Nanoencapsulation increases quinine antimalarial ...

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ARTICLE IN PRESS International Journal of Antimicrobial Agents xxx (2009) xxx–xxx

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Nanoencapsulation increases quinine antimalarial efficacy against Plasmodium berghei in vivo Sandra Elisa Haas a , Clarissa Cassini Bettoni b , Laura Kausburg de Oliveira b , Sílvia Stanisçuaski Guterres a,b , Teresa Dalla Costa a,b,∗ a b

Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul, Brazil Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Brazil

a r t i c l e

i n f o

Article history: Received 7 December 2008 Accepted 13 February 2009 Keywords: Quinine Nanocapsules Plasmodium berghei Antimalarial efficacy Pharmacokinetics Erythrocyte partition coefficient

a b s t r a c t The aims of this work were to develop quinine (QN)-loaded nanocapsules, to evaluate their efficacy in vivo and to determine their pharmacokinetics and erythrocyte partition coefficient. Plasmodium bergheiinfected Wistar rats were used to evaluate the efficacy of QN-loaded nanocapsules using different dosing regimens. Pharmacokinetics was evaluated after intravenous administration of free or nanoencapsulated QN (25 mg/kg) to infected rats. The QN partition coefficient into P. berghei-infected erythrocytes was evaluated. QN-loaded nanocapsules presented an adequate particle size (176 nm), narrow particle distribution (0.19), negative zeta potential (−18 mV) and high drug content and encapsulation efficiency. Intravenous administration of QN-loaded nanocapsules at 75 mg/kg/day to infected rats resulted in 100% survival, representing an almost 30% reduction compared with the free QN effective dose (105 mg/kg/day). The pharmacokinetic parameters of nanoencapsulated QN were not significantly different from those determined for free drug (˛ = 0.05). The QN partition coefficient into infected erythrocytes doubled (6.25 ± 0.25) when the drug was nanoencapsulated compared with the free drug (3.03 ± 0.07). Therefore, nanoencapsulation increased the interaction between QN and the erythrocyte and this mechanism is responsible for the drug’s increased efficacy when nanoencapsulated. © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction Malaria is the most important endemic parasitic infection in humans, accounting for more than 1 million deaths per year, especially in Africa [1]. Besides acquired immune deficiency syndrome (AIDS) and tuberculosis, malaria is one of the world’s biggest public health problems. A total of US$38–45 billion will be spent from 2006 to 2015 for the diagnosis and treatment of malaria [2], mainly in underdeveloped countries, which are the most affected by this disease [3]. Malaria parasites are transmitted by female Anopheles mosquitoes. The sporozoites are inoculated into the human host bloodstream, infect the liver cells and mature into merozoites (exoerythrocytic schizogony), which, after rupture of the liver cell, continue their cycle in red blood cells (erythrocytic phase). The erythrocytic phase is responsible for the clinical manifestations

∗ Corresponding author. Present address: Universidade Federal do Rio Grande do Sul, Faculdade de Farmácia, Programa de Pós-Graduação em Ciências Farmacêuticas, Av. Ipiranga 2752, Porto Alegre, RS, 90610-000, Brazil. Tel.: +55 51 3316 5418; fax: +55 51 3316 5437. E-mail address: [email protected] (T. Dalla Costa).

of the disease [1]. Among the four species of malaria parasite that infect humans, Plasmodium falciparum is the most dangerous because it causes severe malaria that presents unacceptable levels of morbidity and mortality; 45–50% of Plasmodium infections are caused by this species. The first line of treatment for all malaria types is chloroquine, although nowadays treatment failure with this drug is increasing everywhere owing to the development of resistance. In India, for instance, the rate of P. falciparum resistance to chloroquine is >10% [1,4]. This scenario had led to the revival of drugs such as quinine (QN), a second line of choice. QN acts in the erythrocytic phase against all types of Plasmodium. Although QN treatment is generally effective against chloroquine-resistant falciparum malaria [1], its use is limited by its narrow therapeutic index, cardiotoxicity and the development of cinchonism syndrome characterised by neurological, cardiovascular and gastrointestinal toxicity as well as hypoglycaemia and hypersensitivity reactions [5,6]. In this scenario, an improvement of the QN therapeutic index could play an important role in the treatment of drug-resistant malaria. Delivery systems such as liposomes and nanoparticles have been studied for intracellular infections because they are able to deliver the drug to the specific target in the human body where the parasite

