Biodistribution and Trafficking of Hydrogel Nanoparticles in Adult Mosquitoes

Background

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.

Methodology/Principal Findings

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.

Conclusions/Significance

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.     

Protein surface charge effect on 3D domain swapping in cells for c-type cytochromes

Many c-type cytochromes (cyts) can form domain-swapped oligomers. The positively charged Hydrogenobacter thermophilus (HT) cytochrome (cyt) c₅₅₂ forms domain-swapped oligomers during expression in the Escherichia coli (E. coli) expression system, but the factors influencing the oligomerization remain unrevealed. Here, we found that the dimer of the negatively charged Shewanella violacea (SV) cyt c₅ exhibits a domain-swapped structure, in which the N-terminal helix is exchanged between protomers, similar to the structures of the HT cyt c₅₅₂ and Pseudomonas aeruginosa (PA) cyt c₅₅₁ domain-swapped dimers. Positively charged horse cyt c and HT cyt c₅₅₂ domain swapped during expression in E. coli, whereas negatively charged PA cyt c₅₅₁ and SV cyt c₅ did not. Oligomers were formed during expression in E. coli for HT cyt c₅₅₂ attached to either a co- or post-translational signal peptide for transportation through the cytoplasm membrane, but not for PA cyt c₅₅₁ attached to either signal peptide. HT cyt c₅₅₂ formed oligomers in E. coli in the presence and absence of rare codons. More oligomers were obtained from the in vitro folding of horse cyt c and HT cyt c₅₅₂ by the addition of negatively charged liposomes during folding, whereas the amount of oligomers for the in vitro folding of PA cyt c₅₅₁ and SV cyt c₅ did not change significantly by the addition. These results indicate that the protein surface charge affects the oligomerization of c-type cyts in cells; positively charged c-type cyts assemble on a negatively charged membrane, inducing formation of domain-swapped oligomers during folding.     

The role of liposome charge on immune response generated in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63)

Liposomes as a lipid-based system have been shown to be an effective adjuvant formulation. In this study, the role of liposome charge in induction of a Th1 type of immune response and protection against leishmaniasis in BALB/c mice was studied. Liposomes containing rgp63 were prepared by Dehydration-Rehydration Vesicle (DRV) method. Neutral liposomes consisted of dipalmitoylphosphatidylcholine and cholesterol. Positively and negatively charged liposomes were prepared by adding dimethyldioctadecylammonium bromide (DDAB) or dicetyl phosphate (DCP) to the neutral liposome formulation, respectively. Female BALB/c mice were immunized subcutaneously with negatively, positively charged or neutral liposomes encapsulated with rgp63, rgp63 in soluble form or PBS, three times in 3 week intervals. The extent of protection and type of immune response generated were studied in different groups of mice. The group of mice immunized with rgp63 encapsulated in neutral liposomes showed a significantly (P < 0.01) smaller footpad swelling upon challenge with Leishmania major compared with positively or negatively charged liposomes. The mice immunized with neutral liposomes also showed a significantly (P < 0.01) the lowest splenic parasite burden, the highest IgG2a/IgG1 ratio and IFN-γ production and the lowest IL-4 level compared to the other groups. The results indicated that a Th1 type of immune response was induced in mice immunized with neutral liposomes more efficiently than positively charged liposomes and conversely negatively charged liposomes induced a Th2 type of immune response.     

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake

Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO₂ NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO₂ NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays.     

Size-Dependent Toxicity of Silver Nanoparticles to Bacteria,Yeast, Algae,Crustaceans and Mammalian Cells In Vitro

