Nanotechnology

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Nanovesicle-based formulations for photoprotection: a safety and

efficacy approach
To cite this article before publication: Cristal Cerqueira et al 2019 Nanotechnology in press https://doi.org/10.1088/1361-6528/ab177c

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Page 1 of 30 AUTHOR SUBMITTED MANUSCRIPT - NANO-121056.R1

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4 NANOVESICLE-BASED FORMULATIONS FOR
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6 PHOTOPROTECTION: A SAFETY AND EFFICACY APPROACH
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8 Cristal Cerqueira1, Fiammetta Nigro1, Vânia E. B. Campos1, André Rossi2, Ralph

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10 Santos-Oliveira3, Verônica Cardoso4, Alane Beatriz Vermelho4,
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12 Elisabete P. dos Santos5 and Claudia Regina E. Mansur1,6
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15 Federal University of Rio de Janeiro, Institute of Macromolecules, Center of
16 Technology, Ilha do Fundão, Rio de Janeiro, Brazil, ZIP Code: 21945-970
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18 Brazilian Center for Research in Physics, Rio de Janeiro, Brazil
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20 Nuclear Engineering Institute, Rio de Janeiro, Brazil

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BIOINOVAR - Biotechnology Laboratories: Biocatalysis, Bioproducts and Bioenergy,
23 Paulo de Góes Institute of Mirobiology, Federal University of Rio de Janeiro, Rio de
24 Janeiro, Brazil
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Federal University of Rio de Janeiro, Faculty of Pharmacy, Department of Drugs and
Medicines, Laboratório de Desenvolvimento Galênico (LADEG), Ilha do Fundão, Rio de
Janeiro, Brazil
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31 Federal University of Rio de Janeiro, Programa de Engenharia Metalúrgica e de
32 Materiais /COPPE, Ilha do Fundão, Rio de Janeiro, Brazil
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34 Corresponding author: cristalcerqueira@gmail.com
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ABSTRACT
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41 Vesicular nanosystems are versatile and they are able to encapsulate actives with
42
43 different solubilities, such as lipophilic and hydrophilic compounds. The most well-
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45 known vesicular nanosystems are liposomes and niosomes, the last one is formed by
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non-ionic surfactants. In the present work, it was developed photoprotective
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48 niosomes containing sunscreens (octyl methoxycinnamate, diethylamino
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50 hydroxybenzoyl hexyl benzoate and phenylbenzimidazole sulfonic acid), non-ionic
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52 surfactants, cholesterol and stearylamine (positive-charged lipid). Studies based in

53 dynamic light scattering techniques (DLS), entrapment efficiency and morphology by
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55 TEM were performed to characterize the niosomes. In addition, rheology, pH, in vitro
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57 SPF efficacy and toxicity and in vivo and in vitro safety were determined for the
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niosome formulations F-N1 and F-N2. The mean size of the N1 and N2 was 168 ± 5
60 nm and 192 ± 8 nm, respectively, and their morphology were spherical, unilamellar,

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3 with an entrapment efficiency of more than 45% for each sunscreen. Both
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5 formulations, F-N1 and F-N2 presented characteristic of pseudoplastic non-
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7 Newtonian fluids, showing declining viscosity with increasing shear rate applied. SPF
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values were considered satisfactory, 34 ± 8 for formulation F-N1 and 34 ± 5 for F-N2.

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10 The formulations did not presented toxicity when tested in macrophages and pH was
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12 compatible with skin, which minimizes allergies. In vitro safety assay showed
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lipophilic sunscreens’ greater affinity for the epidermis, since this layer contains

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15 natural lipids. In vivo safety assay suggests that the increased skin retention of N2 is
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17 directly correlated with the positive charge of stearylamine. Stable photoprotective
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19 niosomes were obtained and showed to be promising nanostructures to be used
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against solar radiation.
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26 Keywords: Niosomes. Sunscreens. DLS. TEM. In vitro efficacy evaluation. In vivo
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and in vitro safety evaluation.
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3 1. INTRODUCTION
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7 Nanotechnology has been used to improve the properties of active principles,
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such as stability and solubility, with the development of nanoparticles, cyclodextrins,

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10 nanoemulsions and vesicular nanosystems (liposomes and niosomes) (Cerqueira-
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12 Coutinho, Santos-Oliveira et al., 2015). In this context, vesicular nanosystems could
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present versatile structures and are able to encapsulate active compounds with

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15 different natures, both lipophilic and hydrophilic (Manconi et al., 2003; Siddiqui et al.,
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17 2012; Moghassemi e Hadjizadeh, 2014).
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19 Vesicular nanosystems such as niosomes could be applied topically, such as
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for photoprotection. Besides giving stability to sunscreens, they could act as
22 controlled release systems of these compounds, forming a reservoir that releases the
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24 sunscreen gradually into the skin as they fuse with the lipids of the stratum corneum
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26 (Cerqueira-Coutinho et al., 2015). Niosomes could also increase the retention of
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sunscreens in the skin, and thus increase their safety, since these molecules could
be harmful if they permeate through the skin and enter the bloodstream. If they
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31 remain on the skin surface (stratum corneum), the sunscreens will work more
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33 effectively in protecting the skin from the damaging effects of ultraviolet (UV)
34 radiation, such as skin cancer (Shaath, 2007).
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36 The name niosome comes from “nio-” (nonionic) and “-some” (vesicle).
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38 Therefore, all niosomes are formed by nonionic surfactants, polymeric or not (Shen et
39
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al., 2014). Niosomes are biocompatible, biodegradable, nontoxic, nonimmunogenic,
41 noncarcinogenic and resistant to hydrolysis. The properties of niosomes depend on
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43 the composition of the bilayer and the method of preparation. Just like liposomes,
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45 niosomes are designed to mimic cell structures through the arrangement of the
46 bilayer, so they could be considered biomimetic membranes.
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48 Besides a single surfactant, mixtures of surfactants could also be used to
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50 prepare niosomes, where one of them acts as a co-surfactant, helping to stabilize the
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52 nanosystem. Generally this additive is a smaller molecule, which is able to enter the
53 spaces that the main surfactant could not occupy at the interface of the oil and water
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55 phases (Uchegbu and Florence, 1995; Wu and Guy, 2009).
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57 Furthermore, thermodynamically stable vesicles could be formed in the

58
presence of charges, which act as electrostatic stabilizers due to the repulsion
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60 between the nanostructures. To confer the charge to the vesicles, dicetyl phosphate

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3 or phosphatidic acid could be added, giving negative charge to niosomes, or
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5 stearylamine, a cationic lipid, to give positive charge to vesicular nanosystems
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7 (Agarwal et al., 2001).
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The rupture of the vesicles could happen when they are submitted to strong

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10 mechanical forces, such as shear. Excessive heat could also destabilize the lipid
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12 bilayers in the case of liposomes (Wu and Guy, 2009). To prevent dissolution of the
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vesicles, niosomes could be delivered in gels, because even creams, which contain

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15 surfactants, could dissolve the vesicles’ bilayers (Barel et al., 2014). Hyaluronic acid
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17 (HA) is an hydrophilic polymer that could form consistent hydrogel when dispersed in
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19 water. They could form a thin film on the skin surface that protect against TEWL
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(transepidermal water loss). They could also act for controlled release of active
22 substances, reducing the need for multiple administration. Because they are inert,
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24 these gels do not interact with the active substances they carry (Heilmann et al.,
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26 2013).
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The objective of this study was to prepare and characterize vesicular
nanosystems consisting of niosomes, composed of nonionic surfactants based on
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31 poly(ethylene oxide), with or without a positive charge from stearylamine, to be
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33 applied as organic sunscreens. Niosomes were characterized by mean size (DLS),
34 efficiency entrapment (EE%) and TEM, while niosome formulations were evaluated in
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36 terms of pH, reology, TEM, efficacy, toxicity and safety.
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39 2. MATERIALS AND METHODS
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43 2.1. MATERIALS
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46 The following two nonionic surfactants were used: polyoxyethylene sorbitan
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48 monooleate (Tween 80), purchased from Farmos, and the triblock copolymer
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poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronic
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L64), from Sigma-Aldrich.

