INTRODUCTION:

Topically applied minoxidil is one of the most widely used treatment against male and female hair loss, as it is effective in reversing the progressive miniaturization of hair follicles associated with alopecia, by direct effect on the proliferation and apoptosis of dermal papilla cells¹. However, clinical outcomes have demonstrated that the response rates are often limited and efficacy decreases or disappears over time after prolonged use.

Commercial topical minoxidil formulations consist of propylene glycol/water/ethanol solutions, and patients may experience severe adverse reactions, such as irritation, redness, scalp dryness, burning, allergic contact dermatitis¹. To minimize these effects and improve therapeutic efficacy, novel dermatological preparations are required.

The efficacy of a formulation intended for a cutaneous administration, depends on the ability of the drug to be released from the vehicle and diffuse through the skin and especially its outermost layer, the stratum corneum. In order to overcome the skin barrier, different lipid carriers have been proposed for the delivery of minoxidil, such as liposomes, niosomes, ethosomes, nanostructured lipid carriers, microparticles^1-6. In particular, lamellar vesicles have been extensively studied as suitable drug carriers to the skin^7-14. Our group has been studying for years a class of new phospholipid vesicles, called Penetration Enhancer-containing Vesicles (PEVs), as an alternative to conventional liposomes for the (trans) dermal delivery of various drugs^15-19. Penetration Enhancers (PEs) are pharmaceutically inert chemicals able to interact with the stratum corneum facilitating drug passage²⁰. Our results confirmed a synergic effect of PEs and phospholipids, responsible for an enhanced dermal efficacy. In the present investigation, Labrasol^® was selected as the appropriate penetration enhancer to be used to formulate PEVs loading minoxidil. Labrasol is composed by a mixture of caprylcaproyl macrogolglycerides, and is a safe pharmaceutical excipient used as solubility and a bioavailability enhancer, listed in the current editions of the European and the USP Pharmacopoeias. The effect of labrasol concentration and its insertion in the water or lipid phase of PEVs on their physico-chemical features and performances, was assessed. Indeed, mean size, surface charge, incorporation efficiency, and morphology of PEVs were probed, along with their ability to deliver minoxidil to the skin by evaluating drug distribution in the cutaneous major layers and the possible transdermal diffusion.

MATERIAL AND METHODS:

Materials

Soy phospholipid mixture (Phospholipon^® 50, P50, with 45% phosphatidylcholine and 10-18% phosphatidyl ethanolamine; for the complete composition see Ref. 19) was kindly supplied by AVG (Milan, Italy) and Lipoid GmbH (Ludwigshafen, Germany). Minoxidil free base (MX) was purchased from Galeno (Potenza, Italy); oleic acid (OA) was from Sigma-Aldrich (Milan, Italy). Labrasol^® (30% of mono-, di- and triglycerides of C8 and C10 fatty acids, 50% of mono and di-esters of PEG, 20% of free PEG 400; HLB 14; Lab) was a gift from Gattefossè (Saint Priest, France).

Vesicle preparation

PEVs were prepared using P50 (180 mg/ml), OA (10 mg/ml), and loaded with minoxidil (20 mg/ml), according to the thin-film hydration method. The penetration enhancer, labrasol, was added to the lipid phase (0.5, 1, 2% v/v), by dissolving it in chloroform, together with the other components, or to the aqueous phase, by mixing it with water (2, 5, 10% v/v) in the hydration step.

The obtained shaken vesicles were then sonicated (1-minute cycle; 13 mm of probe amplitude) with a high intensity ultrasonic disintegrator (Soniprep 150, MSE Crowley, London, UK), to gain clear opalescent dispersions. Both shaken and sonicated samples were separated from the non-incorporated drug by dialysis: 2 ml were loaded into dialysis tubing (Spectra/Por^® membranes: 12–14 kDa MW cut-off, 3 nm pore size; Spectrum Laboratories Inc., DG Breda, The Netherlands) and dialysed against distilled water (1000 ml) for 2 hours at 5 °C, which was appropriate to allow the dissolution and consequent removal of non-incorporated minoxidil.

