Novel chloroquine loaded curcumin based anionic linear globular dendrimer G2: a metabolomics study on Plasmodium falciparum in vitro using 1H NMR spectroscopy

Abstract

Due to side-effects and inefficiency of the drugs used in malaria treatment, finding alternative medicine with less side-effects has attracted much attention. In this regard, in the present study, nanocomposite synthesized and its effects on the metabolites of P. falciparum were investigated. Subsequent to synthesis of nanocomposites, characterization was carried out using nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC-MS), scanning electron microscopy, dynamic light scattering and Fourier-transform infrared tests. Solubility and drug release were measured and its toxicity on Vero cell was assessed using the MTT assay. The antiparasitic effect of the nanocomposite on the metabolites of P. falciparum was investigated by 1H NMR spectroscopy. Among synthesized nanocomposites, the average size of 239 nm showed suitable solubility in water as well as slow drug release. The MTT assay showed no toxicity for Vero cell lines. Concentrations of 2.5 μg mL-1 of nanocomposite eliminated 82.6% of the total parasites. The most effected metabolic cycles were glyoxylate and dicarboxylate metabolism. In this study, 1H NMR spectroscopy was used with untargeted metabolomics to study the effect of the nanocomposite on P. falciparum. Playing an essential role in understanding drug-target interactions and characterization of mechanism of action or resistance exhibited by novel antiprotozoal drugs, can be achieved by targeting metabolic using LC-MS.

Keywords: Dendrimer G2; Protozoa; malaria; metabolomic; nanocomposite.

Fig. 1.

DLS showing the size distribution of NDC (A) and NDC-CQ (B). Polydispersity index is related to the distribution of the nanocomposite. The polydispersity index (PDI) average was 0.23 ± 0.01 (the PDI value which is close to 0 shows the homogeneous nanoparticle solution while the PDI value above 0.5 indicates the heterogeneous nanoparticle solution).

Fig. 2.

SEM image of NDC-CQ showed proper morphology with magnification 100 and 200k× respectively.

Fig. 3.

LC-MS spectrum of dendrimer G2.

Fig. 4.

¹H NMR spectra to confirm the covalent conjugation of nano-dendrimer to curcumin as the drug carrier (NDC).

Fig. 5.

FTIR spectra of ND (A), NDC (B) and NDC-CQ (C). The distinct peaks for each compound are shown. As the picture shows, CQ is loaded into NDC-CQ.

Fig. 6.

The cumulative release curve of NDC-CQ from NDC. As the graph shows, the pattern of drug-release from nanoparticles follows the slow release pattern.

Fig. 7.

Evaluation of cellular uptake of NDC-CQ (A) and curcumin as a control (B) at different times by FCM.

Fig. 8.

Percentage of survival of Vero cells exposed to synthesized NDC-CQ compared to CQ and the control group within 48 h. The viability effects showed that 20 μg mL⁻¹ NDC-CQ and NDC were perfectly nontoxic on the Vero cells compared with the control group. The values are expressed as mean ± standard deviation from three independent experiments with P < 0.001.

Fig. 9.

Superimposed spectra of NDC-CQ treated P. falciparum.

Fig. 10.

PLS-DA scores plot showing the separation of two groups (the control group and test group receiving the IC₅₀ dose of NDC-CQ).

Fig. 11.

Important features identified by PLS-DA. In VIP scores, the chemical shifts of the metabolites in the test group are shown in green (a decrease in metabolites) and red (an increase in metabolites).

Fig. 12.

Loadings plot for the selected PCs. In the loading plot the categorization of the two control and test groups is indicated by deleting the low value data, without changing the original meaning of the data. The chemical shift of the metabolites, that has the most changes relative to the centre of the plot, is determined.

Fig. 13.

Pathway analysis of the groups receiving NDC-CQ. Pathway analysis showing all matched pathways according to P values from pathway enrichment analysis and pathway impact values from pathway topology analysis.

Fig. 14.

Pathway analysis of the groups receiving CQ.