Development and ex vivo evaluation of 5-aminolevulinic acid-loaded niosomal formulations for topical photodynamic therapy

Accepted Manuscript Title: Development and ex vivo evaluation of 5-aminolevulinic acid-loaded niosomal formulations for topical photodynamic therapy Author: Marco Bragagni Andrea Scozzafava Antonio Mastrolorenzo Claudiu T. Supuran Paola Mura PII: DOI: Reference:

S0378-5173(15)30133-2 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.08.036 IJP 15119

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

29-5-2015 30-7-2015 12-8-2015

Please cite this article as: Bragagni, Marco, Scozzafava, Andrea, Mastrolorenzo, Antonio, Supuran, Claudiu T., Mura, Paola, Development and ex vivo evaluation of 5-aminolevulinic acid-loaded niosomal formulations for topical photodynamic therapy.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.08.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development and ex vivo evaluation of 5-aminolevulinic acidloaded niosomal formulations for topical photodynamic therapy Marco Bragagnia, Andrea Scozzafavaa, Antonio Mastrolorenzob, Claudiu T. Supuran, Paola Muraa* [email protected] a

Department of Chemistry, University of Firenze, Polo Scientifico di Sesto Fiorentino, 50019 Sesto Fiorentino (Firenze), Italy b MST Center, Tuscan Orthopedic Institute Piero Palagi, University of Firenze, Viale Michelangelo 41, 50125 Firenze, Italy c Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Firenze, Polo Scientifico di Sesto Fiorentino, 50019 Sesto Fiorentino (Firenze), Italy *

Corresponding author.: Tel.: 0039 055 4573672

Graphical Abstract

ABSTRACT The objective of this study was the development of a niosomal formulation for improving skin permeation and penetration of 5-aminolevulinic acid (ALA) in the treatment of skin malignancies by photodynamic therapy (PDT). Different niosomal dispersions were prepared, using two different preparation methods. The effect of addition to a classic formulation, consisting in an equimolar Span 60-cholesterol mixture, of two different edge activators, dicethyl-phosphate (DCP) and sodium cholate (SC), and of the presence of ethanol on the vesicle properties and stability was evaluated. Selected formulations were loaded with the drug and evaluated for physicochemical and stability properties and encapsulation efficiency. Classic and elastic DCP-containing niosomes were the only formulations able to effectively incorporate the drug without instability problems. Ex vivo permeation and penetration studies through excised human skin showed that both the niosomal formulations were significantly more effective in improving ALA skin delivery than the simple aqueous drug solution commonly used in clinical practice, allowing, respectively, an increase of about 80 and 40 % of the drug permeated amount and of about 100 and 50% of the drug retained into the skin. These results lead to consider the developed formulations potentially useful for improving

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ALA bioavailability and therapeutic effectiveness in skin malignancies treatment by topical PDT. Keywords: 5-aminolevulinic acid; photodynamic therapy; niosomes; edge activators; ex-vivo skin permeation; ex-vivo skin-penetration.

1. Introduction Photodynamic therapy (PDT) is a non-invasive technique used in the treatment of some epithelial skin tumors (Ericson et al., 2008). Essentially, PDT is based on the use of a photosensitizer which, upon irradiation with light of suitable wavelength, gives rise to tumor cells destruction and failure of tumor vascularization, due to a reduction in oxygen-carrying capacity of the blood (Kalka et al., 2000; Szeimies et al., 2005). Topical administration of 5-aminolevulinic acid (ALA), an endogenous metabolite present in mitochondria, and converted, via the haem cycle, into the photoactive protoporphyrin IX (PpIX), has been widely employed in the PDT treatment of superficial skin carcinomas (De Rosa et al., 2003). According to the commonly employed PDT protocol, a concentrated aqueous solution (20% w/w) of ALA is applied on the lesion, kept 3-6 h in contact, and then removed, and the lesion irradiated (Braathen et al., 2007). ALA-based PDT therapy offers advantages over conventional treatments such as surgery, cryosurgery, or radiotherapy, including low morbility, minimum functional disturbance, good tolerance and excellent cosmetic outcomes (Ericson et al., 2008) However, the major limitation of this therapy is the limited ability of the hydrophilic ALA molecules to cross the skin stratum corneum. Furthermore, the high concentrations used to compensate the low ALA skin penetration ability, cause problems of skin irritations. Recently, several strategies were investigated to enhance ALA skin penetration, evidence of the great interest aimed at improving the clinical success of ALA-based PDT (Zhang et al., 2011). These strategies include the synthesis of lipophilic ALA derivatives (De

