Journal of Pharmaceutical Investigation (2012) 42:279–283 DOI 10.1007/s40005-012-0038-z
RESEARCH ARTICLE
Effect of lipid on physicochemical properties of solid lipid nanoparticle of paclitaxel Jong-Suep Baek • Sang-Chul Shin • Cheong-Weon Cho
Received: 25 July 2012 / Accepted: 29 August 2012 / Published online: 14 September 2012 Ó The Korean Society of Pharmaceutical Sciences and Technology 2012
Abstract The aim of this study was to compare physicochemical properties of solid lipid nanoparticles (SLN) made from different lipids. To make small, stable, uniform and highly encapsulated SLNs, many factors such as the components (lipid, stabilizer) and preparation condition (sonication time, power) can be considered. Out of those, we selected solid lipid as lipid matrix to investigate an effect on SLNs. The SLNs were characterized by particle size, zeta potential, solubility and in vitro release study. In this study, SLNs showed different physicochemical properties and release profiles according to used solid lipid. In case of particle size, M-SLN showed biggest particle size (412.5 ± 29.4 nm) and highest encapsulation efficiency (61.2 ± 4.8 %). And, B-SLN showed highest cumulative drug percentage (85.0 ± 1.7 %, 24 h) in release study. These results suggest that lipids type affect physicochemical properties and release profile of SLN. Keywords Solid lipid nanoparticle Paclitaxel Behenic acid Stearic acid Palmitic acid Myristic acid Introduction Solid lipid nanoparticles (SLNs) made from biodegradable solid lipids exist in the submicron size range have attracted J.-S. Baek C.-W. Cho (&) College of Pharmacy and Institute of Drug Research & Development, Chungnam National University, 220 Gungdong, Yuseonggu, Daejeon 305-764, South Korea e-mail:
[email protected] S.-C. Shin College of Pharmacy, Chonnam National University, Gwangju 500-757, South Korea
increasing attention in recent years. The advantages of SLN are as follows: possibility of controlled drug release and drug targeting, protection of incorporated compound against chemical degradation, no biotoxicity of the carrier, and no problems with respect to large scale production (Marengo et al. 2000; Mahnert and Mader 2001). In this study, paclitaxel (PTX) was chosen as model drug. PTX has been recognized as an effective chemotherapeutic agent for a wide variety of solid tumors. Oral administration of PTX in the treatment of cancer is limited by its poor bioavailability (Shenoy et al. 2009). To prepare PTX loaded-SLNs, many preparation factors such as solid lipid, stabilizer and physical mixer conditions were carefully considered because they have effect on physicochemical properties and release profile (Muller et al. 2000). SLN made from many types solid lipid such as triglyceride (Joseph et al. 2012; Noack et al. 2012; Padois et al. 2011), fatty acid (Carbone et al. 2012; Ghadiri et al. 2012; Spada et al. 2012) and wax (Kheradmandnia et al. 2010; Kumar et al. 2007; Nesseem 2011). Therefore, we assessed the difference of physicochemical properties and release profile of SLNs with different solid lipid matrix. Particle size, polydispersity index, zeta potential, solubility test and in vitro release study were performed using different SLNs in this work.
Materials and methods Materials Paclitaxel (PTX) was a gift sample received from SamyangGenex (Daejeon, Korea). Behenic acid, myristic acid, palmitic acid and stearic acid were purchased from Daejung Chemical (Cheongwon, Korea). Poloxamer 188 was
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obtained from BASF (Ludwigshafen, Germany). Lecithin was purchased from Junsei Chemical Co. (Tokyo, Japan). Mannitol was purchased from Sigma (Steinheim, Switzerland). All other chemicals and solvents were analytical reagent grade and used without further purification. Preparation of SLNs The SLNs were manufactured using a modified hot sonication method (Fig. 1). In case of Behenic acid-SLN (B-SLN), 100 mg of behenic acid was melted at 90 °C in water bath. Five milligrams of PTX was dissolved in 0.25 ml of ethanol and then injected into molten stearic acid under sonication. Lecithin (75 mg) and poloxamer 188 (75 mg) were dispensed into 3 ml of distilled water, sonicated for 10 min at 90 °C in a water bat 3 h using a probe type and then added molten stearic acid and PTX solution under sonication for 10 min at 90 °C (Table 1). Finally, the mixture was injected into 10 % mannitol solution at 4 °C. The final samples were freeze-dried until further use. Myristic acid-SLN (M-SLN), palmitic acid (P-SLN) and stearic acid (S-SLN) were prepared by using the same method described above by adding 100 mg of myristic acid or 100 mg of palmitic acid or 100 mg of stearic acid instead of behenic acid, respectively (Table 1). Analysis of drug loading and encapsulation efficiency of SLNs The SLNs (100 mg) were solubilized with 10 ml of ethanol, heated at 80 °C for 30 min and then cooled down at -20 °C for 30 min. This solution was centrifuged at 3,000 rpm for 5 min to precipitate the undissolved solid stearic acid, filtered through a 0.2 lm filter and injected into the HPLC
