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In particular, the examined therapeutic classes are: antitumoral drugs, i.e. paclitaxel, doxorubicin, tea catechins and hypericin; anti-infective drugs, i.e. Melaleuca ...
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Innovative Drug Delivery Systems for the Administration of Natural Compounds Donatella Paolino, Donato Cosco, Felisa Cilurzo and Massimo Fresta* Department of Pharmacobiological Sciences, Faculty of Pharmacy, University "Magna Græcia" of Catanzaro, Campus Universitario, Building of BioSciences, Viale Europa, I-88100 Germaneto (CZ), Italy Abstract: The use of natural compounds has recently been reconsidered in modern clinical practice. A further advancement of these compounds is represented by the possibility of using innovative drug delivery systems that can improve the biopharmaceutical features of the delivered compounds. This review examines the recent developments in the field of delivery of natural drugs belonging to several therapeutic classes. In particular, the examined therapeutic classes are: antitumoral drugs, i.e. paclitaxel, doxorubicin, tea catechins and hypericin; anti-infective drugs, i.e. Melaleuca alternifolia and Artemisia arborescens L.; and antinflammatory/antioxidant drugs, i.e. cannabinoids and glycyrrhizinates used for topical application. In this review we highlight the utility of a suitable drug delivery system to improve the biopharmaceutical aspects of these drugs. The examined carriers are: vesicular carriers (liposomes, Ethosomes®, ultradeformable liposomes), nano- and microparticles, innovative gels, microemulsions.

Keywords: Natural compounds, antitumoral drugs, anti-infective drugs, antiinflammatory drugs, drug delivery systems, vesicles, nanoparticles, microemulsions, cyclodextrins. 1. INTRODUCTION Over the last few years the use of natural compounds has been re-discovered. In fact, the use of plants extracts is a practice used in traditional medicine. The efficacy of many therapeutic agents depends on their action on target macromolecules located either within or on the surface of particular cells types [1]. It is well known that the therapeutic efficacy of a drug can be improved and its toxic effects can be reduced by augmenting the amount and persistence of drugs in the vicinity of the target cells, while reducing the drug exposure to the non-target cells. This aspect may be improved by means of drug delivery systems. A drug delivery system requires simultaneous consideration of several factors, such as the drug property, route of administration, nature of delivery vehicle, mechanism of drug release, ability of targeting, and biocompatibility [2]. In this review some of innumerable examples of natural drugs administered by means of a drug delivery system will be analyzed. In the first section the principal carriers will be described and in the second section some papers dealing with the delivery of natural drugs with various drug delivery systems will be analyzed. 2. DRUG DELIVERY SYSTEMS A drug delivery system can be defined as a tool to delivery a therapeutic agent into the body. It is well known that the efficacy of many therapeutic agents depends on their action on target macromolecules located either within or on the surface of particular cell types. Over the last few years more sophisticated delivery systems have been developed. In particular, modern technology has produced carriers such as colloidal carriers (liposomes, nanoparticles, Ethosomes® and solid lipid nanoparticles), emulsive systems and molecular carriers (cyclodextrins and hydrogels). 2.1. Colloidal Carriers Colloidal drug delivery devices represent a heterogeneous class of carriers that can be classified into conventional, long circulating and actively targeted systems [3]. Conventional colloidal carriers (liposomes and nanoparticles) are characterized by wide differences both in terms of composition and physicochemical properties, i.e., size, size distribution, surface charge, number and fluidity of phospholipid bilayers, in the case of vesicles, and matrix *Address correspondence to this author at the Department of Pharmacobiological Sciences, Faculty of Pharmacy, University "Magna Græcia" of Catanzaro, Campus Universitario, Building of BioSciences, Viale Europa, I-88100 Germaneto (CZ), Italy; Tel: +39 0961 3694118; Fax: +39 0961 3694237; E-mail: [email protected] 1573-4072/07 $50.00+.00