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Rosa et al., 2003; Fotinos et al., 2006), the use of physical methods (Lopez et al., 2003, Donnelly et al., 2008), the addition of penetration enhancers (Auner et al., 2003), or the development of carriers such as microemulsions (Araujo et al., 2010) nanoparticles (Chung et al.,2013; Shi et al., 2013), or liposomes (Pierre et al., 2001, Kosobe et al., 2005; Casas and Batlle, 2006; Fang et al., 2008a and 2008b; Oh et al., 2011). Among these different approaches, the use of liposomes or niosomes as topical drug carriers can offer several advantages, providing a sustained release and acting as permeation enhancer (Fang et al., 2001). In the case of ALA, an additional benefit could be the protection of the molecule, which suffers a rapid degradation in physiological conditions (Bunke et al., 2000). However, in spite of the promising results in improving drug skin penetration, liposomal formulations of ALA exhibited low entrapment efficiency values, together with typical problems of limited stability (Pierre et al., 2001, Kosobe et al., 2005; Casas and Batlle, 2006; Fang et al., 2008a and 2008b; Oh et al., 2011). Niosomes are amphiphilic vesicles formed by synthetic non-ionic surfactants, which represent an interesting alternative over traditional liposomes, since, although structurally similar, they offer a series of potential advantages, including greater physical and chemical stability, longer shelf-life, greater ease of production and storage, lower cost, and wider formulation versatility (Uchegbu, I.F., Vyas, S.P., 1998; Fang et al., 2001; Sankar et al., 2009; Yadav et al., 2011). However, at the best of our knowledge, encapsulation of ALA into niosomes has not yet been investigated. Based on these premises, the aim of this work was the development and characterization of a niosomal formulation aimed at obtaining an efficient ALA topical delivery and penetration into the skin. Equimolar amounts of sorbitan monostearate and cholesterol were employed as base components for the vesicles preparation, and the effect of addition of sodium cholate or dicetyl phosphate was investigated. In fact, the presence of charged surfactants in the vesicle bilayer should increase the membrane deformability and elasticity

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and promote its interactions with the biological barriers, thus improving the skin permeation of active compounds (Karande and Mitragotri, 2009; Cevc, 2004; El Zaafarany et al., 2010). The effect of ethanol addition to the formulation was also examined, considering the enhancer effect shown by this solvent, when included in colloidal vesicles, in improving permeation of active compounds through the skin by both increasing the vesicle elasticity and fluidizing the lipid domain of the stratum corneum (Ting et al., 2004; Fang et al., 2006 and 2009; Kumar and Rao, 2012). Two distinct methods (thin layer and reverse phase evaporation), were used to prepare the niosomal formulations. The results of this phase of the study allowed a rational selection of the best formulations which were loaded with ALA and fully characterized for particle size, polydispersity, Zeta potential, encapsulation efficacy and stability under storage at room temperature. The skin permeation properties and penetration ability of ALA from the developed niosomal formulations were determined ex vivo, using excised human skin samples, and compared with the simple ALA aqueous solution commonly used in PDT.

2. Materials and methods 2.1. Materials 5-aminolevulinic acid (ALA), sorbitan monostearate (Span 60), cholesterol (CHL), sodium cholate (SC), dicetyl phosphate (DCP) and all the solvents and reagents were purchased from Sigma-Aldrich (Milan, I). Water was obtained from a Milli-Q water purification system Millipore (Billerica, Massachusetts, USA). 2.2. Preparation of niosomes In a preliminary screening, a series of niosomal suspensions were prepared using two different methods. 2.2.1. Thin-layer evaporation method The Span 60-CHL mixture (1:1 mol:mol), in the presence or not of SC or DCP (1:1:0.28 mol:mol), was dissolved in a 2:1 (v/v) chloroform-methanol solution in a round-bottom flask;

