Nanoparticle Toxicology.

Nanoparticles from natural and anthropogenic sources are abundant in the environment, thus human exposure to nanoparticles is inevitable. Due to this constant exposure, it is critically important to understand the potential acute and chronic adverse effects that nanoparticles may cause to humans. In this review, we explore and highlight the current state of nanotoxicology research with a focus on mechanistic understanding of nanoparticle toxicity at organ, tissue, cell, and biomolecular levels. We discuss nanotoxicity mechanisms, including generation of reactive oxygen species, nanoparticle disintegration, modulation of cell signaling pathways, protein corona formation, and poly(ethylene glycol)-mediated immunogenicity. We conclude with a perspective on potential approaches to advance current understanding of nanoparticle toxicity. Such improved understanding may lead to mitigation strategies that could enable safe application of nanoparticles in humans. Advances in nanotoxicity research will ultimately inform efforts to establish standardized regulatory frameworks with the goal of fully exploiting the potential of nanotechnology while minimizing harm to humans.

Targeted Analysis of the Size Distribution of Heavy Chain-Modified Hyaluronan with Solid-State Nanopores.

The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HC•HA) complexes. While limited assessments of HC•HA have been performed previously, determining the size distribution of its HA component remains a challenge. Here, we describe a selective method for extracting HC•HA from mixtures that yields material amenable to MW analysis with a solid-state nanopore sensor. After demonstrating the approach in vitro, we validate extraction of HC•HA from osteoarthritic human synovial fluid as a model complex biological matrix. Finally, we apply our technique to pathophysiology by measuring the size distributions of HC•HA and total HA in an equine model of synovitis.

Hyaluronan synthases; mechanisms, myths, & mysteries of three types of unique bifunctional glycosyltransferases.

Hyaluronan (HA), the essential [-3-GlcNAc-1-ß-4-GlcA-1-ß-]n matrix polysaccharide in vertebrates and molecular camouflage coating in select pathogens, is polymerized by "HA synthase" (HAS) enzymes. The first HAS identified three decades ago opened the window for new insights and biotechnological tools. This review discusses current understanding of HA biosynthesis, its biotechnological utility, and addresses some misconceptions in the literature. HASs are fascinating enzymes that polymerize two different UDP-activated sugars via different glycosidic linkages. Therefore, these catalysts were the first examples to break the "one enzyme/one sugar transferred" dogma. Three distinct types of these bifunctional glycosyltransferases (GTs) with disparate architectures and reaction modes are known. Based on biochemical and structural work, we present an updated classification system. Class I membrane-integrated HASs employ a processive chain elongation mechanism and secrete HA across the plasma membrane. This complex operation is accomplished by functionally integrating a cytosolic catalytic domain with a channel-forming transmembrane region. Class I enzymes, containing a single GT family-2 (GT-2) module that adds both monosaccharide units to the nascent chain, are further subdivided into two groups that construct the polymer with opposite molecular directionalities: Class I-R and I-NR elongate the HA polysaccharide at either the reducing or the non-reducing end, respectively. In contrast, Class II HASs are membrane-associated peripheral synthases with a non-processive, non-reducing end elongation mechanism using two independent GT-2 modules (one for each type of monosaccharide) and require a separate secretion system for HA export. We discuss recent mechanistic insights into HA biosynthesis that promise biotechnological benefits and exciting engineering approaches.

Nanoparticle Surface Engineering with Heparosan Polysaccharide Reduces Serum Protein Adsorption and Enhances Cellular Uptake.

