Uptake of a fluorescently tagged chloroquine analogue is reduced in CQ-resistant compared to CQ-sensitive Plasmodium falciparum parasites

Background Chloroquine (CQ) was the drug of choice for decades in the treatment of falciparum malaria until resistance emerged. CQ is suggested to accumulate in the parasite’s digestive vacuole (DV), where it unfolds its anti-malarial properties. Discrepancies of CQ accumulation in CQ-sensitive (CQS) and CQ-resistant (CQR) strains are thought to play a significant role in drug susceptibility. Analysis of CQ transport and intracellular localization using a fluorescently tagged CQ analogue could provide much needed information to distinguish susceptible from resistant parasite strains. The fluorescently tagged CQ analogue LynxTag-CQ™GREEN (CQGREEN) is commercially available and was assessed for its suitability. Methods IC50 values were determined for both CQ and CQGREEN in two CQS and two CQR Plasmodium falciparum strains. Buffer solutions with varying pH were used to determine pH-dependent localization of CQGREEN in infected red blood cells. Before CQS or CQR parasites were exposed to different pH buffers, they were pre-loaded with varying concentrations of CQGREEN for up to 7 h. Intracellular accumulation was analysed using live cell confocal microscopy. CQGREEN uptake rates were determined for the cytosol and DV in the presence and absence of verapamil. Results In CQS strains, twofold higher IC50 values were determined for the CQGREEN analogue compared to CQ. No significant differences in IC50 values were observed in CQR strains. Addition of verapamil reversed drug resistance of CQR strains to both CQ and CQGREEN. Live cell imaging revealed that CQGREEN fluorescence was mainly seen in the cytosol of most parasites, independent of the concentration used. Incubation periods of up to 7 h did not influence intracellular localization of CQGREEN. Nevertheless, CQGREEN uptake rates in CQR strains were reduced by 50% compared to CQS strains. Conclusion Although fluorescence of CQGREEN was mainly seen in the cytosol of parasites, IC50 assays showed comparable efficacy of CQGREEN and CQ in parasite killing of CQS and CQR strains. Reduced uptake rates of CQGREEN in CQR strains compared to CQS strains indicate parasite-specific responses to CQGREEN exposure. The data contains valuable information when CQGREEN is used as an analogue for CQ.

Background Plasmodium falciparum is the main causative agent of malaria, killing hundreds of thousands of people annually [1]. Resistance to anti-malarial drugs is widespread, and promising anti-malarial drugs that are effective and affordable for people with low income are slow in development. For decades, chloroquine (CQ) was the safest, most affordable and effective drug against malaria, saving the lives of millions of people until resistance emerged [2]. To date, its mode of action is still not fully understood. Some researchers suggest that CQ could have more than one intracellular target, making it more difficult for parasites to develop resistance [3][4][5].
To effectively use CQ as an anti-malarial treatment in areas where CQ resistance has been reported, it is imperative to gain more insight into its mechanism of action. The small size and uncharged form of CQ at neutral pH makes it difficult to use for molecular biological experiments to determine its accumulation in intracellular compartments or its affinity to other molecules.
Chloroquine transport studies have mainly been performed using radiolabelled CQ [6][7][8]. The great advantage of this method is that the intrinsic properties of CQ remain unaltered. However, it is not possible to study intracellular distribution and trafficking of radiolabelled CQ in intact parasites. Therefore, fluorescently labelled CQ analogues provide a new means to evaluate intracellular activity of this drug. To date, there are two fluorescently labelled chloroquine analogues commercially available: LynxTag-CQ ™ BLUE and LynxTag-CQ ™ GREEN (BioLynx Technologies, Singapore, Singapore). While the literature cites that CQ BLUE was mainly found to be fluorescent in the parasite cytosol [9][10][11], only one study has been published with CQ GREEN . This study showed CQ GREEN accumulation in the DV of CQ-sensitive (CQS) P. falciparum parasites and yeast-derived microsomes expressing the P. falciparum chloroquine resistance transporter (PfCRT) [12].
For this study, CQ GREEN was compared to unmodified CQ and investigated whether the fluorescently tagged CQ analogue can be used to obtain insight into intracellular CQ trafficking and localization. A better knowledge of the differences in CQ accumulation, distribution, uptake and efflux between CQS and CQresistant (CQR) strains is imperative to understand drug resistance. The findings show that, although CQ and CQ GREEN show comparable IC 50 values in CQS and CQR parasites, discrepancies were seen between CQ GREEN and unmodified CQ in their expected intracellular localization. A strong CQ GREEN fluorescence was mainly seen in the cytosol of both CQS and CQR strains.

