Formulation and Physicochemical Characterization of Lycopene-Loaded Solid Lipid Nanoparticles

2016 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2016, 6(2), 235-241 doi: 10.15171/apb.2016.032 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
With the increase of different incurable diseases, the nutraceutical scientist should pay more attention to man's feeding system.Lifestyle and nutrition can help preventing cancer.Carotenoids are common natural compounds in food booklet that play a significant role in the prevention of cancers.However, their low bioavailability due to their inadequate intestinal absorption and low solubility in the aqueous medium, and moreover chemical instability are important limiting factors in the food industry.Hence, there is urgent needs to develop new methods to increase their productivity and absorption as well.Carotenoids are fat-soluble pigments mostly appear in plants and microorganisms (algae and some bacteria) which have an essential role in photosynthesis. 1 In addition, the carotenoids play important pharmacological effects in animals including cells protection from free radicals, 2 coping with cancer cells and inhibition of the lipoxygenase activities, 3 and protection of fats against spontaneous oxidation. 4In the food industry carotenoids are used widely in manufacturing of food products and soft/energy drinks as antioxidant, color and flavor modifier (especially bitter). 5e to the unsaturated structure of carotenoids, the compounds are susceptible to oxidative changes.Factors such as temperature, light and pH can also cause different changes in their color and nutritional value. 1 Recently, with the development of nanotechnology in food sciences and technology, investigators try to encapsulate these valuable resources in different nanoparticles to save their nutritional values, bioactivities and antioxidant properties as well as their stability and sustainability.The sparingly solubility of carotenoids hamper good intestinal absorption, thus reduce their bio-efficiency.In the recent decade many attempts have been directed to overcome the solubility issues of carotenoids using their formulation in the lipid based nanoparticles e.g.liposomes, 6-9 micelle, 10-12 solid lipid nanoparticles 13 and nanostructured lipid carriers. 14,15Solid lipid nanoparticles (SINs) are colloidal drug carrier with nanometer-sized, which contains the solid lipid matrix.SLNs consistent high biodegradability and biocompatibility and are good candidates as carriers for both hydrophilic and lipophilic compounds. 16SLNs construction comprise a simple homogenization and solidification process that would allow successful scale up for industry. 17In addition, compared with nanostructured lipid carriers (NLCs), SLNs display more controlled drug release effectiveness. 18Altogether, SLNs possess different advantages in nutraceutical developments including high stability, protection of incorporated compound against chemical degradation, 19 biocompatibility of the carrier, 20 and avoidance of organic solvent during formulation. 21ioactive compounds in the solid core of SLNs have mobility limitation and speed of their distribution to the particle surface are lower than other nano-emulsions. 22hese features of SLNs make them suitable for the decrease of decomposition reactions such as oxidation of bioactive compounds e.g carotenoids.Different procedures are used for the preparation of SLNs including hot/cold homogenization, high pressure homogenization (HPH), ultrasonification, and emulsification and solvent evaporation method.Almost in all methods, emulsions are prepared in the first step, and then by mechanical means large droplets are break down into smaller particles.In this investigation we tried to develop an enhanced lycopene loaded solid lipid nanoparticles (lycopene-SLNs) using simple hot homogenization method with some modification.Although in 2012 Riangjanapatee et al formulated lycopene-NLC and evaluated the surfactant type effects on the stability of the formulation, 23 to the best of our knowledge this investigation is a pioneering attempt in the formulation of lycopene in solid lipid nanoparticles composed of Precirol ® ATO 5 and Compritol 888 ATO as a lipid matrix to improve the pharmacokinetic behavior.Moreover, to enhance zeta potential of the formulated nanoparticles, to obtain a stable formulation, myristic acid was used during all formulation.
Anionic lycopene-SLNs were physicochemically and morphologically characterized by means of zetaseizer and scanning electron microscopy (SEM).
Moreover, we evaluated different physicochemical characterization including encapsulation efficiency (EE%), drug loading (DL) and stability.

Chemicals and reagents
All Chemical substances and compounds used in this investigation were pharmaceutical grade.Precirol® ATO5 and Compritol 888 ATO was gifted from Gattefosse (Nanterre, France).Tween 80, poloxamer 407 and myristic acid were purchased from Sigma-Aldrich (Poole, UK).All solvents used in this study were extra pure and purchased from Merck Co (Darmstadt, Germany).

Lycopene resources and extraction
Tomatoes needed to extract lycopene were purchased from a local market and after washing were air dried under the shade.Dried and ground tomatoes were extracted using petroleum ether aiding laboratory mixers for at least 30 minutes.After then the extract was filtrate using paper filter and solvent was removed in vacuo by rotary evaporator at maximum temperature of 40°C.The lycopene content of the extract was isolated using antisolvent precipitation method.For this, the crude extract was dissolved in ethyl acetate.Then methanol was added dropwise to the extract solution to completely precipitate lycopene.The sediment was dissolved in ethyl acetate and precipitation was repeated once again.Finally, the tartar was filtrate and remaining solvent was evaporated by a flow of nitrogen gas.Eventually dried red sediments were used for the production of solid lipid nanoparticles.