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53 The sunscreens used were: phenylbenzimidazole sulfonic acid (PBSA),
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55 purchased from BASF, diethylamino hydroxybenzoyl hexyl benzoate (DHHB), also
56
from BASF, and octyl methoxycinnamate (OMC), purchased from Fagron.
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58 The cholesterol and stearylamine used were supplied by Sigma-Aldrich and
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60 the hyaluronic acid (HA) was purchased from Pharma Nostra, Brazil.

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2.2. METHODS
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8

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9 2.2.1. Preparation of the niosomes containing sunscreens
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13 The sunscreens, lipophilic and hydrophilic, with protection against UVA and

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15 UVB radiation, were selected based on a survey conducted using an online
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17 application of BASF, the Sunscreen Simulator (BASF, 2010). Phenylbenzimidazole
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sulfonic acid (PBSA) was chosen because it covers the UVB spectrum (290 to 320
20 nm), with λmax absorption at 302 nm, and is ideal for use as the aqueous phase of

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22 sunscreen formulations. In acid form, it presents low solubility, so it was used only in
23
24 salt form, thus requiring neutralization. In turn, diethylamine hydroxybenzoyl hexyl
25
benzoate (DHHB), with λmax absorption at 354 nm, covers the UVA spectrum (320 to
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400 nm) and is a lipophilic sunscreen, as is octyl methoxycinnamate (OMC), but
which covers the UVB spectrum, with λmax absorption at 311 nm (Shaath, 2007). Both
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31 lipophilic sunscreens could be used together, to provide wider coverage against UV
32 radiation.
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34 To prepare the niosomes with (N1) and without positive charge (N2), 0.25 g of
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36 a mixture of the two sunscreens (DHHB and OMC, 1:1) was dissolved in 10 mL of
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dichloromethane and placed in a round-bottom flask, together with the nonionic
39 surfactants Pluronic L64 (0.8 g) and Tween 80 (0.2 g), as well as 0.008 g of
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41 cholesterol. For the niosomes containing positive charge, 0.2 g of stearylamine was
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43 added to and dissolved in this mixture. In both cases, the solution was then
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evaporated under reduced pressure in a rotary evaporator (Büchi R-114) and the film
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46 formed inside the round-bottom flask was hydrated with 10 g of Tris buffer containing
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48 5% (w/w) of PBSA. The surfactant film was hydrated with the buffer to maintain the
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50 osmotic balance and keep the vesicles from rupturing. Furthermore, the addition of
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Tris is important to keep the pH near that of human skin in the final formulation. .
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53 Then, the niosomes were standardized through a polycarbonate membrane with pore
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55 size of 200 nm, to standard niosomes’ size. The pressure in the rotary evaporator
56
was 760 mmHg and the rotation was 120 rpm, sufficient to assure the film formed
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58 evenly (homogeneous thickness) inside the round-bottom flask.
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5 2.2.2. Characterization of the niosomes
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The niosomes were characterized by analyses of long-term stability (by

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10 measuring the mean vesicles size and size distribution of the vesicles by DLS),
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12 encapsulation efficiency (EE%) of each sunscreen and morphology by transmission
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electron microscopy (TEM).

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17 2.2.2.1. Long-term stability assay: determination of mean vesicles size and size
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19 distribution of vesicles
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22 The stability of the niosomes was analyzed by measuring the mean vesicles
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24 size and size distribution of the vesicles, by dynamic light scattering (DLS) technique,
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26 using a Malvern Zetasizer Nano ZS. For this purpose, the niosomes were diluted in
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the proportion of 1:20 in Tris buffer to maintain the osmotic balance. The
measurements were performed at room temperature (25 ºC), using a laser incidence
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31 angle of 173º, with quartz cuvettes of 3.5 mL and optical path of 10 mm.
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33 Niosomes N1 and N2 were analyzed just after being prepared and then 7, 14
34 and 30, 60, 90, 120, 150 and 180 days (for long-term stability).
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36 The mean vesicles size and size distribution of the vesicles were performed in
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38 triplicate and the mean ± standard deviation (SD) of each sample was assessed.
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41 2.2.2.2. Determination of concentration and encapsulation efficiency (EE%)
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45 The concentration and EE% of the organic sunscreens were measured by
46 high-performance liquid chromatography (HPLC), utilizing methanol and water
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48 (80:20) as the mobile phase (Lowe, 2006), with a Gilson chromatograph, with a
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50 model 321 pump, model 152 UV-Vis detector, model 831 temperature regulator,
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52 Shimadzu Rheodyne model 7725i manual injector and Kromasil 100 C18
53 chromatographic column (5 μm, 250 x 4.6 mm), at a flow of 1.2 mL/min and
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55 temperature of 40 ºC. The wavelengths selected were 311 nm (OMC), 302 nm
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57 (PBSA) and 354 nm (DHHB) (Shaath, 2007). The concentrations of the solutions for
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plotting the standard curves were 10, 15, 20, 25, 30 and 35 µg/mL.
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3 To determine the concentration of the three sunscreens in the niosomes, no
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5 purification step was necessary, since the objective was making sure there is no
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7 mass loss within the round-bottom flask, thus the estimated concentration should be
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near 100%.

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10 In turn, to determine the EE% of the lipophilic organic sunscreens (OMC and
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12 DHHB), the vesicles were purified by passing through a gel permeation column
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(GPC) containing Sephadex G50, to remove the portion that was not encapsulated.

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15 Then aliquots were collected to measure the EE% of the OMC and DHHB. However,
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17 for the EE% of the PBSA, the procedure was different, since only molecules of OMC
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19 and DHHB could be removed by purification, not of PBSA (a salt, which is thus
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soluble in the buffer that is present in the composition of the column).
22 To assess the EE% of the PBSA, first the niosome samples were centrifuged
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24 to decant the vesicles and collect the sediment for measurement of EE%, or to
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26 collect the supernatant for indirect determination. This procedure was performed
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initially at a speed of 200 rpm, but the vesicles did not decant, so the speed was
gradually increased (300, 400, 500 and 600 rpm). However, the vesicles started to
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31 rupture, as indicated by dynamic light scattering (DLS) analysis (described further
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33 ahead). Therefore, the method chosen to assess the EE% of the PBSA was to leave
34 the niosomes at rest for one week to allow natural decantation, and then to take
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36 samples from the sediment, followed by dilution in ethanol for subsequent analysis by
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38 HPLC.
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In all cases, 20 mg of niosome obtained after normalization (to assess the
41 concentration of the three sunscreens), purification (for EE% of the DHHB and
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43 OMC), or decantation (for EE% of the PBSA) was weighed in a round-bottom flask
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45 with capacity of 10 mL (DHHB and OMC) or 50 mL (PBSA), and the volume was
46 completed with the mobile phase. For the PBSA, which had the highest concentration
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48 in the niosomes, a larger flask was used, because it was necessary to obtain
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50 concentrations near the central point of the standard curve (around 20 μg/mL). Then
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52 the samples were injected in the HPLC device and the concentration and EE% were
53 determined. The analyses were performed in triplicate and the mean ± standard
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55 deviation (SD) of each sunscreen was assessed.
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2.2.2.3. Evaluation of F-N1 and F-N2 morphology by TEM
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3 To visualize niosome by TEM, they were diluted 500 times in Tris buffer, and
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5 then 20 µL of this dispersion was deposited on a grid coated with carbon and the
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7 excess was removed with filter paper (De Campos et al., 2015). After drying for 2
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hours at room temperature (25 °C), the samples were examined by high-resolution

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10 TEM with the acceleration potential of 120 kV. The TEM images allowed identifying
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12 the number of layers and thus classifying the niosomes as multilamellar or
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unilamellar.

