Mosaic quadrivalent influenza vaccine single nanoparticle characterization.

Recent work by our laboratory and others indicates that co-display of multiple antigens on protein-based nanoparticles may be key to induce cross-reactive antibodies that provide broad protection against disease. To reach the ultimate goal of a universal vaccine for seasonal influenza, a mosaic influenza nanoparticle vaccine (FluMos-v1) was developed for clinical trial (NCT04896086). FluMos-v1 is unique in that it is designed to co-display four recently circulating haemagglutinin (HA) strains; however, current vaccine analysis techniques are limited to nanoparticle population analysis, thus, are unable to determine the valency of an individual nanoparticle. For the first time, we demonstrate by total internal reflection fluorescence microscopy and supportive physical-chemical methods that the co-display of four antigens is indeed achieved in single nanoparticles. Additionally, we have determined percentages of multivalent (mosaic) nanoparticles with four, three, or two HA proteins. The integrated imaging and physicochemical methods we have developed for single nanoparticle multivalency will serve to further understand immunogenicity data from our current FluMos-v1 clinical trial.

Site-Specific Fluorescent Labeling of Hemagglutinin-Specific Antigen Binding Fragment through Amine Chemistry Revealed by Mass Spectrometry.

To capture the structure of assembled hemagglutinin (HA) nanoparticles at single-particle resolution, HA-specific antigen binding fragments (Fabs) were labeled by fluorescent (FLR) dyes as probes to highlight the HA trimers displayed on the assembled tetravalent HA nanoparticles for a qualitative localization microscopic study. The FLR dyes were conjugated to the Fabs through N-hydroxysuccinimide (NHS) ester mediated amine coupling chemistry. The labeling profile, including labeling ratio, distribution, and site-specific labeling occupancy, can affect the imaging results and introduce inconsistency. To evaluate the labeling profile so as to evaluate the labeling efficiency, a combination of intact mass measurement by MALDI-MS and peptide mapping through LC-MS/MS was implemented. At the intact molecular level, the labeling ratio and distribution were determined. Through peptide mapping, the labeled residues were identified and the corresponding site-specific labeling occupancy was measured. A systematic comparative investigation of four different FLR-labeled 1H01-Fabs (generated from H1 strain HA specific mAb 1H01) allowed accurate profiling of the labeling pattern. The data indicate that the labeling was site-specific and semiquantitative. This warrants the consistency of single-particle fluorescent imaging experiments and allows a further imaging characterization of the single nanoparticles.

An etanercept O-glycovariant with enhanced potency.

Most therapeutic proteins are glycosylated with N-glycans and/or O-glycans. N-glycans on therapeutic proteins have been extensively studied for their control strategy and impact on drug product quality. However, knowledge of O-glycosylation in therapeutic protein production and its impact on product quality remains elusive. To address this gap, we generated an O-glycoengineered Chinese Hamster Ovary (CHO) cell line platform to modulate O-glycosylation of therapeutic proteins and investigated the impact of O-glycans on the physicochemical and biological properties of etanercept. Our results demonstrate that this CHO cell line platform produces controlled O-glycosylation profiles containing either truncated O-glycans (sialylTn and/or Tn), or sialylCore 3 alone, or sialylCore 1 with sialylTn or sialylCore 3 O-glycans on endogenous and recombinant proteins. Moreover, the platform demonstrated exclusive modulation of O-glycosylation without affecting N-glycosylation. Importantly, certain O-glycans on etanercept enhanced tumor necrosis factor-α binding affinity and consequent potency. This is the first report that describes the systematic establishment of an O-glycoengineered CHO cell line platform with direct evidence that supports the applicability of the platform in the production of engineered proteins with desired O-glycans. This platform is valuable for identifying O-glycosylation as a critical quality attribute of biotherapeutics using the quality by design principle.

In-Depth Compositional and Structural Characterization of N-Glycans Derived from Human Urinary Exosomes.