Parasite strains and culture conditions
Two CQS (3D7, HB3) and two CQR (FCB, Dd2) strains were used for all experiments. Parasites were cultured continuously, as described by Trager and Jensen [13], with modifications. Briefly, parasites at 5% haematocrit were propagated in culture medium containing RPMI 1640 (Life Technologies, Burlington, ON, Canada) supplemented with 25 mM HEPES, 2 mM l-glutamine, gentamicin (20 µg/ml) (Life Technologies, Burlington, ON, Canada), 100 µM hypoxanthine (Sigma-Aldrich, Oakville, ON, Canada), and 0.5% AlbuMAX I (Life Technologies, Burlington, ON, Canada). Parasites were maintained at 37 °C with an atmosphere of 5% CO 2 , 3% O 2 and 92% N 2 . A + red blood cells were obtained from the Interstate Blood Bank (Memphis, TN, USA). Giemsa-stained blood smears were prepared daily to monitor parasite growth. For synchronization, parasites were treated with 5% d-sorbitol (BioShop Canada, Burlington, ON, Canada) for 10 min at 37 °C; sorbitol was removed and parasites were washed once before returning them back into culture.
Readouts of the assay were performed using the SYBR Green I detection method. For this, plates were thawed at room temperature and 100 µl 2× lysis buffer (20 mM Tris pH 7.5, 5 mM EDTA, 0.008% saponin, 0.08% Triton X-100, and 0.2 µl SYBR Green I/ml) was added to each well. Plates were incubated in the dark for at least 1 h. Fluorescence intensity was determined using a Synergy H4 plate reader (Fisher Scientific, Nepean, ON, Canada) with 485 nm excitation and 520 nm emission wavelengths. IC 50 values were determined by fitting concentration response curves with a custom-made procedure for IGOR Pro 6.2 based on a R script kindly provided by Le Nagard [18,19].

Fluorescence of CQ GREEN at varying pH
To determine if fluorescence intensity of CQ GREEN is altered at varying pH, buffer solutions were prepared ranging from pH 5.0-8.0 based on a modified Ringer's solution (122.5 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl 2 , 0.8 mM MgCl 2 , 11 mM d-glucose). Buffer solutions contained 10 mM MES for pH 5.0-6.5 or 10 mM HEPES for pH 7.0-8.0. In a 96-well plate, 100 µl buffer solutions of varying pH containing 1 µM CQ GREEN were prepared in triplicate. Fluorescence intensity was measured at 37 °C using the Synergy H4 fluorimeter (Bio-Tek, Winooski, VT, USA). Excitation spectra ranging from 400 to 520 nm were measured with fixed emission at 540 nm, and emission spectra ranging from 500 to 630 nm were measured with fixed excitation at 488 nm. Quantification was done using Microsoft Excel 2013.

Live cell imaging
Intracellular CQ GREEN accumulation at different concentration and pH was analysed in intact parasitized red blood cells. For this, 3D7 trophozoite stage parasites were incubated for 30 min at 37 °C with 25, 50, 500 nM or 2.5 µM CQ GREEN in Ringer's solution with pH 7.4 or buffer solutions with pH 7.2 or 5.2. Images were taken using a 488 nm argon laser (12.5 mW, 0.8%) on a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a water-corrected objective (C-apochromat 63×/1.20 W Korr M27). Emission range was set to 500-600 nm. Localization of CQ GREEN within the parasite under various pH conditions was determined using the ZEN 2010 software (Carl Zeiss MicroImaging, Oberkochen, Germany).
For long-term incubation with CQ GREEN , early trophozoite stage 3D7 parasites were incubated with 100 nM, 300 nM and 500 nM CQ GREEN for 7 h in culture medium at 37 °C, 3% O 2 , 5% CO 2 . Parasites were then transferred onto a microscope chamber and imaged using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany), a 63× water corrected objective (C-apochromat 63×/1.20 W Korr M27) and a 488 nm laser (12.5 mW, 0.8%). Emission range was set to 500-600 nm. A constant temperature of 37 °C was maintained during the measurements using a stage-top incubator (Tokai Hit, Shizuoka-ken, Japan). Images were analysed with the ZEN 2010 software (Carl Zeiss MicroImaging, Oberkochen, Germany).
To analyse CQ GREEN uptake of CQS and CQR strains, synchronized trophozoite stage parasites were washed in Ringer's solution and transferred onto a microscope chamber. Parasites were allowed to settle for 5 min, then the solution was aspirated and replaced with new Ringer's solution containing 500 nM CQ GREEN . If verapamil was added, parasites were preincubated with 1 µM VP for 15 min at 37 °C, then transferred onto a microscope chamber. For the time lapse measurement, 500 nM CQ GREEN were added to the Ringer's solution with or without 1 µM VP. Images were taken every 3 s for a time span of 500 s using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany) and a 63× water corrected objective (C-apochromat 63×/1.20 W Korr M27). Excitation was done using a 488 nm laser (12.5 mW, 0.8%), and an emission range from 500 to 600 nm. Regions of interest (ROI) were set for the parasite cytosol, DV, infected RBC cytosol and uninfected RBC. Fluorescence of ROIs was determined for each time point using ImageJ 1.47q (National Institutes of Health, USA). Uptake rates were calculated for the cytosol and DV during the saturation phase and analysed using IGOR Pro 6.2 by fitting influx to ƒ = y 0 + a(1 − e −bx ) and initial rates (v 0 ) to y = mx + b, as described previously [20]. Graphs were created using IGOR Pro 6.2.

