Effects of nanocapsules containing lumefantrine and artemether in an experimental model of cerebral malaria

Abstract

Background

Malaria, a tropical neglected disease, imposes a significant burden on global health, leading to the loss of thousands of lives annually. Its gold standard treatment is a combination therapy of lumefantrine (LUM) and artemether (ART). Nanotechnology holds significant potential for improving drug bioavailability and potency while reducing adverse effects.

Objectives

This study aimed to develop lipid-core nanocapsules containing ART and LUM and evaluate their effects in an experimental cerebral malaria model (ECM).

Methods

The polymeric interfacial deposition method was used to develop lipid-core nanocapsules (LNCs) containing ART and LUM (LNC_ARTLUM) and were characterized using micrometric and nanometric scales. Male C57BL/6 mice were infected with Plasmodium (P.) berghei ANKA (PbA, 1 × 10⁵ PbA-parasitized red blood cells, intraperitoneally). On day 5 post-infection, PbA-infected mice were orally administered with ART + LUM, LNC_ARTLUM, blank nanocapsules (LNC_BL), or ethanol as a control. Parasitemia, clinical scores, and survival rates were monitored throughout the experiment. Organ-to-body weight ratios, cytokine quantification, and intravital microscopy analyses were conducted on day 7 post-infection.

Results

LNCs were successfully developed and characterized. The treatment with LNC_ARTLUM in ECM resulted in complete clearance of parasitemia at 10 dpi, decreased clinical scores, and maintained 100% survival rates. Thereated mice exhibited splenomegaly and reduced TNF-α, IL-1β, and MCP1 levels in the brain. Furthermore, the LNC_ARTLUM treatment protected the brain microvasculature, reducing the number of cells in the rolling process and adherent to the microvasculature endothelium.

Conclusion

Nanoformulations can potentially improve the efficacy of antimalarial drugs and be considered a promising approach to treat malaria.

Supplementary Information

The online version contains supplementary material available at 10.1186/s11671-024-04121-6.

Introduction

Despite recent advancements in reducing the global malaria burden, this disease continues to be a significant global health challenge. Rapid diagnostic tests and artemisinin derivatives play a key role in malaria management. Nonetheless, these strategies alone may not continuously decline the malaria incidence and mortality rates [1]. In 2022, 249 million malaria cases were reported, with approximately 608,000 deaths. The proportion of total malaria-related fatalities among children aged under 5 years was kept at 76% in 2015 and has remained unchanged to date [2]. Malaria, a life-threatening disease, is caused by parasites transmitted via bites of female Anopheles mosquitoes. Despite being preventable and treatable, untreated cases may escalate to severe illness and fatal outcomes. The disease is attributed to five distinct parasite species, with P. falciparum and P. vivax being the most widespread and hazardous [3]. Various host and parasitic factors are involved in cerebral malaria (CM) pathogenesis CM is mainly characterized by trapping the infected red blood cells (RBCs) in the brain’s small blood vessels, a process worsened by the host’s inflammation and the parasite’s infectivity [4]. CM progression is marked by several pathophysiological changes, including adhesion and accumulation of infected RBCs, platelets, and leukocytes in the brain microvasculature, resulting in microvascular obstruction, reduced cerebral blood flow, and impaired neurovascular coupling [5]. These alterations are commonly characterized by brain swelling, intracranial hypertension, retinal changes, and brainstem signs commonly observed in patients with CM [6]. Brain capillaries and post-capillary venules are particularly susceptible to infected RBCs sequestration, resulting in congestion, hypoxia, tissue swelling, coma, and, ultimately, death. Furthermore, the interplay between parasite and host factors, such as endothelial dysfunction, coagulation dysregulation, resident and infiltrating immune cells, and inflamed microenvironment, collectively contribute to infected RBC sequestration and CM development [5].

Artemisinin-based combination therapies (ACTs) are the gold standard treatment for malaria, as advocated by the World Health Organization. These ACTs typically combine a rapid-acting artemisinin derivative (artesunate, artemether, or dihydroartemisinin) with a longer-lasting drug like lumefantrine [7].

Artemether is a lipid-soluble peroxide sesquiterpenoid, classified as an artemisinin derivative [8]. Among the mechanisms described, malaria parasites contain ferrous ions (Fe²⁺) that play a crucial role in activating the artemether. This activation cleavages artemether’s endoperoxide bridge, generating reactive oxygen species and carbon-centered free radicals. These reactive intermediates cause oxidative damage, DNA damage, and the alkylation of multiple parasite proteins, collectively disrupting critical biological functions within the parasite and, ultimately leading to its death [9, 10]. The exact mechanism of action of lumefantrine is not well defined, but it is thought to inhibit β-hematin formation, an important parasitic detoxification pathway. Lumefantrine effectively starves the parasite of a crucial defense mechanism against oxidative stress by preventing the conversion of toxic-free heme into nontoxic hemozoin [11].

