Nanotechnology offers great potential for molecular genetic investigations and potential control of medically important arthropods. Major advances have been made in mammalian systems to define nanoparticle (NP) characteristics that condition trafficking and biodistribution of NPs in the host. Such information is critical for effective delivery of therapeutics and molecules to cells and organs, but little is known about biodistribution of NPs in mosquitoes. PRINT technology was used to construct a library of fluorescently labeled hydrogel NPs of defined size, shape, and surface charge. The biodistribution (organ, tissue, and cell tropisms and trafficking kinetics) of positively and negatively charged 200 nm x 200 nm, 80 nm x 320 nm, and 80 nm x 5000 nm NPs was determined in adult Anopheles gambiae mosquitoes as a function of the route of challenge (ingestion, injection or contact) using whole body imaging and fluorescence microscopy. Mosquitoes readily ingested NPs in sugar solution. Whole body fluorescence imaging revealed substantial NP accumulation (load) in the alimentary tracts of the adult mosquitoes, with the greatest loads in the diverticula, cardia and foregut. Positively and negatively charged NPs differed in their biodistribution and trafficking. Following oral challenge, negatively charged NPs transited the alimentary tract more rapidly than positively charged NPs. Following contact challenge, negatively charged NPs trafficked more efficiently in alimentary tract tissues. Following parenteral challenge, positively and negatively charged NPs differed in tissue tropisms and trafficking in the hemocoel. Injected NPs were also detected in cardia/foregut, suggesting trafficking of NPs from the hemocoel into the alimentary tract. Herein we have developed a tool box of NPs with the biodistribution and tissue tropism characteristics for gene structure/function studies and for delivery of vector lethal cargoes for mosquito control.
Arthropod vectors and pest species are of enormous public health, agricultural, and economic importance. Control of these arthropods is predicated to a large extent on chemical insecticides. Ominously, many vector and pest species have developed or are developing resistance to conventional classes of insecticides. Insecticide resistance is emerging as a major threat for the control of mosquito vectors of human diseases, including Anopheles gambiae, the principal vector of malaria in Africa [1–3]. Identification of new targets and development of new approaches for control of vectors is a public health imperative. Improved and efficient techniques to investigate the molecular biology of and to characterize gene structure and function in arthropods would be of great value to identify novel targets for control. New approaches to deliver effector molecules and compounds to improve vector or pest control would also be of great value. Nanotechnology offers great potential in both of these areas. For example improved nanoparticle (NP) delivery of dsRNA to induce RNAi to silence and functionally characterize genes and to cause insect mortality offers exciting new potential for research as well as insect vector and pest management [4,5]. Physical and chemical properties of natural objects have been refined by nature to optimize biological functions and interactions [6–14]. There are many biological barriers in an organism that condition the efficacy of NP delivery to target tissues and cells [15,16]. In the human host, these include the vascular endothelium and walls of blood vessels, physical entrapment in organs, phagocytosis, and overall clearance of the NPs from the circulatory system. Physicochemical properties, e. g. size, shape, aspect ratio, modulus, and surface charge, are determinants of the biodistribution and trafficking of NPs in vivo in vertebrates and of internalization of NPs into cells [17–21]. The PRINT platform particle technology offers exceptional capability to mimic nature’s handiwork. Particles have been engineered to deliver siRNAs (and other biological molecules) to knock down target gene expression in both in vitro and in vivo systems [22–25]. The NPs containing the siRNAs are effectively delivered to target cells, where the particle is degraded in the endolysosome, and the siRNAs are then released into the cytoplasm to engage the host RNAi machinery. Major advances have been made in the development and optimization of NPs for delivery of drugs, antigens, and RNAs in vitro and in vivo in mammalian systems [26–29]. Microencapsulation techniques have been used to enhance the stability, effectiveness and environmental delivery of effector