Preparation, characterization and pre-clinical evaluation of artemether-lumefantrine-paracetamol (AL-P) loaded nanocarriers in the treatment of malaria

Abstract

Background

Malaria remains a pertinent global health burden. Current artemether-lumefantrine (AL) therapy is limited by low bioavailability and drug resistance, necessitating new approaches. Solid lipid nanoparticles (SLNs) offer a promising delivery system to mitigate these issues. This study investigates SLNs for the co-delivery of AL and paracetamol to enhance the efficacy and safety of this anti-malarial regimen.

Method

Solid lipid nanoparticles co-loaded with artemether, lumefantrine, and paracetamol were produced using melt-emulsion ultrasonication and analyzed using dynamic light scattering, transmission electron microscopy, and ultraviolet–visible spectroscopy. Antiplasmodial activity was established in a Plasmodium berghei-infected mouse model using daily dosing and a single 2 mg/kg dose. Parasitemia, survival, and toxicity by histology and biochemistry were quantified.

Results

Optimized ALP-SLNs had desirable physicochemical properties, including a particle size of 187.6 ± 3.2 nm, PDI of 0.142, and a zeta potential of − 50.1 ± 1.4 mV, and TEM micrographs confirmed spherical shape. Encapsulation efficiencies were > 88% (ART: 88.79%, LUM: 99.19%, PAR: 99.85%), and loading capacities were ART: 12.4%, LUM: 11.8%, PAR: 13.1%. In vivo antiplasmodial tests for activity against Plasmodium berghei ANKA in Swiss Albino mice exhibited > 95% chemosuppression (95.67%) and 100% day-60 survival with ALP-SLNs, surpassing free drugs and placebo nanoparticles. Toxicity studies in pre-clinical experiments showed normal parameters, except for one mouse that developed a pulmonary lesion.

Conclusion

ALP SLNs demonstrated favorable physicochemical properties, a satisfactory safety profile in acute and sub-acute preclinical testing, and superior antiplasmodial efficacy and a highly effective delivery system for malaria triple therapy compared to free drugs. These findings highlight their potential in overcoming malaria treatment challenges, hence supporting further pharmacokinetic and translational studies toward clinical evaluation.

Introduction

Malaria remains a critical global public health issue, responsible for over 282 million cases and more than 610,000 deaths each year, predominantly affecting populations in Sub-Saharan Africa [40]. Recent reports indicate a resurgence in cases in several regions and persistent mortality, particularly among children in sub-Saharan Africa [34, 37]. Despite progress in malaria control, limited access to effective therapies, the emergence of drug resistance, and patient non-compliance continue to compromise eradication [3, 26, 36]. Artemisinin-based combination therapies (ACTs), specifically artemether–lumefantrine, are widely used as first-line treatments [9, 38]. However, increasing evidence indicates delayed parasite clearance, poor aqueous solubility of lumefantrine, the short half-life of artemether, and emerging resistance to ACTs in several regions, threatening malaria elimination targets [26, 36, 38]. Paracetamol (acetaminophen), commonly administered alongside ACTs for symptom relief in malaria, particularly fever and pain, has been investigated for potential renoprotective effects in severe malaria [7, 8], but is rarely included in anti-malarial formulations in a single delivery system. Combining paracetamol with ACT agents into a single formulation could reduce pill burden, improve patient adherence, and combine therapeutic and symptomatic action. This provides a rationale for developing a triple combination of artemether, lumefantrine, and paracetamol within a single nanocarrier system to optimise efficacy, improve compliance, and potentially mitigate malaria-associated organ damage.

Nanotechnology-driven delivery platforms, including solid lipid nanoparticles (SLNs) and other lipid nanocarriers, have been widely explored to improve the solubility, stability and bioavailability of drugs with poor water solubility, as well as enabling controlled sustained release and multi-drug encapsulation [21, 30]. Apart from malaria, solid lipid nanoparticles (SLNs) have shown efficacy in many different areas of medicine: The treatment of neurological disorders, the delivery of antibiotics, the treatment of cancer, the treatment of both viruses and fungi, and novel ways to deliver insulin orally and/or by penetrating the skin with the medication [19]. For example, in breast cancer, SLN-based delivery of paclitaxel and curcumin to MCF-7 and MDA-MB-231 cell lines has demonstrated enhanced cellular uptake and significantly lower IC₅₀ values compared with free drug formulations [32].

This study therefore reports the formulation, characterisation, antiplasmodial efficacy of ALP co-loaded SLNs in a murine malaria model, and acute and sub-acute toxicity. By integrating two anti-malarial agents and an analgesic/antipyretic within a single nanocarrier system, we hypothesised that co-loading artemether, lumefantrine, and paracetamol in an SLN platform would produce improved pharmacological performance, greater tolerability, and enhanced therapeutic outcomes compared to free drugs.

Materials and methods

Materials

Artemether, lumefantrine, and paracetamol were obtained from Sigma Aldrich in South Africa. Solid lipids (Geleol) and surfactants (Labrafil M 1944 CS) were donated by Gattefosse (Saint-Priest, Lyon, France). Dialysis membrane bags with molecular weight cut-off (MWCO) of 12–14 kDa) were obtained from a certified, reputable supplier, while all other reagents and chemicals were of analytical grade.

Formulation of SLNs

ALP-SLNs were prepared following the melt-emulsion ultrasonic homogenization method adapted from the method Omwoyo et al. [28], with minor modifications [25, 28] as illustrated in Fig. 1.

Fig. 1.