0924-8579/$ – see front matter © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2009.02.024

Please cite this article in press as: Haas SE, et al. Nanoencapsulation increases quinine antimalarial efficacy against Plasmodium berghei in vivo. Int J Antimicrob Agents (2009), doi:10.1016/j.ijantimicag.2009.02.024

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is located, such as tissues (spleen and liver) and cells (macrophages and Kupffer cells) [7–12]. Nanocapsules are a specific type of nanoparticle composed by an oil core surrounded by a polymeric membrane, stabilised by surfactants. They have been used successfully for the following purposes: protection of drugs against inactivation in the stomach and improvement of bioavailability [13]; protection of the mucosa from drug toxicity [14]; improvement in the therapeutic index of drugs [11,15,16]; control of drug release [17]; and reduction of drugs side effects [13,18]. In this context, nanocapsules containing Polysorbate 80 can be used as long circulation delivery systems, as they remain in the blood for a long time before being taken up by cells of the mononuclear phagocytic system [19]. Different antiparasitic drugs have been loaded into nanocapsules with the aim of reducing drug toxicity, such as halofantrine [12,18], or to increase the therapeutic potential of drugs such as primaquine [10], atovaquone [7,8,11] and halofantrine [12]. From these reports, only halofantrine-loaded nanocapsules were evaluated in vivo for the treatment of malaria [12]. Halofantrine-loaded poly(d,llactide) (PLA) nanocapsules showed an activity similar to or better than the activity observed for the drug solution when administered as single intravenous (i.v.) doses of 1–100 mg/kg to severely P. berghei-infected mice. Halofantrine-loaded nanocapsules showed an area under the curve (AUC) more than six-fold higher than that observed after the administration of drug solution. This result was related to the faster control of parasitaemia and similar or higher survival rates obtained after nanocapsule administration. The toxic effect of halofantrine PLA nanocapsules on heart rate was reduced in 58% and 75% of rats following 100 mg/kg and 150 mg/kg i.v. dosing, respectively, in comparison with halofantrine administered in solution. This finding showed that nanoencapsulation reduces QT interval prolongation on the electrocardiogram in rats, suggesting that a modification of drug distribution occurred using nanoparticles [18]. QN encapsulated in transferrin-conjugated solid lipid nanoparticles has been investigated in relation to delivery to the brain. Nanoencapsulation promoted an increase in drug penetration in this tissue [20]. The aims of this work were to characterise nanocapsules containing QN prepared with poly(␧-caprolactone) and Polysorbate 80 and to evaluate their efficacy in vivo using a Plasmodium bergheiinfected Wistar rat model as well as QN pharmacokinetics after a single i.v. dose. The ability of nanoencapsulation to interfere with the QN partition coefficient in erythrocytes was also studied. 2. Materials and methods 2.1. Drugs, adjuvants and solvents Poly(␧-caprolactone) (PCL) (65 000 MW), QN base and QN hydrochloride were purchased from Sigma–Aldrich (St Louis, MO), soy phosphatidylcholine (Epikuron 170® ) was obtained from Lucas Meyer (Hamburg, Germany), Polysorbate 80 was obtained from Delaware (Porto Alegre, Brazil) and Miglyol 810® was purchased from Hulls (Puteaux, France). MilliQ® water was used for highperformance liquid chromatography (HPLC) analysis. All other chemicals and solvents were HPLC or analytical grade. 2.2. Preparation and characterisation of quinine-loaded nanocapsules QN-loaded nanocapsules were obtained by interfacial deposition of PCL [21]. Briefly, the organic phase consisted of PCL (0.1 g), Epikuron 170® (0.076 g), Miglyol 810® (0.33 mL) and QN free base (0.2 g) dissolved in acetone (27 mL). This organic phase was added under moderate magnetic stirring to an aqueous solution (53 mL)