molecules, e. g. —insecticides, to control mosquitoes in nature [30], but development of NP-delivered effector molecules, such as dsRNAs, to control mosquitoes is in its infancy. Chitosan NPs have been used to deliver dsRNA to silence chitin synthase in An. gambiae mosquitoes [31], and chitosan-siRNA particles have been used to disrupt expression of an olfactory gene in Ae. aegypti [32]. Other types of NPs have been investigated for vector control [33–36]. The biodistribution of NP-delivered dsRNA to silence a chitinase-like gene in larval Drosophila melanogaster has been reported [37]. However, there is little information about NP physicochemical determinants of trafficking of NPs in mosquitoes following environmental (oral or contact) delivery. Following ingestion by adult mosquitoes, NPs would have to traffic in the alimentary tract, penetrate diverticula, cardia, foregut, or midgut barriers, disseminate into the hemocoel and then be internalized by target tissues and cells. Following contact delivery, NPs must traverse cuticular barriers and then traffic in the insect to be internalized by the appropriate tissues and organs. Determination of the optimal physicochemical characteristics of NPs to negotiate these barriers to deliver their cargoes to target cells is the goal of our research. In this and the accompanying paper [38], we determined the biodistribution and trafficking of poly (ethylene glycol) (PEG) hydrogel particles in vivo in adult and larval An. gambiae mosquitoes following oral, parenteral or contact challenge, and the internalization potential of particles in vitro in cell cultures [38]. PRINT technology was utilized to prepare PEG NPs of defined size, shape, aspect ratio, and surface charge for mosquito challenges. In this paper, fluorescently-labeled NPs without cargoes were used to challenge adult An. gambiae mosquitoes, and the NPs with the preferred biodistribution characteristics (e. g. organ, tissue and cell tropisms and trafficking kinetics) for delivery of molecules to tissues and cells in mosquitoes were identified.
An. gambiae (G3) strain was used in all experiments, and eggs to start the colony were kindly provided by the Malaria Research and Reference Reagent Resource Center (MR4) (http: //www. mr4. org). Rearing and manipulation of mosquitoes generally followed the MR4 recommendations. Briefly, mosquitoes were reared at 27°C (±1°C), 80% (±5%) humidity and a light cycle of 30 min sunrise (at 5: 30 AM), 11. 5h daylight, and 30 min sunset (at 5: 30 PM). Mosquito rearing was conducted in the insectary facilities of the Arthropod-borne and Infectious Diseases Laboratory at Colorado State University. To determine organ, tissue and cell tropisms and to estimate the abundance (load) of the respective NPs, alimentary tract tissues (ventral diverticulum, dorsal diverticula, foregut, midgut, hindgut, and Malpighian tubules) and non-alimentary tract tissues (head, proboscis, salivary glands, thoracic muscles, ovaries, and tracheae) were dissected from mosquitoes (Fig 2) at predetermined days post challenge. Organs and tissues were mounted on slides, covered with PBS-10% glycerol mounting medium, and assayed for the presence and abundance of NPs in tissues up to 7 d post challenge using an Olympus BH2-RFCA fluorescence microscope or a Leica DM4500B fluorescence microscope. NP tropisms were determined by detection of any level of fluorescence signal (1 to 5+) in the respective organ, tissue, or cell. The intensity of the fluorescent signal was subjectively scored from 0 to 5+. NP loads in the respective organ or tissue were assumed to be directly correlated with the relative intensity of the fluorescent signal in the organ or tissue. In some studies, to more accurately determine cell tropisms of selected NPs, alimentary tract tissues were dissected in PBS and fixed in 4% formaldehyde (Electron Microscopy) for at least 1 hr at room temperature (RT). The fixative was then removed, the tissues were rinsed three times with PBS 5 min each at RT and then permeabilized in 1% Triton X-100 for 10 min at RT and then washed three times for 5 min each at RT. The tracts were then incubated in a 1: 40 dilution of—Alexa Fluor 546 phalloidin (high-affinity F-actin probe conjugated to bright, photo stable, orange-fluorescent Alexa Fluor 546, lifetechnologies. com/ product A22283) for 15 min at RT. After rinsing with PBS (three times for 5 min each at RT), alimentary tracts were placed onto slides, covered with Vectashield (Vector Laboratories Inc. , Burlingame, CA), and a coverslip was sealed with nail polish on the slide. Biodistribution of NPs was determined using a Leica DM4500B fluorescence microscope.