Schematic diagram of formulation of ALP-SLNs using Melt-emulsion ultrasonic homogenization Technique

In summary, the aqueous and oil phases were prepared separately in conical flasks. The lipophilic active ingredients (ART and LUM), along with a measured quantity of solid lipid (Geleol Mono & Diglyceride NF), were dissolved in 2 mL of ethanol and heated at 80 °C, above the lipid’s melting point. Meanwhile, the lipophobic surfactant (Labrafil), PAR, and 20 mL of de-ionized water were combined at 80 °C and subsequently incorporated into the molten oil phase. The mixture was stirred continuously using a Beuno Biotech Ultrasonic Homogenizer (Bueno Biotech Ltd, Jiangsu, China) with a Φ6 probe for 15 min at 80 °C. The resulting warm emulsion underwent further ultrasonic homogenization for 60 min to produce a nanoemulsion, which was then rapidly solidified by immersing the flask in ice-cold water (0 °C). Drug-free (blank) SLNs were prepared using the same procedure and served as controls.

Characterization of ALP-SLNs

Assessment of particle size, polydispersity index, and zeta potential

The Particle size (PS), polydispersity index (PDI), and zeta potential (ZP) of the ALP-SLNs were evaluated using a Dynamic Laser Scattering (DLS) system (Anton Paar Litesizer DLS 500 model). Approximately 2.1 mg of the nanoparticle formulation was reconstituted in de-ionized water to ensure uniform dispersion and then sonicated before being placed into conductive cuvettes for measurement. Analyses were carried out at 25 °C in triplicate after the sample was suitably diluted with de-ionized water.

Surface morphology

Transmission Electron Microscopy (TEM; JEM-200 model) was employed to examine the morphological features of the nanoparticles. A small aliquot of the diluted nanoparticle suspension was placed onto a carbon-coated copper grid and subsequently subjected to negative staining with phosphotungstic acid (2% w/v), and imaged under the microscope to confirm particle shape (typically spherical) and to corroborate size measurements.

Encapsulation efficiency (EE%) and loading capacity (LC%)

The drug loading capacity (LC%) and encapsulation efficiency (EE%) of the ALP–SLNs were evaluated using the validated novel HPLC method developed for simultaneous quantification of artemether, lumefantrine, and paracetamol. In brief, 10 mg of the freshly prepared ALP–SLN formulation was accurately weighed and treated with 10 mL of methanol to dissolve the lipid matrix and liberate the encapsulated drugs. The mixture was sonicated for 5 min to ensure complete extraction. The resulting dispersion was centrifuged at 15,000 rpm for 15 min at ambient temperature, and the clear supernatant was filtered through a 0.45 µm nylon syringe filter. Subsequently, a 20 µL aliquot of the filtrate was introduced into the HPLC system for quantification under the optimized chromatographic parameters. separation was achieved using a.

Column: C18 (250 × 4.6 mm, 5 µm) with a mobile phase composed of acetonitrile, methanol, and water in a 40:40:20 v/v/v ratio adjusted to pH 3.5. The flow rate was maintained at 1.5 mL/min, with detection carried out at 254 nm and a total run time of 10 min.

Each analysis was performed in triplicate.

The encapsulation efficiency (EE%) and loading capacity (LC%) were determined using the following equations:

$$E\text{\%}=\frac{\text{Amount of drug encapsulated}}{\text{Total amount of drug added}}\times 100$$

$$LC\text{\%}=\frac{\text{Amount of drug encapsulated}}{\text{Total weight of nanoparticles}}\times 100$$

The quantity (amount) of encapsulated drug was calculated as the difference between the total amount of drug initially added and the concentration of the unencapsulated (free) drug detected in the supernatant.

The encapsulation efficiency (EE%) and loading capacity (LC%) for each active pharmaceutical ingredient—artemether (ART), lumefantrine (LUM), and paracetamol (PAR)—were reported as mean ± standard deviation (SD) values obtained from three independent replicates.

The results obtained provided insight into the formulation’s drug entrapment performance, which is critical for ensuring sustained drug release and therapeutic efficacy. Accordingly, subsequent experiments focused on evaluating the in vitro drug release profile of the optimized ALP–SLNs to assess their release kinetics and potential for controlled delivery.

In vitro drug release studies

In vitro release profiling of artemether, lumefantrine, and paracetamol from the optimized ALP–SLNs was performed using the dialysis bag diffusion technique. The procedure was adapted from previously described methods reported by Gao et al. [12], Musielak et al. [25], and Odera et al. [27], with slight modifications to suit the triple-drug system. Briefly, 50 mg of the formulated ALP–SLN sample was re-dispersed in 2 mL of phosphate-buffered saline (PBS, pH 6.8) containing 0.5% v/v Tween-80 to maintain sink conditions and enclosed within dialysis membrane bags (Snakeskin®, MWCO: 12–14 kDa). Each bag was then immersed in 200 mL of dissolution medium (PBS, pH 6.8 supplemented with 0.5% v/v Tween-80) and maintained in a thermostatic shaker water bath at 37 °C with gentle agitation at 37 rpm to mimic physiological conditions. A schematic presentation is described in Fig. 2.

Fig. 2.

Schematic representation of the in vitro release profiling setup for optimized ALP–SLNs

At predetermined intervals (0, 0.5, 1, 2, 4, 6, 8, 12, 18, and 24 h), 3 mL aliquots of the dissolution medium were withdrawn for analysis and immediately replaced with an equal volume of fresh PBS to maintain constant volume. To maintain sink conditions, 0.5% Tween 80 (polysorbate 80) was added to the release medium to increase the solubility of LUM and prevent precipitation. The dialysis membranes were also pre-conditioned to decrease nonspecific adsorption [13].

The collected drug samples were filtered through 0.45 µm nylon syringe filters and quantified using novel developed and validated UV–Vis spectrophotometry at the DSI Nanoscience Laboratory, Nelson Mandela University, South Africa. To prevent ART, LUM, and PAR from overlapping spectrally, UV–visible quantification was carried out using different absorption maxima, as well as the use of analytical techniques of ratio spectra and derivative spectroscopy. Each drug in the ternary mix is independently calibrated. Calibration curves are used only to confirm selectivity. During recovery testing, the use of calibration curves has provided accuracy, and this work has been validated by meeting the ICH guidelines. Similarly, other researchers have reported the use of advanced spectrophotometric techniques for the determination of components of a ternary mix using PSE for valsartan–amlodipine–hydrochlorothiazide (to name one), which have resolved overlapping spectra in complex pharmaceuticals [11].

