Research paper
Cyclosporine-loaded solid lipid nanoparticles (SLN®): Drug–lipid physicochemical interactions and characterization of drug incorporation

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Abstract

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 β to the α-modification. After production, the drug-free SLN dispersions converted back to β-modification, while the drug-loaded SLN stayed primarily in α-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 α-modification fraction of the loaded SLN.

Introduction

Lipid nanoparticles with a solid matrix, such as solid lipid nanoparticles (SLN™), are an alternative nanoparticulate carrier system to polymeric nanoparticles, liposomes and o/w emulsions [1], [2], [3], [4]. Aqueous SLN dispersions are composed of a lipid which is solid both at body and room temperature, being stabilized by a suitable surfactant. With regard to developing commercial products for the patient, SLN possess distinct advantages compared to other carriers, e.g., polymeric nanoparticles. Especially for topical and oral administration, all lipids can be used as matrix material, which are currently in use for creams, ointments, tablets, and capsule formulations including the long list of different surfactants/stabilizers employed in these traditional formulations. Thus, there is no problem with the regulatory accepted status of excipients [5], [6], [7].

For the production of SLN, different methods can be used such as high pressure homogenization [3], [6], [8], [9], [10], microemulsion technology [11], [12], [13], solvent evaporation [14], [15] and solvent diffusion methods [16], [17]. Large industrial scale production – a fundamental prerequisite for introducing a product to the market – is easily possible when employing high pressure homogenization [18], [19]. Existing industrial production lines used for the production of parenteral o/w emulsions can also be used for lipid nanoparticle production [4]. These production lines are temperature controlled, which is required for production of the lipid nanoparticles at elevated temperature. The aqueous lipid nanoparticle dispersions can be transferred to dry oral dosage forms, e.g., pellets [20]. As in any solid particulate carrier, the release of drugs from lipid nanoparticles can be modulated in order to optimize their blood levels [21]. These features together make lipid nanoparticles an interesting carrier system for optimized oral delivery of drugs.

In this study, SLN were chosen to develop an optimized formulation for cyclosporine (CycA), intended for oral administration of this protein. The low oral bioavailability of CycA is due to its low solubility in water (0.02 mg/ml at 25 °C [22]) and additionally it is a substrate of P-glycoprotein [23]. To increase the solubility and subsequently improve absorption, CycA was administered orally dissolved in a mixture of corn oil and ethanol in the product Sandimmun® capsules [24], [25]. This formulation shows pronounced variations in bioavailability from 10% to 60%, due to differences in the dispersion of the oil in the gut, to more or less finely dispersed oil droplets. The bioavailability was highly dependent on the emulsifying compounds in the gut, i.e., bile salts [24]. The second generation product Sandimmun® microemulsion avoided this problem. After dilution with water in the stomach the microemulsion breaks, i.e., it transforms to an ultrafine o/w emulsion thus eliminating the problem of mechanical dispersion. However, because emulsions do not provide the possibility of prolonged release, an initial plasma peak above 1000 ng/ml occurs. This peak is highly responsible for potential nephrotoxicity [25]. To obtain the same reproducible blood profile as the microemulsion, a new approach is the administration of an ultrafine lipid dispersion (aqueous SLN dispersion), possessing simultaneously controlled release properties due to a solid matrix.

The first step of developing such carrier systems is the preparation of an aqueous SLN dispersion with a sufficiently high loading capacity for CycA. Considering the single doses of 100–200 mg, the loading capacity calculated on the particle mass (lipid + CycA) should be 20% to keep the volume of the final oral formulation (tablet) within an acceptable range. Imwitor®900 was identified as lipid matrix showing sufficient incorporation capability. In previous studies the entrapment efficiency of CycA within these SLN was found to be 96.1% [26].

Drugs can be incorporated differently into the lipid matrix, e.g., molecularly dispersed (solid solution) [27] or in form of amorphous clusters or definite particles, e.g., magnetites [28]. In case of a solid solution, theoretically drugs can be localized in between fatty acid chains or the lamellae of the lipids. This paper investigates the mode of incorporation of CycA into the SLN for better understanding of the delivery system.

Section snippets

Materials

Cyclosporine (CycA) was a gift from the company Pharmatec (Milan, Italy). The solid lipid Imwitor®900 (glycerol monostearate 40–50%) was purchased from Cognis (Dusseldorf, Germany). The emulsifiers Tagat®S (polyoxyethylene glycerol monosteatate) and sodium cholate were obtained from Goldschmidt (Essen, Germany) and Sigma (Deisenhofen, Germany), respectively. Water was collected by the MilliQ system Millipore (Schwalbach, Germany).

Methods

To access the maximum loading capacity of SLN for CycA physical

Characterization of bulk lipid and drug

An important parameter affecting drug incorporation is the polymorphic modification of the lipid particle matrix. In general, the production process of the nanoparticles can change the type of modification of their respective fraction. The bulk lipid Imwitor®900 was analysed by DSC (Fig. 1). Table 1 shows the corresponding DSC data.

The first heating curve revealed an onset temperature of 58.1 °C, corresponding to the β′-modification of Imwitor®900 [29]. To produce SLN, the lipid is melted, the

Conclusions

The drug CycA could be incorporated up to a relatively high loading of 20% into SLN (calculated related to the particle matrix, in case of 10% SLN dispersion this corresponds to 2% CycA in the dispersion itself). The CycA seems to be incorporated in molecularly dispersed form, i.e., as solid solution. In contrast to an o/w emulsion, the increased viscosity of the solid particle matrix in combination with the solid solution character should create a prolonged release. Release in vivo will take

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