INTRODUCTION:

Colloidal, the term refers to a state of subdivision, implying that the molecules or polymolecular particles dispersed in a medium have at least in one direction a dimension roughly between 1 nm and 1 μm¹. Colloidal carriers have attracted increasing attention during recent years. Investigated systems include nanoemulsions, nanosuspensions, micelles, soluble polymer–drug conjugates, polymeric nanoparticles, lipid based carriers such as liposomes and solid lipid nanoparticles².

Colloidal drug carriers offer number of potential advantages as delivery systems, such as better bioavailability for poorly water-soluble drugs³. Looking for drug carrier formulations increasing the bioavailability and consisting of well tolerated excipients, the Solid Lipid Nanoparticles (SLN) are alternative drug carrier systems⁴. In contrast to emulsions and liposomes, the particle matrix of SLN is composed of solid lipids. In the past few years, lipid matrices became extremely popular in controlling release of drugs⁵. General features of SLN are their composition of physiological compounds, possible routes of administration by i.v., oral and topical, the relatively low costs of excipients. The other advantage is easy large scale production by high pressure homogenization which is used, e.g., for parenteral fat emulsions⁶.

Besides, the SLN can be prepared by the microemulsion technique, by high speed stirring and sonication. Solid Lipid Nanoparticles combine the properties of polymer nanoparticles (solid matrix for controlled release) and o/w type emulsions. They can consist of lipids and surfactants which are traditionally used in pharmaceutical preparations, e.g., in tablets or pellets, also can be produced from food lipids. A number of studies have recently been published about production⁷, physico-chemical characterization of particles^{8, 9} and drug incorporation and release¹⁰. Some years ago the first results have been published about the chemical stability of pharmaceutical drugs and cosmetic actives incorporated into SLN formulations¹¹. In addition, intensive analyses regarding lipid crystallization including polymorphic transitions have been performed^12-14. However, in contrast to stability data of active agents incorporated into SLN, nobody looked at the stability of the lipids matrices themselves. This paper presents the first intensive systematic study about the effect of lipid and surfactant on properties of solid lipid nanoparticle, also the stability of solid lipid nanoparticle with lipophile loading and release in lipid nanoparticles.

MATERIALS:

Solid lipid nanoparticles (SLN) were developed at the beginning of the 1990s and attracted increasing attention during recent years as an alternative carrier system. Solid lipid nanoparticles typically are spherical with average diameters between 50 to 500 nanometers. Solid lipid nanoparticles possess a solid lipid core matrix that can solubilize lipophilic molecules. The lipid core is stabilized by surfactants (emulsifiers). For pharmaceutical applications, all formulation excipients must have Generally Recognized as Safe (GRAS) status¹⁵.

General constituents of solid lipid nanoparticles include²—

Solid i.e. drug particle, lipid or lipids, emulsifier(s) and water.

The term Lipid used in a broader sense and Table 1 identifies the types of lipids and surfactants reported in solid lipid nanoparticle formulations. To achieve and maintain a solid lipid particle upon administration, the lipid nanoparticle’s melting point must exceed body temperature (37 °C). High melting point lipids investigated include triacylglycerols (triglycerides), acylglycerols, fatty acids, steroids, waxes, and combinations thereof. Surfactants investigated include biological membrane lipids such as lecithin, bile salts such as sodium taurocholate, and biocompatible nonionics such as ethylene oxide/propylene oxide copolymers, sorbitan esters, fatty acid ethoxylates, and mixtures thereof².

Table 1: Types of lipids and surfactants reported in solid lipid nanoparticle formulations.

Lipids	Surfactant
Triacylglycerols	Phospholipids
Tricarpin	Soy lecithin
Trilaurin	Egg lecithin
Tripalmitin	Phosphatidylcholine
Tristearin	Ethylene oxide/propylene oxide copolymer
Acylglycerol	Poloxamer 188
Glycerol Monostearate	Poloxamer 182
Glycerol behenate	Poloxamer 407
Glycerol palmitostearate	Poloxamer 908
Fatty acids	Sorbitan ethylene oxide
Stearic acid	Polysorbate 20
Palmitic acid	Polysorbate 60
Decanoic acid	Polysorbate 80
Behenic acid	Alkylaryl polyether alcohol polymers
Waxes	Tyloxapol
Cetyl palmitate	Bile slts
Cyclic complexes	Sodium cholate
Cyclodextrin	Sodium glycocholate
Para-acyl-calix-arenes	Sodium taurocholate
	Sodium taurodeoxycholate
	Alcohols
	Ethanol
	Butanol

