Elsevier

Biomaterials

Volume 32, Issue 24, August 2011, Pages 5673-5687
Biomaterials

The use of mitochondrial targeting resveratrol liposomes modified with a dequalinium polyethylene glycol-distearoylphosphatidyl ethanolamine conjugate to induce apoptosis in resistant lung cancer cells

https://doi.org/10.1016/j.biomaterials.2011.04.029Get rights and content

Abstract

Intrinsic multidrug resistance (MDR) of cancers remains a major obstacle to successful chemotherapy. A dequalinium polyethylene glycol-distearoylphosphatidylethanolamine (DQA-PEG2000-DSPE) conjugate was synthesized as a mitochondriotropic molecule, and mitochondrial targeting resveratrol liposomes were developed by modifying DQA-PEG2000-DSPE on the surface of liposomes for overcoming the resistance. Evaluations were performed on the human lung adenocarcinoma A549 cells and resistant A549/cDDP cells, A549 and A549/cDDP tumor spheroids as well as the xenografted resistant A549/cDDP cancers in nude mice. The yield of DQA-PEG2000-DSPE conjugate synthesized was about 87% and the particle size of mitochondrial targeting resveratrol liposomes was approximately 70 nm. The mitochondrial targeting liposomes significantly enhanced the cellular uptake, and selectively accumulated into mitochondria when encapsulating coumarin as the fluorescent probe. Furthermore, mitochondrial targeting resveratrol liposomes induced apoptosis of both non-resistant and resistant cancer cells by dissipating mitochondria membrane potential, releasing cytochrome c and increasing the activities of caspase 9 and 3. They also exhibited significant antitumor efficacy in two kinds of cancer cells, in tumor spheroids by penetrating deeply into the core, and in xenografted resistant A549/cDDP cancers in nude mice. Mitochondrial targeting resveratrol liposomes co-treating with vinorelbine liposomes significantly enhanced the anticancer efficacy against the resistant A549/cDDP cells. In conclusion, mitochondrial targeting resveratrol liposomes would provide a potential strategy to treat the intrinsic resistant lung cancers by inducing apoptosis via mitochondria signaling pathway.

Introduction

The multidrug resistance (MDR) of cancers is a major obstacle to successful cancer chemotherapy, and it results in incomplete therapeutic response, recurrent and metastasis of cancers [1]. Intrinsic MDR plays a crucial role in the drug resistance and is caused by genetic and epigenetic changes of cancer cells through altering function of pro-apoptotic or apoptotic genes encoded proteins such as Bcl-2 family proteins [2], [3] and capase proteins involved in the apoptosis signaling pathway [4], [5].

There are two basic ways to eliminate cancer cells in the chemotherapy. One approach is to kill cancer cells by direct exposure of cancer cells to toxic chemicals. The other one is to induce the suicide of cancer cells, namely, induction of apoptosis which has been regarded as a very important strategy for thoroughly eliminating tumors [6].

Mitochondria are membrane-enclosed organelles found in most eukaryotic cells, and are described as "cellular power plants". Besides supplying cellular energy, mitochondria are involved in a range of other processes, such as cellular differentiation, cell death, as well as the control of the cell cycle and cell growth [7]. Cancer cell mitochondria are structurally and functionally different from their normal counterparts [8]. They exhibit an extensive metabolic and are more susceptible to mitochondrial perturbation than normal cell mitochondria [9].

Cancer cell apoptosis induced by anticancer drugs may involve two independent initiator pathways that lie in upstream of all these effector events: activation of upstream caspase by cross-linking with death receptors on their ligands, and the release of apoptogenic factors by triggering various forms of cellular stress [10]. Both of them seem to be focused on the mitochondrial level, thereby triggering the cell apoptosis. Consequently, induction of apoptosis through direct targeting of mitochondria of cancer cells could be a strategy to circumvent the intrinsic drug resistance in chemotherapy [4]. Based on the above premises, mitochondria-targeting therapy emerges as a promising means to selectively eliminate cancers [11].

A number of experimental drugs have been explored to act primarily on mitochondria to induce apoptosis of cancer cells, such as glucose transporter inhibitors [12], mitochondrial permeability transition (MPT) modulators [13], [14], mitochondrial outer membrane permeabilization (MOMP) modulators [11], and so on. However, these agents are less than ideal due to unfavorable pharmacokinetic property which leads to extensively systemic distribution in the body, hence increasing the systemic toxicity of the agents.

To date, many mitochondrial drug delivery systems have been constructed to solve these problems. For examples, triphenylphosphonium cation (TPP), dequalinium (DQA) and mitochondrial targeting signal peptides (MTSs) have been packed or modified onto the lipid carriers such as mitochondriotropic molecules for targeting mitochondria [15].

Resveratrol is a polyphenol and is widely distributed in plant foods such as grapes wine, and peanuts. Resveratrol has emerged as a potential antitumor agent having the ability to inhibit all three major stages of carcinogenesis, including initiation, promotion and progression [16]. In variety types of cancer cells, resveratrol can stimulate apoptosis through activating the mitochondrial apoptotic pathway [17], [18], [19]. However, the low water solubility, stability and therapeutic index of resveratrol make it unsuccessful in the clinical therapy [20].

