Nanogels conjugated with cell-penetrating peptide as drug delivery vehicle for treating urinary tract infections

Abstract

Among hospital-acquired infections, Pseudomonas aeruginosa-associated urinary tract infections (UTIs) are mainly caused by indwelling urethral catheters (catheter-associated UTIs or CAUTIs) and are difficult to treat, resulting in high rates of morbidity among hospitalized patients. While antibiotics can successfully treat bacteria in the bladder lumen, they are inefficient at crossing stratified urothelium plasma membranes to kill persistent intracellular bacterial communities (IBCs). Herein, we introduce an approach to target UTI IBCs by locally delivering the antibiotic gentamicin via polymeric nanogels conjugated with a cell-penetrating peptide Cys-Gly-Lys-Arg-Lys. This novel approach delivered ~36 % more intracellular gentamicin compared to drug delivered in solution in vitro. In an acute UTI murine model, the nanogel cell-penetrating peptide drug delivery system facilitated the transport of gentamicin into the urothelium and resulted in >90 % clearance of a uropathogenic P. aeruginosa clinical strain in vivo.

Keywords: Cell penetrating peptide; Drug delivery; Nanogel; Urinary tract infection.

Fig. 1.. Overview for the composition and synthesis of HTA-CPP.

Composition of HTA nanogel synthesized after free-radical solution polymerization followed by conjugating the cell-penetrating peptide (CPP) Cys-Gly-Lys-Arg-Lys (CGKRK) onto the nanogel’s surface nanogel (HTA-CPP) for loading and delivering the gentamicin antibiotic in the HTA-CPP.

Fig. 2.. Gentamicin release kinetics from the HTA and HTA-CPP.

Gentamicin released from the HTA-CPP (red, squares) and HTA (blue, circles) at 37 °C in PBS via dialysis method. Gentamicin release was measured by spectroscopy of fluorescently derivatized GEN as described in the Materials and Methods every 2 h to 12 h, 24 h and 96 h. Error bars are the standard deviation for N = 3.

Fig. 3.. Effects of HTA-CPP concentrations on urothelial cell viability.

The metabolic activity of human urothelial cells (HTB-9) was determined by MTT assay after 24 h exposure to the HTA conjugated to the CPP (HTA-CPP). Cells were exposed to 1 % (v/v) Triton X-100 as a positive control. Cells that were not exposed to HTA-CPP (0 μg) was the negative control. Percent metabolic activity was determined as described in Materials and Methods. Error bars are the standard devitation for N = 3. One-way ANOVA woth post-hoc Dunnett’s multiple comparison test; ****, p < 0.0001.

Fig. 4.. Gentamicin absorption by human urothelial cells delivered as free drug vs. encapsulated in HTA-CPP.

The amount of intracellular radioactive ³H-gentamicin in human urothelial cells (HTB-9) by counts-per-minute from lysed cells after ³H-gentamicin was delivered either as a free drug in solution (GEN, green bar) or by HTA-CPP (HTA-CPP + GEN, red bar) after 4 h. Student t-test; **, p < 0.01.

Fig. 5.. Effects of HTA-CPP on murine bladders.

Representative images of hematoxylin and eosin-stained murine bladders that were (A) untreated and treated with HTA-CPP (250 μg), after (B) 24 h, and (C) 48 h (magnification 100×). Representative images of harvested infected murine bladders of (D) untreated (control) and post (E) 15 min and (F) 24 h intravesical treatment with rhodamine B payload from HTA-CPP. Rhodamine channel for rhodamine B molecule (red; nm; nm) and Hoechst channel for nuclei (blue; nm; nm).

Fig. 6.. Intracellular P. aeruginosa within murine bladder.

Representative images of (A) P. aeruginosa UR34-GFP infected murine bladders at 10 dpi and (C) Z-stack images of P. aeruginosa intracellular bacterial community. FITC channel for UR34-GFP and DAPI channel for nuclei of murine bladder.

Fig. 7.. Effect of subcutaneous gentamicin, HTA + GEN and HTA-CPP + GEN on murine P. aeruginosa infected bladders.

Bacterial quantification from uropathogenic P. aeruginosa (UR34-GFP)-infected murine bladders at 3 dpi for (A) untreated (N = 9) and treated subcutaneous (Sub-Q) delivery 1000 μg GEN (N = 9), treated HTA-CPP + GEN (N = 16) by I-VESIC (1000 μg GEN delivered), and for (B) treated HTA + GEN (N = 13) intravesically (I-VESIC) (1000 μg GEN delivered) in comparison to HTA-CPP + GEN group. Box and whisker plot with horizontal line for the median between the interquartile range and minimum/maximum extremes. Kruskal-Wallis one-way ANOVA with post-hoc Dunn’s multiple comparison test against untreated and Mann Whitney test respectively; **, p < 0.01; **** p < 0.0001.