International Journal of Cosmetic Science, 2015, 37, 446–453
doi: 10.1111/ics.12216
Preparation and evaluation of novel octylmethoxycinnamate-loaded solid lipid nanoparticles X.-h. Liu, X.-z. Liang, X. Fang and W.-P. Zhang School of Perfume and Aroma Technology, Shanghai Institute of Technology, No. 100, Haiquan Road, Fengxian District, Shanghai 201418, China
Received 8 October 2014, Accepted 13 February 2015
Keywords: characterization, differential scanning calorimetry, emulsions, octylmethoxycinnamate, solid lipid nanoparticles, spectroscopy, stability
Synopsis OBJECTIVE: Octylmethoxycinnamate (OMC)-loaded solid lipid nanoparticles (SLNs) were prepared by ultrasonic emulsification method. Effects of process variables and formulation composition were investigated on particle size and polydispersity index (PI), and the UV absorbance. Effect of OMC concentration on entrapment efficiency (EE) was also studied. METHODS: The optimal formulation was characterized and evaluated by environment emission scanning electron microscopy (ESEM), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). In addition, the degradations of OMC from SLNs and OMC conventional emulsion were compared. RESULTS: The composition of optimal formulation was determined as 5% (w/w) of solid lipid, 7% (w/w) of emulsifier and 9% of loaded OMC, resulting in a particle size of 392.8 nm, and EE of 88.73%, LD of 38.05% under the preparation condition of 6 min of sonication, 400 W of sonication power. ESEM study showed spherical particles with smooth surface. DSC studies indicated OMC encapsulation within the nanoparticle matrix. The characteristic peaks for OMC-SLNs stood at 1710, 1604, 1513, 1465 and 830.3 cm1. The degradation rate of OMC was decreased when using SLNs formulations compared to conventional emulsion. CONCLUSION: Hence, the developed SLNs can be used as sunscreen carrier for improve the stability. sume Re OBJECTIFS: Les nanoparticules lipidiques solides (NLS) charges en octylmethoxycinnamate (OMC) ont ete prepares par la methode d’emulsification ultrasonique. Les effets des variables du procede et de la composition de la formulation ont ete etudies, sur la taille des particules, sur l’indice de polydispersite (IP) et sur l’absorbance UV. L’effet de la concentration sur l’efficacite de piegeage du methoxycinnamate a egalement ete etudie. METHODES: La formulation optimale a ete caracterisee par balayage a emission d’environnement microscopie electronique a balayage differentiel (DSC) et par spec(ESEM), par calorimetrie a troscopie infrarouge a transformee de Fourier. En outre, les degradations de methoxycinnamate des NLS et d’une emulsion classique et ont ete comparees.
Correspondence: Wan-Ping Zhang, School of Perfume and Aroma Technology, Shanghai Institute of Technology, No. 100, Haiquan Road, Fengxian district, Shanghai 201418, China. Tel.: +86 02160873419; fax: +86 60873373; e-mail: [email protected]
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RESULTATS: La composition de formulation optimale a ete determinee comme 5% (p/p) du lipide solide, 7% (p/p) d’agent emulsifiant, et 9% de charge MOC, resultant en une taille de particule de 392.8nm, une Efficacite d’Encapsulation EE de 88,73%, et 38,05% de LD dans les conditions de preparation de 6 min de sonication, 400W de puissance. L’etude ESEM a montre des particules spheriques avec surface lisse. Les etudes de DSC ont indique l’encapsula l’interieur de la matrice de nanoparticules. Les pics tion d’OMC a 1710, 1604, 1513, caracteristiques pour OMC-NLS se elevent a 1465 et 830,3 cm1. Le taux de degradation du OMC a ete diminue lors de l’utilisation des formulations NLS par rapport a l’emulsion classique. CONCLUSION: Par consequent, les NLS developpes peuvent ^etre utilises comme support de creme solaire pour ameliorer la stabilite. Introduction In the last decade of the last century, solid lipid nanoparticles (SLNs) were developed as an alternative colloidal system to liposome, microemulsions and polymeric nanoparticles [1, 2]. SLNs, the first generation of lipid nanoparticles, are produced by replacing the liquid lipid (oil) of an o/w emulsion by a solid lipid or a blend of solid lipids, that is the lipid particle matrix being solid at both room and body temperature [3]. Compared with the traditional colloidal systems, SLNs avoid the disadvantages [4, 5], such as limited physical stability, aggregation, drug leakage on storage, cytotoxicity of polymers, etc. instead showing low cytotoxicity, drug targeting, excellent biocompatibility and biodegradability [6–8]. SLNs possess some other features that make them promising carriers for cosmetic and pharmaceutical application. SLNs are able to protect the labile compounds against chemical degradation. The feature was proven for many actives such as retinol [9, 10], tocopherol [11], ascorbyl palmitate [12] and coenzyme Q10 [13, 14]. The small size particles also have an occlusive action on skin, so an increased skin hydration effect is observed. SLNs can provide controlled release profiles for many active ingredients [15, 16]. Furthermore, SLNs show a UV-blocking potential, that is they can either solely act as a physical UV blocker and also are able to improve the UV protection by loaded sunscreen [17]. Ultraviolet radiation consists of 2% UVB (290–320 nm) and 98% UVA (320–400 nm) [18]. UVB radiation is mainly responsible for the severe damage: acute damage such as sunburn, and long-term damage including cancer [19]. UVA-light does penetrate to the deeper skin layers causing photoageing [20]. With the
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
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increased public awareness of the side effects of ultraviolet radiation, the use of sunscreen is growing. There are two basic types of sunscreen which are chemical and physical UV filters. Chemical filters, depending on their chemical structures, are used as active ingredients of sunscreen formulation. Inorganic filters such as titanium dioxide and zinc oxide have been said to block UVA/UVB sunlight through reflection and scattering [21]. Currently, the chemical filters are widely used in sunscreen products. However, the photostability is now recognized as a common problem for organic UV absorbers. When the sunscreen molecules absorb the UV-light, the photochemical reaction such as trans-cis isomerization, ketoenol tautomerism may occur [22]. As the result of the reactions, the ability of the sunscreen to absorb the UVA/UVB light will reduce. In addition, more and more people have been aware of the potential irritation caused by the chemical UV filters. Sunscreen formulations are applied to a large skin area and for a long period, producing a constant and high input of the chemical into the viable skin strata and to the systemic circulation. When sunscreens penetrate into the viable layers, they can generate highly reactive oxygen species in the cytoplasm of the nucleated epidermal keratinocytes and the level of reactive oxygen species (ROS) increases above that produced naturally by epidermal chromophores under UV illumination [23]. Octylmethoxycinnamate (OMC) is an oil-soluble chemical sunscreen agent that absorbs UVB radiation and is the most widely used UVB absorber [24]. Some studies have reported OMC is unstable and degraded when exposed to the sunlight [25]. What is more, Elka found that the OMC can permeate the skin and the skin permeation flux was 27 lg cm2 per hour [26]. The molecular formula of OMC was as follows:
O O O So the carriers used to encapsulate them are very important. Solid lipid particles have been widely investigated as carrier systems. For example, glyceryl stearate-based solid lipid particles were used to incorporate benzophenone-3 by Mestres et al. [27]. Santo Scalia and his co-workers incorporated the UVA filter, butyl methoxydibenzoylmethane and the UVB filter octocrylene to the SLNs, which enhanced the UVA filter photostability [28]. The increasing demand of inorganic UV filters as titanium dioxide related to their low potential for producing irritant reactions and their sunscreen efficacy. Carnauba wax-based SLNs were used to incorporate the inorganic filter, titanium dioxide, by M€ uller-Goymann et al. [29]. However, the preparation of the OMC-loaded solid lipid particles and the structure had not been reported. So, in this study, OMC was used as model drug. The influence of the process variables on the particle size and polydispersity index (PI) of SLNs was investigated. The formulation parameters, such as sonication time, sonication power, surfactant concentration, lipid concentration and drug concentration, were optimized for the formulation of SLNs by emulsification–ultrasonication method. The optimal SLNs were physicochemical characterized by environment emission scanning electron microscopy (ESEM), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy. Furthermore, the degradation of OMC from SLNs and conventional emulsion was studied.
