Poly(HPMA-co-NIPAM) copolymer as an alternative to polyethylene glycol-based pharmacokinetic modulation of therapeutic proteins.

PEGylation is the standard approach for prolonging the plasma exposure of protein therapeutics but has limitations. We explored whether polymers prepared by Reversible Addition-Fragmentation chain-Transfer (RAFT) may provide better alternatives to polyethylene glycol (PEG). Four RAFT polymers were synthesised with varying compositions, molar mass (Mn), and structures, including a homopolymer of N-(2-hydroxypropyl)methacrylamide, (pHPMA) and statistical copolymers of HPMA with poly(ethylene glycol methyl ether acrylate) p(HPMA-co-PEGA); HPMA and N-acryloylmorpholine, p(HPMA-co-NAM); and HPMA and N-isopropylacrylamide, p(HPMA-co-NIPAM). The intravenous pharmacokinetics of the polymers were then evaluated in rats. The in vitro activity and in vivo pharmacokinetics of p(HPMA-co-NIPAM)-conjugated trastuzumab Fab' and full length mAb were then evaluated. p(HPMA-co-NIPAM) prolonged plasma exposure more avidly compared to the other p(HPMA) polymers or PEG, irrespective of molecular weight. When conjugated to trastuzumab-Fab', p(HPMA-co-NIPAM) prolonged plasma exposure of the Fab' similar to PEG-Fab'. The generation of anti-PEG IgM in rats 7 days after intravenous and subcutaneous dosing of p(HPMA-co-NIPAM) conjugated trastuzumab mAb was also examined and was shown to exhibit lower immunogenicity than the PEGylated construct. These data suggest that p(HPMA-co-NIPAM) has potential as a promising copolymer for use as an alternative conjugation strategy to PEG, to prolong the plasma exposure of therapeutic proteins.

Proteomics reveals the in vitro protein digestibility of seven transmembrane enzymes from the docosahexaenoic acid biosynthesis pathway.

The measurement of protein digestibility is one of the key steps in determining the safety of a genetically modified crop that has been traditionally accomplished using antibodies. Membrane proteins are often extremely difficult to express with replicated authentic tertiary structure in heterologous systems. As a result raising antibodies for use in safety assessment may not be feasible. In this study, LC-MS based proteomics was used to characterise seven transmembrane enzymes from the docosahexaenoic acid biosynthetic pathway that had been introduced into canola. The application of a two-stage digestion strategy involving simulated gastric fluid followed by trypsin enabled the measurement of protein digestibility in vitro. Tryptic peptide markers that spanned the length of each desaturase protein were monitored and showed that these proteins were readily degraded (>95% within 5 min) and highlighted regions of the elongase enzymes that showed limited resistance to simulated gastric digestion. Traditional gel-based and Western blotting analysis of ω3-desaturase and Δ6-elongase revealed rapid hydrolysis of the intact proteins within seconds and no fragments (>3 kDa) remained after 60 min, complementing the novel approach described herein. The LC-MS approach was sensitive, selective and did not require the use of purified proteins.

Quantitation of seven transmembrane proteins from the DHA biosynthesis pathway in genetically engineered canola by targeted mass spectrometry.

Examining tissue-specific expression and the measurement of protein abundance are important steps when assessing the performance of genetically engineered crops. Liquid chromatography-mass spectrometry offers many advantages over traditional methods for protein quantitation, especially when dealing with transmembrane proteins that are often difficult to express or generate antibodies against. In this study, discovery proteomics was used to detect the seven transgenic membrane-bound enzymes from the docosahexaenoic acid (DHA) biosynthetic pathway that had been engineered into canola. Subsequently, a targeted LC-MS/MS method for absolute quantitation was developed and applied to the simultaneous measurement of the seven DHA biosynthetic pathway enzymes in genetically modified canola grown across three sites. The results of this study demonstrated that the enzymatic proteins that drive the production of DHA using seed-specific promoters were detected only in mature and developing seed of DHA canola. None of the DHA biosynthesis pathway proteins were detected in wild-type canola planted in the same site or in the non-seed tissues of the transgenic canola, irrespective of the sampling time or the tissues tested. This study describes a streamlined approach to simultaneously measure multiple membrane-bound proteins in planta.

