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Abstract: Ferulic acid (FA), a phenolic compound with a significant antioxidant activity in Alzheimer's disease, was entrapped into sev- eral solid lipid ...
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Current Nanoscience, 2009, 5, 26-32

Ferulic Acid-Loaded Lipid Nanostructures as Drug Delivery Systems for Alzheimer’s Disease: Preparation, Characterization and Cytotoxicity Studies M.L. Bondì1,*, , G. Montana2,, E.F. Craparo3, P. Picone3 , G. Capuano3, M. Di Carlo2 and G. Giammona3 1

ISMN-CNR, via Ugo La Malfa, 153, 90146 Palermo; 2IBIM-CNR, via Ugo La Malfa, 153, 90146 Palermo, Italy; 3Dipartimento di Chimica e Tecnologie Farmaceutiche, University of Palermo, via Archirafi 32, 90123 Palermo, Italy Abstract: Ferulic acid (FA), a phenolic compound with a significant antioxidant activity in Alzheimer’s disease, was entrapped into several solid lipid nanoparticles (SLN and NLC) by using the microemulsion technique. Stable SLN and NLC formulations having mean size ranging between 94-140 nm and high zeta potential were obtained. The SLN sample obtained by using as lipid matrix Compritol 888 ATO was chosen for further characterization because, among these particles, showed high Loading Capacity (LC%) and the best characteristics in terms of size, PDI, and drug release profile. Empty SLN showed no cytotoxicity on human neuroblastoma cells (LAN 5) at tested concentrations and the ability to penetrate into these cells. Moreover, cells treated with FA-loaded SLN showed a higher reduced ROS production than cells treated with free FA. These findings demonstrate that FA-loaded SLN possess a higher protective activity than free FA against oxidative stress induced in neurons and suggest that SLN are excellent carriers to transport FA into the cells.

Key Words: Solid lipid nanoparticles, ferulic acid, drug delivery, human neuroblastoma cells, Alzheimer’s disease. 1. INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the extracellular deposition of the amyloid -peptide (A) and the intraneuronal accumulation of neurofibrillary tangles [1], associated with loss of neurons in the brain. Vaccination [2] and secretase inhibitors [3] have been reported as the experimental therapies and for clinical trials. Many researchers favor other therapeutic approaches that target the formation, deposition, and clearance of A from nervous tissue [1]. Having the oxidative injury a key role in the development of AD, many antioxidant compounds, such as vitamin E, nordihydroguaiaretic acid (NDGA), and nicotine, have been demonstrated to protect the brain from A neurotoxicity [4]. Some other molecules, such as phenolic antioxidants, also inhibit Alzheimer’s amyloid fibrils (fA) formation dose dependently in vitro [5,6]. Ferulic acid, (4-hydroxy-3-methoxycinnamic acid) (FA), is a well known antioxidant phenolic compound [4], which long-term administration protects mice against A-induced learning and memory deficits in vivo [7] and neurons against A(1–42)-induced oxidative stress and neurotoxicity [8]. Furthermore FA inhibits the formation of -amyloid fibrils (fA), as well as destabilizes preformed fA in the Central Nervous System (CNS) [4, 9]. For all the therapies targeted to neurodegenerative diseases, the presence of the Blood Brain Barrier (BBB), which regulates the brain homeostasis but limits the penetration of many drugs, is the critical issue encountered. To circumvent the BBB, several delivery systems such as polymeric nanoparticles, niosomes, liposomes, and micelles have been developed [10, 11]. In recent years, the attention has focused to solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), which lipophilic features lead them to CNS by an endocytotic mechanism, overcoming the BBB [10, 12-15]. These nanoparticles are constituted by a solid lipid matrix made from highly purified triglycerides, complex glyceride mixtures or even waxes. In the NLC production, very different lipids are entrapped to form the matrix in order to obtain system with higher storage stability than SLN [15-17]. Their use as drug delivery systems could achieve to many advantages as the possibility of controlled drug release and drug targeting, increased drug stability, high drug payload, the feasibility of incorporating lipophilic and hydrophilic drugs, a lack of biotoxicity of the carrier, avoidance of organic solvents, and possibility to large-scale production and sterilization [15]. *Address correspondence to this author at the ISMN-CNR, Via Ugo la Malfa 153, 90146 Palermo; Tel: +39 091 6809367; Fax: +39 091 6809247; E-mail: [email protected]  These authors contributed equally to this work.