containing Polysorbate 80 (0.076 g). After precipitation, the acetone and part of the water were removed by evaporation under reduced pressure and the final volume was completed with water to 10 mL (2.0 mg/mL of QN). Unloaded nanocapsules were prepared in the same manner, omitting the drug. Nanocapsule size and polydispersity were measured by photon correlation spectroscopy. For these measurements, 20 ␮L of each suspension was diluted with 10 mL of MilliQ® water. Measurements were carried out at room temperature using a Zetasizer® Nano ZS3600 (Malvern Instruments, Westborough, MA). The zeta potentials were determined in samples diluted in 1 mM NaCl aqueous solution. The pH values were determined using a potentiometer (Digimed, Porto Alegre, Brazil). QN nanocapsule concentration was assayed by HPLC using a validated method. A Shimadzu® HPLC system (Shimadzu Corp., Kyoto, Japan) was coupled with a fluorescence detector. The excitation and emission wavelengths were set at 350 nm and 450 nm, respectively. A Waters® ␮Bondapak C18 column (Waters Corp., Milford, MA) (3.9 × 300 mm) and C18 pre-column (37–50 ␮m) were used. The mobile phase consisted of phosphate buffer pH 2.2:acetonitrile:phosphoric acid 1 M:tetrahydrofurane:triethylamine (46:3:2:1:0.8, v/v/v/v/v). QN entrapment in the nanocapsules was determined after ultrafiltration–centrifugation (Ultrafree-MC 10 000 MW; Millipore Corp., Billerica, MA) and total QN was determined after dissolution of nanocapsules with acetonitrile. The entrapped QN was calculated from the difference between the total and free drug concentrations [14]. The formulations were stored at room temperature and at prefixed days (1, 4 and 7) physicochemical characterisation was carried out according to the techniques described above. All the measurements were performed in triplicate using three different formulations. 2.3. Antimalarial efficacy in Plasmodium berghei-infected rats Five-week-old (90–110 g) male Wistar rats (n = 7 per group) were used in a protocol as described previously [22]. On Day 0, rats were infected by the i.v. route with 108 P. berghei-infected erythrocytes from mice in 0.4 mL of saline. The animals were treated from the 7th to the 9th day post infection by i.v. injection with different doses of QN every 8 h: QN free base at 10, 25 and 35 mg/kg; and QN-loaded nanocapsules at 10, 20 and 25 mg/kg. Control groups received 0.6 mL of unloaded nanocapsules or saline solution. Thin blood films were made from the tail blood of all animals every 2 days following infection to determine parasitaemia. The percentage of infected erythrocytes was determined in blood films fixed with methanol and stained with Giemsa after at least 1000 red cells were counted in five microscopic fields (oil immersion, 1000× magnification) [23]. Parasitaemia levels and mortality were monitored up to 4 weeks following inoculation. If the blood films were free of Plasmodium after at least 200 red cells were checked, they were considered cured but were kept under observation to monitor eventual relapses. 2.4. Pharmacokinetic evaluation Pharmacokinetic evaluation of QN (free base or nanoencapsulated form) was conducted in P. berghei-infected Wistar rats with 5–14% parasitaemia levels (n = 5 or 6 per group). The infection was produced in the same manner described previously. Both groups received a single 25 mg/kg i.v. bolus dose of QN hydrochloride solution in water or QN-loaded nanocapsules, injected into the lateral tail vein. The dose was chosen based on the effective dose determined in the pharmacodynamic experiments (total daily dose of 75 mg/kg divided in three administrations).



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Blood samples were collected by lateral tail vein puncture into heparinised tubes at predetermined times (10, 30, 60, 90, 120, 180 and 240 min) after i.v. administration. Plasma was separated by centrifugation at 6800 × g at 21 ± 1 ◦ C for 10 min and quantified by HPLC. A rapid HPLC analytical method was adapted [24] and validated for the determination of QN in rat plasma. Analysis was performed using a Waters® C18 ␮Bondapak column (3.9 × 300 mm) and elution with water:acetonitrile (87:11, v/v) containing 2% triethylamine (pH 2.7). The analyte was monitored using a fluorescence detector with excitation and emission wavelengths set at 350 nm and 450 nm, respectively. Calibration curves in spiked plasma were linear over the concentration range 0.025–2.0 ␮g/mL, with a determination coefficient of >0.99. The accuracy of the method was within 5%. Intraday and interday relative standard deviations were ≤15.4% and ≤3.4%, respectively. Pharmacokinetic characterisation of QN in plasma was performed using non-compartmental analysis [25] with the aid of Excel® v. 2000 software (Microsoft, Chicago, IL). 2.5. Quinine partition coefficient into erythrocytes Blood from P. berghei-infected rats with a mean parasitaemia of 8.3 ± 2.4% was used. The blood was centrifuged at 6800 × g at 21 ± 1 ◦ C for 10 min and the plasma and buffy coat were discarded. The remaining red cells were washed three times with 5% glucose solution (pH 7.4) [26]. Red blood cells were re-suspended in the same medium and the haematocrit was adjusted to 0.48. Free or nanoencapsulated QN was added to the red blood cell suspension to obtain a final concentration of 5 ␮g/mL. The time to reach equilibrium between QN in solution and that bound to erythrocytes was experimentally determined (data not shown). After 30 min incubation at 37 ± 1 ◦ C the samples were centrifuged and the supernatant was quantified by HPLC. The sediment was haemolysed with distilled water (1:1, v/v) and was also quantified. The partition coefficient of QN (D) was determined by the equation [27]: D=