Paclitaxel-Loaded Self-Assembled Lipid Nanoparticles as Targeted Drug Delivery Systems for the Treatment of Aggressive Ovarian Cancer.

Chemotherapy using cytotoxic agents, such as paclitaxel (PTX), is one of the most effective treatments for advanced ovarian cancer. However, due to nonspecific targeting of the drug and the presence of toxic solvents required for dissolving PTX prior to injection, there are several serious side effects associated with this treatment. In this study, we explored self-assembled lipid-based nanoparticles as PTX carriers, which were able to improve its antitumour efficacy against ovarian cancer. The nanoparticles were also functionalized with epidermal growth factor receptor (EGFR) antibody fragments to explore the benefit of tumor active targeting. The formulated bicontinuous cubic- and sponge-phase nanoparticles, which were stabilized by Pluronic F127 and a lipid poly(ethylene glycol) stabilizer, showed a high capacity of PTX loading. These PTX-loaded nanoparticles also showed significantly higher cytotoxicity than a free drug formulation against HEY ovarian cancer cell lines in vitro. More importantly, the nanoparticle-based PTX treatments, with or without EGFR targeting, reduced the tumor burden by 50% compared to PTX or nondrug control in an ovarian cancer mouse xenograft model. In addition, the PTX-loaded nanoparticles were able to extend the survival of the treatment groups by up to 10 days compared to groups receiving free PTX or nondrug control. This proof-of-concept study has demonstrated the potential of these self-assembled lipid nanomaterials as effective drug delivery nanocarriers for poorly soluble chemotherapeutics, such as PTX.

Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis.

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed.

Determining the limit of detection of surface bound antibody.

Determination of a limit of detection (LoD) for surface bound antibodies is crucial for the development and deployment of sensitive bioassays. The measurement of very low concentrations of surface bound antibodies is also important in the manufacturing of pharmaceutical products such as antibody-conjugated pharmaceuticals. Low concentrations are required to avoid an immune response from the target host. Enzyme-linked immunosorbent assay (ELISA), x-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to determine the LoD for the surface bound antibody (antiepidermal growth factor receptor antibody) on silicon substrates. Antibody solution concentrations between 10 µg/ml and 1 ng/ml and a control (antibody-free buffer solution) were employed, and the detection performance of each technique was compared. For this system, the ELISA LoD was 100 ng/ml and the XPS LoD was 1 µg/ml, corresponding to an estimated surface concentration of 49 ± 7 ng/cm2 using a 1 µg/ml solution. Due to the multivariate complexity of ToF-SIMS data, analysis was carried out using three different methods, peak ratio calculations, principal component analysis, and artificial neural network analysis. The use of multivariate analysis with this dataset offers an unbiased analytical approach based on the peaks selected from ToF-SIMS data. The results estimate a ToF-SIMS LoD between applied antibody concentrations of 10 and 100 ng/mL. For surface bound antibodies on a silicon substrate, the LoD is below an estimated surface concentration of 49 ng/cm2. The authors have determined the LoD for this system using ELISA, XPS, and ToF-SIMS with multivariate analyses, with ToF-SIMS offering an order of magnitude better detection over ELISA and 2 orders of magnitude better detection over XPS.

Polypropylene microtitre plates modified with [Cr(OH)6]3- for enhanced ELISA sensitivity.

Chromium solutions have been used as wet chemical modifiers for polymer microtitre plates used in improving immunoassay performance. However, polypropylene has been excluded from the list of potentially modifiable substrates (AnteoTechnologies, 2015). Here we show that untreated polypropylene microtitre plates can indeed be modified using a [Cr(OH)6]3- complex. Compared to unmodified polypropylene, the chromium modified surfaces demonstrate an up to 4-fold improvement in both assay sensitivity and signal intensity in an antigen capture ELISA. Atomic force microscope (AFM) images indicate that the chromium complex facilitates dispersion of the antibody, reducing aggregation.