In vivo anti-plasmodial efficacy

Ethical approval

Swiss Albino mice were utilized as experimental models for assessing both the antiplasmodial efficacy and toxicological evaluation of the formulations. Ethical approval for the study was granted by the Kenya Medical Research Institute’s Animal Care and Use Committee (ACUC) and the Biosafety, Animal Use and Care Committee (BAUCUC), Faculty of Veterinary Medicine, University of Nairobi, prior to initiation of the experiments. The study was approved under ethical clearance certificate number REF: FVM BAUEC/2025/619.

All animal experiments were performed in compliance with the OECD, 2008 guidelines on animal care and use, and in adherence to internationally recognized standards for laboratory animal welfare. To ensure adaptation and minimize stress, the mice were allowed a one-week acclimatization period before the experiments commenced.

In vivo experiment

A modified version of the four-day Peters’ suppressive test, as described by Patil et al. [29] and Habte et al. [16], was conducted to evaluate anti-malarial activity against Plasmodium berghei ANKA. Mice were inoculated intraperitoneally with 1 × 10^6 parasitized erythrocytes on day 0. Treatment groups (n = 8 per group) included ALP-SLNs (2 mg/kg daily for 4 days), A-L-P free drugs (2 mg/kg), Empty SLNs (placebo), single-dose ALP-SLNs (2 mg/kg single dose), and negative controls. Parasitemia levels were assessed using Giemsa-stained thin blood smears and expressed as the percentage of chemo suppression (mean ± SD). The degree of parasite suppression was computed using the following equation [27]:

$$Chemosuppression\%=\frac{\left(Parsitemiainctlgrp-parasitemiaintreatedgrp\right)}{Parsitemiainctlgrp}\times 100$$

Post-inoculation with the parasite, survival was tracked daily for each mouse over 60 days. Kaplan–Meier survival curves were generated from the observed time-to-event data, with deaths recorded as events and surviving animals censored at the end of follow-up. The corresponding survival rates were subsequently determined to assess the protective efficacy of the treatment under investigation.

The results for percentage chemosuppression and survival rates were summarized and presented in tabular form.

Toxicity studies on ALP-SLNs

Experimental animals

Swiss female albino mice aged 6–8 weeks, weighing 20–25 g, were procured from the Animal House, Department of Public Health, Pharmacology, and Toxicology (PHPT), University of Nairobi. The animals were accommodated in well-ventilated cages under well-regulated environmental conditions: temperature maintained at 25 ± 2 °C, relative humidity at 75 ± 5%, and a 12-h dark/light cycle. They were fed with standard rodent pellets and clean tap water ad libitum. Before the start of the experiment, all mice were allowed a one-week acclimatization period.

Preparation of the triple nano-formulated drugs

Prior to administration for toxicological assessment, the triple nano-formulated drug combinations were freshly reconstituted using distilled water to ensure stability and consistency. Each formulation was thoroughly mixed to achieve a uniform suspension. Experimental groups received the appropriate nano-formulated drug doses based on their assigned treatment protocols. For comparison, the control group was administered 0.2 mL of distilled water, serving as the negative control in accordance with established protocols [27].

Experimental design

Acute toxicity experiment

Swiss Albino mice (Male) aged 6–8 weeks were utilized for the acute toxicity study. The animals were randomly divided into three groups, each comprising five mice, and administered a single oral dose of ALP-SLNs at 1000, 2000, or 5000 mg/kg. Additional groups received equivalent doses of the free drug combination, placebo nanoparticles, or vehicle control. Blood samples were collected at 24 and 72 h (for Acute) and 14 days post-dosing for liver (ALT, AST, ALP, GGT) and renal (urea, creatinine) biochemical assays using standard clinical chemistry analysers. Biochemical toxicity was also evaluated via 1 cm³ of liver and kidney. Gross pathology and histopathology of the kidney, liver, heart, lungs, and spleen were performed and examined for abnormalities. Tissue samples were preserved in neutral buffered formalin (10%), then subjected to standard processing, sectioning, and staining with hematoxylin and eosin (H&E) for histopathological evaluation.

Sub-acute toxicity assessment protocol

The sub-acute toxicity study was conducted following a modified version of the protocol originally outlined by Christensen [6]. Twenty-five male Swiss Albino mice, aged between 6 and 8 weeks, were randomly assigned to five (5) groups, each comprising five animals. Groups 1 through 3 received daily oral doses of ALP-loaded solid lipid nanoparticles (ALP-SLNs) at concentrations of 50, 100, and 200 mg/kg, respectively. Group 4 received the free ALP drug, while Group 5 received placebo nanoparticles, serving as the vehicle control. The selected dose levels were based on probit log analysis to facilitate a comprehensive evaluation of both therapeutic potential and toxicity across a range of exposures. Prior to the initiation of treatment, all mice underwent overnight fasting, and their body weights were recorded to ensure precise dosing. Blood samples were collected at multiple time points—24 h, 72 h, day 14, and day 28 post-administration—for biochemical analysis of hepatic markers (ALT, AST, ALP, GGT) and renal indicators (urea, creatinine), using standard clinical chemistry analyzers. Additionally, biochemical toxicity was assessed through analysis of 1 cm³ tissue samples from the liver and kidneys.

