Nanostructured lipid carriers for triamcinolone acetonide delivery to the posterior segment of the eye

Colloids and Surfaces B: Biointerfaces 88 (2011) 150–157

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Nanostructured lipid carriers for triamcinolone acetonide delivery to the posterior segment of the eye Joana Araújo a , Sasha Nikolic a , Maria A. Egea a,b , Eliana B. Souto c,d,∗ , Maria L. Garcia a,b a

Department of Physical Chemistry, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain Institute of Nanoscience and Nanotechnology, University of Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain Institute of Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB-UTAD), P.O. Box 1013, 5000-801 Vila-Real, Portugal d Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia, Nr. 296, P-4200-150 Porto, Portugal b c

a r t i c l e

i n f o

Article history: Received 5 March 2011 Received in revised form 19 June 2011 Accepted 20 June 2011 Available online 25 June 2011 Keywords: Nanostructured lipid carrier Triamcinolone acetonide Nile red Retina Posterior segment Ocular Eye Stability Turbiscan

a b s t r a c t Triamcinolone acetonide (TA) is a corticosteroid drug currently administered by intravitreal injection for a broad spectrum of inflammatory, edematous and angiogenic ocular diseases. To increase the drug’s bioavailability by ocular instillation, TA was encapsulated in nanostructured lipid carriers (NLC), previously optimized by our group using a factorial design approach. In the present paper, nanometric (∼200 nm), unimodal and negatively charged NLC loaded with the fluorescent lipid marker Nile red (NR-NLC) and drug (TA-NLC) were produced by high pressure homogenization. Based on the selected formulations, in vivo tests were carried out by eye-drop instillation of NR-NLC in mice, revealing the systems’ ability of delivering lipophilic actives to the posterior segment of the eye via the corneal and non-corneal pathways. Short and long-term stability of TA-NLC was assessed by high performance stability analysis using the Turbiscan® . The results showed a backscattering of less than 1.5% and during a period of 6 months, anticipated the low tendency of these particles for aggregation during shelf life when stored at room temperature. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Several diseases of the posterior segment of the eye represent the leading causes of vision impairment and blindness, where chronic anterior, intermediate, and posterior uveitis can cause decreased visual acuity [1]. Until recently, treatment of such disorders has been primarily surgical, but over the past few years, improved understanding of the pathophysiology of many retinal diseases has led to the development of effective drug therapies, which replace or complement surgery [2,3]. Ocular injection of triamcinolone acetonide (TA) is a well established procedure for the treatment of these diseases; however, despite the relative success of this approach, the need for repeated injections over the course of months/years creates a significant treatment burden to patients and their families. In addition, because steroidal drugs are almost completely insoluble in water, and effectively excluded

∗ Corresponding author at: Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia, Nr. 296, P-4200-150 Porto, Portugal. Tel.: +351 225 074630; fax: +351 225 074637. E-mail address: [email protected] (E.B. Souto). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.06.025

from the eye by uveal blood-aqueous and blood retinal barriers, administration by traditional liquid eye-drops is not possible and systemic administration fail to reach therapeutic drug levels [4,5]. Having these important shortcomings in mind, improved drug delivery technologies that provide optimal pharmacokinetics, dose intervals, and less invasive routes of administration are needed. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) are interesting systems for the present purposes due to their solid lipid matrices, which are also generally recognized as safe (GRAS) or have a regulatory accepted status [6]. NLC are described as an improved generation of SLN because a controlled nanostructuring of the lipid matrix is performed due to the mixture of solid lipids with spatially incompatible liquids, increasing drugload thus, preventing its expulsion [7]. Nanoparticles promises to be an important part of the new therapeutic armamentarium in ophthalmology because they show the intrinsic capacity to adhere to the ocular surface and interact with the epithelium depending on their physicochemical characteristics, such as size, shape and surface charge [5]. Hironaka et al. [8] have recently developed submicron-sized liposomes loaded with the marker coumarin-6, where fluorescence

