Statistical analysis of solid lipid nanoparticles produced by high-pressure homogenization: a practical prediction approach

Title: Statistical Analysis of Solid Lipid Nanoparticles Produced by High-Pressure Homogenization: A Practical Prediction Approach Authors: Matilde Durán-Lobato, Alicia Enguix-González, Mercedes Fernández-Arévalo, Lucía Martín-Banderas Note: This is the accepted manuscript of the article published in Journal of Nanoparticle Research. This version has been peer-reviewed but has not undergone final editing, formatting, or typesetting by Springer. For the final published version, please refer to: Durán-Lobato, M., Enguix-González, A., Fernández-Arévalo, M., & Martín-Banderas, L. (2013). Statistical Analysis of Solid Lipid Nanoparticles Produced by High-Pressure Homogenization: A Practical Prediction Approach. Journal of Nanoparticle Research, 15(2), Article 1443. https://doi.org/10.1007/s11051-013-1443-6 1 Statistical analysis of solid lipid nanoparticles produced by high-pressure homogenization: a 1 practical prediction approach 2 Matilde Durán-Lobato1*, Alicia Enguix-González2, Mercedes Fernández-Arévalo1, Lucía Martín- 3 Banderas1 4 1Dpto. Farmacia y Tecnología Farmacéutica. Facultad de Farmacia. Universidad de Sevilla. C/Prof. 5 García González nº2. 41012 Sevilla (España). 6 2Dpto. Estadística e Investigación Operativa. Facultad de Matemáticas. Universidad de Sevilla. C/Tarfia 7 s/n. 41012 Sevilla (España). 8 9 *Corresponding author address: Dpto. Farmacia y Tecnología Farmacéutica. Facultad de Farmacia. 10 Universidad de Sevilla. c/Prof. García González nº2. 41012 Sevilla (España); Tel: +34 954556618; Fax: 11 +34 954556085; Email: [email protected] 12 13 14 15 16 17 18 19 20 21 22 23 24 Manuscript Click here to download Manuscript: Manuscript.docx Click here to view linked References 2 Abstract 25 Lipid Nanoparticles (LNP) are a promising carrier for all administration routes due to its safety, small size 26 and high loading of lipophilic compounds. Among the LNP production techniques, the easy scale up, lack 27 of organic solvents and short production times of the high-pressure homogenization technique (HPH) 28 make this method stand out. In this study, a statistical analysis was applied to the production of LNP by 29 HPH. Spherical LNP with mean size ranging from 65 nm to 11.623 m, negative zeta potential (ZP) 30 under -30 mV and smooth surface were produced. Manageable equations based on commonly used 31 parameters in the pharmaceutical field were obtained. The lipid to emulsifier ratio (RL/S) was proved to 32 statistically explain the influence of oil phase and surfactant concentration on final nanoparticles size. 33 Besides, the homogenization pressure was found to ultimately determine LNP size for a given RL/S, while 34 the number of mainly determined polydispersion passes applied. α-Tocopherol was used as a model drug 35 to illustrate release properties of LNP as a function of particle size, what was optimized by the regression 36 models. This study is intended as a first step to optimize production conditions prior to LNP production at 37 both laboratory and industrial scale from an eminently practical approach, based on parameters 38 extensively used in formulation. 39 Keywords 40 Solid lipid nanoparticles, high-pressure homogenization, statistical analysis, regression model, particle 41 size prediction, mathematical model, drug release. 42 Introduction 43 Solid lipid nanoparticles (LNP) consist of carrier systems made from lipids, in which drug compounds 44 can be incorporated. Their mean particle size is in the submicron range, ranging from about 40 to 1000 45 (nm). Particle matrix is made of a solid lipid or a blend of solid lipids, aiming for accurate encapsulation 46 and delivery of compounds (Pardeike et al. 2009). These carrier systems adopted some of the best 47 features of other colloidal carriers such as polymeric nanoparticles and liposomes and can be made of 48 physiological lipids (biocompatible and biodegradable), what supports its safety (Müller et al. 2000b). 49 Their small particle size allows them to be used for all routes of administration (Souto and Müller 2006; 50 Bondì et al. 2010; Müller et al. 1997). In addition, they have shown high encapsulation rates for lipophilic 51 compounds (Das and Chaudhury 2011). 