Materials and methods Materials Hydrogenated castor oil (HR CutinaâHR; BASF, Tarrytown, NY, U.S.A.) was used as solid lipid material of SLNs. The lipophilic surfactant used for the formulation was steareth-21 (S-21 EumulginâS 21, BASF). The caprylic/capric triglyceride (GTCC) was purchased from BASF. Organic UV filters and Uvinul MC80 (OMC) were provided by BASF. Poly oxyethylene [20] sorbitan monooleate (Tween 80, T80), Sorbitan Monooleate (Span80, S80) and methanol were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). The water used throughout the experiments was deionized. All other reagents were of analytical grade, and they were used as supplied. Preparation of formulation Solid lipid nanoparticles were prepared by ultrasonic emulsification method [4]. Briefly, the aqueous phase and the oil phase (castor oil, steareth-21 and OMC) were separately heated at 75°C. Then the oil phase was added to the water phase while being homogenized using a homogenizer (IKAâT-10 basic Ultra-Turraxâ, IKA, Staufen, Germany) at 14 000–15 000 r.p.m. The obtained emulsion was ultrasonicated using a probe sonicator (CP 750; Cole parmer, North America U.S.A.). The resulting nano-emulsion was cooled down under stirring (JB-200S) at 910 Kr min1. Different formulations were prepared by varying the critical process variables. Conventional emulsion was prepared in the same way. Formulation which the oil phase contained Tween 80, Span 80, Caprylic/capric triglyceride and OMC was different from the SLNs, and the process of preparation was the same. Determination of particle size and polydispersity index Particle size (z-average diameter) and PI were measured by photon correlation spectroscopy using Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire U.K.) at 25°C. The instrument contains a 4 mW He–Ne laser operating at 633 nm wavelength. The mean particle size and PI were obtained by averaging three measurements at an angle of 173°. Freeze-drying of SLNs dispersions Solid lipid nanoparticles dispersions were freeze-dried to obtain dry product used for the analytical determination of the thermal analysis. The SLNs dispersions were fast-frozen under 18°C for 24 h, and then the samples were moved to the freeze-drier (FD-LC-50; Xicui Road, Haidian Beijing, China). The drying period was 48 h, and then the SLNs powders were collected for DSC and FTIR measurement. Encapsulation efficiency and drug loading Considering the entrapment efficiency (EE), the amount of OMC that can be incorporated into the particles, and considering the loading capacity (LC), the amount of OMC incorporated per mg of lipid, both parameters were calculated according to the following equations:
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 446–453
EEð%Þ ¼
Wa Ws 100 Wa
ð1Þ
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LCð%Þ ¼
Wa Ws 100 Wa Ws þ Wl
ð2Þ
where Wa stands for the mass of OMC added to the formulation and Ws is the mass of OMC determined in supernatant after separation of the lipid and aqueous phases, Wl is the weight of lipid added in the formulation. The amount of OMC in supernatant was measured by reversephase HPLC. Briefly, 1.000 g of OMC-loaded SLNs was centrifuged at 9000 r.p.m. for 120 min to separate the lipid and aqueous phases. A volume of 0.1 mL of supernatant was then diluted with pure water to 100 mL. Then, samples were filtered and injected into HPLC system. Agilent HPLC (Agilent 1260 series; Santa Clara U.S.A.) attached with a reverse-phase C18 column (Fortis C18; 4.6 mm 9 250 mm, 5 m; Agilent) was used for the assay. Mobile phase used was methano deionized water and (90 : 10) at 1.0 mL min1 isocratic flow. Temperature of the column and detection wavelength were set at 35°C and 311 nm, respectively. Environmental emission scanning electron microscopy Shape and surface morphology of the OMC-SLNs were examined by environmental ESEM (Quanta200 FEG; FEI company, Holland, the Netherlands). Prior to analysis, the sample was diluted with deionized water, placed on a double side carbon tape mounted onto an aluminium stud and air-dried. Then, images were obtained at 950 000 magnification at an excitation voltage of 5 kV. Fourier transform infrared spectroscopy (FT-IR)
Differential scanning calorimetry was measured on a DSC Q2000 apparatus (TA Instruments, New Castle DE U.S.A.). The thermo grams of bulk lipid, steareth-21, and lyophilized unloaded SLNs, OMC-SLNs were recorded. Samples (~2–5 mg) sealed in standard aluminium pans were kept under isothermal condition at 2°C for 10 min. An empty pan was used as a reference. DSC scans were recorded at heating rate of 10°C min1. All samples were heated from 20°C up to 200°C and then cooled back to 20°C. Ultraviolet–visible absorption spectroscopy The absorption spectra of samples were investigated using a SHIMADZU UV-3600 UV–vis–NIR spectrophotometer in the range of 250–400 nm, using 1-mL quartz cuvettes of path length l = 1 cm. It measured the intensity of light passing through the sample (I) and compared it to the intensity of light before it passes through the sample (I0). The ratio (I/I0) was called the transmittance and is usually expressed as a percentage (%T). The absorbance, A, was based on the transmittance and was calculated according to the following equations. Samples suspended in deionized water before measurements were taken. 1 T
ð3Þ
I0 ¼ecl I
ð4Þ
A ¼ log10
A ¼ log10
FTIR spectrometer (VERTEX 70; Ettlingen German) equipped with a deuterated triglycine sulphate (DTGS) as a detector was used to collect the spectroscopy. Freeze-dried sample (~3 mg) was positioned in contact with attenuated total reflectance (ATR) on a multibounce plate of crystal at controlled ambient temperature (25°C). All FTIR spectra were recorded from 4000 to 650 cm1. A total of 64 interferograms (scans) per sample were co-added and averaged for each spectrum. The spectral resolution amounted to 4 cm1. These spectra were subtracted against background air spectrum. After every scan, a new reference air background spectrum was taken. The ATR plate was carefully cleaned in situ by scrubbing with alcohol twice and dried with soft tissue before filling in with the next sample, and made it possible to dry the ATR plate. The plate cleanliness was verified by collecting a background spectrum and compared to the previous one. These spectra were recorded as absorbance values at each data point.
a
Differential scanning calorimetry
Results and discussion The preparation of octylmethoxycinnamate-loaded solid lipid particles The ultrasonic emulsification method was found to be efficient and quick to produce SLNs. The influence of different process variables mainly on particle size and PI was discussed in the following sections. Sonication time Sonication time exerted a huge influence on particle size and PI. As shown in Fig. 1(a), the particle size and PI significantly decreased with increasing sonication time. When the sonication
b
Figure 1 The effect of ultrasonication time: (a) on particle size and polydispersity index (b) the UV absorption.