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emission was detected in the posterior segment of mice eyes after topical administration. To our knowledge, this study is so far the only report on the usage of a colloidal carrier to deliver drugs to the retina via eye-drops. Previously, our research group has developed and characterized an optimized TA-loaded NLC produced by high pressure homogenization [9]. The present study reports the ability of this carrier to deliver lipophilic drugs to the posterior segment of the eye and to avoid the complications associated with intraocular repeated injections. Lipids represent a heterogeneous class of substances, for which insolubility in water is a common property because of the hydrophobic moieties of their molecules mainly composed of fatty acids. The multiple polymorphic modifications of the fatty acids arise from the different molecular conformation of these moieties [10]. Changes in the solid state by effects of temperature, pressure or aging, should be understood and well controlled, since these may influence drug release characteristics of lipid dosage forms. Since the selected solid lipid (Precirol® ATO5) is a mixture of mono, di- and triglycerides, it is likely to suffer these thermodynamic changes. The effect of these thermodynamic parameters, on the short and long-term stability of optimized NLC, is also discussed on the basis of high performance stability analysis and physicochemical characteristic measurements. 2. Materials and methods 2.1. Materials Precirol® ATO5 (solid lipid), a mixture of mono-, di- and triglycerides of palmitic acid (C16 ) and stearic acid (C18 ), was a gift from Gattefosse S.A. (Saint-Priest, France) and Lutrol® F68 (hydrophilic surfactant), a hydrophilic block copolymer of ethylene oxide and propylene oxide, from BASF (Barcelona, Spain). Squalene® (liquid lipid) which is an unsaturated aliphatic hydrocarbon, rac.-1-oleoylglycerol (lipophilic surfactant), Triamcinolone acetonide and the fluorescent lipid marker Nile red (NR), were purchased from Sigma (St. Louis, USA). Double distilled water was used after filtration in a Millipore® system home supplied. All other reagents were of analytical grade.

2.2. Production of NLC NLC were prepared as described elsewhere [11]. Briefly, the lipid phase containing the solid and liquid lipids and the lipophilic surfactant, was heated up to 80 ◦ C to melt the lipids and obtain a homogeneous solution. Following the addition of TA or NR, the hot lipid phase was dispersed in the aqueous solution of hydrophilic surfactant, heated at the same temperature, by high-speed stirring (7000 rpm/30 s) using Ultra Turrax T-10 (IKA, Germany) to form a pre-emulsion, which was passed (3 cycles/600 bar) through a high pressure homogenizer (APV 2000, Denmark). Finally, the resulting hot o/w nanoemulsion was cooled to 4 ◦ C, recrystallizing the lipid and forming the NLC. The NLC composition (Table 1) was previously optimized by factorial design [9].

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Instruments, UK) was used to discard the presence of particles above 3–5 ␮m. 2.3.1.1. Encapsulation parameters. The encapsulation efficiency (EE) and loading capacity (LC) of TA in NLC were assessed indirectly, determining the free TA (non-encapsulated) by reverse-phase high performance liquid chromatography (RP-HPLC), using a modification of the USP method [9], and applying the following equations: EE(%) =

Total amount of TA − Free TA × 100 Total amount of TA

(1)

LC(%) =

Total amount of TA − Free TA × 100 Total amount of lipid

(2)

Free TA was removed by filtration/centrifugation technique using centrifugal filter devices Ultracel YM-100 (Amicon® Millipore Corporation, Bedford, MA) at 3000 rpm for 15 min (Sigma 301 K centrifuge, Spain). Prior to filtration/centrifugation, each sample was diluted suitably with phosphate buffer saline (PBS) solution to avoid deposition of free TA (possibly crystallized in the aqueous phase) onto the NLC surface, and thus measured as encapsulated. If previously diluting the samples, all free TA passes through the filter and is adequately quantified by RP-HPLC. The RP-HPLC system consisted of a Waters 1525 pump (Waters, Milford, MA) with a UV–vis 2487 detector (Waters) set at 254 nm. A reverse-phase column (Hypersil® ODS 5 ␮m, 10 × 0.46) with a flow rate of 1.5 mL/min was used. The mobile phase consisted of acetonitrile:methanol:water (30:10:60). 2.3.2. Differential scanning calorimetry (DSC) Accurately weighted NLC (1–2 mg lipid) were scanned using a Mettler DSC 823e System (Mettler Toledo, Spain) from 25 ◦ C to 85 ◦ C at a rate of 5 ◦ C/min and cooling down to 10 ◦ C in sealed 40 ␮L aluminium pans. An empty pan was used as a reference. Indium (purity > 99.95%; Fluka, Buchs, Switzerland) was employed to check the calibration of the calorimetric system. Data was evaluated from peak areas using the Mettler STARe V 9.01 DB software (Mettler Toledo, Spain). DSC thermograms were recorded for TA-free and TA-loaded NLC stored at different temperatures. The crystallinity index (CI) of NLC was calculated from the heat of fusion according to the following equation: CI(%) =