As − [Csup × Vs × (1 − H)] H × Vs × Csup

where As is the drug concentration added to the medium (5 ␮g/mL), Csup is the drug concentration in the supernatant, Vs is the final suspension volume and H is the haematocrit. 2.6. Statistics Values are expressed as mean ± standard deviation. The value of each characteristic evaluated during the formulation stability study was compared with the values determined on the first day of analysis using Student’s t-test (˛ = 0.05). Statistical comparison of the pharmacokinetic parameters determined for treated and control groups was performed with Student’s t-test assuming unequal variance (˛ = 0.05). 3. Results and discussion 3.1. Physicochemical characteristics and stability of quinine-loaded nanocapsules The nanocapsule suspension containing QN presented a macroscopic homogeneous appearance like a milky bluish opalescent fluid (Tyndall effect), as previously reported for other nanocapsule formulations prepared by interfacial polymer deposition [28]. The nanocapsules showed a high drug content (98.6 ± 2.8%) and an encapsulation efficiency of 95.3 ± 0.4%. These findings can be explained by the lipophilicity of QN in its basic form (octanol/water partition coefficient of 3.4) [29], which presents low water affinity

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Table 1 Physicochemical characteristics of quinine (QN)-loaded nanocapsules (2 mg/mL) after storage at room temperature for up to 7 daysa . Parameter

Day of analysis 1

Drug content (%) pH Polydispersion index Zeta potential (mV) Particle size (nm) a

98.6 8.4 0.19 −18.0 176

4 ± ± ± ± ±

2.8 0.02 0.04 2.2 8

105.3 8.3 0.12 −16.9 201

7 ± ± ± ± ±

4.7 0.06 0.02 2.3 7

96.5 7.3 0.26 −16.5 164

± ± ± ± ±

4.9 0.05 0.04 1.2 7

Results expressed as mean ± standard deviation (n = 9).

and high solubility in caprylic/capric triglycerides that constitute the nanocapsule core. Evaluation of the stability of colloidal suspensions over time is essential because such systems are thermodynamically unstable [14]. The QN content, particle size, zeta potential, polydispersion index and pH were not significantly altered during the investigated period, demonstrating that QN-loaded nanocapsules were stable for 7 days (Table 1). The particle size varied from 176 ± 8 nm to 164 ± 7 nm and the polydispersion from 0.12 ± 0.02 to 0.26 ± 0.04. The particle size was not affected by QN encapsulation because the unloaded nanocapsules exhibited a similar mean diameter of 160 ± 2 nm (polydispersion of 0.16). QN-loaded nanocapsule size was in the range of 200 nm and could be considered suitable for in vivo administration via the i.v. route [30]. Zeta potential values varied between −18.0 ± 2.2 mV and −16.5 ± 1.2 mV. These negative values are due to the presence of PCL and Epikuron 170® in the formulation, as has already been described [28,31]. In summary, QN-loaded nanocapsules were successfully obtained with adequate physicochemical properties for i.v. administration and they were stable over 7 days. For the in vivo experiments, the nanocapsule suspension was prepared and characterised 2 days before its utilisation. 3.2. Antimalarial activity in Plasmodium berghei-infected rats Plasmodium berghei is one of the malaria parasites that infect mammals but not humans. This parasite causes an infection in rodents similar to that produced by Plasmodium species that infect humans. Thus, P. berghei-infected rodents are a suitable model for studying malaria because the infection presents structural, physiological and life cycle analogies with the human disease [32]. In the present investigation, the antimalarial activity of QNloaded nanocapsules was evaluated in Wistar rats infected with P. berghei and treated with different doses of the drug on Days 7–9 after inoculation. In parallel with each experiment, a non-treated group was evaluated. Table 2 shows the results of cure, mortality and peak parasitaemia for all groups investigated. The dose of 30 mg/kg/day of free QN avoided evolution of the infection and extended the period of survival of the rats compared with the control groups treated with saline or unloaded nanocapsules. However, all of the animals in these three groups died between 13 days and 19 days post inoculation, showing parasitaemia levels >40% (Table 2). There was no significant difference in the death profile and parasitaemia levels of animals treated with the unloaded nanocapsules and saline solution, indicating that the unloaded nanoparticles did not contribute to the antimalarial effect. When the animals were treated with free QN at a dose of 75 mg/kg/day, only 42% survival was observed. Cure of all animals was verified only when they were treated with 105 mg/kg/day. For this group, the decrease in parasitaemia took place after the end of treatment, on the 9th day post infection (Fig. 1). On the other


