Innovative Nanotechnology Based Systems For Dermal Application

The present work offers a brief overview about some innovative nanoparticulate delivery systems for dermal application.

In particular solid lipid nanoparticles (SLN), ethosomes and cubosomes are presented as carrier systems alternative to liposomes. It has been demonstrated that that SLN can improve the controlled release of drugs, in addition in vivo studies performed by the use of SLN indicate an increase in skin hydration and a reduction in wrinkle depth.

With regard to ethosomes, their soft structure enables facilitated delivery of the incorporated active agent into the skin lipid bilayers. The major potential of ethosomes in promoting penetration through skin with respect to liposomes is ascribed to the presence of ethanol and to the soft structure of the vesicles, promoting an interaction between ethosomes and skin lipids.

Cubic liquid crystalline materials are an active research topic because their unique structure lends itself well to controlled release and skin care applications. Cubosomes usually have been produced by means of time-consuming methods involving high energy input. Conversely we have recently tested more conventional dispersion techniques demonstrating that the emulsification of monoglyceride/surfactant mixtures in water results in the formation of aqueous dispersions of cubosomes. Organoleptic and morphological features of cubosomes do not change by time, appearing free from phase-separation phenomena for almost 1 year from production. Photon Correlation Spectroscopy studies showed that cubosomes undergo an initial increase in mean diameter within the first month following production; afterwards they generally maintain their dimensions for the next 6 months.

Key words: solid lipid nanoparticles (SLN), liposomes, ethosomes, cubosomes, dermocosmetic application.

Introduction

Nanoparticulate systems able to control release and to improve targeting to skin are innovative common topics both in pharmaceutical and cosmetic field. Liposomes are of course the best known nanoparticulate systems. Several studies demonstrated their efficacy as drug delivery systems both for parenteral and topical administration ways. The well characterized liposome vesicles can host different molecules in the bilayer, on the surface or in the inner of their structure. Since liposome composition and structure strictly resemble to the stratum corneum, percutaneous administration of this vehicle leads to deposition of lipidic components from which liposome load can be slowly release. Actually the major liposome drawback can be ascribed to their limited physical stability (1).

As an alternative to liposome, solid lipid nanoparticles (SLN) represent innovative drug carrier systems firstly designed for i.v. administration and recently investigated for peroral and transdermal application. The solid matrix of SLN should be able to protect chemically labile agents from degradation and to modulate drug release profiles (2, 3).

Ethosomes are lipid vesicular systems embodying ethanol in relatively high concentrations (4). These "soft vesicles", represent novel vesicular carrier for enhanced delivery to/through skin (5). The size of ethosome vesicles can be modulated from tens of nanometers to microns. One main feature of these vesicles is their soft structure which carries the incorporated active agent into the skin lipid bilayers and promotes its delivery (6).

Bicontinuous cubic liquid crystalline materials represent innovative research topic due to their unique structure that lends well to controlled release applications (7). The nanostructured particles of bicontinuous cubic liquid crystalline phase are called cubosomes (8). They possess the same microstructure of the parent cubic phase but have much larger specific surface area, moreover cubosome dispersions have much lower viscosity than the bulk cubic phase (9). Cubosomes dispersion can be incorporated into a number of forms and applied to bodily tissue, namely skin and mucosae. Thereafter, cubosomes material is either absorbed or released via diffusion.

Solid Lipid Nanoparticles

Solid lipid nanoparticles (SLN) offer a number of potential advantages as delivery systems, such as better availability for poorly water-insoluble molecules, the use of physiological lipids and a wide application range (dermal, per os, intravenous) (1) (Table I).

Lipids such as triglycerides (trimyristin, tripalmitin, tristearin), glyceryl behenate (Compritol® 888 ATO), glyceryl monostearate (Imwitor® 900), glyceryl palmitostearate (Precirol® ATO 5), or the wax cetyl palmitate can be used for production of SLN to be topically applied on the skin. Lipid concentration ranges between 5 and 40%. Due to their fluid appearence, the lower concentrated preparations should be incorporated into a viscous forms to be easily applied. On the other hand, higher concentrations of lipids result in formation of SLN of a semisolid appearance, cosmetically acceptable as they are.

