Ethanol (EtOH) abuse induces significant mortality and morbidity worldwide because of detrimental effects on brain function. Defining the contribution of astrocytes to this malfunction is imperative to understanding the overall EtOH effects due to their role in homeostasis and EtOH-seeking behaviors. Using a highly controllable in vitro system, we identify chemical signaling mechanisms through which acute EtOH exposure induces a modulatory feedback loop between neurons and astrocytes. Neuronally-derived purinergic signaling primed a subpopulation of astrocytes to respond to subsequent acute EtOH exposures (SEastrocytes: signal enhanced astrocytes) with greater calcium signal strength. Generation of SEastrocytes arose from astrocytic hemichannel-derived ATP and accumulation of its metabolite adenosine within the astrocyte microenvironment to modulate adenylyl cyclase and phospholipase C activity. These results highlight an important role of astrocytes in shaping the overall physiological responsiveness to EtOH and emphasize the unique plasticity of astrocytes to adapt to single and multiple exposures of EtOH.

Cell-penetrating peptides are a promising therapeutic strategy for a wide variety of degenerative diseases, ageing, and cancer. Among the multitude of cell-penetrating peptides, PepFect14 has been preferentially used in our laboratory for oligonucleotide delivery into cells and in vivo mouse models. However, this activity has mainly been reported towards cytoplasm and nuclei, while the mentioned disorders have been linked to mitochondrial defects. Here, we report a library generated from a combinatorial covalent fusion of a mitochondrial-penetrating peptide, mtCPP1, and PepFect14 in order to deliver therapeutic biomolecules to influence mitochondrial protein expression. The non-covalent complexation of these peptides with oligonucleotides resulted in nano-complexes affecting biological functions in the cytoplasm and on mitochondria. This delivery system proved to efficiently target mitochondrial genes, providing a framework for the development of mitochondrial peptide-based oligonucleotide technologies with the potential to be used as a treatment for patients with mitochondrial disorders.

Recent advances with techniques used for manipulating gene expression have brought us to an era where various gene therapeutic approaches are becoming common therapeutic tools for many previously incurable diseases. The main factor impeding the wider translation of gene therapy is that the active pharmaceutical ingredients used for interfering with gene expression are based on nucleic acids and synthetic oligonucleotides and such molecules do not readily reach their intracellular targets due to their physicochemical properties and therefore they require delivery vectors to cross the cell membrane. 

Cell-penetrating peptides (CPPs) is one such class of delivery vectors that comprise excellent potential for transporting bioactive cargo molecules across cellular membranes, both in vitro and in vivo conditions. CPPs have shown to be very versatile carriers for various types of bioactive cargo, including different nucleic acids such as plasmids (pDNA), splice-correcting oligonucleotides (SCOs), small interfering RNAs (siRNA) and mRNA, or peptides and proteins or even small molecules.

This thesis focuses on characterizing the delivery of various nucleic acids-based molecules with a variety of novel fatty acid modified CPPs. In order to achieve this we utilize the ability of a family of CPPs called PepFects to non-covalently formulate nucleic acids into nanoparticles. More particularly the aim of the thesis is to find and characterize the key parameters of these peptide/nucleic nanoparticles that would improve their potential applicability as a drug formulation and delivery system for future gene therapies.

By simultaneously characterizing the role of N-terminal fatty acid modification and the peptide/nucleic acid ratio in the nanoparticles we were able to show in Papers I and II that increasing the hydrophobicity and reducing unbound free fraction of the peptide improves delivery efficiency and decreases toxicity of these nanoparticles both in vitro and in vivo.

Based on the findings from Paper I regarding the ability of these amphiphilic peptides to self-associate into supramolecular structures we went deeper in Paper III to study the formation, composition and live cell association of these peptide/nucleic acid complexes at single molecule sensitivity.

And finally in Paper IV we enhanced the specificity of these nanoparticles towards in vivo xenograft tumors by incorporating the capacity to be specifically activated in the tumor microenvironment.

Conclusively, these findings contribute to the field with identifying and characterizing some of the key factors in developing efficient and safe peptide-based delivery vectors for gene modulating therapeutics.

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.

In recent decades many new methods have been developed to cure or treat genetical disorders such as cancer, viral infections or inheritable diseases. The problem is that the nucleic acids and their synthetic analogs, oligonucleotides, are not able to cross the cell membrane due to their physicochemical properties like high negative charge and size. Therefore they need assistance to reach their intracellular target.