Enthalpylipid

EnthalpyNLC [J/g] × 100 phase [J/g] × Concentrationlipid

phase [%]

× 100 (3)

2.3.3. Wide angle X-ray scattering (WAXS) WAXS diffractograms were captured on a PANalytical X’Pert PRO power diffractometer (Almelo, The Netherlands) with copper anode (Cu K␣ radiation,  = 1.5418 nm) and X’Celerator detector. The diffractograms were measured at angles 2 = 5–40◦ with step of 0.017◦ and count time of 50 s. WAXS analysis of NLC dispersions was carried out directly without further treatment. 2.4. Animal studies

2.3. Physicochemical characterization 2.3.1. Mean particle size and zeta potential analysis Particle size (Z-ave), polydispersity index (PI) and zeta potential (ZP) were determined by photon correlation spectroscopy (PCS) with a Zetasizer Nano ZS (Malvern Instruments, UK) at 25 ◦ C, after appropriate dilution with ultra-purified water. For Z-ave analysis disposable polystyrene cells were used, whereas disposable plain folded capillary zeta cells were required for ZP analysis. In addition, laser diffraction (LD) with a Mastersizer Hydro 2000MU (Malvern

Mouse experimental models are extremely useful in understanding human retinal conditions. Human and mouse genomes have high homology, with some estimates of up to 95% overlap. Moreover, mice have fast generation times and aging, thus reducing maintenance costs [1]. Unanesthetized female adult CD1 mice weighting 30–35 g were used. The mice were fed with regular diet. A single dose of 4 ␮L NR-NLC formulation or NR solution was dropped onto the surface of the left eye. The contralateral eye was used as the control and received no treatment. The mice were then divided in groups of 3 and sacrificed 8, 20, 40, 70, 110 or 160 min

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Table 1 Composition of the previously optimized triamcinolone acetonide loaded nanostructured lipid carrier (TA-NLC) formulation. TA-NLC composition (%) Solid lipid

Liquid lipid

Hydrophilic surfactant

Lipophilic surfactant

TA

Water

6.300

2.700

1.800

0.200

0.025

Up to 100

after administration of the formulations. Both eyes were enucleated immediately, washed with excess amount of PBS, and then fixed overnight in 3% p-formaldehyde at 4 ◦ C and smooth agitation. Fixed eyes were immersed in 5% sucrose for 2 h, 10% sucrose also for 2 h and 30% sucrose overnight at 4 ◦ C and smooth agitation for cryoprotection. Eyes were embedded in Optimal Cutting Temperature compound (Tissue-Tek® Sakura, USA), frozen at −25 ◦ C, and sliced with a cryostat (Leica CM-3050-S, UK) into sections 50 ␮m thick. The tissue sections were then placed onto slides, previously treated with 2% (3-aminopropyl)-triethoxysilane in acetone for 5 min, and finally covered with 15 ␮L ProlonGold® (Invitrogen, USA) embedding medium and 22 mm × 22 mm cover-slips. Before images were taken, the covering product was left to harden overnight at 4 ◦ C. All experiments were performed according to the Association for Research in Vision and Ophthalmology (ARVO) resolution for the use of animals in research, approved by the animal research ethical committee of the University of Barcelona and responded to the normative of EC Directive 86/609/EEC and Directive 214/97, Gencat. 2.5. Fluorescence microscopy The images of frozen eye sections were observed using confocal microscopy (Leica TCS SPE, Germany) in the wavelength of 550–700 nm. The “blank” was recorded from untreated tissue. The inner plexiform layer (IPL) is reported as a suitable target for evaluating retinal delivery, since it is located very close to the ganglion cell layer (GCL), containing retinal ganglion cells and amacrine cells, where death is a common feature in many ophthalmic disorders (e.g. glaucoma, optic neuropathy and retinal vein occlusions) [8]. In the IPL, the fluorescence intensity of NR was evaluated with appropriately calibrated computerized image analysis (Image Processing and Analysis in Java, Image J, USA), using “median density” as an analytic tool at a constant area (50 ␮m × 50 ␮m). The relative intensity indicates the value of a treated sample when the fluorescence intensity of an untreated sample is estimated as 0. 2.6. Stability studies The prediction of the physical stability of the developed formulations was performed on an optical analyzer Turbiscan® Lab (Formulation, L’Union, France), based on the analysis of the multiple dispersion of the light by concentrated suspensions. The main advantage of Turbiscan is the ability to detect destabilization phenomena much earlier than the naked eye’s operator, especially in the case of opaque systems. Each sample (10 mL) was placed in a cylindrical glass cell. The detection head was composed of a pulsed near-infrared light source ( = 850 nm) and two synchronous transmission (T) and back scattering (BS) detectors. The T detector receives the light, which crosses the sample (at 180◦ from the incident beam), while the BS detector receives the light scattered backwards by the sample (at 45◦ from the incident beam). The detection head scanned the entire height of the sample cell (65 mm longitude), acquiring T and BS each 40 ␮m (1625 acquisitions in each scan), every hour, during a period of 24 h. The measuring principle is based on the variation of the droplet volume fraction (migration) or diameter (coalescence), resulting in a variation of BS and T signals [12,13]. For the long-term stability study, the ini-

