Biodistribution tracks compounds or molecules of interest in vivo to understand a compound's anticipated efficacy and safety. Nanoparticles deliver nucleic acid and drug payloads and enhance tumor permeability due to multiple properties such as high surface area to volume ratio, surface functionalization, and modifications. Studying the in vivo biodistribution of nanoparticles documents the effectiveness and safety of nanoparticles and facilitates a more application-driven approach for nanoparticle development that allows for more successful translation into clinical use. In this study, we present a relatively simple method to determine the biodistribution of magnetic iron nanoparticles in mice. In vitro, cells take up branched amphiphilic peptide-coated magnetic nanobeads (BAPc-MNBs) like their counterparts, i.e., branched amphiphilic peptide capsules (BAPCs) with a hollow water-filled core. Both BAPc-MNBs and BAPCs have widespread applications as a nanodelivery system. We evaluated the BAPc-MNBs tissue distribution in wild-type mice injected intravenously (i.v.), intraperitoneally (i.p.), or orally gavaged to understand the biological interactions and to further the development of branched amphiphilic peptide-based nanoparticles. The magnetic nanoparticles allowed collection of the BAPc-MNBs from multiple organs by magnetic bead sorting, followed by a high-throughput screening for iron content. When injected i.v., nanoparticles were distributed widely to various organs before elimination from the system via the intestines in feces. The spleen accumulated the highest amount of BAPc-MNBs in mice administered NPs via i.v. and i.p. but not via oral gavage. Taken together, these data demonstrate that the magnetic sorting not only allowed quantification of the BAPc-MNBs but also identified the distribution of BAPc-MNBs after distinct administration methods.

The graphite-water interface provides a unique environment for polypeptides that generally favors ordered structures more than in solution. Therefore, systems consisting of designed peptides and graphitic carbon might serve as a convenient medium for controlled self-assembly of functional materials. Here, we computationally designed cyclic peptides that spontaneously fold into a β-sheet-like conformation at the graphite-water interface and self-assemble, and we subsequently observed evidence of such assembly by atomic force microscopy. Using a novel protocol, we screened nearly 2000 sequences, optimizing for formation of a unique folded conformation while discouraging unfolded or misfolded conformations. A head-to-tail cyclic peptide with the sequence GTGSGTGGPGGGCGTGTGSGPG showed the greatest apparent propensity to fold spontaneously, and this optimized sequence was selected for larger scale molecular dynamics simulations, rigorous free-energy calculations, and experimental validation. In simulations ranging from hundreds of nanoseconds to a few microseconds, we observed spontaneous folding of this peptide at the graphite-water interface under many different conditions, including multiple temperatures (295 and 370 K), with different initial orientations relative to the graphite surface, and using different molecular dynamics force fields (CHARMM and Amber). The thermodynamic stability of the folded conformation on graphite over a range of temperatures was verified by replica-exchange simulations and free-energy calculations. On the other hand, in free solution, the folded conformation was found to be unstable, unfolding in tens of picoseconds. Intermolecular hydrogen bonds promoted self-assembly of the folded peptides into linear arrangements where the peptide backbone exhibited a tendency to align along one of the six zigzag directions of the graphite basal plane. For the optimized peptide, atomic force microscopy revealed growth of single-molecule-thick linear patterns of 6-fold symmetry, consistent with the simulations, while no such patterns were observed for a control peptide with the same amino acid composition but a scrambled sequence.