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© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 446–453
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time increased from 0 to 6 min, the particle size and PI decreased from 349.4 nm, 0.416 to 177.5 nm, 0.239, respectively. These observations were reasonable because sonication was responsible for the final particle size of SLNs, as it broke the coarse emulsion drops to nanosize particles. A longer sonication time exerted more sonication energy to the SLNs dispersions, which reduced the size of the emulsion droplets and size distribution. However, the mean droplet size and PI did not change appreciably after about 6 min. On the one hand, 6 min was sufficient to overcome droplet aggregation. On the other hand, with increasing sonication time, the heat generated by sonication could not be balanced by heat radiation, so molecular motion slowed down and particle size did not rearrange at the interface. Therefore, we used a sonication time 6 min in the remaining experiments. As shown in Fig. 1(b), the UV absorption properties were enhanced with increasing sonication time. The OMC concentration was 4% (w/w) in this study which proved that the small particles were in favour of UV absorbance. This finding was expected because of the scattering and high spreadability of small particles. Small particles could stay at the surface of the skin and reflect or scatter UV radiation before it induced cellular damage as the physical sunscreen. Conventional physical sunscreen may produce the undesirable effect of ‘white’ residue which was absent in SLNs containing the lipid. Sonication power The effect of sonication power on the particle size and PI was studied at a sonication time 6 min, and the result was shown in Fig. 2(a). The particle size and PI significantly decreased from 349.4 nm before sonication to 227.7 nm after 6 min of sonication at 80 W. This result indicated that sonication was efficient at forming small droplets n this system. As the sonication power increased from 80 to 400 W, the particle size continued to decrease, but the reductions
were fairly modest. For example, the particle size decreased from 227.7 nm after sonication at 80 W to 200 nm after sonication at 400 W. The trend of particle size was not obvious when the sonication power was continuously enhanced. This finding could be due to the fact that the sample size was small, and 400 W was sufficient to make complete emulsification. Thus, 400 W was selected as the sonication power in the following experiments. As shown in Fig. 2(b), the UV absorption properties were mainly affected by the particle size under the same formulation. The theory that the small particles favoured UV absorbance proved. Emulsifier (S-21) concentration The role of the emulsifier in such systems is threefold [30]: (i) to decrease surface tension; (ii) to render the particles partially wettable and allow adsorption at the oil–water interface; (iii) to promote flocculation of solid particles and stabilize the particles. Therefore, the emulsifier concentration had great effects on the quality of the SLNs dispersion. Analysis of the influence of the emulsifier concentration on the particle size and PI of OMC-SLNs (Fig. 3a) showed that the particle size was lowest at the concentration of 7% (w/w). Emulsifier molecules interacted with each other via Van der Waals interactions, thereby forming a homogeneous layer. These molecules consequently were packed at the oil/water interface. Excessive emulsifier may have formed multilayers around the droplets and aggregated in the continuous phase, which contributed to the light scattering. Lipid (HR) concentration The effect of lipid concentration on the particle size and PI was determined for the system emulsified by 7% (w/w) S21. The particle size showed a remarkable increase on the particle size when the lipid concentration increased from 3% to 8%, but PI showed a decline (Fig. 3b). This result could be attributed to the following
a
b
a
b
Figure 2 The effect of ultrasonication power: (a) on particle size and polydispersity index (b) the UV absorption at 311 nm.
Figure 3 The effect on particle size and polydispersity index: (a) the emulsifier concentration (b) the HR concentration.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 446–453
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reasons. Firstly, the lipid (HR) was at a solid state at room temperature. Its viscosity increased with increasing solid lipid concentration. The theoretical and experimental studies showed that the viscosities altered the efficiency of particle disruption, resulting in a minimum droplet size. Secondly, distribution of sonication energy in the diluted dispersion was better than that of the concentrated dispersion, which was responsible for a marked reduction in efficient particle size. Thirdly, in this study, part of the effects may be due to the limitations of emulsifier concentration. The available emulsifier decreases with increasing lipid content, which favoured oil drop aggregation, thereby increasing the mean droplet diameter. Although particle size was lowest in case of 3% HR, PI was very high. When the HR concentration was 5%, the particle size and PI were appropriate. OMC concentration As previous mentioned, the optimum solid lipid and emulsifier concentrations were 5% and 7%, respectively. Based on the combination of lipid and emulsifier, the effects of OMC concentration on the particle size, PI, EE and the drug loading were studied. The results were shown in Table I. In this study, we investigated the concentrations of 5%, 7% and 9%. We observed a significant increase in the particle size with increasing the concentration. The EE of OMC was also significantly enhanced when the OMC concentration increased from 5% to 7%. However, when the OMC concentration varied from 7% to 9%, the EE only increased by 1.03%. This phenomenon may be explained from the following aspects. The incorporation of liquid lipid (OMC) in the solid lipid structures inhibited the crystallization process by mixing ‘spatially’ different molecules and left behind imperfections in the lattice, which may accommodate OMC. But the defective structures were also limited by the solid lipid concentration. When the OMC concentration increased to 7%, the EE reached the saturation point. Thus, OMC cannot be loaded on the particle and remained only on the surface of the solid lipid particle, which led to an increase in the particle size. Contrary to the particle size and EE, the PI decreased going along with the increase of OMC concentration. This may be related to that the liquid lipid speeded up the ordination of particles. The agglomerate of particles could reduce. From the above, the concentration of OMC set as 9% was fit and the corresponding LD was 38.05%. An optimal SLN formulation [5% (w/w) solid lipid, 7% (w/w) emulsifier and the OMC loaded 9%] with a particle size of 392.8 nm, and EE of 88.73% was thus achieved with 6-min sonication time at 400 W.
Figure 4 Scanning electron micrographs of octylmethoxycinnamate-loaded solid lipid nanoparticles (950 000).
loaded SLNs were almost spherical in shape with regular morphology as seen in Fig. 4. Homogeneous network of spherical micronsized pores with relatively narrow size distribution and smooth walls was observed. In addition, some particles showed bright periphery, representing the coating of surfactants in the external layer. The particles were arranged closely which further accounted for that the system mainly existed in the form of nanoparticles. Fourier transform infrared spectroscopy Pure OMC displayed a stretching vibration band of C=O at 1706 cm1; benzene skeleton vibration bands at 1604.7, 1514.04
c
b
Morphology characterization of nanoparticles The optimized formulation was then subjected to physicochemical characterization examination. SEM studies revealed that OMC-
a
Table I Effect of octylmethoxycinnamate (OMC) concentration on size, PDI entrapment efficiency (EE)
OMC concentration (%)
Size (nm)
Polydispersity index
%EE
5 7 9
267.3 319.6 392.8
0.269 0.251 0.243
78.24 87.76 88.73
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Figure 5 Fourier transform infrared spectroscopy spectra of (a) octylmethoxycinnamate (b) Unloaded solid lipid nanoparticles (c) OMC-SLNs.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 446–453
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Octylmethoxycinnamate, solid lipid nanoparticles and 1463.9 cm1; and a para-orientation peak of CH2 at 829.4 cm1 (Fig. 5a). By contrast, the corresponding characteristic peaks for OMC-SLNs were observed at 1710, 1604, 1513, 1461 and 829.3 cm1 (Fig. 5c). Except for the C=O stretching vibration of OMC-SLNs, in which the spectrum diminished to a small extent possibly because of the reduction in sharpness crystallinity of the drug, other spectra were simply regarded as the superposition of OMC. The FT-IR spectrum of OMC-SLNs showed no significant shift in all the characteristic bands described above. This result suggested the absence of an interaction between components in the formulation, thereby confirming OMC entrapment in the lipid matrix core. Compared with OMC-SLNs, the unloaded SLNs did not exhibit the same characteristic peaks (Fig. 5b). Characterization by differential scanning calorimetry Differential scanning calorimetry is frequently used to measure the heat loss or gain resulting from physical or chemical changes within a sample as a function of the temperature. It has been used to characterize the state and the crystallinity of SLNs. Thermo-dynamics stability of SLNs relies on their existing lipid modification. DSC functions on the principle that different lipid modifications possess different melting points and melting enthalpies. After heated, some hidden information of sample crystal lattice resolution could be showed in DSC curve, such as the melting point, crystallization order, and cocrystal mixture. The crystalline index (CI%), which is defined as the percentage of the lipid matrix that has recrystallized during storage time, was calculated by applying the following equation: CI ¼
DHSLNs 100 DHbulkmaterial Concentration
where DH represents ted the melting enthalpy (J g1) and could be obtained from the area under the DSC curve of the melting endotherm.