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the solvent was then removed at 40 °C by rotavapor (Laborota 4000, Heidolph, D), to form a thin layer on the flask wall. The film was left 20 min under vacuum at 50 °C, to completely remove traces of the solvents, and then hydrated with 10 ml of deionized water or ethanolic solution (20% v/v); 5 cycles of heating (5 min at 60 °C) and vortex mixing (2 min) were performed. The obtained suspension was subjected to 3 cycles of heating (3 min at 50 °C) and bath sonication (2 min) (Eurosonic 44 ultrasonic bath, Anwendungstechnik, Offenbach, D). The suspension was left cooling down and probe sonicated 5 min in a ice bath using a Sonopuls HD2070 (Bandelin GmbH, Berlin, D) equipped with a DS73 titanium probe, setting the instrument at 50% of the power. The suspensions were finally sealed and stored at room temperature. 2.2.2. Reverse phase evaporation method The Span 60-CHL mixture (1:1 mol:mol), in the presence or not of SC or DCP (1:1:0.28 mol:mol), was dissolved in 10 mL of diethyl ether and 5 mL of distilled water were added; the resulting dispersion was probe sonicated 5 min in an ice bath (Sonopuls HD 2070) to form a W/O emulsion. Diethyl ether was then removed by rotary evaporation (5 min at 40 ºC). The vesicles were then hydrated with 5 mL of deionized water and probe sonicated in a ice bath (5 min at 50% power). The suspensions were sealed and stored at room temperature. 2.2.3. Preparation of ALA-loaded niosomes ALA-loaded niosomes were prepared according to the above described methods, under the same conditions used for preparing blank niosomes, by dissolving ALA in the hydrating solution, so that to have a constant drug concentration of 20 mg per mL of niosomal dispersion. 2.3. Characterization of niosomal suspensions

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Both empty and drug-loaded vesicles were characterized for particle size, polydispersity index (PDI) and Zeta potential. For empty vesicles the analyses were carried out before and after probe sonication, to investigate the effect of the treatment. 2.3.1 Particle size, PDI and Zeta potential analysis The average particle size, PDI and Zeta potential of the vesicles were determined by quasielastic light scattering (QELS), using a Zetasizer Nano-ZS90 (Malvern Instruments, Malvern, UK), after a 1:20 dilution of the samples in order to reduce multiscaterring effects. Each sample was measured at least 5 times at 25 ± 0.1 ºC. 2.3.2 Determination of drug encapsulation efficiency (EE%) Encapsulation efficiency (EE%) was determined using the dialysis technique for separating the non-entrapped drug from niosomes. The suitability of this technique (which gave results comparable to those obtained by the ultracentrifugation technique) has been previously demonstrated (Mura et al., 2007; Bragagni et al., 2012a). Briefly, 2.5 mL of the drug-loaded niosomal dispersions was dropped into a cellulose acetate dialysis bag with MW cut-off 12000 (Spectra/Por®, Spectrum Laboratories, CDN), sealed, immersed in 400 mL of distilled water and magnetically stirred at 20 rpm. Samples (2 ml) taken at time intervals from the receiver solution were transferred to assay tubes, and replaced with an equal volume of fresh solvent. From each taken sample, 60 μL were withdrawn, diluted with 1380 μL of acetate buffer and 60 μL of ethylacetylacetate, heated 10 min at 100 ºC, cooled down and analyzed for drug assay by UV-VIS spectrophotometry as described below. The experiment was stopped when constant drug-concentration values were obtained in subsequent withdrawals from the receiver phase (taking into account the progressive dilution of the medium). The percent of encapsulation efficiency (EE%) was calculated according to the following equation:

EE % 

[total.drug ]  [diffused .drug ] .100 [total.drug ]

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Each result is the mean of at least three separate experiments. 2.4 Assay of ALA The quantitative assay of ALA was performed according to a specific colorimetric method (Mauzerall and Granick, 1956; Tomokuni and Hiral, 1986). In this method ALA is condensed by reaction with ethyl acetoacetate to a pyrrole, which reacts with 4(dimethylamino)benzaldehyde (DMAB) (Modified Ehrlich’s reagent), forming a purple colored compound, which is assayed by UV-visible spectrophotometry. Acetate buffer was prepared by dissolving 81.98 g of sodium acetate anhydrous in 57 mL of glacial acetic acid and 900 mL of water. The pH was adjusted to 4.6 and the solution diluted to 1 L with water. For the preparation of the modified Ehrlich’s reagent, 1 g of DMAB was dissolved in 40 mL of glacial acetic acid and 8 mL of 70% perchloric acid was added; the solution was then diluted to 50 mL with glacial acetic acid. Each time, the solution was used the same day of its preparation. For obtaining the calibration curve, a stock solution was made by dissolving 10 mg of ALA in 100 mL of acetate buffer. Then, aliquots of 5 mL acetate buffer and 0.2 mL ethyl acetoacetate were placed in 6 volumetric flasks of 10 mL and mildly agitated. Increasing amounts of stock solution were added to the flasks and the solutions were brought to volume with buffer, to obtain a concentration range of ALA from 1 to 6 mg/L. The solutions were heated 10 min at 100 °C, cooled down and diluted in 1:1 ratio with modified Ehlrich’s reagent, accurately taking note of the time. The solutions progressively turned from transparent to purple. The absorbance was measured 15 min after each dilution, using a UV/VIS 1601 spectrophotometer (Shimatzu, Tokyo, J), set at 545 nm. 2.5. Stability studies Stability studies of empty and drug-loaded niosomal dispersions were carried out by storing samples at room temperature (25 °C) for 3 months and monitoring them every 30 days

