Volkmar Weissig

Cationic bolasomes with delocalized charge centers as mitochondria-specific DNA delivery systems

Since their first discovery during the end of the 1980s, the number of diseases found to be associated with a defect in the mitochondrial genome has grown significantly. However, despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is no satisfactory treatment available for the vast majority of patients. This is largely due to the fact that most of these patients have respiratory chain defects, i.e. defects that involve the final common pathway of oxidative metabolism, making it impossible to bypass the defect by giving alternative metabolic carriers of energy. These objective limitations of conventional biochemical treatment for patients with defects of mtDNA warrant the exploration of gene therapy approaches. However, mitochondrial gene therapy currently appears to be only theoretical and speculative. Any possibility for gene replacement is dependent on the use of a yet unavailable mitochondrial transfection vector. In this review we describe the current state of the development of mitochondrial DNA delivery systems. We also summarize our own efforts in exploring the properties of dequalinium, a cationic bolaamphiphile with delocalized charge centers, for the design of a vector suited for the transport of DNA to mitochondria in living cells.

DQAsome/DNA complexes release DNA upon contact with isolated mouse liver mitochondria.

DQAsomes are mitochondriotropic cationic vesicles, which have been developed by us for the supposed transport of DNA to mitochondria in living cells [Pharm. Res. 15 (1998) 334]. Our strategy for the delivery of DNA into the matrix of mitochondria is based upon the putative transport of a DNA-signal peptide conjugate to mitochondria, the liberation of this conjugate from DQAsomes at the mitochondrial membrane followed by DNA uptake via the mitochondrial protein import machinery. As a first and important step towards delivery of DNA into mitochondria of living cells, we studied the DNA release from DQAsomes upon contact with non-energized mitochondria in vitro. Mitochondria were isolated from mouse liver and characterized by electron microscopy and the determination of mitochondrial marker enzyme activity. DQAsomes were added to DNA in the presence of SYBR Green I resulting in the formation of DQAsome/DNA complex and the complete loss of fluorescence. Following the addition of isolated mitochondria to DQAsome/DNA complex, the fluorescence signal was recovered due to the dissociation of DNA from its cationic carrier. Thus, DQAsome/DNA complexes were shown to release DNA upon contact with the surface of mitochondria thereby meeting a key requirement for our strategy towards mitochondrial DNA delivery.

Selective DNA release from DQAsome/DNA complexes at mitochondria-like membranes.

The number of diseases found to be associated with defects of the mitochondrial genome has grown significantly over the past decade (Wallace 1999). Despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is no satisfactory treatment available for the vast majority of patients and the exploration of gene therapeutic approaches is highly warranted. However, mitochondrial gene therapy still appears only theoretical and speculative. Any possibility for gene replacement depends on the use of a yet unavailable mitochondria-specific transfection vector. Mitochondria-specific vectors must posses two properties: they have to transport DNA to the side of mitochondria; they must not release DNA during endocytosis. Amphiphile compounds with delocalized cationic charge centers such as rhodamine 123 and the bola-amphiphile dequalinium have long been known to accumulate in mitochondria. Sufficient lipophilicity combined with delocalization of the positive charge to reduce the free energy change when moving from an aqueous to a hydrophobic environment are believed to be prerequisite for mitochondrial accumulation in response to the mitochondrial membrane potential. We have recently succeeded in preparing cationic vesicles made of dequalinium that we termed DQAsomes (Weissig et al. 1998a). We have shown that DQAsomes bind and protect DNA against DNase activity (Lasch et al. 1999). Based on the intrinsic property of dequalinium to preferentially accumulate in mitochondria in response to the electrochemical gradient at the mitochondrial membrane, we believe that DQAsomes can serve as a vector to deliver DNA to mitochondria in living cells. As a first step in the development of mitochondria-specific DNA delivery systems, we report here that DQAsome/DNA complexes selectively release DNA at cardiolipin-rich liposomes mimicking both the inner and the outer mitochondrial membrane. We demonstrate that DNA remains tightly associated with DQAsomes in the presence of an excess of anionic lipids other than cardiolipin.