tial physicochemical characteristics of TA-loaded NLC dispersions were analyzed immediately after production, and this batch was then divided into three sample sets, one stored at 4 ◦ C (in a refrigerator), one stored at room temperature (RT) and the other stored at 40 ◦ C (in a temperature-controlled oven). All samples were stored in silanized glass vials that were sealed and wrapped with black paper. Samples were withdrawn after 1, 2, 3 and 6 months, for Z-ave, ZP, PI, RP-HPLC, DSC and WAXS analyses. 3. Results and discussion Optimized NLC (Table 1) were shown to be nanometric, unimodal with a relatively narrow size distribution observed by LD measurements and depicting negatively charged surfaces (Table 2) [9]. Ocular nanoparticle disposition can be evaluated using fluorescent or radio-labelled nanoparticles. For the present study, NR was chosen as marker due to its characteristics. NR is a lipophilic, neutral compound very soluble in organic solvents and strongly fluorescent in a hydrophobic lipid environment, with the emission maximum found near 600 nm. In the case of NR molecules embedded between the relatively long hydrocarbon chain, as those found in Precirol, this location is likely to prevent any contact with adjacent water molecules. On the other hand, due to the shorter chain length of an oily environment, as in the liquid lipid, the accessibility of NR to water molecules results in a lower hydrophobicity and a shift of the corresponding emission maximum. Applying paraelectric spectroscopy, Borgia et al. [14] found that NR is incorporated into the particles or the covering tenside shell and not attached to the surface. To select the maximum NR concentration that is kept within solid and liquid lipid molecules without partitioning the excess to aqueous surrounding media, several NLC formulations ranging from 5 to 50 ␮g NR/g lipid were prepared. Fluorescence microscopy pictures were recorded and 10 ␮g NR/g lipid was the chosen formulation for posterior studies (data not shown). After instillation, the flow of lachrymal fluid always removes a certain amount of instilled compounds from the surface of the eye. Even though the lachrymal turnover rate is only about 1 ␮L/min the excess volume of the instilled fluid is flown to the nasolachrymal duct rapidly in a couple of minutes. Another source of non-productive drug removal is its systemic absorption instead of ocular absorption, either directly from the conjunctival sac via local blood capillaries or after the solution flow to the nasal cavity. Therefore, conventional dosage forms have numerous deficiencies that make the development of alternative delivery strategies desirable. To study the behaviour of NLC after they are dropped into the eye, sections of sliced eye tissue were obtained as described previously and time-course observation was carried out. Fig. 1 shows the magnitude of red emission, quantified using Image J software, in the IPL observed for the samples taken 8, 20, 40, 70, 110 and 160 min after eye-drop administration. The retinal fluorescence of NR gradually increased with time, peaking 40 min after administration, decreasing thereafter and almost disappearing at 160 min after administration. To exclude the hypothesis of retinal fluorescence observation due to NR itself, an aqueous solution with the same concentration of dye was dropped on the mice eyes and neg-