J.-S. Baek et al. Table 1 Composition of various SLNs Unit (mg)
B-SLN
S-SLN
P-SLN
M-SLN
PTX
5
5
5
5
Behenic acid
100
–
–
–
Stearic acid
–
100
–
–
Palmitic acid
–
–
100
–
Myristic acid
–
–
–
100
Lecithin
75
75
75
75
Poloxamer 188
75
75
75
75
Mannitol
25
25
25
25
Total
280
280
280
280
system. An Agient 1100 liquid chromatography system with an autosampler and UV detector were used. The column used was a C18 column (4.0 9 250 mm, 5 lm particle size, SupelcoTM; MetaChem, USA). The flow rate of the mobile phase was 1 ml/min and the detection wavelength was set to 227 nm. The mobile phase was a mixture of water and acetonitrile (60:40 v/v). All procedures were carried out at ambient temperature. Drug loading and encapsulation efficiency (E.E.) were calculated as follows. Drug loading (%) = weight of the drug in particles/ weight of the particles 9 100. E.E. (%) = weight of the drug in particles/weight of the feeding drugs 9 100. Measurements of particle size and polydispersity The particle size and zeta potential analysis of four different SLNs were performed by laser scattering analyzer (ELS-8000, Otasuka Electronics, Osaka, Japan). The lyophilized SLN was dispersed in water, added to the sample dispersion unit and sonificated in order to minimize the inter-particle interactions. The obscuration range was maintained between 20 and 50 %. The instrument was set to measure the sample 50 times and the average volume mean diameter was obtained. Characterization of SLNs using FT-IR The SLNs were characterized using FT-IR (NICOLET 380 FT-IR, Thermo, USA) in order to assess the changes of solid state of the samples. Solubility
Fig. 1 Schematic diagram of the preparation method for SLNs
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Accurately weighed SLNs were added to microcentrifuge tube containing 1 ml of distilled water. The samples were put on an end-to-end lab quake rotator (Barnstead
Lipid effect on physicochemical properties of SLN
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Thermolyne, Sparks, NV, USA) at 8 rpm at ambient temperature for 72 h in order to achieve equilibrium and were then stored at room temperature to investigate the change in solubility according to elapsed time. The samples were filtered with a 0.45 lm membrane filter (Dismic-25; Whatman Ltd., Japan) and the absorbance of the filtrate was measured using HPLC. All solubility determinations were performed in triplicate. In vitro release study In vitro release of SLNs was evaluated using a dialysis bag (molecular weight cut-off of 7000 (Membra-Cel; Viskase, Inc., Chicago, IL, USA)), which was filled with an amount according to 20 lg of PTX and immersed in 30 ml of distilled water including 1 % of sodium lauryl sulfate (SLS). Aliquots of 1 ml samples were withdrawn from the medium and replaced with the same volume of fresh dissolution medium at an indicated time. The withdrawn samples were estimated using the HPLC system.
Results and discussion Particle size, polydispersity and zeta potential of SLNs We compared the physicochemical properties of four different SLNs according to solid lipid by measuring the particle size, polydispersity index and zeta potential (Fig. 2). Figure 2a shows that particle size of various SLNs. This result showed that the size and polydispersity of the SLNs tended to increase with the increase of carbon chain length of fatty acid of lipid. B-SLN showed the biggest particle size of 412.5 ± 29.4 nm, while as M-SLN, P-SLN and S-SLN exhibited 331.5 ± 24.2, 354.2 ± 15.4 and 383.2 ± 20.3 nm, respectively. Behenic acid, stearic acid, palmitic acid and myristic acid have 22, 16, 18, 14 carbon chain length of fatty acid, respectively. And, Fig. 2b showed polydispersity index of four SLNs. Figure 2c showed zeta potential of SLNs. All of zeta potential of SLNs showed about negative 30 mV. Measuring the zeta potential allows predictions of the storage stability (Muller et al. 2000). High zeta potential above 30 mV (in absolute value) is known to prevent nanoparticles from aggregation, ensuring stability of lipid nanoparticles (Muller and Heinemann 1992). The lipid did not affect the zeta potential of SLNs (Fig. 3). Encapsulation efficiency (E.E.) and drug loading of SLNs Table 2 shows E.E. (%) and drug loading of SLNs. The E.E. (%) of B-SLN, P-SLN, S-SLN and M-SLN was found
Fig. 2 Particle size (a), polydispersity index (b) and zeta potential (c) of SLNs. Data are expressed as the mean ± S.D. (n = 3). *P \ 0.05
to be 61.2 ± 4.8, 48.0 ± 3.3, 53.6 ± 5.2 and 56.2 ± 5.9 %, respectively. The increasing order of E.E. (%) was B-SLN, S-SLN, P-SLN and M-SLN. And, the drug loading of B-SLN, P-SLN, S-SLN and M-SLN was found to be 1.2 ± 0.2, 0.9 ± 0.2, 1.1 ± 0.2 and 1.1 ± 0.2 %, respectively. The E.E. (%) and drug loading of SLNs were enhanced with increasing the carbon chain length of fatty acid, because the higher hydrophobicity of the longer chain fatty acids resulted in increased accommodation of lipophilic drugs (Anderson and Ormi 2004).
