4. RESULTS AND DISCUSSION
4.6. Liquid crystalline nanoparticles for topical delivery
Treatment of surgical site infections (SSIs), skin and soft tissue infections (SSTIs) and burn wound infections all rely on efficient antibiotic therapies. Hence, topical delivery of AMPs is of great interest for treatment of various skin infections caused by bacteria. The most common bacteria causing skin related infections are SA and its methicillin resistant analogue MRSA, PA and multi-drug resistant variants, Staphylococcus epidermis (SE) and EC [156-161]. In this part of the thesis, cubosomes prepared by the three methodologies (pre-, post- and hydrotrope) loaded with LL-37 will be discussed. Formulations of cubosomes may be best suitable for topical administration due to the hemolytic and cytotoxic properties reported previously [37, 76], which can be an obstacle e.g. for intravenous administration.
The release of LL-37 from cubosomes prepared by the hydrotrope method was presented and discussed previously; see Figure 15 D in section 4.2.1. No release of LL-37 was observed, and that was also the case for pre- and post-loaded cubosomes (data presented in Paper 5).
Moreover, these cubosomes were found to fully protect LL-37 from proteolysis, as presented in Figure 17 in section 4.3, as a consequence of being fully associated to the cubosomes. The LC structure of the particles discussed in this part of the thesis was investigated by SAXS and can be found in Paper 5.
The skin irritating potential of the cubosomes, with and without LL-37, was investigated using an in vitro human epidermis model. Results are presented in Figure 27 below. A viability >50 % of the keratinocytes predicts no skin irritation of a sample, according to the standardized test OECD TG 439. The viability of the keratinocyte cells was not negatively affected after exposure to any of the cubosome particles, indicating no skin irritation. No significant difference was observed for cubosomes with or without LL-37. Additionally, neither the preparation methodology nor the size of the corresponding cubosomes had any effect on the cells. Due to the very similar chemical compositions of the samples, any big differences would have been surprising. These findings indicate that GMO-based cubosomes can be used for topical administration of drugs, since they do not induce skin irritation. Potential applications within the field of cosmetics may also be possible.
Figure 27. Skin irritation potential of cubosomes presented as viability of keratinocyte cells after exposure to cubosomes with and without LL-37. No skin irritating potential of the samples was found since the viability were always >50 % compared to the negative control.
Results from the ex vivo pig skin wound infection model are presented in Figure 28. All cubosomes displayed a reduced bactericidal effect on SA, compared to pure LL-37 at the same concentrations. Pre-loaded cubosomes appeared to be most efficient at 0.5 mg/mL peptide loading. The hydrotrope particles were found to kill bacteria least effectively. On the other hand, pure LL-37 peptide showed the strongest bacterial killing. More than 95% of the bacteria were killed over the 4 h application period, with a dose-response dependency. The observation that the cubosomes reduced the activity of LL-37 was not expected by the previous in vitro studies. The delivery mechanism and antibacterial properties of LL-37 loaded cubosomes may differ among planktonic bacteria that are used in the MIC and time- kill assays, compared to the bacteria in this ex vivo model. Planktonic bacteria are more easy
accessible for pure LL-37 and while loaded into cubosomes due to the faster diffusion and Brownian motion of the particles. The results here indicate that release of peptide is more important for killing bacteria that are attached to a surface. The ex vivo model shows best antibacterial properties for the pre-loaded cubosomes. The small particle size and narrow size distribution (~130 nm, PDI 0.15) of the pre-loaded cubosomes creates a large surface area that appears to be beneficial in the delivery of LL-37, facilitating many interaction points between the particles and bacteria. Moreover, the smaller particles may diffuse faster into the infected wound, thus increasing the possibility to reach many bacteria. This may explain why the much larger hydrotrope particles do not perform as well. Surprisingly, the cubosomes without any LL-37 affected the SA bacteria negatively, especially in case of pre- and post-loaded cubosomes. This is something that was not observed in MIC-tests previously using similar particles. However, the samples were diluted to a much lower particle concentration in the MIC tests (<1 mg/mL) whereas in the ex vivo model the cubosome dispersions are applied without further dilution (~50 mg/mL), which may explain the difference in activity. Hence, high cubosome concentrations might be suffocating for the bacteria, thereby suppressing their growth. In summary, the use of cubosomes for topical delivery of LL-37 was found to be a trade-off between proteolytic protection and antimicrobial activity.
Figure 28. Survival of SA after sample exposure in the ex-vivo pig skin wound infection model. Data is presented as % of the viability of negative control (approximately 107 CFU/mL). “Reference” refers to cubosome without LL-37 in panel A-C, whilst in D it refers to 20 mM sodium acetate buffer pH 5.5. Negative control is BHI100. Data is presented as mean ± standard error of mean (n=5).