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Mitochondrial Delivery of Doxorubicin by Triphenylphosphonium-Functionalized Hyperbranched Nanocarriers Results in Rapid and Severe Cytotoxicity

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ABSTRACT

Purpose

To develop a novel hyperbranched polymer-based nanocarrier for efficient drug delivery to cell mitochondria. Also to study for the first time the cytotoxic effect of doxorubicin via mitochondria-specific delivery system.

Methods

We introduced alkyltriphenylphosphonium groups (TPP) to a poly(ethylene imine) hyperbranched polymer (PEI). We harnessed the hydrophobic assembly of these alkylTPP functionalized PEI molecules into ~100 nm diameter nanoparticles (PEI-TPP) and further encapsulated the chemotherapy agent doxorubicin (DOX), to produce the mitotropic nanoparticles PEI-TPP-DOX.

Results

By administering PEI-TPP-DOX to human prostate carcinoma cells DU145, we found that: (i) PEI-TPP-DOX specifically localized at cell mitochondria as revealed by the inherent DOX fluorescence; (ii) in contrast to the slow apoptotic cell death incurred by DOX over the period of days at micromolar concentrations, PEI-TPP-DOX triggered rapid and severe cytotoxicity within few hours of incubation and at submicromolar incubation concentrations. This cytotoxicity was mainly found to be of a necrotic nature, not precluding autophagy related death pathways to a smaller extent.

Conclusions

We have elaborated a versatile mitotropic nanocarrier; furthermore, using this platform, we have developed a mitochondrial-doxorubicin formulation with exceptional cytocidal properties, even in nanomolar concentrations.

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Abbreviations

ANT:

Adenine nucleotide translocator

ANTI-A:

Antimycin A

ATR:

Atractyloside potassium salt

CASP8:

Z-IETD-FMK

CASP9:

Z-LEHD-FMK

CCCP:

Carbonyl cyanide 3-chlorophenylhydrazone

CSA:

Cyclosporine A

DIPEA:

N,N-diisopropylethylamine

DLS:

Dynamic light scattering

DOX:

Doxorubicin

EB:

Ethidium bromide

EPR:

Enhanced permeability and retention

ETC:

Electron transport chain

FBS:

Fetal bovine serum

HBTU:

2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium

HOBt:

N-hydroxybenzotriazole

LDH:

Lactate dehydrogenase

L-NAME:

Nω-nitro-L-arginine methyl ester hydrochloride

MnTmPyP:

Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin

MPTP:

Mitochondrial permeability transition pore

MTT:

Thiazolyl blue tetrazolium bromide

MYXO:

Myxothiazol

NADH:

β-Nicotinamide adenine dinucleotide reduced disodium salt

OLIGO:

Oligomycin

PBS:

Phosphate buffer saline

PEI:

Hyperbranched poly(ethylene imine)

PNNAG:

p-nitrophenyl-N-acetyl-β-d-glucosaminide

ROS:

Reactive oxygen species

ROT:

Rotenone

RUR:

Ruthenium red

STS:

Staurosporine

TB:

Trypan blue

TPP:

Triphenylphosphonium cation

TTFA:

Thenoyltrifluoroacetone

UA:

Uric acid

WORT:

Wortmannin

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Authors and Affiliations

  1. Department of Physical Chemistry, IAMPPNM, NCSR “Demokritos”, Patriarchou Gregoriou & Neapoleos, 15310, Ag. Paraskevi, Attiki, Greece

    Theodossis A. Theodossiou, Zili Sideratou, Maria E. Katsarou & Dimitris Tsiourvas

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  1. Theodossis A. Theodossiou
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  2. Zili Sideratou
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  3. Maria E. Katsarou
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  4. Dimitris Tsiourvas
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Correspondence to Theodossis A. Theodossiou.

Electronic supplementary material

Table S1. LDH leakage assay results and parameters for various PEI-TPP-DOX concentrations. Table S2. Inhibitors/modulators co-administered with PEI-TPP-DOX. Table S3. The effects of ATR, ANT1, MYXO and WORT on PEI-TPP-DOX cytotoxicity. Figure S1. 1Η NMR (500 MHz, MeOD-d4) spectrum of PEI-TPP. Figure S2. 13C NMR (125.1 MHz, MeOD-d4) spectrum of PEI-TPP. Figure S3. Representative image of DNA Laddering results for PEI-TPP-DOX.

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Theodossiou, T.A., Sideratou, Z., Katsarou, M.E. et al. Mitochondrial Delivery of Doxorubicin by Triphenylphosphonium-Functionalized Hyperbranched Nanocarriers Results in Rapid and Severe Cytotoxicity. Pharm Res 30, 2832–2842 (2013). https://doi.org/10.1007/s11095-013-1111-7

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