Solid lipid nanoparticles

Advanced Drug Delivery Reviews 64 (2012) 83–101

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Solid lipid nanoparticles☆ Production, characterization and applications Wolfgang Mehnert, Karsten Mäder ⁎ Institute of Pharmacy, Department of Pharmaceutical Technology, Free University of Berlin, Kelchstr. 31, D-12169 Berlin, Germany

a r t i c l e

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Available online 13 September 2012 Keywords: Colloidal drug carriers Stability Drug incorporation Coexistence of different colloidal species Administration routes SLN quality and structure Production parameters

a b s t r a c t Solid lipid nanoparticles (SLN) have attracted increasing attention during recent years. This paper presents an overview about the selection of the ingredients, different ways of SLN production and SLN applications. Aspects of SLN stability and possibilities of SLN stabilization by lyophilization and spray drying are discussed. Special attention is paid to the relation between drug incorporation and the complexity of SLN dispersions, which includes the presence of alternative colloidal structures (liposomes, micelles, drug nanosuspensions, mixed micelles, liquid crystals) and the physical state of the lipid (supercooled melts, different lipid modifications). Appropriate analytical methods are needed for the characterization of SLN. The use of several analytical techniques is a necessity. Alternative structures and dynamic phenomena on the molecular level have to be considered. Aspects of SLN administration and the in vivo fate of the carrier are discussed. © 2012 Published by Elsevier B.V.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aims of solid lipid nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SLN production procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. General ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. SLN preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. High shear homogenization and ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. High pressure homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. SLN prepared by solvent emulsification/evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Microemulsion based SLN preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.5. Comparison of different formulation procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.6. Influence of ingredient composition on product quality . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Sterilization and secondary productions steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Lyophilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Spray drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of SLN quality and structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Defining the goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Measurement of particle size and zeta potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Measurement of crystallinity, lipid modification and assessment of alternative colloidal structures including the time scale of distribution processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Possible problems in SLN preparation and SLN performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. High pressure-induced drug degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Lipid crystallization and drug incorporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Supercooled melts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Lipid modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: CFC, critical flocculation temperature; HPH, high pressure homogenization; PCS, photon correlation spectroscopy; RT, room temperature; SLN, solid lipid nanoparticles; TEM, transmission electron microscopy. ☆ PII of original article: S0169-409X(01)00105-3. The article was originally published in Advanced Drug Delivery Reviews 47 (2001) 165-196. ⁎ Corresponding author. Tel.: +49 61 687 40 24; fax: +49 30 838 50632. E-mail addresses: [email protected] (W. Mehnert), [email protected] (K. Mäder). 0169-409X/$ – see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.addr.2012.09.021

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5.2.3. Particle shape . . . 5.2.4. Gelation phenomena 5.3. Coexistence of several colloidal 6. Drug incorporation and drug release 7. Storage stability . . . . . . . . . . 8. Toxicity aspects and in vivo fate . . 9. Administration routes and in vivo fate 9.1. Peroral administration . . . . 9.2. Parenteral administration . . 9.3. Transdermal application . . . 10. Summary and outlook . . . . . . . Acknowledgements . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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1. Introduction In recent years it has become more and more evident that the development of new drugs alone is not sufficient to ensure progress in drug therapy. Exciting experimental data obtained in vitro are very often followed by disappointing results in vivo. Main reasons for the therapy failure include: • Insufficient drug concentration due to poor absorption, rapid metabolism and elimination (e.g. peptides, proteins). Drug distribution to other tissues combined with high drug toxicity (e.g. cancer drugs) • Poor drug solubility which excludes i.v. injection of aqueous drug solution • High fluctuation of plasma levels due to unpredictable bioavailability after peroral administration, including the influence of food on plasma levels (e.g. cyclosporine) A promising strategy to overcome these problems involves the development of suitable drug carrier systems. The in vivo fate of the drug is no longer mainly determined by the properties of the drug, but by the carrier system, which should permit a controlled and localized release of the active drug according to the specific needs of the therapy. The size of the carrier depends on the desired route of administration and ranges from few nanometers (colloidal carriers), to the micrometer range (microparticles) and to several millimeters (implants). For parenteral administration, it is highly desirable to use biodegradable materials, which avoid surgery to remove the implant after complete drug release and which make the administration of micro- and nanoparticles feasible. The concept has been realized in several commercial products. Implants and microparticles based on biodegradable polyesters permit a controlled drug release over a period of weeks to months after s.c. or i.m. implantation/injection. Commercially available systems have been developed for the treatment of prostate cancer and other GnRH-related diseases [1]. An example of the concept of localized drug release is the development of biodegradable implants for the treatment of gliomas, which ensure very high drug concentrations in the brain and minimize drug concentration in other tissues, including bone marrow [2]. Implants and microparticles are too large for drug targeting and intravenous administration. Therefore, colloidal carriers have attracted increasing attention during recent years. Investigated systems include nanoparticles, nanoemulsions, liposomes, nanosuspensions, micelles, soluble polymer–drug conjugates. The existence of different colloidal carrier systems raises the question as to which of them might be the most suitable for the desired purpose. Of course, there is no simple answer to this question. Aspects to consider include: • Drug loading capacity • Possibility of drug targeting • In vivo fate of the carrier (interaction with the biological surrounding, degradation rate, accumulation in organs) • Acute and chronic toxicity

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• Scaling up of production • Physical and chemical storage stability • Overall costs Polymers from natural [3,4] and synthetic sources [5] have been used. Polymer based systems in the submicron size range include water soluble polymer–drug conjugates [6], polymer nanocapsules [7,8] and nanospheres. A certain advantage of polymer systems is the wealth of possible chemical modifications, including the synthesis of block- and comb-polymers. Problems of polymer based nanoparticles derive from residues from organic solvents used in the production process, polymer cytotoxicity [9] and the scaling up of the production processes. In most production processes, the concentration of nanoparticles is low and does not exceed 2%. Polymer hydrolysis during storage has to be taken into account and lyophilization is often required to prevent polymer degradation. Liposomes are spherical vesicles composed of one or more phospholipid bilayers (in most cases phosphatidylcholine). Lipophilic drugs can be incorporated into the lipid bilayers while hydrophilic drugs are solubilized in the inner aqueous core [10,11]. Drug release, in vivo stability and biodistribution are determined by size, surface charge, surface hydrophobicity and membrane fluidity [12]. Membrane permeability can be adapted by the selection of the phospholipids and the incorporation of additives (e.g. cholesterol). It is possible to prevent a rapid reticuloendothelial uptake of the liposomes by the incorporation of natural compounds (e.g. gangliosides) or by the use of chemical modified polyethylene glycols [13–19]. The development of such sterically stabilized systems (‘stealth liposomes’) permits the practical realization of drug targeting strategies (e.g. by incorporation of specific antibodies) [20,21]. Liposome based drug carriers also permit the intravenous injection of lipophilic drugs with very low water solubility, e.g. amphotericin B (AmBisome®) [22]. The toxicity of the liposome system is 1/10 compared to a commercial micelle-based amphotericin formulation. Chemical and physical stability problems might lead to liposome aggregation and drug degradation during storage and compromise the performance of liposome based drug carriers [23]. Nanosuspensions are colloidal particles which are composed of the drug and the emulsifier only. Possible production procedures include ball milling [24] or high pressure homogenization [25,26]. Lipid nanoemulsions were introduced during the 50s for the purpose of parenteral nutrition. Fatty vegetable oils (e.g. soy oil) or middle chain triglycerides are used for the lipid phase, which amounts to typically 10–20% of the emulsion. Further ingredients include phospholipids (stabilizers, 0.6–1.5%) and glycerol (osmolarity regulation, 2.25%). During recent years it has been recognized that these systems might also be used as drug carriers for lipophilic drugs and several formulations are commercialized [27–35]. Examples include etomidate (Etomidat-Lipuro®) and diazepam (Diazepam-Lipuro®) [36–38]. In comparison to previous, solubilization-based formulations of these drugs, a reduction of the local and systemic side effects (e.g. pain during

W. Mehnert, K. Mäder / Advanced Drug Delivery Reviews 64 (2012) 83–101

injection) has been found. The hemolytic activity of sodium oleate is decreased in lipid emulsions because the lytic agent is restricted at the interface and in the lipophilic core and so the direct contact with erythrocyte membranes is hindered [39]. The possibility of controlled drug release from nanoemulsions is limited due to the small size and the liquid state of the carrier. For most drugs, a rapid release of the drug will be observed [40–42]. It has been estimated, that retarded drug release requires very lipophilic drugs, the octanol/water partition coefficient should be larger than 1 000 000:1 [43]. Advantages of nanoemulsions include toxicological safety and a high content of the lipid phase as well as the possibility of large scale production by high pressure homogenization. The use of solid lipids instead of liquid oils is a very attractive idea to achieve controlled drug release, because drug mobility in a solid lipid should be considerably lower compared with an liquid oil. Solid lipids have been used for several years in the form of pellets in order to achieve a retarded drug release after peroral administration (e.g. Mucosolvan® Retard Capsules). In the beginning of the 80s, Speiser and coworkers developed solid lipid microparticles (by spray drying) [44] and ‘Nanopellets for peroral administration’ [45]. Nanopellets developed by Speiser [45] were produced by dispersing of melted lipids with high speed mixers or ultrasound. The products obtained by this procedure often contain relatively high amounts of microparticles. This might not be a serious problem for peroral administration, but it excludes an intravenous injection. Higher concentrations of the emulsifier result in a reduction of the particle size, but also increase the risk of toxic side effects. Similar systems have been described by Domb as ‘Lipospheres’ [46–48]. They are also produced by means of high shear mixing or ultrasound and often contain considerable amounts of microparticles, too. In the following years, it has been demonstrated that high pressure homogenization is a more effective method for the production of submicron sized dispersions of solid lipids compared to high shear mixing or ultrasound [49–51]. Dispersions obtained in this way are called solid lipid nanoparticles (SLN™). Most SLN dispersions produced by high pressure homogenization (HPH) are characterized by an average particle size below 500 nm and a low microparticle content. Other production procedures are based on the use of organic solvents (HPH/ solvent evaporation) [52] or on dilution of microemulsions [53,54]. 2. Aims of solid lipid nanoparticles It has been claimed that SLN combine the advantages and avoid the disadvantages of other colloidal carriers [55]. Proposed advantages include: • • • • • • •

Possibility of controlled drug release and drug targeting Increased drug stability High drug payload Incorporation of lipophilic and hydrophilic drugs feasible No biotoxicity of the carrier Avoidance of organic solvents No problems with respect to large scale production and sterilization

3. SLN production procedures 3.1. General ingredients General ingredients include solid lipid(s), emulsifier(s) and water. The term lipid is used here in a broader sense and includes triglycerides (e.g. tristearin), partial glycerides (e.g. Imwitor), fatty acids (e.g. stearic acid), steroids (e.g. cholesterol) and waxes (e.g. cetyl palmitate). All classes of emulsifiers (with respect to charge and molecular weight) have been used to stabilize the lipid dispersion. It has been found that the combination of emulsifiers might prevent particle agglomeration more efficiently.

