Delivery of bioactive molecules to mitochondria in vivo

Abstract

Mitochondrial dysfunction contributes to many human degenerative diseases but specific treatments are hampered by the difficulty of delivering bioactive molecules to mitochondria in vivo. To overcome this problem we developed a strategy to target bioactive molecules to mitochondria by attachment to the lipophilic triphenylphosphonium cation through an alkyl linker. These molecules rapidly permeate lipid bilayers and, because of the large mitochondrial membrane potential (negative inside), accumulate several hundredfold inside isolated mitochondria and within mitochondria in cultured cells. To determine whether this strategy could lead to the development of mitochondria-specific therapies, we investigated the administration and tissue distribution in mice of simple alkyltriphenylphosphonium cations and of mitochondria-targeted antioxidants comprising a triphenylphosphonium cation coupled to a coenzyme Q or vitamin E derivative. Significant doses of these compounds could be fed safely to mice over long periods, coming to steady-state distributions within the heart, brain, liver, and muscle. Therefore, mitochondria-targeted bioactive molecules can be administered orally, leading to their accumulation at potentially therapeutic concentrations in those tissues most affected by mitochondrial dysfunction. This finding opens the way to the testing of mitochondria-specific therapies in mouse models of human degenerative diseases.

Figure 1

Uptake of alkyltriphenylphosphonium cations by mitochondria within cells. The lipophilic triphenylphosphonium cation is covalently attached to a biologically active molecule (X) such as an antioxidant or pharmacophore. The lipophilic cation is accumulated 5- to 10-fold into the cytoplasm from the extracellular space by the plasma membrane potential (Δψ_p) and then further accumulated 100- to 500-fold into the mitochondrial matrix by the mitochondrial membrane potential (Δψ_m). As these lipophilic cations pass directly through the lipid bilayer they do not utilize specific uptake systems and have the potential to distribute to mitochondria in all organs, including the brain.

Figure 2

Mitochondria-targeted compounds and their octanol–PBS partition coefficients. The octanol–PBS partition coefficients were from this study or refs. and .

Figure 3

Distribution of alkyltriphenylphosphonium cations within mice. (A) Intraperitoneal injection of TPMP. Mice were injected i.p. with 100 nmol of [³H]TPMP (10 Ci/mol; 1.2 mg of TPMP/kg), and the tissue content of [³H]TPMP was measured at various times after injection by scintillation counting. Data are means ± range for pairs of mice for each time point. (B) Intravenous injection of TPMP. Mice were injected i.v. with 100 nmol of [³H]TPMP (10 Ci/mol; 1.2 mg of TPMP/kg), and the tissue content of [³H]TPMP was determined by scintillation counting. Data are means ± range for pairs of mice for each time point. (C) Feeding TPMP in drinking water. Mice were fed 500 μM [³H]TPMP (0.25 Ci/mol) in their drinking water, and the tissue content of [³H]TPMP was measured at various times after starting feeding by scintillation counting. Data are means ± range for pairs of mice for each time point. (D) Feeding MitoVit E in drinking water. Mice were fed 500 μM [³H]MitoVit E (0.2 Ci/mol) in their drinking water, and the tissue contents of [³H]MitoVit E were measured after 4 days by scintillation counting. Data are means ± range for pairs of mice. (E) Feeding MitoQ in drinking water. Mice were fed 500 μM [³H]MitoQ (0.5 Ci/mol) in their drinking water, and the tissue contents of [³H]MitoQ were measured after 10 days by scintillation counting. Data are means ± range for pairs of mice. Liv, liver; Kid, kidney; Mus, muscle.

Figure 4

Clearance of orally administered alkyltriphenylphosphonium cations. (A) Clearance of [³H]TPMP. Mice were fed 500 μM [³H]TPMP (0.25 Ci/mol) in their drinking water for 7 days, and were then switched to water without TPMP. The tissue content of [³H]TPMP was measured by scintillation counting at various times after withdrawing TPMP. Data are means ± range for pairs of mice for each time point. (B) Log plot of clearance of TPMP from mice. The data shown in A were replotted to determine the first-order rate constant for TPMP clearance.

Figure 5

Accumulation of alkyltriphenylphosphonium cations within mitochondria in vivo. Mice were injected i.v. with 1 μmol IBTP or vehicle and 4 h later heart mitochondria and cytoplasm were prepared. Protein (25 μg) from control and IBTP-treated mitochondria and cytosolic fractions were separated by SDS/PAGE and then immunoblotted with antitriphenylphosphonium antiserum. The mitochondrial band at ≈45 kDa was a nonspecific binding artifact. These data are typical of three independent experiments. Analysis of mitochondrial and cytosolic marker enzymes during our mitochondrial preparations routinely shows negligible cross-contamination of the fractions.

Figure 6

Analysis of accumulated MitoVit E by mass spectrometry. Mice were fed 500 μM MitoVit E in their drinking water for 14 days, then MitoVit E was extracted from tissues and analyzed by electrospray mass spectrometry. Similar analysis of control tissues gave no peaks in the region of interest (data not shown).