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Table 2 Particle size analysis by photon correlation spectroscopy (Z-ave) and laser diffraction (LD) methods, polydispersity index (PI), zeta potential (ZP), encapsulation efficiency (EE) and loading capacity (LC) of the studied formulation. The results are expressed as mean ± S.D (n = 3). LD (nm) Z-ave (nm)

PI

d(0.1)

d(0.5)

d(0.9)

ZP (mV)

EE (%)

LC (%)

173.30 ± 0.32

0.10 ± 0.02

75.00 ± 0.00

116 .00 ± 2.00

180.00 ± 0.00

−46.70 ± 0.91

94.82 ± 1.12

0.26 ± 0.00

ligible fluorescence was found, being this last fluorescent strength similar to the one observed for untreated eyeballs. Although the Image J software could not accurately estimate the amount of NR reaching the IPL, it could evaluate the relative potency with which NLC delivered the hydrophobic compound to the IPL (Fig. 2). In ophthalmic drug delivery systems, nano-sized particles represent a state of matter characterized by higher bioadhesion and

Fig. 1. Accumulated fluorescence intensity variation in the inner plexiform layer after eye-drop administration of Nile red loaded nanostructured lipid carrier (NRNLC) and Nile red solution (NR-solution). The results are expressed as mean ± S.D. (n = 3), * P < 0.05.

greater surface area available for association between cornea and conjunctiva. Strong fluorescence was observed on the surface of the anterior segment for NLC treated eyes in the first 8 min (Fig. 3) and was still detected 160 min later. On the other hand, no fluorescence was detected in the case of eyes treated with NR solution. The longer retention of NLC on the ocular surface increases its association with the tissues and the loaded material absorption. With respect to this, latter three routes can be envisaged: the systemic, corneal and non-corneal pathways. No fluorescence was observed in the optic nerve or in the retina of the untreated contralateral eye during the whole 160 min experiment time. This result evidences that systemic delivery caused by nasolachrymal drainage did not contribute to the observed retinal fluorescence in the treated eyes. Corneal pathway was thought to be one route because fluorescence was observed not only on the surface but also on the corneal tissue (Fig. 3) and non-corneal pathway was thought to be the major route. The physical stability of nanoparticle dispersions is one of the most important desired product characteristics. Lipid nanoparticles are heterogeneous systems and thermodynamically unstable, and therefore, they have a significant tendency to lose physical stability during storage [15]. On the other hand, from the literature, lipid nanoparticle dispersions with optimized stabilizer composition are physically stable on storage for several years [16]. Having this in mind, a combination of lipophilic and hydrophilic surfactants was chosen. The hydrogenated palm oil glyceride is a highly lipophilic emulsifier [17], and the non-ionic hydrophilic emulsifier, offers additional steric stabilization effects by avoiding aggregation of the nanoparticles in the NLC system [18]. Apart from the naked eye, the techniques currently used to detect physical destabilisation include microscopy, spectroscopy,

Fig. 2. Fluorescence microscopic images of the retina 40 min after treatment with (a) Nile red loaded nanostructured lipid carrier (NR-NLC), and (b) Nile red solution (NR-solution).

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Fig. 3. Fluorescence microscopic image of the anterior segment of the eye 8 min after treatment with Nile red loaded nanostructured lipid carrier (NR-NLC).

Fig. 4. Backscattering profile of nanostructured lipid carrier as a function of time (h) and of sample height (mm).

turbidity and particle size analysis. Since formulations are concentrated and opaque, resembling a milky dispersion, previously to measurements, samples need to be diluted. This may severely reduce the accuracy and scope of analytical instruments. The high performance stability analyser has accordingly been developed to fill this gap and to allow the evaluation of the physical destabilisation of particles. The instrument allows distinguishing between the two major destabilisation phenomena affecting the homogeneity of dispersions: particle migration (creaming, sedimentation), which is reversible by mechanical stirring, and particle size variation or aggregation (coalescence, flocculation), which is often irreversible [19]. Fig. 4 shows the BS profile of TA-NLC, which signal can only be analyzed if T signal is nil, otherwise, the partial reflection of the light crossing the sample by the walls of the measurement cell would interfere with the BS signal [13]. A