SLN mean size ranges from 50 to 1000 nm. Depending on the type and concentration of the lipid, 0.5 to 5% emulsifier (surfactant) have to be added for physical stabilisation. In particular poloxamer 188, polysorbate 80, lecithine, tyloxapol, polyglycerol methylglucose distearate (TegoCare® 450), sodium cocoamphoacetate (Miranol® Ultra C32) or saccharose fatty acid ester are very often employed.

SLN can be obtained by different methods, based on solvent emulsification/evaporation or on high pressure homogenization. The latest is an estabilished production method which prevents the need of organic solvents and allows large scale production (2).

SLN dispersions possess interesting features for topical use.

Firstly the solid matrix of SLN can improve chemical stability of drugs protecting molecules from hydrolysis and oxidation (Table I). For instance the chemical stability of tocopherol and retinol improves considerably as compared to an aqueous dispersion (1).

The cutaneous application of SLN can exert occlusive properties. In fact, after application of the lipid nanodispersion to the skin surface, the evaporation of water induce the lipid particles to form an adhesive layer applying occlusion to the surface. As a consequence, an increase in the hydration of the stratum corneum occurs.

The occlusive effects depends to the particle size, in particular it has been demonstrated that nanoparticles are 15fold more occlusive than microparticles (1).

The poor viscosity of SLN makes them little suitable for dermal drug application. The handling of the preparation by the patient can be improved by SLN incorporation into ointments, creams and gels.

It should be noticed that if SLN are incorporated into vehicles, interactions with the vehicle constituents may induce physical instabilities, such as dissolution or aggregation of lipid particles. Therefore, during storage particle sizes and the solid character of the particles have to be followed (3) (Table II).

Retinol incorporated into Compritol®-based SLN has been released more rapidly and to a higher extent as compared to conventional vehicles and a nanoemulsion. This effect appears to result from a burst release from the solid particles following water evaporation on the skin surface and the change of lipid modification.

SLN were already investigated with respect to use in cosmetics. Although adequate controls are difficult to prepare, first experiments indicate an increase in skin hydration and a reduction in wrinkle depth following SLN application . Moreover, cetyl palmitate-nanodispersions act both as particulate UV blockers themselves and as carriers for UV absorbing agents (e.g. 2-hydroxy-4-methoxy benzophenone; Eusolex® 4360) (2). This results in a threefold increase in UV protection which allows reducing the concentration of the UV absorber. This is particularly important since UV absorbers are currently in discussion because of possible estrogenic activity and long-term effects in the environment. SLN may also be suitable for long-lasting perfume and insect repellent formulations(2).

Recently nanoparticulate lipid carriers (NLC) have been developed composed of oily droplets embedded in a solid lipid matrix. Since liquid lipids solubilize lipophilic molecules to a much higher extent than solid lipids, the NLC particles would provide a high incorporation capacity and control of release (1).

Ethosomes

Ethosomes are vesicles constituted of phospholipids, very similar to liposomes but produced in the presence of ethanol. Ethosomes are characterized by a prolonged physical stability with respect to liposomes (Table I). Moreover the use of ethosomal carriers results in delivery of high concentrations of active to/through the skin regulated by system composition and their physical characteristics.

Touitou and colleagues have demonstrated the major potential of ethosomes to promote drug penetration through skin with respect to liposomes (4).