52 3 Several different methods for the production of LNP have been described in the literature. These methods 53 are high-pressure homogenization technique (Liedtke et al. 2000; Mehnert and Mäder 2001), 54 microemulsion technique (Gasco 1997; Priano et al. 2007), emulsification-solvent evaporation method 55 (Sjöström and Bergenstahl 1992), emulsification-solvent diffusion method (Hu et al. 2002; Trotta et al. 56 2003), solvent injection (or solvent displacement) method (Schubert and Müller-Goymann 2003), phase 57 inversion method (Heurtault et al. 2002), multiple emulsion technique (García-Fuentes et al. 2002), 58 ultrasonication technique (Puglia et al. 2008), membrane contactor technique (Charcosset et al. 2005; El- 59 Harati et al. 2006), supercritical fluid technique (supercritical fluid extraction of emulsions (SFEE) 60 (Chattopadhyay et al. 2007) and gas-assisted melting atomization (GAMA) (Salmaso et al. 2009)) and 61 spray drying technique (Sebti and Amighi 2006). 62 However, the lack of a large-scale production method yielding a product of a quality that is acceptable by 63 the regulatory authorities (e.g., Food and Drug Administration) generally hinders the introduction of solid 64 nanoparticles to the market. This lack is due to basic technological problems (e.g. basic scale-up problem, 65 toxicologically problematic residues from the production process) and regulatory aspects such as 66 suitability of the production unit and production process to be qualified and validated (Müller et al. 67 2000a). Considering this, the lack of organic solvents, short production times and easy scale-up provided 68 by the high-pressure homogenization technique (HPH) make this method highly suitable (Liedtke et al. 69 2000; Mehnert and Mäder 2001; Muchow et al. 2008). Either the hot or cold high-pressure 70 homogenization technique can be applied to obtain lipid nanoparticles, enabling to adapt the production 71 according to the physicochemical properties of compounds and the behavior expected from the particles 72 (zur Mühlen et al. 1998; zur Mühlen and Mehnert 1998). Furthermore, high-pressure homogenizers are 73 widely used in many industries including the pharmaceutical industry, e.g. for the production of 74 emulsions for parenteral nutrition. Hence, no regulatory problems exist for the production of LNP using 75 this production technique, which can be considered as being industrially the most feasible one (Müller et 76 al. 2000a). 77 LNP production methods including HPH present however certain hurdles that affect the product quality, 78 i.e. drug degradation induced during the manufacturing process, lipid crystallization, gelation phenomena, 79 supercooled melts, lipid and particle shape modifications and the co-existence of several colloidal species 80 (Mishra et al. 2012; Sinha et al. 2010). Nevertheless, these limitations can be overcome by monitoring the 81 4 production conditions (temperature range, shear stress, light) and improving the selection of the drug 82 carrier, the formulation and the drug loading technique (Mishra et al. 2012; Sinha et al. 2010). Therefore, 83 whatever production technique is used, identifying and optimizing the parameters influencing the final 84 product is of paramount importance, since these determine the drug delivery system properties. 85 Nowadays, the use of experimental designs has become a common method to simultaneously analyze the 86 influence of different variables on the production of particles (Araujo et al. 2010; Varshosaz et al. 2010), 87 especially regarding size (Vitorino et al. 2011). In the case of HPH, many thorough studies focused on 88 emulsions and the homogenization process itself have been carried out (Mohr 1987a; Mohr 1987b; Floury 89 et al. 2000; Qian and McClements 2010; Maindarkar et al. 2012) but fewer attended to LNP (Severino et 90 al. 2012). 