Investigation of follicular and non-follicular pathways for polyarginine and oleic acid-modified nanoparticles

Abstract

Purpose: To investigate the percutaneous permeation pathways of cell penetrating peptide modified lipid nanoparticles and oleic acid modified polymeric nanoparticles.

Methods: Confocal microscopy was performed on skin cultures (EpiDermFT™) for modified and un-modified nanoparticles. Differential stripping was performed following in vitro skin permeation of Ibuprofen (Ibu) encapsulated nanoparticles to estimate Ibu levels in different skin layers and receiver compartment. The hair follicles (HF) were blocked and in vitro skin permeation of nanoparticles was then compared with unblocked HF. The surface modified nanoparticles were investigated for response on allergic contact dermatitis (ACD).

Results: Surface modified nanoparticles showed a significant higher (p < 0.05) in fluorescence in EpiDermFT™ cultures compared to controls. The HF play less than 5% role in total nanoparticle permeation into the skin. The Ibu levels were significantly high (p < 0.05) for surface modified nanoparticles compared to controls. The Ibu levels in skin and receiver compartment were not significantly different when HF were open or closed. Modified nanoparticles showed significant improvement in treatment of ACD compared to solution.

Conclusions: Our studies demonstrate that increased skin permeation of surface modified nanoparticles is not only dependent on a follicular pathway but also occur through non-follicular pathway(s).

Figure 1

EpiDermFT™ permeation & confocal imaging: (A) In vitro EpiDermFT™ permeation of DiO encapsulated modified and un-modified nanoparticles with DiO-Sol. 16 h following application of the indicated nanoparticles, the full thickness skin equivalent sections were cut with a cryotome and observed with a CLSM for associated fluorescence. The top row indicates the NLC formulations and bottom row indicates the NPS formulations. (B) Comparison between the percent fluorescence intensity of the DiO encapsulated surface modified and un-modified nanoparticles for permeated skin equivalent sections. (C) Comparison between the fluorescence intensity of the collected medium. Data represent mean ± SD (n=6); significance DiO-NLC-R11 against DiO-Sol, DiO-NLC and DiO-NLC-YKA, *p<0.05; significance DiO-NPS-OA against DiO-Sol, DiO-NPS and DiO-NPS+OA (PM), **p<0.05.

Figure 2

Differential stripping approach - Distribution of Ibu in the skin: In vitro pig ear skin permeation of surface modified and un-modified nanoparticles (A) lipid nanoparticles: Ibu-NLC Ibu-NLC-R11 and Ibu-NLC-YKA; (B) polymeric nanoparticles: Ibu-NPS Ibu-NPS-OA and Ibu-NPS+OA (PM). Permeation was performed for 24 h was performed on Franz diffusion cells. After permeation, adhesive tape stripping and cyanoacrylate surface biopsies was performed to quantify the Ibu levels in stratum corneum (SC), hair follicle (HF) and remaining skin layers (Epi+Derm). Data represent mean ± SD, n=6, significance Ibu-NLC-R11 against Ibu-Sol, Ibu-NLC and Ibu-NLC-YKA, *p<0.05; significance Ibu-NPS-OA against Ibu-Sol, Ibu-NPS and Ibu-NPS+OA (PM), **p<0.05.

Figure 3

Differential stripping approach – Perpetration of Ibu through the skin: In vitro pig ear skin permeation of surface modified and un-modified nanoparticles (A) lipid nanoparticles: Ibu-NLC Ibu-NLC-R11and Ibu-NLC-YKA; (B) polymeric nanoparticles: Ibu-NPS Ibu-NPS-OA and Ibu-NPS+OA (PM). Permeation was performed for 24 h was performed on Franz diffusion cells. After permeation, the Ibu level in receiver compartment was determined using HPLC. Data represent mean ± SD, n=12, significance Ibu-NLC-R11 against Ibu-Sol, Ibu-NLC and Ibu-NLC-YKA, *p<0.05; significance Ibu-NPS-OA against Ibu-Sol, Ibu-NPS and Ibu-NPS+OA (PM), **p<0.05.

Figure 4

Selective blocking of HF using a microdrop of varnish-wax mixture: Following blocking of HF, DiO-Sol was applied to the pig ear skin and the sections were collected 24 h after application. The top panel represents HF Open: (A) Bright Field, (B) Green Fluorescence Field; and the bottom panel represent HF Close skin: (C) Bright Field; (D) Green Fluorescence Field.

Figure 5

Selective blocking of HF - Distribution of Ibu in the skin: The HF blocked and un-blocked test area of skin was evaluated for in vitro skin permeation for CPP and OA surface modified nanoparticles. The skin biopsies were collected and tested for retention of Ibu in skin layers. The Ibu levels at the end of 24 h (A) when applied in a form of CPP surface modified NLC and (B) when applied as a form of OA surface modified NPS. Data represent mean ± SD, n=6, significance Ibu-NLC-R11 against Ibu-Sol, Ibu-NLC and Ibu-NLC-YKA, *p<0.05; significance Ibu-NPS-OA against Ibu-Sol, Ibu-NPS and Ibu-NPS+OA (PM), **p<0.05.

Figure 6

Selective blocking of HF - Permeated amount of Ibu through the skin: The HF blocked and un-blocked test area of skin was evaluated for in vitro skin permeation for CPP and OA surface modified nanoparticles. The Ibu levels in the collected receiver compartment was evaluated at the end of 24 h (A) when applied as a form of CPP surface modified NLC and (B) when applied as a form of OA surface modified NPS. Data represent mean ± SD, n=12, significance Ibu-NLC-R11 against Ibu-Sol, Ibu-NLC and Ibu-NLC-YKA, *p<0.05; significance Ibu-NPS-OA against Ibu-Sol, Ibu-NPS and Ibu-NPS+OA (PM), **p<0.05.

Figure 7

In vitro drug release of: A) lipid nanoparticles: Ibu-NLC and Ibu-NLC-R11; and B) polymeric nanoparticles: Ibu-NPS and Ibu-NPS-OA in PBS (pH 7.4) containing 0.1% w/v volpo 20. The cumulative amount released was plotted against time. In vitro drug release results show initial burst release followed by steady state release and after 72 h approximately 85 - 95% of the Ibu was released. The Data represent mean ± SD, n=6.

Figure 8

Cytotoxicity of nanoparticles: The surface modified and un-modified NLC and NPS formulations (without drug) showed viability close to 100% against NHEK cells. The Data represent mean ± SD, n=6.

Figure 9

(A) Effect of Ibu-Sol, surface modified nanoparticles containing Ibu and dexamethasone (DXM) on the reduction of ACD in C57/BL mice ears. Data represent mean ± SD, n=4, significance Ibu-NLC-R11 and Ibu-NPS-OA against Ibu-Sol, *p<0.001. (B) H&E staining of inflammation induced by topical application of DNFB, and after 72 h treatment Ibu-Sol, Ibu-NLC-R11 and Ibu-NPS-OA with a positive control, DXM.