In vivo experiments and clinical trials have demonstrated that a range of molecules such as testosterone, acyclovir (Zovirax; Glaxo Wellcome plc) and insulin can be delivered effectively through the cell membranes of animal and human skin. An alteration of the ethosome formulation can modulate the level of penetration (restricting drug delivery to the skin only, as required for herpes labialis treatment with Zovirax, or allowing full dermal penetration as required for insulin therapy) (4). Another molecule, trihexyphenidyl hydrochloride, incorporated in ethosomes is proposed for transdermal administration in Parkinson patients, from which the geriatric population may greatly benefit (5). Transdermal absorption of polypeptides is currently under investigation. The high interest of ethosomes in the design of new therapies has been investigated with other drugs such as propranolol; in this respect ethosomes showed their potential as transdermal dosage forms for prophylaxis of migraine. Moreover the ability of ethosomes to deliver compounds to cells in culture was investigated (6).

The enhanced delivery of actives using ethosomes over liposomes can be ascribed to an interaction between ethosomes and skin lipids. A possible mechanism for this interaction has been proposed. It is thought that the first part of the mechanism is due to the ‘ethanol effect’, whereby intercalation of the ethanol into intercellular lipids enhances lipid fluidity and decreases the density of the lipid multilayer. This is followed by the ‘ethosome effect’, which includes interlipid penetration and permeation by the opening of new pathways due to the malleability and fusion of ethosomes with skin lipids, resulting in the release of the drug in deep layers of the skin (4).

The basic properties and the in vitro release rate kinetics of azelaic acid (AA) alternatively vehiculated in different phospholipid based vesicles, such as ethosomes or liposomes, were investigated (6). Ethosomes were produced by a simple method based on addition of an aqueous phase to an ethanol solution (comprised between 20 and 45 % v/v) of soy phosphatidyl choline (5 % w/w) and AA (0.2 % w/w) under mechanical stirring. Liposomes were obtained by the same composition in the absence of ethanol with the reverse-phase evaporation method. Vesicle size was measured by Photon Correlation Spetroscopy (PCS) evidencing smaller mean diameters and narrower dimensional distributions in the case of ethosomes with respect to liposomes (Table II) . In order to obtain homogeneously sized vesicles, both ethosomal and liposomal dispersions were extruded through polycarbonate membranes with pores of calibrated diameter (400 and 200 nm). Vesicles morphology was characterized by freeze-fracture Scanning Electron Microscopy (SEM) showing the presence of unilamellar vesicles both in liposome and in ethosome based dispersions (Fig.1).

Figure 1. Freeze-fracture electron micrographs of liposome (panel A) and ethosome 20 (panel B) or 40 % (panel C). Liposome and ethosome dispersions were subjected to extrusion through 200 nm pore size membranes. The bar equals 510 nm.

AA diffusion from ethosomal or liposomal dispersions and from ethosomes and liposomes incorporated in a viscous gel was investigated by a Franz cell assembled with synthetic membranes. Release rate was more rapid from ethosomal with respect to liposomal systems, in particular ethosomes produced by the highest ethanol concentration released AA more rapidly, the same trend was found using viscous forms (Table II).

This behavior can be attributed to the presence of ethanol that makes the lipidic membrane packed less tighly than liposomes and confers a softer, more malleable structure to the ethosomes, possibly promoting azelaic acid diffusion through the vehicle (6).

Cubosomes

Unsaturated long-chain monoglycerides such as monoolein are able to form a variety of structures in aqueous media by self-association, depending on water content and temperature. The addition of small amounts of water to the lipids at 37°C results in the initial formation of a reverse micellar solution. As the water content and/or temperature increase, different mesophases such as lamellar, reversed hexagonal, bicontinuous cubic, and isotropic sponge phase are formed (7). In particular cubic liquid crystals are transparent, isotropic viscous phases and physically stable in excess water (8). Cubic phase represents a unique system for the production of pharmaceutical dosage forms (9).

Aqueous dispersions of cubic lipid phases can be used for the development of nanoparticulate drug delivery systems characterized by high biocompatibility, bioadhesivity, and easy production protocol (10). Because of their properties, these versatile delivery systems can be administered orally, parenterally, or percutaneously.

