In vitro antiplasmodial activity of PDDS-coated metal oxide nanoparticles against Plasmodium falciparum

Abstract

Malaria is the most important parasitic disease, leading to annual death of about one million people and the Plasmodium falciparum develops resistant to well-established antimalarial drugs. The newest antiplasmodial drug from metal oxide nanoparticles helps in addressing this problem. Commercial nanoparticles such as Fe₃O₄, MgO, ZrO₂, Al₂O₃ and CeO₂ coated with PDDS and all the coated and non-coated nanoparticles were screened for antiplasmodial activity against P. falciparum. The Al₂O₃ nanoparticles (71.42 ± 0.49 μg ml⁻¹) showed minimum level of IC₅₀ value and followed by MgO (72.33 ± 0.37 μg ml⁻¹) and Fe₃O₄ nanoparticles (77.23 ± 0.42 μg ml⁻¹). The PDDS-Fe₃O₄ showed minimum level of IC₅₀ value (48.66 ± 0.45 μg ml⁻¹), followed by PDDS-MgO (60.28 ± 0.42 μg ml⁻¹) and PDDS-CeO₂ (67.06 ± 0.61 μg ml⁻¹). The PDDS-coated metal oxide nanoparticles showed superior antiplasmodial activity than the non-PDDS-coated metal oxide nanoparticles. Statistical analysis reveals that, significant in vitro antiplasmodial activity (P < 0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes showed no morphological changes in erythrocytes by the nanoparticles after 48 h of incubation. It is concluded from the present study that, the PDDS-Fe₃O₄ showed good antiplasmodial activity and it might be used for the development of antiplasmodial drugs.

Introduction

Malaria caused by parasites of the genus Plasmodium, is one of the leading public health problems in Sub-Saharan Africa. It is estimated that malaria kills over a million annually and some 3.2 billion people living in 107 countries or territories are at risk (WHO 2005). In Sub-Saharan regions, 45 countries were endemic for malaria in 2008 (WHO 2008). Artemisinin combination treatments for falciparum malaria are currently the only first-line antimalarial drugs amenable to widespread use against all chloroquine-resistant malaria parasites (Barnes and Folb 2003). However, artemisinin-resistant malaria parasites were recently detected in Cambodia (Maude et al. 2009). The newest antiplasmodial drugs from biological sources help in addressing this problem (Ravikumar et al. 2011a, b, c, d, e) but it seems to be the loss of biodiversity and cost effective. The nanoparticles posses variety of biological activities (Martinez-Castanon et al. 2008; Lee et al. 2010; Singh et al. 2008) and considered as a rich source of novel antiplasmodial agents and these potential resources were scarcely explored. In the present study, we report the findings of antiplasmodial potential of metal oxide nanoparticles against Plasmodium falciparum.

Materials and methods

Nanoparticles

Commercial nanoparticles of Al₂O₃, Fe₃O₄, CeO₂, ZrO₂, and MgO were procured from Sigma Aldrich Company, India. The characteristics of the nanoparticles are represented in Table 1.

Table 1 Characteristics of nanoparticles used for antiplasmodial assay

Parasite cultivation

The antiplasmodial activity of nanoparticles was assessed against P. falciparum obtained from the Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India. P. falciparum are cultivated in human O Rh⁺ red blood cells using RPMI 1640 medium (HiMedia Laboratories Private Limited, Mumbai, India) (Moore et al. 1967) supplemented with O Rh⁺ serum (10 %), 5 % sodium bicarbonate (HiMedia Laboratories Private Limited, Mumbai, India) and 40 μg ml⁻¹ of gentamicin sulfate (HiMedia Laboratories Private Limited, Mumbai, India). Hematocrits were adjusted at 5 % and parasite cultures were used when they exhibited 2 % parasitaemia (Trager 1987).

In vitro antiplasmodial assay

Different concentrations of filter sterilized nanoparticles (100, 50, 25, 12.5, 6.25 and 3.125 μg ml⁻¹) were incorporated into 96-well tissue culture plate containing 200 μl of P. falciparum culture with fresh red blood cells diluted to 2 % hematocrit in triplicates. Negative control was maintained with fresh red blood cells and 2 % parasitized P. falciparum diluted to 2 % hematocrit, positive control was maintained with parasitized blood cells culture treated with chloroquine and artemether (Azas et al. 2001). Parasitaemia was evaluated after 48 h by Giemsa stain and the average percentage suppression of parasitaemia was calculated by the following formula: Average % suppression of parasitaemia = Average % parasitaemia in control − Average % parasitaemia in test/Average % parasitaemia in control × 100.

Antiplasmodial activity calculation and analysis

The antiplasmodial activities of nanoparticles were expressed by the inhibitory concentrations (IC₅₀) of the drug that induced a 50 % reduction in parasitaemia compared to the control (100 % parasitaemia). The IC₅₀ values were calculated (concentration of extract in X axis and percentage of inhibition in Y axis), using Office XP (SDAS) software with linear regression equation. This activity was analyzed in accordance with the norms of antiplasmodial activity of Rasoanaivo et al. (1992). According to this norms, a nanoparticle is very active if IC₅₀ < 5 μg ml⁻¹, active 5 μg ml⁻¹ < IC₅₀ < 50 μg ml⁻¹, weakly active 50 μg ml⁻¹ < IC₅₀ < 100 μg ml⁻¹ and inactive IC₅₀ > 100 μg ml⁻¹.