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Table 2 Efficacy of free quinine (QN) and QN-loaded nanocapsules (2 mg/mL) against Plasmodium berghei malaria in experimentally infected Wistar rats. Animals were treated intravenously by administration of drug doses every 8 h on Days 7–9 post inoculation. Group (n = 7 per group)

Total daily dose (mg/kg)

Parasitaemia peak (%)

Mortality (days)

Cure (%)

Free QN

30 75 105

43.7 ± 3.8 38.8 ± 38.4 10.9 ± 4.3

15–19 17–19 –

0 42 100

QN-loaded NCs

30 60 75

51.8 ± 19.8 14.6 ± 16.2 11.3 ± 4.8

13–19 17–21 –

28.6 85.7 100

Control groups

Saline Unloaded NCs

61.5 ± 20.8 46.2 ± 7.2

13–17 13–17

0 0

NC: nanocapsules.

hand, nanoencapsulated QN showed an increase in animal survival of almost 30% when the 30 mg/kg dose was used in comparison with administration of the same dose of free QN. QN-loaded nanocapsule doses of 60 mg/kg/day and 75 mg/kg/day led to cure levels of 86% and 100%, respectively. The effective QN doses, free or nanoencapsulated, cured all malaria-infected rats in the same post-inoculation period of time (15 days and 19 days). QN nanoencapsulation reduced the effective dose by almost 30%, from 105 mg/kg/day to 75 mg/kg/day, leading to lower QN tissue exposure and, probably, to less neurological and cardiovascular toxicity, especially considering that these are dose-dependent effects [33]. Furthermore, for QN-loaded nanocapsules the decrease in parasitaemia levels began on the first day of treatment (7th day post infection), in contrast to the free drug for which the decrease in parasitaemia began only at the end of treatment (9th day post infection) (Fig. 1). Indeed, as demonstrated for QN nanocapsules in the present study, nanostructured systems have been reported to reduce the effective dose and increase the survival for different drugs used to treat parasitic infections [11,12,16]. The increase in survival associated with the effective dose reduction indicates that nanoencapsulation of QN brings therapeutic advantages for the treatment of malaria. To elucidate whether this improvement was related to changes in the drug disposition or distribution into infected red blood cells, further experiments were conducted. 3.3. Pharmacokinetic evaluation Pharmacokinetic experiments were performed to investigate whether nanoencapsulation changes QN disposition in animals. Fig. 2 shows the mean plasma profiles obtained in infected rats after i.v. bolus administration of free or nanoencapsulated QN (25 mg/kg). For the free QN group, plasma levels showed a tendency for bicompartmental distribution, similar to that previously

Fig. 2. Mean (±standard deviation) plasma profiles of free quinine (QN) () (n = 5) and QN-loaded nanocapsules (2 mg/mL) () (n = 6) after a single 25 mg/kg intravenous dose.

observed for healthy rats [34], whereas a single decay could be observed in the QN-loaded nanocapsule profile. The QN pharmacokinetic parameters for both evaluated groups are shown in Table 3. None of the QN pharmacokinetic parameters determined in plasma were significantly altered by nanoencapsulation (˛ = 0.05). It has been shown that following i.v. administration, polymeric nanoparticles undergo opsonisation allowing the recognition of the particles by cells of the mononuclear phagocytic system and immediate removal from the circulation to the spleen and liver [35,36]. A similar tendency of increasing clearance (from 7.0 ± 3.5 L/h/kg to 9.5 ± 1.9 L/h/kg) and decreasing half-life (from 1.0 ± 0.5 h to 0.5 ± 0.05 h) was observed for the QN-loaded nanocapsules used in the present study, although the differences were not statistically significant. The absence of a significant difference could be attributed to the high interindividual variability observed for Table 3 Quinine (QN) pharmacokinetic parameters after administration of a single intravenous dose (25 mg/kg) of free or nanoencapsulated drug to Plasmodium berghei-infected Wistar rats (mean ± standard deviation). Parameter −1

Fig. 1. Parasitaemia profiles of the effective doses of quinine (QN) free base (105 mg/kg/day) (), QN-loaded nanocapsules (75 mg/kg/day) (), negative control group (♦) and unloaded nanocapsules () following treatment of Plasmodium berghei-infected Wistar rats (n = 7 per group).