Landh and Larsson have patented the preparation of colloidal dispersions of nonlamellar lyotropic crystalline phases and have termed the particles “cubosomes” (11). Cubosomes usually have been produced by means of time-consuming methods involving high energy input. For instance Gustafsson et al. have investigated the production and structure of aqueous dispersions of lipid-based lyotropic liquid crystalline phases (12). The dispersions were based either on glycerylmonooleate/sunflower oil or glycerylmonooleate/retynilpalmitate mixtures plus a nonionic triblock polymer (Poloxamer 407) in water. Dispersions were produced by dropwise addition of a melt of lipids and poloxamer in water, followed by reduction of size by homogenization under high pressures at 80°C. Recently Seikmann et al. have reported the preparation and characterisation of dispersions constituted of monoolein-rich monoglycerides with or without purified soya phospholipids (13). Dispersions were prepared by equilibration of the monoglyceride/phospholipid/ water cubic phase, subsequent fragmentation by a solution of Poloxamer 407, predispersing by probe sonication and finally high pressure homogenization. Moreover some authors have developed experimental protocols for cubosome production based on the use of organic solvents. In particular Spicer and Hyden have proposed a method based on a dilution process of an ethanolic solution of monooleine with an aqueous solution of Poloxamer. Ethanol was used as a hydrotrope to create a liquid precursor, spontaneously forming cubosomes after dilution (14). Finally Nakano et al. have suggested a method for the production of cubosomes based on hydration of a dry film of monooleine/poloxamer with an aqueous buffer (15). The authors proposed to mix monooleine and poloxamer in chloroform and to dry the mixture by solvent evaporation. After hydration, cubosome were formed by homogenization at 80°C, structure of cubosomes was investigated by small-angle X-ray scattering and ¹³C NMR.

A recent investigation by Esposito et al. has demonstrated the chance to produce cubosome dispersions by a simple processing technique, avoiding time consuming procedures, multiple equilibration steps, intermediate formation of viscous bulk cubic gel, high energy input and use of organic solvents (16). In particular the use of a stirring speed 1500 r.p.m., monooleine 5% w/w (with respect to weight of dispersion ) and Poloxamer 407 10% w/w (with respect to the disperse phase) enabled to produce dispersions presenting 28% of larger irregular particles and cubosomes characterized by spheroidal shape, few aggregates, mean diameter of 193.5 nm and high percentage of recovery (88% w/w).

Figure 2. Cryo-TEM micrographs of monoglyceride based dispersions constituted of monooleine and poloxamer 407 90:10 w/w. The disperse phase/dispersing phase ratio was 5: 95; dispersions were produced by an overhead mechanical stirrer with a 1500 r.p.m. stirring speed. Bar equal 100 nm in panels A and B.

Figure 2 shows two cryo-TEM micrographs evidencing the heterogeneous morphology of the disperse phase. In particular one can observe the coexistence of spherical vesicles and few faceted particles together with well shaped cubosomes exhibiting the typical ordered cubic texture (16). Vesicular structures appear also attached on the surface of cubosomes, as found by other authors, suggesting that by time a transformation may take place from conglomerates of partially fused vesicles to well ordered particles (7,8,12). These results were in agreement with X-ray diffraction data, revealing the coexistence of two different cubic phases, the first being a bicontinuous cubic phase of spatial simmetry Im3m (Q²²⁹) and the second belonging to the P4(3)32 (Q²¹²) spatial symmetry.

Stability studies were performed demonstrating that the organoleptic and morphological aspects of cubsome dispersions do not change by time, cubosomes in fact are free from phase separation phenomena for almost one year from production (16) (Table I).

Moreover PCS studies were conducted at different time intervals (from 0 to 5 months from production) in order to evidence possible variation of mean diameter of cubosomes by time.

Cubosomes undergo an increase in their mean diameters after 30 days from production. and generally maintain their dimensions in the successive 4 months, not exceeding 595 nm after 5 months from their production (16) (Table I).

L’Oreal has patented the use of cubosome particles as oil-in-water emulsion stabilizers and pollutant absorbents in cosmetics. More recently Nivea has introduced cubosome use in personal care product as skin care, hair care, cosmetics, and antiperspirants (17).

