Shape-Dependent Skin Penetration of Silver Nanoparticles: Does It Really Matter?

Abstract

Advancements in nano-structured materials have facilitated several applications of nanoparticles (NPs). Skin penetration of NPs is a crucial factor for designing suitable topical antibacterial agents with low systemic toxicity. Available reports focus on size-dependent skin penetration of NPs, mainly through follicular pathways. Herein, for the first time, we demonstrate a proof-of-concept study that entails variations in skin permeability and diffusion coefficients, penetration rates and depth-of-penetration of differently shaped silver NPs (AgNPs) via intercellular pathways using both in vitro and in vivo models. The antimicrobial activity of AgNPs is known. Different shapes of AgNPs may exhibit diverse antimicrobial activities and skin penetration capabilities depending upon their active metallic facets. Consideration of the shape dependency of AgNPs in antimicrobial formulations could help developing an ideal topical agent with the highest efficacy and low systemic toxicity.

Figure 1. The schematic diagram of possible skin penetration pathways of three differently shaped AgNPs.

(A) Two main possible skin penetration pathways are illustrated: (i) enters via hair follicles (the follicular penetration pathway); and (ii) diffuses through the gaps between corneocytes (the intercellular penetration pathway). (B) Absorption spectra of solutions containing chemically synthesized SNP, TNP, and RNP, showing different peaks of absorbance at certain wavelengths. (C) TEM images showing morphology of synthesized AgNPs. (D) TEM images clearly showing the discrete existence of differently shaped AgNPs after dispersing them in phosphate buffer (pH 7.0), the medium used in the donor compartment of the Franz cell. Dimensions of the AgNPs are almost similar to that of the synthesized ones.

Figure 2. In vitro analysis of shape-dependent skin penetration of AgNPs.

(A) Ultra-thin mouse skin section (surface area 1.64 cm²; thickness 0.7–1.0 mm) was loaded on to the Franz cell system. (B) Mouse skin was exposed to differently shaped AgNPs for 30 h. Amount of silver penetrated from AgNPs through mouse skin was determined and plotted as a function of time. (C) Number density of differently shaped AgNPs permeated through mouse skin, as a function of time in Franz diffusion cell (×10⁹). (D) Graph of versus t (s) of permeability study using mouse skin. Different phases in the permeation of differently shaped AgNPs were identified and the slope of linear fit of the different phases of permeation β_(t) (t = 0 for initial stage and 1, 2 for successive phases, respectively) was employed to determine the corresponding experimental permeability at initial and successive phases of permeation (P_t) (t = 0 for initial stage and 1, 2 for successive phases, respectively). (E) Experimental and theoretical permeability coefficients of differently shaped AgNPs (×10⁻⁶). Experimental permeability coefficients are denoted as (P_t) (t = 0 for initial stage and 1, 2 for successive phases, respectively). Theoretical permeability is represented as P_T. (F) Using the permeability values, experimental diffusion coefficients of AgNPs through mouse skin (D_t) (t = 0 for initial stage and 1, 2 for successive phases, respectively) associated with different phases of permeation were calculated and plotted along with theoretical diffusion coefficients. (G) After 30 h of exposure to differently shaped AgNPs skin tissue samples were subjected to TEM imaging to the locate of the AgNPs (dotted circles) penetrated in the skin. Tissue sections maintained good morphology throughout the experiments. This was assessed by the presence of a stratum corneum (SC) layer (approximately 10–15 μm), a compact viable epidermis (approximately 60–80 μm), and a collagen- and muscle-filled dermis. No major differences were observed in skin samples treated with differently shaped AgNPs, except that SC layer was found absent in tissue treated with SNPs. Scale bar = 10 μm (2,500×), 2 μm (8,000×), 300 nm (40,000×), 200 nm (50,000×), and 150 nm (100,000×).

Figure 3. In vivo skin penetration analysis of three differently shaped AgNPs on hairless mice after 5 days of topical treatment.

Rod-shaped, spherical and triangular AgNPs were topically applied onto the back of the mice covering an area of 2.5 × 2.5 cm² (A). After 5 days, amount of silver in the blood samples collected from their hearts were measured using ICP-MS, and the concentration of silver (B) and number density of differently shaped AgNPs in blood (×10⁸) (C) were calculated and plotted against the different shapes of AgNPs. No significant changes in behavior, health, or treated skin areas were observed in the animals during the experiment (data not shown).