91 In this study, a statistical analysis is applied to the production of LNP by HPH technique from a 92 completely practical approach. A wide range of pressure values, excipient concentrations and number of 93 passes is analyzed. Commonly employed parameters are used to describe the process, leading to 94 manageable equations predicting particle size. The influence of particle size on the final properties of the 95 delivery system is further illustrated using tocopherol as a model drug. 96 Experimental 97 Materials 98 Glycerol monostearate (Monostearin, melting point 63 – 68 ºC, Acofarma, Spain) was used as lipid base. 99 Sorbitan monostearate (Span® 60), polysorbate 80 (Tween® 80) and α-Tocopherol were supplied by 100 Sigma-Aldrich (Spain). Dihydrogen sodium phosphate and phosphoric acid were provided by Panreac 101 (Spain). Acetonitrile (HPLC gradient) was supplied by VWR (Spain). Distilled water was used for all the 102 formulations. 103 LNP preparation 104 LNP were prepared by HPH as described elsewhere (Zur Mühlen et al. 1998). Oil-in-water emulsions 105 were prepared by dispersing 0.5; 2.5; 5.0; 7.5; 10; 15 % w/v of lipid in distilled water with emulsifier at a 106 concentration of 0.12; 0.25; 0.5; 1.0; 1.25; 1.5 % (w/v) for each lipid concentration assayed, yielding a 107 total number of 62 formulations. A dispersing step prior to homogenization was performed with an Ultra- 108 5 Turrax (IKA®-WERKE, Germany) at 75ºC. The systems obtained were passed through a homogenizer 109 (Panda 2K, Gea Niro Soavi, Italy) for different number of passes (1-8) at various homogenization 110 pressures (250-1500 bar). On account of technical recommendation (Gea Niro Soavi) and in order to 111 prevent drop recoalescence, pressure at the second valve was fixed at a relation 1:5 and 1:10 referred to 112 total pressure for values lower and higher than 600 bar of final pressure respectively. Samples 113 temperature variations due to pressure were monitored. In the case of α-tocopherol-loaded LNP, the 114 compound was solubilized in the oil phase of initial emulsions at a concentration of 10 % (w/w) referred 115 to the lipid matrix. 116 LNP characterization 117 Mean particle size and particle size distribution were measured by a laser scattering technique based on 118 Mie theory (LA-950V2 Horiba, Japan) at 25 ± 0.5 ºC. Measurements were carried out under continuous 119 magnetic agitation. Measure range was fixed between 0.01 and 3000 µm. Samples were measured 120 directly or after dilution with distilled water when necessary. 121 LNP surface charge was determined by zeta potential (ZP) measurements. Particles ZP was determined 122 by laser Doppler (Zetamaster 300, Malvern Instruments Ltd, Malvern, UK) at 25 ± 0.5 ºC. ZP 123 measurements were carried out in triplicate. Samples were measured directly or after dilution with 124 distilled water when necessary. 125 LNP morphology and surface were characterized by image analysis obtained by scanning electron 126 microscopy (SEM). For this purpose, a drop of LNP dispersion was spread on carbon tab previously stuck 127 to aluminum stubs and dried overnight. Samples were coated with gold using a sputter coater 128 (EDWARDS Scancoat Six) and examined in a Jeol 6460LV. Besides, particles were also observed by 129 transmission electron microscopy (TEM). In this case, a drop of LNP dispersion was spread on a carbon- 130 coated 200-mesh copper grid and dried overnight. Then, a drop of 2 % (w/v) uranyl acetate in ethanol was 131 placed onto the grid for 10 min, followed by a drop of 2 % (w/v) lead citrate for 15 min. The grid was 132 dried at room temperature and later observed in a Philips CM-10 (Philips, Germany). 133 Statistical analysis 134 6 All measurements were performed by triplicate on freshly samples prepared by triplicate as well. An 135 initial statistical evaluation of data was carried out by one-way analysis of variance (ANOVA). Statistical 136 analysis was considered significant if the p values were lower than 0.05. Further statistical analysis was 137 performed using PASW Statistics 18 (SPSS Inc., 2010). 138 Entrapment efficiency and loading capacity 139 Considering the entrapment efficiency (EE) the amount of drug that can be incorporated into the particles 140 and the loading capacity (LC) the amount of drug incorporated per mg of lipid, both parameters were 141 calculated according to the following equations: 142 The amount of drug contained in the samples was measured by reverse phase high performance liquid 143 chromatography (HPLC) with spectrophotometric detection according to a previously published method 144 (Trombino et al. 2009) with some modification. Briefly, 4-5 mg of LNP were accurately weighed and 145 dissolved in 100 µL DCM. After 10 min of sonication, 900 µL of mobile phase were added and samples 146 were sonicated again for 5 minutes. Then, samples were filtered by 420 nm filter and injected into HPLC 147 system. A 1-cm cartridge precolumn with 5-µm C18 Adsorbosphere packing was used. Mobile phase 148 consisted of 0.01 M dihydrogen sodium phosphate/0.01 M phosphoric acid with acetonitrile (88:12, v/v) 149 pH 2.3, at a flow rate of 0.5 mL/min. Wavelength was fixed at 280 nm on a Jasco UV-2075 detector. 