The concept of nanotechnologies is based on size-dependent properties of particles in the 1–100 nm range. However, the relation between the particle size and biological effects is still unclear. The aim of the current paper was to generate and analyse a homogenous set of experimental toxicity data on Ag nanoparticles (Ag NPs) of similar coating (citrate) but of 5 different primary sizes (10, 20, 40, 60 and 80 nm) to different types of organisms/cells commonly used in toxicity assays: bacterial, yeast and algal cells, crustaceans and mammalian cells in vitro. When possible, the assays were conducted in ultrapure water to minimise the effect of medium components on silver speciation. The toxic effects of NPs to different organisms varied about two orders of magnitude, being the lowest (∼0.1 mg Ag/L) for crustaceans and algae and the highest (∼26 mg Ag/L) for mammalian cells. To quantify the role of Ag ions in the toxicity of Ag NPs, we normalized the EC₅₀ values to Ag ions that dissolved from the NPs. The analysis showed that the toxicity of 20–80 nm Ag NPs could fully be explained by released Ag ions whereas 10 nm Ag NPs proved more toxic than predicted. Using E. coli Ag-biosensor, we demonstrated that 10 nm Ag NPs were more bioavailable to E. coli than silver salt (AgNO₃). Thus, one may infer that 10 nm Ag NPs had more efficient cell-particle contact resulting in higher intracellular bioavailability of silver than in case of bigger NPs. Although the latter conclusion is initially based on one test organism, it may lead to an explanation for “size-dependent“ biological effects of silver NPs. This study, for the first time, investigated the size-dependent toxic effects of a well-characterized library of Ag NPs to several microbial species, protozoans, algae, crustaceans and mammalian cells in vitro.     

Novel Heparan Sulfate-Binding Peptides for Blocking Herpesvirus Entry

Human cytomegalovirus (HCMV) infection can lead to congenital hearing loss and mental retardation. Upon immune suppression, reactivation of latent HCMV or primary infection increases morbidity in cancer, transplantation, and late stage AIDS patients. Current treatments include nucleoside analogues, which have significant toxicities limiting their usefulness. In this study we screened a panel of synthetic heparin-binding peptides for their ability to prevent CMV infection in vitro. A peptide designated, p5+14 exhibited ~ 90% reduction in murine CMV (MCMV) infection. Because negatively charged, cell-surface heparan sulfate proteoglycans (HSPGs), serve as the attachment receptor during the adsorption phase of the CMV infection cycle, we hypothesized that p5+14 effectively competes for CMV adsorption to the cell surface resulting in the reduction in infection. Positively charged Lys residues were required for peptide binding to cell-surface HSPGs and reducing viral infection. We show that this inhibition was not due to a direct neutralizing effect on the virus itself and that the peptide blocked adsorption of the virus. The peptide also inhibited infection of other herpesviruses: HCMV and herpes simplex virus 1 and 2 in vitro, demonstrating it has broad-spectrum antiviral activity. Therefore, this peptide may offer an adjunct therapy for the treatment of herpes viral infections and other viruses that use HSPGs for entry.     

Investigating Fluorescence Quenching of ZnS Quantum Dots by Silver Nanoparticles

Water dispersible zinc sulfide quantum dots (ZnS QDs) with an average diameter of 2.9 nm were synthesized in an environment friendly method using chitosan as stabilizing agent. These nanocrystals displayed characteristic absorption and emission spectra having an absorbance edge at 300 nm and emission maxima (λ _emission) at 427 nm. Citrate-capped silver nanoparticles (Ag NPs) of ca. 37-nm diameter were prepared by modified Turkevich process. The fluorescence of ZnS QDs was significantly quenched in presence of Ag NPs in a concentration-dependent manner with K _sv value of 9 × 10⁹ M⁻¹. The quenching mechanism was analyzed using Stern–Volmer plot which indicated mixed nature of quenching. Static mechanism was evident from the formation of electrostatic complex between positively charged ZnS QDs and negatively charged Ag NPs as confirmed by absorbance study. Due to excellent overlap between ZnS QDs emission and surface plasmon resonance band of Ag NPs, the role of energy transfer process as an additional quenching mechanism was investigated by time-resolved fluorescence measurements. Time-correlated single-photon counting study demonstrated decrease in average lifetime of ZnS QDs fluorescence in presence of Ag NPs. The corresponding F?rster distance for the present QD–NP pair was calculated to be 18.4 nm.     

Morphology of the Yeast Endocytic Pathway

Positively charged Nanogold (Nanoprobes, Stony Brook, NY) has been developed as a new marker to follow the endocytic pathway in yeast. Positively charged Nanogold binds extensively to the surface of yeast spheroplasts and is internalized in an energy-dependent manner. Internalization of gold is blocked in the end3 mutant. During a time course of incubation of yeast spheroplasts with positively charged Nanogold at 15°C, the gold was detected sequentially in small vesicles, a peripheral, vesicular/tubular compartment that we designate as an early endosome, a multivesicular body corresponding to the late endosome near the vacuole, and in the vacuole. Experiments examining endocytosis in the sec18 mutant showed an accumulation of positively charged Nanogold in approximately 30–50 nm diameter vesicles. These vesicles most likely represent the primary endocytic vesicles as no other intermediates were detected in the mutant cells, and they correspond in size to the first vesicles detected in wild-type spheroplasts at 15°C. These data lend strong support to the idea that the internalization step of endocytosis in yeast involves formation of small vesicles of uniform size from the plasma membrane.     