Nanoparticle modification with poly(ethylene glycol) (PEG) is a widely used surface engineering strategy in nanomedicine. However, since the artificial PEG polymer may adversely impact nanomedicine safety and efficacy, alternative surface modifications are needed. Here, we explored the "self" polysaccharide heparosan (HEP) to prepare colloidally stable HEP-coated nanoparticles, including gold and silver nanoparticles and liposomes. We found that the HEP-coating reduced the nanoparticle protein corona formation as efficiently as PEG coatings upon serum incubation. Liquid chromatography-mass spectrometry revealed the protein corona profiles. Heparosan-coated nanoparticles exhibited up to 230-fold higher uptake in certain innate immune cells, but not in other tested cell types, than PEGylated nanoparticles. No noticeable cytotoxicity was observed. Serum proteins did not mediate the high cell uptake of HEP-coated nanoparticles. Our work suggests that HEP polymers may be an effective surface modification technology for nanomedicines to safely and efficiently target certain innate immune cells.

Controlling Nanoparticle Uptake in Innate Immune Cells with Heparosan Polysaccharides.

We used heparosan (HEP) polysaccharides for controlling nanoparticle delivery to innate immune cells. Our results show that HEP-coated nanoparticles were endocytosed in a time-dependent manner by innate immune cells via both clathrin-mediated and macropinocytosis pathways. Upon endocytosis, we observed HEP-coated nanoparticles in intracellular vesicles and the cytoplasm, demonstrating the potential for nanoparticle escape from intracellular vesicles. Competition with other glycosaminoglycan types inhibited the endocytosis of HEP-coated nanoparticles only partially. We further found that nanoparticle uptake into innate immune cells can be controlled by more than 3 orders of magnitude via systematically varying the HEP surface density. Our results suggest a substantial potential for HEP-coated nanoparticles to target innate immune cells for efficient intracellular delivery, including into the cytoplasm. This HEP nanoparticle surface engineering technology may be broadly used to develop efficient nanoscale devices for drug and gene delivery as well as possibly for gene editing and immuno-engineering applications.

Methods for isolating and analyzing physiological hyaluronan: a review.

The carbohydrate hyaluronan (or hyaluronic acid, HA) is found in all human tissues and biofluids where it has wide-ranging functions in health and disease that are dictated by both its abundance and size. Consequently, hyaluronan evaluation in physiological samples has significant translational potential. Although the analytical tools and techniques for probing other biomolecules such as proteins and nucleic acids have become standard approaches in biochemistry, those available for investigating hyaluronan are less well established. In this review, we survey methods related to the assessment of native hyaluronan in biological specimens, including protocols for separating it from biological matrices and technologies for determining its concentration and molecular weight.

Selective isolation of hyaluronan by solid phase adsorption to silica.

A solid phase adsorption method for selective isolation of hyaluronan (HA) from biological samples is presented. Following enzymatic degradation of protein, HA can be separated from sulfated glycosaminoglycans, other unsulfated glycosaminoglycans, nucleic acids, and proteolytic fragments by adsorption to amorphous silica at specific salt concentrations. The adsorbed HA can be released from silica using neutral and basic aqueous solutions. HA ranging in size from ∼9 kDa to MDa polymers has been purified by this method from human serum and conditioned medium of cultured cells.

A primer for initiation of commercialization of glycobiological discoveries.

In this age of translational science, blossoming biotechnology and increasingly elusive federal funding, a working knowledge of commercialization beyond typical academic training is necessary. Here, some issues and initial strategies to help principal investigators, postdoctoral fellows and students considering the route from discovery to invention to commercialization in the glycobiology field. Common myths are addressed, and tips and resources are provided to help start down this path.

Heparan Sulfate Domains Required for Fibroblast Growth Factor 1 and 2 Signaling through Fibroblast Growth Factor Receptor 1c.