Nanoparticles (NPs) have been shown to be a suitable mRNA delivery platform by conferring protection against ribonucleases and facilitating cellular uptake. Several NPs have succeeded in delivering mRNA intranasally, intratracheally, and intramuscularly in preclinical settings. However, intravenous mRNA delivery has been less explored. Only a few NPs have been tested for systemic delivery of mRNA, many of which are formulated with polyethylene glycol (PEG). The incorporation of PEG presents some tradeoffs that must be carefully considered when designing a systemic delivery model. For example, while the addition of PEG may prolong circulation time by preventing early clearance by the mononuclear phagocytic system (MPS), it has also been reported that treating patients with PEGylated drugs can result in hypersensitivity reactions due to anti-PEG antibodies. Thus, it is desirable to have alternative PEG-free delivery methods for mRNA to avoid these adverse effects while preserving the beneficial effects. Our research group developed BAPCs (branched amphiphilic peptide capsules), a peptide-based nanoparticle that resists disruption by chaotropes, proteases, and elevated temperature, thus displaying significant stability and shelf-life. In this study, we demonstrated that similarly to PEG, mRNA shields the BAPC cationic surface to avoid early clearance by the MPS. Multispectral optoacoustic tomography (MSOT) and fluorescence reflectance imaging were imaging techniques used to analyze biodistribution within major MPS organs. Analysis of pro-inflammatory cytokine expression showed that BAPC-mRNA complexes do not cause chronic inflammation. Additionally, BAPCs enhance intracellular delivery of mRNA with negligible cytotoxicity or oxidative stress. These results might pave the way for future therapeutic applications of BAPCs as a delivery platform for systemic mRNA delivery.

Bionanotechnology has developed rapidly over the past two decades, owing to the extensive and versatile, functionalities and applicability of nanoparticles (NPs). Fifty-one nanomedicines have been approved by FDA since 1995, out of the many NPs based formulations developed to date. The general conformation of NPs consists of a core with ligands coating their surface, that stabilizes them and provides them with added functionalities. The physicochemical properties, especially the surface composition of NPs influence their bio-interactions to a large extent. This review discusses recent studies that help understand the nano-bio interactions of iron oxide and gold NPs with different surface compositions. We discuss the influence of the experimental factors on the outcome of the studies and, thus, the importance of standardization in the field of nanotechnology. Recent studies suggest that with careful selection of experimental parameters, it is possible to improve the positive correlation between in vitro and in vivo studies. This provides a fundamental understanding of the NPs which helps in assessing their potential toxic side effects and may aid in manipulating them further to improve their biocompatibility and biosafety.

The use of RNA interference (RNAi) or double‐stranded RNA (dsRNA)‐mediated gene silencing has become one of the most promising tools on the field of pest management. One of the barriers for RNAi efficiency, however, has being the fact that it needs to be rapidly taken up by cells before being degraded by nucleases. In this context, the nanoscale delivery system formed by Branched Amphiphilic Peptides Capsules (BAPCs™) are proposed as a solution for a safer and more efficient cellular uptake of lethal dsRNA constructs. BAPCs can be synthesized in a varied diameters ranging from 10 to 500 nm. BAPCs act as cationic nucleation centers for nucleic acids, generating peptide‐DNA or dsRNA complexes that are readily taken up by eukaryotic cells, escape late endosomes, and lead to the release of the surface‐bound nucleic acids that alter protein expression. Recently, we described the use of BAPCs for suppressing the translation of genes transcripts related to the unfolded protein response (UPR) in Tribolium castaneum and Acyrthosiphon pisum, which led to a premature death with a 50% increase in lethality when compared to dsRNA alone. For this study, BAPCs were combined with 3 different concentrations of a dsRNA supplied by GreenLight Biosciences™. The test sequence contained ~600 bp. Its lethal bio‐efficacy was compared to the dsRNA by itself on Colorado potato beetle. The tests using unformulated dsRNA at 0.00005, 0.0001 and 0.001, mg/mL exhibited a lethal effect of 10%, 30% and 50% lethality after 8 days by contrast of 30%, 60% and 90% of BAPCs‐dsRNA formulations, followed by approximately 40% reduction on leaf consumption in comparison to the unformulated material. The present results have shown that BAPCs‐dsRNA formulations offer a considerable increase in lethality bio‐efficacy over unformulated dsRNA indicating an additive effect in dsRNA delivery over using dsRNA by itself.Support or Funding InformationThis project is a result of a collaboration between GreenLight Biosciences&[trade], Inc. and Phoreus Biotechnology, Inc.