The study of exosomes has become increasingly popular due to their potentially important biological roles. Urine can be used as an effective source of exosomes for noninvasive investigations into the pathophysiological states of the urinary system, but first, detailed characterization of exosomal components in healthy individuals is essential. Here, we significantly extend the number of N-glycan compositions, including sulfated species, identified from urinary exosomes and determine the sialic acid linkages for many of those compositions. Capillary electrophoresis-mass spectrometry (CE-MS), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify N-glycan and sulfated N-glycan compositions. Second, because the alteration of sialylation patterns has been previously implicated in various disease states, ion-exchange chromatography, microfluidic capillary electrophoresis (CE), and MALDI-MS were adopted to resolve positional isomers of sialic acids. Structures of the sialyl-linkage isomers were assigned indirectly through α2-3 sialidase treatment and sialic acid linkage-specific alkylamidation (SALSA). In total, we have identified 219 N-glycan structures that include 175 compositions, 64 sialic acid linkage isomers, 26 structural isomers, and 27 sulfated glycans.

Differential effects of bioreactor process variables and purification on the human recombinant lysosomal enzyme ß-glucuronidase produced from Chinese hamster ovary cells.

ß-Glucuronidase is a lysosomal enzyme and a molecular model of a class of therapeutics approved as enzyme replacement therapies for lysosomal storage diseases. Understanding the effect of bioreactor process variables on the production and quality of the biologics is critical for maintaining quality and efficacy of the biotherapeutics. Here, we have investigated the effect of three process variables, in a head-to-head comparison using a parallel bioreactor system (n = 8), namely 0.25 mM butyrate addition, a temperature shift (from 37 to 32 °C), and a pH shift (from 7.0 to 6.7) along with a control (pH 7, temperature 37 °C, and no additive) on the production and quality of human recombinant ß-glucuronidase (GUS) by a Chinese hamster ovary (CHO) cell line. The study was performed as two independent runs (2 bioreactors per treatment per run; n ≤ 4). Although statistically not significant, protein production slightly increased with either 0.25 mM butyrate addition (13%) or pH shift (7%), whereas temperature shift decreased production (12%, not significant). Further characterization of the purified GUS samples showed that purification selectively enriched the mannose-6-phosphate (M6P)-containing GUS protein. Noticeably, a variation observed for the critical quality attribute (CQA) of the enzyme, namely M6P content, decreased after purification, across treatment replicates and, more so, across different treatments. The dimer content in the purified samples was comparable (~25%), and no significant discrepancy was observed in terms of GUS charge variants by capillary electrophoresis analysis. MALDI-TOF/TOF analysis of released N-glycans from GUS showed a minor variation in glycoforms among the treatment groups. Temperature shift resulted in a slightly increased sialylated glycan content (21.6%) when compared to control (15.5%). These results suggest that bioreactor processes have a differential effect, and better control is required for achieving improved production of GUS enzyme in CHO cells without affecting drastically its CQAs. However, the purification method allowed for enrichment of GUS with similar CQA profiles, regardless of the upstream treatments, indicating for the first time that the effect of slight alterations in upstream process parameters on the CQA profile can be offset with an effective and robust purification method downstream to maintain drug substance uniformity.

Comprehensive analysis of N-glycans in IgG purified from ferrets with or without influenza A virus infection.

Influenza viruses cause contagious respiratory infections, resulting in significant economic burdens to communities. Production of influenza-specific Igs, specifically IgGs, is one of the major protective immune mechanisms against influenza viruses. In humans, N-glycosylation of IgGs plays a critical role in antigen binding and effector functions. The ferret is the most commonly used animal model for studying influenza pathogenesis, virus transmission, and vaccine development, but its IgG structure and functions remain largely undefined. Here we show that ferret IgGs are N-glycosylated and that their N-glycan structures are diverse. Using a comprehensive strategy based on MS and ultra-HPLC analyses in combination with exoglycosidase digestions, we assigned 42 N-glycan structures in ferret IgGs. We observed that N-glycans of ferret IgGs consist mainly of complex-type glycans, including some high-mannose and hybrid glycans, similar to those observed in human IgG. The complex-type glycans of ferret IgGs were primarily core-fucosylated. Furthermore, a fraction of N-glycans carried bisecting GlcNAc. Ferret IgGs also had a minor fraction of glycans carrying α2-6Neu5Ac(s). We noted that, unlike human IgG, ferret IgGs have αGal epitopes on some N-glycans. Interestingly, influenza A infection caused prominent changes in the N-glycans of ferret IgG, mainly because of an increase in bisecting GlcNAc and F1A2G0 and a corresponding decrease in F1A2G1. This suggests that the glycosylation of virus-specific IgG may play a role in its functionality. Our study highlights the need to further elucidate the structure-function relationships of IgGs in universal influenza vaccine development.

