Production of solid lipid nanoparticles (SLN): scaling up feasibilities

Solid lipid nanoparticles (SLN/Lipopearls) are widely discussed as a new colloidal drug carrier system. In contrast to polymeric systems, such as Polylactic copolyol microcapsules, these systems show with a good biocompatibility, if applied parenterally. The solid lipid matrices can be comprised of fats or waxes, and allow protection of incorporated active ingredients against chemical and physical degradation. The SLN can either be produced by 'hot homogenization' of melted lipids at elevated temperatures or by a 'cold homogenization' process. This paper deals with production technologies for SLN formulations, based on non-ethoxylated fat components for topical application and high pressure homogenization. Based on the chosen fat components, a novel and easy manufacturing and scaling-up method was developed to maintain chemical and physical integrity of the encapsulated active ingredients in the carrier.     

Scaling up feasibility of the production of solid lipid nanoparticles (SLN)

Solid lipid nanoparticles (SLN/Lipopearls) are widely discussed as colloidal drug carrier system. In contrast to polymeric systems, such as polylactic copolyol capsules, these systems show up with a good biocompatibility, if applied parenterally. The solid lipid matrices can be comprised of fats or waxes and allow protection of incorporated active ingredients against chemical and physical degradation. The SLN can either be produced by "hot homogenisation" of melted lipids at elevated temperatures or a "cold homogenization" process. This paper deals with production technologies for SLN formulations, based on non-ethoxylated fat components for topical application and high pressure homogenization (APV Deutschland GmbH, D-Lübeck). Based on the chosen fat components, a novel and easy manufacturing and scaling up method was developed to maintain chemical and physical integrity of encapsulated active and carrier.     

Cosmetic applications for solid lipid nanoparticles (SLN)

Solid lipid nanoparticles (SLN) are novel delivery systems for pharmaceutical and cosmetic active ingredients. This paper highlights advantages of SLN for cosmetic applications. The dependence of the occlusive effect on the particle size of SLN due to film formation is presented by in vitro data. An in vivo study showed that addition of 4% SLN to a conventional o/w cream lead to an increase of skin hydration of 31% after 4 weeks. The application of SLN as physical sunscreens and as active carriers for molecular sunscreens has also been investigated. The amount of molecular sunscreen could be decreased by 50% while maintaining the protection level compared to a conventional emulsion.     

Encapsulation of retinoids in solid lipid nanoparticles (SLN)

SLN have been suggested for a broad range of applications, such as intravenous injection, peroral, or dermal administration. The incorporation of the drug in the core of the SLN has to be ensured for these applications, but the inclusion of drugs in SLN is poorly understood. This study is a contribution to further describe the inclusion properties of colloidal lipids and to propose incorporation mechanisms. Besides the well known methods to investigate entrapment of actives in nanoparticles such as DSC or microscopy, the present study focussed on yet a different approach. Based on the different chemical stability of retinoids in water and in a lipid phase, a method to derive information on the distribution of the drug between SLN-lipid and the water phase was established. Comparing different lipids, glyceryl behenate gave superior entrapment compared to tripalmitate, cetyl palmitate and solid paraffin. Comparing three different drugs, entrapment increased with decreasing polarity of the molecule (tretinoin < retinol < retinyl palmitate). The encapsulation efficacy was successfully enhanced by formulating SLN from mixtures of liquid and solid lipids. These particles were solid and provided better protection of the sensitive drugs than an emulsion. X-ray investigations revealed that good encapsulation correlated with a low degree of crystallinity and lattice defects. With highly ordered crystals, as in the case of cetyl palmitate, drug expulsion from the carrier was more pronounced.     