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J.-S. Baek et al.
Fig. 4 Solubility of SLNs at 1, 3 and 7 elapsed days after saturation. Data are expressed as the mean ± S.D. (n = 3). *P \ 0.05 Fig. 3 FT-IR spectra of SLNs
Table 2 Drug loading and E.E. (%) of SLNs Unit (%) Drug loading E.E.
B-SLN
S-SLN
P-SLN
M-SLN
1.2 ± 0.2
1.1 ± 0.2
1.1 ± 0.2
0.9 ± 0.2
61.2 ± 4.8
56.2 ± 5.9
53.6 ± 5.2
48.0 ± 3.3
Data are expressed as the mean ± S.D. (n = 3)
Characterization of SLNs using FT-IR The major peaks of PTX were around 1,250 cm-1 (c–o–c stretching) (Fig. 3). The major peaks of PTX were disappeared in the FT-IR spectra for SLNs, indicated that PTX was incorporated in lipid matrix (Lee et al. 2007).
Fig. 5 In vitro release profiles of PTX from B-SLN, M-SLN, P-SLN and S-SLN in 1 % SLS solution. Data are expressed as the mean ± S.D. (n = 3)
Solubility Figure 4 shows the solubility kinetics according to elapsed day after saturation. The PTX solubility of B-SLN, P-SLN, S-SLN and M-SLN was found to be 3.6 ± 0.3, 3.1 ± 0.2, 3.0 ± 0.5 and 2.8 ± 0.3 lg/mL, respectively. The PTX solubility was enhanced as the particle size of SLNs was decreased because the smaller particle has the higher surface area (Sievens-Figueroa et al. 2012). According to the elapsed day after saturation, the solubility of PTX was decreased. This decrease was due to drug expulsion of drug from lipid matrix (Mukherjee et al. 2009). In vitro release study The dialysis membrane method was chosen to investigate PTX release from different SLNs in 1 % SLS solution. Since 1 % SLS solution containing SLNs was constantly agitated during the dissolution test, collisions among SLNs could first disintegrate the structure of the surfactant layer. The subsequent fluidic shear devastated the particulate
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surfaces and caused the release of a drug (Hurst et al. 2006). The cumulative % release of PTX from B-SLN, S-SLN, P-SLN and M-SLN was found to be 42.4 ± 1.2, 48.9 ± 0.7, 50.9 ± 0.7 and 52.8 ± 1.2 % in 2 h, respectively. And, the cumulative % release of PTX from B-SLN, S-SLN, P-SLN and M-SLN was found to be 77.1 ± 3.1, 79.0 ± 1.5, 83.3 ± 0.6 and 85.0 ± 1.7 %, respectively, in 24 h. The drug release profiles of SLNs showed controlled release of PTX from SLNs (Fig. 5). As the time increased, the rate of release of PTX decreased. This indicated the controlled release of drugs from the SLNs (Song et al. 2008). The prolonged drug release was observed in B-SLN compared to other SLNs. It was reported the release of a drug from the SLN can be influenced by the nature of the lipid matrix, surfactant concentration and production parameters (Wissing et al. 2004) as well as lipid nature, solubility of the drug in lipid and partition coefficient (Hu et al. 2005; Kumar et al. 2007). This clearly confirmed that higher solubility of drug in lipid matrix is sufficient to prolong the drug release for longer period of time.
Lipid effect on physicochemical properties of SLN
Conclusion This study was to examine the effect of solid lipid on the physicochemical properties and release profile of SLNs. PTX-loaded SLNs were prepared using different solid lipids by hot-melted sonication method. We found that the different solid lipid affected the physicochemical properties and release profiles of SLNs. Particle size and E.E. (%) were increased with increasing carbon chain length of fatty acid. In release study, B-SLN (C-22) showed sustained release among SLNs. So, a selection of optimal solid lipid will be major factor to design SLN formulations. Acknowledgments This work was supported by the Basic Science Research Program (2012R1A1B5003358) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology.
