Polyester Nanocapsules for Intravenous Delivery of Artemether: Formulation Development, Antimalarial Efficacy, and Cardioprotective Effects In Vivo

Abstract

Artemether (ATM) is an effective antimalarial drug that also has a short half-life in the blood. Furthermore, ATM is also cardiotoxic and is associated with pro-arrhythmogenic risks. We aimed to develop a delivery system enabling the prolonged release of ATM into the blood coupled with reduced cardiotoxicity. To achieve this, we prepared polymeric nanocapsules (NCs) from different biodegradable polyesters, namely poly(D,L-lactide) (PLA), poly-ε-caprolactone (PCL), and surface-modified NCs, using a monomethoxi-polyethylene glycol-block-poly(D,L-lactide) (PEG5kDa-PLA45kDa) polymer. Using this approach, we were able to encapsulate high yields of ATM (>85%, 0−4 mg/mL) within the oily core of the NCs. The PCL-NCs exhibited the highest percentage of ATM loading as well as a slow release rate. Atomic force microscopy showed nanometric and spherical particles with a narrow size dispersion. We used the PCL NCs loaded with ATM for biological evaluation following IV administration. As with free-ATM, the ATM-PCL-NCs formulation exhibited potent antimalarial efficacy using either the “Four-day test” protocol (ATM total at the end of the 4 daily doses: 40 and 80 mg/kg) in Swiss mice infected with P. berghei or a single low dose (20 mg/kg) of ATM in mice with higher parasitemia (15%). In healthy rats, IV administration of single doses of free-ATM (40 or 80 mg/kg) prolonged cardiac QT and QTc intervals and induced both bradycardia and hypotension. Repeated IV administration of free-ATM (four IV doses at 20 mg/kg every 12 h for 48 h) also prolonged the QT and QTc intervals but, paradoxically, induced tachycardia and hypertension. Remarkably, the incorporation of ATM in ATM-PCL-NCs reduced all adverse effects. In conclusion, the encapsulation of ATM in biodegradable polyester NCs reduces its cardiovascular toxicity without affecting its antimalarial efficacy.

Keywords: QT interval; artemether; cardiotoxicity; drug delivery; malaria; nanocapsules; polylactide; self-assembled polymers.

Figure 1

Experimental protocol of vehicle, blank-NC, free-ATM, and ATM-PCL-NC treatment for ECG and AP signal register.

Figure 2

Morphological characterization of nanocapsules by atomic force microscopy (AFM) in (A–D) images, zeta potential, and size distribution by intensity determined by dynamic light scattering (DLS) in (E–G) graphs. (A) is the AFM height and (B) is the corresponding AFM phase images of ATM-PCL-NC 2 mg/mL and (D) is the 3D AFM image of ATM-PEG-PLA NC 2 mg/mL showing the size dispersion of spherical particles deposited under mica plates. Scheme (C) is the topographical profile of a selected NC in image (A) showing the measurement of diameter at half-height and the values measured by the equipment. In (E): blank-PCL-NC; (F): ATM-PCL-NC 2 mg/mL and (G): ATM-PCL-NC 4 mg/mL formulations.

Figure 3

Profiles of the cumulative release of free-artemether (Free-ATM) and artemether from nanocapsules of polycaprolactone and PEG-PLA polymers following time in PBS pH 7.4 under sink conditions in 37 °C using inverted dialysis sac method. The insert represents the first 2 h. Data shown are means ± standard deviations of n = 3 independent experiments. The assay was made in triplicate (nine measurements in total). ^a is a mean statistically different from free-ATM and ^b statistically different from ATM-PEG-PLA NC. The values at each time point were compared using an unpaired student’s t-test.

Figure 4

Efficacy of the artemether formulations represented in graphs of parasitemia (%) and survival (%) for both protocols (four-day-test) and single low dose (20 mg/kg) with established parasitemia (15%).

Figure 5

Representative ECG signal (lead II) at basal (before treatment) and after treatment with Blank-NC in (A), Free-ATM in (B) and ATM-PCL-NCs in (C), showing the ECG intervals analyzed. The RR interval was used to obtain the heart rate (HR).

Figure 6

QT interval percentual variation from basal (before treatment) until two hours after IV single dose administration of blank-NC, free-ATM, and ATM-PCL-NC, both at 40 (A) and 80 mg/kg (B). * p > 0.05 compared to free-ATM administration. ANOVA followed by Tukey post-test.

Figure 7

Maximal percentual variation of QT and QTc intervals and heart rate (HR) after IV administration of blank-NC, free-ATM, and ATM-PCL-NC, a single dose of 40 (A) and 80 mg/kg (B), and four doses (one every 12 h) of 20 mg/kg (C). * p > 0.05 compared to blank-NC and # p > 0.05 compared to ATM-PCL-NC administration. ANOVA followed by Tukey post-test.

Figure 8

Representative signals of arterial blood pressure of anesthetized rats treated with free-ATM or ATM-PCL-NC, both at 120 mg/kg, showing the severe hypotension produced by the free form and the absence of hypotension with the NC formulation.

Figure 9

Maximal variation of systolic (SAP) and diastolic (DAP) pressure (mmHg) after IV administration of blank-NC, free-ATM, and ATM-PCL-NC, a single dose of 40 (A) and 80 mg/kg (B), and four doses (one every 12 h) of 20 mg/kg (C). * p < 0.05 compared to blank-NC and # p < 0.05 compared to ATM-PCL-NC administration, ⁺ p < 0.05 compared to basal, blank-NC, and ATM-PCL-NC. ANOVA followed by Tukey post-test.