Surface immobilized antibody orientation determined using ToF-SIMS and multivariate analysis.

Antibody orientation at solid phase interfaces plays a critical role in the sensitive detection of biomolecules during immunoassays. Correctly oriented antibodies with solution-facing antigen binding regions have improved antigen capture as compared to their randomly oriented counterparts. Direct characterization of oriented proteins with surface analysis methods still remains a challenge however surface sensitive techniques such as Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) provide information-rich data that can be used to probe antibody orientation. Diethylene glycol dimethyl ether plasma polymers (DGpp) functionalized with chromium (DGpp+Cr) have improved immunoassay performance that is indicative of preferential antibody orientation. Herein, ToF-SIMS data from proteolytic fragments of anti-EGFR antibody bound to DGpp and DGpp+Cr are used to construct artificial neural network (ANN) and principal component analysis (PCA) models indicative of correctly oriented systems. Whole antibody samples (IgG) test against each of the models indicated preferential antibody orientation on DGpp+Cr. Cross-reference between ANN and PCA models yield 20 mass fragments associated with F(ab')2 region representing correct orientation, and 23 mass fragments associated with the Fc region representing incorrect orientation. Mass fragments were then compared to amino acid fragments and amino acid composition in F(ab')2 and Fc regions. A ratio of the sum of the ToF-SIMS ion intensities from the F(ab')2 fragments to the Fc fragments demonstrated a 50% increase in intensity for IgG on DGpp+Cr as compared to DGpp. The systematic data analysis methodology employed herein offers a new approach for the investigation of antibody orientation applicable to a range of substrates. STATEMENT OF SIGNIFICANCE: Controlled orientation of antibodies at solid phases is critical for maximizing antigen detection in biosensors and immunoassays. Surface-sensitive techniques (such as ToF-SIMS), capable of direct characterization of surface immobilized and oriented antibodies, are under-utilized in current practice. Selection of a small number of mass fragments for analysis, typically pertaining to amino acids, is commonplace in literature, leaving the majority of the information-rich spectra unanalyzed. The novelty of this work is the utilization of a comprehensive, unbiased mass fragment list and the employment of principal component analysis (PCA) and artificial neural network (ANN) models in a unique methodology to prove antibody orientation. This methodology is of significant and broad interest to the scientific community as it is applicable to a range of substrates and allows for direct, label-free characterization of surface bound proteins.

Orientation and characterization of immobilized antibodies for improved immunoassays (Review).

Orientation of surface immobilized capture proteins, such as antibodies, plays a critical role in the performance of immunoassays. The sensitivity of immunodiagnostic procedures is dependent on presentation of the antibody, with optimum performance requiring the antigen binding sites be directed toward the solution phase. This review describes the most recent methods for oriented antibody immobilization and the characterization techniques employed for investigation of the antibody state. The introduction describes the importance of oriented antibodies for maximizing biosensor capabilities. Methods for improving antibody binding are discussed, including surface modification and design (with sections on surface treatments, three-dimensional substrates, self-assembled monolayers, and molecular imprinting), covalent attachment (including targeting amine, carboxyl, thiol and carbohydrates, as well as "click" chemistries), and (bio)affinity techniques (with sections on material binding peptides, biotin-streptavidin interaction, DNA directed immobilization, Protein A and G, Fc binding peptides, aptamers, and metal affinity). Characterization techniques for investigating antibody orientation are discussed, including x-ray photoelectron spectroscopy, spectroscopic ellipsometry, dual polarization interferometry, neutron reflectometry, atomic force microscopy, and time-of-flight secondary-ion mass spectrometry. Future perspectives and recommendations are offered in conclusion.

Chromium functionalized diglyme plasma polymer coating enhances enzyme-linked immunosorbent assay performance.