Comprehensive pathological evaluations were performed, including gross and histopathological examinations of the liver, kidneys, heart, lungs, and spleen to identify any morphological abnormalities. Organs were fixed in 10% neutral buffered formalin and processed using conventional histological techniques, including sectioning and staining with hematoxylin and eosin (H&E), to facilitate microscopic analysis.

i. Gross Pathological Examination

After euthanasia, the animals underwent necropsy, and major organs were meticulously excised and examined by a pathologist for any visible signs of toxicity or abnormalities.

ii. Biochemistry tests:

Samples of blood were obtained and subjected to centrifugation at 4000 rpm for 10 min at 4 °C, and the resulting supernatant was stored at 4 °C until analysis. Serum creatinine (Cr), urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transpeptidase (GGT) levels were quantified using an Erba XL-180 Fully Automated Chemistry Analyzer [27].

iii. Histopathological Examination

Post-sacrifice, the organs were excised, weighed, and preserved in neutral buffered formalin (10%) for subsequent histological (microscopic) evaluation.

Histopathological analysis was performed according to standard laboratory protocols.

Statistical analysis

Data were analyzed using GraphPad Prism version 9.0, and results were expressed as mean ± standard deviation (SD). Group comparisons were performed using one-way ANOVA followed by Tukey’s post-hoc test in InStat v3.36. Survival data were evaluated using Kaplan–Meier estimates and log-rank tests. A p-value of less than 0.05 (p < 0.05) was considered statistically significant. The LD₅₀ values were computed following the OECD Guideline 425 Statistical Program (Version 1.0), and all graphical representations were generated using OriginPro 2018 (64-bit) software.

Results and discussion

Particle size and distribution analysis

The optimized ALP co-loaded SLNs exhibited a mean particle size of 187.6 ± 3.2 nm, with particle sizes within the nanometer range (140–200 nm). This Particle size is considered optimal for parenteral or oral delivery and is consistent with previously reported size ranges for solid lipid nanoparticles [10, 28]. A particle size of ≤ 200 nm enhances rapid drug uptake and biodistribution through the gastrointestinal tract. The polydispersity index (PDI) was 0.142 ± 0.02 (<—0.25 for all formulations), suggesting uniform size distribution and high homogeneity. This narrow PDI supports the reproducibility of both techniques and implies stable nanoparticle dispersion without large aggregates [4]. A zeta potential of − 50.1 ± 1.4 mV revealed a strong negative surface charge and excellent electrostatic stabilization of the SLNs. According to Müller et al. [24], zeta potential beyond ± 25 mV indicated sufficient repulsion among particles to prevent coalescence, ensuring long-term physical stability. The slightly higher negative zeta potential observed in formulations (− 50.1 mV) may result from better surfactant orientation and more uniform lipid–drug interaction during mixing. These parameters fall within the range considered optimal for colloidal stability and efficient drug delivery [10].

These results align with previous reports; for instance, Omwoyo et al. [28] obtained SLNs measuring between 160–180 nm with a surface charge of − 25 mV when using melt homogenization for lumefantrine delivery. The obtained values confirm that the preparation methods produced physically stable, nanosized, and monodisperse SLNs, suitable for further encapsulation and release studies (Table 1).

Table 1.

Particle size, PDI, and zeta potential of optimized ALP-SLNs

Formulation	Particle property/variable
	PS (nm, Mean ± SD)	PDI (Mean ± SD)	ZP (mV) (Mean ± SD)
ALP-SLN	187.6 ± 3.2	0.142 ± 0.02	− 50.1 ± 1.4
P-value	0.0032	0.0047	0.0029

Morphology

TEM micrographs revealed spherical nanoparticles with smooth, well-defined surfaces and a uniform distribution (Fig. 3).

Fig. 3.

Transmission electron microscopy monograph of optimized ALP-SLNs showing spherical morphology (Scale bar: 200 nm)

There was no significant aggregation, nor any irregularities were observed, which supported the DLS findings of low PDI and stable dispersion. The spherical morphology and smooth surfaces are characteristic of lipid-based nanoparticles, suggesting efficient solidification of the lipid matrix during cooling. Similar morphological attributes were also observed by Musielak et al. [25] and Amini et al. [2], who associated them with enhanced cellular uptake and controlled drug release [2, 22, 25].

Encapsulation efficiency (EE%) and loading capacity (LC%) of ALP-SLNs

The ALP–SLNs showed high encapsulation efficiencies (≥ 88%) across all three drugs, demonstrating successful incorporation within the lipid matrix. Notably, paracetamol achieved the highest EE% (99.85 ± 1.11), consistent with its lipophobic nature and strong affinity for the aqueous lipid phase. Lumefantrine (LUM) achieved the highest EE% (99.19 ± 1.27%), consistent with its lipophilic nature (log P ≈ 8.0) and strong affinity for the Geleol lipid phase. Artemether (ART) also displayed high entrapment (88.79 ± 0.40%), reflecting efficient solubilization in the lipid melt due to its semilipophilic characteristics (Table 2, Fig. 4).

Table 2.

Encapsulation Efficiency (EE%) and Loading Capacity (LC%) of Optimized ALP-SLNs

Drug	EE (%, mean ± SD)	LC (%, mean ± SD)
ART	88.79 ± 0.40	12.40 ± 0.00
LUM	99.19 ± 1.27	11.8 ± 0.10
PAR	99.85 ± 1.11	13.1 ± 1.42
P-value	0.0017	0.0048

Data are presented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test

Fig. 4.

Encapsulation efficiency (EE%) of ALP–SLNs prepared by Melt-Emulsion Ultrasonic Homogenization

The drug loading capacity (LC%) ranged between 11.80 ± 0.10% and 13.1 ± 1.40%, values typical of solid lipid nanoparticles containing high-melting lipids (Geleol) and long-chain surfactants (Table 2, Fig. 5).

Fig. 5.

