Publications by authors named "Giuseppe Mantovani"

41 Publications

PEG-polyaminoacid based micelles for controlled release of doxorubicin: Rational design, safety and efficacy study.

J Control Release 2021 May 11;335:21-37. Epub 2021 May 11.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F. Marzolo 5, 35131 Padova, Italy.

A library of amphiphilic monomethoxypolyethylene glycol (mPEG) terminating polyaminoacid co-polymers able to self-assemble into colloidal systems was screened for the delivery and controlled release of doxorubicin (Doxo). mPEG-Glu/Leu random co-polymers were generated by Ring Opening Polymerization from 5 kDa mPEG-NH macroinitiator using 16:0:1, 8:8:1, 6:10:1, 4:12:1 γ-benzyl glutamic acid carboxy anhydride monomer/leucine N-carboxy anhydride monomer/PEG molar ratios. Glutamic acid was selected for chemical conjugation of Doxo, while leucine units were introduced in the composition of the polyaminoacid block as spacer between adjacent glutamic repeating units to minimize the steric hindrance that could impede the Doxo conjugation and to promote the polymer self-assembly by virtue of the aminoacid hydrophobicity. The benzyl ester protecting the γ-carboxyl group of glutamic acid was quantitatively displaced with hydrazine to yield mPEG-b-(hydGlu-r-Leu). Doxo was conjugated to the diblock co-polymers through pH-sensitive hydrazone bond. The Doxo derivatized co-polymers obtained with a 16:0:1, 8:8:1, 6:10:1 Glu/Leu/PEG ratios self-assembled into 30-40 nm spherical nanoparticles with neutral zeta-potential and CMC in the range of 4-7 μM. At pH 5.5, mimicking endosome environment, the carriers containing leucine showed a faster Doxo release than at pH 7.4, mimicking the blood conditions. Doxo-loaded colloidal formulations showed a dose dependent cytotoxicity on two cancer cell lines, CT26 murine colorectal carcinoma and 4T1 murine mammary carcinoma with IC slightly higher than those of free Doxo. The carrier assembled with the polymer containing 6:10:1 hydGlu/Leu/PEG molar ratio {mPEG-b-[(Doxo-hydGlu)-r-Leu]} was selected for subsequent in vitro and in vivo investigations. Confocal imaging on CT26 cell line showed that intracellular fate of the carrier involves a lysosomal trafficking pathway. The intratumor or intravenous injection to CT26 and 4T1 subcutaneous tumor bearing mice yielded higher antitumor activity compared to free Doxo. Furthermore, mPEG-b-[(Doxo-hydGlu)-r-Leu] displayed a better safety profile when compared to commercially available Caelyx®.
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http://dx.doi.org/10.1016/j.jconrel.2021.05.010DOI Listing
May 2021

Combining Inducible Lectin Expression and Magnetic Glyconanoparticles for the Selective Isolation of Bacteria from Mixed Populations.

ACS Appl Mater Interfaces 2021 Apr 14;13(16):19230-19243. Epub 2021 Apr 14.

Division of Molecular Therapeutics and Formulation, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, U.K.

The selective isolation of bacteria from mixed populations has been investigated in varied applications ranging from differential pathogen identification in medical diagnostics and food safety to the monitoring of microbial stress dynamics in industrial bioreactors. Selective isolation techniques are generally limited to the confinement of small populations in defined locations, may be unable to target specific bacteria, or rely on immunomagnetic separation, which is not universally applicable. In this proof-of-concept work, we describe a novel strategy combining inducible bacterial lectin expression with magnetic glyconanoparticles (MGNPs) as a platform technology to enable selective bacterial isolation from cocultures. An inducible mutant of the type 1 fimbriae, displaying the mannose-specific lectin FimH, was constructed in allowing for "on-demand" glycan-binding protein presentation following external chemical stimulation. Binding to glycopolymers was only observed upon fimbrial induction and was specific for mannosylated materials. A library of MGNPs was produced via the grafting of well-defined catechol-terminal glycopolymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization to magnetic nanoparticles. Thermal analysis revealed high functionalization (≥85% polymer by weight). Delivery of MGNPs to cocultures of fluorescently labeled bacteria followed by magnetic extraction resulted in efficient depletion of type 1 fimbriated target cells from wild-type or afimbriate . Extraction efficiency was found to be dependent on the molecular weight of the glycopolymers utilized to engineer the nanoparticles, with MGNPs decorated with shorter Dopa-(ManAA) mannosylated glycopolymers found to perform better than those assembled from a longer Dopa-(ManAA) analogue. The extraction efficiency of fimbriated was also improved when the counterpart strain did not harbor the genetic apparatus for the expression of the type 1 fimbriae. Overall, this work suggests that the modulation of the genetic apparatus encoding bacterial surface-associated lectins coupled with capture through MGNPs could be a versatile tool for the extraction of bacteria from mixed populations.
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http://dx.doi.org/10.1021/acsami.1c00907DOI Listing
April 2021