A small library of well defined heparan sulfate (HS) polysaccharides was chemoenzymatically synthesized and used for a detailed structure-activity study of fibroblast growth factor (FGF) 1 and FGF2 signaling through FGF receptor (FGFR) 1c. The HS polysaccharide tested contained both undersulfated (NA) domains and highly sulfated (NS) domains as well as very well defined non-reducing termini. This study examines differences in the HS selectivity of the positive canyons of the FGF12-FGFR1c2 and FGF22-FGFR1c2 HS binding sites of the symmetric FGF2-FGFR2-HS2 signal transduction complex. The results suggest that FGF12-FGFR1c2 binding site prefers a longer NS domain at the non-reducing terminus than FGF22-FGFR1c2 In addition, FGF22-FGFR1c2 can tolerate an HS chain having an N-acetylglucosamine residue at its non-reducing end. These results clearly demonstrate the different specificity of FGF12-FGFR1c2 and FGF22-FGFR1c2 for well defined HS structures and suggest that it is now possible to chemoenzymatically synthesize precise HS polysaccharides that can selectively mediate growth factor signaling. These HS polysaccharides might be useful in both understanding and controlling the growth, proliferation, and differentiation of cells in stem cell therapies, wound healing, and the treatment of cancer.

Identification of a chondroitin synthase from an unexpected source, the green sulfur bacterium Chlorobium phaeobacteroides.

Glycosaminoglycans (GAGs) are known to be present in all animals as well as some pathogenic microbes. Chondroitin sulfate is the most abundant GAG in mammals where it has various structural and adhesion roles. The Gram-negative bacteria Pasteurella multocida Type F and Escherichia coli K4 produce extracellular capsules composed of unsulfated chondroitin or a fructosylated chondroitin, respectively. Such polysaccharides that are structurally related to host molecules do not generally provoke a strong antibody response thus are thought to be employed as molecular camouflage during infection. We observed a sequence from the photosynthetic green sulfur bacteria, Chlorobium phaeobacteroides DSM 266, which was very similar (~62% identical) to the open reading frames of the known bifunctional chondroitin synthases (PmCS and KfoC); some segments are strikingly conserved amongst the three proteins. Recombinant E. coli-derived Chlorobium enzyme preparations were found to possess bona fide chondroitin synthase activity in vitro. This new catalyst, CpCS, however, has a more promiscuous acceptor usage than the prototypical PmCS, which may be of utility in novel chimeric GAG syntheses. The finding of such a similar chondroitin synthase enzyme in C. phaeobacteroides is unexpected for several reasons including (a) a free-living nonpathogenic organism should not "need" an animal self molecule for protection, (b) the Proteobacteria and the green sulfur bacterial lineages diverged ~2.5-3 billion years ago and (c) the ecological niches of these bacteria are not thought to overlap substantially to facilitate horizontal gene transfer. CpCS provides insight into the structure/function relationship of this class of enzymes.

Synthesis and characterization of heparosan-granulocyte-colony stimulating factor conjugates: a natural sugar-based drug delivery system to treat neutropenia.

Many injectable drugs require delivery strategies for enhancing their pharmacokinetics due to rapid loss via renal filtration if possess low molecular weight (<60-70 kDa) and/or clearance by the body's components (e.g., proteases, antibodies, high-efficiency receptors) in their native form. FDA-approved polyethylene glycol (PEG) is a vehicle for improving therapeutics, but artificial polymers have potential biocompatibility and immunogenicity liabilities. Here, we utilized a natural vertebrate carbohydrate, heparosan (HEP), the biosynthetic precursor of heparan sulfate and heparin, to enhance performance of a biologic drug. The HEP polysaccharide was stable with a long half-life (~8 days for 99-kDa chain) in the nonhuman primate bloodstream, but was efficiently degraded to very short oligosaccharides when internalized by cells, and then excreted into urine and feces. Several HEP-modified human granulocyte-colony stimulating factor (G-CSF) conjugates were synthesized with defined quasi-monodisperse HEP polysaccharide chains. Single dosing of 55- or 99-kDa HEP-G-CSF in rats increased blood neutrophil levels comparable to PEG-G-CSF conjugates. Repeated dosing of HEP-G-CSF or HEP alone for 2 weeks did not cause HEP-specific toxic effects in rats. HEP did not possess the anticoagulant behavior of its daughter, heparin, based on testing in rats or clinical diagnostic assays with human plasma. Neither anti-HEP IgG nor IgM antibodies were detected in a long-term (9 doses over 7 months) immunogenicity study of the HEP-drug conjugate with rats. These proof-of-concept experiments with HEP-G-CSF indicate that it is a valid drug candidate for neutropenia and suggest the potential of this HEP-based platform as a safe alternative delivery vehicle for other therapeutics.