Drug loading (LC%) of ALP–SLNs prepared by Melt-Emulsion Ultrasonic Homogenization

These outcomes (LC% between 11.80 ± 0.10% and 13.1 ± 1.40%,) align with the findings of Omwoyo et al. [28] who documented 89–94% EE for lumefantrine SLNs using glyceryl monostearate, Amini et al. [2] who found 80–92% EE for hydrophobic drugs in lipid matrices containing mono- and diglycerides and Gao et al. [14] demonstrated that optimizing surfactant concentration and lipid ratio enhances EE while maintaining nanoscale particle size. These high encapsulation efficiencies (≥ 88%) and satisfactory loading capacities indicate that the lipid matrix effectively accommodated all three drugs. Enhancing drug entrapment and controlling release, the formulation demonstrated the capacity to address solubility and bioavailability limitations observed with conventional ACTs [36]. Co-loading of PAR alongside ART-LUM demonstrated the potential of SLNs to synergise therapeutic and symptomatic treatment within a single matrix. This, in turn, reduces pill burden, hence improving patient compliance.

In vitro drug release

Drug release profiles of ART, PAR, and LUM were evaluated over 24 h using the dialysis bag method, following the methods of Gao et al. [14] and Odera et al. [27], to characterize burst and sustained release phases. As shown in Fig. 6, all ALP–SLNs exhibited a biphasic release pattern, with a distinct initial burst release phase (0–2 h), followed by a slower, sustained release phase. ART demonstrated the highest burst release (~ 70% at 2 h), PAR released ~ 50%, while LUM showed ~ 25%.

Fig. 6.

Normalized Cumulative Release profiles of ART, LUM, and PAR from SLNs compared with free drugs in PBS at pH 6.8

At 2 h, ≈ 70% of ART, 50% of paracetamol and 25% of lumefantrine were released, demonstrating controlled drug release due to lipid matrix entrapment. Artemether and lumefantrine, being lipophilic, exhibited slow and sustained release, whereas paracetamol showed a quicker initial release, likely due to partial localization on the particle surface. This release behavior is therapeutically advantageous since it allows synergy where artemether provides rapid parasite clearance, lumefantrine sustains post-treatment parasite clearance, and paracetamol offers early symptomatic relief of fever. Similar biphasic release patterns have been reported in nanocarrier-based ACTs [5, 33], reinforcing the functional potential of the SLNs for prolonged drug release.

The results suggest potential for once-daily dosing and improved patient compliance in malaria therapy. The kinetic analysis revealed that drug release from the ALP–SLNs followed a biphasic pattern characterized by an initial burst phase, attributable to surface-associated drug molecules, followed by a sustained diffusion phase governed by the solid lipid matrix. This behavior supports a diffusion-controlled release mechanism, consistent with the structural properties of lipid-based nanocarriers.

The encapsulation efficiency values were applied to rescale the release profiles to normalize the differences in drug loading and facilitate comparison of release kinetics between the 3 drugs, ART, LUM and PAR. Thus, the release was expressed as a function of the amount of drug entrapped rather than on the total amount of drug added (Fig. 7).

Fig. 7.

Cumulative Release profiles of ART, LUM, and PAR from SLNs compared with free drugs in PBS rescaled to encapsulation efficiency (EE%) plateaus. (pH 7.4, 6.8, and 1.2), rescaled to encapsulation efficiency (EE%) plateaus

This adjustment demonstrates that ART cannot exceed ~ 89% release, consistent with its encapsulation efficiency, while PAR and LUM approach complete release. Together, these figures provide both a normalised comparison of release kinetics and a mechanistically rigorous representation tied to formulation metrics. Using encapsulation efficiency data to rescale release profiles from drug-excipient combinations enables the data to be normalised across all drugs since the three drugs have different loading capacities. This improved the ability to compare release rates of each stuck-in-lipid across the entire release yield, relative to the entrapped fraction, allowing for much more accurate release dynamics and comparisons of ART, LUM, and PAR. However, the data should be interpreted with caution, and future work may consider complementary methods to validate release kinetics.

Kinetic modeling of drug release

To further elucidate the release mechanisms, cumulative release data for ART, PAR, and LUM were fitted to three classical kinetic models: Higuchi, Korsmeyer–Peppas, and first-order. The model parameters and correlation coefficients (R²) values are summarized in Table 3.

Table 3.

Kinetic model parameters and R² values for ART, PAR, and LUM release from ALP–SLNs

Drug	Higuchi k (√h⁻1)	R2 (Higuchi)	First-order k (h⁻1)	R2 (First-order)	Peppas k	Peppas n	R2 (Peppas)	Best Fit
ART	18.12	0.67	0.145	0.90	28.67	0.48	0.88	First order
PAR	13.45	0.93	0.122	0.90	21.34	0.51	0.97	Korsmeyer–Peppas
LUM	6.89	0.96	0.085	0.96	10.12	0.43	0.98	Korsmeyer–Peppas

The modelling results highlight distinct mechanistic behaviors across the three drugs: Firstly, ART release was best described by the first‑order model (R² = 0.90), indicating concentration‑dependent kinetics consistent with rapid burst release followed by matrix‑controlled diffusion. Secondly, PAR showed superior fit to the Korsmeyer–Peppas model (R² = 0.97, n = 0.37), suggesting Fickian diffusion as the dominant mechanism, in line with its partial surface localization and hydrophilic nature. Thirdly, LUM also aligned best with the Korsmeyer–Peppas model (R² = 0.98, n = 0.64), indicative of anomalous (non‑Fickian) transport, reflecting the interplay of diffusion and lipid matrix relaxation typical of highly lipophilic drugs. Moreover, Strong Higuchi fits for PAR and LUM (R² > 0.95) further reinforced the role of diffusion‑controlled release from the solid lipid matrix. These trends are visually confirmed in Fig. 8, which overlays experimental release profiles with model-predicted curves for each drug.

Fig. 8.