compactness, in the case of nanoparticles. The modulation of these properties can influence technological properties, such as colloidal stability, drug loading, drug release rate, and to a certain extent the in vivo behavior of conventional colloidal carriers (i.e., blood stability, clearance, and distribution). However, some in vivo features are very consistent among different types of conventional colloidal carriers, presenting a short blood circulation time when parenteraly administered due to a rapid RES uptake. A conesquential successful therapeutic use of conventional colloidal carriers characterized by accumulation at the level of the mononuclear phagocyte system is the delivery of antimicrobial agents to infected macrophages [4,5]. Long-circulating colloidal delivery systems allow the therapeutic treatment of a wide range of diseases involving tissues other than the liver and spleen [6]. A common characteristic of all longcirculating systems is the presence along the surface of the colloidal carrier of hydrophilic macromolecular moieties, such as polyethylene glycol (PEG). Highly hydrated macromolecular moieties determine a steric barrier against interactions with some molecular and cellular components in the biological environment, thus avoiding the opsonization phenomenon and hence the RES organ uptake. In this way, actively targeted carriers can be obtained by conjugation of a colloidal drug delivery system to specific antibodies, antibody fragments (e.g., Fab or single-chain antibodies), or small targeting agents (peptides, hormones, specific ligands), thus increasing target site binding and the delivery of the encapsulated drug [7]. 2.1.1. Liposomes Liposomes are self-assembled vesicles that occur naturally and can be prepared artificially, as shown by Bangham and collaborators in the mid-1960. They were first used to study biological membranes but several practical applications, most notably in drug delivery, emerged in the 1970s. An important feature that makes liposomes a unique drug delivery system is their biomimetism; they have the same supramolecular lipid organization of natural membranes of living cells [8]. Liposomes are vesicles with which it is possible to have, at the same time, various microenvironments characterized by different physicochemical properties, namely, a highly hydrophilic region made up of the intravesicular aqueous compartment, a highly hydrophobic region of the bilayer core made up of the alkyl chains of the lipid constituent, and an amphipatic region at the level of the vesicular surface made up of the polar lipid head-groups. These characteristics make liposomes a very versatile drug carrier being able to entrap and delivery hydrophilic (in the intravesicular © 2007 Bentham Science Publishers Ltd.

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aqueous compartment), hydrophobic (in the core of vesicular bilayers), and amphipatic (at the level of vesicular boundary zone) drugs, Fig. (1).

Fig. (1). Schematic representation of a liposomal structure with the characteristic microenvironments and the possible encapsulation of drug as a function of their physicochemical features.

The liposomal carrier has the advantage of also being able to deliver macromolecules, such as enzymes, proteins, and genetic material. From the morphological point of view, liposome systems can be classified as a function of the number of bilayers and the mean size of the carrier in unilamellar, oligolamellar or multilamellar vesicles, and in small (100 nm), medium (100–500 nm) and large (1m) vesicles, respectively. Lecithins and cholesterol are the lipids most commonly used in the preparation of liposomes. Other components can be used in liposome preparation, that is, steroid molecules, charged phospholipids, gangliosides, and polymeric material to modulate the carrier properties as a function of the therapeutic requirements to be achieved [9]. In fact, different components can modify the biodistribution, the surface charge, the release, and the clearance rate of the liposomal drug delivery system [10]. The circulation lifetime of a liposome is also changed by the charge of the liposome surface that can influence the pharmacokinetics of the system [11]. Cholesterol plays a fundamental role in liposome formulations being able to act as a vesicle membrane modulator as concerns membrane fluidity [12]. It has a stabilizing function on the liposome bilayers both in vitro and in vivo, allowing the protection of the vesicular structure by the action of high density blood lipoproteins and hence has the possibility of giving the intact liposomes a prolonged circulation. Similarly to cholesterol, some phospholipid components are also able to influence the physicochemical behaviors of liposomes to obtain a more rigid vesicular structure that is much more resistant to the phospholipid extraction effect mediated by high density blood lipoproteins. In this attempt, both 1,2-distearoyl-3-sn-phosphatidylcholine and sphingomyelin are used to maintain a certain vesicular carrier integrity following intravenous administration. A rigid vesicular structure of liposomes hampers an effective adsorption of opsonines and prolongs the plasmatic level of the drug carrier. A particular class of liposomes, made up of positively char-ged lipids, is used to obtain a vesicular system characterized by a net positive charge, in particular for the potential application as a carrier for genetic material delivery [13].

