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.

Repositioning Dequalinium as Potent Muscarinic Allosteric Ligand by Combining Virtual Screening Campaigns and Experimental Binding Assays.

Structure-based virtual screening is a truly productive repurposing approach provided that reliable target structures are available. Recent progresses in the structural resolution of the G-Protein Coupled Receptors (GPCRs) render these targets amenable for structure-based repurposing studies. Hence, the present study describes structure-based virtual screening campaigns with a view to repurposing known drugs as potential allosteric (and/or orthosteric) ligands for the hM2 muscarinic subtype which was indeed resolved in complex with an allosteric modulator thus allowing a precise identification of this binding cavity. First, a docking protocol was developed and optimized based on binding space concept and enrichment factor optimization algorithm (EFO) consensus approach by using a purposely collected database including known allosteric modulators. The so-developed consensus models were then utilized to virtually screen the DrugBank database. Based on the computational results, six promising molecules were selected and experimentally tested and four of them revealed interesting affinity data; in particular, dequalinium showed a very impressive allosteric modulation for hM2. Based on these results, a second campaign was focused on bis-cationic derivatives and allowed the identification of other two relevant hM2 ligands. Overall, the study enhances the understanding of the factors governing the hM2 allosteric modulation emphasizing the key role of ligand flexibility as well as of arrangement and delocalization of the positively charged moieties.

Rapid mechanochemical synthesis of metal-organic frameworks using exogenous organic base.

Metal-organic frameworks (MOFs) bearing coordinatively unsaturated metal centers, exemplified by the MOF-74 family of frameworks, are promising for applications ranging from gas separations and storage to Lewis acid catalysis. However, the scalable synthesis of MOF-74 analogues remains a significant challenge. Recently, mechanochemistry has emerged as a sustainable strategy for the preparation of MOFs in the solid state with minimal solvent waste. Mechanochemical methods typically rely on metal salts bearing basic anions to deprotonate the conjugate acid of the organic linker and a small amount of organic solvent or water to facilitate liquid assisted grinding. Here, we demonstrate that the liquid exogenous organic base Hünig's base (N,N-diisopropylethylamine) can fulfill both roles, enabling the mechanochemical synthesis of M2(dobdc) analogues (M = Mg, Mn, Co, Ni, Cu, Zn; dobdc4- = 2,5-dioxidobenzene-1,4-dicarboxylate) using metal nitrate salts in only 5 minutes at room temperature. Importantly, we demonstrate that this straightforward method can be generalized to prepare the isomeric framework Mg2(m-dobdc) (m-dobdc4- = 2,4-dioxidobenzene-1,5-dicarboxylate) and the expanded framework Mg2(dobpdc) (dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) under solvent-free conditions for the first time. The MOFs prepared using this method possess high crystallinities and surface areas, with the Mg2(m-dobdc) prepared herein representing the first reported permanently porous variant of this framework. This new sustainable mechanochemical synthesis of MOF-74 analogues should enable their preparation on a large scale for industrial applications.

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.

HIV-1 fusion inhibitors targeting the membrane-proximal external region of Env spikes.

Combination antiretroviral therapy has transformed HIV-1 infection, once a fatal illness, into a manageable chronic condition. Drug resistance, severe side effects and treatment noncompliance bring challenges to combination antiretroviral therapy implementation in clinical settings and indicate the need for additional molecular targets. Here, we have identified several small-molecule fusion inhibitors, guided by a neutralizing antibody, against an extensively studied vaccine target-the membrane proximal external region (MPER) of the HIV-1 envelope spike. These compounds specifically inhibit the HIV-1 envelope-mediated membrane fusion by blocking CD4-induced conformational changes. An NMR structure of one compound complexed with a trimeric MPER construct reveals that the compound partially inserts into a hydrophobic pocket formed exclusively by the MPER residues, thereby stabilizing its prefusion conformation. These results suggest that the MPER is a potential therapeutic target for developing fusion inhibitors and that strategies employing an antibody-guided search for novel therapeutics may be applied to other human diseases.

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.

Interaction of dequalinium chloride with phosphatidylcholine bilayers: A biophysical study with consequences on the development of lipid-based mitochondrial nanomedicines.