EFFECT OF LIPIDS AND SURFACTANTS ON SOLID LIPID NANOPARTICLE ENGINEERING:

SLNs are the lipid particles, the main ingredient in the engineering of SLN are Lipid and surfactant. Many scientists have studied the effect of different lipid on the main criteria of lipid nanoparticles that is its particle size and entrapment efficiency. zur Mühlen A. have studied the effects of lipid type, and average particle size has been shown to increase with increasing lipid melting temperature¹⁶. Mehnert and Mäder suggest this behavior is due to the increased viscosity of the dispersed phased². Alternative explainations could include the increasing molecular size with increasing melting temperature, particularly true for saturated acyl chains, the increasing hydrophobicity of higher melting temperature lipids, varying crystallization rates, and varying lipid crystal Structures². Larger particles and increased polydispersity results when lipid content exceeds 10% of the emulsion/dispersion. Increased emulsion viscosity and increased rate of droplet agglomeration are thought to cause this behavior, which has been observed in lipid nanoemulsions as well^{16, 17}.

Surfactant properties and concentrations greatly affect the quality and efficacy of lipid nanoparticles. By their amphiphilic nature, surfactants lower the interfacial tension between lipid and aqueous phases. In emulsions, the water-lipid interfacial area increases as oil droplets size is reduced, which make the emulsion thermodynamically unfavorable. Without appropriate stabilization of the interface by surfactants, a characteristic phase separation process occurs. So called Ostwald ripening whereby larger oil droplets grow at the expense of smaller droplets occurs first, followed by droplet flocculation, droplet coalescence, and finally, phase separation. Surfactants inhibit the phase separation process by reducing interfacial tension and the imparting steric hindrance. When optimized, the surfactants can ‘stabilize’ emulsions over the useful life.

Few patterns exist in the literature with regard to surfactant composition and quality of solid lipid nanoparticle dispersions. The optimum surfactant concentration must be determined on a case by case situation. For acceptable tribehenin nanoparticles, zur Mühlen determined that 5 % sodium cholate or Poloxamer 188 was required¹⁶. Siekmann et al. determined that 10 % tyloxapol stabilized 85 nm tripalmitin nanoparticles while 2 % tyloxapol failed to stabilize the suspension¹⁷. The varying rates of adsorption onto the interface and hydrophile-lipophile balance (HLB) numbers may account for the phenomena of observed behaviors when surfactants are interchanged. One clear trend is the beneficial role of cosurfactants. Solid lipid nanoparticles stabilized by surfactant mixtures, such as lecithin/Poloxamer 188 and lecithin/tyloxapol, resulted in more stable, smaller particle sizes than formulations of the same lipid and a single surfactant. When using lecithin as the surfactant with taurodeoxycholate and monooctylphosphate as cosurfactants, Cavalli et al. produced stearic acid nanoparticles having 70±2 nm diameters¹⁸. Surfactant mixtures often reduce interfacial tension more than single surfactant formulations on a mole per mole basis, particularly if the cosurfactant head group is significantly smaller than the surfactant head group. This phenomenon is largely due to an increased surfactant concentration at the interface or surface excess, made possible by the minimization of repulsion forces of closely packed like surfactant molecules¹⁹.

STABILITY OF SLN:

Lipid nanoparticle stability must be considered from two perspectives, the particle size distribution and the lipid crystalline state. Particle size is a critical factor for shelf life, as noted previously. Particle size also affects the visible appearance of the product, since the human eye can only detect light scattered by particles greater than ~ 1 μm¹⁹. The degree of polydispersity can impact particle size growth via Ostwald ripening and can impact the overall drug release kinetics. The lipid crystalline state strongly correlates with drug incorporation, drug release, and the particle geometry, i.e. spherical versus prolate².

As noted previously, mechanical shearing forces are primarily responsible for emulsion droplet size reduction. For two immiscible liquids, such as lipids and waters, this reduction in droplet size corresponds to a thermodynamically unfavorable interfacial area expansion. Once the mechanical shearing force is removed, the emulsion droplet’s ability to remain a discrete droplet of constant size depends of the dispersing capability of the surfactants present at the lipid-water interface. An emulsion droplet will appear stable over extended periods of time if the surfactants’ dispersing capability is sufficient, and, consequently an emulsion system will exist for an extended period. However, if the surfactants’ dispersing capability is insufficient for the emulsion system, then characteristic phase separation processes will begin immediately. Phase separation processes include creaming, Ostwald ripening, flocculation, and coalescence. Creaming is the gravity driven process by which a less dense phase rises to the top of a multiphase system.