We proposed here a strategy that mitochondrial targeting resveratrol liposomes could be able to trigger apoptosis by acting on mitochondria of cancer cells, and be used a co-therapy agent with a cytotoxic drug for overcoming the intrinsic resistant cancers.

In the present study, we synthesized a targeting material, dequlinium-polyethylene glycol distearoylphosphatidylethanolamine (DQA-PEG2000-DSPE), which was used as mitochondriotropic molecule for modifying the surface of liposomes. The objectives of the present study were to characterize the mitochondrial targeting resveratrol liposomes, to evaluate their potential apoptosis inducing effect on the resistant lung cancers, and to assess their therapeutic efficacy in combination with a cytotoxic formulation, vinorelbine liposomes.

Section snippets

Materials

Dequalinium (DQA) was purchased from Hangzhou Sanhe Chemicals Co., Ltd. (Hangzhou, China). 1,2-distearoyl-sn-glycero-3-phosphoethamolamine-N-[carboxy (polyethylene glycol) 2000] (COOH-PEG2000-DSPE) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). 1-hydroxy-1H-benzotriazole (HOBt) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Polyethylene glycol-distearoylphosphatidylethanolamine (PEG2000-DSPE) was purchased from NOF Corporation (Japan). Resveratrol was obtained from

Synthesis and characterization of DQA-PEG2000-DSPE

Fig. 1 shows the synthetic scheme for DQA-PEG2000-DSPE. To synthesize DQA-PEG2000-DSPE conjugate, DQA was conjugated through coupling one of the aromatic amino groups of DQA to the carboxylic group on COOH-PEG2000-DSPE, using DCC and HOBt as coupling agents.

Fig. 2 shows the MALDI-TOF-MS spectra of dequalinium (DQA), DQA-PEG2000-DSPE conjugate, and the uncoupled COOH-PEG2000-DSPE. The MALDI-TOF spectra of production after conjugating reaction exhibited the average mass of DQA-PEG2000-DSPE at m/z

Discussion

In the present study, a DQA-PEG2000-DSPE conjugate is first synthesized, and modified onto the mitochondrial targeting resveratrol liposomes, which show apoptosis inducing effect on the resistant lung cancer cells by targeting mitochondria of cancer cells. DQA is a quaternary ammonium cation commonly available as the dichloride salt, and an amphiphile with delocalized cationic charge centers showing selectively mitochondrial accumulation [29]. DQA has been used as mitochondrion-tropic molecule

Conclusions

A DQA-PEG2000-DSPE conjugate was synthesized by acrylation from dequalinium and COOH-PEG2000-DSPE with a yield o 87%, and could be used as mitochondriotropic molecule. The mitochondrial targeting liposomes are able to punctate accumulated in mitochondria of resistant A549/cDDP cells when encapsulating coumarin as the fluorescent probe. Furthermore, mitochondrial targeting resveratrol liposomes exhibit significant antitumor efficacy in resistant A549/cDDP cells by inducing apoptosis through

Acknowledgements

This paper was supported by the Key Grant of Beijing Natural Science Foundation (No. 7091005), and by the National Basic Research Program of China (973 program, 2007CB935801)

References (44)

  • V. Weissig et al.

    DQAsome/DNA complexes release DNA upon contact with isolated mouse liver mitochondria

    J Control Release

    (2001)
  • G.G. D’Souza et al.

    DQAsome-mediated delivery of plasmid DNA toward mitochondria in living cells

    J Control Release

    (2003)
  • X. Li et al.

    A novel stealth liposomal topotecan with amlodipine: apoptotic effect is associated with deletion of intracellular Ca2+ by amlodipine thus leading to an enhanced antitumor activity in leukemia

    J Controlled Release

    (2006)
  • X.M. Sun et al.

    Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis

    J Biol Chem

    (1999)
  • M. Zoratti et al.

    The mitochondrial permeability transition

    Biochim Biophys Acta

    (1995)
  • E. Galeano et al.

    Effects of the antitumoural dequalinium on NB4 and K562 human leukemia cells lines mitochondrial implication in cell death

    Leuk Res

    (2005)
  • A.L. Harris et al.

    Mechanisms of multidrug resistance in cancer treatment

    Acta Oncolog

    (1992)
  • G. Kroemer

    The proto-oncogene Bcl-2 and its role in regulating apoptosis

    Nat Med

    (1997)
  • Y. Ionov et al.

    Mutational inactivation of the proapoptotic gene BAX confers selective advantage during tumor clonal evolution

    Proc Natl Acad Sci

    (2000)
  • O. Tredan et al.

    Drug resistance and the solid tumor microenvironment

    J Natl Cancer Inst

    (2007)
  • K.M. Debatin et al.

    Chemotherapy: targeting the mitochondrial cell death pathway

    Oncogene

    (2002)
  • S. Fulda et al.

    Targeting mitochondria for cancer therapy

    Nat Rev Drug Discov

    (2010)
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