150 Drug release from LNP 151 In order to study the drug release from the particles, LNP samples were suspended in phosphate buffer 152 (pH 7.4) containing Tween® 80 at a concentration of 0.1 % (w/v), maintained at 37 ºC and stirred 153 mechanically (100 rpm) during the release experiments (Unitronic OR, Selecta, Spain). Aliquots (500 µL) 154 were withdrawn at fixed time intervals and filtered upon centrifugation at 8000 rpm. Filtered samples 155 (Millex GV) (10 µL) were injected into the HPLC equipment to quantify the amount of α-tocopherol. 156 Results and discussion 157 7 LNP characterization 158 The influence of lipid concentration, surfactant concentration, applied pressure and number of passes on 159 mean particle size and size distribution was studied. Mean particle size ranged from 65 nm to 11.623 µm. 160 The applied pressure showed to be a high influencing parameter on particle size and size distribution 161 (Table 1), leading to a decrease in LNP size as the pressure was increased. The number of passes applied 162 induced a strong decrease in particle size and size distribution as well, which changed from bimodal to 163 monomodal as the number of cycles was increased (Fig. 1). Temperature was monitored for each pass at 164 every value of pressure (Table 1), remaining constant for each value of pressure regardless of the number 165 of passes applied and formulation composition. An increase in surfactant concentration led to a decrease 166 in LNP size, while increasing both surfactant and lipid concentration in a constant relationship did not 167 induce significant changes on particle size (Fig. 2). Since the influence of these parameters was 168 statistically analyzed and mathematically described, these results will be further commented in the section 169 of statistical analysis. Incorporation of tocopherol did not influence particle size distribution (data not 170 shown). 171 172 Insert Table 1 around here 173 Table 1 Values of applied pressure and corresponding formulation temperature and mean particle size obtained by 174 laser scattering 175 176 Insert Fig. 1 around here 177 Fig. 1 LNP size distributions of formulations produced at 1500 bar of pressure with 0.5 % (w/v) of Monostearin and 178 0.5 % (w/v) of Span® 60 after: - one homogenization cycle; - two homogenization cycles; - three homogenization 179 cycles; - four homogenization cycles; - five homogenization cycles; - six homogenization cycles 180 181 Insert Fig. 2 around here 182 Fig. 2 LNP size distribution of formulations produced with: - 0.50 % (w/v) of Monostearin and 0.25 % (w/v) of 183 Span® 60; - 2.50 % (w/v) of Monostearin and 1.25 % (w/v) of Span® 60; - 10.00 % (w/v) of Monostearin and 5.00 184 % (w/v) of Span® 60 185 8 ZP of all samples was under – 30 mV, thus indicating the suspensions were stable a priori. 186 SEM imaging showed spherical particles with a smooth surface (Fig. 3). However, only large-sized 187 particles could be observed by SEM, since the voltage needed to capture the smallest particles was 188 extremely high and made them melt. Thus, TEM was applied to confirm the size of the smallest particles. 189 Transmission images enabled to observe particles smaller than 500 nm (Fig. 4), confirming therefore the 190 experimental results. No morphological differences were found between empty and tocopherol-loaded 191 LNP. 192 Insert Fig. 3 around here 193 Fig. 3 SEM images of LNP formulations. From left to right, top to bottom: particles produced with 10.00 % (w/v) of 194 lipid and 1.25 % (w/v) of emulsifier at 800 bar and 5 cycles; particles produced with 2.50 % (w/v) of lipid and 0.25 % 195 (w/v) of emulsifier at 800 bar and 5 cycles; particles produced with 2.50 % (w/v) of lipid and 0.12 % (w/v) of 196 emulsifier at 1000 bar and 5 cycles; particles produced with 