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Liposomes are used with success for several applications, in particular a successful therapeutic approach is the both in vitro and in vivo delivery of various anticancer drugs [14,15]; another application is the possibility to administer antibiotic-loaded liposomes in the case of very potent drugs that present a certain toxicity (i.e., nephro- and neurotoxicity) and that can be administered intravenously [16]; they can be used as immunoadjuvants for vaccines and as macrophage activators against tumoral, viral, and microbial cells; liposomes can be efficaciously used for delivery to the central nervous system, in particular under some pathological conditions a hypermeabilization of the blood-brain barrier can occur, thus allowing the passage of very small aggregates (1 m or 4. Statistical significance compared with NTN group: *P < 0.01 (data from reference [78], with permission).

values the lower the availability of the drug to the permeation through the cellular membrane, (ii) the higher the complex solubility the greater the amount of drug reaching the surface of tumoral cells, (iii) the higher the penetration enhancer effect of CDs the greater the amount of drug that is able to permeate through the cellular membrane. Therefore, -CD is able to increase the drug solubility in a similar way to modified CDs, it can allow the transfer of the drug from the cyclodextrin cavity to tumoral cells and it can exert a cellular membrane permeation enhancer effect. 3.1.3. Delivery of Camptothecin Camptothecin is a pentacyclic alkaloid isolated from the wood and bark of Camptotheca acuminate characterized by a highly conjugated pentacyclic ring that presents an a-hydroxylactone moiety that is readily cleavable resulting in the formation of watersoluble salts. It is a potent cytotoxic drug that shows anticancer activity in several tumor models by inhibiting the enzyme topoisomerase I. The use of this natural compound has many disadvantages deriving from its poor aqueous solubility and its toxic effects: neutropenia, nausea, skin rash and alopecia. Additionally, the pharmacologically important lactone ring of camptothecin and its analogs is unstable in the presence of human serum albumin which results in the conversion of the active drug to the inactive carboxylate form bound to albumin. This fact imposes a severe pharmacokinetic limitation on the systemic use of camptothecin and related compounds. One way to reduce the toxicity of the compound and to increase stability is to include it into autogelling systems. Chitosan polymer and glycerol-2-phosphate (b-GP) have been combined to obtain a totally biodegradable and biocompatible hydrogel for the controlled delivery of camptothecin. The formulation was injected directly into a mouse fibrosarcoma (RIF-l) implanted subcutaneously in C3H mice. The characteristic of this gel is its thermosensitivity that allows it to remain liquid at low temperatures and turn into a gel

Fig. (7). Delay of RIF-1 tumor growth after intratumoral (24 mg/kg) and intraperitoneal (6 mg/kg) injections of camptothecin: , no treatment; , blank BST-gel; , intra-peritoneal injection camptothecin, 6 mg/kg; , intra-tumoral implant BST-gel/camptothecin, 24 mg/kg (data from reference [79], with permission).

A new micellar formulation containing camptothecin was recently prepared by using DSPE-PEG2000 (various molar ratios from 0.001:1 to 0.057:1) which was tested in vitro on MCF-7 cells to evaluate the cytotoxicity with respect to the free drug. The sterically stabilized micelles, obtained by the TLE method, were sonicated and characterized. The camptothecin-micelles mean size was about 13-14 nm but during the experiment a second population of large self-aggregating particles (100-300 nm) was observed in the excess of the drug. To eliminate the second micellar population studies were conduced to determine the maximum concentration of solubilized camptothecin without the formation of camptothecin aggregates in a given DSPE-PEG2000 concentration. A critical molar ratio of camptothecin/DSPE-PEG2000 = 0.0063:1 ± 0.0013:1 showed the best camptothecin solubilisation and the micellar formation without the presence of aggregates. The MCF-7 citotoxicity of sterically stabilized micelle-camptothecin with respect to that of the free drug (DMSO 10%) was not different after 24 h of treatment, while after 96 h there was an anticancer effect 3-fold greater with the camptothecin delivered in sterically stabilized micelle. This finding is due to the great stability of the drug loaded into sterically stabilized micelles, to the better cellular uptake of sterically stabilized micelle-camptothecin and to