Electrostatic repulsion results from the formation of an electrical double layer at the lipid-water interface. Ionic surfactants, such as negatively charged lecithin, adsorbed at the interface attract solution counter ions, cations in the case of lecithin, into the interfacial region. The counter ions effectively adsorb onto the oppositely charged interface. The net charge at the interface affects the ion distribution in the nearby region, increasing the concentration of counter ions close to the interface. Thus, an electrical double layer is formed in the interfacial region.

This double layer consists of two parts: an inner region known as the Stern layer that includes ions bound relatively tightly to the interfacial surfactant ions, and an outer region known as the diffuse layer where a balance of electrostatic forces and random thermal motion determines the ion distribution. The electrical potential decreases linearly from the interface to the Stern layer. The potential in the diffuse layer decays exponentially with increasing distance from the Stern layer until eventually reaching the bulk solution value, zero in most instances²⁰.

The particle and adsorbed ions of the Stern layer exist functionally as a single unit when moving through fluid. The surface of shear, or slipping plane, is the boundary at which the relative motion commences between the immobilized layer and the mobile fluid of the surrounding environment. The potential at the surface of shear is known as the zeta potential, ξ, and is measured in millivolts (mV). The magnitude of zeta potential has been correlated to stability of particle and emulsion droplets. As zeta potential increases, electrostatic repulsion between two particles increases. If the electrostatic repulsion exceeds the attractive forces due to van der Waals’ interactions, then the colloidal system will be stable. If not, flocculation followed by coalescence will lead to phase separation²¹. Zeta potential values moreelectronegative than -30 mV generally represent sufficient electrostatic repulsion for stability, and stability is assured in most instances at zeta potentials between -45 to -70 mV²². Frietas et al. demonstrated solid lipid nanoparticle stability and instability when zeta potentials were -25 mV²³.

Steric stabilization prevents two particles from approaching to the short distances required for flocculation and coalescence. Nonionic surfactants operate by steric stabilization, and ethylene oxide/propylene oxide copolymers are routinely employed for their steric stabilization capabilities. The polyoxypropylene chain adsorbs onto the hydrophobic interface, and the polyoxyethylene chain extends into the aqueous phase in a coil configuration. Given sufficient surfactant concentration and hydrophilic chain length, often > 20 ethylene oxide units, the hydrophilic coils extending outward from the surface maintain other particles at distances required for stability¹⁹.

Often, the best stabilization strategy is to invoke both electrostatic and steric approaches. This strategy has been widely used in liposome science^{24, 25}. Several researchers have successfully applied this approach to solid lipid nanoparticles, as well^26-28.

Lipid crystallinity is another dimension of lipid nanoparticle stability, significantly impacting lipid nanoparticle drug incorporation and release characteristics. Crystallization is a balance between attractive intermolecular forces and entropic factors. In lipids, van der Waals forces drive non-polar molecules closer to one another. Entropy favors increased molecular disorder, driving molecules farther apart. As intermolecular attraction increases, or entropy decreases, liquids crystallize more readily. As temperature decreases, entropy decreases and the intermolecular distance decreases. Therefore, intermolecular attraction outweighs entropy, and crystallization will commence at a site of nucleation. Historically, four states of crystallinity are associated with lipids, but more recent research has revealed numerous varieties of crystalline structures in lipids⁵.

Most lipids possess more than one solid phase under normal thermodynamic conditions. These phases result from the multitude of molecular arrangements made possible by the complex interactions of the nonpolar acyl chains and the polar regions of lipids. These solid phases are classified according to polymorphism and polytypism. Polymorphism is critically defined as the ability to reveal different unit cell structures in crystal, originating from a variety of molecular conformations and packings²⁹. Simply put, polymorphism is the existence of multiple crystal forms. Polytypism can be defined as one-dimensional polymorphism where the variation is found in the vertical stacking patterns while the other two dimensions remain constant. The fundamentals of polymorphic and polytypic thermodynamics and kinetics are too complex and lengthy for a thorough discussion in this work. However, understanding the practical implications of polymorphism and polytypism, particularly polymorphism on lipid nanoparticle synthesis and stability. Thermodynamic stability increases, lipid packing density increases, and drug incorporation rates decrease in lipid in the following order of polymorphism; α crystal <β′ crystal < β crystal. Initial crystalline structure is often dependent on the rate of cooling, indicating the kinetic dependency of crystallization. When cooled very slowly, pure lipids will organize themselves into the lowest energy form, the β crystal. When cooled rapidly, pure lipids initially crystallize in the disordered, higher energy α crystal. Intermediate cooling rates yield the intermediate β′ crystal. This behavior can be explained in terms of the activation energy of nucleation, as indicated in Fig. 1. The rapid cooling minimizes the energy input into the system, inhibiting the crystallization of more ordered forms, and vice versa.