Preparation of Lycopene loaded solid lipid nanoparticles
Lycopene-SLNs were prepared by the hot homogenization method according to our previous work with some modification. 20Briefly, lipid phase including lycopene and solid lipid (glyceryl palmitostearate (Precirol® ATO 5) or glyceryl behenate (Compritol® 888 ATO) was simply dispersed by unintended heating at ~10 °C above the lipids melting point.Moreover, myristic acid (< 0.5 % w/w) was added to the lipid phase as zeta enhancer.To prepared aqueous phase, an appropriate concentration of stabilizers (Poloxamer 407) was heated to the same temperature of the oil phase in distilled water.Then the hot aqueous phase was dropped to the oil phase during 30 minutes and homogenization (12 000 rpm and 70 °C).In the next step, the obtained emulsion was further homogenized (at 19 000 rpm) for additional 10 min.Lycopene-SLNs were finally obtained by allowing the hot nanoemulsion to cool down at room temperature, and then were stored at 4 °C.Blank SLNs also were prepared by the same method except instead of lycopene, an equal amount of precirol or compritol was used.

Size, zeta potential, and morphological characteristics of nanoparticles
Getting an appropriate system for lycopene nanoparticles (NPs) preparation, the size of the prepared NPs was measured immediately after fabrication by laser diffraction immediately after preparation (SALD-MS30, Schimadzu, USA).In this context, SLNs was diluted by distilled water to reach appropriate concentration.Moreover, particle size distribution [mean diameter and dispersity index (DI)] and zeta potential of suitable formulation system was determined in pH 8.3 using Malvern zetasizer (3000HS, Malvern Instruments, UK) in the final concentration which recommended by the manufacturer of the instrument.The morphology of the fabricated NPs was observed with Scanning Electron Microscopy (SEM) based on our previous published method. 16,21

Encapsulation efficiency (%) and drug loading
The entrapment efficiency (EE) of the NPs were determined as the percentage of lycopene entrapped in the carriers compared to the total dug which was used for the formulation.For the measurement of the (EE), original suspension containing lycopene-SLNs and 0.3% tween 80 was placed in Ultra free tube with a cutoff of 10,000 Da (Ultrafree, MC Millipore, Bedford, USA) and centrifuged for 8 min at 14,000g (3K30, SIGMA Labrorzentrifugen GmbH, Germany).The filtrate was mixed by hexane (1:1) and hanged for at least 10 min and then the hydrophobe phase was separated by a separatory funnel and the quantity of free lycopene was determined using spectrophotometric method (λ max: 471 nm in hexane).To gain a calibration curve, working standard solutions were prepared by serially dilution with hexane.The stock solutions were prepared by dissolving pure lycopene in hexane at the concentrations of 6000 μg/mL.Each calibration curve consisted of 6 calibration points (600, 300, 100, 80, 40 and 20 μg/mL).Calibration curve was plotted by least square linear regression analysis.The drug EE in the SLNs was calculated from Eq. ( 1) and drug loading (DL) was obtained from Eq. ( 2).

( ) ( )
Eq. ( 1) where W LL is the weight of lycopene loaded in nanoparticles and W NP is the weight of nanoparticles solid mass.

In vitro release studies
Cumulative in vitro release of lycopene-SLNs was carried out based on dialysis method.Regarding to this, 1 mL of lycopene-SLNs was charged into dialysis bag (molecular weight cutoff: 12 kDa).The bag was then inserted in a glass holder containing 10 mL of dialysis buffer (0.3% W/V tween 80 in DW). 20Moreover, intact lycopene also was dialyzed similarly to evaluate the permeability of lycopene to the membrane.During dialysis the media was continuously stirred at RT and 100 rpm and at the fixed time periods, 1 mL of medium was removed and 1 mL of fresh media was added to the receptacle.Drug concentration was determined by the spectrophotometric method mentioned earlier.

Physical stability studies
To evaluate the stability of the formulated SLNs, samples were stored for 2 months at a glass tube at refrigerator (4 °C).Then mean diameter, DI and EE were measured and compared with the fresh ones. 20

Statistical analyses
All data represent the mean of at least three repeated experiments (error bars represent mean ± standard deviation).Independent Student's t-test was utilized to compare mean differences between two independent groups and one-way ANOVA was used to multiple comparisons.Post hoc pairwise comparisons were carried out using Tukey multiple comparison tests for those that showed significant mean differences (SPSS; version 13.0).We used Shapiro-Wilk test to compare the shape of sample distribution with the shape of a normal curve.The statistical significance was defined as p<0.05.