This synergy between two distinct mechanisms of action, targeting various stages of the parasite’s life cycle, ensures the high efficacy of this combination. However, increased parasite resistance poses a significant challenge [12, 13]. Drug delivery systems develop from improving medication’s therapeutic efficacy while reducing their associated side effects [14]. Nanoformulations can potencially play a crucial role in malaria treatment through enhancing drug delivery and treatment efficiency and improving bioavailability while minimizing adverse effects. However, adherence to malaria treatment is challenging because of the high costs and potential adverse effects [15].

The challenges encountered during treatment include poor aqueous solubility, low bioavailability, rapid clearance from the bloodstream, and drug resistance. By encapsulating antimalarial agents into nanoparticles, such as liposomes, polymeric nanoparticles, or solid lipid nanoparticles, their stability can be enhanced, circulation time prolonged, tissue penetration improved, and specific infection sited targeted. Furthermore, nanoformulations effectively controlled release kinetics, allowing sustained drug release and reduced dosing frequency [15].

Considering the small number of innovative antimalarial drugs approved since 1990, the main strategies in fighting against malaria are searching for more efficient and less toxic antimalarials based on nanotechnology. Nanocarriers are useful tools to improve the pharmacokinetic profile of effective drugs that, due to their low water solubility, low bioavailability, and high toxicity, have been employed, to a limited extent, in pharmacotherapy [16, 17]. Furthermore, nanotechnology can potentially restore old and toxic drugs by modifying their biodistribution and reducing toxicity [18]. This advantage is significant in malaria therapy because of the urgent need for new dosage forms to deliver antimalarial drugs to infected cells [19].

Furthermore, the small size of nanoparticles enables them to cross biological barriers more efficiently, including the blood–brain barrier [20], which is crucial for treating CM [21].

CM is a severe and life-threatening form of malaria, frequently associated with significant neurological deficits and long-term cognitive impairments in infected patients. Therefore, this study aimed to develop a novel formulation of existing antimalarial drugs to enhance their therapeutic efficacy by ameliorating the neurological and systemic complications associated with CM. Furthermore, we aimed to develop, characterize, and test the LNCs containing ART and LUM (LNC_ARTLUM) nanoformulation for experimental cerebral malaria model (ECM) by comparing its efficacy to free forms of ART and LUM in ECM parameters, focusing on brain inflammation and microcirculation, which, to use knowledge, has not been previously investigated in preclinical assays. Therefore, we believe this is a key step to foster clinical tests with nanoformulation to treat malaria.

Material and methods

Material

Arthemeter (ART), lumefantrine (LUM), sorbitan monostearate (Span 60®), polysorbate 80 (Tween 80®), and poly(ε-caprolactone) (PCL MW 80.000 g mol-1) were purchased from Sigma-Aldrich (Brazil); caprylic/capric triglyceride was purchased from Brasquim (Brazil). The other reagents and solvents used were of analytical or chromatographic grades.

Preparation of polymeric lipid-core nanocapsules (LNC)

Polymeric lipid-core nanocapsules (LNC) formulation containing ART and LUM was prepared with modifications by the pre-formed polymer deposition method [22, 23]. The polymeric lipid-core nanocapsules were obtained by injecting the organic phase [poly(ε-caprolactone) (0.2 g), monostearate of sorbitan (0.08 g), caprylic/capric triglycerides (0.3 g), LUM (8.5 mg) and ART (1.5 mg) dissolved in acetone (46 mL) under magnetic stirring at 40 °C] into the aqueous phase [polysorbate 80 (0.15 g) and water (0.1 L)] under stirring, remaining in this condition for 10 min. After LNC formation, acetone and part of water were eliminated under reduced pressure in a rotative evaporator at 37 °C (R-200, Buchi, Flawil, Switzerland) until reaching about 9 mL. The formulation was adjusted to a final volume of 10 mL in a volumetric flask. The formulations obtained containing ART and LUM were named LNC_ARTLUM. The formulation contains the same proportion of antimalarial drugs as Coartem® (composed of 20 mg of ART and 120 mg of LUM) (RDC 47/09 + BPL 20.04.12 + Farm Resp 2012). For comparison, an LNC containing caprylic/capric triglycerides as oil, named blank LNC (LNC_BL), was prepared without adding drugs. All formulations were kept at room temperature and protected from light for 30 days.

