Characterization of a targeted nanoparticle functionalized with a urea-based inhibitor of prostate-specific membrane antigen (PSMA)

Abstract

Polymeric nanoparticles represent a form of targeted therapy due to their ability to passively accumulate within the tumor interstitium via the enhanced permeability and retention (EPR) effect. We used a combined approach to decorate the surface of a nanoparticle with a urea-based small-molecule peptidomimetic inhibitor of prostate specific membrane antigen (PSMA). PSMA is expressed by normal and malignant prostate epithelial cells and by the neovasculature of almost all solid tumors. This strategy takes advantage of both the avidity of the functionalized nanoparticle for binding to PSMA and the ability of the nanoparticle to be retained for longer periods of time in the tumor due to enhanced leakage via EPR into the tumor interstitium. As an initial step to introducing the targeting moiety, the amino terminus of the small-molecule PSMA inhibitor was conjugated to PEG (M(n) 3400) bearing an activated carboxyl group to obtain a PEGylated inhibitor. Studies undertaken using a radiolabeled PSMA-substrate based assay established that the PEGylated inhibitor had an IC(50) value similar to the uncomplexed inhibitor. Subsequently, nanoparticles loaded with docetaxel were formulated using a mixture of poly(lactide-beta-ethylene glycol-beta-lactide) and PSMA-inhibitor bound alpha-amino-omega-hydroxy terminated poly (ethylene glycol-beta-epsilon-caprolactone). In vitro studies using these nanoparticles demonstrated selective cytotoxicity against PSMA-producing cells. Binding of fluorescently labeled PSMA-targeted particles to PSMA-producing cells has also been directly observed using fluorescence microscopy and observed in secondary fashion using a PSMA substrate based enzyme inhibition assay. Ongoing in vivo studies address the localization, activity and toxicity of these targeted nanoparticles against PSMA-producing human prostate tumor xenografts.

Figure 1

A general schematic demonstrating the binding of the nanoparticle. PSMA in dimeric form is observed on the cell surface. The polymeric nanoparticle has multiple PEG arms, some of which have the PSMA inhibitor attached to it. Total surface coverage by the inhibitor was computed to be 2.23 × 10¹⁷ molecules/m² of surface area of the nanoparticles (~30,000 inhibitor molecules/nanoparticle).

Figure 2

(A) Inhibition of activity of PSMA by FPPi and PEG. FPPi inhibits the activity of PSMA with an IC₅₀ in the range of 10 to 100 ng/mL. In contrast, PEG inhibits the activity of PSMA by no more than 10% at its highest concentration of FPPi tested suggesting specific inhibition (data not shown). (B) PSMA inhibition by polyPSMAi2 when in free and nanoparticle form. There is a slight increase in the IC₅₀ when bound to the nanoparticle due to steric hindrance.

Figure 3

Size distribution of nanoPSMAi2 as obtained by Light Scattering. The number average of size was estimated to be 222 nm. As observed, the nanoparticles are monodisperse with a polydispersity index of 0.131.

Figure 4

(A) Confocal microscopy using cells stained with Cell Tracker Green shows minimal endocytosis of nontargeted nanoparticles (red). (B) In contrast, PSMA-targeted nanoparticles have been endocytosed (red). (C) Fluoresence microscopy using DAPI nuclear stain also shows minimal binding of red nontargeted nanoparticles. (D) However, in case of targeted nanoparticles, multiple particles (red) are seen bound to the cell surface. Images are representative of the entire field.

Figure 5

In vitro toxicity of nontargeted and targeted nanoparticles after a 15 min incubation at a docetaxel concentration equivalent of 100 nM. After 48 hour incubation, the treated arms show regression in growth. The effect is magnified with the higher incubation time of 96 hours where the controls have expanded considerably, the nontargeted nanoparticles demonstrate regression and the PSMA-targeted nanoparticles not only induce regression, but also reduce the overall cell number. Docetaxel was used as the control.

Figure 6

Scheme 1. Synthesis of the PEGylated PSMA inhibitor, FPPi. Scheme 2. Synthesis of the PSMA inhibitor, PSMAi2 followed by synthesis of the polymeric version of the PSMA inhibitor, polyPSMAi2.