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An overview of ingredients which are commonly used is provided in Table 1. A clear advantage of SLN is the fact, that the lipid matrix is made from physiological lipids which decreases the danger of acute and chronic toxicity. The choice of the emulsifier depends on the administration route and is more limited for parenteral administrations. 3.2. SLN preparation 3.2.1. High shear homogenization and ultrasound High shear homogenization and ultrasound are dispersing techniques which were initially used for the production of solid lipid nanodispersions [45,46]. Both methods are widespread and easy to handle. However, dispersion quality is often compromised by the presence of microparticles. Furthermore, metal contamination has to be considered if ultrasound is used. Ahlin et al. used a Lak Tek rotor–stator homogenizer (Omni International, Gainesville, USA) to produce SLN by melt–emulsification [56]. They investigated the influence of different process parameters, including emulsification time, stirring rate and cooling conditions on the particle size and the zeta potential. Lipids used in this study include trimyristin (Dynasan®114), tripalmitin (Dynasan®116), tristearin (Dynasan®118), a mixture of mono-, di- and triglycerides (Witepsol® W35, Witepsol®H35) and glycerol behenate (Compritol®888 ATO), poloxamer 188 was used as steric stabilizer (0.5 w%). For Witepsol® W35 dispersions the following parameters were found to produce the best SLN quality: stirring for 8 min at 20 000 rpm, the optimum cooling conditions: 10 min at 5000 rpm at room temperature. In contrast, the best conditions for Dynasan®116 dispersions were a 10-min emulsification at 25 000 rpm and 5 min of cooling at 5000 rpm in cool water (T = 16 °C). Higher stirring rates did not significantly change the particle size, but slightly improved the polydispersity index. No general rule can be derived from differences in the established optimum emulsification and cooling conditions. In most cases, average particle sizes in the range of 100–200 nm were obtained in this study. 3.2.2. High pressure homogenization High pressure homogenization (HPH) has emerged as a reliable and powerful technique for the preparation of SLN. Homogenizers of different sizes are commercially available from several manufacturers at reasonable prices. HPH has been used for years for the production of nanoemulsions for parenteral nutrition. In contrast to other techniques, scaling up represents no problem in most cases. High pressure homogenizers push a liquid with high pressure (100–2000 bar) through a narrow gap (in the range of a few microns). The fluid accelerates on a very short distance to very high velocity (over 1000 km/h). Very high shear stress and cavitation forces disrupt the particles down to the submicron range. Typical lipid contents are in the range 5–10% and represent no problem to the homogenizer. Even higher lipid concentrations (up to 40%!) have been homogenized to lipid nanodispersions [57]. Two general approaches of the homogenization step, the hot and the cold homogenization techniques, can be used for the production of SLN (Fig. 1) [51,58,59]. In both cases, a preparatory step involves the drug incorporation into the bulk lipid by dissolving or dispersing the drug in the lipid melt. 3.2.2.1. Hot homogenization. Hot homogenization is carried out at temperatures above the melting point of the lipid and can therefore be regarded as the homogenization of an emulsion. A pre-emulsion of the drug loaded lipid melt and the aqueous emulsifier phase (same temperature) is obtained by high-shear mixing device (Ultra-Turrax). The quality of the pre-emulsion affects the quality of the final product to a large extent and it is desirable to obtain droplets in the size range of a few micrometers. HPH of the pre-emulsion is carried out at temperatures above the melting point of the lipid. In general, higher temperatures result in lower particle sizes due to the decreased viscosity

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Table 1 Lipids and emulsifiers used for preparation of solid lipid nanoparticles. Lipids

Literature

Triglycerides Tricaprin Trilaurin Trimyristin Tripalmitin Tristearin Hydrogenated coco-glycerides (Softisan® 142)

[47] [124,139,47,59,102,87,156] [124,148,139,187,56] [49,62,65,139,56,118,124,135] [124,139,47,56] [129]

Hard fat types Witepsol® W 35 Witepsol® H 35 Witepsol® H 42 Witepsol® E 85

[118,49,56] [148,56] [149] [148,129]

Glyceryl monostearate (Imwitor®900) Glyceryl behenate (Compritol® 888 ATO) Glyceryl palmitostearate (Precirol® ATO 5)

[152] [174,181,136,140,106,187,137,179,56,59,102,115] [181]

Cetyl palmitate

[106,187]

Stearic acid Palmitic acid Decanoic acid Behenic acid

[176,175,71,151,154,149,86,155,152,74,70,153,159] [150] [150] [86]

Acidan N12

[86]

Emulsifiers/coemulsifiers Soybean lecithin (Lipoid® S 75, Lipoid® S 100) Egg lecithin (Lipoid® E 80) Phosphatidylcholine (Epikuron® 170, Epikuron 200) Poloxamer 188

[139,148,59,102,118,124,135] [135] [65,176,175,86,152,74]

Poloxamer 182 Poloxamer 407 Poloxamine 908 Tyloxapol Polysorbate 20 Polysorbate 60 Polysorbate 80

[118,49,65,174,181,136,140,106,137,187,129,179,159, 158,56,59,102,115] [129] [174,181] [174,181] [62,65,139,148,124,135] [74,70] [70] [129]

Sodium cholate Sodium glycocholate

[187,129] [49,65,139,176,175,148,118,124,135]

Taurocholic acid sodium salt Taurodeoxycholic acid sodium salt Butanol Butyric acid Dioctyl sodium sulfosuccinate Monooctylphosphoric acid sodium

[176,175,152] [71,150,151,154,149,86,155,74,70,153] [71,151,155,152,74,153] [154,155] [155] [70]

of the inner phase [60]. However, high temperatures may also increase the degradation rate of the drug and the carrier. The homogenization step can be repeated several times. It should always be kept in mind, that high pressure homogenization increases the temperature of the sample (approximately 10 °C for 500 bar [61]). In most cases, 3–5 homogenization cycles at 500–1500 bar are sufficient. Increasing the homogenization pressure or the number of cycles often results in an increase of the particle size due to particle coalescence which occurs as a result of the high kinetic energy of the particles [62]. The primary product of the hot homogenization is a nanoemulsion due to the liquid state of the lipid. Solid particles are expected to be formed by the following cooling of the sample to room temperature or to temperatures below. Due to the small particle size and the presence of emulsifiers, lipid crystallization may be highly retarded and

the sample may remain as a supercooled melt for several months [63]. 3.2.2.2. Cold homogenization. In contrast, the cold homogenization is carried out with the solid lipid and represents, therefore, a high pressure milling of a suspension (Fig. 1). Effective temperature control and regulation is needed in order to ensure the unmolten state of the lipid due to the increase in temperature during homogenization [61]. Cold homogenization has been developed to overcome the following three problems of the hot homogenization technique: 1. Temperature-induced drug degradation 2. Drug distribution into the aqueous phase during homogenization 3. Complexity of the crystallization step of the nanoemulsion leading to several modifications and/or supercooled melts

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small particles could only be obtained with low fat loads (5 w%) related to the organic solvent. With increasing lipid content the efficiency of the homogenization declines due to the higher viscosity of the dispersed phase. The advantage of this procedure over the cold homogenization process described before is the avoidance of any thermal stress. A clear disadvantage is the use of organic solvents.

The first preparatory step is the same as in the hot homogenization procedure and includes the solubilization or dispersing of the drug in the melt of the bulk lipid. However, the following steps are different. The drug containing melt is rapidly cooled (e.g. by means of dry ice or liquid nitrogen). The high cooling rate favors a homogenous distribution of the drug within the lipid matrix. The solid, drug containing lipid is milled to microparticles. Typical particle sizes obtained by means of ball or mortar milling are in the range 50–100 microns. Low temperatures increase the fragility of the lipid and favor, therefore, particle comminution. The solid lipid microparticles are dispersed in a chilled emulsifier solution. The pre-suspension is subjected to high pressure homogenization at or below room temperature. In general, compared to hot homogenization, larger particle sizes and a broader size distribution are observed in cold homogenized samples [64]. The method of cold homogenization minimizes the thermal exposure of the sample, but it does not avoid it due to the melting of the lipid/drug-mixture in the initial step.

3.2.4. Microemulsion based SLN preparations Gasco and co-workers developed SLN preparation techniques which are based on the dilution of microemulsions [54]. It should be mentioned that there are different opinions in the scientific community about the structure and dynamics of microemulsion. An extended review has recently been published by Moulik and Paul [66]. Gasco and other scientists understand microemulsions as twophase systems composed of an inner and outer phase (e.g. o/wmicroemulsions). They are made by stirring an optically transparent mixture at 65–70 °C which is typically composed of a low melting fatty acid (e.g. stearic acid), an emulsifier (e.g. polysorbate 20, polysorbate 60, soy phosphatidylcholine, taurodeoxycholic acid sodium salt), co-emulsifiers (e.g. butanol, sodium monooctylphosphate) and water. The hot microemulsion is dispersed in cold water (2–3 °C) under stirring. Typical volume ratios of the hot microemulsion to cold water are in the range of 1:25 to 1:50. The dilution process is critically determined by the composition of the microemulsion. According to the literature [67,68], the droplet structure is already contained in the microemulsion and therefore, no energy is required to achieve submicron particle sizes. With respect to the similarities of the production procedure of polymer nanoparticles described by French scientists [69], different mechanisms might be considered. Fessi produced polymer particles by dilution of polymer solutions in water. According to Fessi, the particle size is critically determined by the velocity of the distribution processes. Nanoparticles were produced only with solvents which distribute very rapidly into the aqueous phase (e.g. acetone), while larger particle sizes were obtained with more lipophilic solvents. The hydrophilic cosolvents of the microemulsion might play a similar role in the formation of lipid nanoparticles as the acetone for the formation of polymer nanoparticles. Considering microemulsions, the temperature gradient and the pH value fix the product quality in addition to the composition of the microemulsion. High-temperature gradients facilitate rapid lipid crystallization and prevent aggregation [70,71]. Due to the dilution step, achievable lipid contents are considerably lower compared with the HPH based formulations.