Fig. 1: Activation energy of nucleation for a representative lipid system³⁰

Polymorphism can produce profound effects on physical properties. In addition to the multiple polymorphs, supercooled liquids can result from extremely fast cooling, especially at smaller diameters. If the surface curvature is significant, as in the case of small diameter particles, then the internal bulk phase may be geometrically strained and less shielded from the external environment. The resulting higher energy state contributes to crystallization temperature depression. Westesen et al. showed trilaurin nanoparticles remained in a supercooled state if not artificially induced to crystallize.

In many systems, particularly in single component lipid formulations, the crystallization process continues after initial solidification. Striving for the lowest energy configuration, the lipid molecules will continue to reorganize over time until reaching the β crystal state. This process, known as solid-state phase transformation, undermines solid lipid nanoparticles’ use as a drug delivery technology. As lipid packing density increases, less volume is available for drug molecules. For stearic acid solid-state phase transformations, the reduction in crystal cell unit volume exceeds 50%. More importantly, the presence of drug molecules can disrupt the thermodynamically favored transitions toward the β′ crystal and the β crystal, and, as a result, the drugs can be expelled from the lipid core. However, if solid-state transformations can be controlled, the process could be used as a drug release mechanism. Lipid polymorphism is associated with a change in particle shape. Typically, triglycerides crystallize as a sphere in the α crystal state³¹. Westesen used transmission electron microscopy (TEM) to demonstrate that tripalmitin and tristearin solidified in the α crystal form spheres, but the transformation to the β crystal is accompanied by the onset of a platelet structure. The platelet structure possesses greater surface area than a sphere, thus requiring higher surfactant concentrations to maintain stability. The transformation to the β crystal, therefore, can lead to particle growth. The platelet structure also brings loaded drug molecules closer to the interface, promoting drug release. For these reasons, producing and maintaining the α crystal structure is highly desirable in solid lipid nanoparticle applications. Lipid mixtures, surfactant mixtures, and rapid cooling techniques promote the α crystal structure. Using lipids of dissimilar geometries inhibits closely packed, highly ordered crystal structures. Likewise, introducing surfactants whose hydrophobic tails are geometrically dissimilar to the core lipid inhibits highly ordered crystal formation. Sterols, such as the bile salts like sodium taurocholate, possess a bulky, five ring hydrophobic regions which do not permit close highly ordered crystal formation, at least near the interface. As noted before, rapid cooling does not provide adequate time for the crystallization process to form the more highly ordered β crystal. These techniques provide researchers with opportunities to produce solid lipid nanoparticles in the α crystal form. Despite the stability challenges, optimized solid lipid nanoparticle dispersions can be stable for more than one year³². By photon correlation spectroscopy (PCS) analysis, Müller et al. demonstrated that glycerol palmitostearate and tribehenate nanoparticles were stable for 3 years. Solid lipid nanoparticle stability is a function of formulation and processing parameters, providing several options to researchers and developers.

DRUG LOADING AND RELEASE FROM LIPID NANOPARTICLES

A variety of drugs, including agents for treating cancer, AIDS, fungal infections, high blood pressure, mental illness, skin disease, and imaging have been loaded into solid lipid nanoparticles. For efficiency and efficacy reasons, the amount of drug that can be loaded is very important. Using high pressure homogenization (HPH), Westesen obtained loading capacities up to 50% for Ubidecarenone, 20% for Tetracaine and etomidate, and 25% for cyclosporin^{32, 33}. For HPH, Müller suggests that capacity is determined by the drug solubility in the melted lipid, the miscibility of the melted drug and melted lipid, and the physiochemical structure of the solid lipid⁵.

The drug can locate between fatty acid chains, between lipid layers, in lipid crystal imperfections. As noted previously, the chemical properties of the lipid affect the crystallinity of solid particle. It has been suggested that lipids that form more perfect crystalline solids, such as monoacid triglycerides having a β’ crystal structure, expel solubilized drugs and that those lipids that form less perfect crystalline structures, such as triglyceride mixtures, possess higher loading capacities^{32, 34}.

