Doxorubicin liposomes as an investigative model to study the skin permeation of nanocarriers

Abstract

The objectives of this study were to develop an innovative investigative model using doxorubicin as a fluorophore to evaluate the skin permeation of nanocarriers and the impact of size and surface characteristics on their permeability. Different doxorubicin-loaded liposomes with mean particle size <130 nm and different surface chemistry were prepared by ammonium acetate gradient method using DPPC, DOPE, Cholesterol, DSPE-PEG 2000 and 1,1-Di-((Z)-octadec-9-en-1-yl) pyrrolidin-1-ium chloride (CY5)/DOTAP/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) as the charge modifier. There was minimal release of doxorubicin from the liposomes up to 8h; indicating that fluorescence observed within the skin layers was due to the intact liposomes. Liposomes with particle sizes >600 nm were restricted within the stratum corneum. DOTAP (p<0.01) and CY5 (p<0.05) liposomes demonstrated significant permeation into the skin than DOPA and PEG liposomes. Tape stripping significantly (p<0.01) enhanced the skin permeation of doxorubicin liposomes but TAT-decorated doxorubicin liposomes permeated better (p<0.005). Blockage of the hair follicles resulted in significant reduction in the extent and intensity of fluorescence observed within the skin layers. Overall, doxorubicin liposomes proved to be an ideal fluorophore-based model. The hair follicles were the major route utilized by the liposomes to permeate skin. Surface charge and particle size played vital roles in the extent of permeation.

Keywords: 1,2-Dioleoyl-3-trimethylammonium-propane (chloride salt); 1,2-Dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) imidodiacetic acid) succinyl nickel salt]; 1,2-Dioleoyl-sn-glycero-3-phosphate; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine; 1,2-Dipalmitoyl-sn-glycero-3-Phosphocholine; 1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (ammonium salt); Cell penetrating peptides; Coumarin 6; Doxorubicin HCL; Doxorubicin liposomes; Hair follicle blocking; Polyethylene glycol; Skin permeation; Tape stripping; Topical drug delivery.

Figure 1

A) Permeation of FITC labeled siRNA into rat skin. Green fluorescence depicts the permeation of FITC labeled siRNA solution and FITC labeled siRNA cy5 liposomes in normal and tape stripped skin (4X), respectively after 6 h permeation studies. Tape stripping significantly (p<0.05) increased the permeation of FITC labeled siRNA for solution. B) Permeation of Coumarin-6 into rat skin. Green fluorescence shows the skin permeation of the lipophilic dye entrapped within cy5 and cy5+DOTAP liposomes, respectively after 6 h permeation studies.

Figure 2

A) Graph illustrating the drug release profile of doxorubicin from solution, DOTAP liposomes and cy5 liposomes. B) Bright field microscopic illustration of the cross section of untreated rat skin. Images show the corresponding decrease in the thickness of the stratum corneum with increase in the number of tape stripping.

Figure 3

A) Illustration of the permeation of doxorubicin carrier into rat skin. Red fluorescence indicates the skin penetration of doxorubicin solution and doxorubicin cy5 liposomes after 3 and 6 h permeation studies. B) Effect of tape stripping on the skin permeation of doxorubicin carrier into rat skin. Red fluorescence illustrates the permeation of doxorubicin solution and doxorubicin cy5 liposomes, respectively in normal and tape stripped rat skin (10 times) after 6 h permeation studies.

Figure 4

Effect of tape stripping and hair follicle blocking on the skin permeation of doxorubicin carrier in pigskin. A) Confocal z-stack microscopic analysis showing red fluorescence due to permeated doxorubicin from cy5 liposomes at different skin depth. B) Surface Plot depicting the fluorescence intensity at 61–120 μm for normal skin, tape stripped skin, skin with hair follicles blocked and tape stripped skin with hair follicles blocked, respectively. Results show that the skin hair follicle is the major route of permeation of the doxorubicin liposomes with tape stripping further enhancing permeation of the liposomes via other possible routes.

Figure 4

Effect of tape stripping and hair follicle blocking on the skin permeation of doxorubicin carrier in pigskin. A) Confocal z-stack microscopic analysis showing red fluorescence due to permeated doxorubicin from cy5 liposomes at different skin depth. B) Surface Plot depicting the fluorescence intensity at 61–120 μm for normal skin, tape stripped skin, skin with hair follicles blocked and tape stripped skin with hair follicles blocked, respectively. Results show that the skin hair follicle is the major route of permeation of the doxorubicin liposomes with tape stripping further enhancing permeation of the liposomes via other possible routes.

Figure 5

Effect of charge on the permeation of doxorubicin liposomes into pig skin. Results indicate that the charges present on the surface of the liposomes play significant role in the percutaneous penetration of the doxorubicin liposomes in pig skin.

Figure 6

Effect of size on the permeation of doxorubicin DOTAP liposomes into pig skin. Results indicate that there is limited permeation of doxorubicin for liposomes with sizes ≥ 600 nm.

Figure 7

Effect of surface modification of liposomes (without cy5 and DOTAP lipids) with TAT peptide on their skin permeation into pig skin (open and blocked hair follicles). Results indicate that TAT significantly (p<0.005) enhanced the percutaneous penetration of doxorubicin liposomes in pig skin possibly through the creation of additional channels aside the hair follicles (evident in permeation observed even with hair follicles blocked) and possibly through the disruption of the stratum corneum. But this is not observed in DOX TAT solution and Physical mixture (DOX liposomes with TAT but not DOGS NTA Ni linker).