Physicochemical characterization of the nanossystems

After direct insertion of each sample in the wet unit containing about 150 mL of water, the particle size distribution was assessed using Laser Diffraction Analysis (LD) (Mastersizer 2000, Worcestershire, United Kingdom), and the volume-weighted mean diameter (d4.3), the diameters under the distribution curve at percentiles 10, 50 and 90, respectively d0.1 d0.5 and d0.9, were determined. The polydispersity (SPAN) was calculated for each formulation batch according to the Equation: SPAN = (d0.9−d0.1)/d0.5. The hydrodynamic mean diameter (by intensity) and the polydispersity index (PDI) were determined by photon correlation spectroscopy (Zetasizer® nano-ZS ZEN 3600, Worcestershire, United Kingdom) after diluting (500 times) each sample in purified and filtered water. Electrophoretic mobility was determined (Zetasizer®NanoZS model ZEN 3600, Malvern Instruments, United Kingdom) to calculate the Zeta potential after diluting each sample (500 times) in 10 mmol L⁻¹ NaCl aqueous solution. In addition, the hydrodynamic mean diameter (by number) was determined by nanoparticle tracking analysis (NTA) (NanoSight LM10, Malvern Instruments, United Kingdom) using a sample holder chamber coupled to a 640 nm laser. For this, each sample was diluted in ultrapure water (10.000 times) and inserted (glass syringe) into the sample holder chamber. Video images of the Brownian motion of individual particles are identified and tracked in real time via CCD camera by Analytical Software 2.0 NTA. The morphology of the LNC was evaluated by a transmission electron microscope (TEM, Jeol JEM 1200-ExII, 100 mV, Tokyo, Japan), operating at the Electronic Microscopy using 120 kV. LNC_ARTLUM was diluted in ultrapure water and placed on the grids (formvar-carbon support film, electron microscopy sciences). The uranyl acetate 2% (m/v) was used with negative contrast.

Validation of the analytical method and study of the drug release profiles

The quantification of drugs was performed using high-performance liquid chromatography with UV detection (HPLC–UV, Perkin Elmer®). The formulations were treated with acetonitrile to extract the drugs from the nanocapsules. The analytical validation method was performed on a Perkin Elmer Series 200 chromatograph with an ultraviolet detector at 216 and 335 nm for ART and LUM, respectively [13, 15]. The system consisted of a LiChrosorb 100 RP-18 stainless steel column (5 µm, 150 × 4.5 mm) and a pre-column with LiChrosorb RP-18 stationary phase (5 µm). The mobile phase consisted of a mixture of acetonitrile:water (60:40 v/v), pH 3.5 and flow 1.2 mL.min⁻¹. The formulations were dosed in triplicate. The method was validated according to the Harmonization of Technical Requirements of the Registration of Pharmaceutical for Humane Use and Resolution No. 899 of the National Health Surveillance Agency. The methodology was validated in specificity, linearity, precision, accuracy, detection limits, and quantification. To evaluate the in vitro release profile, the dialysis method was used with an ultraviolet detector at 216 and 335 nm for ART and LUM, respectively. Two solutions were developed: the solution containing 85 mg of lumefantrine dissolved in 10 mL of pH 1.2 solution and 1% polysorbate 80 and the solution containing 15 mg of artemether dissolved in pH 7.4 buffer solution and 1% sodium lauryl sulfate. Dialysis bags (MW cutoff 14,000 Da, mean flat width 10 mm; Sigma-Aldrich) composed of cellulose were filled with LNC_ARTLUM or free drugs and immersed in containers under agitation. To simulate in vivo conditions for lumefantrine, receptor liquid containing buffer pH 1.2 and 1% polysorbate 80 was used. To simulate in vivo conditions for artemether, containers containing phosphate buffer pH 7.4 and sodium lauryl sulfate were used. The samples were agitated at 50 ± 10 rpm, and aliquots of 1 mL were withdrawn at predetermined intervals (30, 60, 120, 240, 360, 480, 720, and 1440 min). After withdrawal, the same volume was replaced with fresh medium. The solutions containing the drugs at the same concentration in the formulations were prepared and immersed in 50 mL of the release medium. After preparing the release profiles versus time, mathematical modeling was performed using different models to evaluate the drug release mechanism from the nanostructures [24, 25].

Pb infection of mice

Two animals (C57BL/6), passage animals, were infected with red blood cells parasitized with PbA, according to previously described [26]. Briefly, inoculation was performed intraperitoneally in the lower left quadrant of both animals at 200 µL/animal. Parasitemia was counted on days 5 and 7 post-infection under an optical microscope at 100 × magnification with immersion oil. Thus, blood was removed from the passage animal using the cardiac puncture method on day 7 after infection. All mice were infected intraperitoneally with 10⁵ red blood cells parasitized by PbA (200 µL/animal diluted in PBS) taken from the passage animals.

Furthermore, cellularity was counted in the Neubauer chamber to establish a relationship between the number of RBCs inoculated into the mouse and the number of infected RBCs. Animals in the uninfected (NI) group received the same amount of RBCs from a healthy animal as the infected animals. The Ethics Committee approved all procedures performed with experimental animal models on the Use of Animals, numbering L-022/2020, and following the current Brazilian legislation from the national council for the control of animal experimentation (Lei 11.794, Ministério de Ciência e Tecnologia e Inovação).

