Formulations of retinyl palmitate included in solid lipid nanoparticles: characterization and influence on light-induced vitamin degradation

J. DRUG DEL. SCI. TECH., 18 (2) 119-124 2008

Formulations of retinyl palmitate included in solid lipid nanoparticles: characterization and influence on light-induced vitamin degradation M. Carafa1*, C. Marianecci2, M. Salvatorelli2†, L. Di Marzio1, F. Cerreto2, G. Lucania3, E. Santucci2 1

Dept. Scienze del Farmaco, University “G. D’Annunzio”, Via dei Vestini, 66100 Chieti, Italy 2 Dept. Studi Chim. Tecnol. Sost. Biol. Attive, University of Rome “Sapienza”, Rome, Italy 3 Dept. Medicina Sperimentale e Patologia, University of Rome “Sapienza”, Rome, Italy *Correspondence: [email protected] †In memory of Marta Salvatorelli, brilliant student and unforgettable friend.

The therapeutic use of retinoids is still limited because of adverse effects and their chemical instability due to moisture, oxygen, acids, metals and light exposure. The purpose of this research was to investigate particulate carrier systems such as solid lipid nanoparticles (SLNs) for formulation of vitamin A palmitate. SLN (Precirol ATO 5, Pluronic F68, sodium cholate) were obtained using the hot homogenization technique and sonication, and were characterized using freeze fracture microscopy, dynamic light scattering, for size and zeta potential measurements and DSC. SLN formulation was optimized by modifying surfactant mixture composition to obtain better SLN physical stability and better vitamin entrapment efficiency. Preferential vitamin disposition on particle surface was evaluated by DSC analyses and release studies. RetP stability studies were carried out using normal phase HPLC on samples exposed continuously, in a darkened room, to the light of a filament lamp (400 lux) and the influence of the inclusion in solid lipid nanoparticles on retinyl palmitate (RetP) light-induced degradation was evaluated in the presence and absence of conservative agent (BHA, 3-tert-butyl-4-idroxi-anisole). The Precirol ATO 5 SLN formulation of RetP offers high entrapment efficiency but does not offer the possibility of preventing photo-degradation of RetP. In the nanoparticulate structures analyzed, RetP is preferentially distributed to the surface of the particles and partially exposed to external aqueous phase, thus no protection from RetP degradation can be evidenced in comparison to reference RetP solution (THF:water 9:1 v:v). Key words: Retinyl palmitate – Isomerization – SLN – BHA – Normal phase HPLC.

water or light, also related to the formation of photodecomposition products, inducing DNA damage and skin photocytotoxicity [10-13]. Therefore, formulations that can increase the chemical stability of these compounds need to be developed [14-16] and SLN have been widely studied for this purpose [17-20]. However, the encapsulation rate of retinoic acid in SLNs is usually low [21]. In contrast, Lim and Kim [22] and Hu et al. [23] described high encapsulation rates for AR in SLN when a high surfactant/lipid ratio was used. In a previous work, we studied the degradation of retinyl palmitate to the less active cis-isomers, in model lipophilic vehicles [21] and after inclusion in phospholipid and surfactant unilamellar vesicles [22]. The aim of the present study was to prepare simple SLN formulations with a high surfactant/lipid ratio, in the presence and absence of a lipophilic conservative agent, BHA, 3-tert-butyl-4-hydroxi-anisole, that can be loaded with retinyl palmitate to evaluate the formulation effect on drug entrapment efficiency and on light-induced vitamin degradation.

