Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics - PubMed (original) (raw)

Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics

Sean Marrache et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Mitochondrial dysfunctions cause numerous human disorders. A platform technology based on biodegradable polymers for carrying bioactive molecules to the mitochondrial matrix could be of enormous potential benefit in treating mitochondrial diseases. 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. An optimized formulation was identified through in vitro screening of a library of charge- and size-varied NPs, and mitochondrial uptake was studied by qualitative and quantitative investigations of cytosolic and mitochondrial fractions of cells treated with blended NPs composed of PLGA-b-PEG-TPP and a triblock copolymer containing a fluorescent quantum dot, PLGA-b-PEG-QD. 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. These biomolecules were loaded into blended NPs with high loading efficiencies. 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. This work represents the potential of a single, programmable NP platform for the diagnosis and targeted delivery of therapeutics for mitochondrial dysfunction-related diseases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

(A) Synthesis of PLGA-_b_-PEG-OH, PLGA-_b_-PEG-TPP, and QD-conjugated polymer. (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. (C) (Upper) Size and zeta potential variation in blended NPs from PLGA-_b_-PEG-TPP and PLGA-COOH. (Lower) Size and zeta potential variation in NPs by blending PLGA-_b_-PEG-TPP with PLGA-_b_-PEG-OH. (D) TEM images of targeted and nontargeted blended NPs. All of the TEM samples except the QD-blended NPs were negatively stained with sterile 2% (wt/vol) uranyl acetate aqueous solution for 15 min. (E) Secretion of IL-6 and TNF-α in the media with charge-varied and size-varied NPs (0.5 mg/mL) after 12 h. *NPs from 100% PLGA-COOH are unstable, and NP diameter varies from 700 nm to 10 μm depending on the batch preparation.

Fig. 2.

Fig. 2.

(A) Subcellular localization of red fluorescent-targeted PLGA-_b_-PEG-TPP/PLGA-_b_-PEG-QD and nontargeted PLGA-_b_-PEG-OH/PLGA-_b_-PEG-QD blended NPs. HeLa cells were exposed to targeted NPs (diameter, 79 nm; zeta potential, 27.4 mV) and nontargeted NPs (diameter, 79 nm; zeta potential, −26.5 mV) at 10 μM or left untreated for 4 h. The cells were then stained with the mitochondrial marker MitoTracker Green (Invitrogen), fixed, and observed by wide-field fluorescence microscopy. The merged images and higher-magnification images show effective overlap of mitochondrial staining (green) and targeted NPs (red). No significant overlap was observed with nontargeted NPs. (B) Confocal images of time-dependent uptake of targeted PLGA-_b_-PEG-TPP/PLGA-_b_-PEG-QD blended NPs and nontargeted PLGA-_b_-PEG-OH/PLGA-_b_-PEG-QD blended NPs in HeLa cells. Lysosomes were stained with CellLight lysosomes-GFP, BacMam 2.0 (Life Technologies) (green).

Fig. 3.

Fig. 3.

Mitochondrial and cytosolic distribution of targeted PLGA-_b_-PEG-TPP/PLGA-_b_-PEG-QD blended NPs in HeLa cells by ICP-MS analysis. (A) Effect of size on uptake of NPs. (B) Overall cellular uptake of size-varying NPs. (C) Effect of zeta potential on cellular trafficking of NPs. (D) Overall cellular uptake of zeta potential-varying NPs.

Fig. 4.

Fig. 4.

(A) Effect on percent survival of IMR-32 neuroblastoma cells after treatment with targeted curcumin NPs, nontargeted curcumin NPs, and free curcumin against Aβ-induced cytotoxicity. The asterisk represents significant differences between targeted curcumin NPs, nontargeted curcumin NPs, and free curcumin according to one-way ANOVA with Tukey’s post hoc test; P < 0.001. (B) Cytotoxicity profiles of targeted LND NPs, nontargeted LND NPs, free LND, targeted α-TOS NPs, nontargeted α-TOS NPs, free α-TOS in HeLa cells, empty targeted NPs, and empty nontargeted NPs in HeLa cells.

Fig. 5.

Fig. 5.

Mouse 3T3-L1 preadipocytes were differentiated into adipocytes in the presence of 1 μM, 4 μM, 25 μM, or 100 μM of targeted 2,4-DNP NPs, nontargeted 2,4-DNP NPs, and free 2,4-DNP for 7 d. Nondifferentiated cells and completely differentiated cells were used as controls. Intracellular lipids were stained with AdipoRed (Lonza), and percent lipid accumulation was calculated. Inhibition of adipocyte differentiation is shown for day 7. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. *P < 0.05; ***P < 0.001. Similar results were obtained from two independent experiments. ns, nonsignificant.

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