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the time that is necessary for camptothecin to have its anti-tumoral effect in the cellular S-phase. In view of a future commercial use and in vivo experiments, the sterically stabilized micellecamptothecin formulation was lyophilized without any modification and no addition of lyoprotectants and cryoprotectants. The results obtained in terms of solubilized camptothecin concentrations, peak fluorescence emission wavelength, micelle size, and phospholipid concentration did not change significantly with respect to the non lyophilized formulation (probably because PEG moieties have a cryoprotectant action) [80]. A variety of ligands (antibodies, growth factors, cytokines) were used as vehicles for introducing drugs, proteins, and nucleic acids to target cells but this approach (with monoclonal antibodies in particular) has several disadvantages, including immunogenecity and the poor diffusion through biological barriers due to the great dimensions of the molecules. A novel camptothecin bioconjugate was recently synthesized using carbodiimide chemistry with a linear poly(ethylene glycol) (PEG) and amino acid glycine as the spacer and the linker, respectively. Folic acid was used as the targeting ligand to take advantage of the FR mediated endocytosis. For an efficient drug delivery, a carrier system should have a specific tumor localization and a high intracellular access. In this study folic acid (whose receptor is over-expressed in a variety of carcinomas) was chosen for its small dimension and for its non immunogenicity. The PEG chain, instead, was used to facilitate the folate/receptor interaction and to increase the camptothecinbioconjugate cellular endocytosis whilst glycine was used as a linker with the added advantage of locking the lactone ring of camptothecin in its active form preventing the esterfication at the 20-OH group. The cytotoxicity of camptothecin-glycine-PEG-folate conjugate was tested on KB cells (human nasopharyngeal carcinoma cell line which over-express the folate receptor) and on CHO cells (Chinese hamster ovary cells that are devoid of the FR receptor), which were used as a negative control in MTT-test. The bioconjugate showed an improved toxicity only on the KB cell line with respect to the free camptothecin probably due to the insufficient interaction and uptake with the second cell line. The tests were repeated using the camptothecin-glycine-folate conjugate obtaining no enhancement of cytotoxicity with respect to the free drug thus confirming the importance of the PEG chain for the flexibility of the bioconjugate that is fundamental for cell interaction [81]. 3.1.4. Delivery of New Antitumoral Natural Drugs In the last years new natural drugs with antitumoral properties have been used experimentally for the treatment of cancer. Tea (Camellia sinensis), the second-most popular beverage next to water, was found to have anticancer and antioxidant properties [82]; in particular, (-)-Epigallocatechin gallate (EGCG), the main active polyphenol in green tea, is responsible for benefits in treating UVinduced photodamage, basal cell carcinomas (BCCs), melanomas, and skin papillomas [83,84]. In a recent work this active compound was loaded into liposomes and its cytotoxic effect was evaluated in vitro on BCC cells by MTT-test and in vivo on BCC, HT-29 cells (colon cancer) and B16 melanoma cells which were injected into the back of nude female mice (Balb/c-nu strain, 6-8 weeks old) measuring the tumor uptake of the drug. Liposomes were made up of egg phosphatidylcholine, cholesterol, deoxycholic acid and the liquid phase was an aqueous ethanol solution (15%) containing 17.2 mM of drug. The same formulations were prepared with (+)catechin and (-)-epicatechin, EGCG derivatives, as active compounds. The loading capacity of EGCG liposomes was about 99% also after the extrusion process (~98%) which is higher than that observed for catechin and epicatechin (53% and 62% respectively). The viability test studied the cytotoxic effect on BCC cells of liposomal EGCG compared to that of an aqueous ethanolic solution (15%) of the active compound. The liposomal formulation produced an inhibition of cellular vitality of ~50% whilst the