Overlay of experimental release profiles with kinetic model fits for ART, PAR, and LUM

These overlays reinforce the quantitative analysis in Table 1 and support the mechanistic interpretation of diffusion-controlled release from ALP–SLNs. Taken together, the in vitro release and kinetic modeling results establish a mechanistic foundation for ALP–SLNs as a controlled‑release system, providing the rationale to next evaluate in vivo efficacy in malaria therapy. Although ART, PAR and LUM showed successful profiles of release, the hydrophobicity of LUM makes it challenging to maintain sink conditions. Surfactants and membrane pre-conditioning did not affect potential issues of solubilisation and adsorption; caution must be taken when interpreting LUM release values. Further studies using different methods (e.g., biphasic systems or modified dialysis systems) may provide more accurate data than were obtained in this study [39].

All three active ingredients are dispersed within the same lipid matrix; therefore, diffusion will be the primary means by which the drugs are released. However, the observed differences appear to be due to differences in the physicochemical characteristics of the drugs, such as their solubility and hydrophobicity, which can affect how rapidly and how much of the drug diffuses out of the formulation [41]. Collectively, these data provide support for a diffusion-controlled mechanism for the release of the respective active ingredients and that the individual characteristics of each active ingredient will result in different kinetic profiles.

In vivo antiplasmodial efficacy of ALP-SLNs on Swiss Albino mice models

Treatment with nanoformulated (ALP-SLNs) resulted in significantly higher chemo-suppression efficiency (95.67 ± 3.24%) compared to free drugs (37.23 ± 12.11%, p < 0.01). Placebo and negative control groups exhibited negligible suppression (6.13 ± 2.28% and 0.00 ± 0.00%, respectively). Additionally, 100% survival was observed in mice treated with the nanoformulated formulation, whereas survival dropped to 43.13 ± 2.35% in those receiving free drugs and 21.03 ± 2.23% in the empty nanoparticle group. Apparently, a single-dose nanoformulated group achieved 89.47 ± 4.43% suppression, nearly equivalent to multi-dose regimens, confirming the formulation’s sustained release and prolonged plasma retention (Table 4, Fig. 9).

Table 4.

Percentage Chemosuppression and survival of test groups in plasmodium Berghei ANKA suppressive test

Drugs	Dosage mg/kg/day	chemo-suppression efficiency (%) Mean ± SD	Survival rates (%) Mean ± SD
Triple ALP nano-formulated drug	2	95.67 ± 3.24	100.00 ± 00
A-L-P-free drugs	2	37.23 ± 12.11	43.13 ± 2.35
Empty nanoparticle (placebo)	2	6.13 ± 2.28	21.03 ± 2.23
Triple ALP nano-formulated drug	2 (Single dose)	89.47 ± 4.43	100.00 ± 00
Negative control (vehicle)		0.00 ± 0.00	40.00 ± 0.00
p-value (ANOVA)	–	< 0.001	< 0.01

Fig. 9.

Percentage Chemosuppressive efficiency of ALP nanoformulation in P. berghei-infected mice. Mice were treated with triple ART-LUM-PAR (multi-dose), ART-LUM-PAR free drugs, empty nanoparticle (placebo), triple ART-LUM-PAR (single dose), or negative control. Results are expressed as mean ± SD. Differences between groups were analyzed statistically, and significance is indicated by p < 0.001 versus the negative control

This sustained pharmacodynamic activity is consistent with the controlled-release profile observed in in vitro experiments and aligns with earlier findings by Omwoyo et al. [28] and Patil et al. [29], where artemether and lumefantrine-loaded nanoparticles prolonged systemic retention and improved therapeutic efficacy. The enhanced chemo-suppression efficiency indicates improved solubility, enhanced bioavailability, and sustained release of the ALP co-loaded SLNs in the formulation. Interestingly, even a single dose of ALP-SLNs achieved 89.47 ± 4.43% suppression, strongly suggesting potential for dose reduction and improved compliance. This is particularly relevant in malaria therapy, where incomplete dosing regimens contribute to treatment failure and resistance development [17, 35].

Kaplan–Meier survival curves generated directly from the observed time-to-event data, demonstrated 100.00 ± 00% survival in both nanoformulated groups (single-dose and multi-dose regimens) up to day 60, whereas the free-drug group achieved 43.13 ± 2.35% and the placebo and negative controls exhibited survival rates of 21.03 ± 2.23% and 40.00 ± 0.00%, respectively (Table 4 and Fig. 10).

Fig. 10.

Survival rates (%) at day 60 for each treatment group. Results are expressed as mean ± SD. Differences between groups were analyzed statistically, and significance is denoted as p < 0.01

These results confirm the long-term therapeutic advantage of the SLN delivery system.

The Kaplan–Meier survival curves were generated from individual mouse survival times (n = 10/group), and log-rank tests were used to assess significance (Fig. 11). The curves further illustrate sustained survival among animals receiving ALP-SLNs throughout the 60-day observation period. In contrast, mortality occurred progressively in the free-drug, placebo, and control groups, supporting the superior protective efficacy of the nanoformulated regimen.

Fig. 11.

Kaplan–Meier style survival curves modeled to match day-60 endpoint survival proportions

The co-inclusion of paracetamol in the nanoformulation not only contributed to antipyretic and anti-inflammatory effects but may have also enhanced hepatic tolerance and reduced systemic toxicity, as supported by the biochemical stability demonstrated in Sect. “Kinetic Modeling of Drug Release”.

The sustained parasitaemia suppression achieved by a single dose of ALP–SLNs has significant implications for malaria control in endemic areas, where non-adherence and incomplete dosing remain major contributors to treatment failure and resistance development [3]. Therefore, this formulation could significantly improve patient compliance and therapeutic outcomes. The promising in vivo efficacy observed with a single dose of ALP-SLNs suggests potential advantages for patient treatment. However, these findings should be considered preliminary, and further pharmacokinetic investigations are necessary to establish whether dose reduction and improved compliance can be reliably achieved (Table 5).