1573-4137/09 $55.00+.00

Moreover, because of their small size, these systems may be injected intravenously and avoid the uptake of macrophages of mononuclear phagocyte system (MPS) [18]. This paper describes the preparation and chemical-physical characterization of empty and FA-loaded SLN and NLC, obtained by using the warm oil-in-water microemulsion technique. In vitro cell viability after treatment with empty and drug-loaded SLN on human neuroblastoma cells (LAN 5) at tested concentrations and the ability of these particles to penetrate into these cells were investigated. The higher protective activity of FA entrapped into SLN than free FA against oxidative stress was demonstrated by monitoring the intracellular ROS formation on LAN5. 2. MATERIALS AND METHODS 2.1. Materials Compritol 888 ATO (mixture of approximately mono-, di- and triglycerides of behenic acid, at 15, 50 and 35% w/w), Compritol HD5 ATO (mixture of PEG-8 behenate and tribehenin), Precirol ATO (mixture of tripalmitin and tristearin, 50% w/w), were obtained from Gattefossè Italia s.r.l. (Milan, Italy); Miglyol 812 (mixture of caprylic and capric triglycerides) was supplied by Sasol GmbH (Germany). Epikuron 200 was a kind gift from Lucas Meyer (Hamburg, Germany); taurocholate sodium salt, Pluronic F68 and dimethyldioctadecylammonium bromide (DDAB), fluorescein free acid, 2’,7’-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma Aldrich. Ferulic acid was purchased from Fluka (Milan, Italy). The other chemicals were obtained from Sigma Aldrich and were of analytical grade. 2.2. Preparation of Empty and FA-Loaded SLN and NLC Several SLN and NLC, empty or drug-loaded, were prepared by using the warm oil-in-water (o/w) microemulsion technique [15]. Briefly, to prepare the lipid phase, the selected lipid or mixture was heated to 5-10 ºC above its melting point. In the case of drugloaded sample preparation, FA (100 mg) was added to the melted lipid phase. Preliminary studies were performed in order to ensure the drug stability above the lipid melting points for a time period required to obtain the hot microemulsions. No degradation process occurs on the drug at tested conditions (data not showed). Successively, an aqueous suspension of surfactant and an aqueous solution of co-surfactant were added, obtaining a clear microemulsion. Moreover, fluorescein-loaded SLN (SLN-f) were obtained by dissolving fluorescein (0.78 mg) into the melted lipid and operating as described above for empty SLN. © 2009 Bentham Science Publishers Ltd.

