Influence of different surfactants on the technological properties and in vivo ocular tolerability of lipid nanoparticles

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Influence of different surfactants on the technological properties and in vivo ocular tolerability of lipid nanoparticles Antonio Leonardi a , Claudio Bucolo b , Giovanni Luca Romano b , Chiara Bianca Maria Platania b , Filippo Drago b , Giovanni Puglisi a,c, Rosario Pignatello a,c, * a

Section of Pharmaceutical Technology, Department of Drug Sciences, University of Catania, Catania, Italy Section of Pharmacology and Biochemistry, Department of Clinical and Molecular Biomedicine, School of Medicine, University of Catania, Catania, Italy c NANO-i, Research Centre on Ocular Nanotechnology, University of Catania, Catania, Italy b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 January 2014 Received in revised form 26 April 2014 Accepted 29 April 2014 Available online xxx

Addition of one or more surfactant agents is often necessary for the production of nanostructured lipid and polymeric systems. The removal of residual surfactants is a required step for technological and toxicological reasons, especially for peculiar applications, such as the ophthalmic field. This study was planned to assess the technological properties of some surfactants, commonly used for the production of lipid nanoparticles, as well as their ocular safety profile. Stable and small-size solid lipid nanoparticles were obtained using Dynasan1 114 as the lipid matrix and all the tested surfactants. However, from a toxicological point of view, the nanocarriers produced using Kolliphor1 P188 were the most valuable, showing no irritant effect on the ocular surface up to the highest tested surfactant concentration (0.4%, w/ v). The SLN produced using Cremophor1 A25 and Lipoid1 S100 were tolerated up to a surfactant concentration of 0.2% by weight, while for Tween1 80 and Kolliphor1 HS 15 a maximum concentration of 0.05% can be considered totally not-irritant. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Solid lipid nanoparticles Ocular drug delivery systems Surfactants Ocular safety Eye tolerability

1. Introduction In the production of nanostructured systems (lipid and polymeric nanoparticles, nanoemulsions, nanocapsules, etc.), surfactants are generally required as stabilizing agents. In fact, they lower the surface tension between the nanosystem and the dispersion liquid (usually an aqueous solution), allowing the formation and stabilization of the nanostructure. This is due to their amphiphilic nature that allows a lipophilic interaction with the lipid or polymeric material and a hydrophilic interaction with the dispersing phase. Hence, the surfactant locates at the interface between nanoparticles and the aqueous solution. The used surfactant or the surfactants mixture is known to strongly influence the properties of the formed LNs (Kovacevic et al., 2011; Kullavadee et al., 2011; Upadhyay et al., 2012), as well as their stability in the bloodstream (Olbrich et al., 2002). Regrettably, therapeutic uses and, in particular, the ophthalmic application of such nanocarriers need surfactant removal, because they usually cause irritation or damage of the tissues. Classical

* Corresponding author at: University of Catania, Department of Drug Sciences, viale A. Doria, 6, Città Universitaria, 95125 Catania, Italy. Tel.: +39 095 738 4005. E-mail address: [email protected] (R. Pignatello).

purification processes used to clear the system from surfactants are ultracentrifugation, ultrafiltration, diafiltration, tangential flow filtration (TFF), and dialysis (De Jaeghere et al., 1999; Lee, 2003; Dalwadi and Sunderland, 2007, 2008). Avoiding such purification step after nanocarrier production would offer some advantages, in terms of time and cost reduction, both at lab and industry level. This would however require to know the maximum surfactant concentration tolerated by the tissues at the site of application, such as the different and complex eye structures. Furthermore, by considering the penetration enhancer activity of most surfactants, their residual presence in the formulation would affect the residence time and tissue permeation of the carried drugs (Liu et al., 2006, 2009, 2011; Xu et al. 2011). We have recently started a wide research plan, aimed at optimizing the preformulation and formulation of lipid-based nanocarriers (LNs) as potential delivery systems for the ophthalmic application of bioactives. In particular, the present study has been focused on the evaluation of the ocular tolerability of different surfactant agents, commonly used for the production of LNs. These systems have been proposed in recent years as suitable carriers for the delivery of drug to both the anterior and posterior eye segments (Attama et al., 2008, 2009; del Pozo-Rodríguez et al., 2008, 2013; Araújo et al., 2009, 2011; Seyfoddin et al., 2010; Souto et al., 2010; Pignatello and Puglisi, 2011; Leonardi et al., 2014a) and

http://dx.doi.org/10.1016/j.ijpharm.2014.04.061 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Leonardi, A., et al., Influence of different surfactants on the technological properties and in vivo ocular tolerability of lipid nanoparticles, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.061