Ensuring the optimum orientation, conformation, and density of substrate-bound antibodies is critical for the success of sandwich enzyme-linked immunosorbent assays (ELISAs). In this work, the authors utilize a diethylene glycol dimethyl ether plasma polymer (DGpp) coating, functionalized with chromium within a 96 well plate for the enhanced immobilization of a capture antibody. For an equivalent amount of bound antibody, a tenfold improvement in the ELISA signal intensity is obtained on the DGpp after incubation with chromium, indicative of improved orientation on this surface. Time-of-flight secondary-ion-mass-spectrometry (ToF-SIMS) and principal component analysis were used to probe the molecular species at the surface and showed ion fragments related to lysine, methionine, histidine, and arginine coupled to chromium indicating candidate antibody binding sites. A combined x-ray photoelectron spectroscopy and ToF-SIMS analysis provided a surface molecular characterization that demonstrates antibody binding via the chromium complex. The DGpp+Cr surface treatment holds great promise for improving the efficacy of ELISAs.

ToF-SIMS and Principal Component Analysis Investigation of Denatured, Surface-Adsorbed Antibodies.

Antibody denaturation at solid-liquid interfaces plays an important role in the sensitivity of protein assays such as enzyme-linked immunosorbent assays (ELISAs). Surface immobilized antibodies must maintain their native state, with their antigen binding (Fab) region intact, to capture antigens from biological samples and permit disease detection. In this work, two identical sample sets were prepared with whole antibody IgG, F(ab')2 and Fc fragments, immobilized to either a silicon wafer or a diethylene glycol dimethyl ether plasma polymer surface. Analysis was conducted on one sample set at day 0, and the second sample set after 14 days in vacuum, with time-of-flight secondary ion mass spectrometry (ToF-SIMS) for molecular species representative of denaturation. A 1003 mass fragment peak list was compiled from ToF-SIMS data and compared to a 35 amino acid mass fragment peak list using principal component analysis. Several ToF-SIMS secondary ions, pertaining to disulfide and thiol species, were identified in the 14 day (presumably denatured) samples. A substrate and primary ion independent marker for denaturation (aging) was then produced using a ratio of mass peak intensities according to denaturation ratio: [I61.9534 + I62.9846 + I122.9547 + I84.9609 + I120.9461]/[I30.9979 + I42.9991 + I73.0660 + I147.0780]. The ratio successfully identifies denaturation on both the silicon and plasma polymer substrates and for spectra generated with Mn+, Bi+, and Bi3+ primary ions. We believe this ratio could be employed to as a marker of denaturation of antibodies on a plethora of substrates.

High-Throughput Production of Chromium(III) Complexes for Antibody Immobilization.

A robot-assisted high-throughput methodology was employed to produce chromium(III) complexes suitable for the surface modification of the commercially available PerkinElmer Optiplate96 well plate for use in enzyme-linked immunosorbent assays (ELISAs). The complexes were immobilized to the native functionality of the well plate and first screened using a horseradish-peroxidase-tagged (HRP) mouse antibody to quantify binding. The top "hits" were further assessed for their ability to present the antibody in a functional state using an ELISA. "Hits" from the second screen yielded four complexes capable of improving the signal intensity of the ELISA by greater than 500%. The metal/base ratio of these complexes was also investigated, and we isolated the most stable and reproducible candidate, [Cr(OH)6]3-, which was formed from chromium(III) perchlorate and pH adjusted with ethylenediamine. This chromium solution was employed in a clinically relevant setting for the detection of bovine TNFα producing up to a 200% increase in signal intensity.

A chemiluminescent sandwich ELISA enhancement method using a chromium (III) coordination complex.

Enzyme linked immunosorbent assays (ELISAs) are employed for the detection and quantification of antigens from biological sources such as serum and cell culture media. A sandwich ELISA is dependent on the immobilization of a capture antibody, or antibody fragment, and the effective presentation of its antigen binding sites. Immobilization to common microtitre plates relies on non-specific interactions of the capture protein with a surface that may result in unfavourable orientation and conformation, compromising ELISA signal strength and performance. We have developed a wet chemical surface activation method that utilizes a chromium (III) solution to immobilize native, non-tagged, capture antibodies on commercially available microtitre plates. Antibodies captured by this method had increased antigen binding, particularly from dilute antibody solutions, relative to antibodies adsorbed directly to the plate surface. A variety of monoclonal antibodies with complementary antigen systems were used to demonstrate improvements in ELISA signal and reproducibility. The simplicity and versatility of this method should enable ELISA enhancement in assays where chemiluminescence is used as the detection method.

Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli.

Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus nucleocapsid protein (HeV N) represents the most abundant viral protein produced by the host cell, and is highly immunogenic with naturally infected humans and horses producing specific antibodies towards this protein. The purpose of this study was to express and purify soluble, functionally active recombinant HeV N, suitable for use as an immunodiagnostic reagent to detect antibodies against HeV. We expressed both full-length HeV N, (HeV NFL), and a C-terminal truncated form, (HeV NCORE), using a bacterial heterologous expression system. Both HeV N constructs were engineered with an N-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified recombinant HeV N proteins self-assembled into soluble higher order oligomers as determined by SEC and negative-stain transmission electron microscopy. Both HeV N proteins were highly immuno-reactive with sera from animals and humans infected with either HeV or the closely related Nipah virus (NiV), but displayed no immuno-reactivity towards sera from animals infected with a non-pathogenic paramyxovirus (CedPV), or animals receiving Equivac® (HeV G glycoprotein subunit vaccine), using a Luminex-based multiplexed microsphere assay.

Epidermal growth factor receptor-targeted lipid nanoparticles retain self-assembled nanostructures and provide high specificity.

Next generation drug delivery utilising nanoparticles incorporates active targeting to specific sites. In this work, we combined targeting with the inherent advantages of self-assembled lipid nanoparticles containing internal nano-structures. Epidermal growth factor receptor (EGFR)-targeting, PEGylated lipid nanoparticles using phytantriol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-maleimide amphiphiles were created. The self-assembled lipid nanoparticles presented here have internal lyotropic liquid crystalline nano-structures, verified by synchrotron small angle X-ray scattering and cryo-transmission electron microscopy, that offer the potential of high drug loading and enhanced cell penetration. Anti-EGFR Fab' fragments were conjugated to the surface of nanoparticles via a maleimide-thiol reaction at a high conjugation efficiency and retained specificity following conjugation to the nanoparticles. The conjugated nanoparticles were demonstrated to have high affinity for an EGFR target in a ligand binding assay.

Gadolinium-DTPA amphiphile nanoassemblies: agents for magnetic resonance imaging and neutron capture therapy.

Engineering biocompatible and physiologically stable nanoscaled therapeutics and imaging agents with the ability to target tumor tissue is a key challenge for the advancement of cancer therapeutics and diagnostic imaging. Here, we present chelating amphiphiles with the capacity to form nanoassembled colloidal particles containing high payloads of gadolinium (Gd) ions. We present the in situ synthesis and complexation of Gd with colloidal nanoassemblies (NAs) based on diethylenetriamine pentaacetic acid (DTPA) amphiphiles. This method allows for facile simultaneous incorporation of several metal ions for applications in multimodal imaging and therapeutics. The diverse internally nanostructured NAs made from sole precursor amphiphiles and their Gd-complexes were investigated by synchrotron small angle X-ray scattering (SAXS) and cryo-TEM. Depending on the molecular structure of the amphiphiles, the structures of NAs range from micelles to liposomes, to colloidal particles of inverse hexagonal (hexosomes) and inverse bicontinuous cubic phases (cubosomes), to multilayered nanospheres. The in vitro contrast activity of these NAs exhibited high relaxivity values as T1-weighted magnetic resonance imaging (MRI) contrast enhancement agents. Further, an α-Flag antibody fragment (Fab') was bioconjugated to the surface of the Gd-complexed NAs. The binding ability of these targeted NAs to a FLAG-tagged protein was confirmed by SDS-PAGE. The in vitro cytotoxicity against two cell lines showed that except for the negatively charged micellar Gd-DTPA amphiphile, liposomal and higher order internally nanostructured NAs had low cell toxicity. The efficient cellular uptake of Gd-NAs by melanoma cancer cells was also investigated.

Biochemical characterization of individual human glycosylated pro-insulin-like growth factor (IGF)-II and big-IGF-II isoforms associated with cancer.

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed "pro" and "big" IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling.