Novel nanosomes for gene delivery to Plasmodium falciparum-infected red blood cells

Abstract

Malaria threatens millions of people annually and is a burden to human health and economic development. Unfortunately in terms of disease control, no effective vaccines are available and the efficacy of treatment is limited by drug resistance. Genetic manipulation in Plasmodium falciparum is hampered due to the absence of robust methods for genetic analyses. Electroporation-based transfection methods have allowed the study of gene function in P. falciparum, with low efficiency. A lipid nanoparticle was developed that allowed nuclear targeting of pDNA with increased efficiency in reporter assay, compared to traditional electroporation method. This method has for the first time, facilitated transfection using both circular and linear DNA in P. falciparum thereby serving as an alternative to electroporation with an increase in transfection efficiency. Availability of a robust method for functional genomic studies in these organisms may be a catalyst for discovery of novel targets for developing drugs and vaccines.

Figure 1. Luciferase activity in P. falciparum- infected erythrocytes transfected by electroporation (EP) and nanosome delivery.

(a) Optimization of K4-DNA mediated P. falciparum transfection. In [A]: K4-DNA was added to parasite pellet and resuspended in culture media. In [B]: K4-DNA was incubated with parasite pellet for 30 min at 37°C and then diluted with culture media. In [C]: K4-DNA was added to parasite pellet, resuspended in 0.5 ml incomplete medium and immediately diluted with culture media. In [D]: K4-DNA was mixed with parasite pellet resuspended in 0.5 ml incomplete media, incubated for 30 min at 37°C and then diluted with culture media. In [E]: K4-DNA was mixed with parasite pellet resuspended in 1 ml incomplete media and diluted with culture media and in [F]: K4-DNA was mixed with parasite pellet resuspended in 1 ml incomplete media, incubated for 30 min at 37°C and diluted with culture media. (b) Luciferase activity was measured 48 h after transfection for EP and four different nanosome formulations K1-K4 using plasmid pHLH-1. Formulations (K1-K4) were added to parasite pellet incubated as described in method [D] above. (c) Luciferase activity was measured 48 h after transfection for EP (50 μg) and K4 using 2.5 μg, 10 μg, 25 μg and 50 μg plasmid pHLH-1, respectively. (d) Luciferase activity was measured 48 h after transfection for EP and K4, (100% to 25% proportional reduction of K4 formulation) with plasmid pHLH-1 using method D. (e) Luciferase activity was measured after transfection by EP and K4 formulation method using 25 μg circular and linear pHLH-1. Aliquots of parasites were withdrawn every 48 hrs for luciferase activity detection and continued for up to 192 h (Day 8). (f) Luciferase activity was measured 48 h after transfection by EP and K4 formulation method using 25 μg of pHLH-1 and pLH plasmid using method D. All experiments (a–f) were performed in triplicates and vertical bars indicate standard deviation. P values were calculated using unpaired t test, and P < 0.05 indicates that there was a statistically significant difference between the formulation and electroporation luciferase activity.

Figure 2. GFP expression and PCR analysis from P. falciparum- infected erythrocytes by electroporation (EP) and K4 formulation methods.

(a) Schematic representation of genomic organization of a disrupted Pfs16 locus. Arrows indicate the location and orientation of various primers used in PCR analyses. Table below indicates the expected size of the various PCR products. (b) K4 (panels A and B) and EP (panels C and D) and parasites transfected with p16FAGFP showing expression of GFP when viewed under fluorescence microscope using 100× magnifications. Bright field (BF) image of the same is shown on the left for each image. The percentage of GFP positive parasites in the K4 group was 75% (range 64 to 86%) as compared to 60% (range 59 to 61%) in the EP group (c) PCR genotype analysis using primer pair A, B and C for wild type and transfected parasites. Numbers on left indicate molecular weight standards (size in base pairs).