Ferulic Acid-Loaded Lipid Nanostructures

The chemical composition of each microemulsion, expressed as % w/w on the total microemulsion weight, is reported in Table 1. In the case of sample SLN-c, the surfactant and the co-surfactant were together suspended in bidistilled water and successively they were added at melted lipid phase containing FA. Successively, the hot microemulsion was dispersed in 100 g of cold water (2-3°C) under mechanical stirring and SLN or NLC were obtained. The colloidal aqueous dispersion of SLN or NLC was centrifuged at 45000 rpm (Centrifuge XL-90 Beckmann) for 1 hr at 4°C. Nanoparticles were then suspended in water, freeze-dried (Modulyo freeze-dryer, Edwards, Crawley, UK) and stored for successive characterization. The aqueous dispersion of SLN-a was purified by a process of exhaustive dialysis and freeze-dried. 2.3. Particle Size Determination The average diameter and polydispersity index (PDI) of empty and FA-loaded SLN and NLC were determined by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instrument, Herrenberg, Germany) that utilizes the Non-Invasive Back-Scattering (NIBS) technique. Each dried sample was appropriately suspended with filtered (0.2 μm) bidistilled water, and the reading was carried at a 173° angle in respect to the incident beam. Each reported value was the average of three measurements. 2.4. Zeta Potential Measurements The zeta potential values were measured using principles of laser Doppler velocitometry and phase analysis light scattering (M3PALS technique). For this purpose, a Zetasizer Nano ZS Malvern Instrument was used (Herrenberg, Germany). Freeze-dried empty and FA-loaded SLN and NLC samples were dispersed in filtered (0.2 μm) bidistilled water. Each sample was analyzed in triplicate. 2.5. HPLC Analysis of FA An adequate HPLC method to study the stability of FA in phosphate saline buffer (PBS) at pH 7.4, the FA Loading Capacity (LC%) and drug release profiles from drug-loaded systems was developed. The HPLC analysis was performed at room temperature using a Shimatzu Instrument equipped with a reversed-phase C18 column (Bondpak, 3 m, 150 x 4.6 mm i.d., Supelco). A mixture of tetrahydrofuran (THF), H2O and CH3COOH (60:35:5 v/v) with a flow rate of 0.2 ml min -1 was used as mobile phase. The HPLC column system was connected with an UV-Vis detector (Shimatzu). The drug peak was measured at a wavelength of 318 nm and quantitatively determined by comparison with a standard curve obtained using FA solutions in THF at known concentrations. The straightline equation was: y = 137.19x ml-1)

(y = peak area x 10 5; x = drug concentration g

The linear regression value was: r = 0.9998. The linearity of the method was studied in the range 6.6-66 μg ml-1. 2.6. FA Stability at pH 7.4 The stability of FA was studied incubating the drug suspension (0.2 mg/ml) at 37°C ± 0.1°C in PBS 0.01 M at pH 7.4. At scheduled time intervals, aliquots were withdrawn and assayed by HPLC using the above-described method to measure FA concentration. 2.7. Determination of Drug Content To determine the amount of FA entrapped into each sample [samples SLN-(b,c,d) and NLC-(a,b)], 5 mg of each freeze-dried system were solubilized in 25 ml of THF. The organic solution was filtered through 0.45 μm (PTFE membrane) filters and analyzed by HPLC. In order to ensure that the drug is not absorbed within the PTFE filter, several THF solutions of drug at known concentrations

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were filtered and the concentration values before and after filtration were evaluated on the solutions by HPLC analysis. No significant differences in drug concentrations were evidenced. Results were expressed as the percentage of the drug amount contained in 100 mg of dried material (LC%). 2.8. Drug Release at pH 7.4 FA release was assayed on samples SLN-(b,c,d) and NLC-(a,b) at eleven prefixed time intervals. For this purpose, eleven dispersions of each batch containing 5 mg of each freeze-dried sample in 10 ml of PBS 0.01 M at pH 7.4 were prepared and kept at 37°C ± 0.1°C under mechanical stirring. At suitable time intervals, samples were ultra-centrifuged, then the supernatant was filtered through 0.45 μm (nylon membrane) filters and analyzed by HPLC. 2.9. Interaction Between Fluorescent Nanoparticles and LAN5 Cells Fluorescein-loaded SLN (SLN-f) were obtained by dissolving fluorescein (0.78 mg) into the melted lipid and operating as described above for SLN-a. In order to study the interaction between fluorescent nanoparticles and LAN5 cells via fluorescence microscopy, Human neuroblastoma cells (LAN5) were plated on slides and cultured with RPMI 1640 medium (CELBIO) supplemented with 10% foetal bovine serum (FBS) (GIBCO) and 1% antibiotics (50 u/ml penicillin and 50 g/ml streptomycin) and antimycotics (SIGMA). Cells were maintained in humidified 5% CO2 atmosphere at 37°C ± 0.1°C. The slides were maintained in humidified 5% CO2 atmosphere at 37°C ± 0.1°C and were incubated with RPMI supplemented with 5l (25 ng/l) of SLN-f for 3 and 24 hrs. Fluorescent or light field images were observed with an Axioscop 2 microscope (Zeiss) and captured with an Axiocam digital camera (Zeiss) interfaced with a computer. In order to ensure the presence of fluorescein into fluoresceinloaded nanoparticles after 24 h of incubation with LAN5 cells, an in vitro diffusion study was performed on SLN-f sample in physiological medium for 24 h. After these time the amount of fluorescein still present into the particles was quantified by UV analysis and resulted to be 88 w/w% of the theoretical value (theoretical amount of fluorescein loaded into SLN-f = 0.43 w/w%). After 24 h, recovered SLN-f sample was also put on a slide and observed with a fluorescence microscope, resulting to be fluorescent. 2.10. Cell Viability Assays 4

