DQAsomes as the Prototype of Mitochondria-Targeted Pharmaceutical Nanocarriers : An Update.

DQAsomes (dequalinium-based liposome-like vesicles) are the prototype for all mitochondria-targeted vesicular pharmaceutical nanocarrier systems. First described in 1998 in a paper which has been cited as of May 2020 over 150 times, DQAsomes have been successfully explored for the delivery of DNA and low-molecular weight molecules to mitochondria within living mammalian cells. Moreover, they also appear to have triggered the design and development of a large variety of similar mitochondria-targeted nanocarriers . Potential areas of application of DQAsomes and of related mitochondria-targeted pharmaceutical nanocarriers involve mitochondrial gene therapy , antioxidant and updated therapy as well as apoptosis-based anticancer chemotherapy. Here, detailed protocols for the preparation, characterization, and application of DQAsomes are given and most recent developments involving the design and use of DQAsome-related particles are highlighted and discussed.

Preparation and antitumor evaluation of hinokiflavone hybrid micelles with mitochondria targeted for lung adenocarcinoma treatment.

Hinokiflavone (HF) is a natural biflavonoid extracted from medicinal plants such as Selaginella tamariscina and Platycladus orientalis. HF plays a crucial role in the treatment of several cancers. However, its poor solubility, instability, and low bioavailability have limited its use. In this study, soluplus/d-α-tocopherol acid polyethylene glycol 1000 succinate (TPGS)/dequalinium (DQA) was applied to improve the solubilization efficiency and stability of HF. HF hybrid micelles were prepared via thin-film hydration method. The physicochemical properties of micelles, including particle size, zeta potential, encapsulation efficiency, drug loading, CMC value, and stability were investigated. The in vitro cytotoxicity assay showed that the cytotoxicity of the HF hybrid micelles was higher than that of free HF. In addition, the HF hybrid micelles improved anticancer efficacy and induced mitochondria-mediated apoptosis, which is associated with the high levels of ROS inducing decreased mitochondrial membrane potential, promoting apoptosis of tumor cells. Furthermore, in vivo tumor suppression, smaller tumor volume and increased expression of pro-apoptotic proteins were found in nude mice treated with HF hybrid micelles, suggesting that HF hybrid micelles had stronger tumor suppressive activity compared with free HF. In summary, HF hybrid micelles developed in this study enhanced antitumor effect, which may be a potential drug delivery system for the treatment of lung adenocarcinoma.

Inhibiting tumour metastasis by DQA modified paclitaxel plus ligustrazine micelles in treatment of non-small-cell lung cancer.

Lung cancer is a kind of malignant tumour characterized as uncontrolled cell growth in lung. These malignant cell growth can spread beyond the lung by process of metastasis into other tissues or parts of the body. In this study, we developed dequalinium (DQA) modified paclitaxel plus ligustrazine micelles to destroy vasculogenic mimicry (VM) channels and inhibit tumour metastasis. In vitro assays showed that the targeting micelles with centralized particle size distribution showed not only vigoroso cytotoxicity on A549 cells but also strong inhibition on VM channels and tumour metastasis. Mechanism studies indicated that the DQA modified paclitaxel plus ligustrazine micelles could down-regulate the expressions of VEGF, MMP2, TGF-ß1 and E-cadherin in A549 cells. In vivo assays indicated that the targeting drug-loaded micelles could enhance the accumulation of chemotherapeutic drugs at tumour sites and exhibit strong tumour inhibitory activity with negligible toxicity. Hence, the DQA modified paclitaxel plus ligustrazine micelles developed in this study may provide a potential strategy for treatment of NSCLC.

Self-assembled nanoparticles composed of glycol chitosan-dequalinium for mitochondria-targeted drug delivery.

Cancer cells divide uncontrollably due to their metabolic imbalance, resistance to mitochondria-mediated apoptosis, and ability to sustain telomere crisis by activating telomere reverse transcriptase. Therefore, mitochondria-mediated cell death has gained considerable attention as an alternative strategy to kill cancer cells. In the present study, an amphiphilic polymer composed of glycol chitosan (GC) and dequalinium (DQA), was synthesized via Michael addition reaction using a methyl acrylate linker and used to target mitochondria. DQA was selected as the mitochondria targeting moiety as well as the lipophilic component of polymer that will self-assemble into nanoparticles in aqueous solvent. GC-DQA nanoparticles were nontoxic compared to positive control when cell viability were assessed in both cancerous and non-cancerous cells. Mitochondria targeting and cell uptake was confirmed by confocal microscopy and flow cytometry, respectively. Curcumin was selected as the anticancer drug and while tested in vitro, the IC50 concentration of the micellar form was 10 µM in cancer cells. These results validate the promising potential of GC-DQA nanoparticles as an efficient mitochondria-targeting drug delivery system for cancer therapy.