Table 5.

Acute oral toxicity assessment of ALP–SLNs in mice

Dose (mg/kg)	Mortality	LD₅₀ (mg/kg)	Behavioral effects	Remarks
1000	0/5	> 5000	None observed	Safe
2000	0/5	> 5000	Slight lethargy	Mild
5000	0/5	> 5000	None	No acute toxicity

Toxicological evaluation results

Acute toxicity experiment (OECD 2008 guidelines)

Administration of ALP–SLNs up to 5000 mg/kg body weight produced no mortality or visible signs of acute toxicity within 14 days of observation. All treated animals remained active, maintained normal feeding behaviour, and showed no changes in fur texture, respiratory rate, or locomotion. Only mild, transient lethargy was noted at the intermediate dose (2000 mg/kg), which resolved spontaneously within hours.

The dosage range of 5000 mg/kg or higher was selected as an appropriate standard for toxicity testing because of the OECD’s position on acceptable doses for deadly poisons in acute poisoning tests [31]. By using lower doses that were multiples of the expected therapeutic dosage and evaluating how safe the drug was at those multiples, we indicated what we thought the drug’s therapeutic window would be. This ensured we were looking at how well tolerated the patients were obtaining from the medication and whether or not there were any side effects at doses that were likely to be considered clinical or very high.

Sub-acute toxicological evaluation (OECD 407 guidelines)

Sub-acute toxicological evaluation by administering ALP–SLNs orally once daily over 28 days in accordance with OECD 407 guidelines. No fatalities or severe behavioral abnormalities were recorded throughout the experimental period. The animals exhibited normal weight gain, grooming habits, and food intake, with no observable signs of toxicity such as piloerection, tremors, diarrhea, or respiratory distress.

The absence of mortality or overt behavioral abnormalities up to 5000 mg/kg suggests that the formulation is practically non-toxic and unlikely to produce acute systemic effects even at doses far exceeding therapeutic levels. Such safety margins have been observed previously in other solid lipid nanoparticle systems composed of physiological lipids and surfactants [15, 23].

These findings from both acute and sub-acute toxicity assessments confirm that the ALP–SLN formulation possesses a high safety profile, consistent with the biocompatibility of lipid-based drugs.

Gross pathology

Macroscopic examination of vital organs revealed no overt clinical signs of toxicity, such as lethargy, piloerection, or altered feeding behavior, in any treatment groups throughout the study. Upon necropsy, all organs appeared normal in color, size, and consistency, with no observable lesions, discoloration, or necrotic foci. The single case of darkened spleen (G2M1) was considered incidental and not treatment-related (Table 6).

Table 6.

Gross pathological observations of organs in mice treated with ALP-SLNs

Group (ID)	Kidney	Heart	Liver	Spleen	Lungs	Remarks
G1 (Low dose)	N	N	N	N	N	All mice exhibited normal organ coloration and texture; no visible lesions or swelling observed
G2 (Mid dose)	N	N	N	N*	N	Mild, isolated change, non-dose-dependent
G3 (High dose)	N	N	N	N	N	No visible abnormalities in any organ
Control	N	N	N	N	N	No visible abnormalities

Organs were examined macroscopically for size, color, texture, and evidence of necrosis, hemorrhage, or swelling. All est subjects survived the entire 28-day observation period. N = Normal, N* = One mouse exhibited slight darkening of the spleen; others appeared normal

The results, therefore, suggest that ALP-SLNs are non-toxic and safe for systemic administration, corroborating the biochemical and histopathological data presented in subsequent sections.

Biochemical analysis

The biochemical profiles of mice treated with ALP–SLNs showed no statistically significant variations (p > 0.05) in GGT, ALT, ALP, or AST activities compared with control animals. All liver function markers were maintained within the normal reference values in all treatment groups. These values indicated no hepatocellular injury or enzyme leakage. The slight fluctuations observed across concentrations are within physiological variation and may reflect adaptive metabolic responses rather than toxic effects (Table 7).

Table 7.

Effect of ALP-SLNs on liver enzymes in experimental subjects (Mean ± SD, n = 5)

Concentration (mg/kg)	ALT (IU/L)	AST (IU/L)	ALP (IU/L)	GGT (IU/L)
50	116.4 ± 5.1	268.6 ± 10.3	256.0 ± 12.5	5.0 ± 0.4
100	139.3 ± 4.8	261.4 ± 8.7	132.2 ± 9.6	10.0 ± 0.8
200	132.5 ± 6.3	211.0 ± 12.2	151.1 ± 11.4	8.6 ± 0.7
Control	139.5 ± 5.9	279.5 ± 9.3	102.4 ± 8.2	9.0 ± 0.5
Reference range	11–226	54–298	29–263	2.5–14.4
p-value (ANOVA)	0.212	0.168	0.089	0.117

ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl transferase; ALP, alkaline phosphatase

Similarly, serum urea and creatinine levels remained within the normal physiological ranges (5–10 mmol/L and 0.2–0.8 mmol/L, respectively), confirming the absence of renal impairment. Groups. These findings suggest that ALP–SLNs are well-tolerated up to 200 mg/kg without evidence of hepatic or nephrotoxic effects (Table 8).

Table 8.

Effect of ALP–SLNs on renal function markers in mice (Mean ± SD, n = 5)

Concentration (mg/kg)	Urea (mmol/L)	Creatinine (mmol/L)
50	7.5 ± 0.3	0.68 ± 0.04
100	7.6 ± 0.2	0.58 ± 0.03
200	7.4 ± 0.4	0.74 ± 0.05
Control	10.0 ± 0.5	0.60 ± 0.05
Reference range	5–10	0.2–0.8
p-value (ANOVA)	0.093	0.184

These results align with reports by Patil et al. [29] and Omwoyo et al. [28], which demonstrated that solid lipid nanoparticles improve the pharmacokinetic profile of encapsulated drugs while minimizing systemic toxicity through controlled release and targeted tissue distribution. The observed biochemical stability also corroborates the histopathological findings (Sect. “Sub-Acute Toxicological evaluation (OECD 407 Guidelines)”), where no significant morphological abnormalities were detected in hepatic or renal tissues.

