Trisodium citrate

Chromatographic separation was performed by preparative TLC Silica gel 60 F254 (0.5 mm thick) (Loba Chemie Pvt. Ltd., Mumbai, India). n-Hexane, chloroform, ethyl acetate and methanol (Loba Chemie Pvt. Ltd., Mumbai, India), trisodium citrate (BDH Chemicals Ltd., London, UK), and Giemsa (ESJAY Chemicals, Maharashtra, India) were all used as received. Pure chloroquine phosphate supplied by Ethiopian Pharmaceuticals Manufacturing Sh. Co. (EPHARM, Addis Ababa, Ethiopia) was used as a reference drug.

The following chemicals, reagents, and drugs were used in the present study: n-butanol (Fresenius Kabi®, India), absolute methanol (Loba chemie®, India), chloroform (Carlo Erba®, France), distilled water (Ethiopian pharmaceutical manufacturing (EPHARM), Ethiopia), normal saline (Addis Pharmaceutical Factory (APF), Ethiopia), Geimsa stain (BDH laboratory supplies Poole Gurr®, England), microscope immersion oil (Neolab Life Science Co., India), trisodium citrate (BDH Chemicals Ltd, England), and active chloroquine phosphate (APF, Ethiopia). All chemicals were of analytical grade and procured from certified suppliers.

The mice infected by P. berghei and with different levels of parasitemia were used as donor mice. The donor mice's parasitemia level was first determined from their blood that is obtained by cutting (0.5 to 1 mm section) the tail of the mice using scissors [47 (link)]. To inoculate and infect the study animals, the donor mouse with a parasitemia of 30 up to 37% [48 ] was sacrificed by a head blown technique, and blood was drained into a test tube containing anticoagulant (3.8% trisodium citrate (BDH Chemicals, England)) through the incision of the jugular vein. The collected blood was then diluted in normal saline to obtain 1 × 10⁷ infected red blood cells (RBCs) in every 0.2 ml suspension [49 (link)]. The dilution was done based on the erythrocyte count of the normal mice and parasitemia of the donor mice in such a way that 1 ml blood contains 5 × 10⁷ infected RBCs [47 (link), 50 (link)]. Therefore, each mouse used was infected by 0.2 ml P. berghei-infected blood (1 × 10⁷ parasitized RBCs) intraperitoneally.

All
aptamers used in this work (Table 6) were acquired from
Integrated DNA Technologies.
Phosphate-buffered saline (PBS) was made up
in milliQ water as
a solution of 10 mM Na₂PO₄ (ECP Labchem), 1.8
mM KH₂PO₄ (ECP Labchem), 137 mM NaCl (Sigma
Aldrich), and 2.7 mM KCl (Sigma Aldrich). To this solution, 0.005%
by volume Tween-20 detergent (ThermoFisher Scientific) was added to
form the SPR buffer (PBS-T). Tris buffer was made up as a 1 M solution
of Tris base (ThermoFisher Scientific) in milliQ and adjusted to pH
8 using hydrochloric acid.
Analytical grade silver nitrate (99.8%,
Aldrich), trisodium citrate
(99.0%, BDH Chemicals), hydrogen peroxide (30% w/w, ThermoFisher Scientific),
potassium bromide (99.0%, Ajax Finechem), and sodium borohydride (98.0%,
Aldrich) were used in nanoparticle preparation.

For each test, the parasitized red blood cells were taken from P. berghei-infected donor mice with a parasitemia of 20 to 30%. The donor mice were surrendered by a head-blown procedure, and venous blood was taken in a Petri dish with 0.5% trisodium citrate (BDH chemicals, England) through the incision of a jugular vein. The venous blood was then thinned with normal saline (0.9%) in 1 to 4 proportions to get a final suspension that holds 1 × 10⁷ infected erythrocytes in every 0.2 ml. The dilution was made based on the parasitemia of donor mice and erythrocyte count of the normal mice in such a way that one ml of blood contains 5 × 10⁷ infected erythrocytes. Every experimental mouse was then infected through the intraperitoneal route with 0.2 ml suspension [48 (link), 50 (link)].

Cells were harvested and plated as described for western blotting. At the time points indicated, cells were trypsinised and transferred to 5 mL BD Falcon tubes (BD Biosciences). Citrate buffer (trisodium citrate (301287F, BDH Laboratory Supplies, Poole, UK), 121 mg Tris Base (T1378, Sigma), 1044 mg spermine tetrahydrochloride (S2876, Sigma) and 2mL Nonidet NP40 (N3516, Sigma) in 2000mL distilled water, pH7.6) was added after centrifugation. The following solutions were added in sequence prior to analysis: 450uL solution A (0.003% trypsin type IX-S (T0303, Sigma) in citrate buffer, pH7.6) for 2 min, solution B (0.05% trypsin inhibitor (T9253, Sigma) and 0.01% RNAse A (R4875, Sigma) in citrate buffer pH7.6) for 10 min, and solution C (0.0416% propidium iodide (81845, Sigma) and 0.1% spermine tetrahydrochloride (S2876, Sigma) in 500 mL citrate buffer pH7.6) for 10 min in the dark. Apoptosis was detected at 24 h using the TACS Annexin V-FITC Kits (R &D Systems) according to the manufacturer's protocol. Flow cytometry was performed using a BD FACSAriaII SORP (Becton Dickinson, Franklin Lakes, NJ). BD FACSDiva software (Becton Dickinson, Version 6.1.2) was used for instrument control and Flowjo software (Version 7.6.5) for Data analysis.

Swiss albino mice, 6–8weeks of age and weighing 27–32 g, obtained from the Ethiopian Public Health Institute (EPHI), were used for the tests. Female mice were used for in vivo acute toxicity test and male mice were used for in vivo antimalarial screening. The mice were maintained in the animal house of ALIPB, AAU, under standard condition at room temperature by exposing them to 12 h light and 12 h dark cycle, with food and water ad libitum. Mice were handled based on internationally accepted guideline [25 ].
Chloroquine sensitive P. berghei (ANKA strain) was obtained from the Ethiopian public Health Institute (EPHI). The parasites were maintained by serial passage of blood from infected mice to non-infected ones on weekly basis [26 ]. Donor mice infected with a rising parasitaemia of 20–30% were used to infect mice in the 4-day suppressive test. The donor mice were sacrificed and blood was pooled together in a petri-dish containing 2% trisodium citrate (BDH chemicals, England) as anticoagulant to avoid variability in parasitaemia. The blood was then diluted with 0.9% normal saline so that each 0.2 ml of blood contained 1x10⁷P. berghei infected erythrocytes. Each mouse used in the experiment was then inoculated intraperitoneally with 0.2 ml of the diluted blood.