Functional paclitaxel plus honokiol micelles destroying tumour metastasis in treatment of non-small-cell lung cancer.

Treatment effect of chemotherapy for aggressive non-small-cell lung cancer (NSCLC) is usually unsatisfactory for non-selective distributions of anticancer drugs, generation of vasculogenic mimicry (VM) channels, high metastasis and recurrence rate. Therefore, we developed a kind of dequalinium (DQA) modified paclitaxel plus honokiol micelles in this study to destroy VM channels and inhibit tumour metastasis. In vitro assays indicated that the targeting paclitaxel micelles with ideal physicochemical characteristics could exhibit not only the powerful cytotoxicity on Lewis lung tumour (LLT) cells but also the effective suppression on VM channels and tumour metastasis. Action mechanism studies manifested that DQA modified paclitaxel plus honokiol micelles could activate apoptotic enzymes caspase-3 and caspase-9 as well as down-regulate FAK, PI3K, MMP-2 and MMP-9. In vivo assays indicated that polymeric micelles could increase selective accumulation of chemotherapeutic drugs at tumour sites and showed a conspicuous antitumour efficacy. Hence, the DQA modified paclitaxel plus honokiol micelles prepared in this study provided a potential treatment strategy for NSCLC.

Fabrication of paclitaxel hybrid nanomicelles to treat resistant breast cancer via oral administration.

AIM: Oral chemotherapy using anticancer drugs would improve the clinical practice and the life quality of patients. The aim of the present study was to develop paclitaxel hybrid nanomicelles for oral administration to treat resistant breast cancer. METHODS: Evaluations were performed on human breast cancer MCF-7 cells, drug-resistant breast cancer MCF-7/Adr cells, and in MCF-7/Adr-xenografted BALB/c nude mice. The nanomicelles were composed of the polymer soluplus, d-α-tocopheryl polyethyleneglycol 1000 succinate (TPGS1000), and dequalinium (DQA). The constructed paclitaxel hybrid nanomicelles were ~65 nm in size. RESULTS: The nanomicelles improved cellular uptake and anticancer efficacy in the resistant breast cancer cells and induced mitochondria-mediated apoptosis. The mechanism of the apoptosis-inducing effect was related to the co-localization of the nanomicelles with mitochondria; the activation of pro-apoptotic protein Bax, cytochrome C, and apoptotic enzymes caspase 9 and 3; and the inhibition of anti-apoptotic proteins Bcl-2 and Mcl-1. Oral administration of paclitaxel hybrid nanomicelles had the same anticancer efficacy as the intravenous injection of taxol in resistant breast cancer-bearing mice. The oral suitability of this formulation was associated with the nanostructure and the actions of TPGS1000 and DQA. CONCLUSION: The fabricated paclitaxel hybrid nanomicelles could provide a promising oral formulation to treat drug-resistant breast cancer.

Dequalinium-based functional nanosomes show increased mitochondria targeting and anticancer effect.

Mitochondria are targets with great potential for therapeutics for many human disorders. However, drug delivery systems for such therapeutics remain in need of more efficient mitochondrial-targeting carriers. In this study, we report that nanosomes composed of Dequalinium/DOTAP (1,2-dioleoyl-3-trimethylammonium-propane)/DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), called DQA80s, can act in the dual role of mitochondrial-targeting carrier and anticancer agent for therapeutic interventions against mitochondrial diseases. In cytotoxicity assays, DQA80s were shown to be more toxic than DQAsomes. The DQA80s showed significantly increased cellular uptake as compared to that of DQAsomes, and DQA80s also showed more efficient escape from the endolysosome to the cytosol. We observed the efficient targeting of DQA80s to mitochondria in living cells using flow cytometry, confocal microscopy, and TEM imaging. We also found evidence of anticancer potential that mitochondrial-targeted DQA80s induced apoptosis by production of reactive oxygen species (ROS) via MAPK signaling pathways, loss of mitochondrial membrane potential, and the caspase-3 activation. The present study demonstrates that DQA80s have excellent dual potential both as a carrier and as an anticancer therapeutic for mitochondria-related disease therapy in vivo.