Solid lipid nanoparticles (SLNs) were developed at the beginning of the 1990s as an alternative carrier system to emulsions, liposomes and polymeric nanoparticles [1]. It has been claimed that SLNs combine the advantages and avoid the disadvantages of other colloidal carriers [2]. Proposed advantages include the possibility of controlled drug release and drug targeting, the absence of biotoxicity of the carrier and of problems with respect to large-scale production, sterilization, along with the possibility of increasing drug stability. For these reasons SLNs have been suggested for a broad range of applications, i.e. parenteral [3], oral [4] and dermal [5-7] routes. Stratum corneum is the main barrier in the percutaneous absorption of topically applied drugs. Small-size and relatively narrow size distribution of SLN give site-specific delivery to the skin [8]. SLNs have great affinity with the stratum corneum, and therefore allow an enhanced bioavailability of the encapsulated material to the skin is achieved. SLNs enhance penetration and transport of active substances, particularly lipophilic agents, and thus increase the concentration of these agents in the skin [1]. All-trans retinol (AR) is a hydrophobic vitamin A compound that exerts a potent influence on cell differentiation, proliferation, and homeostasis [9]. It also helps in maintaining the health of the skin (prevents acne and dermatitis) and surface tissues, especially those with mucous linings. Moreover, AR protects skin against skin-aging by neutralizing unstable oxygen molecules (free radicals) and participating in the process of collagen regeneration. Because of its anti-aging effect, many cosmetic products contain AR as an anti-wrinkle agent. In spite of a wide range of biological and pharmacological effects, the therapeutic and cosmetic uses of AR and its derivatives (i.e. retinyl palmitate) are still limited due to its poor chemical stability when exposed to air,

I. MATERIALS AND METHODS 1. Materials

Analytical grade retinyl palmitate (RetP), sodium cholate and BHA (3-tert-butyl-4-idroxi-anisole) were purchased from Fluka Chemie AG products. Pluronic F68 was purchased from Basf. Precirol ATO 5 was generously donated by Gattefossé. Sepharose 4B was a Pharmacia product. N-hexane, methanol, ethyl ether, tetrahydrofuran (THF) and acetonitrile were all HPLC grade as supplied by Riedel-de Haën GmbH. For irradiation experiments a fluorescent lamp (Circolux EL32 W Osram) and a light meter (LX-103 Lutron) were used. 119

J. DRUG DEL. SCI. TECH., 18 (2) 119-124 2008

Formulations of retinyl palmitate included in solid lipid nanoparticles: characterization and influence on light-induced vitamin degradation M. Carafa, C. Marianecci, M. Salvatorelli, J. Di Marzio, F. Cerreto, G. Lucania, E. Santucci

Beckman) and filtration of the supernatant on glass wool and anhydrous Na2SO4. The vitamin entrapment efficiency (e.e.) was calculated using the following equation, according to Görner et al. [26]:

2. Preparation of SLNs

SLNs were prepared with 0.05 g of RetP, 10.00 g of Precirol ATO5 and 3.5 g of surfactant mixture composed of varying ratios of Pluronic F68 and sodium cholate, in 100 g of aqueous dispersion. Precirol ATO 5 was melted at 70°C and the hot lipid phase was transferred into a hot (65°C) water bath to avoid lipid crystallization. The surfactant mixture (Pluronic F68 and sodium cholate) was dispersed in a filtered warm (65°C) water phase. The warm water phase was then added under homogenization (Ultra Turrax T45, Janke & Kunkel, Ika-Werk, at 10,000 rpm) to the melted lipid phase. The homogenization was performed for 10 min, maintaining the mixture in the water bath. The emulsion obtained was then sonicated (V.C.X 400-Sonics) at 65°C for 20 min to reduce particle dimensions. The dispersions were then quickly cooled by transfer into an ice bath to promote liposphere formation due to thermal shock [13]. The incorporation of retinyl palmitate was performed by adding the drug to the melted lipid phase during the first step of SLN preparation. Particular care was taken that the temperature of the water bath, and of course of the mixture, was exactly 65°C and not higher until the end of the preparation to avoid retinyl palmitate degradation. For samples prepared in the presence of antioxidative agent BHA, this was added to the lipid solid phase and then melted, in the first step of preparation. In order to separate loaded SLN from unentrapped RetP, the SLN dispersion was purified by gel-filtration on Sepharose 4B (glass column 50 × 1.2 cm) after filtration (0.8 µm, cellulose filter, Millipore). Preparation and purification procedures were carried out under dark conditions due to the high light sensitivity of RetP.

e.e. = 100 × (mass of incorporated drug/mass of drug used for vesicle preparation)

