Rational Designs at the Forefront of Mitochondria-Targeted Gene Delivery: Recent Progress and Future Perspectives

Mitochondria play an essential role in cellular metabolism and generate energy in cells. To support these functions, several proteins are encoded in the mitochondrial DNA (mtDNA). The mutation of mtDNA causes mitochondrial dysfunction and ultimately results in a variety of inherited diseases. To date, gene delivery systems targeting mitochondria have been developed to ameliorate mtDNA mutations. However, applications of these strategies in mitochondrial gene therapy are still being explored and optimized. Thus, from this perspective, we herein highlight recent mitochondria-targeting strategies for gene therapy and discuss future directions for effective mitochondria-targeted gene delivery

Molecular dynamics study of the internalization of cell-penetrating peptides containing unnatural amino acids across membranes

Peptide-based delivery systems that deliver target molecules into cells have been gaining traction. These systems need cell-penetrating peptides (CPPs), which have the remarkable ability to penetrate into biological membranes and help internalize different cargoes into cells through the cell membranes. The molecular internalization mechanism and structure–function relationships of CPPs are not clear, although the incorporation of nonproteinogenic amino acids such as α-aminoisobutyric acid (Aib) has been reported to increase their helicity, biostability and penetration efficiencies. Here, we used molecular dynamics to study two Aib-containing CPPs, poly(LysAibAla)3 (KAibA) and poly(LysAibGly)₃ (KAibG), that previously showed high cell internalization efficiency. KAibA and KAibG displayed the lowest internalization energies among the studied CPPs, showing distinct internalization mechanisms depending on the lipid composition of the model membranes. The presence of Aib residues allows these CPPs to adopt amphipathic folding to efficiently penetrate through the membranes. Elucidating how Aib incorporation affects CPP–membrane binding and interactions is beneficial for the design of CPPs for efficient intracellular delivery

All-peptide-based polyion complex vesicles: Facile preparation and encapsulation of the protein in active form

The polyion complex vesicle (PICsome) is a promising platform for bioactive molecule delivery as well as nanoreactor systems. In addition to anionic and cationic charged blocks, a hydrophilic poly(ethylene glycol) (PEG) block is mostly employed for PICsome formation; however, the long-term safety of the PEG component in vivo is yet to be clarified. In this study, we developed novel PEG-free PICsome comprising all peptide components. Instead of the PEG block, we selected the sarcosine (Sar) oligomer as a hydrophilic block and fused it with anionic oligo(l-glutamic acid). Mixing the Sar-containing anionic peptide with cationic oligo(l-lysine) resulted in the formation of stable vesicles. The peptide-based PICsome was able to encapsulate a model protein in its hollow structure. After modification of the surface with a cell-penetrating peptide, the protein-encapsulated PICsome was successfully delivered into plant cells, indicating its promised for application as a biocompatible carrier for protein delivery

Cell wall‐denaturing molecules for plant gene modification

Zwitterionic molecules, such as zwitterionic liquids (ZILs) and polypeptides (ZIPs), are attracting attention for application in new methods that can be used to loosen tight cell wall networks in a biocompatible manner. These novel methods can enhance the cell wall permeability of nanocarriers and increase their transfection efficiency into targeted subcellular organelles in plants. Herein, we provide an overview of the recent progress and future perspectives of such molecules that function as boosters for cell wall-penetrating nanocarriers

Lipid Membrane Interaction of Peptide/DNA Complexes Designed for Gene Delivery

Among gene delivery systems, peptide-based gene carriers have received significant attention because of their selectivity, biocompatibility, and biodegradability. Since cellular membranes function as a barrier toward exogenous molecules, cell-penetrating peptides (CPPs), which are usually cationic and/or amphiphilic, can serve as efficient carriers to deliver cargo into the cytosol. Here, we examined the interactions of carrier peptides and their DNA complexes with lipid membranes using a quartz crystal microbalance (QCM) and high-speed atomic force microscopy (HS-AFM). The carrier peptides are a 12-residue partial presequence of yeast cytochrome c oxidase subunit IV (Cytcox) and BP100, which are a mitochondria-targeting signal peptide and a CPP, respectively. QCM data showed that BP100 has a higher binding affinity than Cytcox to both plasma membrane- and mitochondrial membrane-mimicking lipid bilayers. The DNA complexes with either Cytcox or BP100 exhibited the same tendency. Furthermore, HS-AFM data demonstrated that the DNA complexes of either peptide can disrupt the lipid membranes, forming larger pores in the case of Cytcox. Our results suggest that the binding affinity of the peptide/DNA complex to the plasma membrane is more critical than its membrane disruption ability in enhancing the cellular uptake of DNA

