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.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors.

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide x cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

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 Alzheimer's and Parkinson's 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 cationic bolaamphiphile with delocalized charge centers, for the design of a vector suited for the transport of DNA to mitochondria in living cells.

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.

Mitochondrial DNA metabolism targeting drugs.

Numerous drugs are known to deplete mitochondrial DNA (mtDNA) from mammalian cells. These include DNA polymerase gamma and type II topoisomerase inhibitors, lipophilic cationic compounds, and DNA intercalating and non intercalating agents. The effects of these drugs on mtDNA metabolism will be discussed and potential mechanisms underlying their depletion of mtDNA presented.

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.

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 bolaamphiphile 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.

Mitochondriotropic cationic vesicles: a strategy towards mitochondrial gene therapy.

The number of diseases found to be associated with defects of the mitochondrial genome has grown significantly over the last decade. 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. This is largely due to the fact that almost all mitochondrial DNA defects involve the final common pathway of oxidative metabolism making it impossible to bypass the defect by giving alternative metabolic carriers of energy. These seemingly objective limitations of conventional biochemical treatment for patients with detects of mtDNA warrant the exploration of gene therapeutic approaches. However, mitochondrial gene therapy still appears only theoretical and speculative. Any possibility for gene replacement is dependent on the use of a yet unavailable mitochondria-specific transfection vector. Based upon an analysis of the self-assembly behavior of dequalinium, a cationic single-chain bolaamphiphile which is known to selectively accumulate in mitochondria, we have developed a whole new strategy for mitochondria-specific DNA delivery. We have succeeded in preparing vesicles made of dequalinium, which we termed DQAsomes (U.S. Patent 6,090,619). We have shown that DQAsomes efficiently bind and protect DNA and we could demonstrate that DQAsome/DNA complexes selectively release DNA at cardiolipin-rich liposomes mimicking both, the inner and the outer mitochondrial membrane. Based on the intrinsic property of dequalinium to preferentially accumulate in mitochondria in response to the electrochemical gradient at the mitochondrial membrane and based on the selective DNA release at mitochondria-like membranes we propose DQAsomes as the first mitochondria-specific vector to deliver DNA to mitochondria in living cells.

Dequalinium vesicles form stable complexes with plasmid DNA which are protected from DNase attack.

Upon sonication, the antimicrobial and antineoplastic compound dequalinium forms vesicles (DQAsomes, Weissig et al., 1998). Dequalinium (1,1'-(1,10-decamethylene-bis-[aminoquinaldinium])-chloride) was shown to be a fluorophore with an emission maximum at 366 nm. Addition of DNA results in a characteristic quenching of its intrinsic fluorescence. After density gradient centrifugation a band of dequalinium (DQA) tightly associated with DNA is located between the DNA and DQA bands. DQA/DNA-complexes containing plasmid DNA at a molar ratio of DQA/DNA 6:1 are completely protected against DNase activity. Addition of negatively-charged lipids release intact DNA in the same manner as from cationic lipid/DNA complexes. As regards biological effects, DQAsomes show a differential cytotoxicity for normal and sarcoma cell lines. In vitro incubation with fluorescein-labeled oligodeoxynucleotides (5'-fluorescein-[GATC]5) showed an increased uptake of the tagged oligodeoxynucleotide if complexed with dequalinium. We hypothesize that the DQA/DNA complexes are well-suited for 'DQAsomal gene transfer' in vitro and in vivo. Noteworthy, they display an intrinsic antitumor activity manifested by differential cytotoxicity for normal and sarcoma cells.

Analysis of Plasmodium falciparum mitochondrial six-kilobase DNA by pulse-field electrophoresis.

The 6-kb mtDNA of Plasmodium falciparum is thought to replicate by a recombination-dependent mechanism generating large complex branched structures. For technical reasons, including shearing caused by DNA extraction methods, a meaningful quantitative comparison of large complex mtDNA forms has not been feasible. With the use of pulse-field gel electrophoresis, which minimizes any loss or shearing of DNA, we were able to identify an unusually slow migrating population of mtDNA that was resolved from the 6-23-kb population of linear concatemers. Levels of this slow-migrating population of mtDNA were highest during early schizont stage, suggesting that these forms represent replication intermediates. This approach provides a convenient means to monitor the presence of large mtDNA structures in P. falciparum.

Accumulation of protein-loaded long-circulating micelles and liposomes in subcutaneous Lewis lung carcinoma in mice.