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are considered essentially safe for the ocular structures, because of their composition made by biocompatible lipids (Souto and Müller 2009; del Pozo-Rodríguez et al., 2013). In all the methods proposed for LNs production, even different in their theoretical configuration and mechanism, the surfactants are always needed, often as a combination of two or three of them, to ensure an efficacious dispersion of the lipidic components in the aqueous phase (Müller et al. 2000; Pardeshi et al., 2012; Parhi and Suresh, 2012; Patel et al., 2013). To orientate the choice of the best suitable surfactant(s) for an ocular application of lipid nanosystems, we evaluated a set of amphiphilic compounds, selected on the basis of the materials most often reported in the recent literature for LN production. An overview of them is shown in Fig. 1, while Table S1 (given as Supplementary material) gathers the relevant articles published in recent years (2003–2014). Based on such preliminary search, and merely for the comparative purpose of this study, we selected glyceryl trimyristate (Dynasan1 114) to produce different batches of solid lipid nanoparticles (SLN), a lipid material commonly used for analogous purposes (cf. Section 3 for relevant references). The following

Fig. 1. Number of articles reporting use of surfactants for LNs production (PubMed search, years 2003–2013).

sTable 1 Name, structure and properties of the surfactants tested in the study. HLBa

Acronym

Common name (s)

Chemical name

T

Tweem180

Polysorbate 80

15 Sigma– Aldrich

K

Kolliphor1P188 (Lutrol F 68; Pluronic F68)

Poloxamer 188 (EO–PO block copolymer)

29 BASF

S

SDS

Sodium dodecyl sulphate

40 Sigma– Aldrich

H

Macrogol-15-hydroxystearate (Eur. Ph.); Kolliphor1 HS 15 (Crodasol HS; Polyethylene glycol-15-hydroxystearate Solutol HS 15) (Polyoxyl 15 Hydroxystearate USP)

L

Lipoid1 S100

Soy lecithin

C

Cremophor1 A25 (Ceteareth25) (Emulgin B)

C16–18 fatty alcohol polyoxyethylene ether (Macrogol (25)-cetostearyl ether)

a

Manufacturer Chemical structure

14–16 BASF

4–9 Sigma

15–17 Aldrich

Values taken from Severino et al. (2012) or from the manufactures websites.

Please cite this article in press as: Leonardi, A., et al., Influence of different surfactants on the technological properties and in vivo ocular tolerability of lipid nanoparticles, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.061

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urfactant agents were selected as stabilizers of the lipid systems: Tween1 80 (polysorbate 80), Kolliphor1 P188 (poloxamer 188), SDS (sodium dodecyl sulphate), Kolliphor1 HS 15 (macrogol-15hydroxystearate), Cremophor1 A25 (C16–18 fatty alcohol polyoxyethylene ether) and Lipoid1 S100 (soy lecithin). Molecular weight and HLB values of the tested surfactants are gathered in Table 1. SDS was deliberately enclosed in the set as a positive control, given its proven irritant effects on the eye (Bantseev et al., 2003; see also SDS safety data sheets). The used surfactant concentrations ranged from 0.05 to 0.4% (w/v), a range frequently found in the literature to prepare analogous systems. Among the different production methods, we adopted the quasi-emulsion solvent diffusion one (QESD), as a technique offering interesting advantages in the view of an ocular application of the nanocarriers, especially in terms of low working temperature, low surfactant concentrations and use of highly tolerated (ICH class 3) solvents (Pignatello et al., 2002a, 2002b; Bucolo et al., 2004). While assaying the utility of the chosen surfactants to produce homogeneous and stable SLN populations by the QESD method, as well as in terms of mid-term stability, their ocular tolerability was investigated using a modified Draize test (Giannavola et al., 2003).

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under constant agitation at 20,500 rpm (Ultraturrax T25 equipped with a G-8 accessory; IKA, Germany). The aqueous phase consisted of pro-injection water containing the chosen surfactant at one of the concentrations listed in Table 2. After 15 min, the resulting milky suspension was sonicated (Branson 5002; Branson Ultrasonics, Danbury, CT, USA) for 25 min at 25  C. To evaporate all the organic solvent, samples were then left to stir for 24 h at room temperature, at 500 rpm on a magnetic plate. 2.3. Characterization of SLN

4.1.0.1 Dynasan1 114 was kindly supplied by Sasol Italy S.p.a., Milan, Italy. Kolliphor1 P188 and Kolliphor1 HS 15 were kindly gifted by BASF Italia S.p.a., Cesano Maderno (MB), Italy. Cremophor1 A25, Lipoid1 S100, SDS, Tween1 80, acetone (99% purity) and ethanol (99% purity) were purchased from Sigma–Aldrich Chimica S.r.l., Milan, Italy. Pro-injection water was used throughout the work.