Understanding cellular uptake mechanisms of nanoparticles with therapeutic potential has become critical in the field of drug delivery. Elucidation of cellular entry routes can aid in the dissection of the complex intracellular trafficking and potentially allow for the manipulation of nanoparticle fate after cellular delivery (i.e., avoid lysosomal degradation). Branched amphiphilic peptide capsules (BAPCs) are peptide nanoparticles that have been and are being explored as delivery systems for nucleic acids and other therapeutic molecules in vitro and in vivo. In the present study, we determined the cellular uptake routes of BAPCs with and without a magnetic nanobead core (BAPc-MNBs) in two cell lines: macrophages and intestinal epithelial cells. We also studied the influence of size and growth media composition in this cellular process. Substituting the water-filled core with magnetic nanobeads might provide the peptide bilayer nanocapsules with added functionalities, facilitating their use in bio/immunoassays, magnetic field guided drug delivery, and magnetofection among others. Results suggest that BAPc-MNBs are internalized into the cytosol using more than one endocytic pathway. Flow cytometry and analysis of reactive oxygen and nitrogen species (ROS/RNS) demonstrated that cell viability was minimally impacted by BAPc-MNBs. Cellular uptake pathways of peptide vesicles remain poorly understood, particularly with respect to endocytosis and intracellular trafficking. Outcomes from these studies provide a fundamental understanding of the cellular uptake of this peptide-based delivery system which will allow for strengthening of their delivery capabilities and expanding their applications both in vitro and in vivo.

Self‐assembling peptides based delivery systems are gaining increased importance as they are highly biocompatible, biodegradable and tunable i.e. structure and function can be modified. Branched amphiphilic peptides (BAPs) self‐assemble to form bilayer delimited vesicles, called the Branched Amphiphilic Peptide Capsules (BAPCs), whose molecular architecture is similar to liposomes. BAPCs have been used to date to deliver encapsulated radionuclides and dsRNA and DNA electrostatically bound to the exterior of the capsules. Recently, we have synthesized magnetic nanoparticles with the BAP bilayer coating for studying their interaction with a biological system and for exploring applications in delivery. The magnetic nanoparticles are being used as quantification tools to study some aspects of the peptide bilayer interactions such as their route of uptake by cells in culture and to study their tissue distribution in mice, employing several routes of delivery. Recent in vitro studies using widely used techniques such as confocal imaging and flow cytometry alongside the quantification method using the newly developed BAP‐MNBs, demonstrate that they are readily taken up by cells in culture via more than one endocytic pathway. This study suggests that quantification using BAP‐MNBs is a reliable tool for studying BAPCs delivery system and associated molecules. Current studies involve studying the tissue distribution of BAP‐MNBs when injected intravenously in mice. The BAP‐MNBs are alongside being tested as delivery systems to deliver a therapeutic retro‐inverso D‐peptide (RD‐p9) known to retard the growth of melanoma tumors in mice. Our current studies in mice will provide insight into the fate of BAPCs in vivo, aiding future studies which aim to tailor and use BAPCs as well as BAP‐MNBs for specific applications.Support or Funding InformationPhoreus Biotechology Inc., Olathe, Kansas

Abstract The devastation to the US citrus industries from huanglongbing (HLB or citrus greening disease, caused by 'Candidatus Liberibacter asiaticus') is spread during feeding by the Asian citrus psyllid (ACP) Diaphorina citri. Innovative echnologies, such as RNA suppression by RNAi, morpholino oligos, or gene editing tools, including CRISPR/Cas9, all provide non-transgenic strategies, as well as transgenic solutions to manage arthropod vectors, pests and pathogens. Current results from researchers report that these methods enable suppression of ACP vectors, their endosymbionts and the Liberibacter pathogens in infected citrus trees. This chapter reviews several emerging technologies and strategies that enable direct targeting of pathogenic microbes including bacteria in citrus trees, and the critical genes specific to arthropod vectors, such as ACP and their endosymbionts. RNAi-based approaches continue to make advances to improve persistence and activity, especially when applied as an exogenous spray or as soil-applied treatments. RNA-suppressing biopesticides will soon become common in the protection of crops that are either difficult to transform, such as fruit trees, or that are short-term seasonal crops.