0.25 % of α-tocopherol, 2.50 % (w/v) of lipid and 0.25 % 197 (w/v) of emulsifier at 800 bar and 6 cycles 198 Insert Fig. 4 around here 199 Fig. 4 TEM images of LNP formulations. From top to bottom: particles produced with 2.50 % (w/v) of lipid and 1.25 200 % (w/v) of emulsifier at 1500 bar and 5 cycles; particles produced with 0.50 % (w/v) of lipid and 0.5 % (w/v) of 201 emulsifier at 1500 bar and 8 cycles 202 Statistical analysis 203 Statistical analysis of data was structured in separate stages. First, functional values of production 204 conditions were tested to determine the operating range where the equipment performance, and hence the 205 obtained data, were reliable. Maximal pressure value was fixed at 1500 bar on account of technical 206 recommendation (Gea Niro Soavi). Lipid concentrations were kept under 15 % (w/v), since higher values 207 yielded low-fluid emulsions that could block homogenizer channels. Finally, maximal surfactant 208 concentration was fixed at 1.5 % (w/v) to prevent the formation of bubbles inside the equipment. 209 Following, and since there were no major differences between intermediate values, six values from each 210 of the above variables were selected; 300; 500; 800; 1000; 1300; 1500 bar of pressure; 0.5; 2.5; 5.0; 7.5; 211 10; 15 % (w/v) lipid concentration; 0.12; 0.25; 0.5; 1.0; 1.25; 1.5 % (w/v) emulsifier concentration. A 212 design similar to Latin square was applied to ensure representativeness in the combination of values from 213 production conditions, with 288 measures from 36 batches and 8 passes, which provided a total number 214 of 458 valid measures of particle size. 215 15 Pressures of 500 bar and 1 to 3 passes are typically enough to obtain small particle sizes (Severino et al. 385 2012; zur Mühlen et al. 1998), for which higher pressures and number of cycles were needed in this 386 study. This could be owed to the higher droplet sizes of coarse pre-emulsions that are produced under the 387 conditions employed in this work. Furthermore, maintaining the working temperature under the base lipid 388 melting point could contribute to these differences. HPH of suspensions showed similar behaviors to 389 those previously exposed, regarding pressure and number of cycles influence and the evolution of 390 bimodal distributions (Kluge et al. 2012). 391 In view of all the variables analyzed in this study, it could be deduced that, for a given lipid to surfactant 392 ratio, the homogenization pressure determined the ultimate particle size, while the number of passes 393 applied determined the polydispersity of that size distribution. 394 Entrapment efficiency and loading capacity 395 Drug content of tocopherol-loaded LNP was analyzed in regard to particle size. The results are shown in 396 Table 4. Almost complete loading of tocopherol was achieved for all particle sizes studied, with EE 397 values over 99 % and 10 % of LC. This can be attributed to the fact that tocopherol is a highly lipophilic 398 drug (logP 9.959) and thus has a high affinity towards lipid matrix. In addition, particle size did not 399 influence tocopherol loading capacity for the range of drug concentrations assayed. 400 Insert Table 4 around here 401 Table 4 Tocopherol-loaded LNP size, production conditions (L lipid concentration, RL/S lipid to emulsifier ratio, P 402 homogenization pressure (bar)) and corresponding values of encapsulation efficiency (EE) (%) and loading capacity 403 (LC) (%). 404 Drug release from LNP 405 In order to study how particle size influences drug release from the nanoparticles, samples of tocopherol- 406 loaded LNP with different particle size but equal drug content were compared. Release studies results are 407 illustrated in Fig. 8. All formulations showed an initial burst release, probably due to drug and surfactant 408 adsorbed in particles surface and a drug shell-enriched structure of the nanoparticles (Schäfer-Korting et 409 al. 2007). On one hand, the presence of surfactant was reported to accelerate drug release (R. H. Müller et 410 al. 1994). On the other hand, a drug shell-enriched structure is typically obtained when the drug melting 411 point is under the matrix lipid melting point, as in this case (2 ºC for α-tocopherol; 63-68 ºC for 412 16 Monostearin). In the production process, lipid precipitation takes place first leading to a phase separation 413 during the cooling process. The lipid occupies therefore the particle core while the compound is 414 accumulated within the shell, reaching the release medium faster. Besides, it can be clearly appreciated 415 that smaller particle sizes induced a faster release. This was expected since smaller sizes imply a higher 416 contact surface of the particle with the external medium, so drug diffusion is favored. Consequently, a 417 determined particle size should be aimed for a desired release, what can be easily done by means of the 418 equations presented in this study. 