Conclusion

96 the mentioned studies, the applied curcumin concentration ranged from 10 µmol/l to 100 µmol/l and the encapsulated curcumin revealed a similar activity as the free drug. A pronounced effect of comparable concentrations of free curcuminoids on Caco-2 cells was also confirmed by the present work. However, the drug-loaded nanoparticles did not show any cytotoxic effects on the Caco-2 cells. Even the administration of 680 µmol/l of curcuminoids, encapsulated in the lipid nanoparticles, did not affect the viability of the Caco-2 cells. Free curcuminoids caused cell death at much lower doses, compared to the tolerated dose of encapsulated curcuminoids (see above). The missing immediate cytotoxic effect of the curcuminoids on the Caco-2 cells, was attributed to the association of the drug to the nanoparticles. As already mentioned, the lipid nanoparticles were assumed to be unable to interact with the cell surface, because of the surrounding hydrophilic poloxamer shell. Thus, an interaction of the curcuminoids with the Caco-2 cells was also inhibited, due to the incorporation of the drug in the particle. In addition, the missing effect of the curcuminoids on the cells indicated that the drug was not released during the incubation time. The marginal release of the curcuminoids from the nanoparticles under neutral conditions was already shown by the in vitro release studies (see chapter 4.5.1)

In summary, the curcuminoid-free as well as the curcuminoid loaded nanoparticles revealed no acute cytotoxic effect on the Caco-2 cells. The applied lipid and the emulsifier are known to be non-toxic, e.g. the lipids have been stated as GRAS-substances by the FDA. The curcuminoids, showing a severe effect on the cells in their free form, were thought to be effectively separated from the cells by incorporating them into the nanoparticles. Thus, the nanoparticles rather protected the cells, than supporting the drug in their action. The interaction of the nanoparticles with the cells has to be checked in further experiments with additional analytical methods, e.g. confocal microscopy and by using other cell lines. The long-term effects of the drug-loaded nanoparticles on growth and viability of various cell lines have to be further investigated as well. Especially the induction of apoptosis by the curcuminoid-loaded nanoparticles has to be in the focus of upcoming experiments. It is of distinct interest, whether the drug-loaded nanoparticles are capable of entering the cell and induce apoptotic cell death of cancer cells.

97 (70,73,163). However, these systems had serious drawbacks, such as the insufficient separation of drug and carrier, which have been discussed in detail chapter 4.5.1.

Therefore, a new in vitro method for the determination of the curcuminoid release from the lipid nanoparticles was developed and tested to overcome these problems and to examine the drug-release in a sufficient way. The curcuminoid release was investigated in SGF, phosphate buffer pH 6.8, FaSSIF and FeSSIF. These media were chosen, because they simulate to some extent the path of the formulation after oral administration. The drug release was shown to be dependent on the release medium and the physical state of lipid matrix. The curcuminoids were hardly released in phosphate buffer pH 6.8. A distinct release was detected, when drug-loaded lipid nanoparticles were incubated in SGF and the fastest release was recorded in the simulated intestinal media FaSSIF and FeSSIF, with the release being more accelerated in FeSSIF. The determined release pattern was explained by the different solubility of the curcuminoids in the respective media. In addition, the present bile salts and phospholipids were thought to efficiently “wash-off”

the curcuminoids from the lipid nanoparticles. The influence of the physical state of the lipid on the release properties was also verified by the conducted experiments. It was found that the curcuminoids were released faster from the crystalline TM nanoparticles compared to the supercooled TM nanoemulsions. This finding was explained by the localization of the drug on the surface of the crystalline particles, where the curcuminoids could diffuse easily into the surrounding medium. The fastest diffusion into the medium was found during the incubation of the preparations in FaSSIF or FeSSIF, respectively, because the drug was probably solubilized by the mixed micelles. In contrast, the drug diffusion out of the liquid droplets of the TM nanoemulsion was distinctly slower. The gained results supported the theory, drawn from the fluorescence and Raman measurements, that the curcuminoids are in contact with the surrounding environment and the lipid matrix, either solid or liquid, is not separating the drug. Therefore, a solid lipid matrix was considered to protect a drug only if it is incorporated within the lipid core. The highly crystalline order of the triglycerides prevented this incorporation for the curcuminoids in the developed formulations. It was summarized that the developed model was capable of estimating the release of the curcuminoids from the lipid nanoparticles, but the experimental setup provides options for further optimization and a final conclusion about the applicability, which is not possible yet.