Melanoma, a form of skin cancer, is one of the most common cancers in young men and women. Tumors require angiogenesis to provide oxygen and nutrients for growth. Pro-angiogenic molecules such as VEGF and anti-angiogenic molecules such as sFlt-1 control angiogenesis. In addition, the serum protein, Beta2 Glycoprotein I (β2-GPI) induces or inhibits angiogenesis depending on conformation and concentration. β2-GPI binds to proteins and negatively charged phospholipids on hypoxic endothelial cells present in the tumor microenvironment. We hypothesized that peptides derived from the binding domain of β2-GPI would regulate angiogenesis and melanoma growth. In vitro analyses determined the peptides reduced endothelial cell migration and sFlt-1 secretion. In a syngeneic, immunocompetent mouse melanoma model, β2-GPI-derived peptides also reduced melanoma growth in a dose-dependent response with increased sFlt-1 and attenuated vascular markers compared to negative controls. Importantly, administration of peptide with sFlt-1 antibody resulted in tumor growth. These data demonstrate the therapeutic potential of novel β2-GPI-derived peptides to attenuate tumor growth and endothelial migration is sFlt-1 dependent.

Development of new and specific insect pest management methods is critical for overcoming pesticide resistance and collateral off-target killings. Gene silencing by feeding dsRNA to insects shows promise in this area. Here we described the use of a peptide nano-material, branched amphiphilic peptide capsules (BAPCs), that facilitates cellular uptake of dsRNA by insects through feeding. The insect diets included dsRNA with and without complexation with BAPCs. The selected insect species come from two different orders with different feeding mechanisms: Tribolium castaneum and Acyrthosiphon pisum. The gene transcripts tested (BiP and Armet) are part of the unfolded protein response (UPR) and suppressing their translation resulted in lethality. For Acyrthosiphon pisum, ingestion of BiP-dsRNA associated with BAPCs led to the premature death of the aphids (t1/2=4–5days) compared to ingestion of the same amounts of free BiP-dsRNA (t1/2=11–12days). Tribolium castaneum was effectively killed using a combination of BiP-dsRNA and Armet-dsRNA complexed with BAPCs; most dying as larvae or during eclosion (~75%). Feeding dsRNA alone resulted in fewer deaths (~30%). The results show that complexation of dsRNA with BAPCs enhanced the oral delivery of dsRNA over dsRNA alone.

BACKGROUND:Acute phytic acid intake has been found to decrease iron bioavailability; however, repeated phytic acid consumption leads to iron absorption adaptation. Salivary proline-rich proteins (PRPs) have been shown to inhibit iron chelation to tannins and may mediate similar iron absorption adaptation with phytic acid intake. OBJECTIVES:The objectives of this study were to determine whether salivary proteins bind to phytic acid in vitro, and to explore a proof of concept in a pilot study that examined the impact of 4-wk, daily phytic acid supplementation on individuals' iron status, bioavailability, and salivary PRP concentrations. METHODS:High-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization-time of flight were used to characterize in vitro salivary protein-phytic acid interactions. Nonanemic women (n = 7) consumed 350 mg phytic acid supplements 3 times daily for 4 wk, and meal challenges were employed to determine iron bioavailability, iron status, and salivary protein concentrations before and after supplementation periods. Enzyme-linked immunosorbent assay (ELISA) analysis of purified protein fractions and participant saliva identified proteins bound to phytic acid. RESULTS:In vitro salivary protein-phytic acid interaction identified cystatin SN, a non-proline rich salivary protein, as the specific bound protein to phytic acid. Iron bioavailability (P = 0.32), hemoglobin (P = 0.72), and serum ferritin (P = 0.08) concentrations were not reduced from week 0 to week 4 after phytic acid supplementation. Basic PRPs and cystatin SN concentrations were positively correlated with iron bioavailability at week 4. CONCLUSIONS:Overall, results suggest that phytic acid binds to the non-PRP cystatin SN and that salivary protein production may improve iron bioavailability with phytic acid consumption.