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for physical stability (visual observation and mean size determination) and drug entrapment efficiency. 2.6. Ex vivo permeation studies A sample of human skin was obtained from an abdominal reduction procedure of a single donor. Immediately after the surgical intervention, the subcutaneous fat tissue was removed by a lancet. The remaining tissue, formed by stratum corneum and epidermis, was divided into portions of approximately 1.5 × 1.5 cm which were washed with pH 7.4 phosphate buffer solution (PBS), frozen and stored at -80 °C. Before the experiments each sample was thawed out, gently dried by filter paper, weighted and mounted in a Franz diffusion cell (Rofarma, I) with an orifice diameter of 10 mm. The dermal side of the skin was in contact with the receptor phase, which was PBS (7 ml) maintained at 37 °C under gentle agitation. The donor chamber was loaded with 1 ml of the formulation to study. In all cases, the amount of loaded drug was 2% w/v. Every 30 min, 0.5 ml of solution was removed from the receiving compartment, opportunely diluted with acetate buffer and ethyl acetoacetate and the drug concentration assayed as above described. Each time an equivalent volume of fresh medium was reintroduced. This dilution effect was taken into account in the calculations. Each result is the mean of five separate experiments. After 4 h the samples of human skin were removed, accurately washed with PBS and dried by filter paper, and treated to determine the amount of ALA retained into the tissue. The human skin portions were cut into thin slices (approximately 3 mm wide) which were transferred to a glass vial containing 1 mL of methanol; the vial was closed and the content gently stirred for 12 h. The dispersion was centrifuged (Hermle Z200A, USA) at 6000 rpm for 15 min and the supernatant was separated. The extraction procedure was repeated twice on the precipitate, using 1 mL of fresh methanol each time. The three fractions of supernatant were collected and left 12 h under nitrogen reflux, to completely remove the solvent. The residual

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was finally dissolved in acetate buffer and ethyl acetoacetate and the drug concentration was assayed as above described. Each result is the mean of five separate experiments. 2.7. Statistical analysis Results of vesicle characterization, stability studies and permeation experiments were statistically analyzed by ANOVA (one-way analysis of variance) followed by the StudentNeuwman Keuls multiple comparison post-test (GraphPad Prism, version 4). The differences were considered statistically significant when P<0.05.

3. Results and Discussion 3.1. Selection of the best formulation and preparation method of niosomes The goal of the present work was the development of an ALA niosomal formulation able to assure an effective delivery of the drug into the deeper strata of the skin and overcome the problems of limited stability under storage typical of liposomal formulations. Since in the development of a vesicular formulation there are numerous factors which can sensitively influence the performance of the final preparation, before proceeding to the preparation of ALA-loaded niosomes, a preliminary screening was carried out to opportunely select the most suitable preparation method and component composition in order to obtain vesicles with homogeneous and sufficiently small dimensions. With this aim, nine blank niosomal suspensions were prepared according to the composition and the preparation protocols reported in Table 1. The basic formulations consisted in an equimolar mixture of Span 60 and CHL. Since the skin penetration ability of vesicular systems such as liposomes or niosomes can be improved by the presence in the lipid bilayer of a charged surfactant (Mura et al., 2008; Maestrelli et al., 2010; Bragagni et al., 2012b), the effect of the addition of two potential “edge activators”, such as SC and DCP was investigated. Preliminary experiments showed that addition at the Span 60-CHL mixture of SC at 1:1:0.3 mol:mol ratio resulted in an