DQAsomes as the prototype of mitochondria-targeted pharmaceutical nanocarriers: preparation, characterization, and use.

DQAsomes (dequalinium-based liposome-like vesicles) are the prototype for all mitochondria-targeted vesicular pharmaceutical nanocarrier systems. First described in 1998, they have been successfully explored for the delivery of DNA and low-molecular weight molecules to mitochondria within living mammalian cells. Potential areas of application involve mitochondrial gene therapy, antioxidant therapy as well as apoptosis-based anticancer chemotherapy. Here, detailed protocols for the preparation, characterization, and application of DQAsomes are given.

Towards boron neutron capture therapy: the formulation and preliminary in vitro evaluation of liposomal vehicles for the therapeutic delivery of the dequalinium salt of bis-nido-carborane.

Liposomes of phosphatidylcholine or of dimyristoylphosphatidylcholine that incorporate bis-nido-carborane dequalinium salt are stable in physiologically relevant media and have in vitro toxicity profiles that appear to be compatible with potential therapeutic applications. These features render the structures suitable candidate boron-delivery vehicles for evaluation in the boron neutron capture therapy of cancer.

Transferrin coupled vesicular system for intracellular drug delivery for the treatment of cancer: development and characterization.

OBJECTIVES: In the present study attempt has been made to enhance the selective tumor cell killing in mouse xenograft model using DQAsomes as a mitochondriotropic carrier and transferrin (Tf) as a ligand to target tumor cells. METHODS: Tf modified DQAsomes (Tf-DQAsomes) were prepared by incubating preformed paclitaxel loaded DQAsomes with Tf in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Developed systems were characterized for size and size distribution, entrapment efficiency, and in vitro drug release. Fluorescence microscopy and flow cytometry were performed to evaluate cellular uptake of the carriers. Antitumor activity was determined using HeLa cells. In vivo therapeutic efficacy was determined in xenograft mouse model. KEY FINDINGS: Uptake studies demonstrated that Tf-DQAsomes result in higher fluorescence intensity to the cancer cells as compared to plain DQAsomes. Tf-DQAsomes exhibited better antitumor activity in vitro as compared to plain DQAsomes and paclitaxel solution. In vivo biodistribution study revealed that paclitaxel concentration in the tumor was much higher in the case of Tf-DQAsomes as compared to plain DQAsomes and paclitaxel solution; however in other organs it was much lower than the latter two formulations. Tf-DQAsomes exhibited significant antitumor activity in the mouse xenograft model. CONCLUSIONS: The finding demonstrated that Tf conjugated DQAsomes can effectively be delivered to the tumor in vivo and exhibit significant antitumor activity.

Expression of GFP in the mitochondrial compartment using DQAsome-mediated delivery of an artificial mini-mitochondrial genome.

PURPOSE: We describe a novel strategy for expression of GFP in mammalian mitochondria. METHODS: The key components of the strategy were an artificially created mitochondrial genome pmtGFP and a DQAsome transfection system. RESULTS: Using immunofluorescence and a combination of immunohistochemical and molecular based techniques, we show that DQAsomes are capable of delivering the pmtGFP construct to the mitochondrial compartment of the mouse macrophage cell line RAW264.7, albeit at low efficiency (1-5%), resulting in the expression of GFP mRNA and protein. Similar transfection efficiencies were also demonstrated in a range of other mammalian cell lines. CONCLUSIONS: The DQAsome-transfection technique was able to deliver the exogenous DNA into the cellular mitochondria and the pmtGFP was functional. Further optimization of this strategy would provide a flexible and rapid way to generate mutant cells and useful animal models of mitochondrial disease.

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

Intrinsic multidrug resistance (MDR) of cancers remains a major obstacle to successful chemotherapy. A dequalinium polyethylene glycol-distearoylphosphatidylethanolamine (DQA-PEG(2000)-DSPE) conjugate was synthesized as a mitochondriotropic molecule, and mitochondrial targeting resveratrol liposomes were developed by modifying DQA-PEG(2000)-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-PEG(2000)-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.

Selective targeting of neuroblastoma tumour-initiating cells by compounds identified in stem cell-based small molecule screens.

