPMs, superparamagnetic iron platinum nanoparticles and paclitaxel in a mixture of PEGylated and biotin-functionalized phospholipids.

Figure 4

Cytotoxicity measurements of paclitaxel, SPMs, and SMs in C4-2 prostate cancer cells. The graphs show C4-2 cell viability measured with a WST-1 assay after incubation with paclitaxel or SPMs for (A) 24hours, (B) 48hours, and (C) 72hours. Viability after incubation with SPMs (gray bars) and paclitaxel (black bars) is shown as the percentage of viable cells compared with control samples not incubated with particles or drug. (D) Image showing the lack of cytotoxicity when C4-2 human prostate cancer cells are incubated with SIPP micelles without drug (SMs) for 24hours (black bars) and 48hours (gray bars). Abbreviations: SIPP, superparamagnetic iron platinum nanoparticle; SMs, superparamagnetic iron platinum nanoparticle micelles without drug; SPMs, superparamagnetic iron platinum nanoparticles and paclitaxel in a mixture of PEgylated and biotin-functionalized phospholipids.

Figure 5

In vivo magnetic resonance imaging and contrast measurements of a mouse bearing a C4-2 xenograft. Longitudinal (A) and transverse (B) contrast percentage measured for mice bearing C4-2 xenografts and injected with either J591-SPMs (diamond, solid gray line), J591-SMs (square, dotted gray line), IgG-SPMs (square, dashed gray line), paclitaxel only (triangle, dashed black line), or nothing (square, solid black line). Representative T₂-weighted magnetic resonance images of a mouse injected with J591-SPMs are shown in (C). Notes: The arrows point to areas in the C4-2 xenograft that showed dark contrast enhancement at one hour following injection in the middle frame and an area that still showed contrast enhancement 19hours after injection in the far right frame. *P < 0.05. Abbreviations: SMs, superparamagnetic iron platinum nanoparticle micelles without drug; SPMs, superparamagnetic iron platinum nanoparticles and paclitaxel in a mixture of PEgylated and biotin-functionalized phospholipids.

Figure 6

Tumor volume growth curves for nude mice bearing human C4-2 prostate cancer xenografts treated with various treatments or controls. (A) Black squares, no treatment controls. (B) Red squares, targeted SIPPs without drug, showing no effect on tumor growth. (C) Blue squares, SIPPs containing paclitaxel conjugated to a control Igg antibody, showing no effect on tumor growth. (D) Green triangles, paclitaxel alone, without SIPPs, showing the efficacy of this chemotherapeutic drug by itself. (E) Purple squares, SIPPs containing paclitaxel targeted to PSMA showing that targeting specifically brings the drug to the tumors and prevents tumor growth. Note: ^#P < 0.05. Abbreviations: SIPPs, superparamagnetic iron platinum nanoparticles; PSMA, prostate-specific membrane antigen.

Figure 7

Tumor biodistribution of paclitaxel and platinum in mice bearing C4-2 xenografts and injected with treatments or controls. Mice were injected with paclitaxel alone (black bars), J591-SPMs (gray bars), or IgG-SPMs (open bars) and 20days after injection their tumors were collected and paclitaxel was measured as a percentage of the injected dose. Significantly more paclitaxel was measured in the tumors of mice injected with J591-SPMs, compared with nontargeted SPMs and paclitaxel alone (A). Likewise, the biodistribution of platinum from the SIPPs in the mice injected with J591-SPMs (gray bar), J591-SMs (open bar), or IgG-SPMs (black bar) was measured, using ICP, as percent of the injected dose. Again, significantly more platinum was measured in the tumors of mice injected with J591-SPMs, compared with nontargeted SPMs and paclitaxel alone (B). Notes: ^*,#P < 0.05 compared with paclitaxel alone or Igg-SPMs, respectively. ^**,##P < 0.07 compared with paclitaxel alone or Igg-SPMs, respectively. Abbreviations: SMs, superparamagnetic iron platinum nanoparticle micelles without drug; SPMs, superparamagnetic iron platinum nanoparticles and paclitaxel in a mixture of PEgylated and biotin-functionalized phospholipids.