Encapsulation of retinoids in solid lipid nanoparticles (SLN)

SLN have been suggested for a broad range of applications, such as intravenous injection, peroral, or dermal administration. The incorporation of the drug in the core of the SLN has to be ensured for these applications, but the inclusion of drugs in SLN is poorly understood. This study is a contribution to further describe the inclusion properties of colloidal lipids and to propose incorporation mechanisms. Besides the well known methods to investigate entrapment of actives in nanoparticles such as DSC or microscopy, the present study focussed on yet a different approach. Based on the different chemical stability of retinoids in water and in a lipid phase, a method to derive information on the distribution of the drug between SLN-lipid and the water phase was established. Comparing different lipids, glyceryl behenate gave superior entrapment compared to tripalmitate, cetyl palmitate and solid paraffin. Comparing three different drugs, entrapment increased with decreasing polarity of the molecule (tretinoin < retinol < retinyl palmitate). The encapsulation efficacy was successfully enhanced by formulating SLN from mixtures of liquid and solid lipids. These particles were solid and provided better protection of the sensitive drugs than an emulsion. X-ray investigations revealed that good encapsulation correlated with a low degree of crystallinity and lattice defects. With highly ordered crystals, as in the case of cetyl palmitate, drug expulsion from the carrier was more pronounced.     

Preparation and characterization of n-dodecyl-ferulate-loaded solid lipid nanoparticles (SLN)

Solid lipid nanoparticles (SLN) containing a novel potential sunscreen n-dodecyl-ferulate (ester of ferulic acid) were developed. The preparation and stability parameters of n-dodecyl-ferulate-loaded SLN have been investigated concerning particle size, surface electrical charge (zeta potential) and matrix crystallinity. The chemical stability of n-dodecyl-ferulate at high temperatures was also assessed by thermal gravimetry analysis. For the selection of the appropriated lipid matrix, chemically different lipids were melted with 4% (m/m) of active and lipid nanoparticles were prepared by the so-called high pressure homogenization technique. n-Dodecyl-ferulate-loaded SLN prepared with cetyl palmitate showed the lowest mean particle size and polydispersity index, as well as the highest physical stability during storage time of 21 days at 4, 20 and 40 degrees C. These colloidal dispersions containing the sunscreen also exhibited the common melting behaviour of aqueous SLN dispersions.     

Incorporation of cyclosporin A in solid lipid nanoparticles (SLN)

The cyclic undecapeptide cyclosporin A (CyA) a potent immunosuppressive drug used in many therapies, is extremely hydrophobic. Commercial products employ solubilising agents to improve gastrointestinal absorption. In the present study CyA solid lipid nanoparticles (SLN) are prepared from warm o/w microemulsion, dispersed in cold water. The matrix chiefly consists of stearic acid, phosphatidylcholine and taurocholate; up to 13% of CyA can be incorporated. The average diameter of CyA-loaded SLNs is below 300 nm and transmission electron microscopy (TEM) analysis shows them to be spherical. In vitro release of CyA from SLNs is low. CyA-loaded SLNs can be proposed for most administration routes, in particular for the duodenal route.     

Freeze-drying of drug-free and drug-loaded solid lipid nanoparticles (SLN)

Solid lipid nanoparticles (SLN) of a quality acceptable for i.v. administration were freeze-dried. Dynasan 112 and Compritol ATO 888 were used as lipid matrices for the SLN, stabilisers were Lipoid S 75 and poloxamer 188, respectively. To study the protective effect of various types and concentrations of cryoprotectants (e.g. carbohydrates), freeze-thaw cycles were carried out as a pre-test. The sugar trehalose proved to be most effective in preventing particle growth during freezing and thawing and also in the freeze-drying process. Changes in particle size distribution during lyophilisation could be minimised by optimising the parameters of the lyophilisation process, i.e. freezing velocity and redispersion method. Lyophilised drug-free SLN could be reconstituted in a quality considered suitable for i.v. injection with regard to the size distribution. Loading with model drugs (tetracaine, etomidate) impairs the quality of reconstituted SLN. However, the lyophilisate quality is sufficient for formulations less critical to limited particle growth, e.g. freeze-dried SLN for oral administration.     