Preparation and physicochemical characterization of Lycopene-SLNs
SLNs of lycopene was developed by hot homogenization method without using of organic solvents by means of different solid lipids including Precirol® ATO 5 and Compritol® 888 ATO.The SLNs were stabilized using a surfactant, i.e. Tween 80 and Poloxamer 407.Moreover, myristic acid was used to enhance the zeta quantity of the particles surface.It is well known that the carbonyl carbon of the organic acid such as myristic acid is directly linked to, and in conjugation with, a second electronegative oxygen atom bearing a hydrogen atom.This electronic arrangement allows for loss of a proton and ionization because electron density is "pulled" from the hydroxyl hydrogen through the conjugated carboxyl group, and the negative charge formed upon ionization (in the conjugate base) is stabilized by resonance delocalization. 24he method and composition of the SLNs were optimized, and they were further considered in terms of total DL, EE, particle size, zeta potential, morphology, and stability studies.The characteristics of some formulated lycopene-SLNs and blank SLNs in this investigation are listed in Table 1.Our results showed that the size and DI was generally smaller in the case of SLNs prepared with precirol when compared to SLNs prepared with compritol (Table 1).

Particle size and morphology
The average size of the particles and their shapes may affect the drug release pattern, entrapment efficiency, cytotoxicity and pharmacokinetic behavior. 25The results here showed that the size of the nanoparticles was generally in direct relation with the oil phase surfactant concentration.Clearly seen that nanoparticle size is reduced by increasing the amount of oil phase surfactant.However, our previous results showed that with the increase of tween 80 the cytotoxicity of NPs will increase. 16Moreover, there is an obvious decrease of nanoparticle size with an increase of homogenizing duration time and speed (data not shown).Of the different formulation, F2 (blank SLNs) and F7 (lycopene-SLNs) at 25 °C exhibited mono dispersed characteristics with a mean diameter of 124 ± 5.24 nm and 125 ± 3.89 nm.Besides, F2 and F7 showed acceptable DI of 0.382 ± 0.142 and 0.253 ± 0.105, respectively.The DI values indicated a narrow particle size distribution.These results were approved more by Scanning Electron Microscopy (SEM) images (Figure 1).Moreover, SEM showed spherical and uniform SLNs.Zeta potential as important criteria can predict the particles long-term stability.Data are expressed as mean ± SD (n=3).a: Tween 80 and b: Poloxamer 407 was used as the oil phase and aqueous phase surfactant in all formulations, respectively.0.3% (w/w) myristic acid also was used in all formulation.The zeta potential of higher than −60 mV for a colloidal dispersion forecast physically stable condition for the particles of the dispersion. 26In this investigation, at pH of distilled water (8.3), zeta potential of the selected formulation, F2 and F7, were -14.21 ± 1.14 and -10.08 ± 2.06, respectively (Figure 2).

In vitro release studies
In this study the cumulative drug release was assessed using dialysis method.Figure 3 shows the release profile of lycopene-SLNs in DW containing 0.05% (W/V) tween 80 as co-solubilizer.The release profile showed that lycopene-SLNs exhibited no burst drug release and less than 30% of lycopene was released after 72 h.This type of sustained release profile of sparingly water soluble drug was also reported by others. 20,27 t is may be mainly referring to the low diffusion of the drug from the lipid matrix of SLNs into aqueous media.However, as seen in Figure 3 approximately 23% of lycopene drug release was occurred in the first 24 h.This release may be attributed to those drugs which is located around the surface of the SLNs.Thought, all intact lycopene (>95%) was released to the media in the first 24 h which gives us confidence that lycopene could penetrate into the cellulose pores.

Physical stability studies
Of different formulations, the most suitable one − the formulation composed of 3.5% (w/w) lycopene, a surfactant consisting of 6.07% tween 80 and 46.55% poloxamer 407 in a solid lipid matrix of 43.85% precirol − was placed on long-term stability at 4 ºC for 3 months.After three months, the NPs was checked for any potential aggregation, and SLNs were evaluated in terms of size, DI, and EE.Stability studies showed that in the usual dispersed aqueous medium (distilled water), coacervation and precipitation of lipid did not occur.The result of the stability studies showed that after three months storage of formulation at 4 °C, the mean diameter, entrapment efficiency and DI of lycopene-SLNs displayed no significant differences, as compared with the fresh preparation (p > 0.05) (Table 2).

Conclusion
Here solid lipid nanoparticle containing lycopene successfully fabricated using simple hot homogenization procedure.Lycopene nanoparticles showed good physicochemical characterization in terms of good stability during storage times.The obtained small size of nanoparticles (110 to 130 nm) is hopeful characteristic to candidate lycopene-SLNs formulation to orally usage.There is enough observation revealed that the intestinal absorption of lycopene-SLNs formulated here will be good.Encapsulation efficiency of lycopene-SLNs stored for at least three months showed that an ignorable leakage is occurred in 4 °C.Finally, this investigation could be a pioneer study to propose that using of these types of carotenoid-SLNs (nano-nutraceutical) in the manufacturing of different beverages and dairy products as food supplementary materials.

Figure 3 .
Figure 3. Cumulative release of lycopene.The release of intact lycopene was assessed in a same condition to evaluate the perme ability of the plain drug through the membrane