Treatment with antimalarials

To recover the animals from malaria damage, they were divided into five groups: NI (uninfected—RBCs), ART + LUM (free drugs), and LNC_ARTLUM. The concentrations of 0.6 mg.kg⁻¹ of ART and 3.4 mg.kg⁻¹ of LUM were used in free and nanoencapsulated drug formulations. LNC_BL and EtOH (1%—free drug control group were administrated in the same volume as LNC_ARTLUM (nanoparticles control group) and ART + LUM, respectively. A volume of 100 µL of the solution was administered orally to the animals. This procedure was repeated from days 5 to 7 post-infection. The procedure was extended in survival trials from day 5 post-infection to day 12 (Fig. 1).

Fig. 1.

Light intensity captured by the camera over 60 s as a function of the particle diameter, based on the particle movement tracking technique (NanoSight®), for formulations and photomicrography of LNC_ARTLUM.. A LNC_ARTLUM, B LNC_BL. In detail, the particle diameter distribution by relative intensity is demonstrated. C Photomicrography of LNC_ARTLUM

Survival assay

In the survival assay another set of mice were used, and animals were infected and monitored until day 14 post-infection, with deaths from each group being recorded. The treatment was administered to the animals between days 5 and 12 post-infection. Subsequently, Kaplan–Meier curve analysis was performed, followed by the Mantel-Cox test.

Weighing and spleen/liver/body weight ratio

On the seventh day, the animals were perfused to remove the organs. The animals were inhaled with Isoflurane (5%) in an anesthetic chamber during this procedure. Blood sampling and perfusion were performed through cardiac puncture. The perfusion pump is calibrated for a constant flow of 2 mL per minute. After perfusion, the brain, spleen, and liver were collected. The organs removed after perfusion are weighed to obtain the spleen/liver/body weight correlation.

Assessment of the clinical score

On days 5 and 7 after infection, the cognitive impairment of each animal was assessed according to the measurement of behavioral and physical parameters. In survival trials, the Clinical Score was also evaluated on days 10 and 14, in which one animal at a time was placed inside a rectangular box and behavioral and physical parameters were observed, namely: piloerection, arched body, unsteady gait, convulsions, paw paralysis, coma, breathing rate, skin color, heartbeat, lacrimation, eyelid closure, grip strength, loss of limb, abdominal and body tone, change in body temperature, interest in the environment and escape to the touch. Each clinical sign presented received 1 point, where if the animal reached a score of 12 (indicating severe malaria), before the evaluation of parasitemia, for humane treatment, the animal was euthanized using a lethal dose of Ketamine-Xylazine cocktail.

Assessment of parasitemia

A blood smear was performed on days 5, 7, 10, and 14 after infection to evaluate parasitemia. The parasitemia was evaluated through a small cut made with surgical scissors at the tip of the mouse’s tail and a drop of blood placed on a microscope slide and subsequently stained with panoptic for visualization under an optical microscope (100 × magnification).

Statistical analysis

Regarding statistical analysis, data were tabulated using Microsoft Excel 2007 software and initially evaluated for normality using the Shapiro–Wilk test. Subsequently, ANOVA with Tukey’s test was used to compare experimental groups. All data were expressed as mean and standard deviation. The Prisma 6 GraphPad program was used to construct the graphs and for statistical analysis, and in animal experiments, One-way ANOVA was used, followed by the Newman-Keuls post-test. Significant differences compared to the null hypothesis will be considered when p < 0.05.

Results

Physicochemical characterization of the LNCs

LNC formulations (LNC_BL and LNC_ARTLUM) showed a macroscopically homogeneous white opalescent color without phase separation or precipitate formation. Table 1 depicts the physicochemical parameters obtained for LNCs evaluated by different techniques.

Table 1.

Physicochemical analysis of nanosystems

Formulations
		Day 0		Day 30
		LNCBL	LNCARTLUM	LNCBL	LNCARTLUM
Laser diffraction (LD)	Mean diameter (nm) SPAN	248± 0.002	296± 0.002	247± 0.01	301 ± 0.02
		1.45± 0.02	1.86± 0.02	1.32± 0.02	1.81± 0.03
Dynamic light scattering (DLS)	Z-average diameter (nm) PDI	233± 0.03	248± 0.02
		0.14± 0.02	0.17± 0.01
Zeta potential	Zeta potential (mV)	−14.1± 0.10	−16.4± 0.05
pH		5.98 ± 0.17	6.56 ± 0.14
High-performance liquid chromatography	Drug content (mg mL-1)	–	0.0085* ± 0.2

Analysis of dynamic light scattering demonstrated that all the particles had nanometer diameters (approximately 250 nm) and adequate homogeneity of the size distribution (polydispersity index [PDI] ≤ 0.2). Furthermore, the absence of microparticles in both formulations was confirmed byLD, revealing a 248 and 296 mm diameter, with span of 1.45 and 1.86, respectively. Results of LD analyses revealed no significant difference in the average diameter of the LNC_ARTLUM (Fig. 1 A) and LNC_BL (Fig. 1 B) particles 30 days (p < 0.05). The zeta potential of nanoparticles ranges close to − 15 mV for LNCs. Furthermore, the formulations demonstrated pH values between 5.98 and 6.56, consistent with the composition. The results of the LNC_ARTLUM suspension presented adequate requirements for application in biological models. The morphology and shape of the LNC_ARTLUM nanosystem were analyzed using transmission electron microscopy at 120 kV (Fig. 1 C). The formulation revealed circular particles with sub-micrometer diameters of approximately 250 nm, this was further supported by LD and PCS analysis.