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unstable formulation, rapidly giving rise to evident flocculation phenomena upon film hydration. Therefore a 0:28 mol content of charged surfactant was used as the highest concentration compatible with the used Span 60-CHL mixture. Moreover, in the case of formulations prepared by thin layer evaporation, the effect of using an ethanolic rather than an aqueous solution for the film hydration was examined, considering the enhanced skin delivery of various therapeutic agents mediated by ethanolic vesicles (Fang et al., 2006; Dubey et al., 2007a, 2007b; Manosroi et al., 2008). The efficacy and safety of ethanol as skin permeation enhancer is well documented in the literature (Dayan and Touitou, 2000; Touitou et al., 2000; Lopez Pinto et al., 2005; Fang et al., 2009; Kumar and Rao 2012), and it ha been found that ethanolic vesicles are more efficient than simple hydroalcoholic solutions (Ting et al.,

2004). Both preparation methods used allowed the formation of translucent colloidal dispersions, without sedimentation, flocculation or layer separation phenomena. However, the obtained vesicles were relatively poorly homogeneous, with PDI values in all cases higher than 0.5 and their mean diameter ranged from 830 to 1381 nm (results not shown). The probe sonication (5 min, power 60%) of the dispersions allowed a noticeable reduction of both the mean dimensions and the polydispersity of the vesicles, providing the results presented in Table 2. Therefore, on the basis of these findings, it was established to assume the probe sonication procedure as part of the preparation protocol of the drug-loaded vesicles. As for the preparation method, the reverse phase evaporation technique generally allowed the obtainment of more homogeneous dispersions in comparison with the corresponding formulations prepared by thin layer evaporation, as shown by their PDI values, which represent an estimation of the width of the dimensional distribution of the vesicles (see B vs A, D vs C and F vs H). On the other hand, the thin layer evaporation method gave rise in

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general to vesicles of smaller dimensions. Finally, no effects of the preparation method on the vesicle Zeta potential were found. Regarding the composition of the niosomal dispersions, the formulations not containing edge activators, (i.e. A, B and G), exhibited the highest values in terms of vesicle mean size. Among the other preparations, the smallest vesicles were obtained for the formulation containing DCP and ethanol (formulation E). Instead, no relation was found between the vesicle composition and the PDI. The best value in terms of dispersion homogeneity was exhibited by the formulation containing SC and ethanol (formulation I). Finally, as expected, elastic niosomes showed higher negative Zeta potential values, due to the presence of the anionic surfactants DCP and SC. The physical stability of the nine suspensions of empty niosomes was investigated by both visual observation of possible macroscopic aggregation or phase separation phenomena, and particle size monitoring, through light scattering measurements repeated every 30 days for 3 months. Formulations of classic niosomes (batches A and B) were substantially stable, also in presence of ethanol (batch G), despite their lower Zeta potential values, exhibiting a less than 15% increase in vesicle mean diameter 3 months after their preparation. This was probably due to the more rigid structure of the vesicle membrane. On the contrary, all formulations of elastic niosomes containing SC (batches F, H and I), regardless of the preparation method and the presence or not of ethanol, presented evident flocculation phenomena after few days from the preparation. As for elastic niosomes containing DCP, batches C and D provided the best results in terms of physical stability, showing the lowest increase of the vesicles mean size after the 3 month’s storage (only 6% for C and 8% for D). On the other hand, the presence of ethanol in such formulation (batch E) gave rise to clear flocculation phenomena after a week from the preparation Thus, formulations F, H, I and E were excluded from further studies.

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3.2. Preparation and evaluation of ALA loaded-niosomes On the basis of the results of this initial screening, formulations A, B, C, D and G were selected for the following studies of encapsulation of the drug, which was dissolved in the hydration phase of the vesicles in order to have a final concentration of 2% (w/v). All the obtained niosomal dispersions were probe sonicated (5 min, 60 % power) as in the case of empty vesicles. The characteristics of the ALA-loaded niosomal formulations in terms of mean size, PDI, Zeta potential and encapsulation efficiency (EE%) are presented in Table 3. The best results of encapsulation efficiency, with values around 80 %, were obtained for formulations A and B, which have the same composition but were prepared using two different techniques. In both cases the encapsulation of ALA caused only a limited increase in the vesicle size and PDI with respect to the corresponding blank niosomes (Table 2). Instead, in the case of formulation D, the presence of the drug destabilized the niosomal suspension, causing a marked increase of the vesicle mean size and PDI. Furthermore, the encapsulation efficiency studies evidenced that the drug was not incorporated, or it was very rapidly released from the vesicles during the dialysis. These results seem to be related to the vesicle preparation method, i.e. the reverse phase evaporation method, considering that the formulation C, obtained by the thin layer method, has the same composition but it did not present such problems. Similar negative effects in terms of increase in vesicle dimensions and loss of homogeneity occurred for formulation G, even though in this case ALA was effectively included into the vesicles, with a percentage of encapsulation of 56.1%. The presence in this formulation of ethanol (which is instead absent in the corresponding formulation B) seems to be responsible for such unforeseen result. Finally, in the case of formulation C, the drug encapsulation efficiency was significantly lower with respects to formulations A and B (40.1%), but, unexpectedly, the vesicle mean size and the PDI values were lower than those of the corresponding formulation without the drug. However, this EE%