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2.2.3. Preparation of the niosome formulations and rheological assays
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22 The polymer gels were prepared by adding hyaluronic acid (HA) at a
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concentration of 3% (w/w) in water. Then each solution was dispersed using a
25 mechanical stirrer. Then, niosomes N1 and N2 were manually incorporated in the
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polymer gel dispersion producing niosome formulations F-N1 and F-N2.

The rheological parameters were analyzed in the stationary and oscillatory

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31 states with an AR 2000 rheometer (TA Instruments, UK), using the cone-plate
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measurement geometry, with a cone diameter of 35 cm and angle of 1º, at a
34 temperature of 25 ºC. The rheological characterization was performed in three steps:
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a) The experiments in continuous mode ware performed at a shear rate from 1 to 400
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38 s-1. The flow curves were determined by using two consecutive continuous ramps
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40 with shear rate from 1 to 400 s-1 with ascending and descending cycle.
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42 b) The oscillatory experiments were conducted to determine the region of linear
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44 viscoelasticity, with tension variation between 0.1 and 30 mPa, at a frequency of 1.0
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Hz. The internal structure of the material was altered during the measurements. From
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47 these results it was possible to choose the tension corresponding to the linear
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49 viscoelasticity regime of the samples (0.1 mPa) to conduct the tests described in item
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53 c) The viscous modulus (G’’) and elastic modulus (G’) were determined as a function
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of variation of frequency, from 0.1 to 10 Hz, under constant tension (0.1 mPa), to
56 assure continuation of the linear viscoelasticity regime.
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3 2.2.4. Evaluation of F-N1 and F-N2 morphology by TEM
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7 The morphology of the niosome formulations F-N1 and F-N2 was evaluated by
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transmission electron microscopy (TEM) according to the methodology described in

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10 item 2.2.2.3.
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2.2.5. Evaluation of the in vitro efficacy of the niosome formulations

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17 The in vitro SPF assays were conducted with a transmission
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19 spectrophotometer and integrating sphere (Labsphere UV-2000S SPF analyzer),
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employing quartz plates with area of 25 cm² covered with 3M Transpore™ tape

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22 having the same area, adhered to one of the surfaces, on which the niosome
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24 formulations were deposited and uniformly spread, with application of 2.0 mg/cm 2.
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26 The niosomes were applied using a micropipette, on an analytical scale, and
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were spread by hand using a latex finger cot with standardized movements to obtain
a uniform surface. After protection from light for 15 minutes, the dried samples (in
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31 triplicate) were placed in the analyzer. The mean and standard deviation were
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33 assessed for each sample.
34 Initially a support spectrum with glycerin was obtained for use as a reference
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36 of 100% transmittance (Labsphere, 2008) and then the values were determined of
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38 SPF (obtained by measuring the absorbance and diffuse transmission), UVA/UVB
39
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ratio and critical wavelength (λc) for each formulation. As before, the tests were
41 performed in triplicate and the mean and standard deviation for each sample were
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43 assessed.
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46 2.2.6. In vivo and in vitro evaluation of the safety of the niosome formulations
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48 containing sunscreens
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52 2.2.6.1. In vivo skin permeation assay using technetium-99m

53 2.2.6.1.1. Labeling process
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55 To determine the safety of sunscreens, it is necessary to evaluate the retention
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formulations were labeled with technetium-99m.
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3 The technetium-99m was obtained from a 99Mo/99mTc generator, in the form
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5 of sodium pertechnetate (99mTc-NaTcO4). This technetium has an oxidation number
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7 of 7+, so it is not reactive. Therefore, to enable complexation with other compounds, it
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is necessary to reduce this metal to a lower oxidation state. This reduction was

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10 performed with a stannous chloride solution.
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12 The labeling process was carried out using 100 µL of each sample (F-N1 and
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F-N2) incubated with 30 mg of stannous chloride (SnCl2) (Sigma-Aldrich) for 10

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15 minutes at room temperature. Then this solution was incubated at room temperature
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17 with 100 µCi (approximately 100 µL) of technetium-99m for another 10 minutes in
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19 order to label the structures. Thin layer chromatography (TLC) was performed using
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grade 1 Whatman paper to confirm the correct labeling.
22
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24 2.2.6.1.2. Skin permeation assay
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The skin penetration study was carried out using three male Wistar rats with
approximate weight of 300 g for each labeled sample. The hair was removed from an
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31 area of 16 cm2 on the back of each rat with depilation cream (Veet®) and after 15
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33 minutes, 150 µL of the solution labeled with technetium-99m was applied on the skin
34 with an automatic pipette. One hour later, the rats were sacrificed and the portion of
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36 the solution that did not penetrate the skin was removed. Then the skin where the
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38 formulations had been applied was removed and weighed, to assess the skin
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retention of the samples. The other organs were also removed, and weighed, and the
41 radioactivity captured was quantified with a PerkinElmer 2470 Wizard2 automatic
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43 gamma counter (Mota et al., 2013; Cerqueira-Coutinho, Santos-Oliveira et al., 2015;
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45 Cerqueira-Coutinho et al., 2016).
46 The order in which the organs’ radioactivity was measured was: blood, brain,
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48 stomach, intestines, bladder, kidneys, lungs, liver, spleen and skin (Santos-Oliveira,
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50 2011; De Souza Albernaz et al., 2012; Sá et al., 2013; Albernaz Mde et al., 2014;
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52 Pinto et al., 2014; Machado et al., 2015; Patricio et al., 2015).

53 The mean and standard deviation of the radiation dose of each organ was
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55 assessed for each sample. The results were expressed as percentage of radioactivity
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3 2.2.6.2 In vitro skin permeation assay using Franz cells
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7 Franz cells are vertical diffusion cells made of glass, for the purpose of
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evaluating the release and penetration of substances. The Franz cell has two

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10 compartments, the first containing the substance of interest in solution (donor
11
12 compartment) and the second containing the receptor solution, separated by a
13
synthetic membrane or piece of skin (Franz, 1975).