Female mosquitoes were exposed to the respective NPs (Fig 1) in 10% sucrose (250 μg/mL and 50 μg/mL) for 1 d and then assayed by image analysis and immunofluorescence microscopy for NP biodistribution, trafficking, and kinetics of tissue tropisms. Nearly 100% of An. gambiae females readily ingested the NP-sucrose meals regardless of particle size and charge. NP ingestion exhibited little untoward effect on the mosquitoes; for example, in a typical experiment, the survivorship rates for mosquitoes ingesting positively (N = 30) or negatively (N = 30) charged 80 nm x 320 nm NPs (250 μg/mL) or sucrose (N = 28) were 93,100, and 71%, respectively. Similarly, parenteral challenge of larval mosquitoes or in vitro challenge of mosquito cells in culture (see Figs 6 and 7 in companion paper [38]) caused little differences in larval survivorship or cell viability. Whole body image analysis was used to detect and quantify NPs following parenteral and oral challenges. Adults were injected with positively or negatively charged 200 nm x 200 nm NPs (250 μg/mL) or sucrose. At 1 d post challenge, mosquitoes injected with the negatively charged NPs exhibited the greater fluorescent signal (Fig 3, Row 2). The greatest Mean Fluorescence Intensity (MFI) values were also detected in mosquitoes challenged with negatively charged NPs (Fig 4), and the MFI values at day seven were similar to those at the day of challenge, with the exception of the 80 nm x 5000 nm positively charged NPs (Fig 4). The reasons for the dramatic differences in MFI between positively and negatively charged NPs following parenteral challenge (Fig 4) may be attributable to more efficient internalization of the positively charged NPs [38]. Oral challenge of mosquitoes yielded different results. Following oral challenge with 200 nm x 200 nm negatively charged NPs for 1 d, MFI values remained very low (Fig 5). However, following oral challenge for 2 d, MFI values increased significantly to 3 d post exposure and then declined thereafter (Fig 5). These results were confirmed by oral challenge of mosquitoes with positively and negatively charged 80 nm x 5000 nm and 200 nm x 200 nm NPs (Fig 6), which also increased in abundance following ad libitum oral challenge. The MFI indices declined to essentially undetectable levels at day seven post-initial exposure. The positively charged, but not the negatively charged, 80 nm x 5000 nm NPs exhibited a similar pattern (Fig 6). Adult mosquitoes were challenged by contact with positively or negatively charged 80 nm x 320 nm NPs. A drop of the respective particle solution with NP40 (0. 5%) was placed on the head, thorax, or abdomen. Mosquitoes were dissected at 1 or 2 d post challenge and tissues were examined for fluorescence. Administration of negatively charged NPs to the head resulted in NP detection in head tissues, proboscis and alimentary tract tissues, including diverticula and foregut) in 100% (5/5) of mosquitoes 1 d post challenge (Fig 13A). Fluorescence signal was detected in midgut tissue of 60% (3/5) mosquitoes at 2 d post challenge, but minimal signal was detected in the diverticula at that time. Signal remained intense in certain tissues in the proboscis 2 d post challenge. Following contact challenge to the head with positively charged 80 nm x 320 nm NPs, fluorescence signal was detected in 40% (4/10) of mosquitoes in the alimentary tract tissues and in the proboscis; however the fluorescent signal was much less intense (Fig 13B). than that detected in mosquitoes challenged with the negatively charged NPs (Fig 13A). Contact challenges with positively or negatively charged NPs to the thorax or abdomen were not promising. Following administration of positively or negatively charged NPs to the thorax, very minimal fluorescent signal was detected at 1 or 2 d post challenge in the alimentary tract or proboscis of 43% (12/28) of the challenged mosquitoes. Following administration of positively or negatively charged NPs to the abdomen, very minimal fluorescent signal was detected in tissues of 22% (4/18) of the challenged mosquitoes. The fluorescence signal was typically scored as <1 (on the scale of 0–5+), and it was difficult to differentiate the mosquitoes challenged by these routes from the control mosquitoes (Fig 13C). Clearly trafficking of NPs into mosquitoes following throrax or abdomen contact challenges is inefficient.