3.2.3. SLN prepared by solvent emulsification/evaporation Sjöström and Bergenståhl described a production method to prepare nanoparticle dispersions by precipitation in o/w emulsions [52]. The lipophilic material is dissolved in a water-immiscible organic solvent (e.g. cyclohexane) that is emulsified in an aqueous phase. Upon evaporation of the solvent a nanoparticle dispersion is formed by precipitation of the lipid in the aqueous medium. The mean diameter of the obtained particles was 25 nm with cholesterol acetate as model drug and by using a lecithin/sodium glycocholate blend as emulsifier. The reproducibility of these results is confirmed by Westesen [65]. The cholesterol acetate nanoparticles had a mean particle size of 29 nm (PCS number distribution) prepared according to Sjöström with available equipment. Westesen prepared nanoparticles of tripalmitin by dissolving the triglyceride in chloroform. This solution was emulsified in an aqueous phase by HPH. The organic solvent was removed from the emulsion by evaporation under reduced pressure (40–60 mbar). The mean particle size ranges from approximately 30 to 100 nm depending on the lecithin/co-surfactant blend. Particles with average diameters as small as 30 nm were obtained by using bile salts as co-surfactants. Comparable small particle size distributions are not achievable by melt emulsification of similar composition. The mean particle size depends on the concentration of the lipid in the organic phase. Very

3.2.5. Comparison of different formulation procedures Whenever possible, a direct comparison between the different formulation procedures should be made by the same investigator, using the same ingredients, the same storage conditions and the same equipment for particle sizing. Otherwise, impurities of the ingredients and differences in particle sizing technologies might lead to misleading results. Siekmann et al. investigated the influence of the formulation procedure on the quality of tyloxapol (1.5 w%) and soy lecithin (1 w%) stabilized tripalmitin (3 w%) nanoparticles [62]. They demonstrated the principal possibility to obtain size distributions in the range from 30 to 180 nm by ultrasonification. However, these small particle sizes required long sonication times (>15 min), which raises concerns about metal shed from the probe and contamination of the product. Moreover, it is difficult to disperse higher fat concentrations homogeneously by probe sonication, and therefore, ultrasound is only of limited use for higher lipid concentrations. HPH proved to be a very effective dispersing technique in this study. A reduction of the average particle size from 474 to 155 nm was already obtained after the first homogenization cycle (800 bar). The maximum dispersing grade is observed after 5 homogenization cycles. Results reported by other investigators show similar dependences of the particle size from the homogenization pressure and the number of cycles [51,72].

Fig. 1. Schematic procedure of hot and cold homogenization techniques for SLN production.

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The solvent emulsification/evaporation process was compared to the melt-homogenization method by Westesen [65]. In the case of lecithin/ sodium glycocholate stabilized tripalmitin dispersions, solvent emulsification yielded significantly smaller particles than melt-homogenization of similarly composed systems under the same production conditions. The mean particle size of the melt-homogenized tripalmitin nanoparticles was 124 nm, while that of the solvent-evaporated was 28 nm only. This result might be partly explained by the lower homogenization efficiency of the lecithin/sodium glycocholate blend in the emulsified tripalmitin melt compared to the organic solvent-in-water emulsion, as the mobility of phospholipid molecules is lower in the lipid melt than in the solvent. Moreover, the melt of the lipid/emulsifier blend is much more viscous than the solvent so that the homogenization requires more energy input. However, solvent-emulsification is not always superior to melt-homogenization with respect to the dispersing degree. In contrast, for systems stabilized by phospholipids and nonionic surfactants, melt-homogenization produced smaller particles than the solvent-emulsification procedure. These results show that the attainable particle size is especially dependent on the composition of emulsifiers. In conclusion, solvent-emulsification is a suitable alternative method to prepare small, homogeneously-sized lipid nanoparticles dispersions. An important advantage of that technique is the avoidance of any heat. On the other hand, solvent-emulsified suspensions are relatively diluted (0.5–2.5 w% tripalmitin) due to the limited solubility of the tripalmitin in organic solvents. This is of particular interest regarding the achievable drug load of such lipid dispersions. Furthermore, it has to be considered that, in contrast to melt-homogenization, solvent-emulsification may create toxicological problems arising from solvent residues, since the complete removal of solvent is hardly possible from a technical point of view. Ahlin and Kristl investigated the influence of the emulsifier concentration on the SLN particle size (production by rotor–stator homogenizer) [56]. The mean particle size decreased with increasing emulsifier concentration, reaching the optimum at about 2–3 w%, and then increased again. The increase of particle size and the polydispersity index at higher phospholipid concentrations could be a consequence of increased liposome formation or the formation of lecithin multilayers on the particle surface. A striking different behavior was observed when the same ingredients were used to produce SLN via HPH: increasing amounts of lecithin caused a continuous decrease of the particle size. The reason for the difference of results of the HPH and rotor–stator homogenizer can be attributed to different dispersing degrees of the liposomes which are obtained with both methods. Liposomes produced via HPH were much smaller (50 nm) than the lecithin dispersions subjected to the rotor–stator disperser (200 nm). The dispersing grade depends on the power density and the power distribution in the dispersion volume. High power densities result in more effective particle disruption. High pressure homogenizers reach by far the highest power densities (10 12–10 13 W/m 3). A homogeneous distribution of the power density is necessary to obtain narrow size distributions. Otherwise, particles localized in different volumes of the sample will experience different dispersing forces and therefore, the degree of particle disruption will vary within the sample volume. Inhomogeneous power distributions are observed in highshear homogenizers and ultrasonifiers. High pressure homogenizers are characterized by a homogenous power distribution due to the small size of the homogenizing gap (25–30 μm). 3.2.6. Influence of ingredient composition on product quality 3.2.6.1. Influence of the lipid. Using the hot homogenization, it has been found that the average particle size of SLN dispersions is increasing with higher melting lipids [64,49]. These results are in agreement to the general theory of HPH [61] and can be explained by the higher viscosity of the dispersed phase. However, other critical parameters for nanoparticle formation will be different for different lipids.

Examples include the velocity of lipid crystallization, the lipid hydrophilicity (influence on self-emulsifying properties [49]) and the shape of the lipid crystals (and therefore the surface area). It is also noteworthy, that most of the lipids used represent a mixture of several chemical compounds. The composition might therefore vary from different suppliers and might even vary for different batches from the same supplier. However, small differences in the lipid composition (e.g. impurities) might have considerable impact on the quality of SLN dispersion (e.g. by changing the zeta potential, retarding crystallization processes etc.). For example, lipid nanodispersions made with cetyl palmitate from different suppliers had different particle sizes and storage stabilities (A. Lippacher, personal communication). The influence of lipid composition on particle size was also confirmed on SLN produced via high-shear homogenization [56]. The average particle size of Witepsol®W35 SLN was found to be significantly smaller (117.0 ± 1.8 nm) than the size of Dynasan®118 SLN (175.1 ± 3.5 nm). Witepsol®W35 contains shorter fatty acid chains and considerable amounts of mono- and diglycerides which possess surface active properties. Increasing the lipid content over 5–10% in most cases results in larger particles (including microparticles) and broader particle size distributions [64,62]. Both a decrease of the homogenization efficiency and an increase in particle agglomeration cause this phenomenon which has been observed for lipid nanoemulsions, too [73]. 3.2.6.2. Influence of the emulsifier. The choice of the emulsifiers and their concentration is of great impact on the quality of the SLN dispersion [51,64]. Investigating the influence of the emulsifier concentration on the particle size of Compritol® SLN dispersions, zur Mühlen obtained best results with 5 w% sodium cholate or poloxamer 188. Batches produced with lower concentrations of the emulsifier contained higher amounts of microparticles. Siekmann et al. reported that 2 w% tyloxapol were insufficient to stabilize a 10 w% tripalmitin dispersion. Increasing the tyloxapol concentration to 10 w% resulted in 85-nm particles with unimodal size distribution [62]. High concentrations of the emulsifier reduce the surface tension and facilitate the particle partition during homogenization. The decrease in particle size is connected with a tremendous increase in surface area. The increase of the surface area during HPH occurs very rapidly. Therefore, kinetic aspects have to be considered. The process of a primary coverage of the new surfaces competes with the agglomeration of uncovered lipid surfaces. The primary dispersion must contain excessive emulsifier molecules, which should rapidly cover the new surfaces. The excessive emulsifier molecules might be present in different forms e.g. molecular solubilized (emulsifier monomers), in form of micelles (SDS) or liposomes (lecithin). The time scale of the redistribution processes of emulsifier molecules between particle surfaces, water-solubilized monomers and micelles or liposomes is different. In general, SDS and other micelle-forming low molecular weight surfactants will rapidly achieve the new equilibrium. Redistribution processes will take a longer time for high molecular weight surfactants (poloxamer) and lecithin. However, it is not recommended to exclusively use rapidly distributing surfactants like SDS, because their ability to cover surfaces very rapidly is often combined with considerable water solubility and toxicity. Indeed, it has been reported that SLN stabilized with surfactant mixtures (Lipoid S 75/poloxamer 188 [64] or tyloxapol/lecithin [62]) have lower particle sizes and higher storage stability compared to formulations with only one surfactant. The addition of sodium glycocholate to the aqueous phase as co-emulsifying agent decreases the particle size, too [62]. Different emulsifier compositions might require different homogenization parameters. For example, the maximal degree of dispersing was obtained with 500 bar and three cycles for poloxamer 188 stabilized systems [72]. Homogenization with pressures of 1000 or 1500 bar did not result in further reduction of the particle size. In contrast,

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pressures of 1500 bar proved to be the best for lecithin (Lipoid S 75) stabilized systems. A possible explanation for this observation is the different velocity of the coverage of the new lipid surfaces. The importance of the emulsifier for the quality of the lipid nanodispersion was also demonstrated on microemulsion based SLN dispersions. Cavalli et al. used stearic acid as the lipid phase and compared an ionic surfactant/cosurfactant system composed of Epikuron 100, taurodeoxycholate and monooctylphosphate with a nonionic system composed of Tween 80 and butanol [74]. The particle size of the SLN dispersion produced with the ionic surfactants was considerably smaller (70 ± 2 nm) compared to the nonionic formulation (200± 5 nm). 3.3. Sterilization and secondary productions steps 3.3.1. Sterilization Parenteral administration requires sterile formulations. Aseptic production, filtration, γ-irradiation and heating are normally used to achieve sterility. Filtration sterilization of dispersed systems requires high pressure and is not applicable to particles >0.2 μm. The sterilization should not change the properties of the formulation with respect to physical and chemical stability and the drug release kinetics. Free radicals are formed during γ-sterilization in all samples due to the high energy of the γ-rays. These radicals may recombine with no modification of the sample or undergo secondary reactions which might lead to chemical modifications of the sample. The degree of degradation depends on the general chemical reactivity. Furthermore, high molecular mobility (semisolid or liquid state) and the presence of oxygen favor γ-sterilization-induced degradation reactions. Therefore, it is not surprising that chemical changes of the lipid bilayer components were observed after γ-irradiation [75]. Sterilization by heat is a reliable procedure which is commonly used. It was also applied for liposomes [76,77]. A possible concern is related to temperature-induced changes of the physical stability. Schwarz investigated the impact of different sterilization techniques (steam sterilization at 121 °C (15 min) and 110 °C (15 min), γ-sterilization) on SLN characteristics [72,78,79]. The results indicate that particle aggregation might occur as a result of the treatment. Critical parameters include sterilization temperature and SLN composition. The correct choice of the emulsifier is of significant importance for the physical stability of the sample at high temperatures. Increased temperatures will affect the mobility and the hydrophilicity of all emulsifiers to a different extent. Steam sterilization will cause the formation of an o/w-emulsion due to the melting of the lipid particles. Solid particles are formed after recrystallization. Schwarz found that lecithin is a suitable surfactant for steam sterilization, because only a minor increase in particle size and number of microparticles was observed. In contrast, steam sterilization-induced a significant increase in particle size for poloxamer 188 stabilized Compritol® SLN. This destabilization can be attributed to the decreased steric stabilization by the emulsifier poloxamer. It is well known for emulsifiers of this type, that increased temperatures lead to dehydration of the ethylene glycol chains which means a decrease of the thickness of the protecting layer. It has been demonstrated by 1H-NMR spectroscopy on poloxamer stabilized lipid nanoparticles, that even a moderate temperature increase in RT to 37 °C decreases the mobility of the ethylene glycol chains on the particle surface (Fig. 2) [80]. Temperature-induced flocculation has been observed to occur at CFT = 75.5 °C (CFT, critical flocculation temperature) on poloxamer 188-stabilized polystyrene nanoparticles (60-nm particles in 0.2 M NaSO4 solution) [81]. The presence of ions will strongly influence the CFT in addition to the characteristics of the particles and the emulsifier due to the decrease in the zeta potential. In most cases SLN dispersions do not contain electrolytes and are expected to exhibit higher resistance to temperature-induced flocculation therefore. In the study of Schwarz it was found that a decrease of the sterilization