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15 Many articles describe the use of vertical diffusion cells such as the Franz cell,
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17 equipped with a synthetic membrane to determine release in vitro, or with a biological
18
19 membrane (human skin or hairless skin of animals such as pigs or rats) to measure
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the penetration of topical formulations, such as creams, gels and lotions, and also of
22 transdermal systems.
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24 This experiment was conducted to evaluate the two niosome formulations (F-
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26 N1 and F-N2) regarding release of the sunscreens contained in the niosomes. The
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sample was placed in the upper (donor) compartment and the passage of the
substance through the membrane to the receiver compartment was monitored by
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31 analyzing the liquid collected at different time intervals (Shah et al., 1998; Shah et al.,
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33 1999).
34 The membranes used were skin from the ears of healthy pigs that had been
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36 slaughtered with approximate age of four months, obtained from the abattoir of Rio
37
38 de Janeiro Federal Rural University (UFRRJ). After slaughter, the ears were removed
39
40
and transported under refrigeration to the laboratory, with transport time of less than
41 two hours.
42
43 The ears were washed with tap water to remove blood, and the skin from the
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44
45 back of each ear was removed with a scalpel and tweezers, after which scissors and
46 tweezers were used to remove the hypodermis (innermost layer, replete with fat) and
47
48 blood vessels. After removing the hairs on the epidermis, circular skin membranes
49
50 with approximate area of 11.0 cm2 were cut and wrapped with polyvinyl chloride
51
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52 (PVC) films, labeled and stored at -20 ºC for at most four weeks before use. The
53 excised pig skin disks were placed horizontally with the stratum corneum (SC) facing
54
55 the donor compartment, so that the internal face of the skin remained in contact with
56
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57 the receptor solution. The receiving compartment was filled with 7 mL of the receptor
58
solution, composed of phosphate buffer at pH 7.4 (NaCl 0.850 g, KH 2PO4 0.354 g,
59
60 Na2HPO4 0.725 g and distilled water to complete 100 mL of solution), to mimic

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1
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3 biological fluids, with addition of 5% (w/w) sodium lauryl sulfate (surfactant to dissolve
4
5 the OMC and DHHB sunscreens, which are lipophilic). The use of buffer solutions in
6
7 penetration studies is common, since the idea is to mimic the condition of biological
8
fluids (Monteiro et al., 2012; Santis et al., 2013; Cerqueira-Coutinho et al., 2016).

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10 During the experiment, the receptor solution remained under continuous stir
11
12 with a magnetic stirrer, to keep it homogeneous. The diffusion system was
13
maintained at 32 ºC ± 0.5 with a thermostatically controlled water bath and was

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15 operated for 30 minutes with the skin disks in contact with the receptor solution, to
16
17 establish equilibrium. The experiments were conducted with samples weighing
18
19 approximately 50 mg applied in the donor compartment, in contact with the SC, with
20

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21
an automatic pipette designed for semi-solids. Each hour, a 1 mL aliquot of the
22 receptor solution was collected, for a period of 6 hours. After the removal of each
23
24 aliquot, the volume was completed with new receptor solution. The solution samples
25
26 were placed in Eppendorf tubes and filtered through a disposable filter membrane
27
28
29
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with 0.45 μm pore size, for analysis by high-performance liquid chromatography
(HPLC).
30
31 At the end of the 6-hour experiment, the system was disassembled, the skin
32
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33 disk was removed, from which the test solution was removed with cotton. Then the
34 skin disk was cleaned three times with cotton moistened with water and then dried
35
36 with cotton. The epidermis was separated from the dermis with a scalpel and the
37
38 stratum corneum was submitted to an extraction process with 1 mL of the mobile
39
40
phase. The Eppendorf tubes were vortexed for three 30-second intervals
41 interspersed with 30 seconds at rest. After the third vortexing, the Eppendorf tubes
42
43 were centrifuged at 6,400 rpm for 10 minutes. The samples were filtered through
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44
45 disposable membranes with pore size of 0.45 μm and then placed in the
46 chromatograph to measure the percentage of each sunscreen retained in each skin
47
48 layer.
49
50 The HPLC system consisted of a Gilson chromatograph with model 321 pump,
51
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52 model 152 UV-Vis detector, model 831 temperature regulator, Shimadzu Rheodyne
53 model 7725i manual injector and Kromasil model 100 C18 chromatographic column
54
55 (5 μm, 250 x 4.6 mm). The mobile phases used were methanol:water (80:20) for
56
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57 OMC and PBSA, while methanol:water (90:20) was used for DHHB. The flow rate
58
was 1.2 mL/min and the temperature was 40 ºC. The wavelengths selected in the
59
60 equipment were 311 nm (OMC), 302 nm (PBSA) and 354 nm (DHHB). Finally, the

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3 concentrations of the solutions for plotting the standard curves were 10, 15, 20, 25,
4
5 30 and 35 ug/mL. All tests were performed in sextuplet (n=6) for each formulation.
6
7
8
2.2.7. Evaluation of the toxicity of the niosome formulations containing

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10 sunscreens – cell viability with MTT assay
11
12
13
The cell viability with MTT assay is a calorimetric method that is very popular for

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15 being quick, easy to perform and inexpensive. It is based on the reduction of 3-(4,5-
16
17 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which has a yellow
18
19 color, by the mitochondrial enzyme NADH dehydrogenase, generating formazan
20

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21
crystals, with purple color, which accumulate inside cells. After solubilization of these
22 crystals with dimethyl sulfoxide (DMSO), the wells where the majority of cells are
23
24 viable (with functional mitochondria) take on an intense purple color, unlike what
25
26 happens in the wells with a small number of viable cells. Therefore, by using an
27
28
29
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ELISA microplate reader it is possible to indirectly correlate the intensity of the
absorption of the color purple of the formazan crystals in the visible region with the
30
31 number of viable cells in the control well and each treatment well (Mosmann, 1983;
32
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33 De Campos et al., 2010; He et al., 2011; Sylvester, 2011; Cerqueira-Coutinho et al.,
34 2016).
35
36 After application of the formulations F-N1 and F-N2 (at concentrations of 1.0,
37
38 5.0, 7.5 and 10.0 mg.ml−1 in Tris buffer) in RAW 264.7 macrophage cells in the plate’s
39
40
wells, a solution of MTT at 5 mg/mL in phosphate buffer was prepared. In each well,
41 a volume of 20 µL of MTT diluted in 180 µL of RPMI (culture medium containing
42
43 glutathione and vitamins) was applied. The plates were then wrapped in aluminum
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44
45 foil (for protection from light) and incubated in an oven with atmosphere of 5% CO 2 at
46 37 oC for 4 hours. After this period, the plates were centrifuged at 4,000 rpm for 5
47
48 minutes and then the medium containing MTT was removed from the wells. After this,
49
50 the formazan crystals were dissolved with 100 µL of DMSO/well and the absorbance
51
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52 was read at a wavelength of 570 nm with an ELISA microplate reader.

53 The averages were assessed of the readings of three control wells and three
54
55 wells containing the substances of each treatment, to calculate the difference
56
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57 between the means of the treated and control wells (wells containing only 100 µL of
58
DMSO), according to Equation 1.
59
60

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1
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3 Average Abs.= (Abs. treated1 + Abs. treated2 + Abs. treated3)/3 - (Abs. control1 + Abs. control2
4
5 + Abs. control3)/3 (1)
6
7
8 Then the percentage (%) of viable cells was assessed from the absorbance

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9
10 values obtained from each treatment, according to Equation 2.
11
12
13 % viable cells= (Abs. treated/Abs. control) x 100 (2)

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15
16
17 The cytotoxic concentration (CC50) is the value used to express the
18
19
concentration of a formulation necessary to reduce the number of viable cells by 50%
20 in comparison with the control. The lower the cytotoxic concentration (CC 50) value is,