Neuroblastoma (NB) is the most deadly extra-cranial solid tumour in children necessitating an urgent need for effective and less toxic treatments. One reason for the lack of efficacious treatments may be the inability of existing drugs to target the tumour-initiating or cancer stem cell population responsible for sustaining tumour growth, metastases and relapse. Here, we describe a strategy to identify compounds that selectively target patient-derived cancer stem cell-like tumour-initiating cells (TICs) while sparing normal paediatric stem cells (skin-derived precursors, SKPs) and characterize two therapeutic candidates. DECA-14 and rapamycin were identified as NB TIC-selective agents. Both compounds induced TIC death at nanomolar concentrations in vitro, significantly reduced NB xenograft tumour weight in vivo, and dramatically decreased self-renewal or tumour-initiation capacity in treated tumours. These results demonstrate that differential drug sensitivities between TICs and normal paediatric stem cells can be exploited to identify novel, patient-specific and potentially less toxic therapies.

Disintegration of amyloid fibrils of alpha-synuclein by dequalinium.

alpha-Synuclein is the major amyloidogenic component observed in the Lewy bodies of Parkinson's disease. Amyloid fibrils of alpha-synuclein prepared in vitro were instantaneously disintegrated by dequalinium (DQ). Double-headed cationic amphipathic structure of DQ with two aminoquinaldinium rings at both ends turned out to be crucial to exert the disintegration activity. The defibrillation activity was shown to be selective toward the fibrils of alpha-synuclein and Abeta40 while the other beta2-microglobulin amyloid fibrils were not susceptible so much. Besides the common cross beta-sheet conformation of amyloid fibrils, therefore, additional specific molecular interactions with the target amyloidogenic proteins have been expected to be involved for DQ to exhibit its defibrillation activity. The disintegrating activity of DQ was also evaluated in vivo with the yeast system overexpressing alpha-synuclein-GFP. With the DQ treatment, the intracellular green inclusions turned into green smears, which resulted in the enhanced cell death. Based on the data, the previous observation that DQ led to the predominant protofibril formation of alpha-synuclein could be explained by the dual function of DQ showing both the facilitated self-oligomerization of alpha-synuclein and the instantaneous defibrillation of its amyloid fibrils. In addition, amyloidosis-related cytotoxicity has been demonstrated to be amplified by the fragmentation of mature amyloid fibrils by DQ.

Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump.

Multidrug efflux pumps cause serious problems in cancer chemotherapy and treatment of bacterial infections. Yet high-resolution structures of ligand transporter complexes have previously been unavailable. We obtained x-ray crystallographic structures of the trimeric AcrB pump from Escherichia coli with four structurally diverse ligands. The structures show that three molecules of ligands bind simultaneously to the extremely large central cavity of 5000 cubic angstroms, primarily by hydrophobic, aromatic stacking and van der Waals interactions. Each ligand uses a slightly different subset of AcrB residues for binding. The bound ligand molecules often interact with each other, stabilizing the binding.

Inhibition of protein kinase C(alpha) by dequalinium analogues: dependence on linker length and geometry.

Analogues of a bipartite compound, dequalinium (DECA) (quinolinium, 1,1'-(1,10-decanediyl)bis(4-amino-2-methyl diiodide)), were tested for inhibition of protein kinase C(alpha) (PKC(alpha)). In vitro assays of monomeric and dimeric analogues support a model in which DECA inhibits PKC(alpha) by an obligatory two-point contact, a unique mechanism among PKC inhibitors. The presence of unsaturation in the center of the C(10)-alkyl linker produced geometric isomers with different inhibitory potencies: cis IC(50) = 52 +/- 12 microM and trans IC(50) = 12 +/- 3 microM, where the trans isomer was equipotent to that of the saturated C(10)-DECA. DECA analogues with longer, saturated linkers (C(12), C(14), or C(16)) exhibited enhanced inhibitory potencies which reached a plateau with the C(14)-linker (IC(50) = 2.6 +/- 0.2 microM). Metastatic melanoma cells treated with 250 nM C(12)-, C(14)-, or C(16)-DECA and irradiated with long-wave UV light (which causes irreversible inhibition of PKC(alpha) by DECA) confirmed the linker-dependent inhibition of intracellular PKC(alpha) activity.

Dequalinium vesicles form stable complexes with plasmid DNA which are protected from DNase attack.

