Abstract by Camilla Jantzen Asgreen

Atopic dermatitis, also known as eczema, is a multifactorial inflammatory skin disease that affects up to 30% of children and 10% of adults worldwide. Atopic dermatitis causes dry, itchy, and inflamed skin, which can lead to eczematous lesions that are prone to infection. Reduced levels of host defence peptides in atopic skin might contribute to bacterial colonization. Therefore, restoring these levels could potentially reduce susceptibility to infections and decrease flare-ups. Host defence peptides are small cationic peptides, which exhibit both antimicrobial and immunomodulatory activity. However, as peptides are hydrophilic compounds and prone to proteolytic degradation, the need for an effective drug delivery vehicle is inevitable. Lyotropic liquid crystalline nanoparticles, such as cubosomes and hexosomes, have shown great potential due to their ability to encapsulate peptides without interfering with efficacy. Furthermore, cubosomes have demonstrated to protect peptides from proteolytic degradation.

Therefore, this thesis aimed to investigate the potential of lyotropic liquid crystalline nanoparticles as drug delivery vehicles for host defence peptides for atopic skin infections. To achieve this, Paper 1 set out to investigate sixteen compositions of lyotropic liquid crystalline nanoparticles with different lipids (phytantriol and monoolein) and varying stabilizers (F127 and Citrem). The nanoparticles were assessed according to their structure, size, ζ-potential and colloidal stability. Four selected particles were further investigated for their bactericidal activity against various Gram-positive and Gram-negative strains. Furthermore, the safety and immunomodulatory properties of the lyotropic liquid crystalline nanoparticles were assessed in vitro using both 2D- and 3D cell models. Paper 1 shows that the components used and concentration of the stabilizers affected the assembly of the lyotropic liquid crystalline nanoparticles and their colloidal properties. Nonetheless, all particles were in acceptable size ranges and were stable for at least 6 months. The selected particles all exhibited dosedependent bactericidal activity against Gram-positive strains, however, no effect was observed against Gram-negative strains. The effect was likely caused by the observed membrane disruption, which may be explained by lipoteichoic acid-binding. Binding between lipoteichoic acid and the lyotropic liquid crystalline nanoparticles also resulted in a reduction of lipoteichoic acid-induced NF-κB/AP-1 activation, hence, the particles show direct immunomodulatory properties as well.

The bactericidal activity of the lyotropic liquid crystalline nanoparticles depended on composition, favouring the lipid phytantriol and the stabilizer F127. However both also induced greater toxicity than their counterparts monoolein and Citrem. Interestingly, the observed toxicity for the monoolein-based particle (stabilized with F127) did not translate in a 3D organoid epidermal skin model, where no toxicity was observed.

In Paper 2, three sequence-similar peptides KYE28, KYE21 and WWWKYE21, were encapsulated in monoolein-based lyotropic liquid crystalline nanoparticles using a pre-loading method. The peptideparticle interactions were assessed according to their encapsulation efficiency, size, ζ-potential, structure and colloidal stability. Furthermore, the bactericidal activity against Staphylococcus aureus of KYE28- and WWWKYE21-loaded lyotropic liquid crystalline nanoparticles were assessed at various loading concentrations. Paper 2 shows that peptide encapsulation was effected by peptide sequence, more precisely, their length and hydrophobic moieties, as the peptides were loaded to different extents (WWWKYE21 > KYE28 > KYE21). The structure of the lyotropic liquid crystalline nanoparticles were not significantly affected after encapsulation of KYE28, however it was shown that at higher loading concentrations, WWWKYE21 induced phase transition of the cubic structure to vesicles. Nevertheless, all KYE28- and WWWKYE21-loaded particles resulted in stable lyotropic liquid crystalline nanoparticles in an acceptable size range, which all exhibited bactericidal activity against S. aureus depending on the initial loading concentration. Importantly, after comparison of the bactericidal activity of the loaded host defence peptides against free peptide, it was found that the encapsulation in lyotropic liquid crystalline nanoparticles boosted the antibacterial activity of the peptide at lower concentrations.

In conclusion, this thesis demonstrated that lyotropic liquid crystalline nanoparticles can encapsulate and enhance the activity of host defence peptides, while also exhibiting direct immunomodulatory and bactericidal properties, making them promising drug delivery system for atopic skin infections.