419 Insert Figure 8 around here 420 Fig. 8 Tocopherol release from LNP with 10 % of tocopherol LC as a function of particle size 421 Conclusions 422 LNP are a promising drug delivery system for all administration routes. HPH is a highly advantageous 423 LNP production technique at both laboratory and industrial scale. In this work, a statistical analysis was 424 carried out aiming to understand and control the parameters influencing the production of LNP by HPH, 425 and therefore their final properties. Manageable equations based on commonly used parameters in the 426 pharmaceutical field were obtained. The lipid to emulsifier concentration ratio (RL/S) was proved to 427 statistically explain the influence of oil phase and surfactant concentration on final nanoparticles size. 428 Besides, the homogenization pressure was found to ultimately determine LNP size for a given RL/S, while 429 polydispersion was mainly determined by the number of passes applied. Further studies regarding 430 different lipids and types of emulsifier could extend the conditions covered by these regression models. 431 This study is intended as a first step to optimize production conditions prior to LNP production at both 432 laboratory and industrial scale from an eminently practical approach, based on extensively used 433 parameters in formulation. 434 Acknowledgments 435 M.D.L. thanks University of Seville for a grant from IV Research Plan of University of Seville. L.M.B. is 436 especially grateful to Junta de Andalucía (Spain) for financial support (Project Nr. P09-CTS5029). 437 Microscopy Services (Centro de Investigación, Tecnología e Innovación de la Universidad de Sevilla, 438 CITIUS) technical support is also grateful. Authors also thank Dr. Álvarez-Fuentes for technical support. 439 17 440 References 441 Araujo J, Gonzalez-Mira E, Egea MA, Garcia ML, Souto EB (2010) Optimization and physicochemical 442 characterization of a triamcinolone acetonide-loaded NLC for ocular antiangiogenic applications. 443 Int J Pharm 393:167–175 444 Biasutti M, Venir E, Marchesini G, Innocente N (2010) Rheological properties of model dairy emulsions 445 as affected by high pressure homogenization. Innov Food Sci & Emerg Tech 11:580–586 446 Bondì ML, Craparo EF, Giammona G, Drago F (2010) Brain-targeted solid lipid nanoparticles containing 447 riluzole: preparation, characterization and biodistribution. Nanomed (London, England) 5:25–32 448 Charcosset C, El-Harati A, Fessi H (2005) Preparation of solid lipid nanoparticles using a membrane 449 contactor. J Control Release 108:112–120 450 Chattopadhyay P, Shekunov BY, Yim D, Cipolla D, Boyd B, Farr S (2007) Production of solid lipid 451 nanoparticle suspensions using supercritical fluid extraction of emulsions (SFEE) for pulmonary 452 delivery using the AERx system. Adv Drug Deliv Rev 59:444–453 453 Das S, Chaudhury A (2011) Recent advances in lipid nanoparticle formulations with solid matrix for oral 454 drug delivery. AAPS Pharm Sci Tech 12:62–76 455 El-Harati AA, Charcosset C, Fessi H (2006) Influence of the formulation for solid lipid nanoparticles 456 prepared with a membrane contactor. Pharm Dev Technol 11:153–157 457 Floury J, Desrumaux A, Lardières J (2000) Effect of high-pressure homogenization on droplet size 458 distributions and rheological properties of model oil-in-water emulsions. Innov Food Sci & Emerg 459 Tech 1:127–134 460 García-Fuentes, M.; Torres, D.; Alonso MJ (2002) Design of lipid nanoparticles for the oral delivery of 461 hydrophilic macromolecules. Colloid Surface B 27:159–168. 462 Gasco MR (1997) Solid lipid nanospheres from warm microemulsion. Pharm Technol Europe 9:32–42. 