Mean particle size, polydispersity index (PDI) and Zeta potential values for the prepared SLN were determined. Moreover, the physical stability upon storage was assessed. Mean size and PDI of the formulations were determined by photocorrelation spectroscopy (DLS) using a NanoSizer ZS90 (Malvern Instruments Ltd., Malvern, UK). The following parameters were used for these experiments: temperature: 25  C, medium refractive index: 1.330, medium viscosity: 1.0 mPa  s and dielectric constant value: 80.4. Each sample (100 ml) was diluted to 1 ml with pro injection water to avoid multi-scattering phenomena and placed in a quartz cuvette. The size analysis of a sample consisted of 3 sets of measurements, and the results are expressed as mean size  S.D. (Fig. 2). The Zeta potential was determined by the same instrument using an anemometer Doppler laser technique. Each sample was ten-fold diluted with pro injection water. Up to 100 measurements on each sample were registered at 25  C to calculate the electrophoretic mobility and, using the Smoluchowski constant with a value of 1.5, the corresponding Zeta potential values (Fig. 3). To investigate the stability of the produced SLN, samples were stored at 4  2  C and particle size and PDI were measured each month for up to six months. Results are summarized in Fig. 4.

2.2. SLN preparation

2.4. Ocular tolerability

SLN were prepared according to an adapted QESD method. The procedure includes the dissolution of 100 mg Dynasan1 114 in 1 ml of acetone. The solution was slowly injected, by a thin teflon tube connected to a syringe, into 10 ml of an aqueous phase kept at 0  C

2.4.1. Animals Male New Zealand albino rabbits (Harlan, Italy), weighing 2.0–2.2 kg, free of any sign of ocular inflammation or gross abnormality were used. The animals were maintained on 12 h

2. Material and methods 2.1. Materials

Table 2 Analytical composition of the SLN batches. Sample

SDS (%, w/v)

S04 S02 S01 S005 K04 K02 K01 K005 T04 T02 T01 T005 C04 C02 C01 C005 L04 L02 L01 L005 H04 H02 H01 H005

0.4 0.2 0.1 0.05 – – – – – – – – – – – – – – – – – – – –

Kolliphor1 P188 (%, w/v) – – – – 0.4 0.2 0.1 0.05 – – – – – – – – – – – – – – – –

Tween1 80 (%, w/v) – – – – – – – – 0.4 0.2 0.1 0.05 – – – – – – – – – – – –

Cremophor1 A25 (%, w/v) – – – – – – – – – – – – 0.4 0.2 0.1 0.05 – – – – – – – –

Lipoid1 S100 (%, w/v) – – – – – – – – – – – – – – – – 0.4 0.2 0.1 0.05 – – – –

Kolliphor1 HS 15 (%, w/v) – – – – – – – – – – – – – – – – – – – – 0.4 0.2 0.1 0.05

Please cite this article in press as: Leonardi, A., et al., Influence of different surfactants on the technological properties and in vivo ocular tolerability of lipid nanoparticles, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.061

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Fig. 2. Mean size (S.D.) and PDI of the prepared SLN batches.

dark/12 h light cycle at constant room temperature and humidity. All experiments conformed to the ARVO (Association for Research in Vision and Ophthalmology) resolution on the use of animals in research, and the Animal Care and Use Committee of the University of Catania approved protocols. 2.4.2. Ocular safety The potential ocular irritancy and/or damaging effects of the formulations were evaluated according to a modified Draize test (Giannavola et al., 2003). A slit lamp (mod. 4179T Sbisà, Florence, Italy) was used. Congestion, swelling, and discharge of the conjunctiva were graded on a scale from 0 to 3, 0 to 4, and 0 to 3, respectively (0 means normal). Iris hyperemia and corneal opacity were graded on a scale from 0 to 4. Formulations (30 ml) were topically administered in the right eye every 30 min for 6 h (12 treatments). At the end of the treatment, two observations at 10 min and 6 h were carried out to evaluate the ocular tissues. Observations were made by two independent observers in a masked way. For each relief, values given by the two observers