Thermosensitive "Smart" Surfaces for Biorecognition Based Cell Adhesion and Controlled Detachment.

Macromol Biosci 2021 02 4;21(2):e2000277. Epub 2020 Nov 4.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F. Marzolo 5, Padova, 35131, Italy.

The biorecognition-based control of attachment/detachment of MCF-7 cancer cells from polymer-coated surfaces is demonstrated. A glass surface is coated with a thermoresponsive statistical copolymer of poly(N-isopropylacrylamide-co-acrylamide) [p(NIPAm-co-Am)], which is end-capped with the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide, and the hydrophilic polymer poly(ethylene glycol) (PEG). Below the lower critical solution temperature (LCST) of p(NIPAm-co-Am) (38 °C), the copolymers are in the extended conformation, allowing for accessibility of the GRGDS peptides to membrane-associated integrins thus enabling cell attachment. Above the LCST, the p(NIPAm-co-Am) polymers collapse into globular conformations, resulting in the shielding of the GRGDS peptides into the PEG brush with consequent inaccessibility to cell-surface integrins, causing cell detachment. The surface coating is carried out by a multi-step procedure that included: glass surface amination with 3-aminopropyltriethoxysilane; reaction of mPEG -N-hydroxysuccinimide (NHS) and p(NIPam-co-Am) -bis-NHS with the surface aminopropyl groups and conjugation of GRGDS to the carboxylic acid termini of p(NIPam-co-Am) -COOH. A range of spectrophotometric, surface, and microscopy assays confirmed the identity of the polymer-coated substrates. Competition studies prove that MCF-7 cancer cells are attached via peptide recognition at the coated surfaces according to the mPEG /p(NIPam-co-Am) -GRGDS molar ratio. These data suggest the system can be exploited to modulate cell integrin/GRGDS binding for controlled cell capture and release.
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http://dx.doi.org/10.1002/mabi.202000277DOI Listing
February 2021

Polyvalent Diazonium Polymers Provide Efficient Protection of Oncolytic Adenovirus Enadenotucirev from Neutralizing Antibodies while Maintaining Biological Activity In Vitro and In Vivo.

Bioconjug Chem 2019 04 27;30(4):1244-1257. Epub 2019 Mar 27.

School of Pharmacy , University of Nottingham , Nottingham NG7 2RD , U.K.

Oncolytic viruses offer many advantages for cancer therapy when administered directly to confined solid tumors. However, the systemic delivery of these viruses is problematic because of the host immune response, undesired interactions with blood components, and inherent targeting to the liver. Efficacy of systemically administered viruses has been improved by masking viral surface proteins with polymeric materials resulting in modulation of viral pharmacokinetic profile and accumulation in tumors in vivo. Here we describe a new class of polyvalent reactive polymer based on poly( N-(2-hydroxypropyl)methacrylamide) (polyHPMA) with diazonium reactive groups and their application in the modification of the chimeric group B oncolytic virus enadenotucirev (EnAd). A series of six copolymers with different chain lengths and density of reactive groups was synthesized and used to coat EnAd. Polymer coating was found to be extremely efficient with concentrations as low as 1 mg/mL resulting in complete (>99%) ablation of neutralizing antibody binding. Coating efficiency was found to be dependent on both chain length and reactive group density. Coated viruses were found to have reduced transfection activity both in vitro and in vivo, with greater protection against neutralizing antibodies resulting in lower transgene production. However, in the presence of neutralizing antibodies, some in vivo transgene expression was maintained for coated virus compared to the uncoated control. The decrease in transgene expression was found not to be solely due to lower cellular uptake but due to reduced unpackaging of the virus within the cells and reduced replication, indicating that the polymer coating does not cause permanent inactivation of the virus. These data suggest that virus activity may be modulated by the appropriate design of coating polymers while retaining protection against neutralizing antibodies.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00189DOI Listing
April 2019

Water-soluble substituted chitosan derivatives as technology platform for inhalation delivery of siRNA.