Microbial autotrophic biorefineries: Perspectives for biopolymer production

The use of autotrophic microorganisms to fabricate biochemical products has attracted much attention in both academia and industry. Unlike heterotrophic microorganisms that require carbohydrates and amino acids for growth, autotrophic microorganisms have evolved to utilize either light (photoautotrophs) or chemical compounds (chemolithotrophs) to fix carbon dioxide (CO₂) and drive metabolic processes. Several biotechnological approaches, including synthetic biology and metabolic engineering, have been proposed to harness autotrophic microorganisms as a sustainable/green production platform for commercially essential products such as biofuels, commodity chemicals, and biopolymers. Here, we review the recent advances in natural autotrophic microorganisms (photoautotrophic and chemoautotrophic), focusing on the biopolymer production. We present current state-of-the-art technologies to engineer autotrophic microbial cell factories for efficient biopolymer production

A Screening Method for the Isolation of Polyhydroxyalkanoate-Producing Purple Non-sulfur Photosynthetic Bacteria from Natural Seawater

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters accumulated by a variety of microorganisms as carbon and energy storage under starvation conditions. We focused on marine purple non-sulfur photosynthetic bacteria as host microorganisms for PHA production and developed a method for their isolation from natural seawater. To identify novel PHA-producing marine purple non-sulfur photosynthetic bacteria, natural seawaters were cultured in nutrient-rich medium for purple non-sulfur photosynthetic bacteria, and twelve pink- or red-pigmented colonies were picked up. Gas chromatography mass spectrometry analysis revealed that four isolates synthesized PHA at levels ranging from 0.5 to 24.4 wt% of cell dry weight. The 16S ribosomal RNA sequence analysis revealed that one isolate (HM2) showed 100% identity to marine purple non-sulfur photosynthetic bacteria. In conclusion, we have demonstrated in this study that PHA-producing marine purple non-sulfur photosynthetic bacteria can be isolated from natural seawater under nutrient-rich conditions

Molecular dynamics simulation of complexation between plasmid DNA and cationic peptides

The elucidation of the process by which cationic peptides condense plasmid DNA (pDNA) is important for unraveling the mechanism of peptide/pDNA complex formation, which plays a vital role in gene delivery for the genetic transformation of living cells. We performed atomic MD simulations of the complexation of pDNA in the presence of two cationic peptides, KH9 (with an alternating sequence of lysine and histidine) and Cytcox (functioning as a mitochondria-targeting signal), to investigate the mechanism of pDNA condensation. The simulations revealed that the cationic peptides bound to the pDNA and that defects in pDNA formed in response to the densely packed cationic peptides, presumably initiating the folding of the double-stranded pDNA into a globule morphology. The decrease in the radius of gyration and the number of hydrogen bonds and the increase in the writhe structure, with a slightly higher tendency for the Cytcox/pDNA system, strongly support the formation of pDNA defects leading to the bending of the double helix. The results provided insight into the mechanism of pDNA complexation with cationic peptides, which should contribute to the future design of highly efficient gene delivery systems using peptide-mediated nanocarriers.This work was financially supported by Japan Science and Technology Agency Exploratory Research for Advanced Technology (JST ERATO; Grant No. JPMJER1602). We acknowledge the RIKEN Advanced Center for Computing and Communication (ACCC) for providing access to HOKUSAI BigWaterfall Supercomputer resources.Peer ReviewedPostprint (published version

Organelle-targeted gene delivery in plants by nanomaterials

Genetic engineering of plants has revolutionized agriculture and has had a significant impact on our everyday life. It has allowed for the production of crops with longer shelf lives, enhanced yields and resistance to pests and disease. The application of nanomaterials in plant genetic engineering has further augmented these programs with higher delivery efficiencies, biocompatibility and the potential for plant regeneration. In particular, subcellular targeting using nanomaterials has recently become possible with the cutting-edge developments within nanomaterials, but remains challenging despite the promise in organellar engineering for the introduction of useful traits and the elucidation of subcellular interactions. This feature article provides an overview of nanomaterial delivery within plants and highlights the application of recent progress in nanomaterials for subcellular organelle-targeted delivery