were added and divided by two. Methylene blue staining was used to evaluate the corneal integrity, which allows an accurate determination of the extent of epithelial damage because of its poor diffusion through the stroma layer of the cornea. The data are presented (Fig. 5) as the percent of eyes with a given score. The actual number of eyes with the score is given between parentheses. Asterisks indicate statistically significant (p < 0.05) differences vs. no treatment groups. Analysis was carried out by Kruskal–Wallis test followed by the multiple comparisons after Kruskal test. 3. Results and discussion The goal of the present study was to assess the ocular tolerability of SLN, produced using variable concentrations of the most commonly surfactant agents found in the literature for analogous systems. To this aim, among the various described lipid components proposed for the production of SLN, we chose Dynasan1 114, a commercial glyceryl trimyristate often found in the composition of

Fig. 3. Zeta potential values (S.D.) measured in water on the prepared SLN batches.

Please cite this article in press as: Leonardi, A., et al., Influence of different surfactants on the technological properties and in vivo ocular tolerability of lipid nanoparticles, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.061

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lipid-based nanocarriers (just among the most recent published articles, see Martins et al., 2009; Aditya et al., 2010; Petersen et al., 2011; Joseph and Bunjes, 2012; Martins et al., 2012; Kuo and ShihHuang, 2013; Sun et al., 2013), and whose cytocompatibility has been also reported (Olbrich et al., 2004). In particular, for this study Dynasan1 114 was essentially selected because of its peculiar physico-chemical properties, and mainly its solubility in acetone, that allows to carry out the QESD production method.

3.1. Technological properties of SLN Table 2 reports the composition of the SLN studied in this work. All systems were produced with a constant concentration of Dynasan1 114 (1% by weight). Different concentrations of selected surfactants were tested (Table 1), chosen among the most commonly compounds used in recent literature for the stabilization of LNs (cf. Table S1 and Fig. 1). From a technological point of view, all the used surfactants effectively contributed to obtain colloidal micro- and nanosystems (Fig. 2). As a general trend, using lower surfactant concentrations smaller nanoparticles were generated. Some recent literature data suggest that analogous findings have been obtained for SLN systems based on Dynasan1 114 and using surfactants structurally related to those tested by us (Sanjula et al., 2009), although in other papers contrasting results were reported (e.g., Liu et al., 2012). Most probably, the properties of the produced nanoparticles are basically affected by the technique of production chosen, in turn

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influenced by the presence and concentration of the lipids and surfactants. In particular, however, the SLN produced in the presence of Kolliphor1 P188 (batches K) showed the most interesting technological features, with a mean size around 250 nm and a PDI value around 0.3. The surface charge of the SLN systems was not particularly marked, typically ranging from 10 to +10 mV (Fig. 3). This behavior is justified by the absence of a net charge in both the used lipid matrix and surfactants, a reason why cationic or anionic modifiers are usually required to produce nanocarriers with a more marked surface charge, when necessary for a specific therapeutic goal. On the contrary, the SLN prepared using SDS showed strong negative Zeta potential values; such a marked feature could be also related to the irritant effects possessed by this surfactant. As regards the physical stability of the produced SLN systems, the best results were achieved once more using Kolliphor1 P188 (batches K) that, at all the tested concentrations, allowed to produce small SLN systems which kept constant particle size and homogeneity (PDI) at suitable values for an ocular application (below 300 nm and 0.4, respectively), up to 6 months at 4  C (Fig. 4). All the other tested surfactants, except Cremophor1 A25 (batches C), seemed able to ensure a relative stability of the produced nanosystems when used at low concentration (0.05% by weight); in general, the initial size and PDI values of each SLN batch were maintained along the period of observation, even if with size values (usually over 500 nm) less interesting in the view of an ocular or parenteral administration.

Fig. 4. Changes of mean particle size and PDI values upon storage at 4  C of the prepared SLN.

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Fig. 5. Ocular score (%) after 10 min (a) or 6 h (b) from the last instillation. The actual number of eyes with the score is given between parentheses. Asterisks indicate statistically significant (p < 0.05) differences vs. no treatment group (CTRL).