PURPOSE: The purpose of our work was to compare the biodistribution and tumor accumulation of a liposome- or micelle-incorporated protein in mice bearing subcutaneously-established Lewis lung carcinoma. METHODS: A model protein, soybean trypsin inhibitor (STI) was modified with a hydrophobic residue of N-glutaryl-phosphatidyl-ethanolamine (NGPE) and incorporated into both polyethyleneglycol(MW 5000)-distearoyl phosphatidyl ethanolamine (PEG-DSPE) micelles (< 20 nm) and PEG-DSPE-modified long-circulating liposomes (ca. 100 nm). The protein was labeled with 111In via protein-attached diethylene triamine pentaacetic acid (DTPA), and samples of STI-containing liposomes or micelles were injected via the tail vein into mice bearing subcutaneously-established Lewis lung carcinoma. At appropriate time points, mice were sacrificed and the radioactivity accumulated in the tumor and main organs was determined. RESULTS: STI incorporated into PEG-lipid micelles accumulates in subcutaneously established Lewis lung carcinoma in mice better than the same protein anchored in long-circulating PEG-liposomes. CONCLUSIONS: Small-sized long-circulating delivery systems, such as PEG-lipid micelles, are more efficient in the delivery of protein to Lewis lung carcinoma than larger long-circulating liposomes.

DQAsomes: a novel potential drug and gene delivery system made from Dequalinium.

PURPOSE: Dequalinium, a drug known for over 30 years, is a dicationic amphiphile compound resembling bolaform electrolytes. The purpose of our work was to determine the state of aggregation of dequalinium in aqueous medium and to investigate both, its ability to bind DNA and its potential to serve as a novel non-viral transfection vector. METHODS: The form of aggregation was determined employing electron microscopic techniques. The DNA binding capacity of dequalinium was assayed using SYBR Green I stain. For in vitro cell transfection experiments plasmid DNA encoding for firefly luciferase was used. RESULTS: Dequalinium forms in aqueous medium liposome-like aggregates, which we term DQAsomes. These dequalinium vesicles bind DNA and they are able to transfect cells in vitro with an efficiency comparable to Lipofectin. CONCLUSIONS: Based on the intrinsic properties of dequalinium such as the in vivo selectivity for carcinoma cells and selective accumulation in mitochondria we propose DQAsomes as a novel and unique drug and gene delivery system.

Topoisomerase II inhibitors induce cleavage of nuclear and 35-kb plastid DNAs in the malarial parasite Plasmodium falciparum.

The topoisomerase II-specific inhibitors VP-16 and ciprofloxacin were used to investigate the presence of topoisomerase II activities associated with nuclear and 35-kb plastid DNAs of the malarial parasite Plasmodium falciparum. The eukaryotic topoisomerase II inhibitor VP-16 induced cleavage of both nuclear and 35-kb parasite DNAs. In contrast, ciprofloxacin, a fluoroquinolone drug known to act on the bacterial type II topoisomerase DNA gyrase, only induced cleavage of the Plasmodial 35-kb DNA. Drug-induced cleavage resulted in the protection of the 5'- but not 3'- ends of the cleaved nuclear and 35-kb DNAs from exonuclease digestion, suggesting that the 5'-ends of the broken DNA were protein-linked, a property reminiscent of DNA cleavage mediated by topoisomerase II enzymes. Furthermore, DNA cleavage induced by both VP-16 and ciprofloxacin was heat-reversible. This is the first evidence that P. falciparum contains two distinct topoisomerase II activities that are molecular targets for chemotherapeutic agents.

Delayed cytotoxicity and cleavage of mitochondrial DNA in ciprofloxacin-treated mammalian cells.

We have previously shown that 4-quinolone drugs cause a selective loss of mitochondrial DNA (mtDNA) from mouse L1210 leukemia cells. The loss in mtDNA was associated with a delayed loss in mitochondrial function. Here, we report that the 4-quinolone drug ciprofloxacin is cytotoxic to a variety of cultured mammalian cell lines at concentrations that deplete cells of mtDNA. The IC50 values for ciprofloxacin varied from 40 to 80 micrograms/ml depending on the cell line tested. Cytotoxicity required continuous exposure of cells to drug for 2-4 days, which corresponded to approximately three or four cell doublings. Shorter times of drug exposure did not cause significant cytotoxicity. In addition, cells became drug resistant when they were grown under conditions that bypassed the need for mitochondrial respiration. Resistance was not due to a decrease in cellular drug accumulation, suggesting that ciprofloxacin cytotoxicity is caused by the loss of mtDNA-encoded functions. Analysis of mtDNA from ciprofloxacin-treated cells revealed the presence of site-specific, double-stranded DNA breaks. Furthermore, exonuclease protection studies indicated that the 5'-, but not the 3'-, ends of the drug-induced DNA breaks were tightly associated with protein. These results suggest that ciprofloxacin may be causing cytotoxicity by interfering with a mitochondrial topoisomerase II-like activity, resulting in a loss of mtDNA.