Eq. 1

Samples were diluted to 5 mL with hexane and then analyzed using HPLC direct phase using a silica column able to separate the three RetP isomers: 13cis, 9cis and all-trans. The analytical methods were developed and then validated using the equipment, mobile phases, chromatographic conditions and protocols described below. Equipment: - model 600 solvent delivery system, Water Associates (Milford, MA,USA), - photodiode array detector 991 (DAD), Water Associates (Milford, MA, USA), - model 7125 syringe loading sample injector from Rheodyne (CA, USA) equipped with a 50-µL loop. Chromatographic conditions: - stationary phase: 250 × 4.6 mm LiChrosorb Si 60 (5 µm), - temperature: room temperature, - mobile phase: n-hexane:ethyl ether 99:1, - isocratic flow: 1 mL/min, - detector: 325 nm, - volume injected: 50 µL. The RetP calibration curve was made on solutions of RetP in hexane. The differences in molar extinction coefficients (eM,325 M/cm) of the two isomers and of the all-trans isomer were taken into account calculating the isomer peak areas (9-cis, 13-cis). Then the corrected areas were added to the all-trans area, so only the signals for the alltrans isomer were obtained.

3. SLN characterization

3.1. Freeze-fracture SLNs were examined using the freeze fracture microscopy technique. The samples were impregnated in 30% glycerol and then frozen in partially solidified Freon 22, freeze-fractured in a freeze fracture device (-105°C, 10-6 mmHg) and replicated by evaporation from a platinum/carbon gun. The replicas were extensively washed with distilled water, picked up on Formvar-coated grids and examined with a Philips CM 10 transmission electron microscope.

5. Stability studies

Purified samples on Sepharose 4B (100-mL volumetric flask) were exposed continuously in a darkened room to the light of a filament lamp. The light intensity was set at 400 lux (measured with a Lutron LX-103 light meter) for each sample by adjusting the distance between the sample and the light source. For an appropriate comparison, the same samples were kept under dark conditions as reference samples. A RetP solution in tetrahydrofuran:water 9:1 v:v was used as reference solution to compare vitamin degradation rate in the absence and presence of SLN. At fixed time intervals, 2 mL of each vitamin sample was withdrawn from the volumetric flask; the sample was then freeze-dried to ensure complete loss of water content. The lyophilized sample was then dispersed in a hexane phase at 65°C to destroy SLN. After centrifugation (J-21 B, Beckman), to separate retinyl palmitate from other components, the supernatant was then filtered on glass wool and anhydrous Na2SO4, and the filtered solution was brought to 5 mL with hexane. Sample analyses were performed in triplicate. The samples obtained were then analyzed by means of HPLC using the method described above. Typical chromatograms are shown in Figure 1. The same procedures were also used to evaluate the influence of sonication and lyophilization on RetP stability.

3.2 Size measurements, zeta potential and stability tests Size measurements and evaluation of SLN stability were carried out on aqueous suspensions using dynamic light scattering. The SLN dispersions were diluted 100 times in the same buffer used for their preparation. Vesicle size distribution was measured on a Malvern Nano ZS90 (Malvern) at 25°C, with a scattering angle of 90.0°. The same apparatus was used to evaluate zeta potential using an SLN preparation diluted (1:10 v:v) in distilled water at 25°C. Analyses of empty SLNs were carried out in order to evaluate changes in nanoparticle size and electrophoretic mobility after drug incorporation. The polydispersity index (p.i.) was calculated directly by the software of the apparatus and the values obtained were in agreement with a mono disperse system. SLN stability, in terms of changes in particle dimensions after aggregation, was evaluated using the same technique on samples stored for up to one month at 4, 25 and 37°C, under dark conditions. This type of information was also confirmed using zeta potential measurements.

6. DSC thermal analysis

Differential scanning calorimetry (DSC) was performed using a Setaram DSC 121 instrument, and an empty standard aluminum pan was used as reference. SLN dispersions were lyophilized, placed in a 30-mL aluminum

4. Drug encapsulation efficiency

Drug entrapment within SLNs was assessed by HPLC on samples purified on Sepharose 4B, after freeze-drying, dissolution of the lipophilic moiety in hexane at 65°C, centrifugation at 25°C (J-21 B, 120

Formulations of retinyl palmitate included in solid lipid nanoparticles: characterization and influence on light-induced vitamin degradation M. Carafa, C. Marianecci, M. Salvatorelli, J. Di Marzio, F. Cerreto, G. Lucania, E. Santucci

J. DRUG DEL. SCI. TECH., 18 (2) 119-124 2008

9. Statistical treatment

The results were expressed as the mean ± standard deviation (SD) of at least three experiments. Statistical analyses were used to compare the influence of experimental parameters on kinetics and rates of RetP degradation processes, and were carried out using Student’s unpaired t-test. Significance was taken as P < 0.05. The results were expressed as the mean ± standard deviation (SD) of three experiments.

