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101 micrographs. In the end, the application of both methods was concluded to be needed for providing a comprehensive overview of the particle shape of the developed formulations.
The investigation of the curcuminoid-loaded lipid nanoparticles with different physical methods provided information about the state of the lipid matrix and the curcuminoids.
The crystallization behavior of the lipid nanoparticles was investigated by DSC and XRD. It was shown that the TM nanoparticles did crystallize at 11 °C and therefore supercooled melts were preserved when the TM-based formulations were stored above this temperature. The supercooled TM nanoemulsion stayed liquid during the whole observation period of 16 weeks at a storage temperature of 22 °C. However in some cases a partial recrystallization occurred. Hence, the supercooled state was concluded to be metastable and therefore inappropriate as a pharmaceutical formulation. The solid TM and TS nanoparticles were demonstrated by DSC and XRD to crystallize mainly in the stable β–modification. The TS nanoparticles exhibited also some traces of the α–
modification, which transformed to the β-modification within 16 weeks. The fast crystallization and the highly crystalline order strongly indicated the exclusion of the curcuminoids from the solid lipid matrix. This assumption was supported by the Raman and fluorescence measurements. The curcuminoids were shown to be in an amorphous state within the lipid nanoparticles by the Raman measurements. The fluorescence experiments demonstrated the influence of the physical state of the lipid on the fluorescence properties of the curcuminoids. In addition, the surrounding media also influenced the curcuminoids fluorescence, emphasizing the drug to be localized on the surface of the solid particles. Moreover, fluorescence anisotropy measurements revealed a considerable mobility of the drug associated to the TM nanoparticles, which further pointed to a localization of the curcuminoids apart from the rigid lipid matrix.
The curcuminoid content of the lipid nanoparticles was constant over a storage period of twelve months. In addition, the incubation of the curcuminoid-loaded nanoparticles in physiological media, e.g. SGF and phosphate buffer pH 6.8, revealed no degradation of the drug over an incubation period of eight hours. Thus, the stability of the curcuminoids in vitro makes a pronounced decomposition of the drug after the oral application and its subsequent contact with gastric and intestinal fluids rather improbable.
The curcuminoid release from the lipid nanoparticles was shown to be dependent on the respective release medium and on the physical state of the TM-based lipid matrix. The drug was not released from the nanoparticles during incubation in phosphate buffer pH 6.8. On the contrary, in SGF and in simulated intestinal fluids, a release between 30 % – 80 % was observed. Moreover, the curcuminoids were released faster from the crystalline TM nanoparticles in comparison to the TM nanoemulsion. In connection with the release experiments, the digestion of the lipid nanoparticles in simulated gastric and simulated intestinal fluid was assessed. No degradation of the lipid matrix was observed during the
102 incubation of the TM nanoparticles in SGF. In contrast, the in vitro digestion in simulated intestinal fluid revealed a rapid and complete degradation of the triglyceride particle matrix by the added lipase. The degradation of the lipid was faster in the fed state than in the fasted state. Furthermore, the crystalline TM nanoparticles were digested slower than the TM nanoemulsions. Nevertheless, the digestion was found to be much faster than the previously determined drug release from the nanoparticles. It was therefore concluded that liberation of the drug within the gastro-intestinal tract might be mainly caused by particle decomposition. The released curcuminoids were shown to be transferred to mixed micellar structures within the simulated intestinal fluids. Hence, it was seen as probable that the drug is also transferred to mixed micelles during in vivo digestion. Such a solubilisation of the curcuminoid meets the major prerequisite for the efficient uptake of the drug in the intestine.
The toxicity of the curcuminoid-loaded nanoparticles and the free curcuminoids was tested with Caco-2 cells. The cell culture experiments assessed the tolerance of the cells against the drug-loaded nanoparticles. In contrast, free curcuminoids reduced the viability of the cells in a considerable way. The conducted cell experiments were seen as basic evaluation of the biological activity of the curcuminoid-loaded formulations. The further investigation of the biological activity is considered to be the major challenge of future experiments. For this purpose further cell culture experiments have to be launched to investigate the long-term effects of the curcuminoid-loaded preparations on cells.
Especially, the potential uptake of the particles by the cells and the activity of the incorporated curcuminoids are of certain interest. The next step should be the establishment of an efficient process for extracting the curcminoids out of the blood and a convenient analytical method, allowing the detection of small amounts of drug and its metabolites. Though, the preparation and extraction of curcuminoids out of blood samples and the subsequent quantification have been already described in literature (198-201).
Thereafter, curcuminoid-loaded preparations can be applied in a basic in vivo experiment to determine pharmacokinetic parameters of the drug and to assess potential adverse effects. The final step in the preclinical research would be application of the curcuminoid-loaded lipid nanoparticles in an animal cancer model and the evaluation of its curative or preventive potential.
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