Figure 6 showed DSC curves of hydrogenated castor oil (HR), steareth-21 (S21), and lyophilized SLNs without OMC (unloaded SLNs), and OMC-loaded SLNs (OMC-SLNs). In general, the melting peak of the lipid core of the SLNs was observed at a lower temperature than that of bulk lipid mainly due to the nanocrystalline size, high specific surface area and the presence of emulsifiers. The emulsifier of S21 was solid at room temperature. The DSC curve (Fig. 6a) was characterized by a sharp exothermic peak at about 6.52°C and a sharp endothermic peak at 34.80°C, which was the melting point. Upon cooling a sample containing only HR from 200°C, a narrow exothermic peak (DH = 156.7 J g1, CI % = 100) was observed around 58.8°C (Fig. 6b), which was due to crystallization of the lipid. Upon heating from 20°C, an endothermic peak (DH = 173.7 J g1) was observed. The peak at 80.35°C corresponds to the melting point of the solid lipid. Typical DSC curve for unloaded SLNs was displayed in Fig. 6(c). The thermal behaviour of unloaded SLNs was characterized under the same conditions as for the bulk lipid and the emulsifier. During the heating run, the unloaded SLNs have two endothermic peaks at 36.16°C and 76.44°C, respectively, which can be attributed to the melting of the emulsifier of S21 and solid lipid. The melting point was about 3.91°C lower for the unloaded SLNs than for pure lipid. This effect can be described by the Henderson–Hasselbalch equation, which predicts that the melting point of a high melting point substance decreases in the presence of a low melting point substance (such as the emulsifier). The CI% decreased from 100% to 44.85%. On the cooling curve, the crystallization point of HR declined from 58.8°C to 58.5°C and the peak became narrow. The exothermic peak at around 6.01°C was the signal of the crystalline of the emulsifier (S21). In Fig. 6(d), the DSC profile of SLNs loaded with OMC (OMC-SLNs) was shown as an example of loaded SLNs. The OMC-SLNs showed a thermal behaviour very similar to that of the unloaded SLNs. In the heating process, OMC-SLNs showed a significant peak at 21.08°C, which lowers the skin temperature indicating that OMC can control
a
c
b
d
Figure 6 The differential scanning calorimetry thermograms of (a) the emulsifier of S21 (b) the solid lipid of HR (c) the unloaded solid lipid nanoparticles (d) OMC-SLNs.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 446–453
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1.6
OMC-SLN Emulsion
Absorbance
1.4 1.2 1 0.8 0.6
0
2
4
6
8
10
Time (h) Figure 7 The comparison of degradation of octylmethoxycinnamate from solid lipid nanoparticles and conventional emulsion.
release from the solid lipid and play the role of sunblock. The presence of OMC induced a broadening of emulsifier and lipid fusion peaks. The CI% of lipid decreased from 44.85% to 15.35%. It proved that the incorporation of OMC inside the solid lipid matrix had led to a decrease of the crystallinity and the binding forces between the molecules of the lipid matrix. These results contributed to the improvement of the drug loading, EE and stability of the system. Comparison of degradation of OMC from SLNs and conventional emulsion In this primary study, the degradations of OMC from SLNs and conventional emulsion were investigated over 8 h. Each sample was diluted to 0.025 mg mL1. The obtained samples were exposed to the sun. The samples (~20 g) were obtained every hour, and the absorbance was measured in the UV range. The detection wavelength was set at 311 nm. As shown in Fig. 7, OMC was degraded to a greater extent from the conventional emulsion. The degradation rate of OMC decreased in SLN formulations compared
with that in conventional emulsion, which allowed a reduction in the OMC concentration; this effect was desired. The solid core of the lipid nanoparticles provided a better physical and chemical stability than a liquid or liquid crystalline core of conventional emulsions. The results explained that the solid lipid particles as carriers enhanced the stability of OMC, which may be due to the fact that the solid lipid particles prevented direct sunshine from UVA or visible light, thereby resulting in the instability of UVB sunscreens. This finding was confirmed by Song and Lui [31], who compared the UV absorption properties of 3,4,5-trimethoxybenzochitin-loaded SLNs and free SLNs. Compared with a conventional emulsion, the amount of molecular sunscreen may be reduced by 50% in the SLN formulations while maintaining the protective level of conventional emulsion [32]. Conclusions The main goal of this work was to prepare solid lipid particles loaded with OMC by ultrasonic emulsification method. The optimal formulation was determined as 5% (w/w) of solid lipid, 7% (w/w) of emulsifier and 9% of loaded OMC, resulting in a particle size of 392.8 nm, and EE of 88.73%, LD of 38.05% under the preparation condition of 6 min of sonication, 400 W of sonication power. Environment emission scanning electron microscopy studies revealed that OMC-loaded SLNs were almost spherical in shape. The DSC and FT-IR showed that amount of OMC was loaded in the solid lipid particles. The comparison of degradation rate of OMC from SLNs and conventional emulsion explained that the solid lipid particles were an effective carrier to the sunscreen of OMC. Acknowledgement The authors would like to thank Mrs. Hu Jing for the DSC measurements and Mrs. Hu shaying for the ESEM measurement.
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20. Moyal, D.D. and Fourtanier, A.M. Broad-spectrum sunscreens provide better protection from solar ultraviolet–simulated radiation and natural sunlight–induced immunosuppression in human beings. J. Am. Acad. Dermatol. 58, S149–S154 (2008). 21. Serpone, N., Dondi, D. and Albini, A. Inorganic and organic UV filters: their role and efficacy in sunscreens and suncare products. Inorg. Chim. Acta 360, 794–802 (2007). 22. Shaath, N.A. Ultraviolet filters. Photochem. Photobiol. Sci. 9, 464–469 (2010). 23. Gonzalez, S., Fern andez-Lorente, M. and Gilaberte-Calzada, Y. The latest on skin photoprotection. Clin. Dermatol. 26, 614–626 (2008). 24. Serpone, N., Salinaro, A., Emeline, A.V., Horikoshi, S., Hidaka, H. and Zhao, J. An in vitro systematic spectroscopic examination of the photostabilities of a random set of commercial sunscreen lotions and their chemical UVB/UVA active agents. Photochem. Photobiol. Sci. 1, 970–981 (2002). 25. Yener, G., Inceg€ ul, T. and Yener, N. Importance of using solid lipid microspheres as carriers for UV filters on the example octyl methoxy cinnamate. Int. J. Pharm. 258, 203–207 (2003). 26. Touitou, E. and Godin, B. Skin nonpenetrating sunscreens for cosmetic and pharmaceutical formulations. Clin. Dermatol. 26, 375–379 (2008). 27. Mestres, J., Duracher, L., Baux, C., Vian, L. and Marti-Mestres, G. Benzophenone-3
Supporting Information Additional Supporting Information may be found in the online version of this article:
Figure S1. In vitro penetration profile of OMC from conventional emulsion.