The in vitro digestion of the lipid nanoparticles was monitored by two different methods, the pH-stat method and HPTLC/spectrodensitometry. The experiments were executed to gain more information about the potential fate of the lipid nanoparticles within the gastrointestinal tract. Additionally, it should be determined whether the driving force of the drug liberation is the lipid degradation or the drug release from the particle. The digestion of MCTCurc-NE, TMCurc-NE, TMCurc-NS and TSCurc-NS was conducted in

98 simulated fasted state and simulated fed state intestinal medium and the digestion velocity was recorded by the pH-stat method. It was shown that the major part of the lipid was digested in the first ten minutes of the experiment. The resulting progression of the titration curves and the cumulated consumption of sodium hydroxide were different for the particular formulations. These differences were mainly attributed to the different determinability of the released fatty acids. The release of longer fatty acids, e.g. stearic acids, was only ascertained to about 50 % by the pH-stat method, whereas 100 % of the released caprylic and capric acid were titrable. The underlying cause for this outcome was thought to be the different dissociation of the fatty acids at the given pH of 6.8. The pH-stat method was concluded to be a fast and easy method for the evaluation of the digestability of a lipid formulation, but the quantitative determination of the liberated fatty acids was shown to be not possible.

A comprehensive overview of the digestion process was gained by HPTLC/spectrodensitometry. TMCurc-NE and TMCurc-NS were investigated by this method, because the influence of the physical state of the lipid on the speed of digestion should be illustrated. In contrast to the pH-stat method, all involved compounds, except the diglycerides, were recorded and quantified. The liquid triglyceride matrix of TMCurc-NE was found to be degraded completely within the first ten minutes of the experiment.

Moreover, no difference in digestion velocity was observed between fasted and fed state medium. In contrast, the complete digestion of the crystalline lipid matrix of TMCurc-NS took 30 minutes for FaSSIF and 20 minutes for FeSSIF, respectively. Hence, the examination of the digestion of TMCurc-NE and TMCurc-NS with HPTLC/spectrodensitometry revealed the slower digestion of a crystalline lipid matrix.

Moreover, the accelerated lipid degradation in a simulated fed state was clearly demonstrated. The analysis with HPTLC/spectrodensitometry further allowed a better interpretation of the titration curves of the pH-stat method. It was verified that the steep initial slope of the titration curves was actually caused by the triglyceride degradation, whereas the digestion of the diglycerides and monoglycerides caused the little consumption of sodium hydroxide in the latter stages of the experiment. In summary, a detailed insight of the processes during the in vitro digestion of TM nanoparticles was gained by HPTLC/spectrodensitometry. Thus, the method was concluded to be superior compared to the pH-stat method, but the conduction of the experiments and the evaluation of the raw data is much more time-consuming and costly.

Subsequent to the digestion experiments, the solubilization of the curcuminoids within the simulated intestinal media was determined. The solubilized drug amount after digestion was higher for the curcuminoid-loaded TM nanoparticles compared to the curcuminoid bulk material and the solubilization capacity was higher for the fed state medium than for the fasted state medium.

99 When the aspects of the drug release experiments, the in vitro digestion and the drug solubilization studies are finally summarized, the following conclusions and suggestions for the in vivo fate can be made:

1. The lipid nanoparticles are degraded very fast and only a minor fraction of intact particles might be transferred to the lymphatic system.

2. The absorption of the curcuminoids is would involve the lipid digestion, the subsequent transfer into mixed micelles and the transport of the drug within micelles to the intestinal walls.

3. As a consequence, sole drug release from the nanoparticles plays only a minor role due to the rapid degradation of the particles and should not be used as a single parameter, when investigating the oral bioavailability of curcuminoids.

4. The presence of bile salts and phospholipids is an important prerequisite for the successful solubilization of the curcuminoids and therefore the fed state is to be favored over the fasted state.

The cell culture experiments showed the acute toxicity of curcuminoids on Caco-2 cells.

The death of the cells was attributed to an interaction of the curcuminoids with the cell membranes, resulting in a breakdown of the membrane potential. In contrast, the curcuminoid-loaded lipid nanoparticles were non-toxic to the cells. It was concluded that the cells were not interacting with the nanoparticles, because of the shielding effect of the surrounding poloxamer layer of the nanoparticles. As a result, an uptake of the particles into the cells was not probable within the incubation time and an interaction of the curcuminoids with the cells was prevented. Therefore, the incorporation of the curcuminoids within the nanoparticles protected the cells from the acute toxicity of the drug. However, the performed experiments were designed to investigate the acute effects of free curcuminoids and curcuminoid-loaded nanoparticles on the Caco-2 cells. An assessment of the long-term effects, e.g. induction of apoptosis, is still to be addressed in future experiments. Furthermore, one has to keep in mind that cancer cells were used in the experiments and that such cells are more robust compared to normal cells. So the reaction of non-malignant cells to the curcuminoid-loaded lipid nanoparticles cannot be estimated from the conducted experiments. Though, the basic differences between free drug and drug-loaded nanoparticles were well demonstrated by the performed experiments.

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