Further research on the production, longevity and infectivity of the zoospores of Leptolegnia chapmanii Seymour (Oomycota: Peronosporomycetes)

The effect of temperature on the production, survival and infectivity of zoospores of an Argentinean isolate of Leptolegnia chapmanii was determined under laboratory conditions. Production of zoospores of L. chapmaniiin vitro and in vivo upon first and fourth instars larvae of the mosquito Aedes aegypti was studied at three different temperatures. Zoospores from infected larvae were infective to mosquito larvae for 51, 12, and 5 consecutive days when maintained at 25, 35, and 10 °C, respectively. Maximum zoospore production in infected fourth-instar larvae was 9.6 ± 1.4 × 10⁴ zoosp/larva at 48 h at 25 °C. The average number of zoospores produced by individual fourth-instar Ae. aegypti larvae infected with L. chapmanii was 3.57 ± 0.46 × 10⁵ zoospores during 6 consecutive days at 25 °C. Zoospore production in vitro was also affected by temperature with a maximum of zoospores (n = 47,666/ml) produced at 25 °C. When zoospores produced in vitro were used as inoculum against Ae. aegypti larvae at 25 °C, larval mortality was recorded for 5 consecutive weeks. The encystment process for zoospores took 17-20 min; the germination of cysts (excystment) occurred 5 min after exposure in water to mosquito larvae. The minimal time of contact between zoospores and mosquito larvae to develop infection was two minutes. Infection took place by zoospore attachment onto and then penetration through the larval cuticle or by ingestion of cysts as was confirmed by histological studies. Temperature directly affected infectivity and production of zoospores in vivo and in vitro although L. chapmanii zoospores tolerate a wide range of temperatures.     

Susceptibility of Psorophora columbiae Larvae over Time to Parasitism by Romanomermis culicivorax

The ability of Romanomermis culicivorax preparasites to penetrate and infect Psorophora columbiae decreased substantially after ca. 28 hours. Parasitism at temperatures typical of Louisiana rice fields (i.e., 26, 29, and 32 ± 0.5 C) showed a significant linear decrease (P < 0.01) as the percentage of older larval instars increased at the times of exposure. These data emphasize the need for a synchronous field application of preparasites to challenge the rapid development of early instars of Ps. columbiae. Applications of postparasites rather than insecticide treatments to potential mosquito breeding habitats may offer greater flexibility in larval mosquito control programs.     

Pulsating oocytes and rotating follicles in an insect ovary

The larval ovaries of the paedogenetic gall midge Heteropeza pygmaea can be cultured in vitro throughout ovary development, follicle formation, and egg growth. Time-lapse films of oogenesis in vitro reveal two kinetic phenomena: (1) All oocytes in the ovary display a swift and continuous pulsating movement, bringing about a periodical deformation of their nuclei. (2) Following their formation in the ovaries, the follicles start to rotate slowly and stop rotating only shortly before embryonic development begins.     

Vaccination against the nematode Trichostrongylus colubriformis. I. Vaccination of guinea-pigs with worm homogenates and soluble products released during in vitro maintenance

Vaccination of guineapigs with homogenates of fourth larval stage and adult Trichostrongylus colubriformis and soluble products released by fourth stage larvae during in vitro maintenance, induced a high level of protection against challenge infection with the parasite.     

Charged Amino Acids in a Preprotein Inhibit SecA-Dependent Protein Translocation

Sec translocase catalyzes membrane protein insertion and translocation. We have introduced stretches of charged amino acid residues into the preprotein proOmpA and have analyzed their effect on in vitro protein translocation into Escherichia coli inner membrane vesicles. Both negatively and positively charged amino acid residues inhibit translocation of proOmpA, yielding a partially translocated polypeptide chain that blocks the translocation site and no longer activates preprotein-stimulated SecA ATPase activity. Stretches of positively charged residues are much stronger translocation inhibitors and suppressors of the preprotein-stimulated SecA ATPase activity than negatively charged residues. These results indicate that both clusters of positively and negatively charged amino acids are poor substrates for the Sec translocase and that this is reflected by their inability to stimulate the ATPase activity of SecA.     