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hydroalcholic solution provided no results at 42.5 M concentration after 24 h. The test was repeated on B16 and HT-29 cells and data obtained showed similar results. Moreover, in vivo tests have established that vesicular carrier cause an accumulation of EGCG in the tumor tissues higher than that of the free drug; in particular, in BCC carcinoma, liposome-EGCG showed an uptake of about 56 nmol/g of tumor vs ~3 nmol/g of tumor in the case of treatment with an ethanolic solution of EGCG. The difference in uptake is steady also in melanoma (B16 cells) and colon tumor (HT-29) areas with liposome/solution values of 162/0.04 nmol/g of tumor and 545/8 nmol/g of tumor respectively. The presence of ethanol in the liposomal formulation causes an increase in the uptake of the active compound in the tumor areas because it destabilizes the cellular phospholipids of the bilayers. This phenomenon is also favored by the presence of deoxycholic acid which increases the deformability of colloidal carriers causing a major localization in the carcinoma areas after injection. Moreover, liposomes preserve EGCG from degradation, in fact, it was shown that ~50% of the EGCG still remained at 2 h, while EGCG in the hydroalcoholic solution was unstable, with only 8% of the EGCG remaining [85]. Liposomes were also used for delivery of sclareol (labd-14-ene8,13-diol), which is a member of the labdane-type diterpenes. It was isolated from Cistus incanus and it has shown antimicrobial activity, cytostatic and cytotoxic effects against leukemic cell lines, cell cycle arrest and apoptosis as well as down regulation of protooncogene c-myc expression [86]. Sclareol is a lipophilic drug and was thus enclosed in the lipid phase of the vesicular system. Liposomes were made up of egg-phosphatidylcholine, dipalmitoylphosphatidylglycerol and sclareol (9:0.1:5 molar ratio, respectively) and were prepared by the TLE method. To achieve unilamellar vesicles the liposome mean size was reduced by sonication. The cytotoxic activity of the free and liposome-associated sclareol was tested in vitro on different human cancer cell lines (i.e. MCF-7, PC3, HCT116, OVCAR5) evaluating GI50 (growth inhibiting concentration 50), TGI (total growth inhibition) and LC50 (lethal concentration 50). The results indicate that sclareol induces permanent time and dose dependent damage to cells and the liposomal sclareol showed an enhanced cytotoxicity only in two cell lines (HCT116 and PC3 cells) while it exhibited minimal activity against normal cells (peripheral blood mononuclear cells or PBMCs), suggesting a potential improvement of the therapeutic index of the compound as far as its side effects are concerned. To confirm these results 1106 HCT116 cells were injected subcutaneously in the axillary region of male NOD.CB17 Prkdcscid mice, 9 weeks old, and when the mean tumor volume had reached about 100 mm3, the animals were treated i.p. with free (normal saline/absolute ethanol/PEG400/Tween-40 80:10:0.5:0.5 v/v) and liposomal sclareol. The active natural compound delivered by vesicular carriers exhibited a significant antitumoral activity with a considerable tumor regression and a total animal survival while the drug dispersion caused high toxic effects with the death of all mice during the treatment [87]. Another labdane-type diterpene, the labd-7,13-dien-15-ol, extracted from resin of ladano (Cistus creticus) was tested on different tumor cell lines (MCF-7, SF268, NCI-H460, DMS114, HCT116) in the free form (solubilised in DMSO) and loaded into a liposomal carrier (made up of egg-phosphatidylcholinedipalmitoylphosphatidylglycerol-active compound 9:0.1:5 molar ratio) [88]. The cytotoxicity was evaluated after 48, 72 and 96 h of incubation, measuring GI50, TGI and LC50. It was shown that the labdane-type diterpene compound exhibited a similar activity against all cell lines tested when incubated for 48 and 72 h except for DMS114 cells which showed a significant cytotoxicity only after 96 h. Similar results were obtained after the treatment of cells with the drug loaded in liposomal carriers as occurred previously with sclareol [86]. The liposomal formulations containing the

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labdane derivates were deprived of cholesterol (which stabilizes the structure increasing the cohesion of lipids); this can be easily explained if we compare the chemical structure of cholesterol with that of labdane-type diterpenes because their similarity is evident, Fig. (8). Another common characteristic of the two colloidal formulations is the absence of increased cytotoxicity on the different cancer cell lines with respect to the free drugs. This fact is due to the low release of the natural compounds from vesicular carriers (~20-30%) and, consequently, to the absence of exposure to the total amount of drug present in liposomes for the same period of incubation. On the other hand this phenomenon preserves the diterpenes from degradation because they are protected within liposome structure and are suitable for systemic administration. A

B

OH

HO OH

C

HO Fig. (8). Molecular structure of labd-14-ene,8-13-diol (sclareol) (A), labd7,13-dien-15-ol (B) and cholesterol (C).