Fig. 2. 1H-NMR spectra of SNL kispersion with semisolid lipid matrix obtained at RT and 37 °C. In the same way the peaks of other CH3- and CH2-groups became more visible while warming the sample. These effects visualize the semisolid state of the carrier at RT and a melting (corresponding to higher mobility) at 37 °C [80].

temperature from 121 to 110 °C can reduce sterilization-induced particle aggregation to a large extent. Drug loading might change the resistance to sterilization-induced destabilization. Steam sterilization of 5% tetracaine loaded poloxamer 188/Compritol® SLN induced a broader size distribution (PI from 0.08 to 0.25) and an increase in the mean particle size (from 160 to 260 nm) [78]. Even a larger increase in particle size (> 500 nm) was observed for a higher tetracaine loading of 10%. These results indicate that drug-related phenomena contribute to destabilization processes in addition to changes of the emulsifier film. The destabilizing effect of tetracaine and etomidate has also been observed in steam sterilization of fat emulsions [82]. The observed destabilization was attributed to a distortion of the mechanical properties of the surfactant film. This explanation is also valid for SLN due to the molten state of the lipid at 121 °C. At RT a relatively broad peak around 1.3 ppm was obtained which narrows while measuring the sample at 37 °C. Experiments conducted by Freitas indicated that lowering of the lipid content (to 2%) and surface modification of the glass vials prevent the particle increase to a large extent and avoid gelation [83]. Additionally, it was observed by Freitas that purging the sample with nitrogen showed a protective effect during sterilization. That observation suggests that chemical reactions could contribute to particle destabilization. For example, the removal of solubilized carbon dioxide by nitrogen purging might increase the pH value and decrease the rate of lipid hydrolysis which in turn affects the recrystallization of the lipid [84,85]. However, further studies are necessary to investigate the mechanism of the protective effect of nitrogen purging. Cavalli et al. studied the influence of steam sterilization on particle size and zeta potential of SLN produced via microemulsions [86]. The lipid phase (7.35 w%) was made of stearic acid, behenic acid or Acidan N12 (monostearate monocitrate diglyceride), Epikuron 200 (soy phosphatidylcholine 95%) and taurodeoxycholate were used as stabilizers. SLN were dispersed in aqueous trehalose (2%) or poloxamer 188 solution (2%). Steam sterilization (121 °C, 15 min) did not change the average particle size of Acidan N12 SLN, but increased particle sizes were observed for SLN composed of behenic acid (from 70 to 135 nm) and of stearic acid (from 55 to 110 nm). After 1 year, increased particle sizes were observed for all systems (Acidan N12 SLN: 350 nm, behenic acid SLN: 120 nm, stearic acid SLN: 450 nm). However, no microparticles were observed. Steam sterilization did not cause significant changes of the zeta potential. It is interesting to note that

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particle sizes of diazepam loaded SLN showed similar changes after sterilization as drug free systems. Steam sterilization (121 °C, 20 min) did not cause changes in particle size and zeta potential of azidothymidine palmitate loaded SLN (trilaurin, phospholipid stabilized) [87]. γ-irradiation could be an alternative method to steam sterilization for temperature sensitive samples. Comparative studies on SLN sterilization by steam and γ-rays were conducted by Schwarz [72,78,79]. Compared to lecithin stabilized systems, poloxamer stabilized SLN were found to be more unstable to steam sterilization. However, this difference was not detected for γ-sterilized samples. Compared to steam sterilization at 121 °C, the increase in particle size after γ-irradiation was lower, but comparable to steam sterilization at 110 °C. Unfortunately, most investigators did not search for steam sterilization or irradiation-induced chemical degradation. It should be kept in mind that degradation does not always cause increased particle sizes. In contrast, the formation of species like lysophosphatides or free fatty acids might even preserve small particle sizes, but might cause toxicological problems. Further studies which involve the aspects of chemical stability are clearly necessary to permit valid statements of the possibilities of SLN sterilization.

3.3.2. Lyophilization Storage stability involves chemical and physical aspects and includes the prevention of degradation reactions (e.g. hydrolysis) and the preservation of the initial particle size. It requires that the SLN ingredients have sufficient chemical stability and that the particles have a very narrow size distribution to avoid crystal growth by Ostwald ripening. The SLN formulation should be resistant to temperature changes which will occur during shipping. It has been shown that particle sizes of aqueous SLN dispersions might be stable over 12– 36 months [64]. However, this stability is not a general feature of SLN dispersions and in most cases, an increase in particle size will be observed in a shorter period of time. Lyophilization is a promising way to increase chemical and physical SLN stability over extended periods of time. Transformation into a solid form will prevent Ostwald ripening and avoid hydrolysis reactions. Lyophilization also offers principle possibilities for SLN incorporation into pellets, tablets or capsules. It is expected that the solid state of the lyophilizates will have a better chemical and physical stability than aqueous lipid dispersions. However, two additional transformations between the formulations are necessary which might be the source of additional stability problems. The first transformation — from aqueous dispersion to powder — involves the freezing of the sample and the evaporation of water under vacuum. Freezing of the sample might cause stability problems due to the freezing out effect which results in changes of the osmolarity and the pH. The second transformation — resolubilization — involves, at least in its initial stages, situations which favor particle aggregation (low water and high particle content, high osmotic pressure). The protective effect of the surfactant can be compromised by lyophilization [88]. It has been found, that the lipid content of the SLN dispersion should not exceed 5% to prevent an increase in particle size. Direct contact of lipid particles is decreased in diluted samples. Furthermore, diluted SLN dispersions will also have higher sublimation velocities and a higher specific surface area [89]. The addition of cryoprotectors will be necessary to decrease SLN aggregation and to obtain a better redispersion of the dry product. The influence of the cryoprotectors on the quality of the lyophilizates has been widely investigated in the field of the liposomes [90–100]. Typical cryoprotective agents are sorbitol, mannose, trehalose, glucose and polyvinylpyrrolidone. They decrease the osmotic activity of water and crystallization and favor the glassy state of the frozen sample [88,91,94,95,97,100]. Cryoprotectors are place holders which prevent the contact between discrete lipid nanoparticles. Furthermore, they

interact with the polar head groups of the surfactants and serve as a kind of ‘pseudo hydration shell’ [101]. Schwarz et al. investigated the lyophilization of SLN in great detail [102]. Best results were obtained with the cryoprotectors glucose, mannose, maltose and trehalose in concentrations between 10 and 15%. The observations are in agreement with the results of studies on liposome lyophilization, which indicated that trehalose was the most sufficient substance to prevent liposome fusion and leakage of the incorporated drug [94,103]. Mean particle sizes and size distributions of Compritol® and Dynasan®112 SLN were only slightly increased after freeze–thaw experiments. The particle sizes of reconstituted SLN-lyophilizates were still in the submicron range, but larger in comparison to the original dispersion (Dynasan®112-SLN 360 nm/103 nm; Compritol® SLN: 330 nm/160 nm) [102]. Encouraging results obtained with unloaded SLN cannot predict the quality of drug loaded lyophilizates. Even low concentrations of 1% tetracaine or etomidate caused a significant increase in particle size, which excludes an intravenous administration [102]. The observed instabilities are very likely caused by concentration effects of non-incorporated drug molecules during the freezing procedure, which lead to the formation of high concentrated tetracaine or etomidate solutions. The particle stability will be decreased under these conditions due to the decrease of the zeta potential, the changes in osmolarity and the possible pH effects. Westesen investigated the lyophilization of tripalmitin SLN (surfactants: 4.5% tyloxapol and 3% soy bean lecithin (Lipoid S 100)) [104]. Glucose, sucrose, maltose and trehalose were used as cryoprotective agents in concentrations of 5, 10 and 20%. Handshaking of redispersed samples was an insufficient method, while bath sonification produced better results. Average particle sizes of all lyophilized samples with cryoprotective agents were 1.5–2.4 times higher than the original dispersions. Cryoprotector free samples showed very high particle aggregation. Samples with a lipid content below 10% showed less aggregation than samples of higher concentration. The efficiency of the cryoprotectors decreases in the following order: trehalose >sucrose≫glucose and maltose. The time of the addition of the cryoprotector influences the quality of the final formulation. Best results were obtained when the cryoprotector was added to the sample prior to homogenization. Under these circumstances, average particle size remained almost unchanged. Storage over 1 year caused significant increases in particle sizes. Average particle sizes were 4–6.5 times larger than in the original dispersion. In contrast to the lyophilizates, the aqueous dispersions of tyloxapol/phospholipid stabilized tripalmitin SLN exhibited a remarkable storage stability. The average particle size increased only very slightly from 56 to 65 nm over 1 year. The instability of the SLN lyophilizates can be explained by sintering of the particles. TEM pictures of the tripalmitin SLN show an anisometric, platelet like shape of the particles. Lyophilization changes the properties of the surfactant layer due to removal of water and increases the particle concentration which favor particle aggregation. Cavalli observed increased particle sizes (2.1–4.9 times) after lyophilization, too [86]. A trehalose concentration of 2% was insufficient to prevent lyophilization-induced particle aggregation. Increasing the concentration of trehalose to 15% resulted in average particle sizes of around 100 nm and in polydispersity indices of 0.25 after reconstitution. Heiati compared the influence of four cryoprotectors (trehalose, glucose, lactose and mannitol) on the particle size of azidothymidine palmitate loaded SLN lyophilizates [87]. Trehalose was found to be the most effective cryoprotector for preventing aggregation during lyophilization and subsequent reconstitution of SLN. A sugar/lipid weight ratio of 2.6–3.9 was recommended. The freezing procedure will affect the crystal structure and the properties of the lyophilizate. Rapid cooling leads to small and heterogeneous crystals, improves the formation of amorphous lyophilizates and decreases freezing out effects. The crystal size is a key factor for