A recent cryo-TEM study evidenced that the global cryo-electron density pattern of the stratum corneum keratin intermediate filament network resembles "inverted" cryo-transmission electron micrographs of cubic lipid/water phases with a “cubic-like rod-packing simmetry”(18).

The observation that biological interface itself possesses a cubic architecture appears particular important in the development of cubosome based cosmetic as well as dermal products.

Conclusions

Many studies have been demonstrated the efficiency of liposomes as drug carriers for different administration ways. Nevertheless liposomes are characterized by some drawbacks such as the limited physical stability.

Recently some alternative nanodispersed systems have been developed, such as SLN, Ethosomes and cubosomes.

SLN possess interesting features as nanotechnology systems for a wide spectrum of application. Their solid matrix allows protection of chemically labile agents from degradation. The use of SLN in dermatics seems very attractive due to the lipophilic character of their components Improved skin hydration is conferred due to adhesive and coherent film formation exherting occlusive properties. Moreover, SLN may also enhance the effects of active compounds in cosmetics.

Ethosomes are attracting systems able to solubilize different kind of molecules and to enhance their delivery through the skin due to the presence of ethanol. Moreover their physical stability improves their use with respect to liposomes.

Bicontinuous cubic liquid crystalline phases, either in bulk or cubosome form, offer unique properties of particular interest to the personal care industry. Cubic phase materials can be formed by simple combination of biologically compatible lipids and water and are thus well-suited for use in treatments of skin, hair, and other body tissue. Dermal application of personal care products containing cubosomes is particularly interesting due to the possible stratum corneum-cubosome interaction recently demonstrated.

References

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(2) Wissing SA, Müller RH (2001) Intern. Symp. Control.Rel. Bioact. Mater. 28, 522-523 .

(3) Westesen K, Bunjes H, Koch MHJ (1997) Physicochemical characterizationm of lipid nanoparticles and evaluation of their drug loading capacity and sustained release potential. J. Control. Rel. 48: 223-236.

(4) Touitou E, Dayan N, Bergelson L, Godin B, Eliaz M (2000) Ethosomes-novel vesicular carriers for enhanced delivery: characterization and skin penetration properties. J. Contr. Rel. 65: 403-418.

(5) Dayan N, Touitou E (2000) Carriers for skin deliveryof trihexyphenidyl HCl: Ethosomes vs. Liposomes. Biomaterials 21: 1879-1885.

(6) Esposito E, Menegatti E, Cortesi R (2004) Ethosomes and liposomes as topical vehicles for azelaic acid: a preformulation study. The Journal of Cosmetic Sciences 55: 253-264.

(7) Gustafsson J, Ljusberg-Wharen H, Almgrem M, Larsson K (1996) Cubic lipid-water phase dispersed into submicron particles. Langmuir 12: 4611-4613.

(8) Engstroem S, Norden TP, Nyquist H (1999) Cubic phases for studies of drug partition into lipid bilayers. Eur. J. Pharm. 8: 243-254.

(9) Shah JC, Sadhale Y, Chilukuri DM. (2001) Cubic phase gels as drug delivery systems. Advanced Drug Delivery Reviews 47: 229– 250.

(10) Larsson K (2000) Aqueous dispersion of cubic lipid-water phases. Curr Opin Colloid In 5:64-69.

(11) Landh T, Larsson K (1996) Particles, method of preparing said particles and uses thereof. US patent N° 5531925..

(12) Gustafsson J, Ljusberg-Wharen H, Almgrem M, Larsson K (1997) Submicronparticles of reversed lipid phases in water stabilized by a nonionic amphiphilic polymer. Langmuir 13: 6964-6971.

(13) Siekmann B, Bunjes H, Koch M H J, Westesen K (2002) Preparation and structural investigations of colloidal dispersions prepared from cubic monoglyceride–water phases. Int. J. Pharm. 244:33-43.

(14) Spicer PT, Hayden KL (2001) Novel process for producing cubic liquid cristalline nanoparticles (cubosomes). Langmuir 17: 5748-5756.