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value can be considered still satisfying, being higher than values previously obtained for ALA liposomal formulations (Pierre et al., 2001, Fang et al., 2008, Oh et al., 2011) On the basis of these findings, formulations D and G were discarded, while formulations A, B and C were selected and submitted to stability studies on storage. All the three selected formulations demonstrated good physical stability, showing the absence of evident macroscopic effects of flocculation and an increase in mean diameter and PDI values less than 15% after 3 months storage at room temperature (25 °C). The reduction of drug encapsulation efficiency of these systems after 3 months storage at ambient temperature was not significant (P>0.05) with respect to the corresponding freshly prepared samples. 3.3. Ex vivo skin permeation and penetration studies Considering that formulations A and B had identical compositions and comparable dimensional distributions as well as analogous stability under storage, only the latter was selected for the following ex vivo permeation studies, considering its slightly higher encapsulation efficiency and greater homogeneity. Also formulation C was chosen for ex vivo permeation studies, since, although the drug encapsulation of these vesicles was significantly inferior compared to B, the presence of the edge activator in the vesicles membrane, and their significantly (P<0.05) lower dimensions could be of substantial importance for favoring the penetration of ALA into the deepest strata of the skin. Ex vivo penetration and permeation studies through excised human skin of ALA from the selected niosomal formulations were performed at 37°C, using as receiving phase a pH 7.4 buffered aqueous solution, simulating the plasmatic compartment. A simple aqueous solution of ALA at the same concentration as in the niosomal dispersions was used as control, as commonly made in this kind of studies (Pierre et al., 2001; Fang et al., 2009; Araujo et al., 2010), since this is the ALA preparation usually utilized in clinical procedures. On the other hand, it was not possible a comparison of our results with those obtained by other authors

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using ALA liposomal formulations, because widely varying experimental conditions from study to study have been observed. The results of the permeation experiments are presented in Figure 1. It is evident that both the classic (batch B) and elastic (batch C) niosomal formulations of ALA were able to significantly (P<<0.05) improve the drug permeation properties through the skin, with respect to its simple aqueous solution, allowing an about 80 and 40 % increase, respectively, of the drug permeated amount at the end of the experiment. However, elastic niosomes (batch C), in spite their lower dimensions and the presence of DCP, which should enhance their ability to penetrate through the skin, were less effective than classic niosomes in improving the ALA skin permeation rate. Such unexpected result could be reasonably attributed to the about 50% lower drug encapsulation efficiency of elastic niosomes with respect to the classic ones. On the other hand, separation of non-entrapped drug from the drug-loaded niosomes was considered not economically viable. In fact, it would result in the loss of about 60 % of the drug initially added to the preparation, with a consequent strong increase in the final formulation price, owing to the considerable cost of the drug. The results of studies of the drug penetration ability through the skin showed the same trend observed in the permeation studies. Figure 2 shows the amounts of ALA retained in the human skin samples at the end of permeation experiments from the different formulations tested. As expected, both classic and elastic niosomal formulations significantly (P<<0.05) improved the drug penetration ability into the skin layers with respect to the simple aqueous suspension, allowing an about 100% and 50% increase, respectively, of the drug amount retained. This positive effect of niosomal carriers can be explained as a probable consequence of vesicles-skin interactions, providing a deposit effect of ALA in the skin. However, analogously to that observed in the permeation studies, the ALA retention in the human skin

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was significantly (P<0.05) higher in the case of classic niosomes (batch B) compared to the elastic niosomes (batch C), reasonably for the same reason above illustrated.