A

II. RESULTS AND DISCUSSION

It can be underlined that in all analyzed samples under dark conditions the RetP degradation shows a zero order kinetic, while under light irradiation it shows a first order kinetic (data not shown), according to data in the literature [21, 22, 31, 32]. According to the above statement, degradation curves are reported as all-trans concentration or log all-trans concentration versus time respectively.

B

1. Optimization of RetP-loaded SLN formulation

For the preparation of RetP-loaded SLN, Precirol was selected as a solid lipid component that would constitute the core of SLN because the lipid, used in the pharmaceutical field, shows a phase transition from solid to liquid under 65°C (degradation temperature of RetP). Pluronic and sodium cholate were chosen as components of surfactant mixture to stabilize SLN because they are acceptable surfactants even in pharmaceutical formulation. SLNs were prepared using high shear mixing and ultrasound, because both methods are easy to handle and were used for the production of solid lipid nanodispersions [33-35]. A high surfactant/lipid ratio was used to enhance RetP entrapment efficiency in SLN [22, 23]. Since SLNs prepared with combination of surfactants generally tend to have smaller particle diameter and higher storage stability by preventing particle agglomeration more efficiently [36, 37], we prepared RetP-loaded SLN with surfactant mixture composed of varying ratios of Pluronic F68 and Na cholate, and we investigated the effect of surfactant composition on the size and zeta potential of the resulting SLN. The mean particle diameter of SLNs prepared with 100% Pluronic F68 or 100% Na cholate was 194 and 210 nm, respectively (Table I). The combination of Na cholate and Pluronic F68 reduced the particle diameter, resulting in 171 nm in SLN with 75:25 weight ratio of Pluronic and Na cholate. The P.I. values of RetP-loaded SLN prepared with 100% Pluronic F68 and 100% Na cholate were 0.33 and 0.31. Similar to particle diameter, the P.I. value of SLN was lowered with the 75:25 weight ratio of Pluronic and Na cholate resulting in 0.241, suggesting narrow size distribution. The zeta potential of SLN was modified by varying the mixing ratio of surfactants (Table I). Previous studies have shown that a minimum zeta potential of -60 mV is required for excellent physical stability, but good physical stability of colloidal carrier systems can be achieved

Figure 1 - Typical chromatograms of RetP samples in hexane analyzed using normal phase HPLC at time = 0 (A) and after exposure to light (B).

pan (sample weight 8-10 mg) and then analyzed at heating rate of 10°C/min.

7. In vitro release experiments

Vitamin A palmitate is practically insoluble in water, so its in-vitro release kinetics were analyzed by studying the remaining amount of RetP in the SLN (27). For this purpose, several samples of SLN (10 mg) were suspended in isotonic PBS pH 7.4 containing 3% polysorbate 80 [28] (3 mL). At fixed time intervals, the samples were centrifuged, the supernatant was removed and the remaining SLNs were washed three times in distilled water (100 mL) and freeze-dried [29]. The lyophilized sample were treated and analyzed by HPLC as described above. The release kinetics was assessed using the following equation [30]: Mt/M∞ = Ktn

Eq. 2

where Mt is the amount of drug released at time t, M∞ is the loading drug amount, K is the kinetic constant and n is the release exponent, related to the release mechanism. Values for K and n were calculated by regression analysis.

Table I - Effect of content of Na cholate in the surfactant mixture composed of Pluronic F68 and Na cholate on the mean particle diameter, polydispersity index, and zeta potential of RetP-loaded SLNs.

8. Validation of the analytical method

In the validation of the analytical method the following points were determined: linearity and replicability, calculated as described by M. Scalzo et al. [25]. The limit of detection (LoD) was established by analyzing RetP standards in six replicate injections at different concentration levels in decreasing order until signal-to-noise (S/N) ratio reached about 3. The limit of quantification (LoQ) was established in the same way as LoD, but the S/N ratio was about 10. The LoD and LoQ of the analytical method were 0.003 and 0.015 µg/mL, respectively.