Branched Amphiphilic Peptides Capsules (BAPCs™) are a novel class of nano‐carriers constituted from the spontaneous co‐assembly of two unique, engineered peptide sequences. We recently reported on a thermally induced variant of BAPC™ that demonstrates promise as a vehicle for gene delivery and transfection. These BAPCs™ display a uniform size of 20–30 nm; they are easy to synthesize, stable and produce minimal immunogenic and inflammatory responses, in contrast to those commonly observed with viral and cationic lipid based approaches for gene delivery. BAPCs™ act as cationic nucleation centers allowing nucleic to wrap around them in a nucleosome like fashion, generating peptide‐nucleic complexes with sizes ranging from 50 to 250 nm. In this study, we combine the pre‐constituted BAPCs™ with a plasmid DNA construct encoding the green fluorescent protein (eGFP) in order to generate DNA/peptide complexes. We utilize these complexes to successfully transfect mammalian cells in vitro , yielding higher transfections rates than the commercially available lipid based reagent Lipofectin®, with minimal cytotoxicity. We then test the transfection system for the in vivo delivery of a DNA vaccine, previously designed to activate immune responses capable of controlling tumors induced by type 16 human papilloma virus (HPV‐16). We successfully demonstrate the efficacy of the DNA/BAPCs™ complexes in the in vitro activation of murine dendritic cells, and the enhancement of the associated vaccine's anti‐tumor potency, without significant toxic effects in vaccinated mice. Together these results indicate that the interaction of double stranded DNA to the cationic BAPCs™ nanoparticles represents a promising new in vitro and in vivo non‐viral gene delivery system. Support or Funding Information Partial support for this project was provided by the Terry Johnson Cancer Center at Kansas State University, The Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD) at Kansas State University This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Branched amphiphilic peptide capsules (BAPCs) are an efficient transport system that can deliver nucleic acids, small proteins, and solutes. The ability of BAPCs to break down is essential to their adoption as a delivery vehicle for human and agricultural applications. Until now, however, BAPCs were shown to be inert to mammalian degradation systems. Here, we demonstrate, using BAPCs encapsulating the toxic urea analogue thiourea, that the common soil fungus Aspergillus nidulans can degrade BAPCs. We provide evidence that this degradation is extracellular through the action of secreted proteases. Our data indicate that BAPCs are likely biodegradable in the environment.

New method for heritable gene editing in Insects, Hemiptera. Addition of Branched Amphiphilic Peptide Capsules, BAPC, improves delivery of CRISPR components, plasmids, and dsRNA for heritable gene editing and gene targeting in insect nymphs and adults (Psyllids- Diaphorina citri, Leafhoppers- Homalodisca vitripennis, Whitefly- Bemisia argentifolii). First heritable gene Knockouts, KO, using BAPC-assisted-CRISPR-Cas9, produced G2 mutants from injected adult females. The KO target was the thioredoxin gene, resulting in 550 nt deletion in gDNA of target insects. In psyllids the thioredoxin gene, TXT, and Vermillion, Vm, KO produced changes in physiology and eye color, respectively. Strategies are alter the insect vectors from vector into non-vectors. Psyllids transmit pathogenic bacteria, Liberibacter asiaticus, which threatens citrus worldwide, Leafhoppers transmit, Xylella fastidiosa, bacteria, which threatens tree crops and grapevines worldwide; Whiteflies transmit Begomovirus, which threaten global food security. Previous attempts with embryonic injections failed. To bypass the problem we used BAPC with CRISPR components injected in 3rd – 5th instars, and adult females near ovaries. One function of TXT is to promote development. The result is slower development, reduced lifespan, and reduce fecundity as observed in TXT-KO psyllid mutants. Reduce fecundity and slow development are traits that would reduce insect populations. Parasitoids and predators would also have more time to attack nymphal stages as pests take 2 to 3 weeks longer to go from egg to adult. Emerging adults have a reduced lifespan. For the Vm-KO, eye phenotype had color break, white regions. The BAPC-assisted delivery system advances gene editing efforts across all hemipteran pests by permitting the use of nymphs and adults. BAPC-assisted CRISPR delivery revolutionizes strategies to protect citrus and food crops from insect vectors and pathogens [citrusgreening.org]. Support or Funding Information Funding in part: USDA-NIFA 2014-70016-23028. Developing an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Disease. Micro-CT scan of Adult Female Psyllid (Alba-Tercedor & Hunter 2016) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

This chapter will discuss cationic polymers containing ɛ-amine groups and immine or amide linkages that are being used as nanocarriers for nonviral gene delivery. We will focus particularly on cationic peptides. Cationic peptides, free or complexed with self-assembling moieties, have been proposed as carriers of small-molecule drugs as well as nucleic acids and are emerging as an alternative tool for gene delivery. This chapter is organized into two basic sections: cationic polymeric nanocarriers that are chemically synthesized as monomers and those nanocarriers that self-assemble into active structures from smaller synthetic elements. While these chemically synthesized molecules form numerous structures and have been intensely studied for more two decades, very few have entered clinical trials and just one is available to clinicians. In this chapter, we will highlight the unmet needs in this field and also provide insight into the most promising systems for gene delivery.