Vesicle characterization

Vesicle formation and morphology were checked by transmission electron microscopy (TEM) using a JEM-1010 (Jeol Europe, Croissy-sur-Seine, France) microscope, equipped with a digital camera MegaView III and Software ‘‘AnalySIS’’, at an accelerating voltage of 80 kV. Vesicles were examined using a negative staining technique: samples were adsorbed on a carbon grid and stained with 1% phosphotungstic acid.

The average diameter, polydispersity index (P.I.; a measure of the width of size distribution) and zeta potential of samples were determined by Dynamic and Electrophoretic Light Scattering using a Zetasizer nano-ZS (Malvern Instruments, Worcestershire UK). Samples diluted (1:100) with the hydration medium used for their preparation, were analysed 24 hours after their production, at 25 °C.

Drug loading efficiency (E%), expressed as the percentage of the amount of drug initially used, was determined by high performance liquid chromatography (HPLC) after disruption of vesicles with 0.025% non-ionic Triton X-100. Minoxidil content was assayed at 231 nm using a chromatograph Alliance 2690 (Waters, Milan, Italy). The analytical column was an SunFire C18, 3.5 mm, 4.6 mm × 150 mm (Waters, Milan, Italy). The mobile phase was a mixture of water:acetonitrile (20:80, v/v) at a flow rate of 1 ml/min.

A stability study was performed by monitoring vesicle average size and zeta potential over 90 days at 4±1 °C.

Ex vivo skin penetration/permeation studies

Experiments were performed non-occlusively by means of Franz diffusion vertical cells with an effective diffusion area of 0.785 cm², using new born pig skin. Goland–Pietrain hybrid pigs (1-1.5 kg), died by natural causes, were provided by a local slaughterhouse. The skin, stored at -80 °C, was pre-equilibrated in saline (NaCl 0.9% w/v) at 25 °C. Skin specimens (n = 6 per formulation) were sandwiched securely between donor and receptor compartments of the Franz cells, with the stratum corneum (SC) side facing the donor compartment.

The receptor compartment was filled with 5.5 ml of saline, which was continuously stirred and thermostated at 37±1 °C. 100 μl of the tested vesicle suspensions, both non-dialysed and dialysed, was placed onto the skin surface. At regular intervals, after 2, 4, 6 and 8 hours, the receiving solution was withdrawn and analyzed by HPLC for drug content (as described above).

After 8 hours, the skin surface of specimens was gently washed and the SC removed by stripping with adhesive tape. The epidermis was separated from the dermis with a surgical sterile scalpel¹⁸. Tape strips, epidermis, and dermis were placed each in methanol, sonicated to extract the drug and then assayed for drug content by HPLC.

Statistical analysis of data

Data analysis was carried out with the software package R, version 2.10.1. Results are expressed as the mean ± standard deviation. Multiple comparisons of means (Tukey test) were used to substantiate statistical differences between groups, while Student’s t-test was used for comparison between two samples. Significance was tested at the 0.05 and 0.01 levels of probability (p).

RESULTS:

Vesicle characterization: physico-chemical and structural features

A series of minoxidil-loaded vesicles containing labrasol were developed, using increasing concentration of the PE and varying the experimental step of its addition. In detail, PEVs were prepared by the film hydration method, followed by sonication, when appropriate, and labrasol was added at 0.5, 1, 2% to the lipophilic components of the formulation, while at 2, 5, 10% to the hydration water. Samples were compared in terms of size, polydispersity, surface charge, incorporation efficiency, lamellar structure, and stability. TEM provided evidence of vesicle formation and showed almost spherical vesicles, with different size and lamellarity (i.e., number of bilayers) depending on the preparation process (with or without sonication). Figure 1 A and B shows multilamellar non-sonicated vesicles and oligolamellar sonicated vesicles, respectively.