A new method to treat cancer is based on the administration of drugs known as photosensitizers that are preferentially taken up and retained by malignant tissues (photodynamic therapy) [89]. Illumination of these compounds with light at the appropriate wavelength induces photochemical reactions that product tissue destruction. A natural compound used in this therapy is the hypericin, a natural compound extracted from the plant Hypericum perforatum belonging to the class of phenanthroperylenequinones, which is characterized by a high lipophilicity and which has an absorption spectrum with two major peaks at 545 and 590 nm [90]. In a recent study hypericin was encapsulated in nanoparticles, produced by the nanoprecipitation method and made up of polylactic acid (PLA), and its phototoxicity was evaluated on the ovarian cancer cell model derived from Fischer 344 rats (NuTu-19 cells). After the treatment with free and loaded hypericin in nanoparticles, the cells were irradiated and cell death was assessed by the MTT-test. After 24 h of light exposure at a drug concentration of 0.025 mg/l, hypericin-loaded PLA nanoparticles exhibited a 3-fold higher activity than the free drug. In these tests also the influence of light exposition time and drug phototossicity were evaluated. The NuTu-19 cells, after treatment with free and nanoparticle hypericin, were incubated for increasing times (from 15 min to 2 h) and irradiated at a light dose of 2.3 J/cm2 and the cytotoxicity was evaluated by the MTT-test after 24 h following light exposure. For both investigated formulations, photoactivity increased with increasing incubation times and light doses and the most important data is obtained after 1 h of light exposition 1.6 J/cm2 in which hypericin-nanoparticles exhibited a 4-fold higher phototoxicity than the free drug. The different efficacy of hypericin

formulations could be explained by two considerations: firstly, the encapsulation of hypericin in nanoparticles increases the amount of drug in NuTu-19 cells (probably by an endocytosis process) and, secondly, the better uptake may modify the intracellular distribution in the different compartments of the compound with a specific subcellular localization which could determine the mechanism of cell death [91]. 3.2. Antioxidant and Anti-Inflammatory Drugs Glycyrrhizinates, are triterpene saponins extracted from Glycyrrhiza glabra L. that present an anti-inflammatory activity, due to an indirect strengthening of the glucocorticoid activity. Various salts are present in commerce and these compounds have been delivered by means of colloidal carriers. Trotta et al. [92] evaluated the possibility of using a particular class of liposomes for skin delivery of dipotassium glycyrrhizinate for the treatment of acute and chronic dermatitis. Dipotassium glycyrrhizinate was mixed with soy lecithin or hydrogenated soy lecithin for the preparation of liposomes by means of the solvent evaporation method and the vesicles were then passed through a high pressure homogenizer. Liposome size and entrapment efficiency were determined and the interaction between dipotassium glycyrrhizinate and soy lecithin was investigated using differential scanning calorimetry. Transepidermal permeation through intact pig skin, skin deposition of dipotassium glycyrrhizinate from liposomes and O/W emulsion containing liposomes were assessed and compared with values for aqueous control solutions. The results showed that the entrapment efficiency depended on the lipid: dipotassium glycyrrhizinate ratio; the maximum efficiency was obtained at 4:1 w/w. Dipotassium glycyrrhizinate interacted with liposomes disrupting and fluidizing the lipid bilayer, forming elastic liposomes able to penetrate through membrane pores of diameter much smaller than their own diameter. The skin fluxes were less than the HPLC detection limit for all systems, while skin deposition increased 4.5-fold compared with aqueous solutions when dipotassium glycyrrhizinate was formulated in liposomes (Table 2). Another salt of this compound, ammonium glycyrrhizinate, was loaded into Ethosomes® [93]. Some formulations at different ethanol and soybean phosphatidylcholine percentages were prepared to evaluate the effect of the composition on both the mean size and size distribution of this colloidal carrier. Ethosomes® showed a narrow particle size distribution, in particular the ethosomal formulation prepared with the greatest amount of ethanol. While, the concentration of lecithin used for Ethosome® preparation influenced the vesicle mean size in a different way, namely the higher the lecithin concentration the larger the Ethosome® mean size. Photon correlation spectroscopy analysis was also carried out on ammonium glycyrrhizinate/Ethosomes®. In both cases, at the concentrations investigated no significant (p