IC 50 determination of CQ and CQ GREEN in CQS and CQR parasite strains
To evaluate the efficacy of the fluorescently tagged chloroquine analogue CQ GREEN , the half maximal inhibitory concentrations (IC 50 ) were determined using the SYBR Green I detection assay and a standardized calculation method for data analysis in two CQS and two CQR P. falciparum strains (Table 1) [19]. The two CQS strains 3D7 and HB3 had a CQ IC 50 of 24 ± 6 nM and 14 ± 1 nM, respectively. Compared to exposure to CQ, IC 50 values doubled in both CQ GREEN treated 3D7 (48 ± 3 nM, p = 0.02) and in HB3 parasites (

Fluorescence intensity of CQ GREEN is dependent on pH
pH is known to affect the fluorescence intensity and excitability of a molecule [21]. While some fluorophores have stable emission at different pH, others are more pH sensitive [22,23]. This must be considered when comparing fluorescence intensity of a fluorochrome in intracellular compartments with significantly different pH, as is the case for the parasite's cytosol (at approx. pH 7.1) and DV (at approx. pH 5.2) [24]. CQ GREEN consists of a chloroquine analogue tagged with a Bodipy fluorophore, which is relatively insensitive to solvent polarity and pH [25].
To determine if this fluorescently tagged CQ is pH dependent, CQ GREEN fluorescence was tested using buffer solutions containing HEPES or MES to obtain pH values ranging from 5.0 to 8.0. These buffer solutions were used to evaluate possible changes in fluorescence intensity seen in the parasite cytosol, having a physiological pH of 7.2, and the digestive vacuole, maintaining a pH of 5.2 [24]. Excitation spectra for CQ GREEN showed constant fluorescence intensity at a fixed emission of 540 nm (Fig. 1a). The CQ GREEN excitation peak was determined at 508 nm. Since live cell imaging will be performed using a 488 nm laser for excitation, CQ GREEN emission spectra were measured at different pH with a fixed excitation wavelength of 488 nm. CQ GREEN emission intensity peaked at 522 nm and increased 1.6-fold from 13,698 RFU at pH 7.5 to 22,419 RFU at pH 5.5 (Fig. 1b). Therefore, somewhat higher CQ GREEN fluorescence is emitted in acidic compartments compared to neutral compartments at equal intracellular CQ GREEN concentrations.