463 Håkansson A, Fuchs L, Innings F, Revstedt J, Trägårdh C (2011) On flow-fields in a high pressure 464 homogenizer and its implication on drop fragmentation. Procedia Food Sci 1:1353–1358 465 Heurtault B, Saulnier P, Pech B, Proust JE, Benoit JP (2002) A novel phase inversion-based process for 466 the preparation of lipid nanocarriers. Pharm Res 19:875–880 467 Hu FQ, Yuan H, Zhang HH, Fang M (2002) Preparation of solid lipid nanoparticles with clobetasol 468 propionate by a novel solvent diffusion method in aqueous system and physicochemical 469 characterization. Int J Pharm 239:121–128 470 Innocente N, Biasutti M, Venir E, Spaziani M, Marchesini E (2009) Effect of high-pressure 471 homogenization on droplet size distribution and rheological properties of ice cream mixes. J Dairy 472 Sci 92:1864–1875 473 Jafari S, Assadpoor E, He Y, Bhandari B (2008) Re-coalescence of emulsion droplets during high-energy 474 emulsification. Food Hydrocoll 22:1191–1202 475 18 Kluge J, Muhrer G, Mazzotti M (2012) High pressure homogenization of pharmaceutical solids. J 476 Supercrit Fluids 66:380–388 477 Liedtke S, Wissing S, Müller RH, Mäder K (2000) Influence of high pressure homogenisation equipment 478 on nanodispersions characteristics. Int J Pharm 196:183–185 479 Liu J, Hu W, Chen H, Ni Q, Xu H, Yang X (2007) Isotretinoin-loaded solid lipid nanoparticles with skin 480 targeting for topical delivery. Int J Pharm 328:191–195 481 Maindarkar SN, Raikar NB, Bongers P, Henson MA (2012) Incorporating emulsion drop coalescence into 482 population balance equation models of high pressure homogenization. Colloids Surf A 396:63–73 483 McClements DJ (2005) Food Emulsions Principles, Practices, and Techniques (Second Edition). CRC 484 Press 485 Mehnert W, Mäder K (2001) Solid lipid nanoparticles: production, characterization and applications. Adv 486 Drug Deliv Rev 47:165–196 487 Mishra DK, Dhote V, Bhatnagar P, Mishra PK (2012) Engineering solid lipid nanoparticles for improved 488 drug delivery: promises and challenges of translational research. Drug Deliv Transl Res 2:238–253 489 Mohr K-H (1987a) High-pressure homogenization. Part I. Liquid-liquid dispersion in turbulence fields of 490 high energy density. J Food Eng 6:177–186 491 Mohr K-H (1987b) The influence of cavitation on liquid-liquid dispersion in turbulence fields of high 492 energy density. J Food Eng 6:311–324 493 Mucho M, Maincent P, Muller RH (2008) Lipid nanoparticles with a solid matrix (LNP, NLC, LDC) for 494 oral drug delivery. Drug Dev Ind Pharm 34:1394–1405 495 zur Mühlen, Mehnert W (1998) Drug release and release mechanism of prednisolone loaded solid lipid 496 nanoparticles. Pharmazie 53:552–555 497 zur Mühlen A, Schwarz C, Mehnert W (1998) Solid lipid nanoparticles (LNP) for controlled drug 498 delivery-drug release and release mechanism. Eur J Pharm Biopharm 45:149–155 499 Müller R, Maassen S, Schwarz C, Mehnert W (1997) Solid lipid nanoparticles (LNP) as potential carrier 500 for human use: interaction with human granulocytes. J Controlled Release 47:261–269 501 Müller RH, Dingler A, Schneppe T, Gohla S (2000) Large Scale Production of Solid Lipid Nanoparticles 502 (SLN) and Nanosuspensions (DissoCubes). In: Wise D (ed) Handbook Of Pharmaceutical 503 Controlled Release Technology [e-book], Marcel Dekker, pp 359–376 504 Müller RH, Mäder K, Gohla S (2000) Solid lipid nanoparticles (LNP) for controlled drug delivery - a 505 review of the state of the art. Eur J Pharm Biopharm 50:161–177 506 Müller RH, Schwarz C, zur Mühlen A, Mehnert W (1994) Incorporation of lipophilic Drugs and Drug 507 Release Profiles of Solid Lipid Nanoparticles (LNP). Proceed Intern Symp Control Rel Bioact 508 Mater 21:146 509 Pardeike J, Hommoss A, Müller RH (2009) Lipid nanoparticles (LNP, NLC) in cosmetic and 510 pharmaceutical dermal products. Int J Pharm 366:170–184 511 Priano L, Esposti D, Castagna G, De Medici C, Fraschini F, Gasco MR, Mauro A (2007) Solid lipid 512 nanoparticles incorporating melatonin as new model for sustained oral and transdermal delivery 513 systems. J Nanosci Nanotechnol 7:3596–3601. 