LAN5 were plated onto 96-well plates at a density of 1,5x10 per well and cultured as described above. For the evaluation of SLN-a on LAN5 cell viability, cells were incubated in fresh medium (100 l) with different aqueous suspensions (5, 10, 20 l) of SLN-a (280 g/ml), for 24 hrs. For the evaluation of free FA and FA-loaded SLN (sample SLN-b), cells were incubated with different concentrations (14 and 28 M) of FA, free in DMSO solution or entrapped into SLN-b, for 24 hrs. The treated cultured cells and the controls were morphological analysed by microscopy inspection or utilized for specific assays. Cell viability was measured by MTS assay (PROMEGA). MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium] was utilized according to the manufacturer’s instructions. After 24 hrs of incubation, the culture medium was replaced with fresh medium and 20 l of the MTS solution were added to each well, and the incubation was continued for 4 hrs at 37°C ± 0.1°C, 5% CO2. The absorbance was read at 490 nm on the Microplate reader wallacVic2 tor 1420 Multilabel Counter (Perkin Elmer). Results were expressed as the percent MTS reduction of control cells. 2.11. JC-1 Assay Mitochondrial membrane potential was measured with the JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyani-

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Table 1. Hot Microemulsion Chemical Composition (w/w%) of Developed FA-Free and FA-Loaded Formulations Sample

Lipid Matrix (w/w%)

FA (w/w%)

SLN-a

Compritol ATO 888 (13.4)

---

SLN-b

Compritol ATO 888 (12.4)

7.0

SLN-c

Compritol ATO 888 (6.0)

SLN-d SLN-f *

NLC-a

NLC-b

Surfactant (w/w%)

Cosurfactant (w/w%)

Epikuron (2.7) Epikuron (2.5)

NaTaurocholate (9.7) NaTaurocholate (9.0)

3.3

DDAB (0.4)

Pluronic F68 (6.7)

83.6

Compritol HD5 ATO (8.8)

4.9

Epikuron (4.1)

NaTaurocholate (3.7)

78.5

Compritol ATO 888 (13.4)

---

Epikuron (2.7)

NaTaurocholate (9.7)

74.2

7.0

Epikuron (2.5)

NaTaurocholate (9.0)

8.0

Epikuron (2.4)

NaTaurocholate (6.7)

Compritol ATO 888 (9.7) Miglyol 812 (2.7) Precirol ATO (14.1)

Hot Water (w/w%) 74.2 69.1

69.1

66.7

* SLN-f is fluorescein-loaded sample. This batch contains a percentage value of fluorescein on the total microemulsion weight equal to 0.06 w/w%.

