Poly-N-Vinylcaprolactam (PNVCL) is a thermoresponsive polymer that exhibits lower critical solution temperature (LCST) between 25 and 50 °C. Due to its alleged biocompatibility, this polymer is ...becoming popular for biomedical and environmental applications. PNVCL with carboxyl terminations has been widely used for the preparation of thermoresponsive copolymers, micro- and nanogels for drug delivery and oncological therapies. However, the fabrication of such specific targeting devices needs standardized and reproducible preparation methods. This requires a deep understanding of how the miscibility behavior of the polymer is affected by its structural properties and the solution environment. In this work, PNVCL-COOH polymers were prepared via free radical polymerization (FRP) in order to exhibit LCST between 33 and 42 °C. The structural properties were investigated with NMR, FT-IR and conductimetric titration and the LCST was calculated via UV-VIS and DLS. The LCST is influenced by the molecular mass, as shown by both DLS and viscosimetric values. Finally, the behavior of the polymer was described as function of its concentration and in presence of different biologically relevant environments, such as aqueous buffers, NaCl solutions and human plasma.
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•The combination of several analytical methods to characterize PPS is reported.•An in-depth characterization of PPS is reported for the first time.•The integrative approach reveals major and minor ...quality attributes of PPS.•This approach represents a tool for comparative studies of Pentosan samples.
Rapid advances have been made in developing analytical technologies for characterization of highly heterogeneous active ingredients of complex drugs, such as pentosan polysulfate (PPS), active ingredient of the drug Elmiron®, approved by the Food and Drug Administration and marketed in the United States to treat interstitial cystitis. PPS sulfated polysaccharides comprise of a repeat unit of β(1–4)‐D‐xylopyranoses randomly substituted by 4‐O-methyl-glucopyranosyluronic acid. To define the critical quality attributes (CQAs) of such a complex drug, it is critical to develop an approach that integrates data from orthogonal analytical methodologies. Here, we developed an approach integrating diverse analytical tools including gel permeation chromatography, LC/ESI-MS and NMR to measure CQAs of PPS. The proposed mathematical framework integrates the data from these diverse analytical methods as function of PPS chain length and building blocks. Our approach would facilitate in establishing a scientific foundation for comparative characterization of drug products with complex active ingredients.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
As part of a more extensive investigation on structural features of different low-molecular-weight heparins (LMWHs) that can affect their biological activities, Enoxaparin, Tinzaparin and Dalteparin ...were characterised with regards to the distribution of different chain length oligosaccharides as determined by size-exclusion (SE) chromatography, as well as their structure as defined by 2D-NMR spectra (HSQC). The three LMWHs were also fractionated into high affinity (HA) and no affinity (NA) pools with regards to their ability to bind antithrombin (AT). The HA fractions were further subfractionated and characterised. For the parent LMWHs and selected fractions, molecular weight parameters were measured using a SE chromatographic system with a triple detector (TDA) to obtain absolute molecular weights. The SE chromatograms clearly indicate that Enoxaparin is consistently richer in shorter oligosaccharides than Tinzaparin and Dalteparin. Besides providing the content of terminal groups and individual glucosamine and uronic acid residues with different sulfate substituents, the HSQC-NMR spectra permitted us to evaluate and correlate the content of the pentasaccharide, AT-binding sequence A-G-A*-I-A (AT-bs) through quantification of signals of the disaccharide sequence G-A*. Whereas the percent content of HA species is approximately the same for the three LMWHs, substantial differences were observed for the chain distribution of AT-bs as a function of length, with the AT-bs being preferentially contained in the longest chains of each LMWH. The above information will be useful in establishing structure-activity relationships currently under way. This study is therefore critical for establishing correlations between structural features of LMWHs and their AT-mediated anticoagulant activity.