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the speed of sublimation during freeze drying and for the reconstitution process. A large number of small crystals and the presence of large amorphous regions might cause a slow sublimation due to the formation of a very dense lyophilizate network. Slow freezing leads to the formation of large crystals and results in unrestricted sublimation, but it increases the risk of freezing-related destabilization phenomena. Comparing the results of two studies shows that the freezing process needs to be optimized to a particular sample. Schwarz recommended rapid freezing in liquid nitrogen [102]. In contrast, other researchers observed the best results after a slow freezing process. Zimmermann found that optimization of the lyophilization parameters results in formulations which are i.v. injectable with regard to particle size [105]. Again, best results were obtained with samples of low lipid content and with the cryoprotector trehalose. Slow freezing in a deep freeze (−70 °C) was superior to rapid cooling in liquid nitrogen. Furthermore, introduction of an additional thermal treatment to the frozen SLN dispersion (2 h at −22 °C followed by a 2-h temperature decrease to −40 °C) was found to improve the quality of the lyophilizate. 3.3.3. Spray drying Spray drying might be an alternative procedure to lyophilization in order to transform an aqueous SLN dispersion into a dry product. This method has been used scarcely for SLN formulation, although spray drying is cheaper compared to lyophilization. Freitas obtained a redispersable powder by spray drying, which complies with the general requirements of i.v. injections with regard to the particle size and the selection of the ingredients [106]. Spray drying might potentially cause particle aggregation due to high temperatures, shear forces and partial melting of the particles. Freitas recommends the use of lipids with melting points >70 °C for spray drying. Furthermore, the addition of carbohydrates and low lipid content favor the preservation of the colloidal particle size in spray drying. The melting of the lipid can be minimized by using ethanol–water mixtures as a dispersion medium instead of pure water due to the lower inlet temperatures. The best result was obtained with SLN concentrations of 1% in solutions of 30% trehalose in water or 20% trehalose in ethanol– water mixtures (10/90 v/v). 4. Characterization of SLN quality and structure 4.1. Defining the goals An adequate characterization of the solid lipid nanodispersion is a necessity for the control of the quality of the product. The characterization methods should be sensitive to the key parameters of SLN performance and should avoid artifacts. However, characterization of SLN is a serious challenge due to the colloidal size of the particles and the complexity of the system, which includes also dynamic phenomena. One statement of Laggner about lipids should always be kept in mind [107]: “Lipids and fats, as soft condensed material in general, are very complex systems, which not only in their static structures but also with respect to their kinetics of supramolecular formation. Hysteresis phenomena or supercooling can gravely complicate the task of defining the underlying structures and boundaries in a phase diagram.” This is especially true for lipids in the colloidal size range. Many analytical tools do not permit direct measurement in the undiluted SLN dispersion. Therefore, possible artifacts caused by sample preparation (removal of emulsifier from particle surface by dilution, induction of crystallization processes, changes of lipid modifications) should be kept in mind. For example, the exposure of an SLN dispersion to a syringe needle might result in spontaneous transformation of the low viscous SLN dispersion into a viscous gel. In this case, the artifact caused by sample preparation is clearly visible, in other cases it will not be.

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What are the most important key factors which have a direct impact on the stability and release kinetics? Several parameters have to be considered: 1. Particle size and zeta potential 2. Degree of crystallinity and lipid modification 3. Coexistence of additional colloidal structures (micelles, liposomes, supercooled melts, drug-nanoparticles) and time scale of distribution processes 4.2. Measurement of particle size and zeta potential Photon correlation spectroscopy (PCS) and laser diffraction (LD) are the most powerful techniques for routine measurements of particle size. The Coulter Counter method is rarely used to measure SLN particle size because of difficulties in the assessment of small nanoparticles and the need of electrolytes which may destabilize colloidal dispersions. PCS (also known as dynamic light scattering) measures the fluctuation of the intensity of the scattered light which is caused by particle movement. This method covers a size range from a few nanometers to about 3 microns. This means that PCS is a good tool to characterize nanoparticles, but it is not able to detect larger microparticles. They can be visualized by means of LD measurements. This method is based on the dependence of the diffraction angle on the particle radius (Fraunhofer spectra). Smaller particles cause more intense scattering at high angles compared to the larger ones. A clear advantage of LD is the coverage of a broad size range from the nanometer to the lower millimeter range. The development of PIDS technology (Polarization Intensity Differential Scattering) greatly enhanced the sensitivity of LD to smaller particles. However, despite this progress, it is highly recommended to use PCS and LD simultaneously. It should be kept in mind that both methods do not ‘measure’ particle sizes. Rather, they detect light scattering effects which are used to calculate particle sizes. For example, uncertainties may result from nonspherical particle shapes. Platelet structures commonly occur during lipid crystallization [108] and have also been suggested in the SLN literature [49]. Furthermore, difficulties may arise both in PCS and LD measurements for samples which contain several populations of different size. Therefore, additional techniques might be useful. For example, light microscopy is recommended, although it is not sensitive to the nanometer size range. It gives a fast indication of the presence and character of microparticles (microparticles of unit form or microparticles consisting of aggregates of smaller particles). Electron Microscopy provides, in contrast to PCS and LD, direct information on the particle shape. However, the investigator should pay special attention to possible artifacts which may be caused by the sample preparation. For example, solvent removal may cause modifications which will influence the particle shape [108]. Atomic force microscopy (AFM) is attracting increasing attention. For example, imaging of fibrinogen polymerization [109], the budding of a virus of an infected cell [110], the in vitro degradation of polymer surfaces [111] and polymer nanoparticles [112,113] were performed. This technique utilizes the force acting between a surface and a probing tip resulting in a spatial resolution of up to 0.01 nm for imaging. Striking advantages of AFM are the simplicity of sample preparation, as no vacuum is needed during operation and that the sample does not need to be conductive. Therefore, it has the potential for the direct analysis of the originally hydrated, solvent containing samples. It has been reported for biological compounds that it is sufficient to prepare samples by placing a drop of solution or dispersion of a sample on a washed microscope slide or on a mica substrate [109,114]. The atomic force microscope obtains images quickly enough (about 20 s per image) to allow the observation of in situ processes occurring at interfaces. A cautionary note applies to the use of AFM in the field of nanoparticles, because an immobilization of the SLN is required to

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assess their shape by the very tiny AFM tip. Immobilization will be found for microparticles due to sedimentation of the particles. However, submicron particles are moving rapidly (see particle sizing by PCS) and can therefore not be assessed without preparatory steps such as solvent removal which may cause substantial changes of the molecular structure of the particles. zur Mühlen demonstrated the ability of AFM — especially by operating in the non-contact mode — to image the morphological structure of solid lipid nanoparticles [115]. The size of the visualized particles was of the same magnitude compared with the results of PCS measurements. The AFM investigations revealed the disk-like structure of the particles. AFM images indicate that the crystalline particles are surrounded by soft layers. The softness of these layers was proved by form alterations, which occurred if they were imaged successively by contact AFM. By monitoring force distance curves the hardness of the particle surface compared to the silicon surface was quantified. In contrast to the silicon surface a form deformation of about 1 nm occurred when the tip was pushed into the particle. Dingler investigated cetyl palmitate SLN (stabilized by polyglycerol methylglucose distearate, Tego Care 450®) by electron microscopy and AFM [116,117]. Both methods suggest an almost spherical form of the particles. Different SLN shapes, such as cubical or platelet-like patterns, were reported by Westesen for SLN made of well defined lipids of high purity (e.g. pure triglycerides) [49,118]. The chemically homogenous lipid tends to form more or less perfect crystals with the typical platelet-like pattern of the β-modification. The use of chemically heterogeneous lipids in combination with heterogeneous surfactants favors the formation of ideally spherical lipid nanoparticles. Rapid progress in the development of field–flow-fractionation (FFF) has been observed during recent years. The separation principle of FFF is based on the different effect of a perpendicular applied field on particles in a laminar flow [119–122]. The separation principle corresponds to the nature of the perpendicular field and may for example be based on different mass (sedimentation FFF), size (cross-flow FFF) or charge (electric field FFF). A combination of different FFF separation principles may give unique resolution. A certain advantage of FFF over PCS is the high resolution of small particle size differences. Pilot studies with lattices of different size demonstrate that particles with a size difference of 30 nm are well resolved. Furthermore, FFF leads to a separation of the particles, which means that the separated particles may be subjected to further characterization. The high dilution of the sample by FFF may cause potential problems because it may disturb the sample characteristics (for example, dilution with pure water may cause removal of the surfactant from the particle surface). Current studies at the Free University Berlin investigate the influence of the dilution media on the particle characteristics. Due to the advantages of FFF and the development of commercial FFF product lines it can be anticipated that FFF will be a key tool for the characterization of colloidal dispersions like SLN in the future. The measurement of the zeta potential allows for predictions about the storage stability of colloidal dispersion [123]. In general, particle aggregation is less likely to occur for charged particles (high zeta potential) due to electric repulsion. However, this rule cannot strictly be applied for systems which contain steric stabilizers, because the adsorption of steric stabilizers will decrease the zeta potential due to the shift in the shear plane of the particle (for detailed discussion see [123]).

4.3. Measurement of crystallinity, lipid modification and assessment of alternative colloidal structures including the time scale of distribution processes Particle size analysis is a necessary, but not a sufficient step to characterize SLN quality. Special attention must be paid to the characterization of the degree of lipid crystallinity and the modification of the lipid, because these parameters are strongly correlated with drug

incorporation and release rates. Both thermodynamic stability, lipid packing density increase, while drug incorporation rates decrease in the following order: Supercooled melt; α-modification; β′-modification; β-modification Due to the small size of the particles and the presence of emulsifiers, lipid crystallization and modification changes might be highly retarded. For example, it has been observed, that polymorphic transitions might occur very slowly and that Dynasan®112 SLN — if crystallization is not artificially induced — may remain as a supercooled melt over several months [118,124]. Differential scanning calorimetry (DSC) and X-ray scattering are widely used to investigate the status of the lipid. DSC uses the fact that different lipid modifications possess different melting points and melting enthalpies. By means of X-ray scattering it is possible to assess the length of the long and short spacings of the lipid lattice. It is highly recommended to measure the SLN dispersions themselves because solvent removal will lead to modification changes. Sensitivity problems and long measurement times of conventional X-ray sources might be overcome by synchrotron irradiation [118]. In addition, this method means that time-resolved experiments can be conducted and it permits the detection of intermediate states of colloidal systems which will be undetectable by conventional X-ray methods [107]. Unfortunately, this source has limited accessibility for most investigators. Infrared and Raman spectroscopy are useful tools for investigating structural properties of lipids [125]. Their potential to characterize SLN dispersions has yet to be explored. Rheometry might be particularly useful for the characterization of the viscoelastic properties of SLN dispersions. Studies by Lippacher show that the SLN dispersion possesses higher elastic properties than emulsions of comparable lipid content [57]. Furthermore, a sharp increase of the elastic module is observed at a certain lipid content. This point indicates the transformation from a low viscous lipid dispersion to an elastic system with a continuous network of lipid nanocrystals. The coexistence of additional colloidal structures (micelles, liposomes, mixed micelles, supercooled melts, drug nanoparticles) has to be taken into account for all SLN dispersions. The characterization and quantification are a serious challenge due to the similarities in size combined with the low resolution of PCS to detect multimodal distributions. Furthermore, the sample preparation will modify the equilibrium of the complex colloidal system. Dilution of the original SLN dispersion with water might cause the removal of surfactant molecules from the particle surface and induce further changes such as crystallization or changes of the lipid modification. Therefore, it would be highly desirable to use methods which are sensitive to the simultaneous detection of different colloidal species and which do not require preparatory steps. Both nuclear magnetic resonance (NMR) and electron spin resonance (ESR) meet these requirements. They are powerful tools for investigating dynamic phenomena and the characteristics of nanocompartments in colloidal lipid dispersions. Due to the non-invasiveness of both methods, repeated measurements of the same sample are possible. NMR active nuclei of interest are 1H, 13C, 19F and 31P. Due to the different chemical shifts it is possible to attribute the NMR signals to particular molecules or their segments. For example, lipid methyl protons give signals at 0.9 ppm while protons of the polyethylene glycol chains give signals at 3.7 ppm (Fig. 2). Simple 1H-NMR spectroscopy permits an easy and rapid detection of supercooled melts due to the low linewidths of the lipid protons [126]. This method is based on the different proton relaxation times in the liquid and semisolid/solid state. Protons in the liquid state give sharp signals with high signal amplitudes, while semisolid/solid protons give weak and broad NMR signals under these circumstances (Fig. 2). It also allows for the characterization of liquid nanocompartments in recently developed lipid particles, which are made from blends of solid and liquid lipids [127]. The great potential of NMR with its variety of different approaches (solid-state NMR, determination of self-diffusion coefficients etc.) has