The studies provided important information for the potential use of and preferred physical characteristics of hydrogel NPs for delivery of cargoes (e. g. dsRNA) to silence mosquito genes for functional analyses and for mosquito control. Both adult and larval mosquitoes [38] readily ingested the NPs tested. Following 1 d oral challenge of adults positively and negatively charged NPs were detected in dorsal and ventral diverticula, cardia, foregut, and midgut at day one post ingestion (Fig 7). By day two post ingestion, signal decreased, especially for negatively charged NPs in both the number of tissues exhibiting fluorescence and in fluorescence intensity (Fig 7). When orally challenged for 1 d, there was minimal detection of the respective NPs in non-alimentary tract tissues (Fig 7). However, when mosquitoes were challenged for 2 d or more, the biodistribution of NPs changed dramatically (Figs 5,6 and 7). Multiple day challenges resulted in dramatic increases in MFI values, which were nearly undetectable in whole body images of mosquitoes that were orally challenged for just 1 d (Fig 5). The reason for this is unclear. The NP load is clearly increased in the mosquitoes challenged for multiple days (Fig 5), and both positively and negatively charged NPs are detected abundantly and persist in the alimentary tract (Fig 7). Multiple day challenges also results in NP dissemination to non-alimentary tract tissues (Fig 7), and the resulting biodistribution of NPs was similar to that seen with NPs following parenteral challenge where NPs were detected in or associated with tracheae and tracheoles, cardia, proboscis, etc. (Fig 12). The decline in MFI values beginning at day two (Figs 5 and 6) was unexpected. Sugar pads were placed on the cages after 1 d, and perhaps mosquitoes began to feed preferentially on the sugar pads. The anatomic basis for trafficking of NPs out of the alimentary tract remains to be determined. Multiple ingestions of NPs may somehow perturb tissue barriers and promote accumulating and trafficking of NPs. However, trafficking of NPs from the hemocoel into the alimentary tract also occurs following parenteral challenge; NPs were detected in the cardia, foregut, and midgut of mosquitoes (Fig 12B). The large accumulation of particles in the cardia and foregut (Figs 8 and 12) following oral or parenteral challenge suggests that this organ may be involved in trafficking. Arboviruses, which must disseminate from the vector gut to infect salivary glands to be transmitted, have been detected in the cardia of infected mosquitoes. Investigators have proposed that the intussusception of the foregut and esophagus, which may be only a cell or two thick, is a likely anatomic mechanism for arbovirus dissemination [41]. The abundant accumulation of NPs in the cardia could result in trafficking of the particles between the body compartments. Arboviruses can also traffic from the midgut into the hemocoel via tracheae [42,43]. The association of NPs with tracheae (Fig 12A) is also provocative in this regard. The exact mechanism conditioning the trafficking remains to be determined. There were major differences in the biodistribution and trafficking of positively and negatively charged NPs. Following parenteral challenge, positively charged NPs seemed to coat the basal lamina of multiple organs; negatively charged NPs exhibited more punctate fluorescence associated with cells or tissues (Fig 12B). The negatively charged NPs seemed to transit the alimentary tract more rapidly than positively charged NPs. Indeed, negatively charged NPs were expelled and detected on the filter papers lining the plastic containers more frequently and abundantly (Fig 9). Negatively charged NPs were also detected more frequently in proboscis and head tissues of injected mosquitoes (Fig 10), suggesting that they trafficked more in the hemolymph than positively charged NPs. Whole body imaging analyses of parenterally challenged mosquitoes (Figs 2 and 3) revealed that negatively charged NPs exhibited greater MFI values and persisted longer in mosquitoes than positively charged NPs. It is noteworthy that the positively and negatively charged NPs exhibited the same phenotype in parenterally challenged larval mosquitoes (see Fig 5 in companion paper) [38]. In this regard, positively