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Data S1. The SPF of conventional emulsion and OMC-SLNs.
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doi: 10.1111/ics.12216
Preparation and evaluation of novel octylmethoxycinnamate-loaded solid lipid nanoparticles X.-h. Liu, X.-z. Liang, X. Fang and W.-P. Zhang School of Perfume and Aroma Technology, Shanghai Institute of Technology, No. 100, Haiquan Road, Fengxian District, Shanghai 201418, China
Received 8 October 2014, Accepted 13 February 2015
Keywords: characterization, differential scanning calorimetry, emulsions, octylmethoxycinnamate, solid lipid nanoparticles, spectroscopy, stability
Synopsis OBJECTIVE: Octylmethoxycinnamate (OMC)-loaded solid lipid nanoparticles (SLNs) were prepared by ultrasonic emulsification method. Effects of process variables and formulation composition were investigated on particle size and polydispersity index (PI), and the UV absorbance. Effect of OMC concentration on entrapment efficiency (EE) was also studied. METHODS: The optimal formulation was characterized and evaluated by environment emission scanning electron microscopy (ESEM), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). In addition, the degradations of OMC from SLNs and OMC conventional emulsion were compared. RESULTS: The composition of optimal formulation was determined as 5% (w/w) of solid lipid, 7% (w/w) of emulsifier and 9% of loaded OMC, resulting in a particle size of 392.8 nm, and EE of 88.73%, LD of 38.05% under the preparation condition of 6 min of sonication, 400 W of sonication power. ESEM study showed spherical particles with smooth surface. DSC studies indicated OMC encapsulation within the nanoparticle matrix. The characteristic peaks for OMC-SLNs stood at 1710, 1604, 1513, 1465 and 830.3 cm1. The degradation rate of OMC was decreased when using SLNs formulations compared to conventional emulsion. CONCLUSION: Hence, the developed SLNs can be used as sunscreen carrier for improve the stability. sume Re OBJECTIFS: Les nanoparticules lipidiques solides (NLS) charges en octylmethoxycinnamate (OMC) ont ete prepares par la methode d’emulsification ultrasonique. Les effets des variables du procede et de la composition de la formulation ont ete etudies, sur la taille des particules, sur l’indice de polydispersite (IP) et sur l’absorbance UV. L’effet de la concentration sur l’efficacite de piegeage du methoxycinnamate a egalement ete etudie. METHODES: La formulation optimale a ete caracterisee par balayage a emission d’environnement microscopie electronique a balayage differentiel (DSC) et par spec(ESEM), par calorimetrie a troscopie infrarouge a transformee de Fourier. En outre, les degradations de methoxycinnamate des NLS et d’une emulsion classique et ont ete comparees.
Correspondence: Wan-Ping Zhang, School of Perfume and Aroma Technology, Shanghai Institute of Technology, No. 100, Haiquan Road, Fengxian district, Shanghai 201418, China. Tel.: +86 02160873419; fax: +86 60873373; e-mail: [email protected]
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RESULTATS: La composition de formulation optimale a ete determinee comme 5% (p/p) du lipide solide, 7% (p/p) d’agent emulsifiant, et 9% de charge MOC, resultant en une taille de particule de 392.8nm, une Efficacite d’Encapsulation EE de 88,73%, et 38,05% de LD dans les conditions de preparation de 6 min de sonication, 400W de puissance. L’etude ESEM a montre des particules spheriques avec surface lisse. Les etudes de DSC ont indique l’encapsula l’interieur de la matrice de nanoparticules. Les pics tion d’OMC a 1710, 1604, 1513, caracteristiques pour OMC-NLS se elevent a 1465 et 830,3 cm1. Le taux de degradation du OMC a ete diminue lors de l’utilisation des formulations NLS par rapport a l’emulsion classique. CONCLUSION: Par consequent, les NLS developpes peuvent ^etre utilises comme support de creme solaire pour ameliorer la stabilite. Introduction In the last decade of the last century, solid lipid nanoparticles (SLNs) were developed as an alternative colloidal system to liposome, microemulsions and polymeric nanoparticles [1, 2]. SLNs, the first generation of lipid nanoparticles, are produced by replacing the liquid lipid (oil) of an o/w emulsion by a solid lipid or a blend of solid lipids, that is the lipid particle matrix being solid at both room and body temperature [3]. Compared with the traditional colloidal systems, SLNs avoid the disadvantages [4, 5], such as limited physical stability, aggregation, drug leakage on storage, cytotoxicity of polymers, etc. instead showing low cytotoxicity, drug targeting, excellent biocompatibility and biodegradability [6–8]. SLNs possess some other features that make them promising carriers for cosmetic and pharmaceutical application. SLNs are able to protect the labile compounds against chemical degradation. The feature was proven for many actives such as retinol [9, 10], tocopherol [11], ascorbyl palmitate [12] and coenzyme Q10 [13, 14]. The small size particles also have an occlusive action on skin, so an increased skin hydration effect is observed. SLNs can provide controlled release profiles for many active ingredients [15, 16]. Furthermore, SLNs show a UV-blocking potential, that is they can either solely act as a physical UV blocker and also are able to improve the UV protection by loaded sunscreen [17]. Ultraviolet radiation consists of 2% UVB (290–320 nm) and 98% UVA (320–400 nm) [18]. UVB radiation is mainly responsible for the severe damage: acute damage such as sunburn, and long-term damage including cancer [19]. UVA-light does penetrate to the deeper skin layers causing photoageing [20]. With the
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increased public awareness of the side effects of ultraviolet radiation, the use of sunscreen is growing. There are two basic types of sunscreen which are chemical and physical UV filters. Chemical filters, depending on their chemical structures, are used as active ingredients of sunscreen formulation. Inorganic filters such as titanium dioxide and zinc oxide have been said to block UVA/UVB sunlight through reflection and scattering [21]. Currently, the chemical filters are widely used in sunscreen products. However, the photostability is now recognized as a common problem for organic UV absorbers. When the sunscreen molecules absorb the UV-light, the photochemical reaction such as trans-cis isomerization, ketoenol tautomerism may occur [22]. As the result of the reactions, the ability of the sunscreen to absorb the UVA/UVB light will reduce. In addition, more and more people have been aware of the potential irritation caused by the chemical UV filters. Sunscreen formulations are applied to a large skin area and for a long period, producing a constant and high input of the chemical into the viable skin strata and to the systemic circulation. When sunscreens penetrate into the viable layers, they can generate highly reactive oxygen species in the cytoplasm of the nucleated epidermal keratinocytes and the level of reactive oxygen species (ROS) increases above that produced naturally by epidermal chromophores under UV illumination [23]. Octylmethoxycinnamate (OMC) is an oil-soluble chemical sunscreen agent that absorbs UVB radiation and is the most widely used UVB absorber [24]. Some studies have reported OMC is unstable and degraded when exposed to the sunlight [25]. What is more, Elka found that the OMC can permeate the skin and the skin permeation flux was 27 lg cm2 per hour [26]. The molecular formula of OMC was as follows:
O O O So the carriers used to encapsulate them are very important. Solid lipid particles have been widely investigated as carrier systems. For example, glyceryl stearate-based solid lipid particles were used to incorporate benzophenone-3 by Mestres et al. [27]. Santo Scalia and his co-workers incorporated the UVA filter, butyl methoxydibenzoylmethane and the UVB filter octocrylene to the SLNs, which enhanced the UVA filter photostability [28]. The increasing demand of inorganic UV filters as titanium dioxide related to their low potential for producing irritant reactions and their sunscreen efficacy. Carnauba wax-based SLNs were used to incorporate the inorganic filter, titanium dioxide, by M€ uller-Goymann et al. [29]. However, the preparation of the OMC-loaded solid lipid particles and the structure had not been reported. So, in this study, OMC was used as model drug. The influence of the process variables on the particle size and polydispersity index (PI) of SLNs was investigated. The formulation parameters, such as sonication time, sonication power, surfactant concentration, lipid concentration and drug concentration, were optimized for the formulation of SLNs by emulsification–ultrasonication method. The optimal SLNs were physicochemical characterized by environment emission scanning electron microscopy (ESEM), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy. Furthermore, the degradation of OMC from SLNs and conventional emulsion was studied.
