Development of Peptide-Based Nanoparticles for Mitochondrial Plasmid DNA Delivery

Abstract

A mitochondrion is a cellular organelle able to produce cellular energy in the form of adenosine triphosphate (ATP). As in the nucleus, mitochondria contain their own genome: the mitochondrial DNA (mtDNA). This genome is particularly susceptible to mutations that are at the basis of a multitude of disorders, especially those affecting the heart, the central nervous system and muscles. Conventional clinical practice applied to mitochondrial diseases is very limited and ineffective; a clear need for innovative therapies is demonstrated. Gene therapy seems to be a promising approach. The use of mitochondrial DNA as a therapeutic, optimized by peptide-based complexes with mitochondrial targeting, can be seen as a powerful tool in the reestablishment of normal mitochondrial function. In line with this requirement, in this work and for the first time, a mitochondrial-targeting sequence (MTS) has been incorporated into previously researched peptides, to confer on them a targeting ability. These peptides were then considered to complex a plasmid DNA (pDNA) which contains the mitochondrial gene ND1 (mitochondrially encoded NADH dehydrogenase 1 protein), aiming at the formation of peptide-based nanoparticles. Currently, the ND1 plasmid is one of the most advanced bioengineered vectors for conducting research on mitochondrial gene expression. The formed complexes were characterized in terms of pDNA complexation capacity, morphology, size, surface charge and cytotoxic profile. These data revealed that the developed carriers possess suitable properties for pDNA delivery. Furthermore, in vitro studies illustrated the mitochondrial targeting ability of the novel peptide/pDNA complexes. A comparison between the different complexes revealed the most promising ones that complex pDNA and target mitochondria. This may contribute to the optimization of peptide-based non-viral systems to target mitochondria, instigating progress in mitochondrial gene therapy.

Peptides vs. Polymers: Searching for the Most Efficient Delivery System for Mitochondrial Gene Therapy

“…The peptides used in this work were synthesized according to the protocol described in a previous work [18]. The synthesis of the PEI-DQA polymer was carried out in two steps: The first step was the synthesis of PEI-SA.…”

“…2.2.3. Formulation of Peptide/pDNA and PEI-DQA/TAT/pDNA Complexes Peptide-based complexes were formulated according to the procedure described in a recent publication by our team [18]. PEI-DQA, TAT, and plasmid DNA stock solutions were prepared in sodium acetate buffer (0.1 mM sodium acetate/0.1 M acetic acid, pH 4.5) at a concentration of 0.5 mg/mL for PEI-DQA and TAT solutions, and 100 µg/mL for pDNA solutions.…”

“…In line with this, the need to develop new, effective therapies arises. Since most mitochondrial diseases are associated with mutations in mtDNA, mitochondrial gene therapy emerges as a very promising approach to treat the problem at its root [16][17][18]. This type of therapy is focused on the delivery of genetic material to the mitochondria to suppress, alter, or complement the effect of defective genes [16].…”

Peptide-Assisted Nucleic Acid Delivery Systems on the Rise

“…For example, a library of fusion peptides with mitochondria targeting (mtCPP1) and cellpenetrating properties (Pepfect14, a stearylated CPP forming ASO nanocomplexes with splice-correction activity in cells [241]) that self-assembled with antisense oligonucleotides (ASO) into complexes was successful in knocking down mitochondrial mRNA [242]. A combinatorial approach to develop mitochondrial gene expression was also pursued by incorporating an MTS into WRAP peptides (short tryptophan/arginine rich peptides; [243]) which formed nanocomplexes with plasmid DNA encoding the mitochondrial ND1 gene that were taken up by cells and targeted to mitochondria [244]. Systematic analysis of CPPs and MTSs revealed that while both types of peptides were rich in Ala and Arg, the latter included Leu, suggesting a role for Leu in targeting to mitochondria [245].…”

Design of Experiments to Achieve an Efficient Chitosan-Based DNA Vaccine Delivery System

“…However, the transfection efficiency of eukaryotic cells with naked nucleic acids, such as plasmid DNA, is disfavored by the negative repulsion of cell membrane. In this field, several biomaterials based on cationic liposomes [9,10], peptides [11,12] and polymers [13,14] have been shown to be able to efficiently condense, protect, carry, and deliver nucleic acids to eukaryotic cells. For instance, chitosan and chitosan derivatives have been used as DNA delivery systems due to their cationic charge, biocompatibility, biodegradability, mucoadhesiveness and permeability-enhancing properties [15][16][17][18][19].…”