charged NPs are more efficiently internalized by mosquito cells (see Fig 1 in companion paper) [38]. Perhaps inefficient internalization of the negatively charged NPs conditions their persistence in the closed system of the hemocoel (Figs 2 and 3), which is in contrast to their rapid transit through the alimentary tract (Fig 9). The preferred charge of NPs for environmental challenge of mosquitoes remains to be determined. Positively charged particles are more efficiently internalized in vector cells [38], but negatively charged NPs were detected abundantly in tissues in the proboscis, regardless of the mode of challenge. Cells in the labella of the proboscis of mosquitoes frequently contained or were associated with large accumulations of NPs (Figs 10B and 13). Sensory cells in the labella are of particular interest in terms of potential contact delivery of NPs. These organs are sampling the environment and could be a portal of entry of NP through oral or contact delivery of negatively charged NPs to control mosquitoes. In addition, negatively charged NPs more efficiently trafficked from the cuticle to mosquito organs than positively charged particles (Fig 13). Future studies will incorporate effector molecules in the NPs, which will then be used to challenge mosquitoes. Such studies will be most informative in selecting the optimal NPs for oral, contact, and parenteral delivery of effector molecules. The large accumulation of NPs in the diverticula of mosquitoes is potentially important in terms of environmental delivery of NPs and their cargoes for vector control. Upon emergence, adult females typically ingest sugar meals to provide energy reserves for mating, host seeking, and other behaviors. Following ingestion, the sugar meal accumulates in the diverticula and is slowly released into the alimentary tract. The large NP load in the diverticula and their subsequent release into the alimentary tract provide ongoing opportunities for NP internalization by gut cells. Importantly, the sugar meal does not induce peritrophic matrix formation, which could serve as a barrier to NP contact with target cells. Sugar baited stations [44], which have proven to be very useful for arbovirus surveillance in mosquito populations, would thus be a potentially fruitful approach for delivering NPs and their cargoes to mosquitoes in nature. Our results provide insights into NP design that could facilitate insect gene structure and function studies. The ability to deliver effector molecules through oral or contact challenge for gene structure function studies would be of great value and would preclude confounding effects of injection on gene regulation (e. g. induction of innate immune genes by penetration of the cuticle) and would also minimize mortality in experimental insects due to injection. Even with parenteral challenge, optimal internalization of NPs (e. g. positively charged particles) and delivery of their cargoes into target cells could greatly increase efficiency of gene silencing. Our studies also provide important information for exploiting NP technology for development of new insecticides for mosquito vector control. Studies are in progress to define the preferred physicochemical properties of NPs for environmental delivery of effector molecules for gene silencing and vector lethality. The power of PRINT technology provides unparalleled capacity in this regard and for the development of a new generation of insecticides for insect vector and pest control.
Emerging insecticide resistance in disease vectors is of great public health concern. Discovery of new targets and novel strategies for insecticidal interventions to control vector borne diseases is a public health imperative. Nanotechnology offers great potential for molecular genetic investigations and for delivery of effector molecules for control of disease vectors. We have developed a tool box of hydrogel nanoparticles (NPs) with the biodistribution and tissue tropism characteristics for gene structure/function studies and for delivery of vector lethal cargoes to adult mosquitoes. Nanotechnology will likely be useful for molecular investigations and potential control of the arthropod vectors of other neglected tropical diseases.
Biodistribution and Trafficking of Hydrogel Nanoparticles in Adult Mosquitoes