CQ GREEN accumulates in the parasite cytosol
The weak base properties of chloroquine and its diprotonation at low pH result in its high accumulation in the parasite's digestive vacuole [26]. Addition of a fluorescent group (here Bodipy) to chloroquine could alter the intracellular distribution of this molecule. In this study, the CQS strain 3D7 was treated with 25 nM, 50 nM, 500 nM and 2.5 µM CQ GREEN in Ringer's solution, or buffer solutions with pH 7.2 and pH 5.2, respectively. CQ GREEN uptake and intracellular distribution was monitored in 5 min intervals for a total of 30 min at 37 °C. Increasing CQ GREEN concentrations resulted in stronger fluorescence signals, independent of the buffer solution used (Fig. 2). The fluorescence signal was always stronger in the cytosol compared to the DV. Faint accumulation of CQ GREEN in the digestive vacuole was observed with all buffer solutions and CQ GREEN concentrations. Although CQ is expected to accumulate in the parasite's DV within minutes, addition of CQ GREEN was extended for up to 7 h in 3D7 parasites at 37 °C, 3% O 2 , 5% CO 2 , 92% N 2 . It was previously reported that incubation of the chloroquine analogue CQ BLUE at a 3 µM concentration for 8 h resulted in nonspecific localization throughout the parasite's cytosol, while accumulation of CQ BLUE in the DV was detected using a concentration of 300 nM [9]. Therefore, for this study, CQ GREEN concentrations ranging from 100 to 500 nM were used. Fluorescence signals obtained after 7 h incubation were low for all tested CQ GREEN concentrations (Fig. 3). Accumulation of CQ GREEN in the DV could be detected in very few parasites (approx. 5%) (Fig. 3b + d). Furthermore, accumulation of CQ GREEN in the DV was only seen in one parasite of a double-infected red blood cell (RBC) (Fig. 3b), Fig. 1 CQ GREEN excitation and emission spectra at varying pH. Solutions with varying pH ranging from 5 to 8, each containing 1 µM CQ GREEN were prepared in triplicate and measured using a fluorimeter. Temperature was set to 37 °C during measurements. a Excitation spectra with fixed emission at 540 ± 4.5 nm. b Emission spectra with fixed excitation at 488 ± 4.5 nm suggesting that CQ GREEN accumulation in the DV is not equally achieved in individual parasites. Moreover, the CQ GREEN fluorescence in the DV of the parasite in Fig. 3b is only 2.8-fold higher compared to the cytosol, which is much lower than expected.

Stability of CQ GREEN compound
Next, it was investigated if the Bodipy moiety of CQ GREEN is cleaved and remains in the cytosol while CQ reaches the DV, where it can unfold its anti-malarial properties. It was previously demonstrated that cleavage of fluorescent molecules through proteases changes the environment of the reactive center loop, resulting in a spectral shift of the fluorescence peak [27,28]. To evaluate if a spectral shift of the fluorescence peak occurs in CQ GREEN after exposure to parasite proteases, uninfected or 3D7-infected cultures with a parasitaemia of 2.5%, 5% or 10% were loaded with 500 nM CQ GREEN for 1 h. Parasites were then washed to remove excess CQ GREEN in the supernatant and were lysed before measuring fluorescence. Since live cells were loaded with the dye, any detected signal should be derived from CQ GREEN molecules taken up by the parasites. As a control, parasites were with free acid Bodipy-FL were analysed using the same conditions. The fluorescence peak for the free acid Bodipy-FL control was measured at 512 nm, while the peak for CQ GREEN was measured at 520 nm (Fig. 4). This fluorescence shift is large enough to differentiate between free Bodipy and Bodipy conjugated to CQ. Treatment of uninfected RBCs (uRBCs) and infected RBCs (iRBCs) with 500 nM Bodipy-FL for 1 h resulted in low fluorescence in all samples, indicating that Bodipy-FL is not readily accumulating in uRBCs or iRBCs. When parasites were treated with 500 nM CQ GREEN , a proportional increase in CQ GREEN fluorescence was observed with increasing parasitaemia from 0% (uRBCs) to 10% (iRBCs), as expected. The fluorescence peak was measured at 520 nm for CQ GREEN in Ringer's solution alone (control) and remained constant Long term incubation of CQ GREEN treated parasites. Early trophozoite stage 3D7 parasites were incubated with 100 nM, 300 nM or 500 nM CQ GREEN for 7 h in culture medium at 37 °C, 3% O 2 , 5% CO 2 , and then transferred onto a microscope chamber. Faint fluorescence accumulation of CQ GREEN in the DV could be detected in few parasites (approx. 5%). Experiments were done in triplicate. a-c Images of parasites exposed to 100 nM CQ GREEN for 7 h. d-f Images of parasites exposed to 300 nM CQ GREEN for 7 h. g-i Images of parasites exposed to 500 nM CQ GREEN for 7 h. Scale bar, 5 µm