Persistent adult zebrafish behavioral deficits results from acute embryonic exposure to gold nanoparticles

As the number of products containing nanomaterials increase, human exposure to nanoparticles (NPs) is unavoidable. Presently, few studies focus on the potential long-term consequences of developmental NP exposure. In this study, zebrafish embryos were acutely exposed to three gold NPs that possess functional groups with differing surface charge. Embryos were exposed to 50 μg/mL of 1.5 nm gold nanoparticles (AuNPs) possessing negatively charged 2-mercaptoethanesulfonic acid (MES) or neutral 2-(2-(2-mercaptoethoxy)ethoxy)ethanol (MEEE) ligands or 10 μg/mL of the AuNPs possessing positively charged trimethylammoniumethanethiol (TMAT). Both MES- and TMAT-AuNP exposed embryos exhibited hypo-locomotor activity, while those exposed to MEEE-AuNPs did not. A subset of embryos that were exposed to 1.5 nm MES- and TMAT-AuNPs during development from 6 to 120 h post fertilization was raised to adulthood. Behavioral abnormalities and the number of survivors into adulthood were evaluated at 122 days post fertilization. We found that both treatments induced abnormal startle behavior following a tap stimulus. However, the MES-AuNPs exposed group also exhibited abnormal adult behavior in the light and had a lower survivorship into adulthood. This study demonstrates that acute, developmental exposure to 1.5 nm MES- and TMAT-AuNPs, two NPs differing only in the functional group, affects larval behavior, with behavioral effects persisting into adulthood.     

Chromobacterium Csp_P Reduces Malaria and Dengue Infection in Vector Mosquitoes and Has Entomopathogenic and In Vitro Anti-pathogen Activities

Plasmodium and dengue virus, the causative agents of the two most devastating vector-borne diseases, malaria and dengue, are transmitted by the two most important mosquito vectors, Anopheles gambiae and Aedes aegypti, respectively. Insect-bacteria associations have been shown to influence vector competence for human pathogens through multi-faceted actions that include the elicitation of the insect immune system, pathogen sequestration by microbes, and bacteria-produced anti-pathogenic factors. These influences make the mosquito microbiota highly interesting from a disease control perspective. Here we present a bacterium of the genus Chromobacterium (Csp_P), which was isolated from the midgut of field-caught Aedes aegypti. Csp_P can effectively colonize the mosquito midgut when introduced through an artificial nectar meal, and it also inhibits the growth of other members of the midgut microbiota. Csp_P colonization of the midgut tissue activates mosquito immune responses, and Csp_P exposure dramatically reduces the survival of both the larval and adult stages. Ingestion of Csp_P by the mosquito significantly reduces its susceptibility to Plasmodium falciparum and dengue virus infection, thereby compromising the mosquito''s vector competence. This bacterium also exerts in vitro anti-Plasmodium and anti-dengue activities, which appear to be mediated through Csp_P -produced stable bioactive factors with transmission-blocking and therapeutic potential. The anti-pathogen and entomopathogenic properties of Csp_P render it a potential candidate for the development of malaria and dengue control strategies.     

Biodegradable PLGA microcarriers for injectable delivery of chondrocytes: effect of surface modification on cell attachment and function

Poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres were prepared by an oil/water emulsion solvent evaporation method to use as an injectable microcarrier for cell delivery. Three different kinds of PLGA microspheres having hydrophobic, negatively charged, and positively charged surfaces were prepared. Hydrophobic and negatively charged PLGA microspheres were prepared by using terminally capped and uncapped PLGA polymer, respectively. Positively charged PLGA microspheres were prepared by blending PLGA with PLGA-g-poly(L-lysine) graft copolymer as a surface modifying agent. Bovine chondrocytes were cultured on the three PLGA microspheres under serum conditions to comparatively evaluate cell attachment, cell proliferation, and cell function with respect to surface properties. Positively charged PLGA microspheres showed the highest cell attachment, growth, and function compared to hydrophobic and negatively charged microspheres. Surface-modified PLGA microspheres can potentially be used as an injectable delivery system for cells into a tissue defect site.     