Materials and methods Materials Hydrogenated castor oil (HR CutinaâHR; BASF, Tarrytown, NY, U.S.A.) was used as solid lipid material of SLNs. The lipophilic surfactant used for the formulation was steareth-21 (S-21 EumulginâS 21, BASF). The caprylic/capric triglyceride (GTCC) was purchased from BASF. Organic UV filters and Uvinul MC80 (OMC) were provided by BASF. Poly oxyethylene [20] sorbitan monooleate (Tween 80, T80), Sorbitan Monooleate (Span80, S80) and methanol were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). The water used throughout the experiments was deionized. All other reagents were of analytical grade, and they were used as supplied. Preparation of formulation Solid lipid nanoparticles were prepared by ultrasonic emulsification method [4]. Briefly, the aqueous phase and the oil phase (castor oil, steareth-21 and OMC) were separately heated at 75°C. Then the oil phase was added to the water phase while being homogenized using a homogenizer (IKAâT-10 basic Ultra-Turraxâ, IKA, Staufen, Germany) at 14 000–15 000 r.p.m. The obtained emulsion was ultrasonicated using a probe sonicator (CP 750; Cole parmer, North America U.S.A.). The resulting nano-emulsion was cooled down under stirring (JB-200S) at 910 Kr min1. Different formulations were prepared by varying the critical process variables. Conventional emulsion was prepared in the same way. Formulation which the oil phase contained Tween 80, Span 80, Caprylic/capric triglyceride and OMC was different from the SLNs, and the process of preparation was the same. Determination of particle size and polydispersity index Particle size (z-average diameter) and PI were measured by photon correlation spectroscopy using Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire U.K.) at 25°C. The instrument contains a 4 mW He–Ne laser operating at 633 nm wavelength. The mean particle size and PI were obtained by averaging three measurements at an angle of 173°. Freeze-drying of SLNs dispersions Solid lipid nanoparticles dispersions were freeze-dried to obtain dry product used for the analytical determination of the thermal analysis. The SLNs dispersions were fast-frozen under 18°C for 24 h, and then the samples were moved to the freeze-drier (FD-LC-50; Xicui Road, Haidian Beijing, China). The drying period was 48 h, and then the SLNs powders were collected for DSC and FTIR measurement. Encapsulation efficiency and drug loading Considering the entrapment efficiency (EE), the amount of OMC that can be incorporated into the particles, and considering the loading capacity (LC), the amount of OMC incorporated per mg of lipid, both parameters were calculated according to the following equations:
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 446–453
EEð%Þ ¼
Wa Ws 100 Wa
ð1Þ
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LCð%Þ ¼
Wa Ws 100 Wa Ws þ Wl
ð2Þ
where Wa stands for the mass of OMC added to the formulation and Ws is the mass of OMC determined in supernatant after separation of the lipid and aqueous phases, Wl is the weight of lipid added in the formulation. The amount of OMC in supernatant was measured by reversephase HPLC. Briefly, 1.000 g of OMC-loaded SLNs was centrifuged at 9000 r.p.m. for 120 min to separate the lipid and aqueous phases. A volume of 0.1 mL of supernatant was then diluted with pure water to 100 mL. Then, samples were filtered and injected into HPLC system. Agilent HPLC (Agilent 1260 series; Santa Clara U.S.A.) attached with a reverse-phase C18 column (Fortis C18; 4.6 mm 9 250 mm, 5 m; Agilent) was used for the assay. Mobile phase used was methano deionized water and (90 : 10) at 1.0 mL min1 isocratic flow. Temperature of the column and detection wavelength were set at 35°C and 311 nm, respectively. Environmental emission scanning electron microscopy Shape and surface morphology of the OMC-SLNs were examined by environmental ESEM (Quanta200 FEG; FEI company, Holland, the Netherlands). Prior to analysis, the sample was diluted with deionized water, placed on a double side carbon tape mounted onto an aluminium stud and air-dried. Then, images were obtained at 950 000 magnification at an excitation voltage of 5 kV. Fourier transform infrared spectroscopy (FT-IR)
Differential scanning calorimetry was measured on a DSC Q2000 apparatus (TA Instruments, New Castle DE U.S.A.). The thermo grams of bulk lipid, steareth-21, and lyophilized unloaded SLNs, OMC-SLNs were recorded. Samples (~2–5 mg) sealed in standard aluminium pans were kept under isothermal condition at 2°C for 10 min. An empty pan was used as a reference. DSC scans were recorded at heating rate of 10°C min1. All samples were heated from 20°C up to 200°C and then cooled back to 20°C. Ultraviolet–visible absorption spectroscopy The absorption spectra of samples were investigated using a SHIMADZU UV-3600 UV–vis–NIR spectrophotometer in the range of 250–400 nm, using 1-mL quartz cuvettes of path length l = 1 cm. It measured the intensity of light passing through the sample (I) and compared it to the intensity of light before it passes through the sample (I0). The ratio (I/I0) was called the transmittance and is usually expressed as a percentage (%T). The absorbance, A, was based on the transmittance and was calculated according to the following equations. Samples suspended in deionized water before measurements were taken. 1 T
ð3Þ
I0 ¼ecl I
ð4Þ
A ¼ log10
A ¼ log10
FTIR spectrometer (VERTEX 70; Ettlingen German) equipped with a deuterated triglycine sulphate (DTGS) as a detector was used to collect the spectroscopy. Freeze-dried sample (~3 mg) was positioned in contact with attenuated total reflectance (ATR) on a multibounce plate of crystal at controlled ambient temperature (25°C). All FTIR spectra were recorded from 4000 to 650 cm1. A total of 64 interferograms (scans) per sample were co-added and averaged for each spectrum. The spectral resolution amounted to 4 cm1. These spectra were subtracted against background air spectrum. After every scan, a new reference air background spectrum was taken. The ATR plate was carefully cleaned in situ by scrubbing with alcohol twice and dried with soft tissue before filling in with the next sample, and made it possible to dry the ATR plate. The plate cleanliness was verified by collecting a background spectrum and compared to the previous one. These spectra were recorded as absorbance values at each data point.
a
Differential scanning calorimetry
Results and discussion The preparation of octylmethoxycinnamate-loaded solid lipid particles The ultrasonic emulsification method was found to be efficient and quick to produce SLNs. The influence of different process variables mainly on particle size and PI was discussed in the following sections. Sonication time Sonication time exerted a huge influence on particle size and PI. As shown in Fig. 1(a), the particle size and PI significantly decreased with increasing sonication time. When the sonication
b
Figure 1 The effect of ultrasonication time: (a) on particle size and polydispersity index (b) the UV absorption.