Heparosan-coated liposomes for drug delivery.

Liposomal encapsulation is a useful drug delivery strategy for small molecules, especially chemotherapeutic agents such as doxorubicin. Doxil® is a doxorubicin-containing liposome ("dox-liposome") that passively targets drug to tumors while reducing side effects caused by free drug permeating and poisoning healthy tissues. Polyethylene glycol (PEG) is the hydrophilic coating of Doxil® that protects the formulation from triggering the mononuclear phagocyte system (MPS). Evading the MPS prolongs dox-liposome circulation time thus increasing drug deposition at the tumor site. However, multiple doses of Doxil® sometimes activate an anti-PEG immune response that enhances liposome clearance from circulation and causes hypersensitivity, further limiting its effectiveness against disease. These side effects constrain the utility of PEG-coated liposomes in certain populations, justifying the need for investigation into alternative coatings that could improve drug delivery for better patient quality of life and outcome. We hypothesized that heparosan (HEP; [-4-GlcA-ß1-4-GlcNAc-α1-]n) may serve as a PEG alternative for coating liposomes. HEP is a natural precursor to heparin biosynthesis in mammals. Also, bacteria expressing an HEP extracellular capsule during infection escape detection and are recognized as "self," not a foreign threat. By analogy, coating drug-carrying liposomes with HEP should camouflage the delivery vehicle from the MPS, extending circulation time and potentially avoiding immune-mediated clearance. In this study, we characterize the postmodification insertion of HEP-lipids into liposomes by dynamic light scattering and coarse-grain computer modeling, test HEP-lipid immunogenicity in rats, and compare the efficacy of drug delivered by HEP-coated liposomes to PEG-coated liposomes in a human breast cancer xenograft mouse model.

Expanding glycosaminoglycan chemical space: towards the creation of sulfated analogs, novel polymers and chimeric constructs.

Glycosaminoglycans (GAGs) have therapeutic potential in areas ranging from angiogenesis, inflammation, hemostasis and cancer. GAG bioactivity is conferred by intrinsic structural features, such as disaccharide composition, glycosidic linkages and sulfation pattern. Unfortunately, the in vitro enzymatic synthesis of defined GAGs is quite restricted by a limited understanding of current GAG synthases and modifying enzymes. Our work provides insights into GAG-active enzymes through the creation of sulfated oligosaccharides, a new polysaccharide and chimeric polymers. We show that a C6-sulfonated uridine diphospho (UDP)-glucose (Glc) derivative, sulfoquinovose, can be used as an uronic acid donor, but not as a hexosamine donor, to cap hyaluronan (HA) chains by the HA synthase from the microbe Pasteurella multocida. However, the two heparosan (HEP) synthases from the same species, PmHS1 and PmHS2, could not employ the UDP-sulfoquinovose under similar conditions. Serendipitously, we found that PmHS2 co-polymerized Glc with glucuronic acid (GlcA), creating a novel HEP-like polymer we named hepbiuronic acid [-4-GlcAß1-4-Glcα1-]n. In addition, we created chimeric block polymers composed of both HA and HEP segments; in these reactions GlcA-, but not N-acetylglucosamine-(GlcNAc), terminated GAG acceptors were recognized by their noncognate synthase for further extension, likely due to the common ß-linkage connecting GlcA to GlcNAc in both of these GAGs. Overall, these GAG constructs provide new tools for studying biology and offer potential for future sugar-based therapeutics.

Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine Uridine Diphosphate Donors: Chain Terminators in Glycosaminoglycan Synthesis.

Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). The resulting unnatural UDP-sugar donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various synthases. UDP-4FGlcNAc was transferred onto an acceptor by Pastuerella multocida heparosan synthase 1 and subsequently served as a chain terminator.

Synthesis of 4-Azido-N-acetylhexosamine Uridine Diphosphate Donors: Clickable Glycosaminoglycans.

Unnatural chemically modified nucleotide sugars UDP-4-N3-GlcNAc and UDP-4-N3-GalNAc were chemically synthesized for the first time. These unnatural UDP sugar products were then tested for incorporation into hyaluronan, heparosan, or chondroitin using polysaccharide synthases. UDP-4-N3-GlcNAc served as a chain termination substrate for hyaluronan or heparosan synthases; the resulting 4-N3-GlcNAc-terminated hyaluronan and heparosan were then successfully conjugated with Alexa Fluor 488 DIBO alkyne, demonstrating that this approach is generally applicable for labeling and detection of suitable glycosaminoglycans.

Irreversible heavy chain transfer to chondroitin.

We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture.

Fibroblast growth factor-based signaling through synthetic heparan sulfate blocks copolymers studied using high cell density three-dimensional cell printing.

Four well-defined heparan sulfate (HS) block copolymers containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ~40 saccharide units. Microtiter 96-well and three-dimensional cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR) but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2-HS2-FGFR2 ternary complex model and the asymmetric FGF2-HS1-FGFR2 ternary complex model. In the symmetric FGF2-HS2-FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2-HS1-FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2-HS2-FGFR2 ternary complex model.

A refined model for the TSG-6 link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was (13)C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a D-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.

Irreversible heavy chain transfer to hyaluronan oligosaccharides by tumor necrosis factor-stimulated gene-6.

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).

CSPG4-dependent cytotoxicity for C. difficile TcdB is influenced by extracellular calcium and chondroitin sulfate.

TcdB is an intracellular bacterial toxin indispensable to Clostridioides difficile infections. The ability to use chondroitin sulfate proteoglycan 4 (CSPG4) as a primary cell surface receptor is evolutionarily conserved by the two major variants of TcdB. As CSPG4 does not typically undergo receptor-mediated endocytosis, we sought to identify environmental factors that stabilize interactions between TcdB and CSPG4 to promote cell binding and entry into the cytosol. Using a series of TcdB receptor-binding mutants and cell lines with various receptor expression profiles, we discovered that extracellular Ca2+ promotes receptor-specific interactions with TcdB. Specifically, TcdB exhibits preferential binding to CSPG4 in the presence of Ca2+, with the absence of Ca2+ resulting in CSPG4-independent cell surface interactions. Furthermore, Ca2+ did not enhance TcdB binding to chondroitin sulfate (CS), the sole glycosaminoglycan of CSPG4. Instead, CS was found to impact the rate of cell entry by TcdB. Collectively, results from this study indicate that Ca2+ enhances cell binding by TcdB and CS interactions contribute to subsequent steps in cell entry. IMPORTANCE: Clostridioides difficile is a leading cause of antibiotic-associated gastrointestinal illness, and many disease pathologies are caused by the toxin TcdB. TcdB engages multiple cell surface receptors, with receptor tropisms differing among the variants of the toxin. Chondroitin sulfate proteoglycan 4 (CSPG4) is a critical receptor for multiple forms of TcdB, and insights into TcdB-CSPG4 interactions are applicable to many disease-causing strains of C. difficile. CSPG4 is modified by chondroitin sulfate (CS) and contains laminin-G repeats stabilized by Ca2+, yet the relative contributions of CS and Ca2+ to TcdB cytotoxicity have not been determined. This study demonstrates distinct roles in TcdB cell binding and cell entry for Ca2+ and CS, respectively. These effects are specific to CSPG4 and contribute to the activities of a prominent isoform of TcdB that utilizes this receptor. These findings advance an understanding of factors contributing to TcdB's mechanism of action and contribution to C. difficile disease.