Na cholate in the surfactant mixture (%)

Mean diameter (nm)

Polydispersity index (P.I.)

Zeta potential (mV)

0 15 25 100

194±4.56 190±3.96 171±4.43 210±3.87

0.330 0.310 0.241 0.331

-12.675± 0.197 -17.548± 0.432 -21.903± 0.253 -23.872±0.372

SLNs were prepared with 0.05 g of RetP, 10.00 g of Precirol ATO5 and 3.5 g of surfactant mixture composed of varying ratios of Pluronic F68 and Na cholate, in 100 g of aqueous dispersion. At 50:50% of surfactant mixture, no relevant SLN formation was obtained. Data represented means ± SD (n = 3). 121

Formulations of retinyl palmitate included in solid lipid nanoparticles: characterization and influence on light-induced vitamin degradation M. Carafa, C. Marianecci, M. Salvatorelli, J. Di Marzio, F. Cerreto, G. Lucania, E. Santucci

J. DRUG DEL. SCI. TECH., 18 (2) 119-124 2008

with zeta potential values of -30 mV [38]. In this context, the zeta potential of SLN prepared with the ratio of 75:25 (w/w) (-22 mV) was only slightly lower than that required to get good electrostatic stabilization. Taken together, the smallest mean particle diameter and P.I. value and an acceptable negative value of zeta potential of SLN could be obtained by a combination of Na cholate and Pluronic at the ratio of 25:75 (w/w). From these results, the surfactant ratio was optimized at this value in the subsequent studies.

HeatFlow/mW 2.5

A

Exo

0.0

-2.5

-5.0

-7.5

-10.0

-12.5

-15.0

Peak :56.0388 °C Onset Point :48.0944 °C Enthalpy /J/g : 128.1380 (Endothermic effect)

-17.5

2. Nanoparticle characterization

-20.0

Evaluated SLN sizes in the range of 100-200 nm are in agreement with a previous study, reporting the same preparation procedure [39]. The freeze fracture microscopy technique evidenced that analyzed samples show quite regular structure both in the presence and absence of BHA (Figure 2). These data are in agreement with those obtained by DLS and it can be evidenced that in both formulations the encapsulation of retinyl palmitate did not modify the particle size distribution and the zeta potential values. Particle size analysis is a necessary but not in itself sufficient step to characterize SLN quality. Special attention must be paid to the characterization of the degree of lipid crystallinity and to lipid modification, because these parameters correlate closely with drug incorporation and release rates. For this reason the solid state of SLN was investigated using thermal analysis: melting peak of vitamin free SLN of Pluronic F68 and sodium cholate proved the solid character of the lipid matrix at room temperature (melting temperature higher than room temperature) [19]. The thermogram reported as an example in Figure 3 shows that the introduction of retinyl palmitate in SLN slightly decreased the nanoparticle transition temperature from 56.04 to 55.20°C. The slight lowering of the melting temperature after the addition of the active substance could suggest that the drug with a melting point below that of the lipid matrix (Table II) preferentially distributes to the surface of the particles [21, 40, 41]. This in agreement with the observation that a high surfactant/lipid ratio favors the interface location of Retinoic acid [42], consequently diminishing the benefits obtained by the encapsulation in lipid matrix (increased stability, controlled release, targeting effect), as also evidenced by in vitro release studies (Figure 4), according to zur Mühlen et al. [42] who report that factors contributing to a fast release are the large surface area and a short diffusion distance for the drug (i.e. release from outer surface region of the nanoparticle). SLN formulation did not influence the RetP entrapment efficiency, which is above 98% for all tested formulations.

-22.5

-25.0 30

50

60

70

80

90

100

Sample temperature/°C

B

HeatFlow/mW Exo 0.0

-2.5

-5.0

-7.5

-10.0

-12.5 Peak :55.2342 °C Onset Point :49.9942 °C Enthalpy /J/g : 134.6797 (Endothermic effect) -15.0

-17.5

-20.0

30

35

40

45

50

55

60

65

70

Sample temperature/°C

Figure 2 - DSC thermogram of empty SLN and retinyl palmitate-loaded SLN. A

B

Figure 3 - Transmission electron micrographs of SLN after freeze-fracture: SLN (A) and SLN-BHA (B). Bar = 300 nm.