(Patho)physiologic pathways to drug targeting: artificial viral envelopes.

The goal of this study was to exploit molecular recognition of cell surface receptors by viral surface glycoproteins as a means for the selective intracellular delivery of macromolecules. To accomplish this, artificial viral envelopes (AVE) resembling the human immunodeficiency virus-1 (HIV-1) were designed as a model system. Recombinant HIV-1 surface glycoprotein gp160 (HIV-1 rgp160) was inserted in the artificial envelope by a two-step detergent dialysis process. The artificial HIV-1 envelope recognized the CD4 cell surface receptor. FITC-dextran and ricin A were employed as model macromolecules as they cannot passively diffuse across cell membranes. Selective transfer of FITC-dextran encapsulated in HIV-1 rgp160 AVE into a CD4-positive cell line (REX-1B) versus a CD4-negative cell line (KG-1) was demonstrated. Ricin A at concentrations as low as 2 ng/ml arrested cell growth of CD4-positive MOLT-4 cells, whereas 8 ng/ml ricin A in solution had no effect on cell growth. The arrest of cell growth was reverted in the presence of excess anti-gp120 monoclonal antibody. Naked envelopes (without HIV-1 rgp160 inserted) were also found to interact with cells and transfer material, although less efficiently and in a non-specific manner. Viral mimicry using AVE may be a means for targeted intracellular delivery of peptides, proteins, enzymes, toxins, oligodeoxynucleotides, gene constructs, and other non-diffusive, labile or toxic macromolecules.

Carboxyacyl derivatives of cardiolipin as four-tailed hydrophobic anchors for the covalent coupling of hydrophilic proteins to liposomes.

Two carboxyacyl derivatives of cardiolipin, O-succinyl- and O-glutarylcardiolipin, were synthesized with the aim of using them as artificial membrane anchors for the immobilization of hydrophilic proteins to liposomes. Four adjacent fatty acid residues can be introduced into a protein with only one single amino group being blocked, by reacting the cardiolipin derivatives with the protein amino groups after carbodiimide activation. alpha-Chymotrypsin, used as a model protein, and modified with on average two molecules of O-succinylcardiolipin was incorporated into liposomes, which had been prepared by different methods, with very high yield. If incorporated in preformed liposomes, the carboxyacyl cardiolipin anchors were also efficient in binding proteins to liposomal surfaces. Up to 350 micrograms chymotrypsin/mumol lipid were coupled to small unilamellar vesicles, preserving reactivity of the enzyme towards specific macromolecular inhibitors. Human IgG could also be bound to anchor-containing liposomes with high protein to lipid coupling ratio as well as high coupling yield.

Comparison of the immune response against polio peptides covalently-surface-linked to and internally-entrapped in liposomes.

The adjuvanticity of liposomes on two different modes of presentation of polio virus subunit peptides was demonstrated by incorporating the poorly immunogenic synthetic polio peptides, W1 and W2, into the internal space of and covalently-linked to the surface of dehydration-rehydration vesicles (DRV). It was found that for both peptides, liposome association in either mode boosted the primary and secondary IgG1 responses against 5 micrograms peptide as compared to controls in which free peptide was administered. Surface-linkage of peptides (both W1 and W2) exhibited an initially more rapid rise in antibody levels, as compared to internal entrapment of the peptides, but elicited no observable secondary response. However, although encapsulated W1 showed a milder primary response when compared to the surface-linked formulation, it later elicited a strong secondary response. These results suggested that it may be advantageous to administer liposomal virus subunit vaccines in both surface-linked and internally entrapped formulations to achieve adequate initial antibody levels followed by an anamnestic response.

Covalent coupling of sugars to liposomes.

A procedure is described for the covalent coupling of p-aminophenyl-alpha-D-mannopyranoside, in the presence of carbodiimide, to a derivative of phosphatidylethanolamine (N-glutarylphosphatidylethanolamine) incorporated into the bilayers of multilamellar liposomes prepared by the dehydration-rehydration method. It appears that much of the phospholipid derivative exposed on the surface of the outer liposomal bilayer interacts with the aminosugar. The procedure is simple, does not destabilize liposomes and could be applied to other receptor-specific aminosugars.