Drug Deliv 2018 Nov;25(1):644-653

a Division of Molecular Therapeutics and Formulation, School of Pharmacy , University of Nottingham, University Park , Nottingham , United Kingdom.

Despite research efforts full potential of siRNA-based therapeutics has not yet been fully realized due to a need for suitable, effective delivery formulations. Here, we examine a potential of a new class of water-soluble chitosans as siRNA platform for pulmonary delivery. The system is based on piperazine-substituted chitosans, a material designed to integrate established, safe application of chitosan for mucosal administration with novel properties: the piperazine-substituted chitosans are freely water-soluble at physiological pH, possess low cytotoxicity (no significant reduction in cell viability up to 0.1 mg/ml), and provide efficient incorporation of siRNA into sub-300 nm colloidal complexes (at relatively low polymer/siRNA ratio of 5:1). In vitro, the complexes achieved silencing of a model gene at a level of 40-80%, when tested in a panel of lung epithelial cells. Considering the formulation 'developability', there were no significant changes in the complexes' size and integrity on aerosolisation by microsprayer (PenCentury™) device. Following intratracheal aerolisation, the complexes deposited throughout the lung, although relatively inhomogeneously, as judged from IVIS imaging of the isolated mouse lung (visualizing DY647-siRNA). In vivo data illustrate absence of adverse effects on repeated administration of complexes and significant tumor reduction in atopical lung cancer model in mice. Altogether, the data illustrates potential of substituted chitosan derivatives to be utilized as a safe system for inhalation delivery of siRNA.
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http://dx.doi.org/10.1080/10717544.2018.1440668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058492PMC
November 2018

Switching of Macromolecular Ligand Display by Thermoresponsive Polymers Mediates Endocytosis of Multiconjugate Nanoparticles.

Bioconjug Chem 2018 04 14;29(4):1030-1046. Epub 2018 Mar 14.

School of Pharmacy , University of Nottingham , University Park , Nottingham NG72RD , United Kingdom.

Ligand-mediated targeting and internalization of plasma membrane receptors is central to cellular function. These types of receptors have accordingly been investigated as targets to facilitate entry of diagnostic and therapeutic constructs into cells. However, there remains a need to characterize how receptor targeting agents on nanoparticles interact at surface receptors and whether it is possible to control these interactions via exogenous stimuli. Here, we describe the switchable display of the iron-transporting protein, transferrin (Tf), at the surface of thermoresponsive polymer-coated gold nanoparticles and show that internalization of the coated nanoparticles into target cells changes across temperature ranges over which transferrin is expected to be sterically "hidden" by an extended polymer chain and then "revealed" by polymer chain collapse. The switching process is dependent on the numbers of transferrin molecules and thermoresponsive polymer chains attached and whether the assay temperature is above or below the transition temperatures of the responsive polymers at the nanoparticle surfaces. Significantly, however, the control of internalization is critically reliant on overall nanoparticle colloidal stability while the thermoresponsive component of the surface undergoes conformational change. The data show that the cell entry function of complex and large biomolecule ligands can be modulated by polymer-induced accessibility change but that a simple "hide and reveal" mechanism for ligand display following polymer chain collapse is insufficient to account for nanoparticle uptake and subsequent intracellular trafficking.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00704DOI Listing
April 2018

Dry-powder formulations of non-covalent protein complexes with linear or miktoarm copolymers for pulmonary delivery.

Int J Pharm 2018 Apr 6;540(1-2):78-88. Epub 2018 Feb 6.

