Enter multiple addresses on separate lines or separate them with commas. One of the most challenging problems in the development of drug therapy to treat mitochondrial dysfunction is not the creation of the drugs themselves, but rather the distribution of these drugs to the mitochondria of cells (2). To examine whether encapsulation of 2,4-DNP in targeted NPs can suppress the induced differentiation of 3T3-L1 preadipocytes at a low dosage that is insufficient to exert cytotoxicity, we continuously exposed 3T3-L1 cells to 1 μM, 4 μM, 25 μM, and 100 μM targeted 2,4-DNP NPs (Fig. Evaluation of the mitochondrial uptake of NPs of 80–330 nm diameter showed a trend toward a maximum uptake of 80- to 100-nm-diameter particles. To further support the findings that our NPs have high endosomal escapability and that some of the targeted NPs are not engaged in trafficking to lysosomes, we studied a time-dependent colocalization of the NPs with lysosomes (Fig. A recent study found that 2,4-DNP linked to TPP in a covalent manner is ineffective at uncoupling (30). (B) Construction of targeted and nontargeted NPs by blending PLGA-b-PEG-OH and PLGA-COOH with PLGA-b-PEG-TPP, with mitochondria-acting therapeutics used as payloads. wrote the paper. Then 50 mL of cold diethyl ether was added to the resulting mixture to precipitate the polymer. A comparison of fluorescence intensities indicated a significantly greater overall uptake of the positively charged targeted NPs compared with nontargeted NPs. We evaluated immune responses from size- and charge-varied NPs in terms of the production of proinflammatory cytokines IL-6 and TNF-α in RAW 264.7 macrophages by ELISA, with LPS used as a control. Any use of this site constitutes your agreement to the Terms and Conditions and Privacy Policy linked below. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1210096109/-/DCSupplemental. Quantitative analysis using the ImageJ “colocalization finder” plug-in revealed significant colocalization of the targeted NPs with MitoTracker Green (Invitrogen) in the mitochondria of cells (Pearson’s correlation coefficient, ρ = 0.53; SI Appendix, Fig. The Mitochondrial Neurobiology and Therapeutics Laboratory of Eugenia Trushina, Ph.D., at Mayo Clinic studies the mitochondria-based molecular mechanisms of neurological conditions. The mitochondria acquired a novel morphological phenotype, mitochondria-on-a-string (MOAS), which allows cells to protect mitochondrial functions under stress conditions, such as during disease or aging. The versatility of this platform was demonstrated by studying various mitochondria-acting therapeutics for different applications, including the mitochondria-targeting chemotherapeutics lonidamine and α-tocopheryl succinate for cancer, the mitochondrial antioxidant curcumin for Alzheimer’s disease, and the mitochondrial uncoupler 2,4-dinitrophenol for obesity. 2B). 1D), allowing us to explore the effects of NP size and charge on mitochondrial uptake. Mayo Clinic is a nonprofit organization and proceeds from Web advertising help support our mission. We synthesized the blended NPs using a nanoprecipitation method (18, 28) (Fig. Keep up to date on mitochondrial neurobiology and therapeutics research at Mayo Clinic with published articles from Dr. Trushina's laboratory. ↵*This Direct Submission article had a prearranged editor.

These biomolecules were loaded into blended NPs with high loading efficiencies. As a proof-of-concept demonstration of versatility of this system, we studied the delivery of mitochondria-acting therapeutics for the management of neurodegeneration, obesity, and cancer. To explore the effect of surface charge on the mitochondrial uptake, we blended predefined amounts of PLGA-b-PEG-OH with PLGA-b-PEG-TPP.

An equally important consideration is whether the same NPs can deliver the therapeutic payload to the intended target inside the cells. The immunofluorescence procedure is described in detail in SI Appendix. Mitobridge Inc. was founded in 2013 with financing from MPM Capital, Longwood Fund, and Astellas Pharma’s Venture Management team, all sharing a vision for the promise of mitochondrial-targeted therapeutics.
(C) Effect of zeta potential on cellular trafficking of NPs. 2A). The mixture was warmed to room temperature and stirred overnight. Existing drugs are not effective enough to reverse the trend. We anticipated that the complicated structures of the tubular, vesicular, and flat cristae and their slight connections to the inner mitochondrial membrane might impose constraints on NP mobility and make their diffusion a very complicated, size-dependent process. Proton absorbance by buffering of positively charged PEG prevents acidification of endosomal vesicles, thereby increasing the ATPase-mediated influx of protons and counter ions, which in turn leads to osmotic swelling, endosomal membrane rupture, and eventual leakage of the NPs into the cytosol, making them accessible for mitochondrial uptake. Mitochondrial dysfunctions cause numerous human disorders.

It will activate the protein that mitochondria naturally use to produce heat. S9). These NP libraries exhibited homogenous populations of similar shape (TEM images; Fig. The mixture was stirred overnight at room temperature, after which any dicyclohexylurea formed was filtered off. Over time, the targeted NPs exhibited complete endosomal escape and localized in the mitochondria of cells (Fig. Curcumin is known to inhibit Aβ and the associated mitochondrial oxidative stress; however, its low bioavailability and photodegradation are major concerns. The lab studies mitochondrial dynamics in health and disease and develops biomarker panels, tools to visualize mitochondria and mitochondria-targeted therapeutics. P < 0.05 was considered to indicate statistical significance. Considering efficacy, the targeted PLGA-b-PEG-TPP NP provides a remarkable improvement in the drug therapeutic index for cancer, Alzheimer’s disease, and obesity compared with the nontargeted construct or the therapeutics in their free form. Here we report a rationally designed mitochondria-targeted polymeric nanoparticle (NP) system and its optimization for efficient delivery of various mitochondria-acting therapeutics by blending a targeted poly(d,l-lactic-co-glycolic acid)-block (PLGA-b)-poly(ethylene glycol) (PEG)-triphenylphosphonium (TPP) polymer (PLGA-b-PEG-TPP) with either nontargeted PLGA-b-PEG-OH or PLGA-COOH.