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scarcely been used in the SLN field, although it will provide unique insights into the structure and dynamics of SLN dispersions. ESR requires the addition of paramagnetic spin probes to investigate SLN dispersions. A large variety of spin probes is commercially available. The corresponding ESR spectra give information about the microviscosity and micropolarity. ESR permits the direct, repeatable and non-invasive characterization of the distribution of the spin probe between the aqueous and the lipid phase. Experimental results demonstrate that storage-induced crystallization of SLN leads to an expulsion of the probe out of the lipid into the aqueous phase (Fig. 3) [80]. Furthermore, using an ascorbic acid reduction assay it is possible to monitor the time scale of the exchange between the aqueous and the lipid phase. The development of low-frequency ESR permits non-invasive measurements on small mammals. ESR spectroscopy and imaging will give new insights about the fate of SLN in vivo. 5. Possible problems in SLN preparation and SLN performance SLN offer several advantages compared to other systems (easy scaling up, avoidance of organic solvents, high content of nanoparticles). These advantages have been discussed in a variety of papers. However, less attention has been paid to the detailed and appropriate investigation of the limitations of this carrier system. Therefore, these aspects will be discussed in the following part of the article. Points to consider include high pressure-induced drug degradation, the coexistence of different lipid modifications and different colloidal species, the low drug-loading capacity and the kinetics of distribution processes.

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polyacrylamide gel electrophoresis and the rate of lysis of Micrococcus lysolideikticus [129]. He reported that the peptide remained in its active form after cold homogenization. According to the data in the literature, it can be stated that HPH-induced drug degradation will not be a serious problem for the majority of the drugs. However, HPH might be not suitable for the processing of shear sensitive compounds (DNA, albumin, erythropoietin). 5.2. Lipid crystallization and drug incorporation Lipid crystallization is an important point for the performance of the SLN carriers. The relation between lipid modification and drug incorporation has been investigated for decades. The characterization of lipid modifications is well established. Methods are mainly based on X-ray and DSC measurements. However, most of the data have been extracted from investigations on bulk lipids. The behavior of SLN might differ considerably due to the very small size of the particles and the high amount of surfactant molecules which are necessary to stabilize the colloidal lipid dispersion. Therefore, surface-related phenomena and lipid–surfactant interactions may contribute to a great extent to the properties of the lipid particle. The following four key aspects should be considered in the discussion of drug incorporation into SLN: 1. 2. 3. 4.

The existence of supercooled melts The presence of several lipid modifications The shape of lipid nanodispersions Gelation phenomena

5.1. High pressure-induced drug degradation HPH has been shown to decrease the molecular weight of polymers [60]. High shear stress has been assumed to be the major cause and evidence of free radical formation was reported. This study also indicated that cavitation is less important for the mechanism of polymer degradation. Cavitation can be suppressed by the application of back pressure without significant changes of the homogenization efficiency. The molecular weight and the general molecular structure are the most important parameters for predicting the drug degradation. High molecular weight compounds and long chain molecules are more sensitive than low molecular weight drugs or molecules with a spherical shape. For example, it was found that HPH causes degradation of DNA and albumin [128]. Almeida investigated the influence of HPH on the activity of the peptide lysozyme by means of sodium dodecyl sulphate–

Fig. 3. ESR spectra of TEMPO loaded SLN dispersion stored at RT and 5 °C. Experimental 2.1-GHz ESR spectra of TEMPO loaded (0.2 mmol/l) aqueous dispersions: (a) stored at RT; (b) stored at 5 °C. Quantitative assessment was done by spectral simulation of the lipophilic component (c) aN = 0.156 mT) and the hydrophilic component (d) aN = 0.168 mT). Low temperature storage doubles the percentage (50% vs. 25%) of the nitroxide molecules excluded from the lipophilic drug carrier).

5.2.1. Supercooled melts Supercooled melts are not unusual in SLN systems [63]. They describe the phenomenon that lipid crystallization may not occur although the sample is stored at a temperature below the melting point of the lipid. Supercooled melts are not lipid nanosuspensions but emulsions. Special attention should be paid to supercooled melts, because the potential advantages of SLN over nanoemulsions are linked to the solid state of the lipid. The main reason for the formation of supercooled melts is the size dependence of crystallization processes. Crystallization requires a critical number of crystallization nuclei to start [130]. This critical number of molecules is less likely to be formed in small droplets and therefore, the tendency of the formation of supercooled melts increases with decreasing droplet size. The range of supercooling (temperature difference between the melting and crystallization points) can reach 30–40 °C in lipid dispersions. For example, the melting temperature of trilaurin is > 40 °C, but in phospholipid/tyloxapol stabilized nanodispersions the lipid recrystallizes at temperatures below the freezing point of water [124]. In addition to size, crystallization can be affected by emulsifiers, incorporated drugs and other factors. It is therefore necessary to proof the solid state of the lipid by appropriate analytical techniques such as NMR, X-ray or DSC. Among these, NMR permits a very rapid and nondestructive analysis of the presence of supercooled melts. 5.2.2. Lipid modifications It is not sufficient to describe the physical state of the lipid as crystallized or non-crystallized, because the crystallized lipid may be present in several modifications of the crystal lattice. In general, lipid molecules have a higher mobility in thermodynamically unstable configurations. Therefore, these configurations have a lower density and ultimately, a higher capability to incorporate guest molecules (e.g. drugs). The advantage of higher incorporation rates in unstable modifications is paid off by an increased mobility of the drug. During storage, rearrangement of the crystal lattice might occur in favor of thermodynamically stable configurations and this is often connected with expulsion of the drug molecules. The performance of the SLN system will be determined to

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a large extent by the lipid modification, because this parameter triggers drug incorporation and drug release. Aspects of reproducibility and drug safety demand the assurance of systems with defined and reliable characteristics. Therefore, the utilization of the higher drugloading capacity in unstable configurations requires the development of strategies to prevent modification during storage. Further opportunities of modified drug release profiles will be open, if this problem will be solved. For example, Jenning has shown in vitro on skin that the evaporation of water leads to modification changes of SLN dispersions which cause drug expulsion from the lipid and result in increased penetration of the drug into the skin [131]. It has to be mentioned that the problem of lipid modifications is not always solved with the assignment to α, β, or β′ form. Complexity further increases due to several subspecies and the interactions of the lipid with the emulsifiers. Furthermore, recent work of Westesen’s group demonstrates that the particle size itself is the decisive factor for the physical properties of SLN [132]. Several sharp peaks were observed in DSC thermograms which correspond to a different number of lipid layers of the SLN particles. 5.2.3. Particle shape The shape of lipid nanoparticles may significantly differ from a sphere. Lipids prefer to crystallize in the platelet form [49,108]. Differences in shape between nanoemulsion (spheres) and SLN (platelets) have been observed by TEM [49,133]. The shape of the lipid crystals is not only a theoretical matter of particle sizing (PCS and LD assume spherical shapes). Platelet shapes have much larger surface areas compared to spheres, therefore, higher amounts of surfactants are needed for stabilization. Particle sizes of 100 nm (measured by PCS or LD) translate into 20 lipid layers assuming if a spherical shape. However, they translate into smaller values if a platelet structure exists. Cryo transmission electron microscopy studies of trimyristin nanoparticles give clear evidence that PCS sizes of 130 nm correspond to only 1–5 (!!) lipid layers [132]. Therefore, a much higher amount of the drug will be localized directly on the surface of the particles, which is in conflict with the general aim of the SLN systems (drug protection and controlled release due to the incorporation of the drug in the solid lipid). 5.2.4. Gelation phenomena Gelation phenomena describe the transformation of a low-viscosity SLN dispersion into a viscous gel. This process may occur very rapidly and unpredictably. In most cases, gel formation is an irreversible process which involves the loss of the colloidal particle size. It can be stimulated by intense contact of the SLN dispersion with other surfaces and shear forces. A typical example is a syringe needle. If gelation occurs in vitro during preparatory steps of SLN characterization, results will be influenced by artifact generation. However, if this happens during the i.v. injection into a living species the life of this organism is put at risk. Siekmann suggested that gel formation is connected with crystallization processes. Strange surfaces induce crystallization or change of modification of the lipid crystals. This process is connected with an increase of the particle surface due to the preferred formation of platelets (in β-modification). The surfactant molecules cannot longer provide sufficient coverage of the new surfaces and therefore, particle aggregation is observed [134]. Gelation can be retarded or prevented by the addition of co-emulsifying surfactants with high mobility (e.g. glycocholate) [135]. Freitas found that high temperatures, exposure to light and mechanical stress promote gelation in SLN (10% Compritol® and 1.2% poloxamer 188) [136]. Storage in darkness at 8°C prevented particle growth. In addition it was found that fat samples stored under nitrogen atmosphere were more stable than samples filled under regular air. High lipid concentrations and high ionic strengths promote gelation [137]. The zeta potential was a good predictor of gelation phenomena. Stable samples had a zeta potential of − 25 mV, while a

