The application of local hypobaric pressure - A novel means to enhance macromolecule entry into the skin

Abstract

The local application of controlled hypobaric stress represents a novel means to facilitate drug delivery into the skin. The aims of this work were to understand how hypobaric stress modified the properties of the skin and assess if this penetration enhancement strategy could improve the percutaneous penetration of a macromolecule. Measurements of skin thickness demonstrated that the topical application of hypobaric stress thinned the tissue (p<0.05), atomic force microscopy showed that it shrunk the corneocytes in the stratum corneum (p<0.001) and the imaging of the skin hair follicles using multiphoton microscopy showed that it opened the follicular infundibula (p<0.001). Together, these changes contributed to a 19.6-fold increase in in vitro percutaneous penetration of a 10,000 molecular weight dextran molecule, which was shown using fluorescence microscopy to be localized around the hair follicles, when applied to the skin using hypobaric stress. In vivo, in the rat, a local hemodynamic response (i.e. a significant increase in blood flow, p<0.001) was shown to contribute to the increase in follicular transport of the dextran to produce a systemic absorption of 7.2±2.81fg·mL(-1). When hypobaric stress was not applied to the rat there was no detectable absorption of dextran and this provided evidence that this novel penetration enhancement technique can improve the percutaneous penetration of macromolecules after topical application to the skin.

Keywords: Bioavailability; Blood flow; Drug delivery; Follicle; Hypobaric pressure; In vivo; Macromolecule; Penetration enhancement; Skin.

Graphical abstract

Fig. 1

Permeation of FD-4 (a) and FD-10S (b) into rat skin under atmospheric (1010 mBar) and hypobaric conditions (500 mBar). Each point represents mean ± standard deviation (n = 4) for atmospheric conditions and (n = 5) for hypobaric conditions. ER (Enhancement Ratio) represents the ratio between the amount of drug in the tissue under hypobaric and atmospheric conditions. Student's t-test with **p < 0.01 and ***p < 0.001.

Fig. 2

Fluorescence microscopic examination after topical administration of FD-4 and FD-10S dextran under atmospheric conditions (1010 mBar) and hypobaric stress conditions (500 mBar): control samples at atmospheric (a) and after hypobaric treatment (b); topical FD-4 delivery under atmospheric conditions (c) and upon hypobaric stress treatment (d); topical FD-10S delivery under atmospheric conditions (e) and upon hypobaric stress treatment (f). Green: FITC, blue: DAPI. Original magnification × 10. SC, stratum corneum; E, epidermis and D, dermis.

Fig. 3

Multiphoton microscopic images of porcine follicular infundibula. a) 3D reconstruction under atmospheric conditions (1010 mBar), b) top view (singe Z-stack cross-section at 1010 mBar), c) side view at 1010 mBar with an average length of 151 ± 40.5 μm and depth of 190 ± 30.1 μm, d) 3D reconstruction after applying hypobaric pressure (500 mBar), e) top view after hypobaric pressure (Z-stack cross-section), f) side view after hypobaric pressure with an average length of 243 ± 23.9 μm and depth of 159 ± 14.5 μm.

Fig. 4

Atomic force microscopy analysis of in vitro porcine skin corneocytes at a) atmospheric conditions (1010 mBar) with average length of 41.5 ± 5.5 μm and width of 37.2 ± 7.2 μm and b) within 25 min of applying hypobaric pressure (500 mBar) with average length of 31.5 ± 8.2 μm and width of 26.2 ± 6.8 μm.

Fig. 5

Porcine and rat skin histology a), e) and b), f) control under atmospheric conditions 4 × and 40 × respectively c) and d) porcine skin after hypobaric treatment of 500 mBar for 7 h at 4 × and 40 × respectively g) and h) rat skin after hypobaric treatment of 500 mBar for 1 h at 4 × and 40 × respectively. SC, stratum corneum, E, epidermis, D, dermis and arrow indicates dermal–epidermal detachment.

Fig. 6

Hypobaric stress induced vascular response a) representative % change in contralateral and ipsilateral (control) hind paw blood flow from baseline over a 15 min period following hypobaric stress treatment b) % change in contralateral and ipsilateral (control) paw blood flow from baseline 3 min after hypobaric treatment (maximum vasodilatation). Student's t-test with ***p < 0.001 c) representative full-field laser perfusion imaging pictures alongside grey scale picture showing blood flow at baseline, 2 and 15 min after hypobaric stress treatment of the contralateral hind paw. Arrow indicates the site of topical hypobaric treatment.

Fig. 7

Blood concentration vs. time profile of ¹⁴C-labeled 10 kDa dextran in phosphate buffer (0.79 μCi equivalent to 1.428 pM) applied topically under atmospheric (1010 mBar) and hypobaric conditions (500 mBar). Each point represents mean ± standard deviation (n = 5). ^#Values below limit of detection (< 3 × background level measurements).

Fig. 8

in vivo percutaneous penetration studies under atmospheric (1010 mBar) and hypobaric (500 mBar) pressure conditions. a) in vitro vs. in vivo 10 kDa dextran rat skin deposition b) biodistribution of ¹⁴C labeled 10 kDa dextran. Each point represents mean ± standard deviation (n = 5). ER (Enhancement ratio) represents the ratio between the amount of drug found under hypobaric and atmospheric conditions. Students t-test with *p < 0.05, **p < 0.01 and ***p < 0.001.