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21
22 the more toxic the sample is, since a lower concentration is necessary to reduce the
23
24 number of viable cells by 50% (Lebeau et al., 2006).
25
The value of CC50 for each product was assessed by the average of three
26
27
28
29
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independent texts. The significance of the means of the different parameters of the
experimental groups was determined by analysis of variance (ANOVA), followed by
30
31 the Newman Keuls test, with 95% interval (p<0.05). The means and standard
32 deviations of the absorbance readings of three repetitions were compared.
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33
34
35
36 2.2.8. Statistical analysis
37
38
39 The experimental results were expressed as mean ± standard deviation and
40
41 were submitted to analysis of variance (ANOVA) with 95% significance (α= 0.05). The
42
43 calculations were performed with Origin® version 8.0 for Windows.
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44
45
46 3. RESULTS
47
48
49
50
3.1. Long-term stability assay: determination of mean vesicles size and size
51
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distribution of vesicles
52
53
54
55 The mean vesicles size and size distribution remained constant during 6
56
months for both nanosystems with a mean size of 168 ± 5 nm (N1) and 192 ± 8 nm
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57
58 (N2) in the sixth month, indicating the stability of both niosomes (Figure 1).
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60

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5 b)
a)
6
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8

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Figure 1: Size distribution of the niosomes N1 (a) and N2 (b), prepared by hydration of the nonionic
25
surfactant film and analyzed immediately after preparation (T0) and 7, 14, 30, 60, 90, 120, 150 and
26
27
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29
180 days.
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31 3.2. Determination of concentration and encapsulation efficiency (EE%)
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34 The retention times of the OMC, PBSA and DHHB in the analysis by HPLC
35
36 were 15, 8 and 7 minutes respectively.
37
38 The niosomes N1 and N2 presented concentrations between 98.1 and 99.4%
39
40 for the sunscreens OMC, DHHB and PBSA. Values slightly under 100% are
41 acceptable, since some material is always lost in the flask when preparing niosomes.
42
43 The encapsulation efficiency (EE%) values for the lipophilic organic
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44
45 sunscreens OMC and DHHB in N1 and N2 were: OMCN1 54 ± 3%; OMCN2 51 ± 2%;
46
DHHBN1 48 ± 3%; and DHHBN2 50 ± 4%. No statistical differences were found
47
48 between the EE% values obtained for OMC and DHHB. This was expected, since
49
50 mixtures of these sunscreens were used in 1:1 proportion and in the same
51
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52 concentration for both niosomes N1 and N2.

53 In turn, the sunscreen PBSA presented higher EE% values (PBSAN1 90 ± 2%
54
55 and PBSAN2 87 ± 4%). This better EE% performance of niosomes with PBSA could
56
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57 be attributed to the capacity of these niosomes to encapsulate hydrophilic

58
59
compounds. Another characteristic to explain the high EE% obtained for PBSA is the
60 possibility that the niosomes were unilamellar, making the amount of hydrophilic

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1
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3 space (interior of the niosome) greater than the lipophilic space (interior of the
4
5 bilayer).
6
7 Besides these aspects, the decantation might not have been complete during
8
the experiment, so that the aliquot collected as a sample did not only contain

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10 vesicles, but also some of the medium bearing the vesicles (a mixture of Tris buffer
11
12 and PBSA), used for hydration of the film formed by the surfactants. Hao and Li
13
(2011) and Gupta et al. (2015) also obtained high EE% values for hydrophilic

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15 compounds.
16
17
18
19
20

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21 3.3. Evaluation of N1 and N2 morphology by TEM
22
23
24 Figures 2-a and 2-b show the images obtained by TEM for the niosomes N1
25
26 and N2. The dilutions of N1 and N2 were performed in Tris buffer in the proportion of
27
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29
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1:1. The ImageJ software was also used to calculate the mean vesicles size and the

30
size distribution of the vesicles present in the TEM images. The bar graphs showed
31 the respective size distributions of Figures 2-a and 2-b. N1 presented average
32
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33 vesicle size of 208 ± 77 nm, while N2 mean vesicles size was 112 ± 28 nm. Tavano
34
35 et al. (2011) and Gupta et al. (2015) also found that vesicles of niosomes have
36 spherical morphology and homogeneous size distribution when analyzed by TEM. It
37
38 is interesting to note that other authors have also obtained similar TEM images of
39
40 niosomes (Manosroi et al., 2008; Tavano et al., 2010; Antunes et al., 2011;
41
42 Muzzalupo et al., 2011; Sakai et al., 2011; Muzzalupo et al., 2013; Tavan et al.,
43
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2013).
44
45 Both the N1 and N2 niosomes were spherical, with regular edges and smooth
46
47 surfaces. No statistical difference was found for the mean vesicles size of the
48
vesicles obtained by TEM for N1 and N2 (p> 0.05).
49
50 The results obtained by TEM were complementary to those from the size
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52 analysis by DLS (168 ± 5 nm and 192 ± 8 nm, respectively). It is important to mention

53
54 that TEM images analyzed by the ImageJ software are photographs of only a small
55 area of the sample. Thus, the images are representative of the samples analyzed,
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57 but the values obtained for the mean size and standard deviation will not be exactly
58
59 equal to those obtained by DLS, since the intrinsic error of size analysis by
60

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3 microscopy is greater. Nevertheless, the values found in this study are close and
4
5 correlated.
6
7
8

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12 a) b)
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41
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43
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Figure 2: TEM images of the niosomes: a) N1; b) N2. Samples diluted 1:1 in Tris buffer. Bar graphs
46
shows the size distribution of the niosomes for each sample.
47
48
49
50 3.4. Rheological assays
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52
53
54 3.4.1. Flow curves
55
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57 To obtain the niosome formulations F-N1 and F-N2, the niosomes N1 and N2
58
59 were added to the HA hydrogel at 3% (w/w) containing 5% (w/w) PBSA, and then the
60

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 AUTHOR SUBMITTED MANUSCRIPT - NANO-121056.R1 Page 18 of 30

1
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3 samples were homogenized by manual stirring to preserve the bilayers of the
4
5 niosomes. The viscosity of formulation F-N1 increased after incorporation of the
6
7 niosomes in the HA gel at 3% (w/w) with PBSA at 5% (w/w).
8
Figure 3 present the flow curves of the formulations F-N1 and F-N2, each

pt
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10 containing 5% (w/w) PBSA. In all cases, graphs were obtained with decreasing
11
12 viscosity and similar hyperbolic shapes, characteristic of pseudoplastic non-
13
Newtonian fluids, showing declining viscosity with increasing shear rate applied .

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34
35
36 Figure 3: Non-Newtonian behavior of the niosome formulations F-N1 and F-N2
37
38
39 3.4.2. Variation of tension
40
41
42 As can be seen in Figure 4, there is inversion of the values of G’ and G’’ for the
43
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44 gels where the curves meet, demonstrating the breakdown of the hydrogels at 3%
45
46 (w/w) with PBSA at 5% (w/w). In contrast, for the niosome formulations (Figure 4),
47
48 which contained niosomes N1 or N2 in the hydrogel (in 3:1 proportion), this
49 phenomenon did not occur. The reason is that after the niosomes were incorporated
50
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in the hydrogel, the formulations no longer behaved as gels.

52
53
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b)
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5 a)
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8

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21 Figure 4: Rheological behavior of the niosome formulations, where the elastic modulus (G’) is in black
22 and the viscous modulus (G’’) is in red, versus variation of tension, with fixed frequency of 1 Hz: a) F-
23 N1 and b) F-N2
24
25
26 Therefore, formulations F-N1 and F-N2 were not analyzed regarding variation
27
28
29
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of frequency, since the results obtained regarding variation of tension did not showed

30 gel behavior even though visually appearing to have high viscosity.