Comparison of wax and glyceride solid lipid nanoparticles (SLN)

The present study compares solid lipid nanoparticles (SLN) formulated with either wax or glyceride bulk material. While most published data deal with glyceride SLN, little knowledge is reported on wax carriers. The two types were compared with respect to drug encapsulation efficacy, particle size distribution after production and storage, and crystal packing. The inclusion of retinol as a model drug was investigated. Retinol is chemically unstable in water and rather stable in lipid phases. Thus, rapid degradation of retinol indicates rapid drug expulsion from the carrier. Good stability indicates an effective drug encapsulation in the lipid phase of the nanoparticles. Particle size distribution was measured by laser diffractometry. Subcell packing and assignment of polymorphic forms was investigated by WAXS measurements. Glyceride SLN showed good drug encapsulation, while physical stability was poor. In contrast, wax SLN possessed good physical stability but lacked sufficient drug encapsulation in the solidified state. These differences were attributed in part to different crystal packing. Less ordered crystal lattices favour successful drug inclusion, as in the case of glyceryl monosterate and glyceryl behenate SLN. The highly ordered crystal packing of wax SLN comprised of beeswax or cetyl palmitate, for instance, leads to drug expulsion, but also to superior physical stability.     

Oral bioavailability of cyclosporine: solid lipid nanoparticles (SLN) versus drug nanocrystals

For the development of an optimized oral formulation for cyclosporine A, 2% of this drug has been formulated in solid lipid nanoparticles (SLN, mean size 157 nm) and as nanocrystals (mean size 962 nm). The encapsulation rate of SLN was found to be 96.1%. Nanocrystals are composed of 100% of drug. For the assessment of the pharmacokinetic parameters the developed formulations have been administered via oral route to three young pigs. Comparison studies with a commercial Sandimmun Neoral/Optoral used as reference have been performed. The blood profiles observed after oral administration of the commercial microemulsion Sandimmun revealed a fast absorption of drug leading to the observation of a plasma peak above 1,000 ng/ml within the first 2 h. For drug nanocrystals most of the blood concentrations were in the range between 30 and 70 ng/ml over a period of 14 h. These values were very low, showing huge differences between the measuring time points and between the tested animals. On the contrary, administration of cyclosporine-loaded SLN led to a mean plasma profile with almost similarly low variations in comparison to the reference microemulsion, however with no initial blood peak as observed with the Sandimmun Neoral/Optoral. Comparing the area under the curves (AUC) obtained with the tested animals it could be stated that the SLN formulation avoids side effects by lacking blood concentrations higher than 1,000 ng/ml. In this study it has been proved that using SLN as a drug carrier for oral administration of cyclosporine A a low variation in bioavailability of the drug and simultaneously avoiding the plasma peak typical of the first Sandimmun formulation can be achieved.     

Lopinavir loaded solid lipid nanoparticles (SLN) for intestinal lymphatic targeting

The poor orally available lopinavir was successfully encapsulated in glyceryl behenate based solid lipid nanoparticles (Lo-SLN) for its ultimate use to target intestinal lymphatic vessels in combined chemotherapy—the so-called Highly Active Anti-Retroviral Therapy (HAART). SLN with mean particle size of 230 nm (polydispersity index, PDI < 0.27) and surface electrical charge of approx. ?27 mV, were produced by hot homogenization process followed by ultrasonication. Particles were characterized using differential scanning calorimetry (DSC), wide angle X-ray scattering (WAXS) and atomic force microscopy (AFM) to confirm their solid character and the homogeneous distribution of drug within the lipid matrix. In vitro release studies at pH 6.8 phosphate buffer (PBS) and at pH 1.2 HCl 0.1 N showed a slow release in both media. From the intestinal lymphatic transport study it became evident that SLN increased the cumulative percentage dose of lopinavir secreted into the lymph, which was 4.91-fold higher when compared with a conventional drug solution in methyl cellulose 0.5% (w/v) as suspending agent (Lo-MC). The percentage bioavailability was significantly enhanced. The AUC for the Lo-SLN was 2.13-fold higher than that obtained for the Lo-MC of similar concentration. The accelerated stability studies showed that there was no significant change in the mean particle size and PDI after storage at 25 ± 2 °C/60 ± 5% RH. The shelf life of optimized formulation was assessed based on the remained drug content in the stabilized formulation and was shown to be 21.46 months.     