ne iodide) fluorescent dye. LAN5 treated with 10 l of SNL-a aqueous dispersion (280 g/ml) were incubated with JC-1 (2 M f. c.) in PBS at pH 7.4 for 30 min. Fluorescence intensity was measured with 570 nm emission wavelength (TRITC filter), or with 528 nm emission wavelength (FITC filter). Fluorescent or light field images were observed with an Axioscop 2 microscope (Zeiss) and captured with an Axiocam digital camera (Zeiss) interfaced with a computer. 2.12. Reactive Oxygen Species (ROS) Detection LAN5 cells were plated and cultured as described above. The standard control was treated with H2O2 as free radical generator. 28 M of free FA or entrapped into SLN-b were provided to the cells 24 hrs before the experiment. On the day of the experiment, after removal of the medium, the cells were washed with PBS at pH 7.4 and then incubated with 1 M DCFH-DA final solution in PBS at pH 7.4 at room temperature for 10 min. After, DCFH-DA was removed; the cells were dissolved in PBS at pH 7.4 and then submitted to F.A.C.S. analysis. 2.13. Statistical Analysis All experiments were repeated at least three times. Each experiment was performed in triplicate. The results are presented as mean ± SD. Statistical evaluation was conducted by ANOVA for comparisons among groups, followed by Student’s t-test for analysis of significance. Differences were considered to be significant at p value < 0.05 or < 0.02.

3. RESULTS AND DISCUSSION In this work, several solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), potentially useful as drug delivery systems for ferulic acid (FA), were prepared successfully by using the microemulsion technique [15]. FA, a phenolic compound, is well known to be an important antioxidant [4] which protects neurons against A(1–42)-induced oxidative stress and neurotoxicity [8]. In Table 1, the chemical composition (w/w%) of the hot microemulsion prepared for obtaining drug-free and drug-loaded SLN and NLC, as described in the experimental part, is reported. In particular, Compritol 888 ATO was used as lipid matrix for obtaining SLN-a (drug-empty sample), SLN-b (drug-loaded sample) and SLN-c (cationic drug-loaded sample). SLN-d sample was obtained by using as lipid matrix Compritol HD5 ATO, which contains a certain amount of pegylated fatty ester. The NLC-a sample was obtained by using as lipid matrix a mixture of Compritol 888 ATO and the liquid lipid Miglyol 812, while NLC-b sample was obtained by using as lipid component Precirol ATO, which is a mixture of spatially different lipids. After preparation and purification, all obtained samples freezedried and then characterized in terms of mean particle size, polydispersity index (PDI) and zeta potential, after dispersion in bidistilled water. Table 2 shows mean size, PDI and zeta potential values of all obtained systems. Data indicate that the average diameter of empty and drugloaded nanoparticle batches were in the nanometer scale, ranging

Table 2. Mean size, PDI, Zeta Potential and LC% Values of Samples SLN-(a,b,c,d,f) and NLC-(a,b) Sample

Mean Size (Nm)

PDI

SLN-a

94.4

0.256

SLN-b

96.2

SLN-c

129.2

SLN-d

Zeta Potential (mV) (± S.D.)

LC% (w/w)

- 30.00

± 1.26

/

0.196

- 36.40

± 1.38

20.0%

0.289

+ 49.11

± 1.45

26.0%

139.1

0.312

- 33.49

± 1.71

20.6%

SLN-f *

100.0

0.282

- 34.49

± 1.71

/

NLC-a

124.4

0.252

- 34.24

± 1.53

25.0%

NLC-b

95.9

0.174

- 25.50

± 1.23

27.3%

* SLN-f is fluorescein-loaded sample.