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time increased from 0 to 6 min, the particle size and PI decreased from 349.4 nm, 0.416 to 177.5 nm, 0.239, respectively. These observations were reasonable because sonication was responsible for the final particle size of SLNs, as it broke the coarse emulsion drops to nanosize particles. A longer sonication time exerted more sonication energy to the SLNs dispersions, which reduced the size of the emulsion droplets and size distribution. However, the mean droplet size and PI did not change appreciably after about 6 min. On the one hand, 6 min was sufficient to overcome droplet aggregation. On the other hand, with increasing sonication time, the heat generated by sonication could not be balanced by heat radiation, so molecular motion slowed down and particle size did not rearrange at the interface. Therefore, we used a sonication time 6 min in the remaining experiments. As shown in Fig. 1(b), the UV absorption properties were enhanced with increasing sonication time. The OMC concentration was 4% (w/w) in this study which proved that the small particles were in favour of UV absorbance. This finding was expected because of the scattering and high spreadability of small particles. Small particles could stay at the surface of the skin and reflect or scatter UV radiation before it induced cellular damage as the physical sunscreen. Conventional physical sunscreen may produce the undesirable effect of ‘white’ residue which was absent in SLNs containing the lipid. Sonication power The effect of sonication power on the particle size and PI was studied at a sonication time 6 min, and the result was shown in Fig. 2(a). The particle size and PI significantly decreased from 349.4 nm before sonication to 227.7 nm after 6 min of sonication at 80 W. This result indicated that sonication was efficient at forming small droplets n this system. As the sonication power increased from 80 to 400 W, the particle size continued to decrease, but the reductions
were fairly modest. For example, the particle size decreased from 227.7 nm after sonication at 80 W to 200 nm after sonication at 400 W. The trend of particle size was not obvious when the sonication power was continuously enhanced. This finding could be due to the fact that the sample size was small, and 400 W was sufficient to make complete emulsification. Thus, 400 W was selected as the sonication power in the following experiments. As shown in Fig. 2(b), the UV absorption properties were mainly affected by the particle size under the same formulation. The theory that the small particles favoured UV absorbance proved. Emulsifier (S-21) concentration The role of the emulsifier in such systems is threefold [30]: (i) to decrease surface tension; (ii) to render the particles partially wettable and allow adsorption at the oil–water interface; (iii) to promote flocculation of solid particles and stabilize the particles. Therefore, the emulsifier concentration had great effects on the quality of the SLNs dispersion. Analysis of the influence of the emulsifier concentration on the particle size and PI of OMC-SLNs (Fig. 3a) showed that the particle size was lowest at the concentration of 7% (w/w). Emulsifier molecules interacted with each other via Van der Waals interactions, thereby forming a homogeneous layer. These molecules consequently were packed at the oil/water interface. Excessive emulsifier may have formed multilayers around the droplets and aggregated in the continuous phase, which contributed to the light scattering. Lipid (HR) concentration The effect of lipid concentration on the particle size and PI was determined for the system emulsified by 7% (w/w) S21. The particle size showed a remarkable increase on the particle size when the lipid concentration increased from 3% to 8%, but PI showed a decline (Fig. 3b). This result could be attributed to the following
a
b
a
b
Figure 2 The effect of ultrasonication power: (a) on particle size and polydispersity index (b) the UV absorption at 311 nm.
Figure 3 The effect on particle size and polydispersity index: (a) the emulsifier concentration (b) the HR concentration.
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reasons. Firstly, the lipid (HR) was at a solid state at room temperature. Its viscosity increased with increasing solid lipid concentration. The theoretical and experimental studies showed that the viscosities altered the efficiency of particle disruption, resulting in a minimum droplet size. Secondly, distribution of sonication energy in the diluted dispersion was better than that of the concentrated dispersion, which was responsible for a marked reduction in efficient particle size. Thirdly, in this study, part of the effects may be due to the limitations of emulsifier concentration. The available emulsifier decreases with increasing lipid content, which favoured oil drop aggregation, thereby increasing the mean droplet diameter. Although particle size was lowest in case of 3% HR, PI was very high. When the HR concentration was 5%, the particle size and PI were appropriate. OMC concentration As previous mentioned, the optimum solid lipid and emulsifier concentrations were 5% and 7%, respectively. Based on the combination of lipid and emulsifier, the effects of OMC concentration on the particle size, PI, EE and the drug loading were studied. The results were shown in Table I. In this study, we investigated the concentrations of 5%, 7% and 9%. We observed a significant increase in the particle size with increasing the concentration. The EE of OMC was also significantly enhanced when the OMC concentration increased from 5% to 7%. However, when the OMC concentration varied from 7% to 9%, the EE only increased by 1.03%. This phenomenon may be explained from the following aspects. The incorporation of liquid lipid (OMC) in the solid lipid structures inhibited the crystallization process by mixing ‘spatially’ different molecules and left behind imperfections in the lattice, which may accommodate OMC. But the defective structures were also limited by the solid lipid concentration. When the OMC concentration increased to 7%, the EE reached the saturation point. Thus, OMC cannot be loaded on the particle and remained only on the surface of the solid lipid particle, which led to an increase in the particle size. Contrary to the particle size and EE, the PI decreased going along with the increase of OMC concentration. This may be related to that the liquid lipid speeded up the ordination of particles. The agglomerate of particles could reduce. From the above, the concentration of OMC set as 9% was fit and the corresponding LD was 38.05%. An optimal SLN formulation [5% (w/w) solid lipid, 7% (w/w) emulsifier and the OMC loaded 9%] with a particle size of 392.8 nm, and EE of 88.73% was thus achieved with 6-min sonication time at 400 W.
Figure 4 Scanning electron micrographs of octylmethoxycinnamate-loaded solid lipid nanoparticles (950 000).
loaded SLNs were almost spherical in shape with regular morphology as seen in Fig. 4. Homogeneous network of spherical micronsized pores with relatively narrow size distribution and smooth walls was observed. In addition, some particles showed bright periphery, representing the coating of surfactants in the external layer. The particles were arranged closely which further accounted for that the system mainly existed in the form of nanoparticles. Fourier transform infrared spectroscopy Pure OMC displayed a stretching vibration band of C=O at 1706 cm1; benzene skeleton vibration bands at 1604.7, 1514.04
c
b
Morphology characterization of nanoparticles The optimized formulation was then subjected to physicochemical characterization examination. SEM studies revealed that OMC-
a
Table I Effect of octylmethoxycinnamate (OMC) concentration on size, PDI entrapment efficiency (EE)
OMC concentration (%)
Size (nm)
Polydispersity index
%EE
5 7 9
267.3 319.6 392.8
0.269 0.251 0.243
78.24 87.76 88.73
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Figure 5 Fourier transform infrared spectroscopy spectra of (a) octylmethoxycinnamate (b) Unloaded solid lipid nanoparticles (c) OMC-SLNs.