31
32
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33 3.5. Evaluation of F-N1 and F-N2 morphology by TEM
34
35
36
37 Figures 5-a and 5-b contain the images obtained by transmission electron
38
39 microscopy (TEM) for the formulations containing niosomes N1 and N2 (a and b,
40
41 respectively). F-N1 and F-N2 were diluted in Tris buffer at 1:1. The images show the
42 presence of hydrogels, where the niosome spheres share space with structures
43
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44 having varied forms that are similar to “networks” and fragments of polymeric gel,
45
46 possibly indicating rupture of the gel when dried on a copper grid coated with formvar
47
(thermoplastic resin that gives adherence of the formulation to the grid used for
48
49 microscopic observation).
50
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8 a) b)

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35
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Figure 5: TEM images of the niosomes incorporated in hydrogel of hyaluronic acid at 3% (w/w)
41
containing 5% (w/w) PBSA, in 1:3 proportion; a) F-N1 and b) F-N2; Dilution of the samples 1:1 in tris
42
buffer. Scales of 1 µm and 0.5 µm
43
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44
45
46
47
48 Processing of the TEM images with Image J software allowed estimating the
49 average sizes and size distribution of the vesicles contained in the HA hydrogel. The
50
51
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mean size of the F-N1 niosomes was 209 ± 51 nm and for the F-N2 niosomes it was
52
53 134 ± 39 nm. There was no statistical difference between these two values (p> 0.05).
54
55
The reason the average size tended to be larger for F-N1 could be related to
56 the absence of a positive charge, which might have increased the coalescence
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57
58 and/or agglomeration of the vesicles when formulation F-N1 was placed on the TEM
59
60 grid. It seems that in formulation F-N2, the presence of a positive charge causes the

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3 vesicles to repel each other, reducing agglomerations after placement of sample F-
4
5 N2 on the TEM grid.
6
7
8
3.6. Evaluation of the in vitro efficacy of the niosome formulations

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10
11
12 The SPF value of the sunscreen formulations were considered satisfactory,
13
since a sunscreen product with SPF around 30 (34 ± 8 for formulation F-N1 and 34 ±

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15 5 for F-N2), when used at the concentration (2 mg/cm 2) recommended by the U.S.
16
17 Food and Drug Administration (FDA) reduces by 60% the dose necessary to
18
19 generate erythema on the skin surface after exposure for 2 hours to sunlight at the
20

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21
level of the Equator, where incidence of UV radiation is the strongest. If this time is
22 shortened, or the sunscreen is reapplied, the risk of erythema is lower .
23
24 The transmission spectrophotometry with integrating sphere method allows
25
26 obtaining SPF values that could be correlated with values obtained in tests with
27
28
29
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volunteers, and has the advantage of being noninvasive, besides being less
expensive than in vivo trials. It also does not require approval by an ethics committee
30
31 .
32
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33 There was no statistical difference between the SPF results (p>0.05). These
34 were basically the same because the sunscreens and their concentration were the
35
36 same in each formulation. The standard deviation found could be considered high for
37
38 each of the samples. This pattern is common for this type of analysis, since the
39
40
sample is applied manually in the quartz plate and the film may not be uniform on all
41 parts of the plate.
42
43 The higher standard deviation observed for the mean SPF value of F-N1 might
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44
45 be related to the absence of positive charge, meaning absence of repulsion of the
46 vesicles, leading to their greater accumulation at specific points of the plate. This was
47
48 noted visually when spreading the formulations on the plates: the F-N2 formulation,
49
50 which contains the positively charged stearylamine, spread easily and smoothly, with
51
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52 the appearance being more evenly distributed on the surface compared to

53 formulation F-N1, without stearylamine.
54
55
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4
5 3.7. In vivo and in vitro evaluation of the safety of the niosome formulations
6
7 containing sunscreens
8

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10 3.7.1. In vivo skin permeation assay using technetium-99m
11
12 Before conducting the in vivo skin penetration test with radioactive labeling, it
13
was necessary to investigate whether the element technetium-99m bonds with the

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15 niosome formulations and the formulation containing free sunscreens (gel with free
16
17 sunscreens), if not, the test could not be conducted. The thin-layer chromatography
18
19 confirmed that the niosome formulations F-N1 and F-N2 and the gel with free
20

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sunscreens were labeled with technetium-99m, with labeling efficiency near 100% for
22 both samples.
23
24 The formulations for photoprotection must be retained in the skin to exert their
25
26 action against UV radiation, and should not pass into the bloodstream, since the raw
27
28
29
materials of sunscreens are toxic.
an
The skin penetration test showed that formulation F-N2 was retained in the rat
30
31 skin longer than formulation F-N1 (Figure 6). The figure also reveals that the main
32
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33 organ of deposition of N1 was the small intestine (1.1%), followed by the stomach
34 (0.9%). This result demonstrates the niosomes penetrated the skin, indicating high
35
36 cutaneous absorption and fecal excretion. For F-N1, the skin was the organ with
37
38 lowest retention, at 0.7%. Even lower levels of niosomes were found in the brain and
39
40
right kidney of the rats.
41 With respect to the niosomes in formulation F-N2, Figure 6 shows a higher
42
43 concentration retained in the skin (3.0%, roughly four times higher than F-N1), along
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45 with retention in the stomach (0.8%) and small intestine (0.2%), indicating that skin
46 penetration also occurred.
47
48 The difference between the levels of niosome retained in the stomach between
49
50 formulations F-N1 and F-N2 was not statistically significant (p> 0.05). The differences
51
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52 were significant for the amounts retained in the skin and small intestine (p< 0.05).
53 Figure 6 also shows the results of the third sample, in which the free
54
55 sunscreens OMC and DHHB were incorporated in a gel of HA at 3% (w/w) containing
56
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57 PBSA at 5% (w/w) (gel with free sunscreens). In this case there was little interaction
58
of the sunscreens present in the gel with the skin, since the retention was only 0.5%,
59
60 statistically lower compared to levels of the niosomes N1 and N2 (p< 0.05). The

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3 quantities present in the stomach and small intestine were also lower for this
4
5 formulation, indicating a low penetration rate. However, this result could be directly
6
7 connected to the low retention of the sunscreens in the skin. Since the sunscreens
8
had low retention on the skin surface, their penetration would be lower, which would

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9
10 be positive in relation to safety of the formulation. On the other hand, the fact the
11
12 sunscreens had low retention on the skin is a negative aspect.
13
The main organ for deposition of the N2 niosomes with stearylamine was the

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15 skin, suggesting its greater affinity for this organ. The retention rate of the niosomes
16
17 present in F-N2, with positive charge was about four times (3.0%) that of the
18
19 niosomes of F-N1 (0.7%). This suggests that the increased skin retention of N2 is
20

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21
directly correlated with the positive charge conveyed by stearylamine, which is
22 opposite to the negative charge of the stratum corneum, increasing the nanosystem’s
23
24 adherence to the skin .
25
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43
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54 Figure 6: Concentration (%) of the sunscreens of formulations F-N1 and F-N2 and free sunscreens
55 (FS), labeled with technetium-99-m, in the organs evaluated
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5 3.7.2. In vitro skin permeation assay using Franz cells
6
7
8
Through the in vitro penetration test using Franz vertical diffusion cells, it was

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9
10 possible to verify the absence of penetration of the sunscreens contained in the
11
12 niosomes of formulations F-N1 and F-N2 to the receptor medium during the 6 hours
13
of the experiment. The sunscreens were retained, in different concentrations, in the