School of Pharmacy, University of Nottingham, Nottingham, United Kingdom. Electronic address:

Pulmonary delivery of protein therapeutics has considerable clinical potential for treating both local and systemic diseases. However, poor protein conformational stability, immunogenicity and protein degradation by proteolytic enzymes in the lung are major challenges to overcome for the development of effective therapeutics. To address these, a family of structurally related copolymers comprising polyethylene glycol, mPEG, and poly(glutamic acid) with linear A-B (mPEG-lin-GA) and miktoarm A-B (mPEG-mik-(GA)) macromolecular architectures was investigated as potential protein stabilisers. These copolymers form non-covalent nanocomplexes with a model protein (lysozyme) which can be formulated into dry powders by spray-drying using common aerosol excipients (mannitol, trehalose and leucine). Powder formulations with excellent aerodynamic properties (fine particle fraction of up to 68%) were obtained with particle size (D) in the 2.5 µm range, low moisture content (<5%), and high glass transitions temperatures, i.e. formulation attributes all suitable for inhalation application. In aqueous medium, dry powders rapidly disintegrated into the original polymer-protein nanocomplexes which provided protection towards proteolytic degradation. Taken together, the present study shows that dry powders based on (mPEG-polyGA)-protein nanocomplexes possess potentials as an inhalation delivery system.
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http://dx.doi.org/10.1016/j.ijpharm.2018.02.008DOI Listing
April 2018

Properties of acyl modified poly(glycerol-adipate) comb-like polymers and their self-assembly into nanoparticles.

J Polym Sci A Polym Chem 2016 10 8;54(20):3267-3278. Epub 2016 Jul 8.

School of Pharmacy, University of Nottingham, University Park Nottingham NG7 2RD United Kingdom.

There is an increasing need to develop bio-compatible polymers with an increased range of different physicochemical properties. Poly(glycerol-adipate) (PGA) is a biocompatible, biodegradable amphiphilic polyester routinely produced from divinyl adipate and unprotected glycerol by an enzymatic route, bearing a hydroxyl group that can be further functionalized. Polymers with an average Mn of ∼13 kDa can be synthesized without any post-polymerization deprotection reactions. Acylated polymers with fatty acid chain length of C, C, and C (PGAB, PGAO, and PGAS, respectively) at different degrees of substitution were prepared. These modifications yield comb-like polymers that modulate the amphiphilic characteristics of PGA. This novel class of biocompatible polymers has been characterized through various techniques such as FT-IR, H NMR, surface, thermal analysis, and their ability to self-assemble into colloidal structures was evaluated by using DLS. The highly tunable properties of PGA reported herein demonstrate a biodegradable polymer platform, ideal for engineering solid dispersions, nanoemulsions, or nanoparticles for healthcare applications. © 2016 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. , , 3267-3278.
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http://dx.doi.org/10.1002/pola.28215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516180PMC
October 2016

Polymers for binding of the gram-positive oral pathogen Streptococcus mutans.

PLoS One 2017 3;12(7):e0180087. Epub 2017 Jul 3.

School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.

Streptococcus mutans is the most significant pathogenic bacterium implicated in the formation of dental caries and, both directly and indirectly, has been associated with severe conditions such as multiple sclerosis, cerebrovascular and peripheral artery disease. Polymers able to selectively bind S. mutans and/or inhibit its adhesion to oral tissue in a non-lethal manner would offer possibilities for addressing pathogenicity without selecting for populations resistant against bactericidal agents. In the present work two libraries of 2-(dimethylamino)ethyl methacrylate (pDMAEMA)-based polymers were synthesized with various proportions of either N,N,N-trimethylethanaminium cationic- or sulfobetaine zwitterionic groups. These copolymers where initially tested as potential macromolecular ligands for S. mutans NCTC 10449, whilst Escherichia coli MG1655 was used as Gram-negative control bacteria. pDMAEMA-derived materials with high proportions of zwitterionic repeating units were found to be selective for S. mutans, in both isolated and S. mutans-E. coli mixed bacterial cultures. Fully sulfobetainized pDMAEMA was subsequently found to bind/cluster preferentially Gram-positive S. mutans and S. aureus compared to Gram negative E. coli and V. harveyi. A key initial stage of S. mutans pathogenesis involves a lectin-mediated adhesion to the tooth surface, thus the range of potential macromolecular ligands was further expanded by investigating two glycopolymers bearing α-mannopyranoside and β-galactopyranoside pendant units. Results with these polymers indicated that preferential binding to either S. mutans or E. coli can be obtained by modulating the glycosylation pattern of the chosen multivalent ligands without incurring unacceptable cytotoxicity in a model gastrointestinal cell line. Overall, our results allowed to identify a structure-property relationship for the potential antimicrobial polymers investigated, and suggest that preferential binding to Gram-positive S. mutans could be achieved by fine-tuning of the recognition elements in the polymer ligands.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180087PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495209PMC
October 2017