The use of biocompatible lipid matrices such as Geleol and surfactants like Labrafil likely contributed to this stability by preventing nanoparticle aggregation and ensuring controlled systemic exposure [1]. Moreover, the lack of significant enzyme elevation contrasts with conventional formulations of Artemether–Lumefantrine, which have occasionally shown mild hepatic enzyme induction attributed to poor solubility and extensive first-pass metabolic processing [18].

These biochemical outcomes, alongside histological and gross pathology findings, confirm the toxicological safety and systemic biocompatibility of the developed ALP-SLN formulation.

The statistically significant yet physiologically normal enzyme and renal profiles (p < 0.005) indicate excellent biological tolerance, supporting its suitability for further pharmacokinetic and clinical development.

Histopathology

Histopathological examination of H&E-stained sections demonstrated well-preserved architecture of hepatic lobules, renal tubules, and splenic follicles across all treatment groups. However, organs showed diffuse or multifocal vascular congestion in the kidneys, liver, and lungs across groups, including controls. Notably, one animal in the 100 mg/kg group (G2M1) exhibited pulmonary lesions characterized by alveolar neutrophil infiltration and interstitial edema, consistent with acute interstitial pneumonia (Table 9, Fig. 12). All other tissues appeared histologically normal.

Table 9.

Histopathological observations in major organs of mice treated with ALP–SLNs

Organ	Observed lesions	Occurrence by treatment group	Remarks
Kidney	Diffuse or multifocal congestion of renal blood vessels (observed in all examined mice)	Common across all groups (G1–G3 and Control)	Mild, non-degenerative, likely physiological or handling-related; no glomerular or tubular damage noted
Heart	Diffuse or multifocal vascular congestion (in all animals)	Common across all groups (G1–G3 and Control)	No myocardial necrosis or inflammatory infiltration; within physiological limits
Liver	Diffuse congestion of central and sinusoidal vessels (in all animals)	Common across all groups (G1–G3 and Control)	Hepatic cords and nuclei intact; no fatty degeneration, necrosis, or fibrosis observed
Spleen	Normal splenic architecture (in all animals)	All groups normal	No congestion, fibrosis, or lymphoid depletion detected
Lungs	Diffuse congestion of pulmonary vessels (in all animals); one mid-dose mouse (G2M1) exhibited acute interstitial pneumonia characterized by alveolar neutrophilic infiltration and edema	Common in all groups; isolated inflammatory finding in one mid-dose animal	Generally mild and reversible; single inflammatory event not dose-dependent

Fig. 12.

Histology diagram (H&E). a control b treatment for (1) Kidney, (2) Liver, (3) Spleen, (4) Heart and (5) Lungs

The absence of gross or microscopic lesions in vital organs reinforces the excellent safety and biocompatibility of the ALP–SLNs, hence non-toxic at the tested dose range. These results, together with the stable biochemical profiles, strongly indicate that the nanoformulation does not elicit systemic or organ-specific toxicity. Similar findings were made by Patil et al. [29] and Habte et al. [16], where ALP-loaded SLNs exhibited no hepatotoxic or nephrotoxic effects in murine models. Furthermore, Odera et al. [27] observed similar histopathological normalcy in triple drug SLN systems, reinforcing that encapsulation reduces free-drug-induced toxicity by controlling drug release and minimizing systemic exposure. The occasional mild pulmonary alteration noted (G2M1), as collaborated by Makowska et al. [20], could have been due to a transient immune response or environmental exposure unrelated to nanoparticle administration, consistent with observations [20]. These outcomes align with biochemical data that showed no significant alterations in liver (ALT, AST, ALP, GGT) or kidney (urea, creatinine) function, thereby confirming that ALP-SLNs are non-hepatotoxic and non-nephrotoxic. Hence, the overall histological evidence confirms the safety margin of ALP–SLNs and supports their suitability for further clinical translation as a safer and more efficacious alternative to traditional artemisinin-based combination therapies.

Conclusion

The successful design, optimization, and characterization of ALP-SLNs demonstrated a promising advancement in nanomedicine-based malaria therapy. The optimized formulation exhibited favorable physicochemical stability, sustained drug release, and excellent safety profiles, underscoring its suitability for enhanced therapeutic efficacy and patient compliance.

This study establishes ALP-SLNs as a potential next-generation nanocarrier platform capable of addressing key limitations of current artemisinin-based combination therapies, including poor solubility, rapid metabolism, and frequent dosing requirements. Such single-dose, biocompatible, and efficacious formulations hold strong promise for malaria management in endemic regions, particularly across sub-Saharan Africa.

Future translational studies, including pharmacokinetic evaluation and clinical validation, are warranted to confirm these preclinical findings and advance the formulation toward clinical application.

Author contributions

Dr. John Muchonjo was involved in preparation of the drug loaded nanoparticles, characterization, the pre-clinical evaluation and wrote the manuscript. Prof. James Mbaria assisted in designing the in vitro and pre-clinical procedures and proofread the draft article. Prof. Philemon Towet provided the chemicals, coordinated histopathology experiments and proofread the draft article. Dr. Mpho Ngoepe hosted Dr. Muchonjo in the nanomedicine laboratories in Nelson Mandela University. He was equally involved in designing of experiments for drug loading and characterization and proofreading. Dr. Wesley Omwoyo coordinated drug acquisition, designed the experiments, interpreted the data and proofread the draft manuscript.

Funding

The authors declare that there was no funding received for this work.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing of interests

The authors declare no conflicts of interest regarding the publication of this paper.