Campos, Débora

Gomes, Ana M. P.

Madureira, Raquel

Pintado, M. E.

Sarmento, Bruno

English

Repositório Institucional da Universidade Católica Portuguesa

USE OF CARNAUBA WAX FOR THE FORMULATION OF ROSMARINIC ACID LOADED SOLID LIPID NANOPARTICLES Débora Campos1, Ana Raquel Madureira1*, Ana Maria Gomes1, Bruno Sarmento2,3, Maria Manuela Pintado1 1CBQF, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal 2CICS, Health Sciences Research Center, Instituto Superior de Ciências da Saúde-Norte, Gandra, Portugal 3Faculdade de Farmácia, Universidade do Porto, Porto, Portugal * e-mail: rmadureira@porto.ucp.pt MATERIALS & METHODS INTRODUCTION RESULTS & DISCUSSION REFERENCES Parnham, M., et al. (1985) Drug of  the Future 10:756-757. Parhi, R. et al. (2010) J Chem Pharm Res 2(1):211–227. Fontes, P. et al. (2011) Drug Deliv and Transl Res 1:299–308.  The inclusion of  antioxidants, or rich sources thereof  (fruits, aromatic herbs, etc.) in foods is becoming a common procedure of  the food industry. Rosmarinic acid (RA) is a phenolic compound with several biological activities, such as antioxidant, anti-inflammatory, antimutagenic, antibacterial and antiviral properties (Parnham and Kesselring, 1985) However, these compounds need protection from the interactions to which they are exposed when incorporated in food matrices. Hence, the formulation of  loaded polyphenols nanoparticles can offer a way to protect such compounds from these interactions, in particular with dairy proteins. The use of  lipid nanoparticles (SLN) has been extensively reported and represents an alternative carrier system to traditional colloidal carriers (Parhi and Suresh, 2010). Furthermore, SLNs combine advantages such as biocompatibility and biodegradability, physical stability, protection of  incorporated compounds, controlled release and specific targeting (Parhi and Suresh, 2010). The increasing demand for functional foods with beneficial effects for human health, has opened the doors to the use of  SLN in the food industry, with the incorporation of  natural compounds with beneficial purposes.  Thus, SLNs were prepared by a hot melt ultrasonication method using carnauba wax as lipid and Tween 80 as surfactant. The effects of  lipid proportion in the lipid mixture and surfactant concentration were evaluated. Also, the stability of  the nanoparticles during 28 days was tested in aqueous solution stored at 4 °C, tracking the mean particle size of  the different formulations by photon correlation spectroscopy using a ZetaPALS, Zeta Potential Analyzer. Thermal analyses of  the nanoparticles were also performed using DSC (Differential Scanning Calorimetry). The loading capacity and loading efficiency were calculated by measuring the concentration of  RA in supernatants by HPLC. Surface properties and morphology were observed by Scanning Electron Microscopy (SEM).  CONCLUSION • The smallest, physical and thermally stable SLNs were those for H formulation (1.5% lipid and 2 % tween 80). The zeta potential was low in absolute values, meaning that possible aggregation could occur in the nanosuspensions. The formulations do not affect the polyphenol entrapment. Preparation of  SLNs The SLNs were prepared by hot melt ultrasonication method using rosmarinic acid (RA) (0.15 mg/mL) and carnauba wax as lipid and running a 32 factorial design. As surfactant, tween 80 at different percentages (viz. 1, 2 and 3%, v/v) and carnauba wax (0.5, 1 and 1.5%, w/v) were used to optimize the formulation. Carnauba wax was warmed to a temperature 5 °C above the melting point value of  the lipid (i.e. 90 °C). The surfactant solution was added to the lipid and polyphenol solution and then homogenized during 1 min at 70% of  intensity in sonicator. The resulting solution was left to cool at room temperature. SLNs were stored at 4 °C during 28 days until further use.  Particle size and zeta potential analyses The average particle size (PS), polydispersity (PI) and zeta potential (ZP) were analyzed by phase analysis light scattering using ZetaPALS, Zeta Potential Analyzer (Holtsville, New York, USA); samples were diluted with MilliQ-water to suitable concentration and were carried with an angle of  90 degrees at 25 °C.  Thermal properties determination  DSC thermograms of  the materials used and final SLNs were obtained using differential scanning calorimetry (DSC-60, Shimadzu, Columbia, USA). The measurements were taken on freeze-dried SLN, and 3 mg of  SLN were placed on an aluminum pan and the thermal behavior determined in the range of  20-100 °C at a heating rate of  10 °C/min. Enthalpies were calculated by equipment software.  