Results from particle sizing showed that shaken vesicles with labrasol at the lowest concentrations (0.5 and 1%) were around 150 nm; at 2%, the mean diameter increased up to 200 nm, decreasing again at the highest PE concentrations (5 and 10%) (Table 1). Further, when labrasol was inserted in the lipid phase, PEVs disclosed a broad size distribution (P.I. ~0.3), whereas it was ≤0.24 when labrasol was added to the water phase. On the other hand, sonicated vesicles were smaller in size, around 100 nm, with a narrower size distribution (P.I. <0.27), indicative of an acceptable homogeneity (Table 2). It is noteworthy that Lab10%-PEVs were not affected by sonication, maintaining the same characteristics as for shaken sample.

Table 1. Characteristics of non-dialysed (ND) and dialysed (D) minoxidil-loaded shaken PEVs: mean diameter, polydispersity index (P.I.), zeta potential (ζ) and incorporation efficiency (E%). Values are the means ± standard deviation (n = 3). * P < 0.05.

	*Shaken sample*		Mean size (nm)	P.I.	ζ (mV)	E%
Lab in the lipid phase	Lab 0.5%	ND	155 ± 13.3	0.36	-70 ± 1.9
		D	154 ± 11.4	0.30	-77 ± 2.3	63 ± 4.8
	Lab 1%	ND	158 ± 16.2	0.39	-76 ± 3.3
		D	156 ± 12.7	0.34	-80 ± 0.8	68 ± 9.1
	Lab 2%	ND	*200 ± 8.7	0.33	-73 ± 1.1
		D	*199 ± 13.2	0.34	-75 ± 3.1	76 ± 8.5
Lab in the aqueous phase	Lab 2%	ND	196 ± 20.5	0.24	-74 ± 2.5
		D	199 ± 33.3	0.24	-78 ± 1.5	68 ± 9.1
	Lab 5%	ND	151 ± 9.5	0.19	-68 ± 5.6
		D	155 ± 8.1	0.22	-76 ± 7.2	84 ± 6.9
	Lab 10%	ND	*117 ± 13.8	0.17	-60 ± 2.4
		D	*125 ± 4.1	0.15	-59 ± 3.3	86 ± 7.7

Table 2. Characteristics of non-dialysed (ND) and dialysed (D) minoxidil-loaded sonicated PEVs: mean diameter, polydispersity index (P.I.), zeta potential (ζ) and incorporation efficiency (E%). Values are the means ± standard deviation (n = 3). *,° P < 0.05 between pairs of samples.

	*Sonicated sample*		Mean size (nm)	P.I.	ζ (mV)	E%
Lab in the lipid phase	Lab 0.5%	ND	99 ± 4.4	0.25	-58 ± 4.2
		D	106 ± 4.6	0.26	-63 ± 1.5	66 ± 6.9
	Lab 1%	ND	97 ± 5.8	0.27	-60 ± 6.6
		D	107 ± 6.2	0.26	-69 ± 6.8	62 ± 5.7
	Lab 2%	ND	107 ± 18.0	0.25	-61 ± 8.8
		D	122 ± 12.0	0.23	-68 ± 10.0	61 ± 7.3
Lab in the aqueous phase	Lab 2%	ND	*96 ± 12.2	0.23	-62 ± 3.8
		D	111 ± 13.7	0.25	-68 ± 8.5	62 ± 8.6
	Lab 5%	ND	°84 ± 13.4	0.26	-64 ± 6.0
		D	101 ± 10.1	0.27	-71 ± 4.6	74 ± 2.6
	Lab 10%	ND	*°118 ± 12.4	0.15	-57 ± 2.5
		D	125 ± 2.1	0.16	-62 ± 3.9	69 ± 2.9

Table 3. Results from ex vivo permeation study with PEVs loading minoxidil. Amount of minoxidil accumulated into the whole pig skin and permeated through it after 8 h-experiment; Local Accumulation Capacity (LAC) values: drug accumulated into the skin/drug permeated through the skin ratio; transdermal flux (J) and lag time prior to minoxidil transdermal release.

