Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter
Introduction
Mutations and defects in mitochondrial DNA (mtDNA) have been associated with various mitochondrial dysfunctions, which form the basis of a variety of human disorders including neurodegenerative and neuromuscular diseases [1], [2], [3], [4], [5], [6], [7]. For example, a mutation in mtDNA in the region that encodes for tRNA causes mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) [8] and myoclonic epilepsy with ragged-red fibers (MERRF) [9]. In addition, deletions in mtDNA have been discovered in the majority of cases of chronic progressive external ophthalmoplegia (CPEO) and cases of the Kearns-Sayre syndrome (KSS) [10]. Therefore, mitochondrial gene therapy and diagnosis promises to be useful and productive in the treatment of many patients suffering from these intractable diseases.
To achieve such a strategy to target mitochondrial genome, it will be ultimately necessary to develop an optimal drug delivery system, which will likely be achieved through innovation associated with the nanotechnology of intracellular trafficking. In addition, for the strategy to be successful, it will be necessary to deliver therapeutic/diagnostic agents into the innermost mitochondrial space, the mitochondrial matrix, where the mtDNA pool is located. A number of systems for delivering cargoes to mitochondria have been reported to date [11], [12], [13]. However, reports of mitochondrial genome targeting by means of an artificial gene vector are limited, although endogenous mitochondrial importing signal RNA can deliver exogenous RNA to mitochondria in mammalian cells [14], [15]. Weissig and coworkers reported on the development of DQAsomes, which are mitochondriotropic and cationic vesicles designed for mitochondrial-targeted DNA delivery [16], [17]. They showed that DQAsomes specifically release pDNA proximal to mitochondria in living cells [17], [18], although the delivery of cargoes into the interior of mitochondria has not been validated.
In previous studies, we proposed the use of a MITO-Porter, which is a liposome-based nano carrier that delivers cargos to mitochondria via membrane fusion [19]. Using the green fluorescent protein as a model macromolecule and analysis by confocal laser scanning microscopy (CLSM), we were able to confirm mitochondrial macromolecule delivery of a macromolecule by the MITO-Porter. Moreover, we verified that the MITO-Porter could deliver cargos into the mitochondrial matrix, which contains the mtDNA pool [20]. We utilized propidium iodide, PI, an impermeable red fluorescence-dye for nucleic acids, as a probe to detect mtDNA. When the MITO-Porter was added to living cells, strong red-signals were detected within mitochondria, suggesting that the carrier had the ability to deliver PI to the mitochondrial matrix in living cells.
In this study, we report on an approach for the mitochondrial delivery of bioactive molecules using a Dual Function (DF)-MITO-Porter, aimed at mtDNA. The DF-MITO-Porter, which possesses mitochondria-fusogenic inner and endosome-fusogenic outer envelopes, has the ability to pass through endosomal and mitochondrial membranes via step-wise membrane fusion (Fig. 1) [21]. Octaarginine (R8) functions as a cell-uptake device [22], [23] in the outer envelope and as a mitochondrial targeting device [12], [24], [25] in the inner envelope. In this experiment, we attempted to deliver DNase I protein to mitochondria in living cells. It was expected that mtDNA would be digested, resulting in a reduction in mitochondrial activity, when the mitochondrial delivery of DNase I progressed, as shown in Fig. 1.
We first constructed a DF-MITO-Porter encapsulating DNase I and observed the intracellular trafficking of the DNase I delivered by DF-MITO-Porter using CLSM. We then measured the activity of mitochondrial dehydrogenase to confirm that the digestion of mtDNA by DNase I influenced mitochondrial activity. Moreover, we quantified the levels of mtDNA and nuclear DNA after the mitochondrial delivery of DNase I to demonstrate its potential use in therapies that are aimed selectively at mtDNA.
Section snippets
Materials
1, 2-Dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE) was purchased from Avanti Polar lipids (Alabaster, AL, USA). Egg yolk phosphatidyl choline (EPC) was obtained from Nippon Oil and Fats Co. (Tokyo, Japan). Cholesteryl hemisuccinate (5-cholesten-3-ol 3-hemisuccinate; CHEMS), phosphatidic acid (PA) and sphingomyelin (SM) were purchased from Sigma (St. Louis, MO, USA). Stearyl octaarginine (STR-R8) [26] was obtained from KURABO INDUSTRIES LTD (Osaka, Japan). DNase I protein (from bovine
Construction of DF-MITO-Porter encapsulating DNase I
The DF-MITO-Porter encapsulating DNase I was constructed as described in our previous reports (see Figure S1 in supplementary data for details) [21]. Complexed DNase I particles were first prepared with STR-R8, and were then coated with a mitochondria-fusogenic envelope. Finally, the envelopes were further coated with an endosome-fusogenic envelope. The size and ζ-potential of the DF-MITO-Porter and control carrier are summarized in Table S3. Particle diameters were around 150 nm and the
Discussion
In this study, we attempted to package a bioactive molecule via the use of a multifunctional envelope-type nano-device (MEND). The MEND consisted of a condensed pDNA core and a lipid envelope equipped with various functional devices that mimic envelope-type viruses [28], [29]. To date, we have been successful in efficiently packaging, not only pDNA, but also oligo DNAs, proteins and other substances into a MEND [30], [31], [32], [33]. Various candidates for cargoes that can be used in
Conclusion
In this study, we attempted to validate the possibility of mitochondrial genome targeting using a DF-MITO-Porter. The findings described here showed that the mitochondrial delivery of DNase I protein by the DF-MITO-Porter caused a substantial decrease in mitochondrial activity, suggesting that a multi-structured particle with a different lipid composition can be useful for mitochondrial delivery. In addition, we demonstrated that the mitochondrial delivery of DNase I by the DF-MITO-Porter
Acknowledgements
This work was supported, in part by, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, Japan (NIBIO), a Grant-in-Aid for Young Scientists (A) and a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japanese Government (MEXT). We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.
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