Neutralization of Pseudomonas auruginosa Exotoxin A by human neutrophil peptide 1.

Pseudomonas aeruginosa produces a large number of virulence factors, including the extracellular protein, Exotoxin A (ETA). Human Neutrophil Peptide 1 (HNP1) neutralizes the Exotoxin A. HNP1 belongs to the family of α-defensins, small effector peptides of the innate immune system that combat against microbial infections. Neutralization of bacterial toxins such as ETA by HNP1 is a novel biological function in addition to direct killing of bacteria. In this study, we report on the interaction between HNP-1 and Exotoxin A at the molecular level to allow for the design and development of potent antibacterial peptides as alternatives to classical antibiotics.

Comprehensive Analytical Approach toward Glycomic Characterization and Profiling in Urinary Exosomes.

Exosomes are extracellular nanosized vesicles with lipid bilayers encapsulating nucleic acids and proteins, both with and without glycosylation. While exosomal nucleic acids and proteins have previously been explored to identify cancer biomarkers with some promising results, little information has been available concerning their glycoconjugate content. Exosomes were isolated from normal urine samples through multistep differential centrifugation. The isolated exosomes have an average size of 146 nm and a spherical shape, as determined by dynamic light scattering and transmission electron microscopy, respectively. N-Glycans were enzymatically released from the isolated vesicles. After being reduced and permethylated, N-glycans were measured by MALDI mass spectrometry. Paucimannosidic, high-mannose, and complex type glycans were identified and their relative abundances were determined. Some detailed structures of these glycans were revealed through liquid chromatography/tandem mass spectrometry (LC/MS-MS). The reduced N-glycans, without being permethylated, were also separated and analyzed by LC/MS-MS, and their structures were further detailed through isomeric separation on porous graphitized carbon (PGC) packed in long capillaries. Using microfractionation before LC/MS-MS, minor multiantennary N-glycans were preconcentrated as based on hydrophobicity or charge. Preconcentration of the reduced and permethylated glycans on a C18 cartridge revealed numerous large glycans, whereas fractionation of the reduced N-glycans by ion-exchange cartridges facilitated detection of sulfated glycans. After removing N-glycans from the original sample aliquot, O-glycans were chemically released from urinary exosomes and profiled, revealing some unusual structures.

Dynamically-enhanced retention of gold nanoclusters in HeLa cells following X-rays exposure: A cell cycle phase-dependent targeting approach.

BACKGROUND AND PURPOSE: Cell cycle phase could affect the cellular uptake of nanoparticles. Based on the fact that ionizing radiation exposure can delay cell cycle progression including inducing G2/M phase arrest, we propose that ionizing radiation exposure is a cell cycle phase-dependent targeting approach for intracellular delivery of nano-agents in tumor cells. MATERIALS AND METHODS: We synthesized luminescent gold nanoclusters (AuNCs) using a one-pot green synthetic method. Subsequently, we used the as-prepared AuNCs as both "nano-agents" and fluorescent trafficking probes for our study using human cervical carcinoma HeLa cells. Estimating the cellular uptake of AuNCs and cell cycle analysis were performed following X-rays irradiation and cell synchronization. RESULTS: Our work showed that X-rays irradiation could delay the division of HeLa cells and thereby enhance the retention of AuNCs in HeLa cells, which is a reverse strategy compared with other studies on synergistic nano-radiotherapy. Our results demonstrated that the cell cycle synchronization influenced the cellular uptake processes of AuNCs, suggesting that dynamic cell cycle progression could affect the cellular uptake kinetics of AuNCs. CONCLUSION: We consider that the radiation-induced cell division delay might provide a possible mechanism underlying the enhanced effect for the cellular uptake of AuNCs in irradiated HeLa cells.

Probe-inspired nano-prodrug with dual-color fluorogenic property reveals spatiotemporal drug release in living cells.

The versatility of the fluorescent probes inspires us to design fluorescently traceable prodrugs, which enables tracking the drug delivery kinetics in living cells. Herein, we constructed a self-indicating nanoprodrug with two fluorescent moieties, an aggregation-induced emission molecule (tetraphenylethylene, TPE) and a luminant anticancer drug (doxorubicin, DOX), with a pH-responsive linker between them. Except when a low pH environment is encountered, an energy-transfer relay (ETR) occurs and inactivates the fluorescence of both, showing a dark background. Otherwise, the ETR would be interrupted and evoke a dual-color fluorogenic process, giving distinct fluorogenic read out. By observing the dual-color fluorogenic scenario, we captured the kinetics of the drug release process in living cells. Because the separated TPE and DOX are both fluorescent but have a distinct spectrum, by examining the spatiotemporal pattern of TPE and DOX, we were able to precisely disclose the drug-releasing site, the releasing time, the destinations of the carriers, and the executing site of the drugs at subcellular level. Furthermore, different intracellular drug release kinetics between free doxorubicin and its nanoformulations were also observed in a real-time manner.