Cell-penetrating peptides (CPPs) are a class of versatile delivery vectors that can be used to transport various types of bioactive molecules inside the cells, including proteins, small molecules and also nucleic acids like plasmid DNA (pDNA), splice-correcting oligonucleotides (SCO), small interfering RNA (siRNA) and messenger RNA (mRNA).

A well-known method to improve CPPs in non-covalent delivery of nucleic acids is to modify them N-terminally with fatty acids such as stearic acid (C18:0). In this thesis we have studied the role of N-terminal acylation and the length of the carbon chain in the delivery of short SCO as well as larger plasmid DNA. In paper I we varied the N-terminal acyl chain length of a well-studied stearylated CPP, PepFect14, from 2-22 carbons. The delivery efficiency of SCO was dependent on the acyl chain length and it was found to be proportional to the increased association of peptide/oligonucleotide complexes to the cell membrane. In paper II the versatility of PepFect14 as a non-covalent nucleic acid delivery vector was validated using plasmid DNA. Compared to its non-stearylated counterpart, PepFect14 was able to condense pDNA into stable nanoparticles and mediate high gene expression both in regular adherent cell lines as well as difficult-to-transfect primary cells.

Non-viral gene delivery systems have gained considerable attention as a promising alternative to viral delivery to treat diseases associated with aberrant gene expression. However, regardless of extensive research, only a little is known about the parameters that underline in vivo use of the nanoparticle-based delivery vectors. The modest efficacy and low safety of non-viral delivery are the two central issues that need to be addressed. We have previously characterized an efficient cell penetrating peptide, PF14, for in vivo applications. In the current work, we first develop an optimized formulation of PF14/pDNA nanocomplexes, which allows removal of the side-effects without compromising the bioefficacy in vivo. Secondly, based on the physicochemical complex formation studies and biological efficacy assessments, we develop a series of PF14 modifications with altered charge and fatty acid content. We show that with an optimal combination of overall charge and hydrophobicity in the peptide backbone, in vivo gene delivery can be augmented. Further combined with the safe formulation, systemic gene delivery lacking any side effects can be achieved.

Cell-penetrating peptide (CPP) based transfection systems (PBTS) are a promising class of drug delivery vectors. CPPs are short mainly cationic peptides capable of delivering cell non-permeant cargo to the interior of the cell. Some CPPs have the ability to form non-covalent complexes with oligonucleotides for gene therapy applications. In this study, we use quantitative structure–activity relationships (QSAR), a statistical method based on regression data analysis. Here, a fragment QSAR (FQSAR) model is developed to predict new peptides based on standard alpha helical conformers and Assisted Model Building with Energy Refinement molecular mechanics simulations of previous peptides. These new peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were capable of achieving plasmid transfection with significant improvement compared to the previous generation of peptides. Our results demonstrate that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity.

Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well studied CPP, PepFectl4, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.

Gene therapy has great potential to treat a range of different diseases, such as cancer. For that therapeutic gene can be inserted into a plasmid vector and delivered specifically to tumor cells. The most frequently used applications utilize lipoplex and polyplex approaches where DNA is non-covalently condensed into nanoparticles. However, lack of in vivo efficacy is the major concern that hinders translation of such gene therapeutic applications into clinics. In this work we introduce a novel method for in vivo delivery of plasmid DNA (pDNA) and efficient tumor-specific gene induction using intravenous (i.v) administration route. To achieve this, we utilize a cell penetrating peptide (CPP), PepFect14 (PF14), double functionalized with polyethylene glycol (PEG) and a matrix metalloprotease (MMP) substrate. We show that this delivery vector effectively forms nanoparticles, where the condensed CPP and pDNA are shielded by the PEG, in an MMP-reversible manner. Administration of the complexes results in efficient induction of gene expression specifically in tumors, avoiding normal tissues. This strategy is a potent gene delivery platform that can be used for tumor-specific induction of a therapeutic gene.

Peptides and peptide-cargo complexes have been used for drug delivery and gene therapy. One of the most used delivery vectors are cell-penetrating peptides, due to their ability to be taken up by a variety of cell types and deliver a large variety of cargoes through the cell membrane with low cytotoxicity. In vitro and in vivo studies have shown their possibility and full effectiveness to deliver oligonucleotides, plasmid DNA, small interfering RNAs, antibodies, and drugs. We report in this review some of the latest strategies for peptide-mediated delivery of nucleic acids. It focuses on peptide-based vectors for therapeutic molecules and on nucleic acid delivery. In addition, we discuss recent applications and clinical trials.