CQ GREEN uptake differs in CQS and CQR strains
Most researchers focus on CQ accumulation in the DV, suggesting that enhanced CQ efflux from the DV through PfCRT confers drug resistance [29,30]. CQ uptake in intact iRBCs has mainly been described as passive diffusion and subsequent accumulation in the DV due to the drug's weak base properties [31,32]. Molecular mechanisms that influence CQ uptake and, therefore, reduce the drug influx in CQR strains compared to CQS strains may have gone unrecognized. CQ GREEN offers the possibility to measure uptake in live cells. Although CQ GREEN mainly accumulates and fluoresces in the parasite's cytosol, with weak fluorescence in the DV, it was still possible to measure its uptake rate in both compartments. The rate of CQ GREEN uptake was analysed in two CQS strains (3D7 and HB3) and compared with two CQR strains (Dd2 and FCB). Fluorescence was measured from the parasite cytosol, DV, iRBC cytoplasm and uRBC. No increase in CQ GREEN fluorescence beyond background levels was observed in the cytoplasm of infected or uninfected RBCs (Fig. 5a). There was a rapid increase in fluorescence in the parasite cytosol and DV in the initial 150 s, followed by a slower, almost linear CQ GREEN uptake. This is in agreement with previous studies where they showed that CQ uptake can be separated into a short period of very rapid uptake (< 30 s) followed by a long, slower phase [33], and addition of verapamil did not influence CQ uptake during the initial phase [34].
CQ GREEN uptake rate for the parasite cytosol was approx. twofold higher in CQS strains compared to CQR strains (Fig. 5b). For the DV, CQ GREEN uptake rates were approx. 2.5-fold higher in CQS strains compared to CQR strains. CQ GREEN uptake rates between CQS and CQR strains were statistically highly significant for both the cytosol (p < 0.0001) and DV (p < 0.0001). Increased CQ GREEN uptake rates for the DV of CQS strains suggest that active transport of CQ into the DV occurred in addition to diffusion, which is absent in CQR strains. As expected, addition of verapamil did not influence CQ GREEN uptake in the parasite cytosol or DV in all CQR strains tested.