How important are aquatic predators to mosquito larval populations in natural wetlands? A case study from Carolina bays in Georgia

Predation is believed to be an important natural control on larval mosquito populations. However, empirical evidence for predator impacts is lacking, especially from natural wetlands (swamps and marshes). Over a 2-year period, we sampled larval mosquito populations and naturally co-occurring predator assemblages (aquatic invertebrates, fishes) from ten depressional wetlands (Carolina bays) located on a wildlife management area in east central Georgia. We collected a diversity of mosquito larvae and predators (odonates, bugs, beetles, flies, and fishes) from the wetlands, with predator numbers substantially exceeding mosquito larval numbers. However, using a community ecology approach with multivariate ordination and correlation techniques, we found no compelling evidence that these predators were controlling mosquito larval distributions (i.e. significant negative statistical associations were not detected). Those mosquitoes that successfully breed in Carolina bay wetlands (Culiseta melanura, Coquillettidia perturbans, Anopheles crucians) appear well adapted to co-exist with a plethora of naturally occurring predators.     

Microbial larvicides for mosquito control: Impact of long lasting formulations of Bacillus thuringiensis var. israelensis and Bacillus sphaericus on non‐target organisms in western Kenya highlands

The microbial larvicides Bacillus thuringiensis var. israelensis and Bacillus sphaericus have been used extensively for mosquito control and have been found to be effective and safe to non‐target organisms cohabiting with mosquito larvae. Recently developed long lasting microbial larvicides (LLML), although evading the previous challenge of short duration of activity, increase the risk of persistence of toxins in the treated larval habitats. This study monitored the impact of LLML FourStar^® and LL3 on non‐target organisms cohabiting with mosquito larvae in an operational study to control malaria vectors in western Kenya highlands. A total of 300 larval habitats were selected in three highland villages. The habitats were first monitored for 5 weeks to collect baseline data on non‐target organisms cohabiting with mosquito larvae and then randomized into two treatment arms (respective FourStar^® and LL3) and one control arm. Non‐target organisms were sampled weekly for 5 months after treatment to assess the impact of LLML intervention. Before treatment, the mean density of all non‐target organisms combined in the control, LL3 and FourStar^® treated habitats was 1.42, 1.39 and 1.49 individuals per habitat per sampling occasion, respectively. Following treatment, this density remained fairly unchanged for 21 weeks at which time it was 1.82, 2.11, and 2.05 for the respective control, LL3 and FourStar^® treated habitats. Statistical analysis revealed that LL3 and FourStar^® did not significantly alter abundance, richness or diversity of the 11 taxa studied, when comparing the intervention and control larval habitats. However, both FourStar^® and LL3 significantly reduced the density of malaria vectors. In conclusion, one round of label rate application of FourStar^® or LL3 in natural larval habitats did not alter richness, abundance or diversity of the monitored aquatic non‐target organisms cohabiting with mosquito larvae to an ecologically significant level.     

Bacterial Communities Associated with Culex Mosquito Larvae and Two Emergent Aquatic Plants of Bioremediation Importance

Microbes are important for mosquito nutrition, growth, reproduction and control. In this study, we examined bacterial communities associated with larval mosquitoes and their habitats. Specifically, we characterized bacterial communities associated with late larval instars of the western encephalitis mosquito ( Culex tarsalis ), the submerged portions of two emergent macrophytes (California bulrush, Schoenoplectus californicus and alkali bulrush, Schoenoplectus maritimus ), and the associated water columns to investigate potential differential use of resources by mosquitoes in different wetland habitats. Using next-generation sequence data from 16S rRNA gene hypervariable regions, the alpha diversity of mosquito gut microbial communities did not differ between pond mesocosms containing distinct monotypic plants. Proteobacteria, dominated by the genus Thorsellia (Enterobacteriaceae), was the most abundant phylum recovered from C . tarsalis larvae. Approximately 49% of bacterial OTUs found in larval mosquitoes were identical to OTUs recovered from the water column and submerged portions of the two bulrushes. Plant and water samples were similar to one another, both being dominated by Actinobacteria, Bacteroidetes, Cyanobacteria , Proteobacteria and Verrucomicrobia phyla. Overall, the bacterial communities within C . tarsalis larvae were conserved and did not change across sampling dates and between two distinct plant habitats. Although Thorsellia spp. dominated mosquito gut communities, overlap of mosquito gut, plant and water-column OTUs likely reveal the effects of larval feeding. Future research will investigate the role of the key indicator groups of bacteria across the different developmental stages of this mosquito species.