zeta potential of − 15 mV indicated the beginning of gelation phenomena. Similar values have been reported for the stability of parenteral fat emulsions [138]. Several mechanisms might be involved in the gelation process. All promoters of gelation (high temperature, light, shear stress) increase the kinetic energy of the particles and favor collision of the particles. The surfactant film might change his performance with temperature (especially PEG-surfactants!). Further aspects relate to the kinetics of crystallization and transformation between the lipid modifications which will be influenced by the factors mentioned above. Rapid crystallization of the lipid increases the gelation process [134,139]. The crystallinity of Compritol® SLN is comparatively high (about 70–80%) on the day of production. The liquid parts crystallize during storage. Stable SLN systems do not completely recrystallize during the storage time of 3 years and contain several lipid modifications (unstable α and subα, more stable β′) [140]. Semisolid samples contain only β′ and α modification, while complete gelation leads to the complete transformation to the β′ modification. The presence of liquid phases promotes the crystallization in the stable form because unstable crystals may redissolve and crystallize in the stable modification [141]. In this way, it is possible to accelerate the α→β′ transformation during storage at RT without melting of the Compritol. In most cases, triglycerides will crystallize in the α modification. The α→β′ transformation can be retarded by surfactants, e.g. poloxamer [134,142]. Freitas observed that a nitrogen atmosphere had similar effects. The retardation by nitrogen was attributed to the inhibition of the lipid hydrolysis (pH effect) [83]. The crystal size of the α modification of polycrystalline lipid aggregates is few microns or less. A change of the modification is associated with an increase in particle size (β′: less than 5 μm; β: 20–100 μm) [108]. Westesen demonstrated by TEM that tripalmitin and tristearin crystals have a spherical shape in the α modification. The β′ modification is built up from stapled spheroids and the stable β modification is built up from long, coagulated platelets [135]. 5.3. Coexistence of several colloidal species The presence of several colloidal species is an important point to consider. Unfortunately, this aspect has been ignored in the majority of the SLN literature. Stabilizing agents are not localized exclusively on the lipid surface, but also in the aqueous phase. Therefore, micelle forming surfactant molecules (e.g. SDS) will be present in three different forms, namely: (i) on the lipid surface; (ii) as micelle; and (iii) as surfactant monomer. Lecithin will form liposomes, which have also been detected in nanoemulsions for parenteral nutrition [143]. Mixed micelles have to be considered in glycocholate/lecithin stabilized and related systems. Micelles, mixed micelles and liposomes are known to solubilize drugs and are, therefore, alternative drug incorporation sites. In a recent ESR study on cetyl palmitate SLN, we found that lipophilic nitroxides were almost exclusively localized in the micelles and not in the wax matrix. We are currently establishing NMR-based methods to determine drug localization in undiluted samples. Nondilution is a requirement for the avoidance of artifacts. For example, mixed micelles are sensitive to dilution [144] and the structure of poloxamer micelles is concentration dependent [145]. Only the detection of the presence of several colloidal species is not sufficient to describe the structure of colloidal lipid dispersions, because dynamic phenomena are very important for drug stability and drug release. Therefore, the kinetics of distribution processes have to be considered. For example, the degradation of hydrolysable drugs will be faster for water solubilized and surface localized molecules compared to molecules in the lipid matrix. The kinetics of the degradation will be determined by: (i) the chemical reactivity of the drug; and (ii) the concentration of the drug in the aqueous medium or at the lipid/water interface. Unstable drugs will hydrolyze rapidly in contact

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with water and, therefore, the distribution equilibrium of the drug between the different environments will be distorted. Carrier systems will be protective only if they prevent the redistribution of the drug. Of course, increasing the matrix viscosity will decrease the diffusion coefficient of the drug inside the carrier and, therefore, SLN are expected to be superior to lipid nanoemulsions. However, drug stabilization is a very challenging task for colloidal drug carriers, because of the very high surface area and the short diffusion pathways. It should also be kept in mind that the surface of SLN dispersions might be greater than that of a nanoemulsion with comparable lipid content due to the non-spherical shape of the particles. Comparative ESR studies between polylactide–co-glycolide polymers and several lipid matrices (conducted in our laboratory) indicate a much higher mobility in the lipid bulk materials. Therefore, it can be expected that polymeric nanoparticles will be more effective in immobilizing drug molecules. From the ESR spectra, it can be estimated that the diffusion coefficient is in the range of 10−11 cm2 s−1 or even higher. It means that incorporated molecules will need approximately a few seconds at most to reach the interface. It does not mean that SLN may not be able to decrease drug degradation, because an appropriate surfactant film may protect very well. However, it suggests that the protective effect of SLN cannot be anticipated and, if observed, not solely attributed to the increased immobilization of the incorporated drug molecules. Indeed, protection of tocopherol in SLN was only moderate in comparison to Lipofundin® nanoemulsions [116]. The kinetics of distribution processes can also be studied with an ESR ascorbic acid reduction assay. Ascorbic acid reduces nitroxide molecules only in water or at interfaces to ESR silent hydroxylamines. Our recent results indicate a higher nitroxide protection in polymer nanoparticles compared to SLN. Ahlin conducted similar studies on lecithin stabilized SLN and liposomes. These results indicate, that the protection of the nitroxide was best in the liposome system [146]. In both studies, nitroxide reduction took place a matter of minutes and not hours or days. An interesting finding of Ahlin was that the reduction rate decreases with storage time. This change represents most likely distribution phenomena of the nitroxide from the SLN into the liposomes. 6. Drug incorporation and drug release A large number of drugs with a great variety of lipophilicity and the general structure have been studied with regard to their incorporation into SLN [147], e.g. oxazepam, diazepam, cortisone, betamethasone valerate, retinol, prednisolone, retinol, menadione, ubidecarenone [148], timolol [149,150], desoxycortisone [71], pilocarpine [151], progesterone [152], hydrocortisone [152], idarubicin [53], doxorubicin [53], thymopentin [153], [D-Trp-6]LHRH [154], gadolinium (III) complexes [155], 3′-azido-3′-deoxythymidine palmitate [156,157], azidothymidine palmitate [87], camptothecin [158,159], aciclovir [160,161], cyclosporine [162], vitamin E palmitate [117], etomidate, tetracaine [59]. As discussed before, drug loading might result in strong changes of the SLN characteristics (particle size distribution, zeta potential, lipid modification etc.). Drug incorporation implies the localization of the drug in the solid lipid matrix. However, several alternative incorporation sites (micelles, mixed micelles, liposomes, drug-nanosuspensions) exist in addition to the complex physicochemical status of the lipid (supercooled melt and several modifications). Unfortunately, these aspects have been neglected in the design and discussion of most SLN experiments. The following precautionary remarks should be kept in mind during the interpretation of the published results: Particle size measurements alone are not sufficient for the characterization of SLN dispersions. The modification of the lipid (and the drug) should be characterized by DSC, X-ray and NMR techniques. The selection of the control sample deserves special attention in order to prove that the observed result was due to the colloidal lipid carrier and not due to other colloidal structures

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which are present in the sample. For example, an appropriate control to a lecithin-stabilized SLN dispersion would be the liposome formulation produced under the same conditions. Glycocholate/lecithin stabilized SLN should be compared to mixed micelles. A large number of drugs, including very hydrophilic molecules, has been postulated to be incorporated into SLN. However, few data exist on the localization site and the physical state of the drug molecule. Lukowski observed, by electron diffraction, that aciclovir is not molecularly dissolved in the lipid matrix [160]. Most likely, the lipid and aciclovir nanoparticles are coexisting. Bunjes used NMR techniques to characterize the physicochemical state of diazepam, the lipid and the emulsifier. The published NMR spectra of diazepam indicate a high mobility of the drug [163]. Ahlin and Kristl [146,164] as well as our group [165] used ESR techniques to monitor the physical state of model drugs. Independently, it was found that a high percentage of lipophilic nitroxides is localized in the polar environment and that distribution processes occur rapidly. It can be anticipated, that new NMR, ESR and synchrotron irradiation studies will shed more light on the incorporation and release processes of SLN. Especially solid state NMR techniques will be very powerful for the characterization of the molecular arrangement of the drug and the lipid. In most cases, burst release is observed from SLN. For example, both hot and cold homogenization produced SLN released tetracaine and etomidate immediately [59]. In contrast, it was possible to retard the release of prednisolone by the cold homogenization technique [58,59]. An appropriate selection of the homogenization temperature permitted the modification of the release profile. The release kinetics depend on the release conditions (sink or non-sink conditions, release medium etc). Due to the colloidal size, release studies are not trivial experiments. Unfortunately, release experiments were conducted under several conditions (with sample separation by filtration or centrifugation or by dialysis) and therefore, it is not easy to compare the results. Every technique has its own advantages (simplicity, time) and drawbacks (possible artifacts due to separation; retardation of release by dialysis bag). Further experiments have to be done to elucidate the contribution of enzymes to drug release from SLN. It has been shown that matrix degradation by lipase depends on the lipid and the emulsifier. The balance between steric stabilizers and other surfactants can possibly be used to modify the release and particle degradation, because lipases need a lipid interface for activation of the enzyme (lid opening) [166]. Therefore, PEG-coated SLN are not easily recognized by these enzymes.

7. Storage stability SLN and nanoemulsions have remarkable similarities with respect to their composition and production methods. However, SLN cannot simply be regarded as colloidal lipid dispersions with solidified droplets. The problems connected with the presence of additional colloidal structures (micelles, mixed micelles, liposomes) exist for both carrier systems. However, SLN have additional features (supercooled melts, different modifications, non-spherical shapes) which are contributing to or determining the stability of the colloidal lipid suspension. Gelation phenomena, increase in particle sizes and drug expulsion from the lipid carrier are the major problems of storage stability. As described above, there is a close relation between the modification of the lipid, gelation, particle aggregation and drug expulsion. A supercooled melt, which is the first product formed after hot homogenization, represents a nanoemulsion. It is characterized by spherical lipid droplets and a high incorporation rate for guest molecules (e.g. drugs). The transformation of the lipid melt to lipid crystals results in an increase of particle surface, a decrease of the loading capacity of the lipid and therefore, it leads to increased stability problems. Stability of the lipid dispersions decreases as stability of the lipid modification increases.