Discussion
The exact mechanisms responsible for chloroquine resistance have eluded researchers for decades. It has long been described that CQR strains accumulate two to sevenfold less CQ than CQS strains [35]. Nevertheless, CQR strains are still able to tolerate higher intracellular CQ concentrations than CQS strains before irreversible cell damage occurs [3,7,33,36]. Researchers have advocated that inhibition of haemozoin formation by CQ is not sufficient to explain its effect on parasite killing [37]. Furthermore, a possible role of CQ in the parasite's cytosol has also been proposed [5]. A fluorescently labelled CQ analogue could provide insight into the intracellular distribution of CQ that is not attributed to diffusion alone. This study set out to examine CQ uptake in live parasites using CQ GREEN .
IC 50 values were compared in CQS and CQR strains after exposure to CQ or CQ GREEN . A decrease in the efficacy for CQ GREEN may suggest that the CQ analogue does not reach its site of action as efficiently as its native form or binds to its target less efficiently. Calculated IC 50 values in CQS strains showed that CQ was twice as effective as CQ GREEN in parasite growth inhibition. In comparison, similar IC 50 values for CQ and CQ GREEN were determined in CQR strains. In a previous study by Loh and colleagues [12], IC 50 values in 3D7 were approx. fivefold higher for CQ GREEN compared to CQ, and nearly doubled in the CQR strain K1. Thus, the parasites used for this study seemed to be more sensitive to CQ GREEN exposure than reported in earlier publications. In both studies, drug resistance could be reversed by chemosensitizers such as verapamil, suggesting that CQ GREEN 's mode of action is similar to CQ.
Although CQ GREEN seemed slightly less effective than CQ in the IC 50 assays for CQS strains, its Bodipy fluorescent tag makes it suitable for live cell imaging. This was confirmed by fluorometric readings, where a strong fluorescence emission signal for CQ GREEN was measured at a 488 nm excitation wavelength. Spectral scans showed a strong fluorescence signal at the measured pH spectrum ranging from pH 5-8 with a moderate increase in fluorescence at acidic pH.
Accumulation of CQ GREEN fluorescence was found in only 5% of the parasite's DV compared to the cytosol during live cell imaging at any of the tested CQ GREEN concentrations, ranging from 25 nM to 2.5 µM. Considering that the CQ GREEN IC 50 was determined at 24 nM, any of the tested concentrations for live cell imaging would have been sufficient to kill the parasites. If CQ and CQ GREEN had its primary target in the parasite's DV, as suggested by several studies [38][39][40][41][42], then we would have expected a higher CQ GREEN fluorescence in the DV.
Protonation of CQ, or its analogues, influence their membrane permeability and thus their intracellular distribution [43,44]. Treatment of intact P. falciparuminfected erythrocytes with pH buffered solutions ranging from pH 5-8 showed that protonation did not play a role in the intraparasitic CQ GREEN distribution. One study used microsomes to resemble events occurring in the DV and reported accumulation of CQ GREEN fluorescence in these microsomes [12]. They did not use intact parasites in their experiments and the data does not support their assumption that CQ GREEN accumulates equally well in the DV of live parasites as it does in microsomes [12].
Fluorometric measurements were performed to verify CQ GREEN fluorescence at low pH. Slightly higher fluorescence was observed for CQ GREEN at acidic pH (5.0) compared to neutral pH (7.0). Therefore, if CQ GREEN accumulated in the DV, a fluorescent signal would be expected. The absence of fluorescence in the DV suggests that CQ GREEN does not reach the DV. Two reasons for this are possible. First, cleavage of the fluorescent Bodipy moiety from CQ may already occur in the parasite cytosol. This would allow CQ to accumulate in the DV, while the Bodipy moiety remains in the cytosol. In this case, the fluorescence signal does not specify the subcellular localization of CQ but rather only the fluorochrome. Second, the Bodipy moiety alters the intrinsic properties of the substrate and, therefore, prevents its accumulation in the DV. A simple way to elucidate whether the Bodipy moiety gets cleaved is determining fluorescence properties of conjugated versus free acid Bodipy-FL. Since there is a fluorescence shift between free acid Bodipy-FL alone (peak at 512 nm) and the Bodipy-tagged CQ GREEN (peak Trophozoite stage parasites were incubated with CQ GREEN and fluorescence was recorded every 3 s. a Fluorescence was measured in the parasite cytosol, DV, infected RBC (iRBC) cytoplasm and uninfected RBC (uRBC). No accumulation of CQ GREEN was observed for the uRBC and iRBC cytoplasm. Uptake rates were calculated for the parasite cytosol and DV during the saturation phase (black curve), which was followed by a linear uptake phase. b CQR strains showed a slower CQ GREEN uptake rate compared to CQS strains in both the cytosol and the DV of the parasite. Addition of verapamil (VP) did not influence the CQ GREEN uptake rate in CQR strains. Experiments were done in triplicate at 520 nm), cleavage of the Bodipy moiety from CQ GREEN during the cellular uptake would result in a fluorescence shift to 512 nm when parasites are treated with CQ GREEN . For this study, intact P. falciparum-infected RBCs were treated with CQ GREEN to analyse the potential cleavage of the Bodipy moiety. Although a proportional increase in fluorescence with higher parasitaemia was measured, no shift in the fluorescence peak was observed. Thus, CQ GREEN remained intact and functional in live parasites prior to parasite lysis for fluorescence measurements.
Despite the unexpected localization of CQ GREEN fluorescence mainly in the parasite cytosol and to a lesser extent in the DV, uptake rates were analysed in two CQS and two CQR strains. Although the fluorescent signal obtained from the DV was weak compared to the cytosol, it was sufficient to calculate the uptake rate for this compartment. CQS strains had approx. twofold higher CQ GREEN uptake rates for the cytosol and 2.5-fold higher rates for the DV compared to CQR strains. Addition of verapamil to the CQR strains did not alter CQ GREEN uptake rates, suggesting that PfCRT does not play a role in its uptake. This is consistent with the hypothesis that PfCRT is involved in CQ efflux from the DV and is not involved in its uptake [20]. Decreased CQ uptake rates in CQR strains compared to CQS strains may be explained through reduced availability or affinity to an intracellular target, such as free haem [4]. Thus, accumulation of CQ in the DV may contribute to parasite killing, but additional drug targets in the parasite cytosol could also play a role.

Conclusion
Live cell imaging using fluorescently tagged anti-malarial drugs provides a great tool to elucidate their intracellular localization and give insights into their uptake or efflux rates. The challenge using fluorescently labelled CQ analogues is their sensitivity to pH and altered intracellular localization, compared to native CQ. Finding fluorescent groups that are more stable at different pH and do not influence the diffusion properties of the protein would provide a powerful tool in studying the activity of antimalarial drugs. Alterations between drug-sensitive and -resistance strains can be closely monitored to enhance our understanding of resistance mechanisms. Moreover, affinity of CQ, or its analogues, to cytosolic proteins may direct research on new anti-malarial drug design away from the DV and increase the focus on cellular pathways in the parasite cytosol.