Liposomes and Liposome-like Vesicles for Drug and DNA Delivery to Mitochondria

Mitochondrial research is presently one of the fastest growing disciplines in biomedicine. Since the early 1990s, it has become increasingly evident that mitochondrial dysfunction contributes to a large variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Most remarkably, mitochondria, the “power house” of the cell, have also become accepted as the “motor of cell death” reflecting their recognized key role during apoptosis. Based on these recent exciting developments in mitochondrial research, increasing pharmacological efforts have been made leading to the emergence of “Mitochondrial Medicine” as a whole new field of biomedical research. The identification of molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch a multitude of new therapies for the treatment of mitochondria-related diseases, which are based either on the selective protection, repair, or eradication of cells. Yet, while tremendous efforts are being undertaken to identify new mitochondrial drugs and drug targets, the development of mitochondria-specific drug carrier systems is lagging behind. To ensure a high efficiency of current and future mitochondrial therapeutics, colloidal vectors, i.e., delivery systems, need to be developed able to selectively transport biologically active molecules to and into mitochondria within living human cells. Here we review ongoing efforts in our laboratory directed toward the development of different phospholipid- and non-phospholipid-based mitochondriotropic drug carrier systems.     

Mitochondrial drug delivery and mitochondrial disease therapy--an approach to liposome-based delivery targeted to mitochondria

Recent progress in genetics and molecular biology has provided useful information regarding the molecular mechanisms associated with the mitochondrial diseases. Genetic approaches were initiated in the late 1980s to clarify the gene responsible for various mitochondrial diseases, and information concerning genetic mutations is currently used in the diagnosis of mitochondrial diseases. Moreover, it was also revealed that mitochondria play a central role in apoptosis, or programmed cell death, which is closely related to the loss of physiological functions of tissues. Therefore, drug therapies targeted to the mitochondria would be highly desirable. In spite of the huge amount of mechanism-based studies of mitochondrial diseases, effective therapies have not yet been established mainly because of the lack of an adequate delivery system. To date, numerous investigators have attempted to establish a mitochondrial drug delivery system. However, many problems remain to be overcome before a clinical application can be achieved. To fulfill a drug delivery targeted to mitochondria, we first need to establish a method to encapsulate various drugs, proteins, peptides, and genes into a drug carrier depending on their physical characteristics. Second, we need to target it to a specific cell. Finally, multi-processes of intracellular trafficking should be sophisticatedly regulated so as to release a drug carrier from the endosome to the cytosol, and thereafter to deliver to the mitochondria. In this review, we describe the current state of the development of mitochondrial drug delivery systems, and discuss the advantage and disadvantage of each system. Our current efforts to develop an efficient method for the packaging of macromolecules and regulating intracellular trafficking are also summarized. Furthermore, novel concept of "Regulation of intramitochondrial trafficking" is proposed herein as a future challenge to the development of a mitochondrial drug delivery system.     

Liposomes and liposome-like vesicles for drug and DNA delivery to mitochondria

Mitochondrial research is presently one of the fastest growing disciplines in biomedicine. Since the early 1990s, it has become increasingly evident that mitochondrial dysfunction contributes to a large variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Most remarkably, mitochondria, the "power house" of the cell, have also become accepted as the "motor of cell death" reflecting their recognized key role during apoptosis. Based on these recent exciting developments in mitochondrial research, increasing pharmacological efforts have been made leading to the emergence of "Mitochondrial Medicine" as a whole new field of biomedical research. The identification of molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch a multitude of new therapies for the treatment of mitochondria-related diseases, which are based either on the selective protection, repair, or eradication of cells. Yet, while tremendous efforts are being undertaken to identify new mitochondrial drugs and drug targets, the development of mitochondria-specific drug carrier systems is lagging behind. To ensure a high efficiency of current and future mitochondrial therapeutics, colloidal vectors, i.e., delivery systems, need to be developed able to selectively transport biologically active molecules to and into mitochondria within living human cells. Here we review ongoing efforts in our laboratory directed toward the development of different phospholipid- and non-phospholipid-based mitochondriotropic drug carrier systems.     