Loading efficiency The loading efficiency was calculated by measuring the concentration of  rosmarinic acid in supernatants by HPLC according the method described by Fonte, et al., (2011). The calculation was performed according to the following formula:          Morphology properties of  SLNs The morphology of  nanoparticles was investigated by SEM (Scanning Electron Microscopy). Briefly, an amount of  freeze-dried SLNs were placed in proper supports and coated with gold using a Sputter Coater (Polaron).  ACKNOWLEDGEMENTS Authors acknowledge FCT- Fundação para a Ciência e Tecnologia for funding research work., trough project (PTDC/AGR-ALI/117808/2010 and project PEst-OE/EQB/LA0016/2011 SLN  A B C D E F G H I Particle size (nm) 0 43 ± 3 722 ± 41 887 ± 426 907 ± 438 582 ± 392 587 ± 200 945 ± 211 897 ± 189 35 ± 2 28 124 ± 72 892 ± 60 542 ± 380 585 ± 307 438 ± 338 527 ± 285 605 ± 248 491 ± 210 49 ± 6 Polydispersity index 0,243 0,172 0,169 0,227 0,186 0,114 0,393 0,399 0,251 Zeta potential -6,14 -5,67 -1,49 -1,77 0,35 -6,45 -4,89 -9,34 -7,61 Loading efficiency (%) 99,91 99,97 99,96 99,97 99,92 99,94 99,88 99,91 99,89 Thermal properties Enthalpy (J/g) -27,56 -3,69 -10,57 -36,50 -29,11 -1,35 -15,56 -48,67 -3,77 Melting T (°C) 80,86 79,59 80,92 80,07 81,18 80,30 80,76 80,24 80,26 Table 1: Physical properties of  the SLNs throughout storage time,  formulated with the different % of  lipid (w/v) and percentage of  tween 80 (v/v), (A) 0.5% : 1%, (B) 0.5% : 2%, (C) 0.5% : 3%, (D) 1.0% : 1%, (E) 1.0% : 2%, (F) 1.0% : 3%, (G) 1.5% : 1%, (H) 1.5% : 2%,  (I) 1.5% : 3%.  Fig. 1: Differential scanning calorimetry thermograms of   the ingredients (a) and the SLNs (b). (a) Raw materials: (A) Carnauba wax (B) Rosmarinic acid (C) Tween 80  (D) 1.0% carnauba wax : 2% Tween 80 without RA. (b) Percentage of  carnauba wax (w/v) and percentage of  Tween 80 (v/v), (A) 0.5% : 1%, (B) 0.5% : 2%, (C) 0.5% : 3%, (D) 1.0% : 1%, (E) 1.0% : 2%, (F) 1.0% : 3%, (G) 1.5% : 1%, (H) 1,5% : 2%, (I) 1.5% : 3%. Fig. 2: Photographs of  SLNs by scanning electron microscope (SEM). Percentage of  carnauba wax (w/v) and percentage of  Tween 80 (v/v), (a) 0.5% : 2%,  5000x (b) 1.0% : 2%, 5000x (c) 1.5% : 2%, 2000x.   In general, increasing lipid content increases the mean particle size, as expected, with exception of  formulation I. When using 0.5% of  carnauba wax (A, B and C formulations), the average PS increased, with the use of  higher contents of  tween 80; in contrast, for the other formulations (D-I) with higher lipid %, the average PS decreased when using higher tween 80 content, as expected;  There were no significant differences in the PI (P>0.05). Nevertheless, low PI indicates better stability of  SLN over time, as examples are formulations B, E and F; in formulations E and I, the PS of  SLNs was maintained after 28 days of  storage, and with low PI;  In general, all formulations resulted in negative ZP, with exception of  formulation E, but, all with low absolute values with indicates a possibility of  occurrence of  aggregation (Muller, 1996). The higher value of  ZP was obtained for formulation H, using 1.5% of  lipid and 2 % of  tween 80, suggesting lower probability of  aggregation. In general, these results indicate that a study of  a new surfactant has to be considered in the future;  The percentage of  loading efficiency is high for all formulations (≈ 99.9%), which means that the polyphenol entrapment does not change with the different formulations tested;  The melting temperature is similar for all the formulations, range of  temperature between 79.6 - 81.2 °C, including the formulation without AR (81.0 ºC; Fig 1aD), but their values were smaller than that for carnauba wax alone (84.4 ºC; Fig 1aB). Formulation I showed a lower enthalpy value, which suggests that the SLNs have lower particle arrangement, in contrast with H, which showed a high value. Even not showing a maintenance of  the size after 28 d this formulation is the most stable.  Figure 2 shows the SLNs micrographs of  chosen formulations. Here it is possible to confirm the cylindrical shape which is characteristic of  carnauba wax nanoparticles. a) b) c) -4-3-2-1012320 30 40 50 60 70 80 90 100DSC (mW)Temperature (°C)A B C D E F G H I-6-5-4-3-2-101220 30 40 50 60 70 80 90 100DSC (mW)Temperature (°C)A B C Da) 84.37 °C - 169.51 J/g  b) 80.99 °C - 91.00 J/g  

Use of carnauba wax for the formulation of rosmarinic acid loaded solid lipid nanoparticles

https://repositorio.ucp.pt/bitstream/10400.14/10093/1/PI2.pdf

Use of carnauba wax for the formulation of rosmarinic acid loaded solid lipid nanoparticles

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