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15 two skin layers (epidermis and dermis) (Figure 7). Both the lipophilic sunscreens
16
17 (OMC and DHHB) and the PBSA were found in higher concentration in the epidermis
18
19 than the dermis, for both formulations (F-N1 and F-N2). The result is interesting,
20

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21
since the action mechanism of sunscreens occurs on the skin surface, the epidermis,
22 affected by the UV radiation reaches, making it necessary for the photoprotection
23
24 product to be retained in this layer.
25
26 The sunscreens OMC and DHHB, which were used in the same proportions to
27
28
29
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prepare the niosomes, showed similar retentions in the dermis and epidermis, with
both F-N1 and F-N2. The average retentions of OMC in epidermis were 34.6% (144.1
30
31 µg) for F-N1 and 39.2% (163.1 µg) for F-N2. The mean retentions of DHHB in this
32
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33 same layer were 29.6% (123.4 µg) for F-N1 and 22% (91.5 µg) for F-N2. With
34 respect to the dermis, the average retentions of OMC were 1.8% (7.3 µg) for F-N1
35
36 and 1.5% (6.1 µg) for F-N2. The average penetration rates of DHHB in the dermis
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38 were 0.8% (3.5 µg) for F-N1 and 0.7% (2.9 µg) for F-N2. There was no statistical
39
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difference between the retention values of OMC and DHHB in the epidermis for F-N1
41 and F-N2 (p>0.05). There also was no statistical difference between the retention of
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43 OMC and DHHB in the dermis for F-N1 and F-N2 (p>0.05). However, the sunscreens
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45 OMC and DHHB were retained more in the epidermis than the dermis, both when
46 contained in F-N1 and F-N2 niosomes. This behavior indicates that the lipophilic
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48 character of those two sunscreens causes them to have greater affinity for the
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50 epidermis, since this layer contains ceramides, cholesterol and free fatty acids, all of
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52 them lipids present among the corneocytes of the corneum layer of the epidermis .
53 The lipophilic sunscreens interact with the lipids present in large quantity in the
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55 outermost skin layer, the epidermis, and are more strongly retained there. The more
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57 lipophilic characteristic of the epidermis is important for the isolation and protection of
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the skin against the external environment .
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3 In contrast, PBSA, the only hydrophilic sunscreen, presented greater retention
4
5 in terms of concentration in the dermis than did the other sunscreens. Between the
6
7 epidermis and dermis for PBSA, both contained in the F-N1 and F-N2 formulations,
8
thee was greater retention in the epidermis than the dermis (p<0.05). This could

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10 possibly be attributed to the fact that the formulations were applied on the skin
11
12 surface (epidermis). On that layer, the mean retention values of PBSA were 19.0%
13
(436.2 µg) for F-N1 and 12.4% (284.4 µg) for F-N2, while in the dermis the rates

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15 were 7,2% (164.5 µg) for F-N1 and 6.5% (148.4 µg) for F-N2.
16
17 It was also noted that in absolute values (µg), PBSA was always present in the
18
19 highest quantity in the two layers when compared with the other sunscreens. This
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could be explained by the fact that PBSA was present in the largest quantity in
22 preparations F-N1 and F-N2, but at the end of the experiment, the excess of the
23
24 formulation on the surface (that was not absorbed) was carefully removed with cotton
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26 moistened with distilled water. Therefore, the lower concentration of PBSA in the
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epidermis could be attributed to its weaker interaction with that skin layer (p<0.05)
compared to the lipophilic sunscreens.
30
31 In the comparison of the lipophilic sunscreens OMC and DHHB with PBSA, the
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33 former two had greater affinity for the epidermis due to the presence of abundant
34 lipophilic components while hydrophilic PBSA had less affinity for the epidermis
35
36 because of the lack of solubility of most of its components. This possibly explains
37
38 why it reached the dermis in greater quantity than the other two sunscreens. The
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dermis is composed of collagen, elastin and other elements of the extracellular
41 matrix, such as glycosaminoglycans (e.g., hyaluronic acid), water and soluble
42
43 substances like electrolytes . Therefore, because PBSA did not interact strongly with
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45 the epidermis, a greater quantity reached the underlying tissue, the dermis, than in
46 the case of OMC and DHHB. The dermis contains a quantity of water, which could
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48 have facilitated the passage of PBSA to a site with a larger quantity of this solvent,
49
50 where it is soluble. Therefore, the concentration of PBSA found in the dermis was
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52 statistically greater (p<0.05) than the concentrations of OMC and DHHB found in this
53 layer.
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55 No statistical differences were noted between the formulations F-N1 and F-N2
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57 in any of the cases (when comparing the sunscreens with each other or when
58
comparing the epidermis and dermis). We expected to observe greater retention of F-
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60 N2 in the epidermis than in the dermis, since F-N2 has a positive charge from the

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1
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3 stearylamine. Nevertheless, as mentioned before, in the in vivo biodistribution
4
5 experiments with radioactive labeling, there was a statistical difference in the
6
7 retention of the sunscreens between the epidermis and dermis. Those experiments
8
demonstrated that the concentration of F-N2 retained in the skin was three times that

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10 of F-N1. In vivo evaluations tend to produce results nearer to reality, but it is
11
12 necessary to consider both types of tests, since the pig ear skin ustilized in the in
13
vitro test has characteristics near those of human skin, so the importance of the in

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15 vitro penetration and retention assay with Franz vertical diffusion cells could not be
16
17 disregarded .
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Figure 7: Percentage (%) of each sunscreen (OMC, DHHB and PBSA) retained after 6 hours in the
44 epidermis and dermis. Blue bars are related to formulation F-N1 and red bars F-N2
45
46
47 3.8. Evaluation of the toxicity of the niosome formulations containing
48
49
sunscreens – cell viability with MTT assay
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52 The macrophages present in the dermis act to protect the skin and thus the
53
54 organism against the entry of pathogenic microorganisms that could reach the
55 bloodstream if passing the cutaneous barrier. The cells used in this assay were
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57 macrophages and the CC50 values found were 3.81 ± 1.29 mg/mL for F-N1 and 0.83
58
59 ± 0.24 mg/mL for F-N2, in both cases statistically significant differences (p<0.05). The
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1
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3 cell viability for positive control was 100.18 ± 7.24 %.
4
5 The lower the CC50 value, the greater the cytotoxicity is, so that the F-N2
6
7 formulation was more toxic than F-N1. The positive charge from the presence of
8
stearylamine might have caused this increase of cytotoxicity, since some authors

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9
10 have reported increased cytotoxicity of nanosystems designed to carry cationic lipids
11
12 .
13
Jonas and Speller (1989) observed that liposomes containing stearylamine