(15) Nakano M, Sugita A, Matsuoka H, Handa T (2001) Small Angle X-ray scattering and ¹³C NMR investigation on the internal structure of “cubosomes”. Langmuir 17: 3917-3922.

(16) Esposito E, Eblovi N, Rasi S, Drechsler M, Di Gregorio GM, Menegatti E, Cortesi R (2003) Lipid-Based Supramolecular Systems for Topical Application: A Preformulatory Study. AAPS PharmSci, 5 (4) 30.

(17) Spicer PT, Lynch ML, Steven BH (2003) Bicontinuous Cubic Liquid Crystalline Phase and Cubosome Personal Care Delivery Systems. The Procter & Gamble Company.

(18) Norlén, Al-Amoudi (2004) Stratum Corneum Keratin Structure, Function, and Formation: The Cubic Rod-Packing and Membrane Templating Model The Journal of Investigative Dermatology 123, 4: 715.

Table I. Comparison of the features of different nanoparticulate systems

-: not determined

Table II. Vesicles mean diameter and polydispersity of azelaic acid containing liposomes and ethosomes before and after extrusion through 200 nm pore size membranes, as determined by PCS

LIPO: liposomal suspension

ETHO 20: ethosomal suspension produced by the use of ethanol 20%

ETHO 40: ethosomal suspension produced by the use of ethanol 40 %

ex 200: vesicles extruded through 400 nm pore size polycarbonate membranes and through 200 nm pore size polycarbonate membranes

Data were the mean of four determinations on different dispersions, SD were always comprised between ± 5%.

Table III. "In vitro" release rate coefficients of azelaic acid incorporated in different topical forms.

EtOH / Carbomer gel: ethanol solution incorporated in Carbomer based gel; LIPO: liposome suspension; LIPO gel: LIPO incorporated in Carbomer based gel; ETHO 20: ethosomal suspension produced by the use of ethanol 20%; ETHO 20 gel: ETHO 20 incorporated in Carbomer based gel; ETHO 40: ethosomal suspension produced by the use of ethanol 40 %; ETHO 40 gel: ETHO 40 incorporated in Carbomer based gel;

°Azelaic acid concentration was always 12 mg/ml

Experiments were performed by a Franz release rate cell assembled with a cellulose ester membrane (0.6 mm pore size) and IPB / ethanol 70:30 v/v as receptor phase.

Data were the mean of six determinations, SD were always comprised between ± 8%.

Table IV. X-ray diffraction data for monooleine/poloxamer dispersions

Dispersions were produced by a disperse phase constituted of monooleine/Poloxamer 407 90:10 w/w in water with a 5:95 disperse phase/dispersing phase ratio. The stirring speed was 1500 r.p.m.

Authors : Elisabetta Esposito*, Enea Menegatti, Rita Cortesi

* for correspondence

About Elisabetta Esposito

Dr Elisabetta Esposito earned her degree on Pharmaceutical Chemistry and Technology in 1991 at the University of Ferrara (Italy) and in 1996 she obtained her Ph.D. in Pharmaceutical Sciences with a dissertation on microparticulate systems for controlled release of drug. From 1997 up to now she has been working at the department of Pharmaceutical Sciences of the University of Ferrara as post-doc fellow, studying innovative formulations for dermatologic and cosmetic applications as well as nanotechnologies for gene therapy. Dr. Esposito's research efforts enabled to carry out 2 patents with pharmaceutical italian companies. Dr Esposito has published more than 60 papers in international journals and has presented more than 50 contributions to national and international congresses. Dr Esposito is a member of the CoReS Techno enterprise

Dr Elisabetta Esposito, PhD

Dipartimento di Scienze Farmaceutiche

Università di Ferrara

Via Fossato di Mortara,19

44100 Ferrara

tel. 0532/291259

fax. 0532/291296

e-mail: ese@unife.it

http://web.unife.it/ricerca/corestechno