Ferulic Acid-Loaded Lipid Nanostructures

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between 90 and 140 nm. Moreover, all these systems possessed a low PDI values which indicate a good dimensional homogeneity of the prepared systems. SLN-a and SLN-b samples, corresponding to empty or drugloaded nanoparticles with the same composition respectively, did not significantly differ in terms of diameter. Only the zeta potential seemed to be affect by the presence of the drug. Sample SLN-c resulted to be larger than SLN-a and SLN-b and with a strongly positive zeta potential value, which could be attributed to the presence of DDAB as surfactant. The use of Compritol HD5 ATO instead of Compritol 888 ATO gave larger nanoparticles (sample SLN-d) in the same experimental conditions, probably attributed to the presence of PEG chains. Sample NLC-a, obtained by mixing Miglyol 812 to Compritol 888 ATO, was constituted by larger nanoparticles compared to SLN-b, the former obtained by using a mixture of liquid and solid lipids. The high surface charge of all the obtained samples could reduce the occurrence of aggregation phenomena and could assure a considerable stability to the aqueous dispersion of these systems. The amount of FA entrapped into drug-loaded samples, expressed as the weight percentage ratio between the entrapped FA and the total sample weight (Loading Capacity, LC%), resulted to be above the 20 % for all the obtained systems. Thanks to their chemical–physical characteristics, these systems could be administered by all the possible routes, so also intravenously. For these reasons, all the obtained systems were submitted to drug release studies in PBS at pH 7.4, in order to investigate the drug release profiles, under experimental conditions mimicking extracellular fluids. These release kinetics are reported in Fig. (1). For ensuring that the drug was stable in the investigated medium, a stability study on FA was carried out in PBS at pH 7.4 and the Fig. (2) shows the absence of FA degradation at pH 7.4. By comparing the drug release profiles (Fig. (1)), an initial burst effect and a complete drug release within 10 hrs can be seen for all the tested samples. These profiles can be considered acceptable for the targeted release to the CNS, considering that, once in the blood stream the rate of accumulation into the brain could be very fast. In fact these systems, thanks to their small size, minimize the uptake of macrophages of mononuclear phagocyte system (MPS) [18, 19] and quickly reach the CNS, overcoming the BBB [10, 12-15].

Fig. (2). Profile of FA degradation at 37°C ± 0.1°C in PBS aqueous solutions at pH 7.4. Each value is the mean of three experiments.

for an in vitro further investigation in order to evaluate its interaction with cells, its effect on cell viability and its potential as drug delivery system for FA. To investigate whether these SLN were able to be absorbed by membrane cell surface, fluorescein-loaded nanoparticles were prepared (sample SLN-f). For this aim, human neuroblastoma cells (LAN5) were incubated with SLN-f for 3 and 24 hrs. In Fig. (3), the light field microscopic and fluorescent microscopic images of LAN5 cells incubated with SLN-f for 24 hrs are reported. As can be seen by microscopic inspection, an intense fluorescence inside the cells is clearly detectable (Fig. (3B)), indicating that the nanoparticles are able to cross cell membrane and enter into these cells. To evaluate the potential effects of the nanoparticle chemical composition on the tested cells and to ascertain the possibility to utilize these systems as drug carriers, the dose dependence viability of empty nanoparticles (sample SLN-a) was evaluated on LAN5 cell line. In Fig. (4), the LAN5 cell viability (%) as function of SLN-a aqueous dispersion concentration is reported. As can be seen, no significant toxicity was detected for all the employed amounts of SLN-a respect to the control, indicating that tested SLN sample is composed by physiological well tolerated ingredients and can be suitable as pharmacological carrier. Another evidence that SLN did not produce any damage into the cells was provided by JC-1 assay. This assay permits to investigate about the integrity of membrane potential of the mitochondrion (m), a cellular organelle very sensitive to different stress. At physiological conditions, JC-1 forms aggregates into mitochondria and emits a red-fluorescent light. When a cell is under stress, the mitochondrial membrane potential is lower and JC-1, under monomer form, is unable to penetrate into mitochondria, and emits a green-fluorescent light, which can be visualized by microscopic inspection. The Fig. (5) shows, the fluorescence images of cells treated with SLN-a.

Fig. (1). Release profiles of FA in PBS at pH 7.4 and at 37°C ± 0.1°C from samples SLN-b, SLN-c, SLN-d, NLC-a and NLC-b. Each value is the mean of three experiments.