Obtaining controlled drug release from lipid nanoparticles remains elusive as, more often than not, burst release kinetics have been observed⁵. Müller reported the first controlled release of a drug, prednisolone, from HPH produced solid lipid nanoparticles. In vitro drug release was obtained for 7 weeks. Release kinetics were dependent on the lipid matrix, surfactant concentration, and HPH production parameters, but were independent of particle size. The size independence suggests the mass transfer was not diffusion limited. Burst release increased with increasing processing temperature and increasing surfactant concentration, leading Müller to suggest that drug partitioning into the aqueous phase during homogenization negatively affects sustained release. As temperature and surfactant levels increase, drug solubility in water increases. As temperature decreases, Müller rationalizes that the lipid crystallizes initially in the center, and the drug tends to repartition into the lipid; however, because the lipid core has already crystallized, the core is unavailable to the drug. As the system continues to cool, the drug solubility in the water diminishes, and the drug is concentrated in the lipid ‘shell’ region. This enriched shell profile then promotes drug burst release. These events are depicted in Fig. 2.

During During

Homogenization Cooling

Fig. 2: Proposed redistribution of drug from molecularly dispersed state to enriched shell state, postulated as a cause of drug burst release phenomena observed in lipid nanoparticles (modified from¹⁸).

To obtain sustained drug release, Müller suggests a diffusion controlled release mechanism and a uniform drug profile throughout the lipid shell or an enriched ‘core’¹⁸. If a delayed release profile is desirable, the proposed drug-enriched core/lipid shell model represents an interested option.

Fig. 3: Proposed structural models for drug loading profiles in lipid nanoparticles

Fig. 4: Possible models for drug entrapment depending upon melting point of drug and lipid.

Fig. 3 and Fig. 4 depict the commonly proposed drug loading profiles thought to be possible in lipid nanoparticles. Existing synthesis techniques do not provide precise control, for constructing prescribed desired drug profiles. The process remains a trial-and-error based approach in which changing drugs, lipids, surfactants, concentrations, and process parameters lead to unpredictable results. An improved production technology is required to provide this level of control.

CONCLUSION:

Solid lipid nanoparticle is very promising drug carrier system which combines the advantage and avoids the disadvantages of other colloidal carrier systems. SLNs are nanoparticles made up of solid lipid which are evaluated on the basis of particle size, entrapment efficiency and stability. The main parameter that affect the SLNs properties are lipid and surfactant. Stability of SLN is dependent on polymorphism and lipid crystallinity. The drug loading profile is according to the melting point of lipid and drug and homogeneous matrix or drug enriched core or shell forms, accordingly the release profile as burst or sustained release observed.

REFERENCES:

1. Almeida AJ. Solid Lipid Nanoparticles as Colloidal Drug Carrier Systems. Research Institute for Medicines and Pharmaceutical Sciences, Universidade de Lisboa, Portugal, 2007.

2. Mehnert W and Mader K. Solid lipid nanoparticles-Production, characterization and applications. Adv Drug Deliv. 2001; 47: 165-196.

3. Reithmeier H, Hermann J and Gapferich A. Structure investigations on lipid nanoparticles containing high amounts of lecithin. J Control Rel. 2001; 73: 339-350.

4. Peters K and Müller RH. Nanosuspensions for the oral administration of poorly soluble drugs. European Symposium on formulation of Poorly available Drugs for Oral Administration, APGI, Paris, 1996; 330–333.

5. Műller RH, Mäder K and Gohla S. Solid Lipid Nanoparticles (SLN) for controlled drug delivery - a review of the state of the art. Eur J Pharm Biopharm. 2000; 50: 161–177.

6. Műller RH and Mehnert K. Solid Lipid Nanoparticles (SLN)—an alternative colloidal carrier system for controlled drug delivery. Eur J Pharm Biopharm. 1995; 41: 62–69.

7. Műller RH, Weyhers H, Műhlen zur A, Dingler A and Mehnert W. Solid Lipid Nanoparticles (SLN)- ein neuartigerWirkstoff-Carrier für Kosmetika und Pharmazeutika I. Systemeingeschaften, Herstellung und scaling up. Pharm Ind. 1997; 59: 423–427.

8. Westesen K, Siekmann B and Koch MHJ. Investigations on the physical state of lipid nanoparticles by synchrotron radiation x-ray diffraction. Int J Pharm. 1997; 93: 189–199.

9. Mühlen zur A, Mühlen zur E, Niehaus E and Mehnert W. Atomic force microscopy studies of solid lipid nanoparticles. Pharm Res. 1996; 13: 1411–1416.