2. Influence of sample preparation procedure on RetP degradation-isomerization pathway

1

To evaluate whether sample preparation technique (e.g. sonication and lyophilization) could accelerate RetP degradation, we compared the degradation curves of RetP samples in THF:H2O 9:1 stored under dark conditions, before and after the above-mentioned technique. Sonication was used to modify SLN dimensions, with no variations in SLN composition [33-35]. In Figure 5, it can be seen that the difference between degradation rates is not significant. Since sample preparation did not affect the chemical stability of SLN components (data not shown), it can

SLN

Mt/Minf

0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0

Table II - Melting temperature (Tm) of SLN components.

0

Tm Precirol ATO5 Retinyl Palmitate BHA

40

5

10

15

20

25

30

Hours

50-60°C 29°C 48.55°C

Figure 4 - Drug release profiles of RetP-loaded SLN as a function of time. The values are the mean of three determinations; SD were < 3% of the mean values. 122

Formulations of retinyl palmitate included in solid lipid nanoparticles: characterization and influence on light-induced vitamin degradation M. Carafa, C. Marianecci, M. Salvatorelli, J. Di Marzio, F. Cerreto, G. Lucania, E. Santucci

J. DRUG DEL. SCI. TECH., 18 (2) 119-124 2008

THF/water 9/1

1

SLN

SLN-BHA

1,2 1

co/ct

ct/co

0,8

0,5

0,6 0,4 0,2 0

RetP (THF/water 9/1)

0

sonication lyophilization

0

1,2

0

20

40

60

1

Days logco/logct

Figure 5 - Influence of sonication and lyophilization on vitamin degradation rate, expressed as All Trans concentration at fixed time/All Trans concentration at time 0 vs time (days) in dark condition.

be reasonably asserted that the degradation-isomerization of RetP included in SLN depends only on light irradiation.

0,6 0,4

0 0

The RetP disposition on the SLN surface could explain the results of the preliminary stability studies on analyzed vitamin formulations: in dark conditions and under light irradiation, the RetP inclusion in SLN does not lead to statistically different (p > 0.05) degradation rates (Figure 6), comparing formulation to reference solution in THF:water 9:1. Partially, according to Lee et al. [18], reporting that SLN inclusion did not modify vitamin degradation rate. These data are in agreement with those reported in our previous work [25] on the influence of RetP stability after inclusion in: RetP, preferentially located in vesicle bilayer, showed an increase in degradation rate compared to reference solution. The addition of BHA to SLN formulation did not enhance RetP stability (Figure 6), and these data are not in agreement with the observation of Lee’s study [18] in which the stabilization of All-trans retinoic acid was achieved by the addition of a mixture of antioxidant agents to SLN formulations. This probably related to smaller nanoparticle size in our samples, offering a greater surface to degradative process.

50

100

Days

Figure 6 - Influence of the inclusion in SLN on vitamin degradation rate, in the presence and absence of BHA, expressed as All Trans concentration at fixed time/All Trans concentration at time 0 vs time (days), in dark conditions (A) and under light irradiation (B).

3. 4. 5.

6.

* The studied Precirol ATO 5 SLN formulation of RetP offers high entrapment efficiency but does not offer the possibility of preventing photo-degradation of RetP. In the analyzed nanoparticulate structures, RetP is preferentially distributed on the surface of the particles and partially exposed to external aqueous phase, thus no protection from RetP degradation can be evidenced in comparison to reference RetP solution (THF:water 9:1). Other studies are in progress to evaluate the protective effect on RetP degradation of polymer coating the preformed SLN.

7.

8. 9. 10.

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0,8

0,2

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1.

50 Days

11.

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Formulations of retinyl palmitate included in solid lipid nanoparticles: characterization and influence on light-induced vitamin degradation M. Carafa, C. Marianecci, M. Salvatorelli, J. Di Marzio, F. Cerreto, G. Lucania, E. Santucci

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Manuscript Received 5 October 2007, accepted for publication 1 February 2008.

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