Mitochondrial dysfunction in breast cancer cells prevents tumor growth

Metformin is a well-established diabetes drug that prevents the onset of most types of human cancers in diabetic patients, especially by targeting cancer stem cells. Metformin exerts its protective effects by functioning as a weak “mitochondrial poison,” as it acts as a complex I inhibitor and prevents oxidative mitochondrial metabolism (OXPHOS). Thus, mitochondrial metabolism must play an essential role in promoting tumor growth. To determine the functional role of “mitochondrial health” in breast cancer pathogenesis, here we used mitochondrial uncoupling proteins (UCPs) to genetically induce mitochondrial dysfunction in either human breast cancer cells (MDA-MB-231) or cancer-associated fibroblasts (hTERT-BJ1 cells). Our results directly show that all three UCP family members (UCP-1/2/3) induce autophagy and mitochondrial dysfunction in human breast cancer cells, which results in significant reductions in tumor growth. Conversely, induction of mitochondrial dysfunction in cancer-associated fibroblasts has just the opposite effect. More specifically, overexpression of UCP-1 in stromal fibroblasts increases β-oxidation, ketone body production and the release of ATP-rich vesicles, which “fuels” tumor growth by providing high-energy nutrients in a paracrine fashion to epithelial cancer cells. Hence, the effects of mitochondrial dysfunction are truly compartment-specific. Thus, we conclude that the beneficial anticancer effects of mitochondrial inhibitors (such as metformin) may be attributed to the induction of mitochondrial dysfunction in the epithelial cancer cell compartment. Our studies identify cancer cell mitochondria as a clear target for drug discovery and for novel therapeutic interventions.     

Mitochondriotropic Liposomes

Mitochondrial dysfunction contributes to a large variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Increasing pharmacological efforts toward therapeutic interventions have been made leading to the emergence of “Mitochondrial Medicine” as a new field of biomedical research. The identification of molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch a multitude of new therapies for the treatment of mitochondria-related diseases, which are based either on the selective protection, repair, or eradication of cells. Yet, while tremendous efforts are being undertaken to identify new mitochondrial drugs and drug targets, the development of mitochondria-specific drug carrier systems is lagging behind. To ensure a high efficiency of current and future mitochondrial therapeutics, delivery systems need to be developed, which are able to selectively transport biologically active molecules to and into mitochondria within living human cells. In this study we present the first data demonstrating that conventional liposomes can be rendered mitochondria-specific via the attachment of known mitochondriotropic residues to the liposomal surface.     

Quantitative and qualitative 2D electrophoretic analysis of differentially expressed mitochondrial proteins from five mouse organs

Mitochondria fulfill many tissue‐specific functions in cell metabolism. We set out to identify differences in the protein composition of mitochondria from five tissues frequently affected by mitochondrial disorders. The proteome of highly purified mitochondria from five mouse organs was separated by high‐resolution 2DE. Tissue‐specific spots were identified through nano‐LC/ESI‐MS/MS and quantified by densitometry in ten biological replicates. We identified 87 consistently deviating spots representing 48 proteins. The percentage of variant spots ranged between 4.2% and 6.0%; 21 proteins having tissue‐specific isospots. Consistent tissue‐specific processing/regulation was seen for carbamoyl‐phosphate‐synthase, aldehyde‐dehydrogenase 2, ATP‐synthase α‐chain, and isocitrate‐dehydrogenase α‐subunit. Thirty tissue‐specific proteins were associated with mitochondrial disorders in humans. We further identified alcohol‐dehydrogenase, catalase, quinone‐oxidoreductase, cyclophilin‐A, and Upf0317, a potential biotin‐carboxyl‐carrier protein, which had not been annotated as “mitochondrial” in Gene Ontology or MitoCarta databases. Their targeting to the mitochondria was verified by transfection of full‐length GFP‐tagged plasmids. Given the high evolutionary conservation of mitochondrial metabolic pathways, these data further annotate the mitochondrial proteome and advance our understanding of the pathophysiology and tissue‐specificity of symptoms seen in patients with mitochondrial disorders. The generation of 2D electrophoretic maps of the mitochondrial proteome using tissue specimens in the milligram range facilitates this technique for clinical applications and biomarker research.     