10. Mühlen zur A and Mehnert W. Drug release and release mechanism of prednisolone loaded solid lipid nanoparticles. Pharmazie 1998; 53: 552.

11. Jenning V. Feste Lipid Nanopartikel als kolloidaleWirkstofftrager- systeme zur dermalen Applikation. PhD Thesis. Freie University of Berlin. 1998.

12. Freitas C and Műller RH. Correlation between long-term stability of Solid Lipid Nanoparticles (SLN) and crystallinity of the lipid phase. Eur J Pharm Biopharm. 1999; 47: 125–132.

13. Wissing SA and Műller RH. The influence of the crystallinity of lipid nanoparticles on their occlusive properties. Int J Pharm. 2002; 242: 377–379.

14. Wissing SA and Műller RH. The development of an improved carrier system for sunscreen formulations based on crystalline lipid nanoparticles. Int J Pharm. 2002; 242: 373–375.

15. Food and Drugs. In United States Code Of federal Regulations. Anonymous (2004) Title 21.

16. Mühlen zur A. Feste Lipid-Nanopartikel mit prolongierter Wirkstoffliberation: Herstellung, Langzeitstabilität, Charaktersierung, Freisetzungsverhalten and mechanismen. In, Free University of Berlin, Berlin, Germany. 1996.

17. Siekmann B and Westesen K. Melt-homogenized solid lipid nanoparticles stabilized by the nonionic surfactant tyloxapol. Preparation and particle size determination. Pharm Pharmacol. 1994; 3: 194-197.

18. Cavalli R. The effect of the components of microemulsions on both size and crystalline structure of solid lipid nanoparticles (SLN) containing a number of model molecules. Pharmazie 1998; 53: 392-396.

19. Porter MR. Handbook of Surfactants, Chapman and Hall, London. 1994.

20. http://www.bic.com/WhatisZetaPotential.html

21. Heimenz PC. Principles of Colloid and Surface Chemistry. Marcel Dekker. Inc., New York. 1986.

22. http://www.zetareader.com/pages/overview.html

23. Freitas C and Müller RH. Stability determinatin of solid lipid nanoparticles (SLN) in aqueous dispersion after addition of electrolyte. J Microencapsulation 1999; 16: 59-71

24. Gregoriadis G. Targeting of drugs: strategies for stealth therapeutic systems. Plenum Press, New York. 1998.

25. Srinath P and Diwan PV. Stealth Liposomes - An Overview. Ind J Pharmacol. 1994; 26: 176-184.

26. Bocca C, Caputo O, Cavalli R, Gabriel L, Miglietta A and Gasco MR. Phagocytic uptake of fluorescent stealth and non-stealth solid lipid nanoparticles. Int J Pharm.1998; 175: 185-193.

27. Cavalli R, Caputo O and Gasco MR. Preparation and characterization of solid lipid nanospheres containing paclitaxel. Eur J Pharm Sci. 2000; 10: 305-309.

28. Fundarò A, Cavalli R, Bargoni A, Vighetto D, Zara GP, Paolo G and Gasco MR. Non-stealth and stealth solid lipid nanoparticles carrying doxorubicin: pharmacokinetics and tissue distribution after i.v. administration to rats. Pharmacological Research. 2000; 42: 337-343.

29. Small DM. The Physical Chemistry of Lipids. In Handbook of Lipid Research (Hanahan, D. J., ed) Vol. 4, Plenum, New York, 1986.

30. Westesen K, Siekmann B and Koch MHJ. Investigations on the physical state of lipid nanoparticles by synchrotron radiation X-ray diffraction. Int J Pharm. 1993; 93: 189-199.

31. Siekmann B and Westesen K. Thermoanalysis of the recrystallization of melthomogenized glyceride nanoparticles. Colloids and surfaces B: Biointerfaces. 1994; 3: 159-175.

32. Westesen K, Bunjes H and Koch MHJ. Physiochemical characterization of lipid nanoparticles and evaluation of their drug loading capacity and sustained release potential. J Control Rel. 1997; 48: 223-236.

33. Schwarz C. Feste Lipidnanopartikel: Herstellung, Charakterisierung, Arzneistoffinkorporation und -freisetzung, Sterilisation und Lyophilisation. In, Free University of Berlin, Germany. 1995.

34. Runge SA. Feste Lipid-Nanopartikel (SLN) als kolloidaler Arzneistoffträger zur Cyclosporin. In, Free University of Berlin, Germany. 1998.