Design and Optimization of Membrane Active Peptides and Lipid Vesicles for Triggered Release

• Liposomes can reduce toxic side effects and improve the efficacy of drugs and several liposome-based drug formulations are approved for clinical use. The therapeutic effect is dependent on the bioavailability of the drug and a slow drug release from liposomes can reduce their efficacy. Multiple strategies have been proposed to control the release of drugs from liposomes using both external stimuli such as light, heat and ultrasound, and endogenous factors such as changes in pH or enzymatic activity. However, because of the difficulties to efficiently modulate lipid membrane permeability and the challenges to trigger drug release in the target tissue, no stimuli responsive liposomes have so far been approved. There is consequently a great need for new means to tune lipid membrane integrity for liposome cargo release to improve the development of new advanced drug delivery systems for better and safer treatment of patients.  The aim of this thesis was to design and explore synthetic membrane active peptides for triggered release from liposomes and to expand the knowledge on how peptide-lipid conjugation strategies and lipid properties affect the membrane activity of the peptides. This work was based on two different de novo designed cationic and amphipathic, conjugation-dependent membrane active peptides (CKV4 and JR2KC). Both peptides fold and adopt α-helical structures upon conjugation to liposomes, triggering lipid membrane destabilization. Addition of cholesterol in the lipid membrane greatly enhanced the release efficiency of JR2KC due to a peptide-triggered lipid phase separation, resulting in domains with high local peptide concentrations. Additionally, both peptide surface concentrations and lipid net charge were found to be important factors for efficient release. However, when the zeta potential decreased below -75 mV, conjugation-independent release mechanisms were triggered. Liposome size was shown to only have minor effects on the release kinetics for both sets of peptides while a mixture of saturated and unsaturated lipids was beneficial for the peptide-triggered membrane destabilization, possibly due to increased propensity for lipid phase separation.  In addition to changing lipid properties, peptide-lipid conjugation strategies proved to highly affect the release kinetics, where the Michael addition reaction between a cysteine in the peptide and maleimide-lipids was much more efficient in causing peptide-triggered membrane destabilization than strain-promoted alkyne azide cycloaddition reactions using azide-modified peptides and DBCO-functionalized lipids. However, thiols tend to oxidize under ambient conditions which complicates peptide-lipid conjugation. This was addressed by synthesizing a peptide with a cysteine modified with an enzyme labile thiol protection group. Enzymatic deprotection allowed efficient peptide-lipid conjugation, reducing the risk of peptide oxidation.  To further find means to tailor peptide-lipid interactions, we explored the effect of a competing peptide heterodimerization process on lipid membrane destabilization. Addition of a charge complementary peptide to CKV4 resulted in heterodimerization and folding into a coiled coil, which inhibited its membrane activity. However, when the two peptides were synthesized as a single sequence, the membrane activity was altered, most likely due to the induced preorganization increasing membrane affinity. The results presented in this thesis provide new understandings of the complex peptide-lipid interactions that govern peptide-induced release from liposomes and will facilitate further optimization in peptide design for the future development of advanced liposome-based drug delivery systems. 

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

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