	*Sample*		Accumulated MXD (µg/cm² ± SD)	Permeated MXD (µg/cm² ± SD)	LAC	J (µg/cm²/h ±SD)	Lag time (h)
Lab in the lipid phase	Lab 0.5%	ND	242 ± 31.4	11 ± 0.2	21.0	2.7 ± 0.22	4.2
		D	153 ± 19.6	8 ± 0.6	16.5	2.0 ± 0.16	4.3
	Lab 1%	ND	351 ± 68.1	14 ± 1.2	25.1	3.4 ± 0.35	4.2
		D	194 ± 61.2	13 ± 0.8	13.2	3.2 ± 0.28	4.2
	Lab 2%	ND	501 ± 111.7	18 ± 0.6	27.8	3.0 ± 0.25	2.6
		D	335 ± 64.8	20 ± 2.9	16.7	3.2 ± 0.17	2.8
Lab in the aqueous phase	Lab 2%	ND	202 ± 41.4	13 ± 0.4	15.5	3.3 ± 0.10	4.0
		D	113 ± 42.1	14 ± 0.1	8.1	3.4 ± 0.11	4.0
	Lab 5%	ND	132 ± 61.8	6 ± 0.2	22.0	3.2 ± 0.19	6.0
		D	134 ± 31.4	11 ± 0.4	11.1	2.8 ± 0.19	4.1
	Lab 10%	ND	171 ± 41.0	151 ± 24.4	1.1	20.3 ± 1.52	0.6
		D	105 ± 24.5	40 ± 2.4	2.6	5.1 ± 0.27	0.2

Figure 2. Cumulative amount of minoxidil (MX) accumulated into stratum corneum (SC), epidermis (Ep) and dermis (D) of full-thickness pig skin after 8 h of topical application of non-dialysed (A) and dialysed (B) PEVs.

Lab 2% l = labrasol added to the lipid phase; Lab 2% w = labrasol added to the water phase. Data represent the mean ± standard deviation of at least six experimental determinations. ** P < 0.01, * P < 0.05 among values in the group SC (with non-dialysed Lab 0.5% and Lab 1% equal to each other); ° P < 0.05 among values in the group Ep; ⁺ P < 0.05 among values in the group D.

DISCUSSION:

PEVs loading minoxidil were formulated as a function of the concentration of the penetration enhancer labrasol, and of its addition to the lipid or water phase of the vesicular dispersions. Labrasol is a physical mixture of mono-, di- and triglycerides, mono and di-esters of PEG, and free PEG 400, forming a monophasic water-miscible system. It can be assumed that at low concentrations labrasol, if mixed with phospholipid organic solution, it can locate at the bilayer interface, which becomes saturated at high PE concentrations, thus preferably occupying the aqueous phase, tightly binding to water molecules. In the light of this, a series of minoxidil-entrapped Lab-PEV formulations were developed keeping constant the drug and P50 amount and varying the labrasol concentration or the experimental step of labrasol addition aiming at determining the influence of such variables on vesicle characteristics². This pre-formulation study showed that 2% labrasol is the critical concentration at which it is possible to add the hydrophilic PE to the phospholipid organic phase. On the basis of this outcome, we added labrasol at 0.5, 1, 2% to the lipophilic components of the formulation, while at 2, 5, 10% to the hydration water in order to find the most suitable preparation method, as well as labrasol concentration to decrease vesicle size and improve system stability.

Almost spherical vesicles were obtained, multilamellar or oligolamellar, depending on the preparation procedure (without or with sonication). As expected, sonicated vesicles were smaller in size and with better homogeneity. In fact, the sonication technique is an effective method to reduce the dimensions of lamellar vesicles²². It is noteworthy that Lab10%-PEVs were not affected by sonication, maintaining the same characteristics as for shaken sample. Among the sonicated vesicles, no relevant differences in size and size distribution, surface charge and loading capacity were noticed, indicating that neither labrasol concentration or the step of its addition were critical for PEVs’ colloidal characteristics.