The analytical methods developed were validated and met the International Conference on Harmonization (ICH, 2003) and resolution No. 899 of the National Health Surveillance Agency requirements. The validated methodologies of LUM proved specific, linear, precise, and accurate. The developed HPLC method was validated through linearity, precision, accuracy, and robustness assessments. The obtained r-value from five concentration data points was 0.999, confirming the equation linearity. The intraday variability test, revealed that the RSD values were < 0.05%. The interday variability test, showed 0.45% and 0.51% (for days 1 and 2, respectively). Precision assay RSD values indicated that the developed method is precise, aligns with regulatory requirements, and is accurate. The method exhibited robustness during evaluation, as no significant changes were observed with altered analytical column and flow rate. The drug content of LUM in LNC_ARTLUM was 0.0085 mg.mL⁻¹, corresponding to 100.0 ± 0.2% (Table 1).

As for the results of in vitro assays, the results obtained from LNC_ARTLUM are promising, demonstrating the controlled release capacity of LNC (Fig. 2). The in vitro drug release profile is a crucial tool for developing and quality-controlling pharmaceutical forms containing polymeric nanoparticles, where greater control over the release of substances is sought in comparison with conventional pharmaceutical forms [27, 28]. The developed LNC is made from the biodegradable and biocompatible polymer PCL, forming the nanocapsule wall and significantly influencing drug release control [29]. The oily core is responsible for core viscosity and modulates the in vitro drug release [18].

Fig. 2.

In vitro release profile of non-nanoencapsulated lumefantrine (LUM) and lipid-core nanocapsules (LNCs) containing artemether and lumefantrine (LNC_ARTLUM)

Cumulative percentages of LUM released in vitro from non-nanoencapsulation LUM (free LUM) and LNC_ARTLUM were evaluated over 24 h using the dialysis bag method. The in vitro release assay revealed that 47% of free LUM was released within 1 h, reaching 99.27% after 12 h. Conversely, nanoencapsulated LUM released 18.9% within the first hour, 62,51% after 12 h, and 65.60% after 24 h of the study. The results obtained in this study revealed the controlled release of the drug LUM, by the type and composition of the developed nanosystem.

LNCARTLUM treatment effects on ECM

Results obtained through the LNC_ARTLUM treatment of PbA-infected mice demonstrated significant potential. To assess the effects of LNC_ARTLUM treatment on ECM, all groups started treatment on day 5 post-infection. A schematic representation of the experimental design is presented in Fig. 3. In the survival analysis, the only infected group with all animals that survived was LNC_ARTLUM, contrasting with ART + LUM, with a survival rate of 40% at day 14 post-infection. The negative control groups ethanol and LNC_BL had 40% and 30% survival rates, respectively (Fig. 4).

Fig. 3.

Schematic representation of experimental design. A Mice were infected with PbA and divided into 4 groups: ART + LUM, ethanol, LNCBL, and LNC_ARTLUM. B Parasitemia, Clinical Score, and survival were assessed on 5–14 dpi. The organ weight, cerebral cytokines levels, and microcirculation analysis were evaluated on 7 dpi

Fig. 4.

The survival rate of C57Bl-6 mice infected with PbA and treated with LNC_ARTLUM and ART + LUM. Post-infection, mice were followed daily for 14 days. LNC_ARTLUM treatment had a survival of 100% mice compared to non-infected mice, whereas ART + LUM had a 60% survival rate. Analysis was performed using the Kaplan–Mayer curve followed by the Mantel–Cox test *p < 0.05; **p < 0.005 (n = 10)

On day 7, when parasite outbreaks occur, many untreated animals die of hyperparasitemia. Animals treated with LNC_ARTLUM had complete parasite clearance on 10 dpi 5 days of post-treatment (Fig. 5A). Similar observations were seen in the clinical score. LNC_ARTLUM-treated mice did not evolve to CM and, at 10 dpi, stopped presenting clinical signs of the disease (Fig. 5B). The mice treated with ART + LUM exhibited reduced clinical signs compared with the control group receiving ethanol. However, after the treatment discontinuation at 7 dpi, clinical signs persisted until 14 dpi (Fig. 5B). We then evaluated whether a positive correlation existed between parasitemia and clinical scores in LNC_ARTLUM and LNC_BL. Using linear regression analysis, Figs. 5C and 5D demonstrate a positive correlation between LNC_ARTLUM (p < 0.0001) and LNC_BL (p < 0.03), respectively.