Upon sonication, the antimicrobial and antineoplastic compound dequalinium forms vesicles (DQAsomes, Weissig et al., 1998). Dequalinium (1,1'-(1,10-decamethylene-bis-[aminoquinaldinium])-chloride) was shown to be a fluorophore with an emission maximum at 366 nm. Addition of DNA results in a characteristic quenching of its intrinsic fluorescence. After density gradient centrifugation a band of dequalinium (DQA) tightly associated with DNA is located between the DNA and DQA bands. DQA/DNA-complexes containing plasmid DNA at a molar ratio of DQA/DNA 6:1 are completely protected against DNase activity. Addition of negatively-charged lipids release intact DNA in the same manner as from cationic lipid/DNA complexes. As regards biological effects, DQAsomes show a differential cytotoxicity for normal and sarcoma cell lines. In vitro incubation with fluorescein-labeled oligodeoxynucleotides (5'-fluorescein-[GATC]5) showed an increased uptake of the tagged oligodeoxynucleotide if complexed with dequalinium. We hypothesize that the DQA/DNA complexes are well-suited for 'DQAsomal gene transfer' in vitro and in vivo. Noteworthy, they display an intrinsic antitumor activity manifested by differential cytotoxicity for normal and sarcoma cells.

Dequalinium induces a selective depletion of mitochondrial DNA from HeLa human cervical carcinoma cells.

Treatment of cultured human cervical carcinoma cells with the anticancer drug dequalinium (DEQ) was found to cause a delayed inhibition of cell growth. This inhibition was preceded by a loss of mitochondrial DNA (mtDNA), a decrease in cytochrome c oxidase activity, and an increase in the level of lactate, indicating that growth inhibition was due to the loss of mtDNA-encoded functions. There was a progressive two-fold loss of mtDNA following each cell division in the presence of DEQ, suggesting that this drug was acting by inhibiting some aspect of mtDNA synthesis. Furthermore, cells became resistant to the growth inhibitory and cytotoxic affects of DEQ when they were grown under conditions that bypassed the need for mtDNA-encoded functions. Resistance was not associated with significant changes in drug accumulation. These results suggest that the DEQ-induced depletion of mtDNA plays an important role in drug cytotoxicity.

Synthesis and evaluation of novel rhodacyanine dyes that exhibit antitumor activity.

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) = 50 nM, IC50(CV-1) = 17.3 microM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agents candidates.

Photoinactivation of the bovine heart mitochondrial F1-ATPase by [14C]dequalinium cross-links phenylalanine-403 or phenylalanine-406 of an alpha subunit to a site or sites contained within residues 440-459 of a beta subunit.

Synthesis of [14C]dequalinium, 1,1'-(1,10-[1,10-14C]decanediyl)bis[4-amino-2-methylquinolinium ], is described, which photoinactivates the bovine heart mitochondrial F1-ATPase (MF1). Maximal photoinactivation occurs on incorporation of about 1.5 mol of [14C]dequalinium/mol of MF1. Three radioactive species were resolved when photoinactivated enzyme was submitted to polyacrylamide gel electrophoresis at pH 4.0 in the presence of tetradecyltrimethylammonium bromide, which correspond to the alpha and beta subunits and a cross-linked species with an M(r) of 116,000. Fractionation of a tryptic digest of photoinactivated enzyme by high-performance liquid chromatography led to isolation of a radioactive peptide which contains residues 399-420 of a alpha subunit. Two fragments containing equal amounts of radioactivity were obtained on fractionation of an endoproteinase Asp-N digest of the isolated radioactive tryptic peptide by high-performance liquid chromatography. Amino acid sequence analysis showed that both fragments contained residues 399-408 of the alpha subunit, but one was missing Phe-alpha 403 and the other was lacking Phe-alpha 406. Fractionation of a cyanogen bromide digest of photoinactivated enzyme followed by trypsin digestion of partially purified cyanogen bromide fragments and fractionation of the resulting radioactive tryptic fragments yielded several radioactive species comprised of residues 399-420 of the alpha subunit cross-linked to residues 440-459 of the beta subunit and a radioactive fragment containing residues 399-420 of the alpha subunit. Partial sequence analyses of the cross-linked fragments suggest that Phe-alpha 403 and Phe-alpha 406 participate in cross-links, whereas no information was obtained on the site or sites of cross-linking in the beta subunit fragment.(ABSTRACT TRUNCATED AT 250 WORDS)