Topically Applied Curcumin-Loaded Nanoparticles Treat Erectile Dysfunction in a Rat Model of Type-2 Diabetes

Abstract

Background: Curcumin, a naturally occurring anti-inflammatory compound, has shown promise in pre-clinical studies to treat erectile dysfunction (ED) associated with type-1 diabetes. However, poor bioavailability following oral administration limits its efficacy. The present study evaluated the potential of topical application of curcumin-loaded nanoparticles (curc-np) to treat ED in a rat model of type-2 diabetes (T2D).

Aim: Determine if topical application of curc-np treats ED in a T2D rat model and modulates expression of inflammatory markers.

Methods: Curc-np (4 mg curcumin) or blank nanoparticles were applied every 2 days for 2 weeks to the shaved abdomen of 20-week-old Zucker diabetic fatty male rats (N = 5 per group). Lean Zucker diabetic fatty male rat controls were treated with blank nanoparticles (N = 5). Penetration of nanoparticles and curcumin release were confirmed by 2-photon fluorescence microscopy and histology. Erectile function was determined by measuring intracorporal pressure (ICP) normalized to systemic blood pressure (ICP/BP) following cavernous nerve stimulation. Corporal tissue was excised and reverse transcription and quantitative polymerase chain reaction used to determine expression of the following markers: nuclear factor (NF)-κβ, NF-κβ-activating protein (Nkap), NF erythroid 2-related factor-2, Kelch-like enoyl-CoA hydratase-associated protein-1, heme oxygenase-1 (HO-1), variable coding sequence-A1, phosphodiesterase-5, endothelial and neuronal nitric oxide synthase, Ras homolog gene family member A, and Rho-associated coiled-coil containing protein kinases-1 and -2.

Outcomes: Erectile function by determination of ICP/BP and expression of molecular markers in corporal tissue by RT-qPCR.

Results: Nanoparticles penetrated the abdominal epidermis and persisted in hair follicles for 24 hours. Curc-np-treated animals exhibited higher average ICP/BP than animals treated with blank nanoparticles at all levels of stimulation and this was statistically significant (P < .05) at 0.75 mA. In corporal tissue, Nkap expression decreased 60% and heme oxygenase-1 expression increased 60% in curc-np- compared to blank nanoparticle-treated animals. ICP/BP values inversely correlated with Nkap and directly correlated with HO-1 expression levels.

Clinical translation: These studies demonstrate the potential for topical application of curc-np as a treatment for ED in T2D patients.

Conclusions: The T2D animal model of ED represents a more prevalent disease than the more commonly studied type-1 diabetes model. Although there is improved erectile response in curc-np-treated animals, only at the lower levels of stimulation (0.75 mA) was this significant compared to the blank nanoparticle-treated animals, suggesting more studies are needed to optimize protocols and evaluate toxicity. Topical application of curc-np to a rat model of T2D can systemically deliver curcumin, treat ED, and modulate corporal expression of inflammatory markers. Draganski A, Tar MT, Villegas G, et al. Topically Applied Curcumin-Loaded Nanoparticles Treat Erectile Dysfunction in a Rat Model of Type-2 Diabetes. J Sex Med 2018;15:645-653.

Keywords: Curcumin; Diabetes; Erectile Dysfunction; Inflammation; Nanoparticle.

Figure 1. Two-photon microscopy of nanoparticles topically applied to rat abdomen

(A) Image generated 1-hour post-application of nanoparticles to the shaved abdomen of a ZDF rat. Red fluorescently labeled nanoparticles are seen to reside in skin furrows between the white collagen fibers that make up dermal papillae at a depth of 45µm. (B) Image generated 24-hours post-application of nanoparticles show residence in hair follicles. (C) Image generated 2-hours post-application of curc-np at 12µm depth and (D) 45 µm depth; the yellow color is due to the fluorescence of curcumin.

Figure 2. Epifluorescent histologic image of ZDF rat skin treated with fluorescent nanoparticles

Images generated 1-hour post-application of red labeled nanoparticles loaded with green colored lipip in an 8µm thick slice of ZDF rat abdominal skin. (A) is an overlay of the phase and red channels showing labeled nanoparticles have penetrated the thin epidermis and have begun to collect in hair follicles. (B) is the green channel of the same image showing the labeled lipid diffusing out from nanoparticles across the epidermis as well as deeper at the base of hair follicles.

Figure 3. Erectile function: intracavernosal pressure (ICP) normalized by systemic blood pressure (BP)

Representative traces of ICP in response to electrostimulation of the cavernous nerve at 0.75, 1, 2, 4, 6, and 10mA current for (A) lean rats treated with blank-np, (B) Diabetic ZDF treated with blank-np, and (C) Diabetic ZDF treated with curc-np. The lean rats exhibited high ICP response to electrostimution, not seen in the ZDF fatty animals; this loss was recovered in the curc-np treated ZDF fatty rats. (D) Shows a representative trace of systemic BP (BP was not significantly different between the different groups of animals). The average ICP/BP ratio is shown for 0.75 mA (E) and for all level of stimulation (F), demonstrating improvement in erectile function in ZDF rats treated with curc-np (*P<0.05; **P <0.01).

Figure 4. Quantitative reverse transcription PCR analysis of corporal tissue

(A) Comparison of gene expression by RT-qPCR between ZDF fatty and lean rats expressed in fold change. RPL19 was used as the reference gene. Red bars denote upregulated genes and blue bars denote downregulated genes. The solid line represents relative gene expression in the lean animals (control). (B) Comparison of fold-change in gene expression between curc-np and blank-np (control) treated diabetic ZDF rats. (C) Linear regression analysis between gene expression of ho-1 and (D) nkap and ICP/BP following CN stimulation between 0.75 and 1.0mA. The y-axis (ΔCt) represents the amount of target gene normalized by the reference gene (RPL19). Ct (threshold cycle) values are inverse to the amount of RNA in the sample. Dotted line represents 95% confidence intervals. P< 0.05 denotes statistical significance.