The penetration ability of vesicular minoxidil was evaluated on intact full-thickness pig skin. Since sonicated vesicles had shown the best physico-chemical properties, they were chosen for this study. The amount of drug accumulated into and permeated through the main skin layers is expressed both as the percentage of the drug applied onto the skin and as mg/cm² of skin. Results showed improved minoxidil local accumulation, influenced by labrasol concentration and insertion in the aqueous or lipid phase of the vesicular formulations. Among non-dialysed vesicles, Lab2%-PEVs (labrasol in the lipophilic phase) provided the highest drug accumulation in the skin with low transdermal flux. This indicates the good capability of this formulation of favouring the local accumulation, rather than reaching the deep soft tissues and the systemic circulation^{16, 18}.

In order to evaluate PEVs’ ability as carriers for the skin delivery of minoxidil, permeation experiments were also carried out applying dialysed formulations. Taking into account that dialysed formulations contain only entrapped minoxidil, outcomes from this study demonstrate the actual ability of the vesicles to carry their content through the skin. Overall, the amount of drug found in the skin was proportional to the amount applied to the skin, despite the lower amount of minoxidil present in the dialysed systems (~65%). In non-dialysed dispersions the guest molecules are partially loaded inside the vesicular structures and partially solubilized outside the vesicles²³. Therefore, when non-dialysed PEVs were used, the highest amounts of drug deposited into the skin were also due to the presence of unentrapped drug that may diffuse thanks to the penetration enhancing effect of labrasol and P50 phospholipids. Some authors have hypothesized that vesicles acted only as true carriers^24-27, or only by a penetration enhancing mechanism^27-29. Nevertheless, the present results are in agreement with our previous findings disclosing that both mechanisms occurred: intact PEVs enter the SC carrying their content, but a part of them fuse with the intercellular lipid bilayers, creating an improved pathway for free drug molecules throughout the skin^{16, 30}.

Therefore, it can be concluded that minoxidil accumulation was closely related to labrasol concentration and its insertion in the lipid or water compartments of PEV formulations. Indeed, as said, Lab2%-PEVs (labrasol in the lipophilic phase) gave the highest values of LAC, almost 2-fold higher than that obtained using PEVs with the same amount of labrasol (2% in the aqueous phase), but added in a different step of vesicle preparation.

As a conclusion, we can deduce that PEVs prepared adding low concentrations of labrasol to the lipid phase can be regarded as the optimal formulations for the cutaneous delivery of minoxidil, especially Lab2%-PEVs (labrasol in the lipophilic phase), as they provided the highest local accumulation of the drug into the skin. Therefore, it is critical not only labrasol concentration, but also the step of its addition during PEVs’ preparation. Indeed, as reported above, Lab2%-PEVs prepared adding labrasol to the aqueous phase, were not as effective as Lab2%-PEVs prepared adding labrasol to the lipid phase, since they were not able to provide a comparable level of drug accumulation (2.5-fold lower).

CONCLUSIONS:

This work shows that innovative vesicular systems are eligible for the use as suitable carriers for minoxidil to the skin. Ex vivo studies disclosed that the concentration of labrasol and its location in the lipophilic phase of the formulation is crucial for improving minoxidil cutaneous delivery, enhancing local accumulation and reducing transdermal permeation. Hence, the present therapeutic approach may be promising in the treatment of hair loss.

ACKNOWLEDGEMENTS:

Authors gratefully thank Allevado (Allevatori Associati del Parteolla) Soc. Coop. A.R.L. for kindly supplying new born pig skin. Sardegna Ricerche Scientific Park (Pula, CA, Italy) is acknowledged for free access to facilities of the Nanobiotechnology Laboratory.

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Received on 23.10.2012 Modified on 30.10.2012

Research J. Pharm. and Tech. 5(12): Dec. 2012; Page 1563-1569