Mitochondrial leader sequence--plasmid DNA conjugates delivered into mammalian cells by DQAsomes co-localize with mitochondria

In the last decade the increase in therapeutic strategies aimed at mitochondrial targets has resulted in the need for novel delivery systems for the selective delivery of drugs and DNA into mitochondria. In this study, we have continued our efforts towards the development of the first mitochondriotropic drug and DNA delivery system (DQAsomes). Prepared from derivatives of the self-assembling mitochondriotropic bola-amphiphile dequalinium chloride, these vesicles bind and transport DNA to mitochondria in living mammalian cells where upon they have been shown to release the DNA on contact with mitochondrial membranes. We present data to demonstrate that oligonucleotides as well as plasmid DNA conjugated to a mitochondrial leader sequence (MLS) co-localize with mitochondria when delivered into mammalian cells by DQAsomes. In contrast to a commercially available DNA delivery vector, our vesicles appear to have a pronounced specificity for mitochondria. Further, the data strongly suggest that linear conjugates might be better suited to delivery into mitochondria and that in the absence of a mitochondria specific vector, the presence of a MLS-peptide conjugated to the DNA is alone not sufficient to direct the accumulation of DNA at mitochondria.     

MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.     

MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.     

Exploiting endobiotic metabolic pathways to target xenobiotic antioxidants to mitochondria

Oxidative stress plays a role in a range of human disease entities. Hence, strategies to target antioxidants to mitochondria are an active area of investigation. Triphenylphosphonium cation-based antioxidants and SS-peptides have been described and show significant uptake by mitochondria and effectiveness in animal models of conditions linked to oxidative stress. We tested the hypothesis that the mitochondrial β-oxidation pathway could be exploited to activate the antioxidant phenolic and methimazole prodrugs. Most compounds studied underwent mitochondrial biotransformation to release their antioxidant moieties, and some were cytoprotective in a hypoxia–reoxygenation model in rat cardiomyocytes. These results demonstrate the feasibility of exploiting mitochondrial bioactivation reactions for targeted drug delivery.     

Smart materials on the way to theranostic nanorobots: Molecular machines and nanomotors,advanced biosensors,and intelligent vehicles for drug delivery

BackgroundTheranostics, a fusion of two key parts of modern medicine - diagnostics and therapy of the organism's disorders, promises to bring the efficacy of medical treatment to a fundamentally new level and to become the basis of personalized medicine. Extrapolating today's progress in the field of smart materials to the long-run prospect, we can imagine future intelligent agents capable of performing complex analysis of different physiological factors inside the living organism and implementing a built-in program thereby triggering a series of therapeutic actions. These agents, by analogy with their macroscopic counterparts, can be called nanorobots. It is quite obscure what these devices are going to look like but they will be more or less based on today's achievements in nanobiotechnology.Scope of ReviewThe present Review is an attempt to systematize highly diverse nanomaterials, which may potentially serve as modules for theranostic nanorobotics, e.g., nanomotors, sensing units, and payload carriers.Major ConclusionsBiocomputing-based sensing, externally actuated or chemically “fueled” autonomous movement, swarm inter-agent communication behavior are just a few inspiring examples that nanobiotechnology can offer today for construction of truly intelligent drug delivery systems.General SignificanceThe progress of smart nanomaterials toward fully autonomous drug delivery nanorobots is an exciting prospect for disease treatment. Synergistic combination of the available approaches and their further development may produce intelligent drugs of unmatched functionality.     