A smart pH-switchable luminescent hydrogel.

Here we report a novel example of a luminescent hydrogel, which is formed from silent individual molecules simply by altering the pH of the system. Formation of the emissive nanostructure is fully and repeatedly reversible. This hydrogel, with switchable luminescence, can potentially be used as a nano pH sensor.

Phenylboronic acid-functionalized magnetic nanoparticles for one-step saccharides enrichment and mass spectrometry analysis.

ABSTRACT: In this work, 2-(2-aminoethoxy) ethanol-blocked phenylboronic acid-functionalized magnetic nanoparticles (blocked PMNPs) were fabricated for selective enrichment of different types of saccharides. The phenylboronic acid was designed for capturing the cis-diols moieties on saccharides molecules, and the 2-(2-aminoethoxy) ethanol can deplete the nonspecific absorption of peptides and proteins which always coexisted with saccharides. For mass spectrometry analysis, the PMNPs bound saccharides can be directly applied onto the MALDI plate with matrix without removing the PMNPs. By PMNPs, the simple saccharide (glucose) could be detected in pmol level. The complex saccharides can also be reliably purified and analyzed; 16 different N-glycans were successfully identified from ovalbumin, and the high-abundance N-glycans can be detected even when the ovalbumin was in very low concentration (2 µg). In human milk, ten different oligosaccharides were identified, and the lactose can still be detected when the human milk concentration was low to 0.01 µL.

Self-assembled Peptide nanofibers designed as biological enzymes for catalyzing ester hydrolysis.

The structural arrangement of amino acid residues in a native enzyme provides a blueprint for the design of artificial enzymes. One challenge of mimicking the catalytic center of a native enzyme is how to arrange the essential amino acid residues in an appropriate position. In this study, we designed an artificial hydrolase via self-assembly of short peptides to catalyze ester hydrolysis. When the assembled hydrolase catalytic sites were embedded in a matrix of peptide nanofibers, they exhibited much higher catalytic efficiency than the peptide nanofibers without the catalytic sites, suggesting that this well-ordered nanostructure is an attractive scaffold for developing new artificial enzymes. Furthermore, the cytotoxicity of the assembled hydrolase was evaluated with human cells, and the novel artificial biological enzyme showed excellent biocompatibility.

Cell membrane tracker based on restriction of intramolecular rotation.

The fluorescence of tetraphenylethylene (TPE), an archetypal luminogen, is induced by restriction of intramolecular rotation (RIR). TPE was grafted with palmitic acid (PA) onto a hydrophilic peptide to yield a cell membrane tracker named TR4. TR4 was incorporated into liposomes, where it showed significant RIR characteristics. When cells were incubated with TR4, cytoplasmic membranes were specifically labeled. TR4 shows excellent photostability and low cytotoxicity.

A fluorescent probe with restricted intramolecular rotation-induced emission for label-free detection of mercury ions.

Formation of T-Hg(2+)-T complexes changes the configuration of a single-stranded DNA, leading to enhanced fluorescence of an anchored cyanine-based probe that displays restricted intramolecular rotation (RIR)-induced emission. This label-free system can be used as a sensor for mercury ions with a detection limit of 4 nM.

Highly Sensitive Simultaneous Detection of Mercury and Copper Ions by Ultrasmall Fluorescent DNA-Ag Nanoclusters.

Fluorescent metal nanoclusters (NCs) have given rise to a new class of fluorescent nanomaterials for the detection of heavy metals. Here, we design a simple, rapid and highly sensitive sensing nanosystem for the detection of Hg2+ and Cu2+ based on fluorescence quenching of ultrasmall DNA-Ag NCs. The fluorescence intensity of DNA-Ag NCs was selectively quenched by Hg2+ and Cu2+, and the limit of detection (LOD) was found to be 5 nM and 10 nM, respectively. The technique was renewable employment by EDTA addition and successfully applied to detection of Hg2+ and Cu2+ in domestic water samples. The quantum yield (QY) of DNA-Ag NCs was significantly higher to ~30% compared to traditional water-soluble fluorescent metal NCs. The DNA-Ag NC detection system make it potentially suitable for detecting Hg2+ and Cu2+ and monitoring water quality in a wide range of samples regulated under the Environmental Protection Agency.