References Anderson M, Ormi A (2004) The effect of different lipid components on the in vitro stability and release kinetics of liposome formulations. Drug Deliv 11(1):33–39 Carbone C, Tomasello B, Ruozi B, Renis M, Pulglisi G (2012) Preparation and optimization of PIT solid lipid nanoparticles via statistical factorial design. Eur J Med Chem 49:110–117 Ghadiri M, Fatemi S, Vatanara A, Doroud D, Najafabadi AR, Darabi M, Rahimi AA (2012) Loading hydrophilic drug in solid lipid media as nanoparticles : statistical modeling of entrapment efficiency and particle size. Int J Pharm 424(1–2):128–137 HuFQ JiangSP, Du YZ (2005) Preparation and characterization of stearic acid nanostructured lipid carriers by solvent diffusion method in an aqueous system. Colloids Surf B Biointerfaces 45:167–173 Hurst SJ, Lytton-Jean AK, Mirkin CA (2006) Maximizing DNA loading on a range of gold nanoparticle sizes. Anal Chem 78(24):13–18 Joseph S, Bunjes H (2012) Preparation of nanoemulsions an solid lipid nanoparticles by premix membrane emulsification. J Pharm Sci 101(7):2479–2489 Kheradmandnia S, Vasheghani-Farahani E, Nosrati M, Atyabi F (2010) Preparation and characterization of ketoprofen-loaded solid lipid nanoparticles made from beeswax and carnauba wax. Nanomedicine 6(6):753–759 Kumar VV, Chandrasekar D, Ramakrishna S, Kishan V, Rao YM, Diwan PV (2007) Development and evaluation of nitrendipine
283 loaded solid lipid nanoparticles: influence of wax and glyceride lipids on plasma pharmacokinetics. Int J Pharm 335(1–2): 167–175 Lee MK, Lim SJ, Kim CK (2007) Preparation, characterization and in vitro cytotoxicity of paclitaxel loaded sterically stabilized solid lipid nanoparticles. Biomaterials 28:2137–2146 Marengo E, Cavalli R, Caputo O, Rodriguez L, Gasco M (2000) Scale-up of preparation process of solid lipid nanospheres. Part I. Int J Pharm 205(1–2):3–13 Mahnert W, Mader K (2001) Solid lipid nanoparticles: production, characterization and applications. Adv Drug Deliv Rev 47(2–3): 165–196 Mukherjee S, Ray S, Thakur RS (2009) Solid lipid nanoparticles: a modern formulation approach in drug delivery system. Indian J Pharm Sci 71(4):349–358 Muller RH, Heinemann S (1992) Fat emulsions for parenteral nutrition. I. Evaluation of microscopic and laser light scattering methods for the determination of the physical stability. Clin Nutr 11:223–272 Muller RH, Mader K, Gohla S (2000) Solid lipid nanoparticles (SLN) for controlled drug delivery—a review of the state of the art. Eur J Pharm Biopharm 50(1):161–177 Nesseem D (2011) Formulation of sunscreens with enhancement sun protection factor response based on solid lipid nanoparticles. Int J Cosmet Sci 33(1):70–79 Noack A, Hause G, Mader K (2012) Physicochemical characterization of curcuminoid-loaded solid lipid nanoparticles. Int J Pharm 423(2):440–451 Padois K, Cantieni C, Bertholle V, Bardel C, Pirot F, Falson F (2011) Solid lipid nanoparticles suspension versus commercial solutions for dermal delivery of minoxidil. Int J Pharm 416(1):300–304 Shenoy VS, Gude RP, Murthy RS (2009) Paclitaxel-loaded glyceryl palmitostearate nanoparticles: in vitro release and cytotoxici activity. J Drug Target 17(4):304–310 Sievens-Figueroa L, Bhakay A, Jerez-Rozo JI, Pandya N, Romanach RJ, Michniak-Kohn B, Iqbal Z, Bilgili E, Dave RN (2012) Preparation and characterization of hydroxypropyl methyl cellulose films containing stable BCS Class II drug nanoparticles for pharmaceutical applications. Int J Pharm 423(2):496–508 Song X, Zhao Y, Hou S, Xu F, Zhao R, He J, Cai Z, Li Y, Chen Q (2008) Dual agents loaded PLGA nanoparticles: systematic study of particle size and drug entrapment efficiency. Eur J Pharm Biopharm 69(2):445–453 Spada G, Gavini E, Cossu M, Rassu G, Giunchedi P (2012) Solid lipid nanoparticles with and without hydroxypropyl-b-cyclodextrin: a comparative study of nanoparticles designed for colonic drug delivery. Nanotechnology 23(9):095101 Wissing SA, Kayser O, Muller RH (2004) Solid lipid nanoparticles for parenteral drug delivery. Adv Drug Deliv Rev 56:1257–1272
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