Medium scale production of solid lipid nanoparticles (SLN) by high pressure homogenization.

Solid lipid nanoparticles (SLN) were produced by high pressure homogenization using piston-gap homogenizers. Batch sizes varied between 40 ml and 50 l. Because of the different batch sizes, different homogenizer types were used, but the same functional principles were maintained, and the change from 40 ml to 50 l was not critical. With increasing batch sizes, the product quality in terms of particle size distribution and physical storage stability improved. Medium scale (30 l and 50 l) drug-free and drug-loaded SLN batches could be produced reproducibly and batch-to-batch uniformity was proven: within one batch particle sizes were homogeneous. This study revealed the influence of pressure and temperature for the hot homogenization technique A change of pressure between 300-500 bars induced only minor differences in particle size, but some influence of the heating temperature was found. More important than control of the heating process was the control of the cooling process of the final product. A too rapid cooling deteriorated the product quality: cooling with water of 18 degrees C proved to be the optimum cooling condition.     

Characterisation of surface-modified solid lipid nanoparticles (SLN): influence of lecithin and nonionic emulsifier.

Solid lipid nanoparticles (SLN), an alternative colloidal drug delivery system to polymer nanoparticles, emulsions and liposomes, are generally produced by high pressure melt-emulsification. However, the harsh production process is not applicable for formulations containing shear and temperature sensitive compounds. For that reason, subsequent adsorptive SLN loading might be a promising alternative. The aim of the present study was the development and characterisation of surface-modified SLN for adsorptive protein loading by variation of both the lipid matrix and the emulsifier concentration in the continuous phase. Variations in SLN composition resulted in particle sizes between 674 and 61 nm corresponding to specific surfaces of 4.5 m(2)/g and 48.9 m(2)/g and zeta potentials between -23.4 mV and -0.9 mV. In dependence of SLN surface properties, albumin payload ranged from 2.5 to 15%. Thermoanalysis, X-ray diffraction and electron microscopy revealed anisometrical and crystalline particles. In vitro cytotoxicity was low in terms of both haemolysis, which was between 1 and 2%, and neutral red test (NRT) showing a half lethal dose between 1.1 and 4.6%.     

Correlation between long-term stability of solid lipid nanoparticles (SLN) and crystallinity of the lipid phase.

Aqueous dispersions of solid lipid nanoparticles (SLN) are usually physically stable for more than 3 years. However, in some systems gelation occurred leading to solid gels due to an unknown mechanism. To elucidate this mechanism, Compritol SLN were stored at different temperatures, varying light exposure, in different packing materials and stressed by shear forces in short-term tests and a long-term study of 3 years. The SLN were analyzed by differential scanning calorimetry and sizing techniques. After production by hot homogenization of the melted lipid, the Compritol SLN crystallize in a mixture of stable beta' with unstable polymorphs (alpha, sub alpha). The destabilizing factors light, temperature and shear forces cause a distinct increase in the recrystallization index by transformation of the lipid to the beta' modification being accompanied by gel formation. Physically stable SLN remain as a mixture of modifications, increase in crystallinity index during storage is slow and crystallization occurs mainly in unstable modifications. From this, stabilization of physically critical SLN dispersions seems possible by inhibition of the transformation of the lipid to the stable modification.     

Cyclosporine-loaded solid lipid nanoparticles (SLN): drug-lipid physicochemical interactions and characterization of drug incorporation.