It is however worthy to note that, according to the basic aim of this study, the SLN systems presented here have not been optimized, e.g., in terms of components, size uniformity and charge, all qualities that could be easily varied by adding lipid modifiers or co-surfactants during the production phase, or through post-production steps. For the same reason, no drug or model active compound was loaded in these systems, to not alter the evaluation of their tolerability profile. However, in parallel researches we have demonstrated the ability of SLN based on Dynasan1 114 and made using some of the above tested surfactants to efficiently carry active ingredients, such as melatonin (Leonardi et al., 2014a), idebenone (Leonardi et al., 2014b), and a siRNA (Pignatello et al., unpublished results), and to form technologically valid and stable delivery systems. 3.2. Ocular tolerability To quantify the ocular irritation potential of the chosen surfactants, and the related SLN formulations, the parameters of

congestion, swelling and discharge of conjunctiva, iris hyperaemia and corneal opacity were evaluated in rabbits. After the instillation to rabbit’s eye, all the SLN suspensions made with Kolliphor1 P188 (batches K) caused no sign of ocular inflammation or tissue alteration in the eye tissues, with “zero” score as regards conjunctival congestion, swelling, and discharge both at 10 min (Fig. 5a) and 6 h measurements (Fig. 5b). Iris hyperemia and corneal opacity scores were also equal to zero at all observations, up to a surfactant concentration of 0.4% by weight. As expected, the formulations produced using 2% SDS (S02) caused a severe inflammation in terms of congestion, swelling and discharge of the conjunctiva, even though corneal opacity was never observed. The SLN formulated with 2% Tween 80 (T02) also elicited some irritancy, particularly in the conjunctival tissue; some evidences of irritation were also observed at a lower concentration (0.05% Tween 80). Similar results were observed using Kolliphor1 HS 15 (H). The SLN suspensions containing 2% Cremophor1 A25 (C02) were well tolerated, with some signs of conjunctival irritancy with

Please cite this article in press as: Leonardi, A., et al., Influence of different surfactants on the technological properties and in vivo ocular tolerability of lipid nanoparticles, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.061

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a double concentration and for a long contact time (C04). The SLN obtained using Lipoid1 S100 were well tolerated at the lowest tested concentration (2%, sample L02), with some conjunctival irritancy at the highest dose (4%, sample L04). No corneal opacity was observed for all the tested formulations (data not shown). Comparing the above experimental findings with other recent published data, some confirmations can be founds. For instance, Gökçe et al. (2009) showed that Tween1 80 and Pluronic1 F68 (a Poloxamer 188), used for the production of SLN made of Compritol1 888 ATO, where safe on the tissues in a 0.1–0.4% by weight concentration range. In different articles, Souto and coworkers have tested the ocular toxicity of LNs made using different lipids and surfactants. For instance, Precirol1 ATO5 SLN associated to Lutrol1 F68 were shown to be perfectly compatible with the ocular surface (Araújo et al., 2010), as well as flurbiprofenloaded NLC containing Tween1 80 (Gonzalez-Mira et al., 2011). 4. Conclusions The main aim of this study was to assess whether some surfactants, commonly described in the literature as ingredients for the production of LNs, have a suitable tolerability profile to be used in nanocarrier systems for ocular application. In fact, the biocompatibility assessment of many and well-known nanocarriers is often lacking, particularly for ophthalmic use. Our experimental data showed that stable SLN systems, with good technological features can be obtained using Dynasan1 114 in the presence of different surfactant agents. In particular, addition of Kolliphor1 P188 gave small and uniform nanoparticle dispersions, with a long physical stability upon storage. Also from a toxicological point of view, the nanocarriers produced using the latter surfactant were the most valuable, showing no irritant effect on the ocular surface up to the highest tested surfactant concentration (0.4%, w/v). The SLN produced with Lipoid1 S100 and Cremophor1 A25 were flawlessly tolerated up to 0.2% (w/v), while for Tween1 80 and Kolliphor1 HS 15 a maximum concentration of 0.05% by weight could be considered safe. The results obtained in this study can be considered useful not only for SLN preparations, but also for polymeric nanoparticles, nano-emulsions and generally for nano- or micro-structured systems with an ocular application, and whose preparation process requires the presence of surfactants. In particular, on the basis of the results obtained in this preliminary study, the more safe and stable nanocarriers will be further evaluated for other aspects linked to the type and concentration of surfactant(s), such as the permeability of loaded drugs through the cornea or other epithelial or mucosal barriers of the body. Acknowledgements This work was partially supported by Italian Minister of University (PRIN2009 project no. 2009BM7LJC and PON01-00110 project). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijpharm. 2014.04.061. References Aditya, N.P., Patankar, S., Madhusudhan, B., Murthy, R.S.R., Souto, E.B., 2010. Arthemeter-loaded lipid nanoparticles produced by modified thin-film hydra-

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Please cite this article in press as: Leonardi, A., et al., Influence of different surfactants on the technological properties and in vivo ocular tolerability of lipid nanoparticles, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.04.061