Fig. 5.

Evaluation of ART + LUM and LNC_{ARTLUM on} the experimental CM model. A. Parasitemia in mice at 5, 7, 10, and 14 dpi (n = 10–20). * LNC_BL compared with LNC_ARTLUM and # LNC_ARTLUM compared with ART + LUM. #p < 0.01, **p < 0.006, ***p < 0.0003. B. Clinical Score in mice at 7, 10, and 14 dpi. *p < 0.05; ***p < 0.001, ****p < 0.0001. C. Linear regression analysis between the clinical scores of LNC_ARTLUM mice at 7 dpi and parasitemia. D. Linear regression analysis between clinical score of LNC_BL and parasitemia. Data are presented as means ± standard deviations

Hepatosplenomegaly, characterized by liver and spleen enlargement, is a prevalent characteristic of acute and severe malaria. This enlargement is primarily caused by reticuloendothelial and lymphoid hyperplasia, related to the immune response, pigment deposition, and sinusoidal dilatation [30, 31] caused by an intense RBC clearance with reduced deformability and infected RBCs [32]. The size of the spleen is used to identify the burden of malaria transmission in endemic areas [33]. Similar processes are observed in the liver, i.e., to intravascular hemolysis and disseminated intravascular coagulation [34], besides retention of hemozoin pigment and direct correlation with the pRBC load [34, 35]. To assess this phenomenon, the spleen and liver indices were calculated by correlating the organ and body weights (Fig. 6). The treated groups revealed an increased index, probably due to the more intense parasite clearing in those groups.

Fig. 6.

Evaluation of ART + LUM and LNC_ARTLUM on the experimental CM mode. Spleen and liver indices were measured relative to the body weight on 7 dpi. A. Liver/ body weight ratio. **p < 0.008. B. Spleen/ body weight ratio. *p < 0.05, **p < 0.001, ***p < 0.0003(n = 4–11)

Conversely, the control groups maintained a higher parasitemia throughout the disease course. Despite increased liver weight\in all groups, previous studies demonstrated that liver pathology is not essential in PbA infection [36]. In our case, both treatments did not alter liver weight at 7dpi (Fig. 6).

LNC_ARTLUM treatment effects on neuroinflammation in ECM

To explore the therapeutic potential of LNC_ARTLUM in ECM, brain tissue analysis of PbA-infected mice were performed on 7th dpi. Inflammatory markers were determined to elucidate the impact of LNC_ARTLUM on mitigating brain inflammation in this model. Specifically, levels of pro-inflammatory cytokines, such as TNF-α, IL-6, IL-1β, MCP1, and KC were measured. CM was significantly associated with increased cytokine levels compared to NI controls. Treatment with LNC_ARTLUM markedly reduced TNF-α, IL-6, and MCP1 levels, indicating its anti-inflammatory effects in ECM (Fig. 7).

Fig. 7.

LNC_ARTLUM treatment results in decreased inflammatory markers in the brain tissue. Cytokines were measured by ELISA. A TNF-α, B IL-6, C IL-1β, D MCP-1, E KC. *p < 0.01, **p < 0.001, ***p < 0.0005, ****p < 0.0001(n = 5–8)

Microvascular dysfunction, characterized by impaired reactivity and tissue perfusion, is closely associated with the severity and outcome of severe malaria. Sequestration of infected RBCs within the microvasculature and heightened endothelial activation characterize CM. Endothelial activation further contributes to blood–brain barrier permeability, exacerbating neurological complications in ECM. To assess endothelial activation and brain inflammation, leukocyte rolling and adhesionin brain microcirculation were evaluated (Fig. 8). Videos from brain intravital microcirculation experiments were provided as supplementary material (supplementary videos). Remarkably, LNC_ARTLUM treatment effectively prevented leukocyte adhesion (Fig. 8C), demonstrating a significant improvement over the free ART + LUM treatment.

Fig. 8.

LNC_ARTLUM treatment increased rolling velocity and severely impaired neutrophil adhesion in the brain microvasculature of PbA-infected mice on 7 dpi. Brain intravital microscopy was performed to assess leukocyte rolling and adhesion to the wall’s vasculature. A Representative images of brain microcirculation of NI and PbA-infected mice treated with LNC_BL, ART + LUM and LNC_ARTLUM under intravital microscopy. B The number of rolling cells/min. C Adherent cells were assessed over 1 min of observation. *p < 0.02, **p < 0.003, ***p < 0.0005, ****p < 0.0001

Additionally, LNC_ARTLUM reduced leukocyte rolling (Fig. 8B), a crucial inflammatory response process. These findings highlight the superior efficacy of LNC_ARTLUM in mitigating leukocyte rolling and adhesion and underscore its potential as a therapeutic agent for managing CM-associated inflammation and endothelial dysfunction. The optimized control of the inflammatory processes indicates that LNC_ARTLUM could be a potential alternative to treating and managing CM.