Long-circulating gadolinium-loaded liposomes: potential use for magnetic resonance imaging of the blood pool.

In our previous paper, we reported a method of liposome loading with Gadolinium (Gd) via so called polychelating amphiphilic polymer (PAP). A novel Gd-containing polymeric probe, suitable for the incorporation into the liposomal membrane, was prepared from a low-molecular-weight DTPA-polylysine by linking its N-terminus to a lipid anchor, NGPE-PE. When compared with known membranotropic MR probes, such as Gd-DTPA-SA and Gd-DTPA-PE, liposomes containing new membrane-bound polychelator possess enhanced relaxivity for water protons resulting in an increase of tissue signal intensity on MR images. In this study, we developed the optimized protocol to prepare a liposomal MR contrast agent with high relaxivity and narrow size distribution. Gd-containing liposomes were additionally modified with PEG to provide longevity in vivo. We also demonstrated that upon intravenous administration in rabbit and dog, the new preparation causes a prolonged decrease in the blood T(1) value (reflecting the proton relaxation rate in the blood) and may be considered as a potential contrast agent for MRI of the blood pool.

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.

Towards mitochondrial gene therapy: DQAsomes as a strategy.

Mitochondrial dysfunction is a cause, or major contributing factor in the development, of degenerative diseases, aging, cancer, many cases of Alzheimers and Parkinsons disease and Type II diabetes (D. C. Wallace, Science 283, 1482–1488, 1999). Despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is no satisfactory treatment for the vast majority of patients available. Objective limitations of conventional biochemical treatment for patients with defects of mtDNA warrant the exploration of gene therapeutic approaches. However, mitochondrial gene therapy has been elusive, due to the lack of any mitochondria-specific transfection vector. We review here the current state of the development of mitochondrial DNA delivery systems. In particular, we are summarizing our own efforts in exploring the mitochondriotropic properties of dequalinium, a canonic bolaam-phiphile with delocalized charge centers, for the design of a vector suited for the transport of DNA to mitochondria in living cells.

Targeted drug delivery to mammalian mitochondria in living cells

Mitochondrial dysfunction causes or contributes to a large number of human disorders including neuromuscular and neurodegenerative diseases, diabetes, ischaemia–reperfusion injury and cancer. Increasing efforts are being made towards mitochondria-directed pharmacological intervention, leading to the emergence of ‘mitochondrial medicine’ as a new field of biomedical research. The identification of new 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 new therapies for the treatment of mitochondria-related diseases, based either on the selective protection, repair or eradication of cells. This review discusses the need for the development of mitochondria-specific drug and DNA delivery systems, and evaluates the currently employed strategies for mitochondrial drug targeting, including some of their potential therapeutic applications.

Intracellular targets for DNA delivery: nuclei and mitochondria.

All discussions on the intracellular delivery of DNA are based on a seemingly evident assumption that the key task is to bring the intact DNA into the cell cytoplasmic compartment, and then the DNA will find its way to a right place. The nuclear genome is usually considered to be this “right place.” However, until recently, in numerous experiments on the intracellular DNA delivery, it has been almost completely neglected that cells contain another genome, the mitochondrial one. And, in many cases, this genome should become a therapeutic target. Being delivered inside the cell, DNA actually has two ways to go—to nuclei and to mitchondria, and the proper choice between these two ways may be decisive for the success of gene therapy. Certainly, nuclear DNA delivery is far more advanced than mitochondrial delivery one. In addition, free DNA from the cytoplasm has a strong tendency to spontaneously associate with the nuclear genome. Mitochondria as a target for DNA have much less accessibility, still remaining an important site to reach. Whereas the nuclear delivery of DNA is under active investigation and just awaits better protocols to be elaborated, practically applicable mitochondrial DNA delivery is at its early stage and must be developed almost from scratch. In our studies on intracellular DNA delivery, we have attempted to develop new protocols for targeting DNA to nuclei and to mitochondria. In this chapter we provide a brief description of our recent experiments in both of these important areas.