Ke (h ) t1/2 (h) AUC0–∞ (ng h/mL) MRT (h) CLtotal (L/h/kg) Vdss (L/kg)

Free QN (n = 5) 0.856 1.0 4824 0.8 7.0 5.4

± ± ± ± ± ±

0.435 0.5 2880 0.3 3.5 2.5

QN-loaded nanocapsules (n = 6) 1.364 0.5 2706 0.7 9.5 6.5

± ± ± ± ± ±

0.126 0.05 453 0.1 1.9 1.7

Ke , elimination rate constant; t1/2 , half-life; AUC0–∞ , area under the curve; MRT, mean residence time; CLtotal, total plasma clearance; Vdss , volume of distribution at steady state.



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the drug when administered in the free form. QN is metabolised to 3-hydroxyquinine predominantly by the hepatic CYP3A4 system. Since expression of this enzyme system exhibits considerable interindividual variation, ranging between 10% and 60% of total hepatic cytochrome P450 activity in humans, genetic factors may contribute to the variable disposition of QN [34,37]. Nanoencapsulation decreases the intrinsic variability of QN pharmacokinetics. The variability in the kinetic parameters determined after the administration of QN-loaded nanocapsules was 2–5-fold smaller than that observed after the administration of free QN. This can be observed for the AUC0–∞ , for instance, where the coefficient of variation of 59.7% after free QN dosing was reduced to 16.7% after injection of nanoencapsulated drug. A decreased pharmacokinetic variability was also reported by Fang et al. [38] for the lipid nanoemulsion of flurbiprofen. 3.4. Erythrocyte partition coefficient Despite nanoencapsulated QN presenting a similar plasma profile to that observed for the free drug, nanoencapsulation increased QN efficacy by almost 30%. A second hypothesis to explain the pharmacodynamic findings could be that nanoencapsulation increases the intraerythrocyte concentration of QN. In other words, nanocapsules could have increased drug concentration at the site of action. To investigate this hypothesis, the QN in vitro partition coefficient into erythrocytes of P. berghei-infected rats was determined. The partition coefficient of free QN into erythrocytes (3.03 ± 0.07) was increased to 6.253 ± 0.25 when QN-loaded nanocapsules were used. Nanoencapsulation doubled the drug penetration into red blood cells of P. berghei-infected rats, justifying the improvement in QN efficacy when nanoencapsulated. However, the experiment did not allow it to be determined whether the mechanism of increasing partition coefficient was due to bioadhesion of the nanocapsules to the erythrocyte membrane, facilitating drug penetration, or increased internalisation of the nanostructures, or both [39–41]. Since expansion of the antimalarial therapeutic arsenal, including drugs that efficiently treat resistant P. falciparum malaria, has not occurred so far, alternatives to reduce the drawbacks of QN and to improve its clinical use are important owing to the revival of its use when chloroquine is not effective. The present work showed that nanotechnology can play a decisive role in this scenario by decreasing the effective dose of this traditional drug. Whether the nanocapsules developed in this study will maintain the observed effect following oral administration, which is a more suitable route for antimalarial dosing, remains to be proven. Nevertheless, the results are promising and encourage further development in this line of investigation. In conclusion, the present work showed that it is possible to produce QN-loaded nanocapsules (2 mg/mL) with adequate physicochemical characteristics for i.v. administration and that this formulation allowed for the reduction of the QN effective dose by almost 30%. The improvement in efficacy may be a consequence of the higher QN partition coefficient into erythrocytes. In summary, nanoencapsulation may be an interesting approach to improve the efficacy of drugs such as QN, used to treat malaria, owing to the increase in penetration of the drug’s target, the red blood cells. Funding: This project was financed by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil (# 47831/2006-3). The authors thank the CNPq and CAPES, Brazil, for individual grants received. Competing interests: None declared. Ethical approval: The protocols for animal experiments were approved by the Universidade Federal do Rio Grande do Sul Ethics in Research Committee (protocol # 2005477), Porto Alegre, Brazil. Animals were treated according to recommendations of the Canadian

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