decrease of only 1.5% BS signal was detected, demonstrating that the sample does not show significant tendency to aggregate. Few changes were detected in particle migration rate, noticed by 6% decrease in the scattered light on the top of the cell. This discrete sedimentation easily disappeared under stirring. The long-term physical stability of the colloidal systems is an important parameter since it controls the validity and the required storage conditions. Particle size distribution is one of the most important characteristics for the evaluation of the stability of colloidal systems, since it plays an important role in their ability to interact, in particular, with the ocular mucosa. As such, the particle size parameters have been evaluated immediately after production of NLC, and during 6 months of storage at three different temperatures (4 ◦ C, RT and 40 ◦ C), to challenge the systems for stress conditions.

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Fig. 5. X-ray diffraction patterns of nanostructured lipid carrier within the day of production (initial profile) and after 6 months of storage at 40 ◦ C, room temperature (RT) and 4 ◦ C.

With time-course observation studies, particle size growth was observed to be a function of time and storage temperature. The NLC stored at 4 ◦ C and RT remained with a milky-like appearance and the Zave was maintained in the nanometric range, whereas the Z-ave of the NLC stored at 40 ◦ C increased from ∼200 nm to the micrometer range, creaming after 2 months. For 4 ◦ C and RT storage temperatures, PI depicted the same magnitude as the values obtained immediately after production, with a slight decrease after 6 months attributed to the fusion of the smaller particles to form larger ones (Table 3).

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It has been reported that lipid molecules recrystallize in different polymorphic forms, i.e. unstable ␣, metastable ␤ , and the most stable the ␤ modification, existing additional intermediate ␤i forms, between ␤ and ␤, for mixtures of glycerides [20]. Although the ZP values did not vary notably, keeping the particles a negative surface charge, it was found to increase with longer storage time and higher temperature. The formation of long ␤-crystals takes place during storage and since different sides of a crystal show different charge density, this crystalline reorientation results in modification of the surface ratio of differently charged crystal sides and, consequently, ZP changes. Similar results were observed by Vivek et al. [21] when studying the stability of lipid nanoparticles with different matrix materials. The crystalline structure of the lipid particles, which is related to the chemical nature of the used lipids, is a key factor to decide in determining whether a drug will be expelled or firmly incorporated for longer time. The polymorphic form is also a parameter determining drug incorporation. The initial EE of ∼95% was significantly reduced during the 6 months of storage, by about 7% and 4% at 4 ◦ C and RT, respectively. In the case of LC, the values were maintained at 0.26% for RT and slightly decreased for 4 ◦ C. Despite not being significant, the decrease of encapsulation parameters could be explained by the creation of a perfect crystalline ␤-modification of solid lipid that takes place during storage and leads to drug expulsion. Similar results were reported for hydroxycampothecinloaded NLC [22]. Crystallization of the bulk lipid is different to the lipid in nanoparticles, bulk lipids recrystallize preferentially in the ␤ modification and transforming rapidly into the ␤-form [23], whereas NLC recrystallize at least partially in the ␣-form. With time and stress conditions, there is an increased tendency to form the more stable ␤ /␤i-modifications, which present a more perfect lattice and consequently promote the previously referred drug expulsion. In general, the transformation is slower for long-chain than for short-chain triglycerides [24], and the solid lipid with its C16 and C18 chains reveals a promising long-term stability for NLC. This carrier was produced in a controlled way by creating a certain fraction of ␣-form, which gave the characteristic amorphous profile in both DSC and WAXS previous studies [9]. This is clearly preserved during the storage time of 6 months for 4 ◦ C and RT, with no significant changes in both WAXS profiles, while in the case of NLC stored at 40 ◦ C the carriers lost their regular crystalline structure (Fig. 5). The stability during storage can also be monitored by looking at the changes in melting enthalpy and crystallinity of colloidal NLC matrices. From Fig. 6 no significant changes in crystallinity index (CI) and melting point (MP) are depicted for NLC stored at

Fig. 6. Crystallinity indices (CI) and melting points (MP) of nanostructured lipid carrier after 7 days, 1, 2, 3 and 6 months of storage at 4 ◦ C, room temperature (RT) and 40 ◦ C.