N‐(3‐Oxo‐acyl)‐homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts

N‐(3‐Oxododecanoyl)‐l ‐homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum‐sensing molecule for bacteria–bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12‐triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in “initiator” caspases or “effector” caspases. Our data indicate that C12 selectively induces the mitochondria‐dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both “initiator” and “effector” caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.     

Mitochondrial groups of germinal cells of teleost Cyprinidae. II. High resolution autoradiographic study of the incorporation of 3H phenylalanine and 3H uridine

A high resolution autoradiographic study of the incorporation of tritiated uridine and amino acids by the mitochondrial groups, which are typical of most of germ cells at the beginning of gametogenesis, has been made on tench spermatocytes. By this technique, we confirm the partially proteinacious composition of the intermitochondrial “cement”; and for the first time, the presence of RNA in the “cement” is demonstrated by autoradiography. Moreover, a study of the kinetics of the incorporation of both precursors makes likely the hypothesis that at least a part of the “cement” derived from nucleocytoplasmic transfer. Since the biogenesis of mitochondria results from the complementary functioning of the two protein synthesic systems, the cytoplasmic one, which is preponderant, and the mitochondrial one, the mitochondrial groups seem to be the direct visualization of the contribution of the nuclear genome to the edification of new mitochondria.     

Intermembrane electron transport in the absence of added water-soluble carriers

Electron transport from untreated to mersalyzed microsomal vesicles at the level of NADH-cytochrome b₅ reductase or cytochrome b₅ has been demonstrated in the absence of added water-soluble electron carriers. A similar effect was shown in the systems “intact mitochondria — mersalyzed microsomes” and “mersalyzed mitochondria— untreated microsomes”. No measurable electron transport between intact and mersalyzed particles of inner mitochondrial membrane was found. The obtained data suggest that the capability to carry out intermembrane electron transfer is specific for NADH-cytochrome b₅ reductase and/or cytochrome b₅, localized in microsomal and outer mitochondrial membranes.     

Enhancement in selective mitochondrial association by direct modification of a mitochondrial targeting signal peptide on a liposomal based nanocarrier

The focus of this study was on the development of a nano carrier targeted to mitochondria, a promising therapeutic drug target. We synthesized a lipid derivative that is conjugated with a mitochondrial targeting signal peptide (MTS), which permits the selective delivery of certain types of proteins to mitochondria. We then explored the use of an innovative technology in which MTS and the MITO-Porter were integrated. The latter is a liposome that delivers cargos to mitochondria via membrane fusion. The results indicate that the combination of MTS and the MITO-Porter would be useful for selective mitochondrial delivery via membrane fusion.     

Targeting mitochondria: Strategies,innovations and challenges: The future of medicine will come through mitochondria

Mitochondrial dysfunction has been associated with the aging process and a large variety of human disorders, such as cardiovascular and neurodegenerative diseases, cancer, migraine, infertility, kidney and liver diseases, toxicity of drugs and many more. It is well recognized that the physiological role of mitochondria widely exceeds that of solely being the biochemical power plant of our cells. Over the recent years, mitochondria have become an interesting target for drug therapy, and the research field aimed at “targeting mitochondria” is active and expanding as witnessed by this already third edition of the world congress on targeting mitochondria. It is becoming a necessity and an urge to know why and how to target mitochondria with bioactive molecules and drugs in order to treat and prevent mitochondria-based pathologies and chronic diseases. This special issue covers a variety of new strategies and innovations as well as clinical applications in mitochondrial medicine.     

Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development

Mfn2 is a mitochondrial fusion protein with bioenergetic functions implicated in the pathophysiology of neuronal and metabolic disorders. Understanding the bioenergetic mechanism of Mfn2 may aid in designing therapeutic approaches for these disorders. Here we show using endoplasmic reticulum (ER) or mitochondria‐targeted Mfn2 that Mfn2 stimulation of the mitochondrial metabolism requires its localization in the ER, which is independent of its fusion function. ER‐located Mfn2 interacts with mitochondrial Mfn1/2 to tether the ER and mitochondria together, allowing Ca²⁺ transfer from the ER to mitochondria to enhance mitochondrial bioenergetics. The physiological relevance of these findings is shown during neurite outgrowth, when there is an increase in Mfn2‐dependent ER‐mitochondria contact that is necessary for correct neuronal arbor growth. Reduced neuritic growth in Mfn2 KO neurons is recovered by the expression of ER‐targeted Mfn2 or an artificial ER‐mitochondria tether, indicating that manipulation of ER‐mitochondria contacts could be used to treat pathologic conditions involving Mfn2.     

5-Fluorouracil Nanoparticles Inhibit Hepatocellular Carcinoma via Activation of the p53 Pathway in the Orthotopic Transplant Mouse Model

Biodegradable polymer nanoparticle drug delivery systems provide targeted drug delivery, improved pharmacokinetic and biodistribution, enhanced drug stability and fewer side effects. These drug delivery systems are widely used for delivering cytotoxic agents. In the present study, we synthesized GC/5-FU nanoparticles by combining galactosylated chitosan (GC) material with 5-FU, and tested its effect on liver cancer in vitro and in vivo. The in vitro anti-cancer effects of this sustained release system were both dose- and time-dependent, and demonstrated higher cytotoxicity against hepatic cancer cells than against other cell types. The distribution of GC/5-FU in vivo revealed the greatest accumulation in hepatic cancer tissues. GC/5-FU significantly inhibited tumor growth in an orthotropic liver cancer mouse model, resulting in a significant reduction in tumor weight and increased survival time in comparison to 5-FU alone. Flow cytometry and TUNEL assays in hepatic cancer cells showed that GC/5-FU was associated with higher rates of G0–G1 arrest and apoptosis than 5-FU. Analysis of apoptosis pathways indicated that GC/5-FU upregulates p53 expression at both protein and mRNA levels. This in turn lowers Bcl-2/Bax expression resulting in mitochondrial release of cytochrome C into the cytosol with subsequent caspase-3 activation. Upregulation of caspase-3 expression decreased poly ADP-ribose polymerase 1 (PARP-1) at mRNA and protein levels, further promoting apoptosis. These findings indicate that sustained release of GC/5-FU nanoparticles are more effective at targeting hepatic cancer cells than 5-FU monotherapy in the mouse orthotropic liver cancer mouse model.     

Synthesis of a Smart Gold Nano‐vehicle for Liver Specific Drug Delivery

Targeting drug formulations to specific tissues and releasing the bioactive content in response to a certain stimuli remains a significant challenge in the field of biomedical science. We have developed a nanovehicle that can be used to deliver “drugs” to “specific” tissues. For this, we have simultaneously modified the surface of the nanovehicle with “drugs” and “tissue-specific ligands”. The “tissue-specific ligands” will target the nanovehicle to the correct tissue and release the “drug” of interest in response to specific stimuli. We have synthesised a “lactose surface-modified gold nanovehicle” to target liver cells and release the model fluorescent drug (coumarin derivative) in response to the differential glutathione concentration (between blood plasma and liver cells). Lactose is used as the liver-specific targeting ligand given the abundance of l-galactose receptors in hepatic cells. The coumarin derivative is used as a fluorescent tag as well as a linker for the attachment of various biologically relevant molecules. The model delivery system is compatible with a host of different ligands and hence could be used to target other tissues as well in future. The synthesised nanovehicle was found to be non-toxic to cultured human cell lines even at elevated non-physiological concentrations as high as 100 μg/mL. We discover that the synthesised gold-based nanovehicle shows considerable stability at low extracellular glutathione concentrations; however coumarin is selectively released at high hepatic glutathione concentration.