Chloroquine: a brand-new scenario for an old drug

Chloroquine (CQ), most commonly known as an anti-malaria drug, has a long history of use in the prevention and treatment of malaria, amebiasis and further found effective in some autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus [1,2]. In recent years, the drug has attracted scientists’ interest as an anti-cancer agent used as a monotherapy or adjunct therapy in several types of cancer.

Different mechanisms have been demonstrated in studies regarding the anti-cancer properties of CQ. The drug has proven to act as an autophagy inhibitor where autophagy is linked with cancer initiation and development through promoting survival of starving cancer cells, or degrading apoptotic mediators [3,4]. Another proposed anti-cancer mechanism for the drug is related to its role in the inhibition of the TLR9/nuclear factor kappa B signaling pathway [5]. High expression of TLR9 in lung cancer cells is associated with poor prognosis [6]. Moreover, both in vitro and in vivo research showed CQ can stabilize p53 protein and increase transcription of p53-related pro-apoptotic genes [7,8]. The drug has also been proposed to affect glutamate dehydrogenase activity, which has a strong correlation with cancer growth and homeostasis [9,10].

In recent years, the advent of nanotechnology has considerably changed the available options for the cancer cure. Nanotechnology offers treatments with enhanced anti-cancer effects with minimal damage to the normal cells. Drugs and genes of interest can be incorporated within a nano-scale carrier or absorbed onto the surface of a nano vehicle to be specifically delivered into the site of interest. However, the practical application of many delivery systems is hardened by the effective cellular uptake [11,12].

Endocytosis is the primary pathway for cellular entry and the dominant mechanism for uptake of the drug/gene-loaded nanoparticles. During the endocytosis process, nanocarriers to be internalized are surrounded by a small piece of plasma membrane, which then invaginates to form a vesicle containing the entered substances. The most accepted model of endocytosis proposes that the internalization is followed by the formation of early endosomes, late endosomes and, eventually, lysosomes [13]. The early endosome containing the introduced materials ripens to become the late endosome in which the interior environment becomes acidic causing physicochemical change in the therapeutic agent. Afterward, lisosomes join the endosomes to digest the trapped material resulting in irreversible damage and ineffectiveness of the treatment. Hence, endosomal escape can be thought as the critical bottleneck in effective drug/gene delivery.

Attempts have been made to find a solution to facilitate the endosomal escape of therapeutic agents and several options have been proposed to interfere with the natural process of endosomes acidification during endocytosis. Viruses, as one of the first found ones, disrupt the endosome membrane through various mechanisms dependent on the type of the employed virus as the pH falls. Influenza and adenoviruses act through generating fusion peptides and hydrophobicity-induced membrane disruption, respectively. With that in mind, application of viruses is associated with safety concern and complicated manufacturing process [14]. Peptides such as KALA, GALA and histidine are another available option, however price and long-term stability are their serious pitfalls limiting their practical application [15,16].

In addition to its excellent role in malaria care and the potentials in cancer treatment, CQ has been the focus of research for its contribution in endosomal escape process. CQ acts its function as a molecule increasing endosomal escape through several proposed mechanisms: (1) it has a basic nature and as a weak base efficiently enters the cell, sequesters the available H+ by becoming protonated itself, accumulates in acidic endosomes and increase the pH of the within environment which in turn inhibits hydrolytic activity of lysosomal enzymes such as proteases and nucleases. Moreover, the increased pH deactivates P-gp in lysosomal membrane and finally leads to the drug efflux from lysosomes [17,18], (2) it can act as an osmotic agent increasing the osmotic pressure of the endosomes resulting in swelling and rupture of endosomal membrane [16]. When CQ enters the lysosome it causes a substantial increase in the intralysosomal pH in which, as evidences demonstrate, most lysosmoal enzymes are not able to function [19]. At neutral pHs, CQ is unportonated and relatively lipophilic, thus easily penetrate the cells and endosomes. On entering the endosome, in the presence of acidic environment CQ changes to its protonated form which is too polar to readily diffuse across the endosome membrane, and thus contributes to the endosomal escape thorough increase in osmotic pressure which in turn can rupture the endosomes [20]. Additionally, in case of gene delivery, another mechanism is involved in the enhancement of the overall delivery efficiency. CQ is reported to play a role in polyplexes unpacking within the cytosol through its ability to bind nucleic acids by its quinoline moiety and the positive charges in its protonated form resulting in improvement in gene transfection [21]. The amount of CQ in polyplexes in the delivery of anti-miR-210 into the cancer cells, was associated with the efficient miRNA delivery as higher content of chloroquine caused increased endosomal escape [22]. In another study conducted by Yang Hu et al., in the presence of 75 μM concentration of CQ, polyethyleneimine (PEI)/pDNA polyplex at its optimal N/P showed 2.2 times higher transfection efficiency than PEI-pDNA polyplex only, which was attributed to the enhanced buffering capacity of PEI in the presence of CQ. The same observation was made in case of pDNA delivery using dextran–peptide vector at distinct proportion in which CQ concentration of 75 μM significantly enhanced the transfection efficiency [23]. CQ is reported to increase expression levels of CD22 following receptor-mediated endocytosis by preventing acidification of the endosomes resulting in the prevention of the receptor degradation and thus enabling higher amounts of CD22 to return back to the surface of the cell [24]. Intravenous injection of CQ combined with chemotherapy drugs resulted in significantly prolonged survival time in mice. Furthermore, encapsulated doxorubicin-CQ and paclitaxel-CQ in methoxy poly(ethylene glycol)-poly(l-lactic acid) (MPEG-PLA) nanoparticles demonstrated enhanced tumor inhibition and prolonged survival time [25]. The same result was observed in a recent study in which 25 μM concentration of CQ increased the functional delivery of extracellular vesicles cargos [26]. Retinal gene delivery using chloroquine-containing noisome showed a significant impact of CQ on effective gene delivery to rat retina while the final concentration of CQ of 25 μg/ml did not induce any significant cytotoxicity [27]. Similar results were reported in other studies using CQ as an endosomal disrupting molecule [22,28–30].

Endosomal escape remains one of the serious challenges in nucleic acid therapy. Among current solutions for enhanced endosomal escape, chloroquine is one of the promising candidates by being inexpensive, physicochemically stable and effective. The drug also possesses direct and indirect anti-cancer effects through several mechanisms. Moreover, in some chemoresistant cancer models, CQ might be an effective therapeutic option by inhibiting the lysosomal functions [31]. Although, many studies argues against the clinical use of the drug in cancer cure mainly due to its toxicity, the needed and studied doses for endosomal escape purposes are not high enough to be a concern for the successful therapy. The advantages offered by CQ make it a multipurpose therapeutic option in cancer treatment and the research is ongoing upon the practical use of CQ in drug/gene delivery in vitro and in vivo brightening the hope for brand-new solutions against cancer in the near future.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Funding

This paper was not funded.