Heparin has been conjugated to Fe3O4, Co3O4, and NiO nanoparticles (NPs) through electrostatic interactions, producing colloidal suspensions of hybrid metal oxide heparin NPs that are stable in ...water. Negative zeta potentials and retention of heparin's ability to capture toluidine blue indicate that heparin's negative charges are exposed on the surface of the coated NPs. IR results confirmed the formation of nanohybrids as did NMR experiments, which were also interpreted on the basis of toluidine blue tests. Transmission electron microscopy results revealed that the heparin coating does not modify the shape or dimension of the NPs. Dynamic light scattering and negative zeta potential measurements confirmed that heparin surface functionalisation is an effective strategy to prevent NP aggregation.
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Several publications have recently proposed NMR spectroscopy to evaluate the critical quality attributes (CQA) of pentosan polysulfate sodium (PPS), the active ingredient of Elmiron™ approved to ...treat interstitial cystitis. PPS is a polymer of sulfated β(1—4)‐d‐xylopyranose residues randomly substituted by 4–O-methyl-glucopyranosyluronic acid, containing, beyond the main xylose-2,3-O-disulfate repetitive unit, some minor residues that can be marker of both the starting material and preparation process. In the present study we assigned some previously unknown cross-peaks in 1H–13C HSQC NMR of PPS related to its minor sequences adding additional details to its CQA. Four anomeric cross-peaks related to glucuronate-branched xylose and different sulfation pattern as well as the preceding xyloses were identified. Two minor process-related signals of monosulfated xyloses (unsubstituted in position 2 or 3) were also assigned. The isolation of a disaccharide fraction allowed the assignment of the reducing end xylose-α/β as well as the preceding xylose residues to be corrected. Additionally, the oversulfation of PPS allowed detection of the reducing end xylose-tri-1,2,3-O-sulfate. The newly identified cross-peaks were integrated into an updated quantitative NMR method. Finally, we demonstrated that an in-depth PPS analysis can be obtained using NMR instruments at medium magnetic fields (500 MHz/600 MHz), commonly available in pharmaceutical industries.
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•Updated structural characterization of the complex drug Pentosan Polysulfate PPS.•Assignment of previously unknown resonances in HSQC NMR spectra of Elmiron® PPS.•Improved NMR analysis of the PPS monosaccharide composition and method validation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•A new PHA extraction process from mixed microbial biomass is proposed.•Non-ionic surfactants were used as pretreatment to increase biomass disruption.•Extraction yields increased in 50% compared ...with non-pretreated samples.•Extracted polymer had a high purity (>90%) and low molecular weight loss (<24%)•Economic and carbon footprint analysis supported the selection of the method.
Polyhydroxyalkanoates (PHA) are biodegradable plastics of microbial origin, whose biodegradability and thermochemical properties make them greener alternatives to conventional plastics. Despite their high industrial potential, the PHA’ high production costs still hinder their application. Mixed microbial biomass combined with agro-industrial wastes are being used to strategically reduce these costs. However, it is still necessary to optimize the downstream processing, where the extraction process amounts to 30–50% of the total costs. Conventional processes apply chlorinated solvents to recover PHA from microbial biomass but cannot be implemented industrially due to environmental regulations. Alternative solvents, with good results of purity and recovery yields, usually have a negative impact on the molecular weight of the final polymer. In this work, the addition of a pre-treatment based on non-ionic surfactants (Tween® 20, Brij® L4, and Triton™ X-114) to extract PHA from mixed microbial biomass selected on fermented agro-industrial wastes was investigated. The best results were obtained with Tween® 20 allowing for an increase in 50% compared with the use of dimethylcarbonate without any pre-treatment (from 38.4 ± 0.8% to 53 ± 2%) and very close to those obtained with chloroform (63%). The extracted polymer was analysed and characterized, revealing a PHA of high purity (>90%) and low molecular weight loss (under 24%). Additionally, a material-focused economic and a carbon footprint analysis were performed and supported the selection of the method as one of the cheapest options and with the lowest carbon footprint.