Dequalinium (DQ) has been proposed as a mitochondrial targeting ligand for nanomedicines, including liposomes, given the implication of these organelles in many diseases. This original study focuses on the interactions of DQ with phosphatidylcholine bilayers during the formation of liposomes. Firstly, PEGylated liposomes suitable for drug delivery were studied and were found to be more stable when made in water than in phosphate-buffered saline, emphasizing the role of electrostatic interactions between positive charges on DQ and the polar head groups of the lipids. To gain more information, differential scanning calorimetry, small- and wide-angle X-ray scattering and diffraction, 31P and 2H NMR spectroscopy and freeze-fracture electron microscopy were performed on dimyristoylphosphatidylcholine (DMPC) model membranes in the presence of DQ. This molecule was shown to be located at the level of polar head groups and to induce electrostatic repulsions between adjacent lipid bilayers leading to membrane budding in water. These findings indicate that DQ is not completely inert towards lipid membranes and therefore is not an ideal candidate for encapsulation in liposomes. Overall, our work stresses the necessity for thorough physico-chemical characterization to better understand the mechanisms underlying the development of nanomedicines.

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.

Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.

Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe the novel application of cationic bolasomes to deliver ASOs into bacteria. We also report the first successful in vitro antisense treatment to inhibit the growth of C. difficile.

Mechanisms and Consequences of Macromolecular Phase Separation.

Over a century ago, colloidal phase separation of matter into non-membranous bodies was recognized as a fundamental organizing principal of cell "protoplasm." Recent insights into the molecular properties of such phase-separated bodies present challenges to our understanding of cellular protein interaction networks, as well as opportunities for interpreting and understanding of native and pathological genetic and molecular interactions. Here, we briefly review examples of and discuss physical principles of phase-separated cellular bodies and then reflect on how knowledge of these principles may direct future research on their functions.

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.

Export of a single drug molecule in two transport cycles by a multidrug efflux pump.

Secondary multidrug transporters use ion concentration gradients to energize the removal from cells of various antibiotics. The Escherichia coli multidrug transporter MdfA exchanges a single proton with a single monovalent cationic drug molecule. This stoichiometry renders the efflux of divalent cationic drugs energetically unfavourable, as it requires exchange with at least two protons. Here we show that surprisingly, MdfA catalyses efflux of divalent cations, provided that they have a unique architecture: the two charged moieties must be separated by a long linker. These drugs are exchanged for two protons despite the apparent inability of MdfA to exchange two protons for a single drug molecule. Our results suggest that these drugs are transported in two consecutive transport cycles, where each cationic moiety is transported as if it were a separate substrate. We propose that secondary transport can adopt a processive-like mode of action, thus expanding the substrate spectrum of multidrug transporters.

Design and development of novel mitochondrial targeted nanocarriers, DQAsomes for curcumin inhalation.

Curcumin has potent antioxidant and anti-inflammatory properties but poor absorption following oral administration owing to its low aqueous solubility. Development of novel formulations to improve its in vivo efficacy is therefore challenging. In this study, formulation of curcumin-loaded DQAsomes (vesicles formed from the amphiphile, dequalinium) for pulmonary delivery is presented for the first time. The vesicles demonstrated mean hydrodynamic diameters between 170 and 200 nm, with a ζ potential of approximately +50 mV, high drug loading (up to 61%) and encapsulation efficiency (90%), resulting in enhanced curcumin aqueous solubility. Curcumin encapsulation in DQAsomes in the amorphous state was confirmed by X-ray diffraction and differential scanning calorimetry analysis. The existence of hydrogen bonds and cation-π interaction between curcumin and vesicle building blocks, namely dequalinium molecules, were shown in lyophilized DQAsomes using FT-IR analysis. Encapsulation of curcumin in DQAsomes enhanced the antioxidant activity of curcumin compared to free curcumin. DQAsome dispersion was successfully nebulized with the majority of the delivered dose deposited in the second stage of the twin-stage impinger. The vesicles showed potential for mitochondrial targeting. Curcumin-loaded DQAsomes thus represent a promising inhalation formulation with improved stability characteristics and mitochondrial targeting ability, indicating a novel approach for efficient curcumin delivery for effective treatment of acute lung injury and the rationale for future in vivo studies.

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.