514 19 Puglia C, Blasi P, Rizza L, Schoubben A, Bonina F, Rossi C, Ricci M (2008) Lipid nanoparticles for 515 prolonged topical delivery: an in vitro and in vivo investigation. Int Pharm 357:295–304 516 Qian C, McClements DJ (2010) Formation of nanoemulsions stabilized by model food-grade emulsifiers 517 using high-pressure homogenization: Factors affecting particle size. Food Hydrocoll 25:1000–1008 518 Saheki A, Seki J, Nakanishi T, Tamai I (2012) Effect of back pressure on emulsification of lipid 519 nanodispersions in a high-pressure homogenizer. Int J Pharm 422:489–494 520 Salmaso S, Elvassore N, Bertucco A, Caliceti P (2009) Production of solid lipid submicron particles for 521 protein delivery using a novel supercritical gas-assisted melting atomization process. J Pharm Sci 522 98:640–650 523 Schubert MA., Müller-Goymann CC (2003) Solvent injection as a new approach for manufacturing lipid 524 nanoparticles – evaluation of the method and process parameters. Eur J Pharm Biopharm 55:125– 525 131 526 Schäfer-Korting M, Mehnert W, Korting HC (2007) Lipid nanoparticles for improved topical application 527 of drugs for skin diseases. Adv Drug Deliv Rev 59:427–443 528 Sebti T, Amighi K (2006) Preparation and in vitro evaluation of lipidic carriers and fillers for inhalation. 529 Eur J Pharm Biopharm 63:51–58 530 Severino P, Santana MHA, Souto EB (2012) Optimizing LNP and NLC by 22 full factorial design: Effect 531 of homogenization technique. Mater Eng C 32:1375-1379 532 Sinha VR, Srivastava S, Goel H, Jindal V (2010) Solid Lipid Nanoparticles ( SLN’S ) – Trends and 533 Implications in Drug Targeting. Int J Adv Pharm Sci 1:212–238 534 Sjöstrom B.; Bergenstahl B. (1992) Preparation of submicron drug particles in lecithin-stabilized o / w 535 emulsions : I . Model studies of the precipitation of cholesteryl acetate. 84:107–116. 536 Souto EB, Müller RH (2006) Investigation of the factors influencing the incorporation of clotrimazole in 537 LNP and NLC prepared by hot high-pressure homogenization. J Microencapsul 23:377–388 538 Trombino S, Cassano R, Muzzalupo R, Pingitore A, Cione E, Picci N (2009) Stearyl ferulate-based solid 539 lipid nanoparticles for the encapsulation and stabilization beta-carotene and alpha-tocopherol. 540 Colloids Surf B 72:181–187 541 Trotta M, Debernardi F, Caputo O (2003) Preparation of solid lipid nanoparticles by a solvent 542 emulsification–diffusion technique. Int J Pharm 257:153–160 543 Varshosaz J, Ghaffari S, Khoshayand MR, Atyabi F, Azarmi S, Kobarfard F (2010) Development and 544 optimization of solid lipid nanoparticles of amikacin by central composite design. J Liposome Res 545 20:97–104 546 Vitorino C, Carvalho FA, Almeida AJ, Sousa JJ, Pais AACC (2011) The size of solid lipid nanoparticles: 547 an interpretation from experimental design. Colloids Surf B 84:117–130 548 Walstra P (1993) Principles of emulsion formation. Chem Eng Sci 48:333–349 549 Wooster TJ, Golding M, Sanguansri P (2008) Impact of Oil Type on Nanoemulsion Formation and 550 Ostwald Ripening Stability. Langmuir 24:12758–12765 551 Figure Click here to download Figure: Fig1.docx Figure Click here to download Figure: Fig2.docx Figure Click here to download Figure: Fig3.docx Figure Click here to download Figure: Fig4.docx Figure Click here to download Figure: Fig5.docx Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle 8 Variable r Variable r Variable r Variable r Variable r Variable r Variable r Variable r 0.758 -0.652 -0.803 -0.778 -0.873 0.912 0.927 0.925 -0.751 -0.644 -0.785 -0.769 0.852 -0.895 -0.915 -0.908 0.733 0.618 0.773 0.739 -0.847 0.892 0.904 0.907 -0.71 -0.612 -0.745 -0.733 -0.802 0.863 0.871 0.879 0.699 -0.609 -0.74 -0.729 -0.796 0.862 0.87 0.88 0.698 -0.567 0.71 -0.683 0.794 -0.84 -0.86 0.855 0.668 0.555 -0.69 0.668 -0.743 0.835 0.842 -0.85 -0.656 -0.53 0.646 -0.641 0.731 -0.78 -0.799 -0.786 -0.664 0.493 -0.646 0.598 0.731 -0.771 -0.789 -0.776 -0.607 0.492 0.645 0.597 0.683 -0.71 -0.726 -0.709 -0.566 0.447 0.597 0.545 -0.697 -0.663 -0.675 -0.656 -0.376 0.33 0.148 0.157 0.149 0.226 0.223 0.271 -0.002 0.011 -0.009 -0.003 0.004 -0.019 -0.024 -0.025 table Click here to download table: Table3.docx L (% w/v) RL/S P* (bar) Cycle Mean size (nm) EE (%) LC (%) 0.50 1 1500 8 71 ± 8 91 ± 9 9 ± 1 0.50 2 1300 5 188 ± 10 96 ± 5 8 ± 2 0.50 2 1000 6 305 ± 12 94 ± 9 10 ± 2 2.50 20 1000 6 487 ± 15 98 ± 6 10 ± 1 2.50 20 1000 5 608 ± 14 97 ± 7 9 ± 2 2.50 10 800 6 886 ± 18 92 ± 10 10 ± 2 0.50 2 800 6 994 ± 22 94 ± 7 7 ± 2 table Click here to download table: Table4.docx