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Octylmethoxycinnamate, solid lipid nanoparticles and 1463.9 cm1; and a para-orientation peak of CH2 at 829.4 cm1 (Fig. 5a). By contrast, the corresponding characteristic peaks for OMC-SLNs were observed at 1710, 1604, 1513, 1461 and 829.3 cm1 (Fig. 5c). Except for the C=O stretching vibration of OMC-SLNs, in which the spectrum diminished to a small extent possibly because of the reduction in sharpness crystallinity of the drug, other spectra were simply regarded as the superposition of OMC. The FT-IR spectrum of OMC-SLNs showed no significant shift in all the characteristic bands described above. This result suggested the absence of an interaction between components in the formulation, thereby confirming OMC entrapment in the lipid matrix core. Compared with OMC-SLNs, the unloaded SLNs did not exhibit the same characteristic peaks (Fig. 5b). Characterization by differential scanning calorimetry Differential scanning calorimetry is frequently used to measure the heat loss or gain resulting from physical or chemical changes within a sample as a function of the temperature. It has been used to characterize the state and the crystallinity of SLNs. Thermo-dynamics stability of SLNs relies on their existing lipid modification. DSC functions on the principle that different lipid modifications possess different melting points and melting enthalpies. After heated, some hidden information of sample crystal lattice resolution could be showed in DSC curve, such as the melting point, crystallization order, and cocrystal mixture. The crystalline index (CI%), which is defined as the percentage of the lipid matrix that has recrystallized during storage time, was calculated by applying the following equation: CI ¼
DHSLNs 100 DHbulkmaterial Concentration
where DH represents ted the melting enthalpy (J g1) and could be obtained from the area under the DSC curve of the melting endotherm.
Figure 6 showed DSC curves of hydrogenated castor oil (HR), steareth-21 (S21), and lyophilized SLNs without OMC (unloaded SLNs), and OMC-loaded SLNs (OMC-SLNs). In general, the melting peak of the lipid core of the SLNs was observed at a lower temperature than that of bulk lipid mainly due to the nanocrystalline size, high specific surface area and the presence of emulsifiers. The emulsifier of S21 was solid at room temperature. The DSC curve (Fig. 6a) was characterized by a sharp exothermic peak at about 6.52°C and a sharp endothermic peak at 34.80°C, which was the melting point. Upon cooling a sample containing only HR from 200°C, a narrow exothermic peak (DH = 156.7 J g1, CI % = 100) was observed around 58.8°C (Fig. 6b), which was due to crystallization of the lipid. Upon heating from 20°C, an endothermic peak (DH = 173.7 J g1) was observed. The peak at 80.35°C corresponds to the melting point of the solid lipid. Typical DSC curve for unloaded SLNs was displayed in Fig. 6(c). The thermal behaviour of unloaded SLNs was characterized under the same conditions as for the bulk lipid and the emulsifier. During the heating run, the unloaded SLNs have two endothermic peaks at 36.16°C and 76.44°C, respectively, which can be attributed to the melting of the emulsifier of S21 and solid lipid. The melting point was about 3.91°C lower for the unloaded SLNs than for pure lipid. This effect can be described by the Henderson–Hasselbalch equation, which predicts that the melting point of a high melting point substance decreases in the presence of a low melting point substance (such as the emulsifier). The CI% decreased from 100% to 44.85%. On the cooling curve, the crystallization point of HR declined from 58.8°C to 58.5°C and the peak became narrow. The exothermic peak at around 6.01°C was the signal of the crystalline of the emulsifier (S21). In Fig. 6(d), the DSC profile of SLNs loaded with OMC (OMC-SLNs) was shown as an example of loaded SLNs. The OMC-SLNs showed a thermal behaviour very similar to that of the unloaded SLNs. In the heating process, OMC-SLNs showed a significant peak at 21.08°C, which lowers the skin temperature indicating that OMC can control
a
c
b
d
Figure 6 The differential scanning calorimetry thermograms of (a) the emulsifier of S21 (b) the solid lipid of HR (c) the unloaded solid lipid nanoparticles (d) OMC-SLNs.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 446–453
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Octylmethoxycinnamate, solid lipid nanoparticles
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OMC-SLN Emulsion
Absorbance
1.4 1.2 1 0.8 0.6
0
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Time (h) Figure 7 The comparison of degradation of octylmethoxycinnamate from solid lipid nanoparticles and conventional emulsion.
release from the solid lipid and play the role of sunblock. The presence of OMC induced a broadening of emulsifier and lipid fusion peaks. The CI% of lipid decreased from 44.85% to 15.35%. It proved that the incorporation of OMC inside the solid lipid matrix had led to a decrease of the crystallinity and the binding forces between the molecules of the lipid matrix. These results contributed to the improvement of the drug loading, EE and stability of the system. Comparison of degradation of OMC from SLNs and conventional emulsion In this primary study, the degradations of OMC from SLNs and conventional emulsion were investigated over 8 h. Each sample was diluted to 0.025 mg mL1. The obtained samples were exposed to the sun. The samples (~20 g) were obtained every hour, and the absorbance was measured in the UV range. The detection wavelength was set at 311 nm. As shown in Fig. 7, OMC was degraded to a greater extent from the conventional emulsion. The degradation rate of OMC decreased in SLN formulations compared
with that in conventional emulsion, which allowed a reduction in the OMC concentration; this effect was desired. The solid core of the lipid nanoparticles provided a better physical and chemical stability than a liquid or liquid crystalline core of conventional emulsions. The results explained that the solid lipid particles as carriers enhanced the stability of OMC, which may be due to the fact that the solid lipid particles prevented direct sunshine from UVA or visible light, thereby resulting in the instability of UVB sunscreens. This finding was confirmed by Song and Lui [31], who compared the UV absorption properties of 3,4,5-trimethoxybenzochitin-loaded SLNs and free SLNs. Compared with a conventional emulsion, the amount of molecular sunscreen may be reduced by 50% in the SLN formulations while maintaining the protective level of conventional emulsion [32]. Conclusions The main goal of this work was to prepare solid lipid particles loaded with OMC by ultrasonic emulsification method. The optimal formulation was determined as 5% (w/w) of solid lipid, 7% (w/w) of emulsifier and 9% of loaded OMC, resulting in a particle size of 392.8 nm, and EE of 88.73%, LD of 38.05% under the preparation condition of 6 min of sonication, 400 W of sonication power. Environment emission scanning electron microscopy studies revealed that OMC-loaded SLNs were almost spherical in shape. The DSC and FT-IR showed that amount of OMC was loaded in the solid lipid particles. The comparison of degradation rate of OMC from SLNs and conventional emulsion explained that the solid lipid particles were an effective carrier to the sunscreen of OMC. Acknowledgement The authors would like to thank Mrs. Hu Jing for the DSC measurements and Mrs. Hu shaying for the ESEM measurement.
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Supporting Information Additional Supporting Information may be found in the online version of this article:
Figure S1. In vitro penetration profile of OMC from conventional emulsion.
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Data S1. The SPF of conventional emulsion and OMC-SLNs.
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