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Two years since the outbreak of the novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic, there remain few clinically effective drugs to complement ...vaccines. One is the anticoagulant, heparin, which in 2004 was found able to inhibit invasion of SARS-CoV (CoV-1) and which has been employed during the current pandemic to prevent thromboembolic complications and moderate potentially damaging inflammation. Heparin has also been shown experimentally to inhibit SARS-CoV-2 attachment and infection in susceptible cells. At high therapeutic doses however, heparin increases the risk of bleeding and prolonged use can cause heparin-induced thrombocytopenia, a serious side effect. One alternative, with structural similarities to heparin, is the plant-derived, semi-synthetic polysaccharide, pentosan polysulfate (PPS). PPS is an established drug for the oral treatment of interstitial cystitis, is well-tolerated, and exhibits weaker anticoagulant effects than heparin. In an established Vero cell model, PPS and its fractions of varying molecular weights inhibited invasion by SARS-CoV-2. Intact PPS and its size-defined fractions were characterized by molecular weight distribution and chemical structure using nuclear magnetic resonance spectroscopy and liquid chromatography–mass spectrometry, then employed to explore the structural basis of interactions with SARS-CoV-2 spike protein receptor-binding domain (S1 RBD) and the inhibition of Vero cell invasion. PPS was as effective as unfractionated heparin, but more effective in inhibiting cell infection than low-molecular-weight heparin (on a weight/volume basis). Isothermal titration calorimetry and viral plaque-forming assays demonstrated size-dependent binding to S1 RBD and inhibition of Vero cell invasion, suggesting the potential application of PPS as a novel inhibitor of SARS-CoV-2 infection.
A highly productive Arundo donax L. clone (Clone AD-20) was produced at full field to give 54.6 Mg total solids biomass Ha–1. Biomass was chemically and enzymatically pretreated, recovering 13.9 Mg ...Ha–1 of glucose and 3.6 Mg Ha–1 of xylose, i.e., 3.5–4.5 more than yield typically obtained from corn stover or switchgrass. The subsequent fermentation of the liberated sugars to organic acids (OA) by dark fermentation generated yields of 3850 Nm3 Ha–1 of biohydrogen and 14.2 Mg Ha–1 of OAs. OAs were then used as a feed to produce polyhydroxyalkanoates (PHA), with 3-hydroxybutyrate the major monomer present (PHB > 95% PHA), from a biological process using mixed microbial culture producing 5.04 Mg Ha–1 of PHA. An initial economic analysis indicated that this multistage biorefinery approach would result in a net revenue of 10,415 € Ha–1, which is approximately 9-fold greater than that obtained by a traditional biorefinery producing bioethanol.
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In Europe, almost 87.6 million tonnes of food waste are produced. Despite the high biological value of food waste, traditional management solutions do not consider it as a precious resource. Many ...studies have reported the use of food waste for the production of high added value molecules. Polyhydroxyalkanoates (PHAs) represent a class of interesting bio-polyesters accumulated by different bacterial cells, and has been proposed for production from the organic fraction of municipal solid waste (OFMSW). Nevertheless, until now, no attention has been paid to the entire biological process leading to the transformation of food waste to organic acids (OA) and then to PHA, getting high PHA yield per food waste unit. In particular, the acid-generating process needs to be optimized, maximizing OA production from OFMSW. To do so, a pilot-scale Anaerobic Percolation Biocell Reactor (100 L in volume) was used to produce an OA-rich percolate from OFMSW which was used subsequently to produce PHA.
The optimized acidogenic process resulted in an OA production of 151 g kg
from fresh OFMSW. The subsequent optimization of PHA production from OA gave a PHA production, on average, of 223 ± 28 g kg
total OA fed. Total mass balance indicated, for the best case studied, a PHA production per OFMSW weight unit of 33.22 ± 4.2 g kg
from fresh OFMSW, corresponding to 114.4 ± 14.5 g kg
of total solids from OFMSW. PHA composition revealed a hydroxybutyrate/hydroxyvalerate (%) ratio of 53/47 and Mw of 8∙10
kDa with a low polydispersity index, i.e. 1.4.
This work showed how by optimizing acidic fermentation it could be possible to get a large amount of OA from OFMSW to be then transformed into PHA. This step is important as it greatly affects the total final PHA yield. Data obtained in this work can be useful as the starting point for considering the economic feasibility of PHA production from OFMSW by using mixed culture.
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