Fig 1.
Physicochemical characteristics of nanoparticles manufactured and successfully used to challenge Anopheles gambiae mosquitoes.
Representative scanning electron micrographs and dynamic light scattering characterization of (A) 80 nm x 320 nm, (B) 200 nm x 200 nm, and (C) 80 nm x 5000 nm PRINT Particles. Bars in micrographs = 1μm. (NP—nanoparticle; Z avg—average hydrodynamic particle diameter; PdI—polydispersity index: ZP—zeta potential)
Fig 2.
Anopheles gambaie adult female alimentary tract and dissected principal organ systems.
A. Sagittal plane diagram of organ systems in situ in An. gambiae. Illustration by R. Isaì Madriz. B. Dissected alimentary tract from female An. gambiae. prob = proboscis,dr. dv = dorsal diverticulum, v. dv = ventral diverticulum,. car = cardia, for.g = foregut, mid.g = midgut, hin.g = hind gut, Mal.tu = Malpighian tubules, Bar = 500 μM.
Fig 3.
Whole body imaging of Anopheles gambiae females parenterally challenged with 200 nm x 200 nm hydrogel nanoparticles.
Mosquitoes were sacrificed and imaged 1 day post challenge. Fluorescence signal was detected in the thorax and abdomen of NP challenged mosquitoes and was visibly brighter for females challenged with negatively charged NPs (row 2) compared to those challenged with positively charged particles (row 3). Fluorescence signal was not detected in non-challenged control mosquitoes (row 1).
Fig 4.
Whole body mean fluorescence intensity (MFI) values of Anopheles gambiae females parenterally challenged with 80nm x 320 nm, 200 nm x 200 nm and 80 nm x 5000 nm hydrogel nanoparticles.
Negatively charged NPs consistently produced MFI values that were significantly higher than positively charged NPs.
Fig 5.
Whole body mean fluorescence intensity (MFI) values of Anopheles gambiae females orally challenged with negatively charged 200 nm x 200 nm hydrogel particles for 1 and 2 day(s).
MFI values for females orally challenged for 1 d with NPs were low compared to MFI values for females orally challenged for 2 d in which MFI values increased and peaked at approximately 3 d post challenge and decreased thereafter. Statistical difference (p<0.05) from controls and 1 day oral challenge groups are indicated by * and #.
Fig 6.
Whole body mean fluorescence intensity (MFI) values of Anopheles gambiae females orally challenged ad libitum with positively and negatively charged 80nm x 320 nm, 200 nm x 200 nm and 80 nm x 5000 nm nanoparticles.
MFI values for females orally challenged with 80 nm x 320 nm and 200 nm x 200 nm NPs showed similar patterns over time with an increase in MFI values the first two days of challenge and a decrease thereafter. However, MFI values for females orally challenged with 80 nm x 5000 nm NPs showed a different pattern with lower MFI values and the MFI values for negatively charged NPs declined after 1 d post oral challenge.
Fig 7.
Tissue tropisms and fluorescence intensity of positively and negatively charged 80 nm x 320 nm hydrogel nanoparticles in Anopheles gambiae females following 1 and 3 day(s) oral challenges.
Tissue tropisms (percent of mosquitoes with NP fluorescent signal of any intensity detected in the respective organ or tissue) of positively and negatively charged 80 x 320nm NPs in An. gambiae following 1 day (A) and 3 day (C) oral challenge (250 μg/mL). Fluorescence intensity (mean level of fluorescence intensity (NP load) in organs and tissues containing NPs) of positively and negatively charged 80 x 320 nm NPs in An. gambiae following 1 day (B) and 3 day (D) oral challenge. In mosquitoes challenged for 1 d, tissue tropisms and fluorescence intensity decreased substantially by 1 or 2 d post challenge. However, some organs/tissues still contained fluorescence signal at 7 d post challenge. In mosquitoes challenged for 3 d, tissue tropisms and fluorescence intensity were greater and of longer duration than in mosquitoes challenged for only 1 d. VD = ventral diverticulum; DD = dorsal diverticula; FG = foregut; MG = midgut; HG = hindgut; MT = Malpighian tubules; TM = thoracic muscles.