Chemical injury to erythrocytes

To assess any chemical injury to erythrocytes that might be attributed to the nanoparticles, 200 μl of erythrocytes were incubated with 100 μg ml⁻¹ of the nanoparticles at a dose equal to the highest used in the antiplasmodial assay. The conditions of the experiment were maintained as in the case of antiplasmodial assay. After 48 h of incubation, thin blood smears were stained with Giemsa stain and observed for morphological changes under high-power light microscopy. The morphological findings were compared with those in erythrocytes that were uninfected and not exposed to nanoparticles (Waako et al. 2007).

Results

A total of 11 samples were screened for antiplasmodial activity and the IC₅₀ values were represented in Table 2. Among the metal oxide nanoparticles, Al₂O₃ (71.42 ± 0.49 μg ml⁻¹) showed minimum level of IC₅₀ value and followed by MgO (72.33 ± 0.37 μg ml⁻¹) and Fe₃O₄ (77.23 ± 0.42 μg ml⁻¹). However, the PDDS-coated Fe₃O₄ showed minimum level of IC₅₀ value (48.66 ± 0.45 μg ml⁻¹) and followed by PDDS-coated MgO (60.28 ± 0.42 μg ml⁻¹) and CeO₂ (67.06 ± 0.61 μg ml⁻¹). The PDDS alone showed IC₅₀ value of 49.63 ± 0.38 μg ml⁻¹ and the PDDS-coated metal oxide nanoparticles showed better antiplasmodial activity than the non-PDDS-coated metal oxide nanoparticles. The microscopic observation of uninfected erythrocytes added with all the test samples and uninfected erythrocytes from the blank column of the 96-well plate showed no morphological differences after 48 h of incubation (Fig. 1).

Table 2 Antiplasmodial (IC₅₀) activity of synthesised nanoparticles

Fig. 1

Microscopical images of erythrocytes treated with synthesized nanoparticles. a Al₂O₃-treated erythrocytes; b CeO₂-treated erythrocytes; c Fe₃O₄-treated erythrocytes; d ZrO₂-treated erythrocytes; e MgO-treated erythrocytes; f PDDS-coated Al₂O₃-treated erythrocytes; g PDDS-coated CeO₂-treated erythrocytes; h PDDS coated Fe₃O₄-treated erythrocytes; i PDDS-coated ZrO₂-treated erythrocytes; j PDDS-coated MgO-treated erythrocytes; k PDDS-treated erythrocytes; l Uninfected and non-treated erythrocytes

Discussion

Metal nanoparticles have attracted much attention in the fields of physics, chemistry, electronics and biology (Lanje et al. 2010a; Schmid 1994; Henglein 1989) because of their unique electrical (Peto et al. 2002), chemical (Kumar et al. 2003) and optical (Krolikowska et al. 2003) properties, which are strongly dependent on the sizes and shapes of metal nanomaterials (Creighton and Eadon 1991; Zhang et al. 2000; Liu et al. 1995; Lanje et al. 2010a, b). Metal nanoparticles have a high specific surface area and a high surface-to-volume ratio. Nanostructured noble metals are potentially used in catalysis, optoelectronics and microelectronics. Metal nanoparticles are particularly interesting systems because of the ease with which they can be synthesized and modified chemically (Ying et al. 2005). However, the emergence of strains of P. falciparum resistant to chloroquine and many other drugs in succession and antimicrobial nature of nanoparticles has stimulated us to identify new antiplasmodial agents from nanoparticles.

The present study has tested 10 nanoparticles samples and a PDDS alone for antiplasmodial activity. Among the 10 nanoparticles, the PDDS-coated Fe₃O₄ showed minimum level of IC₅₀ value (48.66 ± 0.45 μg ml⁻¹) and the concentration is 2.5-fold higher than the positive control chloroquine (19.59 ± 0.29 μg ml⁻¹). According to Rasoanaivo et al. (1992), 10 % of the nanoparticles used by the present study are classified as active and 90 % of the samples are classified as weakly active (Fig. 2). The intracellular malarial parasite penetrates into the host red blood cell membrane through new permeation pathways (Go et al. 2004). It is already reported that, macromolecules like dextrans, protein A and IgG2a antibody also gain access to react with the parasite through the new permeation pathways (Pouvelle 1991). Foger et al. (2006) reported that, the antisense nanoparticles inhibited the malarial topoisomerase II in P. falciparum. The antisense oligodeoxynucleotides inhibit the growth of parasites in a non-specific manner by the polyanionic properties of oligonucleotides which interfere with the merozoite invasion into red blood cells (Noonpakdee et al. 2003; Barker et al. 1998). Moreover, the metal nanoparticles exhibited many antimicrobial activities (Kvitek et al. 2008; Marini et al. 2007; Holt and Bard 2005). Smaller size nanoparticles having the large surface area available for interaction and it might be a one of the reason for better antiplasmodial activity of PDDS-coated Fe₃O₄. It is concluded from the present study that, the PDDS-coated Fe₃O₄ nanoparticles could be used as an effective antiplasmodial agent for the management of malaria after successful completion of in vivo and clinical studies.

Fig. 2

Percentage of antiplasmodial activity IC₅₀ values of metal oxide nanoparticles against Plasmodium falciparum