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8. Toxicity aspects and in vivo fate

9. Administration routes and in vivo fate

One can anticipate that SLN are well tolerated in living systems because they are made from physiological compounds and therefore, metabolic pathways exist. Of course, the toxicity of the emulsifiers has to be considered, but their potential toxicity is relevant for other carrier systems, too. No problems should be observed for peroral or transdermal administration and i.m. or s.c. injection if appropriate surfactants are used. The particle size is not a very critical issue for these administration routes, because a low content of microparticles might decrease the performance of the SLN system, but will not cause toxic events. The absence of pyrogens must be checked for parenteral administration. Problems may arise, because SLN may interfere with the pyrogen tests (limulus test) and cause gelation in any case. SLN were originally designed for the controlled release of drugs after intravenous injection. The change of the lipid droplet (nanoemulsions) to a solid core (SLN) should decrease drug delivery due to decreased drug diffusion coefficients. Particle size distribution is a key issue for i.v. injection due to the danger of capillary blockage which could result in death due to fat embolism. The diameter of the fine capillaries is about 9 microns. For safety reasons, the particle size should be completely in the submicron range. However, microparticles exceeding the size of the fine capillaries have been found in commercial nanoemulsions for parenteral nutrition [167,168]. Obviously, a certain amount of larger microdroplets is tolerated by the human body. This statement should not be transferred to SLN, because a solid lipid is not deformable as an oil, and therefore, in contrast to nanoemulsions, capillary blockage will occur if the particle size exceeds the size of the diameter of the blood vessel. Gelation of the low-viscosity SLN dispersion in the syringe needle might occur, which will immediately form a viscous suspension with unacceptable particle sizes. Both the solid state of the lipid and the danger of injection-induced gelation are serious hurdles for the development of SLN dispersions suitable for i.v. injection in the clinical practice. The interaction of SLN with phagocytizing cells has been studied in vitro on human granulocytes [169,170]. A luminol-based chemoluminescence was used to compare SLN with polymer particles and to assess the influence of the SLN composition on the phagocytosis rate. It was found that phagocytosis rate of poloxamer stabilized Compritol® and cetyl palmitate SLN was lower in comparison to polystyrene nanoparticles [170–172]. An indirect chemoluminescence assay was developed in order to distinguish the small differences in SLN phagocytosis [173]. The results indicate that poloxamine 908 prevents the uptake of Compritol®

The in vivo fate of the SLN particles will depend mainly on the following points:

SLN more efficiently than poloxamer 407 [174]. Bocca et al. used dipalmitoyl–phosphatidylethanolamine–PEG and stearic acid–PEG 2000 to produce stealth SLN [175]. The steric stabilizers reduced the SLN uptake in murine macrophages to a large extent. It has also been demonstrated that PEG chains prevent the interaction of SLN with human serum albumin [176]. According to the mechanism proposed by Torchilin, surface grafted chains of flexible and hydrophilic polymers (e.g. polyethylene glycol) form dense ‘conformational clouds’ preventing macrophages from interacting with nanoparticle surface even at a low concentration of the protecting polymer [177]. The results of cytotoxicity studies (MTT test) indicated that SLN are less toxic than polymeric nanoparticles [178,179]. The viability of human granulocytes was 84% after incubation with 2.5% poloxamer188–cetyl palmitate SLN and 72% after incubation with 5% poloxamer188–Compritol® SLN. Poloxamer stabilized polylactide/ polyglycolide particles reduced the cell viability to 50% at a concentration of 0.1%. Higher concentrations (0.5%) led to complete cell death [180]. Similarly, low cytotoxicity of SLN has been observed in a comparable study on poloxamine 908 and poloxamer 407 stabilized SLN [181]. The experimental data of the toxicity studies on cells in vitro confirm the expected low toxicity of the carrier.

Peroral administration forms of SLN may include aqueous dispersions or SLN loaded traditional dosage forms, e.g. tablets, pellets or capsules. The microclimate of the stomach favors particle aggregation due to the acidity and high ionic strength. It can be expected, that food will have a large impact on SLN performance, however, no experimental data have been published on this issue to our knowledge. The question concerning the influence of the stomach and pancreatic lipases on SLN degradation in vivo remains open, too. Unfortunately, only few in vivo studies have been performed yet. Recently, camptothecin (CA)-containing SLN were produced from stearic acid (2%), lecithin (1.5%) and poloxamer 188 (0.5%) [159]. The claimed encapsulation efficiency of CA was 99.6%. The zeta potential (−45 mV) was remarkably high for poloxamer stabilized dispersions. Unfortunately, the authors provided neither X-ray nor DSC data of the dispersion. With respect to the high concentrations of the stabilizers (same weight as stearic acid) and the method of determination of encapsulation efficiency (ultrafiltration) it is not possible to conclude that CA was molecular dispersed in solid stearic acid nanocrystals. It is likely that CA forms nanocrystals or that it is incorporated in micelles. The plasma levels and body distribution were

(a) administration route (b) interactions of the SLN with the biological surroundings including: (b1) distribution processes (adsorption of biological material on the particle surface and desorption of SLN components into the biological surrounding) (b2) enzymatic processes (e.g. lipid degradation by lipases and esterases) SLN are composed of physiological or physiologically related lipids or waxes. Therefore, pathways for transportation and metabolism are present in the body which may contribute to a large extent to the in vivo fate of the carrier. Probably the most important enzymes of SLN degradation are lipases, which are present in various organs and tissues. Lipases split the ester linkage and form partial glycerides or glycerol and free fatty acids. Most lipases require activation by an oil/water interface, which opens the catalytic center (lid opening) [182–184]. In vitro experiments indicate that solid lipid nanoparticles show different degradation velocities by the lipolytic enzyme pancreatic lipase as a function of their composition (lipid matrix, stabilizing surfactant) [166,185–187]. As a measure to follow the degradation, the free fatty acids formed have been analyzed using an enzymatic test [188]. In these studies SLN degradation showed dependences in relation to the length of the fatty acid chains in the triglycerides and the surfactants. The longer the fatty acid chains in the glycerides, the slower the degradation was. The effect of the surfactants on the degradation effect can be to accelerate it (e.g. cholic acid sodium salt) or to hinder it/slow it down due to steric stabilization (e.g. poloxamer 407, poloxamer 188). As a second steric stabilizer, Tween 80 has been used and the results showed that the hindering effect on the degradation process was less pronounced than that of poloxamer 407. This result seems to be correlated to the number of ethylene glycol chains in the molecule, which suppress the anchoring of the lipase/colipase complex and consequently the degradation of the SLN. The result could be used to adjust degradation of SLN and consequently drug release in a controlled way. The degradation of SLN based on waxes (e.g. cetyl palmitate) was found to be slower compared to glyceride matrices [185]. 9.1. Peroral administration

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determined after administration of CA–SLN suspension versus a CA solution (CA-SOL). Two plasma peaks were observed after administration of CA–SLN. The first peak was attributed to the presence of free drug, the second peak can be attributed to controlled release or potential gut uptake of SLN. These two peaks were also found in the total CA concentration–time profiles of all measured organs. It was also found that the incorporation into SLN protected CA from hydrolysis. The conclusion from this study was that SLN are a promising sustained release system for CA and other lipophilic drugs after oral administration. Increased bioavailability and prolonged plasma levels have been described after peroral administration of cyclosporine containing lipid nanodispersions to animals [189]. An increased uptake of SLN into the lymph has been described by Bargoni after intraduodenal administration [190].

9.2. Parenteral administration SLN have been administered intravenously to animals. Pharmacokinetic studies of doxorubicin incorporated into SLN showed higher blood levels in comparison to a commercial drug solution after i.v. injection in rats. Concerning the body distribution, SLN were found to cause higher drug concentrations in lung, spleen and brain, while the solution led to a distribution more into liver and kidneys [191]. Yang reported on the pharmacokinetics and body distribution of camptothecin after i.v. injection in mice. In comparison to a drug solution SLN were found to lead to much higher AUC/dose and mean residence times (MRT) especially in brain, heart and reticuloendothelial cells containing organs. The highest AUC ratio of SLN to drug solution among the tested organs was found in the brain [158]. The adsorption of a blood protein onto particle surfaces is supposed to be responsible for the uptake of SLN by the brain by mediating the adherence to the endothelial cells of the blood–brain barrier [192,193].

9.3. Transdermal application The smallest particle sizes are observed for SLN dispersions with low lipid content (up to 5%). Both the low concentration of the dispersed lipid and the low viscosity are disadvantageous for dermal administration. In most cases, the incorporation of the SLN dispersion in an ointment or gel is necessary in order to achieve a formulation which can be administered to the skin. The incorporation step implies a further reduction of the lipid content. An increase of the solid lipid content of the SLN dispersion results in semisolid, gel-like systems, which might be acceptable for direct application on the skin. Unfortunately, in most cases, the increase in lipid content is connected with a large increase of the particle size. Surprisingly it has been found that very high concentrated (30–40%), semisolid cetyl palmitate formulation preserve the colloidal particle size. A dramatic increase of the elastic properties was observed with increasing lipid content [57]. The rheological properties are comparable to typical dermal formulations. The results indicate that it is possible to produce high concentrated lipid dispersions in the submicron size range in a one-step production. Therefore, further formulation steps (e.g. SLN dilution in cream or gel) can be avoided. The cosmetic field offers interesting applications. It has been found in vitro that SLN have UV reflecting properties [194]. The UV reflectance is related to the solid state of the lipid and was not evident in nanoemulsions of comparable composition. These observations open the possibility of the development of SLN-based UV protective systems. The use of physiological components in SLN is a clear advantage over existing UV protective systems (UV blockers or TiO2) with respect to skin penetration and potential of skin toxicity.

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SLN have also been found to modulate drug release into the skin [126] and to improve drug delivery to particular skin layers [195] in vitro. The loss of water after application on the skin causes changes of the lipid modification and SLN structure. Electron microscopy indicates that dense films are formed after drying (32 °C) of SLN dispersions in contrast to spherical structures which have been proposed previously [116]. The formation of the dense structure will favor occlusive effects on skin. It is interesting to note that the films made from melts of the lipid bulk do not form close films as dried SLN dispersions do. The surfactant plays a significant role in preventing pore formation. 10. Summary and outlook Solid lipid nanoparticles do not, as proposed, “combine the advantages of other colloidal drug carriers and avoid the disadvantages of them”. They cannot simply be regarded as nanoemulsions with a solid core. Clear advantages of SLN include the composition (physiological compounds), the rapid and effective production process including the possibility of large scale production, the avoidance of organic solvents and the possibility to produce high concentrated lipid suspensions. Disadvantages include low drug-loading capacities, the presence of alternative colloidal structures (micelles, liposomes, mixed micelles, drug nanocrystals), the complexity of the physical state of the lipid (transformation between different modifications, possibility of supercooled melts) which cause stability problems during storage or administration (gelation, particle size increase, drug expulsion). Sample dilution or water removal might significantly change the equilibria between the different colloidal species and the physical state of the lipid. The appropriate characterization of the complex surfactant/lipid dispersions requires several analytical methods in addition to the determination of the particle size. Kinetic aspects have to be taken into account. Further work needs to be done to characterize SLN on the molecular level. NMR, ESR and synchrotron irradiation will help to answer the question whether the drug is really incorporated in the solid lipid or whether lipid and drug nanosuspensions coexist in the sample. Unfortunately, these aspects have not always been considered and the terminus ‘drug incorporation’ in the SLN literature is often misleading. The lipid matrix of solid lipid nanodispersions is more mobile compared to polylactide–co-glycolide based nanoparticles and therefore, controlled release due to restricted diffusion of the drug within the lipid matrix is questionable because of the drug mobility and the short way length. Controlled release is, however, not impossible to achieve, but other factors will determine the release rate (e.g. kinetics to reach equilibrium of the drug between lipid and aqueous phase might be slow). Further work needs to be done to understand the interaction of SLN with their biological surrounding (adsorption/desorption processes, enzymatic degradation, agglomeration, interaction with endogenous lipid carrier systems). Several administration routes are feasible for SLN administration. The most challenging route will be i.v. injection which requires absolute control of the particle size. The results obtained with dermal application are encouraging and probably, this will be the main application of SLN. In summary, SLN are very complex systems with clear advantages and disadvantages to other colloidal carriers. Further work needs to be done to understand the structure and dynamics of SLN on a molecular level in vitro and in vivo. Acknowledgements The authors would like to thank Susan Liedtke and Katja Jores for their support in the preparation of this manuscript.

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