Fig 8.
Biodistribution of hydrogel nanoparticles in the alimentary tract of Anopheles gambiae females following a 1 day oral challenge.
Following oral challenge with the respective NPs (250 μg/mL), fluorescence signal was detected in all alimentary tract tissues, including Ventral diverticulum (VD), dorsal diverticulum (DD), foregut (FG), midgut (MG) and cardia (arrow). Fluorescence was not detected in the alimentary tracts of the control, sucrose-fed females. Bright field micrographs show the outline of the tissues from control mosquitoes that were not challenged with NPs. Bar = 1 μm
Fig 9.
Excretion of 80 nm x 320 nm hydrogel nanoparticles from the alimentary tract of Anopheles gambiae females following a 1 d oral challenge.
Excretion of negatively charged NPs resulted in larger and brighter fluorescence spots on filter paper lining the cages than excreted positively charged NPs. Bar = 1 μm.
Fig 10.
Biodistribution of hydrogel nanoparticles in tissues of Anopheles gambiae females following a 3 d oral challenge.
(A) Alimentary tract tropisms: Fluorescence was present in many tissues in alimentary tract tissues and organs including the ventral diverticulum (VD), dorsal diverticula (DD), and MG (midgut). (B) Hemocoel associated tropisms: Fluorescent signal was present in tissues associated the hemocoel including ommatidia (OM) and proboscis. Bar = 1 μm
Fig 11.
Tissue tropisms and fluorescence intensity of positively and negatively charged 80 nm x 320 nm hydrogel nanoparticles in Anopheles gambiae females following parenteral challenge.
(A) Tissue tropisms (percent of mosquitoes with NP fluorescent signal of any intensity detected in the respective organ or tissue) of positively and negatively charged 80 x 320nm NPs in An. gambiae following parenteral challenge (250 μg/ml). (B) Fluorescence intensity (mean level of fluorescence intensity (NP load) in organs and tissues containing NPs) of positively and negatively charged 80 x 320 nm NPs in An. gambiae organs and tissues following parenteral challenge. NP tissue tropisms and loads were greater and of longer duration in most tissues females injected with positively charged NPs than in those injected with negatively charged NPs. Negatively charged NPs were most frequently associated with head and proboscis tissues. VD = ventral diverticulum; DD = dorsal diverticula; FG = foregut; MG = midgut; HG = hindgut; MT = Malpighian tubules; TM = thoracic muscle.
Fig 12.
Biodistribution of positively and negatively charged 80 nm x 320 nm hydrogel particles in Anopheles gambiae mosquitoes 1 d following parenteral challenge.
(A) NP biodistribution in cells and tissues associated with the hemocoel: NPs were detected in or associated with many tissues following parenteral challenge, including Malpighian tubules (MT), salivary glands (SA), and trachea (TR) associated tissues, and in tissues on the surface of organs exposed to the hemocoele such as ventral diverticulum (VD), foregut (FG), and midgut (MG). (B) NP detection in alimentary tract associated organs and tissues: NPs also disseminated from the hemocoele into the alimentary tract and were detected in cardia (CA), foregut (FG), midgut (MG), ventral diverticulm (VD) and dorsal diverticula (DD). Bar = 1 μm
Fig 13.
Biodistribution of 80 nm x 320 nm hydrogel nanoparticles in the alimentary tracts of Anopheles gambiae females 1 d following contact challenge.
Female mosquitoes were challenged by administration of NPs to the head. (A) Negatively charged NPs. (B) Positively charged NPs. (C) Control mosquitoes. Negatively charged NPs trafficked more effectively and were detected more frequently and abundantly in alimentary tract tissues and the proboscis than positively charged NPs. VD = ventral diverticulum; DD = dorsal diverticula; FG = foregut; Bar = 1 μm