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Table 3 Particle size analysis by photon correlation spectroscopy (Z-ave) and laser diffraction (LD) methods, polydispersity index (PI) and zeta potential (ZP) of optimized NLC, monitored for 6 months at 4 ◦ C, room temperature (RT) and 40 ◦ C. The results are expressed as mean ± S.D. (n = 3).. Storage

Z-ave (nm)

PI

ZP (mV)

LD d(0.9) (nm)

0 days

173.30 ± 0.32

0.10 ± 0.02

−46.70 ± 0.91

180.00 ± 0.00

7 days

4 ◦C RT 40 ◦ C

218.70 ± 1.55 179.70 ± 1.12 232.70 ± 0.93

0.11 ± 0.03 0.10 ± 0.01 0.12 ± 0.01

−41.50 ± 0.81 −43.10 ± 1.01 −39.60 ± 0.41

223.00 ± 1.00 202.00 ± 0.00 228.00 ± 0.00

1 month

4 ◦C RT 40 ◦ C

222.90 ± 0.45 189.90 ± 1.59 252.60 ± 4.18

0.13 ± 0.00 0.10 ± 0.01 0.14 ± 0.02

−41.50 ± 0.81 −41.20 ± 0.35 −39.20 ± 0.10

225.00 ± 1.00 209.00 ± 3.00 514.00 ± 5.00

2 months

4 ◦C RT 40 ◦ C

227.50 ± 1.36 188.00 ± 1.19 −

0.13 ± 0.03 0.10 ± 0.02 1

−38.40 ± 0.45 −42.10 ± 0.64 −35.20 ± 0.44

234.00 ± 1.00 217.00 ± 1.00 654.00 ± 9.00

3 months

4 ◦C RT 40 ◦ C

243.40 ± 2.37 226.10 ± 2.35 −

0.12 ± 0.04 0.10 ± 0.01 −

−39.70 ± 0.40 −41.90 ± 1.01 −

237.00 ± 1.00 218.00 ± 0.00 −

6 months

4 ◦C RT 40 ◦ C

312.40 ± 3.39 294.10 ± 1.79 −

0.09 ± 0.01 0.08 ± 0.02 −

−35.60 ± 0.36 −42.30 ± 0.81 −

540.00 ± 8.00 522.00 ± 6.00 −

4 ◦ C and RT, which is a good sign for anticipating long-term-stability [18].

4. Conclusions Research on the posterior segment diseases is of high clinical significance and there is an urgent requirement for efficient drug delivery systems. Another important issue that can be envisaged is the route of administration, representing ocular topical instillation the most convenient and less invasive. Results from this study in mice, evidence the possibility of drug delivery to posterior segment by NLC carriers, making these nanoparticles a promising approach to provide selective and prolonged drug concentration in the eye and avoid the problematic intravitreal injections. Of the various new drug delivery systems, colloidal nanoparticle carriers studied in this work appear to be useful for ocular absorption enhancement of drugs possibly acting via multidimensional mechanisms, namely, by prolonged drug residence time in the ocular surface and conjunctival sac, by sustained drug release from the delivery system, and/or by reduced precorneal drug loss [25]. An extrapolation from animal data to human data should be carefully carried out because of species differences. However, a better understanding about the behaviour of instilled drugs and a thorough knowledge of the physiology of the eye will result in development of useful drug delivery systems. The data provided by the stability studies show that higher temperature has an important role to play. NLC kept at RT revealed higher stability and lower rates of loss of encapsulated corticoid in comparison with those stored at 4 ◦ C. In both cases, the size was kept in the nanometric range, while in the case of storage at 40 ◦ C, NLC completely lost their crystalline structure and creamed after 2 months.

Acknowledgements The authors wish to acknowledge the sponsorship of the “Spanish-Portuguese Integrated Actions” (Ref: HP2008-0015) and of Fundac¸ão para a Ciência e Tecnologia do Ministério da Ciência e Tecnologia (PTDC/SAU-FAR/113100/2009). The kind help of Dr. Marta Taulés and Dr. Manel Bosch from Serveis Cientificotècnics of University of Barcelona, Spain, in the confocal microscopy studies, is also acknowledged.

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