We provide strong chemical and biophysical evidence that documents that branched amphiphilic peptides, BAPs, known to assemble into spherical nanoassemblies in solution, do assemble as peptide-bilayer-delimited capsules. These nanoassemblies are termed branched amphiphilic peptide capsules (BAPCs). BAPCs are taken up by cells and accumulate in the perinuclear region to persist there without apparent degradation. BAPCs also entrap small proteins and solutes and stably encapsulate a-particle-emitting radionuclides. We have devised a method utilizing thiol chemistry to conjugate these peptide sequences onto gold nanoparticles (<= 5 nm) with the objective of demonstrating the assembly of these peptides into a bilayer. The peptides are initially assembled as a monolayer on the gold surface via interaction with cysteine residues on the peptide C-terminus in an organic solvent. The subsequent transition of these peptide-monolayer-protected gold nanoparticles to an aqueous solution in the presence of excess peptides led to the formation of the peptide bilayer on the gold surface. The approach was exploited further to produce bilayer-coated magnetic nanoparticles. The innovation described in this study provides a stable metallic nanoparticle-peptide conjugate system that will help to determine interactions of BAPs in a biological system, with relative ease, important for developing future applications such as simultaneous delivery and imaging of surface-bound molecules of interest.

Innovative gene targeting strategies are often limited in application across arthropod species due to problems with successful delivery. In hemipterans, embryonic injections often used to deliver CRISPR components fail due to nearly complete embryo mortality. The Asian citrus psyllid, Diaphorina citri , Kuwayama, (Hemiptera: Liviidae), is the vector for a pathogenic bacterium, Candidatus Liberibacter asiaticus, CLas, which is devastating the U.S. citrus industries. The disease called, Huanglongbing, HLB, (aka. Citrus greening disease), is transmitted during psyllid feeding. Infection causes severe tree decline, loss of fruits, and eventually tree death. The citrus tree pathogen, CLas, is a fastidious alpha-proteobacterium, which has spread into all citrus growing regions worldwide. The economic losses are estimated in the billions of dollars, in U.S.A., Brazil, and China. Innovative technologies aimed at reducing psyllid populations using targeting RNA suppression, like RNAi, or gene-editing tools, like CRISPR/Cas9 have potential to reduce psyllid vectors and the pathogen in a highly specific manner. Breakthroughs that improve gene editing in psyllids, such as the BAPC-assisted -CRISPR/Cas9 System, enabled delivery by injection of CRISPR/Cas9 components directly into nymphs and adult females. Injection near ovaries produced heritable germline gene editing in subsequent generations. This method opens the world of gene editing across arthropods and bypasses the need for microinjection of eggs. Effective development of therapeutic treatments to reduce insect vectors, and stop pathogen transmission would provide sustainable citrus and grapevine industries.

The colony of eusocial bee Apis mellifera has a reproductive queen and sterile workers performing tasks such as brood care and foraging. Chemical communication plays a crucial role in the maintenance of sociability in bees with many compounds released by the exocrine glands. The Dufour's gland is a non-paired gland associated with the sting apparatus with important functions in the communication between members of the colony, releasing volatile chemicals that influence workers roles and tasks. However, the protein content in this gland is not well studied. This study identified differentially expressed proteins in the Dufour's glands of nurse and forager workers of A. mellifera through 2D-gel electrophoresis and mass spectrometry. A total of 131 spots showed different expression between nurse and forager bees, and 28 proteins were identified. The identified proteins were categorized into different functions groups including protein, carbohydrate, energy and lipid metabolisms, cytoskeleton-associated proteins, detoxification, homeostasis, cell communication, constitutive and allergen. This study provides new insights of the protein content in the Dufour's gland contributing to a more complete understanding of the biological functions of this gland in honeybees.