Solid lipid nanoparticles (SLN) were produced loaded with cyclosporine A in order to develop an improved oral formulation. In this study, the particles were characterized with regard to the structure of the lipid particle matrix, being a determining factor for mode of drug incorporation and drug release. Differential scanning calorimetry (DSC) and wide-angle X-ray scattering (WAXS) measurements were employed for the analysis of the polymorphic modifications and mode of drug incorporation. Particles were produced using Imwitor 900 as lipid matrix (the suspension consisted of 10% particles, 8% Imwitor 900, 2% cyclosporine A), 2.5% Tagat S, 0.5% sodium cholate and 87% water. DSC and WAXS were used to analyse bulk lipid, bulk drug, drug incorporated in the bulk and unloaded and drug-loaded SLN dispersions. The processing of the bulk lipid into nanoparticles was accompanied by a polymorphic transformation from the beta to the alpha-modification. After production, the drug-free SLN dispersions converted back to beta-modification, while the drug-loaded SLN stayed primarily in alpha-modification. After incorporation of cyclosporine A into SLN, the peptide lost its crystalline character. Based on WAXS data, it could be concluded that cyclosporine is molecularly dispersed in between the fatty acid chains of the liquid-crystalline alpha-modification fraction of the loaded SLN.     

Protein adsorption patterns on poloxamer- and poloxamine-stabilized solid lipid nanoparticles (SLN)

Solid lipid nanoparticles (SLN) were produced using a full range of poloxamer polymers and poloxamine 908 for stabilization. The protein adsorption pattern acquired on the surface of these particles after intravenous injection is the key factor determining the organ distribution. Two-dimensional polyacrylamide gel electrophoresis (2-DE) was employed for determination of particle interactions with human plasma proteins. The objective of this study was to investigate changes in the plasma protein adsorption patterns in the course of variation of the polymers stabilizing the SLN. Considerable differences in the protein adsorption with regard to preferential adsorbed proteins were detected for the different stabilizers. Possible correlations between the polyethylene oxide (PEO) chain length and the adsorption of various proteins (first of all apolipoproteins) are shown and discussed. Besides the study of protein adsorption patterns, the total protein mass adsorbed to the SLN was also evaluated using the bicinchoninic acid (BCA)-protein assay. The knowledge concerning the interactions of proteins and nanoparticles can be used for a rational development of particulate drug carriers. Based on the findings presented in this paper, we anticipate that the in vivo well-tolerable SLN are a promising site-specific drug delivery system for intravenous injection.     

Investigations on 5-fluorouracil solid lipid nanoparticles (SLN) prepared by hot homogenization

The purpose of this study was to investigate the effect of different formulation factors on the properties of solid lipid nanoparticles (SLN) prepared by a hot homogenization method. Using the particle size, physical stability constant (K(e)) and zeta-potential as standards, the stability of SLNs was investigated as a function of phospholipid and poloxamer contents. It was demonstrated that the content of phospholipid had a significant influence on the zeta-potential, which increased considerably with increasing phospholipid content. However, the particle size increased remarkably when the phospholipid content was as high as 1.5% due to the increased viscosity. Poloxamer 188 exhibited no remarkable influence on particle size when the concentration was as low as 1.0%. The influence of the phospholipid and poloxamer content on the embedding ratios of drug substances was further studied using 5-fluorouracil (5-Fu) as a model drug. It was shown that the embedding ratio increased considerably with phospholipid content and independent of poloxamer content, implying that 5-Fu was incorporated into the phospholipid bilayer membrane.     

Preparation and characterization of solid lipid nanoparticles (SLN) made of cacao butter and curdlan.

Solid lipid nanoparticles (SLN) were prepared using cacao butter, as the lipid core, and curdlan, as the shell material. Tween 80 was used as a co-surfactant in order to prevent aggregation and gelling of the curdlan. Mannitol was used as a cryoprotectant in order to prevent aggregation during redispersion. No significant change in the size of the SLN was observed up to a lipid concentration of 1.0%, and the particle size ranged from 140 to 200 nm with a unimodal distribution. When an alternating pH between 7 and 11 was used to test the physical stability of an SLN solution, the change in the particle size remained within a narrow range up to a lipid concentration of 0.5%. Above 0.5%, the particles began to aggregate due to the insufficient amount of the coating material, curdlan and Tween 80. The critical aggregation concentration at pH 7.4 was found to be 6.95 x 10(-4) mg/ml. Pyrene was used as a fluorescence probe. As the temperature increased, pyrene was gradually released from the SLN. The loading efficiency was >75% when the verapamil to lipid ratios were 1:10 and 1:5 and decreased significantly as the ratio became 1:1. The release rate was significantly delayed when verapamil was loaded into the SLN.     

5-Fluorouracil shell-enriched solid lipid nanoparticles (SLN) for effective skin carcinoma treatment

Context: The effective treatment of skin carcinoma is warranted for targeting the chemotherapeutic agents into tumor cells and avoiding unwanted systemic absorption.

Objective: This work was dedicated to the purpose of engineering highly penetrating shell-enriched nanoparticles that were loaded with a hydrophilic chemotherapeutic agent, 5-fluorouracil (5-FU).

Methods: Varying ratios of lecithin and poloxamer188 were used to produce shell-enriched nanoparticles by enabling the formation of reversed micelles within this region of the SLN. The localization of 5-FU within the shell region of the SLN, was confirmed using 5-FU nanogold particles as a tracer. SLN were introduced within sodium carboxy methylcellulose hydrogel, and then applied onto the skin of mice-bearing Ehrlich’s ascites carcinoma. The mice were treated with the gel twice daily for 6 weeks.

Results: The transmission electron microscope (TEM) revealed the formation of uniform nanoparticles, which captured reversed micelles within their shell region. The SLNs’ had particle size that ranged from 137?±?5.5?nm to 800?±?53.6, zeta potential of ?19.70?±?0.40?mV and entrapment efficiency of 47.92?±?2.34%. The diffusion of the drug-loaded SLN (269.37?±?10.92?μg/cm²) was doubled when compared with the free drug (122?±?3.09?μg/cm²) when both diffused through a hydrophobic membrane. SLN-treated mice exhibited reduced inflammatory reactions, with reduced degrees of keratosis, in addition to reduced symptoms of angiogenesis compared to 5-FU-treated mice.

Conclusion: SLN possesses the capacity to be manipulated to entrap and release hydrophilic antitumor drugs with ease.     

Effect of lipid matrix and size of solid lipid nanoparticles (SLN) on the viability and cytokine production of macrophages.

Solid lipid nanoparticles (SLN) interact with mononuclear cells following intravenous injection. Little is known about the interaction of SLN with these cells, including cytotoxic effects and a possible up-regulation of pro-inflammatory cytokines. Therefore, we investigated the influence of lipid matrix, concentration, and size of SLN on murine peritoneal macrophages (mphi). mphi were incubated with SLN consisting of different lipid matrices and coated with the same surfactant. Cytotoxicity as assessed by MTT test was found to be concentration-dependent and was dramatically influenced by the lipid matrix. Marked cytotoxic effects were observed when cells were incubated with SLN consisting of stearic acid (STE) or dimethyl-dioctadecylammonium bromide (DDA) at concentrations of 0.01%, whereas SLN consisting of triglycerides, cetylpalmitate or paraffin did not exert major cytotoxic effects at the same concentrations. Cytotoxic effects were most likely caused by products of enzymatic degradation including free stearic acid. Analysis of cytokine production by mphi following incubation with SLN revealed concentration-dependent decreases in IL-6 production. These decreases seemed to be associated with cytotoxic effects. IL-12 and TNF-alpha production was neither detected in supernatants of mphi treated with SLN at any concentration nor in those of untreated cells. The size of SLN did neither affect cytotoxicity of SLN nor resulted in induction or digression of cytokine production by mphi. In conclusion, results of the present study revealed that the nature of the lipid matrix and the concentration of SLN dramatically impact cytotoxicity of SLN on mononuclear cells. Lipid matrices of SLN should therefore be carefully chosen and tested for later intravenous use.