Discussion

Nanoparticles were synthesized by nanoprecipitation of the preformed polymer [22]. This method, widely employed for its simplicity and reproducibility, yields an opalescent liquid [28]. The bluish-white hue observed in nanosystems is attributed to the Tyndall effect observed in concentrated colloidal solutions, a phenomenon arising from the Brownian motion of nanoparticles. The analysis of sample diameters revealed nanometric particles without macroscopic particles. Consistent with these findings, the low polydispersion values indicated uniformity in the analyzed sample’s diameters. These characteristics persisted throughout the evaluated period (30 days) without significant deviation (p < 0.05). The pH values align with the composition of nanosystems in this study, representing the pH of the aqueous phase rather than that of the particle–water interface because of the presence of carboxylate groups (–COO–) at the interface and ester groups of the PCL. Negative zeta potential values ranged between − 14.10 and − 16.43, sufficient to prevent particle aggregation, as the used nonionic surfactants used form a highly stable complex at the interface between the internal and external phases, employing a steric hindrance stabilization mechanism [37]. Based on these parameters, LNC_ARTLUM and LNC_BL formulations are deemed suitable for biological models. Particle motion tracking analysis (NTA), obtained with the NanoSight® equipment, was used to verify the presence of non-encapsulated drug nanocrystals in the formulations. The scattered light intensity detected across various frames of the LNC_ARTLUM formulation, captured using the equipment’s camera, remained unchanged in the drugs. This indicated the absence of anisotropic structures in the formulations and proofs of drug encapsulation, justifying an increased particle diameter from 233 to 248 nm. Therefore, ARTLUM nanocrystals were not formed concurrently with the nanocapsules. Lumefantrine in LNCs was detected, and its release rate was a function of time for 24 h. However, artemether was not quantified because its concentration was too low for the sensitivity of the equipment.

Co-loaded nanostructured formulations containing LUM and ART were developed and evaluated across in vivo malaria models. Nanostructured lipid carriers (NLCs) containing ART–LUM were administered once daily at 1/5 of the standard therapeutic dose (16 mg ART and 96 mg LUM). These ART–LUM NLCs exhibited a globule size of 68.80 ± 4.32 nm and a PDI of 0.41 ± 0.02, smaller than the nanostructures in this study (248 nm ± 0.02) due to the nanostructures differences [38]. The co-loaded NLCs showed a hydrodynamic diameter of 145 nm and a surface charge of –66 mV. Our formulation also exhibited a negative zeta potential, indicating a favorable surface charge. Remarkably, the treatment in the referenced study was administered at a higher dose (ART: 4 mg/kg + LUM: 24 mg/kg) [39]. The same dose was used in another study of ART–LUM co-loaded NLCs that showed 188.6 nm and was evaluated in the P. berghei malaria infection model [40]. Notably, our LNC_ARTLUM formulation was the only to successfully eliminated the parasites in an experimental model of CM. Moreover, parasites were successfully killed with a smaller dose compared to other studies. The ECM induced by infection with P. berghei ANKA has been used to investigate the role of single genes, proteins, and neuroinflammation in CM pathogenesis [41]. Our investigation revealed that mice treated with ART + LUM and LNC_ARTLUM at 5–7 dpi exhibited remarkable outcomes, particularly regarding survival rates. Furthermore, none of the mices administered with LNC_ARTLUM succumbed to the infection within the 14-day observation period, a notable improvement compared to the non-infected (NI) control group. Conversely, the group treated with the free form of ART and LUM exhibited a survival rate of only 60%. Previous studies utilizing NLC co-loaded with artemether and lumefantrine demonstrated similar results, with > 50% of mice initiating treatment on day 3 dpi surviving beyond the 28-day mark [39], a lower rate as compared to LNC_ARTLUM.

Parasitemia was reduced by 7 dpi, with P. berghei ANKA elimination achieved by 10 dpi. ART and LUM target different stages of P. berghei ANKA life cycle a possible explanation of the enhanced early efficacy in parasite clearance of our nanostructured formulation in a murine model of Plasmodium yoelii infection, applying the ART and LUM dissolving microneedle patches yielded a 99.5% decrease in parasitemia observed lately, only by day 12 post-infection [42].