SLN-b sample, showing the slower drug release profile and the lower PDI, was chosen as model among all the obtained samples

As can be clearly seen in Fig. (5B), SLN-a treated LAN5 cells, visualized with TRITC filter, showed an intense red fluorescence. When the same sample was visualized with FITC filter (Fig. (5C)), as control, a slight green fluorescence was detected, indicating that SNL-a did not induce any damage in mitochondrial membrane. Being these systems produced as potential carrier for FA, this drug was incorporated into these systems and the effect of FAloaded SLN on cell viability was tested. In particular, cell viability experiments were carried out incubating LAN5 cells with different

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concentrations (14 and 28 M) of FA, free in DMSO solution or entrapped into SLN-b, and then cultured for 24 hrs. In Fig. (6), the LAN5 cell viability (%) in the presence of free FA in DMSO solution or entrapped into SLN-b is reported. No significant difference in cell viability was detected for the sample treated with a 14 M concentration of FA both free or entrapped into SLN respect to the untreated control. Furthermore, when the highest concentration of FA (28 M) was tested, it was even detected, both in LAN5 treated with free FA or with SLN-b, a light increase of vitality. This result could be explained considering the probable FA induction of cell proliferation, as described by other authors [20, 21]. In addiction, the increase of vitality was lightly higher for the SLN-b than free FA, suggesting that the nanoparticles act as nano-vectors which increase the amount of FA into the cells. Being FA a good antioxidant for its ability to reduce free radical damage [20, 21], the percentage of reactive oxygen species (ROS) produced after incubation of LAN5 cells with free FA (28 M) and FA-loaded SLN (SLN-b) corresponding to 28 M of FA, respect to the untreated cells, was measured in order to evaluate the potential of these SLN as delivery systems for FA. Intracellular ROS was measured by 2’,7’-dichlorofluorescein (DCFH) fluorometric assay. DCFH-DA is cell permeable and, once inside the cell, is hydrolyzed by cellular esterase in dichlorofluorescein (DCFH). DCFH in turn is converted upon oxidation to highly fluorescent dichlorofluorescein (DCF). Thus, the emitted fluorescence is directly proportional to the concentration of hydrogen peroxide inside the cell. In Fig. (7), the percentage of ROS formation in LAN5 cells after incubation for 24 hrs with free FA, H2O2 (positive control), empty SLN (sample SLN-a) and FA-loaded SLN (sample SLN-b) is reported.

Fig. (4). Dose dependence viabili.ty of SLN-a. LAN5 cells were incubated in the presence of 5, 10 and 20 l of a SLN-a aqueous dispersion (280 g/ml) for 24 hrs and submitted together with untreated cells as control to viability MTS assay. On the left, the percentage of viability is indicated. The blank control value was subtracted from each.

As shown in Fig. (7), LAN5 cells control displayed a fluorescence related to metabolic activity of mitochondria, corresponding to about 25% of ROS production. A high percentage of ROS (about 60%) was instead measured for the cells treated with H2O2, a standard free radical generator. FA-loaded SLN (sample SLN-b) caused a reduction of ROS production in LAN5 cells, more evident than for cells treated with free FA.

Fig. (3). Interaction between fluorescent SLN (sample SLN-f) and LAN5 cells. (A) Light field microscopic and (B) fluorescent microscopic images of LAN5 cells incubated with SLN-f for 24 hrs. The bar represents 20 m.

Fig. (5). SLNs do not change mitochondrial membrane potential. LAN5 cells treated with 10 l of SLN-a aqueous dispersion (280 g/ml) were incubated with JC-1. (A) Light field microscopic image, (B) fluorescent microscopic image at 570 nm emission wavelength, and (C) fluorescent microscopic image at 528 nm emission wavelength. The bar represents 20 m.

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Fig. (6). Dose dependence viability of LAN5 cell line of A) free FA (14 M), A1) FA-loaded SLN-b corresponding to 14 M, B) free FA (28 M) and B1) FA-loaded SLN-b corresponding to 28 M. Cells were incubated in the presence of each sample for 24 hrs and then cell viability was assessed by MTS assay. On the left, the percentage of viability is indicated. The blank control value was subtracted from each. Data are the mean ± SD of three separate experiments, expressed as percentage of control value (* p