Neuropilin-1-targeted gold nanoparticles enhance therapeutic efficacy of platinum(IV) drug for prostate cancer treatment.

Platinum-based anticancer drugs such as cisplatin, oxaliplatin, and carboplatin are some of the most potent chemotherapeutic agents but have limited applications due to severe dose-limiting side effects and a tendency for cancer cells to rapidly develop resistance. The therapeutic index can be improved through use of nanocarrier systems to target cancer cells efficiently. We developed a unique strategy to deliver a platinum(IV) drug to prostate cancer cells by constructing glutathione-stabilized (Au@GSH) gold nanoparticles. Glutathione (GSH) has well-known antioxidant properties, which lead to cancer regression. Here, we exploit the advantages of both the antioxidant properties and high surface-area-to-volume ratio of Au@GSH NPs to demonstrate their potential for delivery of a platinum(IV) drug by targeting the neuropilin-1 receptor (Nrp-1). A lethal dose of a platinum(IV) drug functionalized with the Nrp-1-targeting peptide (CRGDK) was delivered specifically to prostate cancer cells in vitro. Targeted peptide ensures specific binding to the Nrp-1 receptor, leading to enhanced cellular uptake level and cell toxicity. The nanocarriers were themselves nontoxic, but exhibited high cytotoxicity and increased efficacy when functionalized with the targeting peptide and drug. The uptake of drug-loaded nanocarriers is dependent on the interaction with Nrp-1 in cell lines expressing high (PC-3) and low (DU-145) levels of Nrp-1, as confirmed through inductively coupled plasma mass spectrometry and confocal microscopy. The nanocarriers have effective anticancer activity, through upregulation of nuclear factor kappa-B (NF-κB) protein (p50 and p65) expression and activation of NF-κB-DNA-binding activity. Our preliminary investigations with platinum(IV)-functionalized gold nanoparticles along with a targeting peptide hold significant promise for future cancer treatment.

Imaging intracellular anticancer drug delivery by self-assembly micelles with aggregation-induced emission (AIE micelles).

Nanoformulations show many therapeutic advantages over conventional formulations. We seek to develop traceable nanoformulations in order to closely monitor delivery. Herein, we developed a new drug delivery system (DDS) using tetraphenylethene (TPE) to fabricate a self-assembly micelle with aggregation-induced emission (AIE micelle). AIE makes the nanocarriers visible for high-quality imaging, and the switching on and off of the AIE is intrinsically controlled by the assembly and disassembly of the micelles. This DDS was tested for doxorubicin (DOX) delivery and intracellular imaging. For the DOX-loaded micelles (TPED), the DOX content reached as much as 15.3% by weight, and the anticancer efficiency was higher than for free DOX. Meanwhile, high-quality imaging was obtained to trace the intracellular delivery of the TPED.

Spatiotemporal drug release visualized through a drug delivery system with tunable aggregation-induced emission.

Tetraphenylethene and doxorubicin are assembled into a self-indicating drug delivery system (TD NPs). TD NPs are decomposed into DOX and TPE NPs in lysosome. Since TD NPs, TPE NPs and DOX are all fluorescent, the detachment of DOX from TPE NPs is accompanied by fluorescence changing. By observing the fluorescence changes, the spatiotemporal drug release is visualized.

In situ self-assembly of peptides in glucan particles for macrophage-targeted oral delivery.

In this study, an orally administered macrophage-targeting peptide delivery system was constructed through in situ self-assembly of Q11 peptide inside hollow glucan particles (GPs), which are approved by the FDA. The glucan shell efficiently protected the encapsulated peptide from enzymatic degradation in the gastrointestinal tract. ß-1,3-(d)-Glucan is recognized by the membrane receptor dectin-1, which is highly expressed by intestinal antigen-presenting cells, including macrophages. GPs are thus efficiently phagocytized by intestinal macrophages. This study is applicable to the pharmaceutical industry for the development of orally delivered macrophage-targeting systems for effective and personalized remedies like immunotherapeutic vaccines.