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BACKGROUND: At the time of diagnosis about 75% of patients are at a noninvasive stage of bladder cancer. If treatment is ineffective many will progress from noninvasive stage to an invasive stage. Thus, we are addressing methods of improving local therapeutic modalities, which are crucial for the treatment of the disease at a non-muscle invasive stage. Improved delivery of active agents across the bladder permeability barrier (BPB) is needed as the urothelium restricts transport of many drugs and poses a challenge for local therapy. In addition, there are issues with short term drug exposure and low bladder drug uptake. To overcome these obstacles, we designed a poly(lactide-co-glycolide) (PLGA) surface modified nanoparticle (NP) with a novel cell penetrating cationic polymer, poly (guanidinium oxanorbornene) (PGON) that can efficiently attach to the bladder urothelium and be internalized into normal urothelial and bladder cancer cells. As a therapeutic agent, we chose to deliver belinostat (Bel), a histone deacetylase inhibitor with anti-cancer properties. Belinostat has been shown to induce growth inhibition and cell cycle arrest in vitro, and has efficacy against non-muscle invasive urothelial cancers in transgenic and xenograft models.
METHODS: Fluorophores (Nile red, NR; courmin-6, C6) and Bel were encapsulated in biodegradable PLGA, and surface modified with PGON. Bladder cancer cell lines (UM-UC-3 and T24) were treated with Bel or NP-Bel-PGON and assessed for acetyl-histone expression. In vivo mouse bladder models were treated with NP-NR-PGON and evaluated using fluorescence microscopy for urothelial penetration, whereas ex vivo human ureter models were treated with NP-C6-PGON and evaluated for uptake using tissue extraction and fluorescence-activated cell sorting (FACS). Fluorescently labeled UM-UC-3 cells were injected subcutaneously into the flank of female Foxn1 nu/nu mice. The tumors were treated biweekly with NP-Bel-PGON or appropriate controls.
RESULTS: Mouse bladders intravesically instilled with NP-NR-PGON demonstrated a significant increase in penetration of these NPs into the urothelium compared to unmodified NP-NR. Ex-vivo human ureters showed a 10-fold greater uptake of NP-C6-PGON than of control NPs. In vitro bladder cancer cells, at 12 hr post wash, showed an 8-fold increase in histone hyperacetylation upon treatment with NP-Bel-PGON compared to treatment with Bel alone. In the xenograft bladder cancer models, there was a 70% reduction in tumor volume, which was accompanied by a 2.5-fold higher intratumoral acetyl-H4, in the NP-Bel-PGON treated tumors compared to the tumors treated with unloaded NP-PGON.
CONCLUSIONS: PGON nanoparticles encapsulated with belinostat have been shown to increase urothelium penetration, prolong histone hyperacetylation, and impede bladder tumor growth. The PGON nanoparticle system has the potential to be an effective intravestical delivery system for the treatment bladder cancers.