A study utilizing the microemulsion template technique to develop NLCs loaded with artemether–lumefantrine (ARM–LFN), revealed that the efficacy of these formulations was evaluated in two clinical simulation protocols. The ARM–LFN NLC formulation demonstrated complete clearance of parasitemia and achieved 100% survival rates following once-daily oral administration at a dose equivalent to 1/5 of the standard combination dose for four consecutive days. Conversely, mice treated with marketed ART and LUM tablets, administered twice daily at the therapeutic dose for the same duration, exhibited late-stage recrudescence in two out of six cases [38]. Another study employing the same nanostructure significantly reduced parasitemia among PbA-infected mice [39]. The clinical score represents a set of behavioral characteristics exhibited by infected mice, which serve as predictive indicators of the infection outcome. The clinical scoring system is vital for assessing the pathogenesis and outcomes of PbA infection, especially CM. In our model, treatment with LNC_ARTLUM significantly reduced clinical scores in infected mice. However, another study on prophylactic piperine administration did not affect the rectal temperature or clinical scores in PbA-infected mice, suggesting its limited efficacy in alleviating infection severity [43]. Our study, revealed that the clinical signs were significantly reduced on day 7 post-infection and completely extinguished on days 10–14 post-infection, showing complete recovery.

Mice infected with PbA and left untreated exhibited splenomegaly and hepatomegaly, as evidenced by enlarged spleen and liver. Although the treatment did not show a difference in the liver wet weight/body weight ratio, the spleen enlarged possibly due to the high requirement of this organ for the clearance of infected RBCs.

Increased cytokines in the brain are one of the hallmarks of CM, contributing to worsening the outcome [44]. LNC_ARTLUM reduced TNF-α, IL-1β, and MCP1 in brain tissues. Artesunate and other natural extracts also inhibited the expression of TNF-α, IL-1β, IL-6, CXCR4, and CXCR10 [45]. The effects of LNC_ARTLUM in reducing the production of inflammatory mediators in the brain can be a result of its effect on improving parasite elimination.

There is significant evidence that brain-infiltrating CD8⁺ T leukocytes and other leukocytes, and arrest in perivascular compartments of the brain play a central role in ECM during PbA infection of C57BL/6 mice [46]. Herein, leukocyte rolling and adhesion notably increased within the brain vasculature of PbA-infected mice. Remarkably, LNC_ARTLUM treatment significantly reduced leukocyte rolling and adhesion, surpassing the efficacy even of free-form ARTLUM. As evaluated through intravital microscopy, the treatment with mesenchymal stromal cells demonstrated promising outcomes by preserving microvascular integrity and reducing leukocyte adhesion in brain venules [47]. The preventive effect of LNC_ARTLUM for characteristic neuroinflammation events such as cytokine production and leukocyte-endothelial interaction is beneficial, in reducing the encephalopathy, CM progression severity, clinical symptoms, subsequent neurological deficits of survivors and long-term cognitive decline. LNC_ARTLUM may potentially improve survival and prevent sequalae after recovery.

Although the animal model employed in our study effectively replicates the symptomatic profile of malaria, certain limitations, such as a simplified parasite life cycle relative to the complexity of human malaria, are inevitable. This discrepancy may influence how our findings can be extrapolated to human cases. Despite these limitations, the observations presented collectively highlight the potential of nanocapsules as antimalarial agents in improving treatment outcomes and warrant further investigation into their therapeutic applications.

Conclusion

The lipid–core nanocapsules were successfully developed and characterized. The treatment with LNC_ARTLUM in ECM decreased parasitemia and clinical scores and protected the animals. Moreover, splenomegaly was observed in the treated mice, along with decreased TNF-α, IL1β, and MCP1 levels in the brain. The treatment with LNC_ARTLUM improved the brain microvasculature, preventing adhesion and leukocyte rolling. Thus, the nanoformulation tested can potentially improve and protect mice from CM and may represent a promising approach to treat malaria and its consequences, atrributing to the findings of future research and clinical trials so that they can ensure promising results.

Author contributions

Conceptualization, methodology, research, data curation S.R.F., B.P.T.M., K.P.S., I.M.S., S.O.R., K.F.C.S.S., C.M.C.C., M.A.P.A., C.F.G.A., andA.R.S.; analytical validation, S.R.F., and A.P.S; characterization of nanosystems S.R.F., K.P.S., S.S.G., A.R.P., and K.P.; project management, S.R.F., A.R.S., and C.F.G.A.; financing acquisition, S.R.F., P.T.B., H.C.C.F.N., A.R.S., and C.F.G.A.; Written, S.R.F., B.P.T.M., K.P.S., and C.F.G.A. All authors read and agreed with the published version of the manuscript.

Funding

This work was supported by grants from Universidade Federal Fluminense (PROPPI/UFF), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Grant [001], Programa de Biotecnologia da Universidade Federal Fluminense (UFF), Programa de Pós Graduação em Biologia Molecular Celular (UNIRIO), Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) Grants (E-26/010.000983/2019, E-26/203.290/2017, and E-26/2010.592/2019, E-26/201.448/2021), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Instituto Oswaldo Cruz, FIOCRUZ.

Availability of data and material

The authors declare that the data supporting this study’s findings are available within the paper and its Supplementary Information files. Should raw data files be needed in another format, they are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no conflict of interest.