4. Conclusions An initial screening on different blank niosomal formulations allowed a rational selection of the best preparation method and vesicle composition for obtaining stable and homogeneous nanometric niosomal dispersions, suitable to incorporate the PDT agent ALA. Classic niosomes based on Span 60 and CHL gave the highest encapsulation efficiency, greater than 80%, while elastic niosomes containing DCP exhibited a lower but always good encapsulation efficiency, near to 40%. The developed formulations proved to be more effective than previously developed ALA liposomal formulations in terms of EE%, and stability, resulting perfectly stable after 3 months storage at room temperature Ex vivo skin permeation and penetration studies, performed using excised human skin, showed that both the selected niosomal formulations allowed a significant improvement of ALA permeation and penetration properties through the skin with respect to the simple drug aqueous solution usually applied in clinical practice. However, unlike what expected, classic niosomes were significantly more effective than the elastic ones, probably in virtue of their higher encapsulation efficiency. The ex vivo delivery performance showed by classic niosomes lead to consider this formulation useful for improving ALA bioavailability and therapeutic effectiveness in skin malignancies treatment by topical PDT. Further studies are in progress at the Piero Palagi Hospital of Florence (I) to evaluate the actual efficacy of the developed ALA niosomal formulation in PDT therapy on human volunteers.

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Figures Captions Fig. 1 - Permeation studies through excised human skin of ALA from a simple solution of the drug (), a dispersion of classic niosomes (batch B, ) and a dispersion of elastic niosomes (batch C, ), all containing the same drug concentration (2% w/v). See Table 1 for batch formulation composition; each result is the mean of five separate experiments.

Fig. 2 - Amount of ALA penetrated into excised human skin after 240 min application as aqueous solution, classic niosomal dispersion (batch B), and elastic niosomal dispersion (batch C), all containing the same drug concentration (2% w/v). See Table 1 for batch formulation composition; each result is the mean of five separate experiments.

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Tables Table 1. Components and preparation protocols of blank niosomes. sample Components (mmol) Components molar ratio A B C D E F G H I

Span 60 (10), CHL (10) Span 60 (10), CHL (10) Span 60 (8.77), CHL (8.77), DCP (2.46) Span 60 (8.77), CHL (8.77), DCP (2.46) Span 60 (8.77), CHL (8.77), DCP (2.46) Span 60 (8.77), CHL(8.77), SC (2.46) Span 60 (10) : CHL (10) Span 60 (8.77), CHL(8.77), SC (2.46) Span 60 (8.77), CHL(8.77), SC (2.46)

1:1 1:1 1:1:0.28 1:1:0.28 1:1:0.28 1:1:0.28 1:1 1:1:0.28 1:1:0.28

Preparation technique Thin layer Reverse phase Thin layer Reverse phase Thin layer + EtOH Reverse phase Thin layer + EtOH Thin layer Thin layer + EtOH

Table 2. Mean size, PDI and Z-potential of the different niosomal formulations (see Table 1 for batch formulation composition) Sample Size (nm) PDI Z-pot (mV) A

603.9 ±91.7

0.344 ±0.044

-12.1±1.4

B

555.2 ±32.1

0.270 ±0.026

-13.3±0.8

C

256.8 ±34.4

0.358 ±0.038

-31.9±0.9

D

328.3 ±47.7

0.295 ±0.015

-35.8±0.5

E

217.5 ±38.4

0.481 ±0.079

-30.9±0.4

F

492.0 ±83.0

0.373 ±0.023

-24.8±0.9

G

601.9 ±90.1

0.451 ±0.036

-9.0±1.0

H

295.4 ±25.9

0.421 ±0.048

-25.3±0.3

I

356.2 ±26.8

0.252 ±0.010

-25.0±0.4

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Table 3. Mean size, PDI, Z-potential and encapsulation efficiency (EE%) of the selected ALA-loaded niosomal formulations (see Table 1 for batch formulation composition). Sample Size (nm) PDI Z-pot (mV) EE% A

655.2 ±68.9

0.402 ±0.180

-15.3±0.3

74.8±2.1

B

611.2 ±63.3

0.340 ±0.110

-12.4±1.4

81.1±2.3

C

221.1 ±26.0

0.266 ±0.011

-28.4±0.6

40.1±1.6

D

1515 ±211.3

0.579 ±0.220

-31.9±1.2

0.90±0.3

G

1849 ±163.4

0.564 ±0.235

-8.6±0.6

56.1±1.8

Fig 1

Fig 2

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