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15 were highly toxic to dermal fibroblasts, which according to them limits use of this
16
17 substance. In turn, Nishiya et al. (1995) reported that liposomes containing
18
19 stearylamine led to hemolysins when in contact with blood, a characteristic that would
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be negative for intravenous application of preparation.
22 The formulations tested in this study only presented some toxicity to the
23
24 macrophages when the quantity in formulation reached a concentration on the order
25
26 of milligrams per milliliter in relation to the sunscreen content. The results of this
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toxicity assay could be correlated with those of the in vitro penetration test using
Franz cells (item 5.7.), since permeation assay allows quantifying how much of each
30
31 sunscreen was retained in the dermis, allowing comparison with the toxicity to
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33 macrophages, which are cells present in the dermis.
34 Therefore, formulations F-N1 and F-N2 were not considered toxic because the
35
36 amount of each sunscreen retained in the dermis (164.5 µg/mL of PBSA in F-N1 and
37
38 148.4 µg/mL in F-N2; 7.3 µg/mL of OMC in F-N1 and 6.1 µg/mL in F-N2; 3.5 µg/mL of
39
40
DHHB in F-N1 and 2.9 µg/mL in F-N2) were significantly lower than the CC50 values
41 found for both formulations (3.81 mg/mL for F-N1 and 0.83 mg/mL for F-N2).
42
43
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46 4. CONCLUSIONS
47
48
49
50 N1 and N2 niosomes presented at least a six-month stability, with sizes of 168
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52 ± 5 nm and 192 ± 8 nm, respectively, and monomodal mean size distributions. The
53 encapsulation efficiency (EE%) values for the lipophilic sunscreens OMC and DHHB
54
55 in N1 and N2 were: OMCN1 54 ± 3%; OMCN2 51 ± 2%; DHHBN1 48 ± 3%; and
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57 DHHBN2 50 ± 4%. While hydrophilic PBSA presented higher EE% values (PBSA N1 90
58
± 2% and PBSAN2 87 ± 4%), which could be attributed to the capacity of these
59
60 niosomes to encapsulate hydrophilic compounds. SPF values were considered

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1
2
3 satisfactory, 34 ± 8 for formulation F-N1 and 34 ± 5 for F-N2. In vitro toxicity
4
5 suggested that both formulations were non-toxic to the skin. In vitro safety assay
6
7 showed lipophilic sunscreens’ greater affinity for the epidermis, since this layer
8
contains natural lipids. In vivo safety assay suggests that the increased skin retention

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10 of N2 is directly correlated with the positive charge of stearylamine. Photoprotective
11
12 niosomes showed to be promising in solar protection, once they were an efficient
13
shield against UV light and did not present toxicity, also they remained retained to the

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15 upper layer of the skin, epidermis.
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19 ACKNOWLEDGEMENTS
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22 We thank the National Scientific and Technological Research Council (CNPQ)
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24 and the Rio de Janeiro State Research Foundation (FAPERJ) for funding.
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The authors declare no conflict of interest.

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1
2
3 REFERENCES
4
5
6 Abaee A and Madadlou A 2016 Food Chemistry 196 106
7
8 Agarwal R, Katare O P and Vyas S P 2001 Int J Pharm 228 43

pt
9
10
11 Antunes F E, Gentile L, Rossi C O, Tavano L and Ranieri G A 2011 Colloids Surf B 87
12 42
13

cri
14 Bansal S, Aggarwal G, Chandel P and Harikumar S L 2013 J Pharm Bioall Sci 5 318
15
16 Barradas T N, De Campos V E B, Senna J P, Coutinho C D S C, Tebaldi B S, E Silva
17
18
K G H and Mansur C R E 2015 Colloids Surf., A 480 214
19
20 Cerqueira-Coutinho C S, Santos-Oliveira R, Dos Santos E P and Mansur C E 2015

us
21 Eng Life Sci, 15 593
22
23 Colliex C 2014 C R Phys 15 2
24
25
26
De Campos V E, Silva J A, Ricci-Júnior E, Mansur C R , Conti D S and da Rocha S R
an
27 2016 Curr Drug Deliv 13 287
28
29 Ertekin Z C, Bayindir Z S and Yuksel N 2015 Curr Drug Deliv 12 192
30
31 Fraenza C C, Maledandri C J, Anoardo E, Brougham D F 2014 Chemphyschem 15
32
dM
425
33
34
35 Gupta A, Singh S, Kotla N G and Webster T J 2014 Int J Nanomedicine 10 171
36
37 Hao Y M and Li K 2011 Int J Pharm 403 245
38
39 Jurkiewicz P, Olżyńska A, Cwiklik L, Conte E, Jungwirth P, Megli F M and Hof M 2012
40
41 Biochim Biophys Acta 1818 2388
42
43
pte

Kumar G P and Rajeshwarrao P 2011 Acta Pharm Sin B 1 208

44
45
Lintner K 2009 Global Regulatory Issues for the Cosmetics Industry (Boston: William
46
47 Andrew Publishing)
48
49 Lowe N J 2006 Dermatol Clin 24 9
50
51
ce

Mahale N B, Thakkar P D, Mali R G, Waluni D R and Chaudhari S R 2012 Adv

52 Colloid Interface Sci 183-184 46
53
54
55 Manconi M, Valenti D, Sinico C, Lai F, Loy G and Fadda A M 2003 Int J Pharm 260
56 261
Ac

57
58 Manosroi A. Jantrawut P and Manosroi J. 2008 Int J Pharm 360 1
59
60

29
 AUTHOR SUBMITTED MANUSCRIPT - NANO-121056.R1 Page 30 of 30

1
2
3 Marianecci C, Di Marzio L, Rinaldi F, Celia C, Paolino D, Alhaique F, Esposito S and
4
Carafa M 2014 Adv Colloid Interface Sci 205 187
5
6
7 Moghassemi S and Hadjizadeh A 2014 J Control Release 185 22
8

pt
9 Mota A C, De Freitas Z M, Ricci-Júnior E, Dellamora-Ortiz G M, Santos-Oliveira R,
10 Ozzetti R A, Vergnanini A L, Ribeiro V L, Silva R S and Dos Santos E P 2013 Int J
11 Nanomedicine 8 4689
12
13
Muzzalupo R, Tavano L, Cassano R, Trombino S, Ferrarelli T and Picci N 2011 Eur J

cri
14
15 Pharm Biopharm 79 28
16
17 Muzzalupo R, Tavano L, La Mesa C 2013 Int J Pharm 458 224
18
19 Muzzalupo R, Tavano L, Trombino S, Cassano R, Picci N and La Mesa C 2008
20

us
21
Colloids Surf B 64 200
22
23 Nejadmansouri M, Hosseini S M H, Niakosari M, Yousefi G H and Golmakani M T
24 2016 Food Hydrocolloids 61 801
25
26 Pardakhty A and Moazeni E 2013 Nanomedicine Journal 1 1
27
28
29
an
Ritwiset A, Krongsuk S and Johns J R 2016 App Surf Sci 380 23
30
31 Sakai T, Kurosawa H, Okada T and Mishima S 2011 Coll Surf A 389 82
32
dM
33 Shaath N A 2007 The Encyclopedia of Ultraviolet Filters (New York: Allured)
34
35
Shi L, Shan J, Ju Y, Aikens P and Prud’homme R K 2012 Coll Surf A 396 122
36
37
38 Siddiqui A, Gupta V, Liu Y Y and Nazzal S 2012 Int J Pharm 431 222
39
40 Tavano L, Alfano P, Muzzalupo R and De Cindio B 2011 Coll Surf B 87 333
41
42 Tavano L, Gentile L, Oliviero R C and Muzzalupo R 2013 Coll Surf B 110 281
43
pte

44
45 Tavano L, Muzzalupo R, Trombino S, Cassano R, Pingitore A and Picci N 2010 Coll
46 Surf B 79 227
47
48 Tavano L, Vivacqua M, Carito V, Muzzalupo R, Caroleo M C, Nicoletta F 2013 Coll
49 Surf B 102 803
50
51
ce

52 Uchegbu I F and Florence A T 1995 Adv Colloid Interface Sci 58 1

53
54 Uchegbu I F and Vyas, S P 1998 Int J Pharm 172 33
55
56
Ac

57
58
59
60

30