Amphiphilic block copolymers from a renewable ε-decalactone monomer: prediction and characterization of micellar core effects on drug encapsulation and release.

J Mater Chem B 2016 Nov 20;4(44):7119-7129. Epub 2016 Oct 20.

School of Pharmacy, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.

Here we describe a methoxy poly(ethyleneglycol)-b-poly(ε-decalactone) (mPEG-b-PεDL) copolymer and investigate the potential of the copolymer as a vehicle for solubilisation and sustained release of indomethacin (IND). The indomethacin loading and release from mPEG-b-PεDL micelles (amorphous cores) was compared against methoxy poly(ethyleneglycol)-b-poly(ε-caprolactone)(mPEG-b-PCL) micelles (semicrystalline cores). The drug-polymer compatibility was determined through a theoretical approach to predict drug incorporation into hydrated micelles. Polymer micelles were prepared by solvent evaporation and characterised for size, morphology, indomethacin loading and release. All the formulations generated spherical micelles but significantly larger mPEG-b-PεDL micelles were observed compared to mPEG-b-PCL micelles. A higher compatibility of the drug was predicted for PCL cores based on Flory-Huggins interaction parameters (χ) using the Hansen solubility parameter (HSP) approach, but higher measured drug loadings were found in micelles with PεDL cores compared to PCL cores. This we attribute to the higher amorphous content in the PεDL-rich regions which generated higher micellar core volumes. Drug release studies showed that the semicrystalline PCL core was able to release IND over a longer period (80% drug release in 110 h) compared to PεDL core micelles (80% drug release in 72 h).
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http://dx.doi.org/10.1039/c6tb01839dDOI Listing
November 2016

Synthesis, characterization and evaluation of in vitro toxicity in hepatocytes of linear polyesters with varied aromatic and aliphatic co-monomers.

J Control Release 2016 12 4;244(Pt B):214-228. Epub 2016 Aug 4.

School of Pharmacy, University of Nottingham, University Park, Nottingham NG72RD, UK. Electronic address:

Polyesters are extensively used in drug delivery because of their controllable biodegradation properties and perceived favorable cytocompatibility. However, new ester-based materials are continually being sought which can be produced from readily accessible monomers, which can be tuned for drug encapsulation and which retain good cellular compatibilities. In this study, 5 polyesters of similar molar mass were synthesized by reacting 1,10-decanediol with different ratios of succinic acid/phenylsuccinic acid and the effect of the phenyl side-chain group addition on polymer properties relevant to drug delivery was investigated. A polymer with a 70/30 ratio of succinic acid and phenylsuccinic acid was selected based on its ability to encapsulate a model dye in nanoparticle (NP) formulations, and was found to be slowly degradable in phosphate buffered saline (PBS) but more rapidly degraded in the presence of a lipase. The compatibility of NP formulations of this polymer either with or without a Pluronic F68 stabilizing coating was assessed in vitro using the C3A hepatocyte cell line. Cell viability was assessed, at NP concentrations ranging from 4.68-300μgmL 24h post-exposure, using the Alamar Blue, CDFA and Neutral Red assays. C3A cells internalized both coated and uncoated polyester NPs to a similar extent, with uptake observed to increase over time (10-1440min). Although cell viability was >80% at the concentrations tested, in all assays, it was found that a Pluronic F68 coated poly (decanediol-phenylsuccinate-co-succinate) stimulated significant DNA damage driven by an oxidant mechanism, whereas the non-coated polyester analogue and the Pluronic F68 alone had no effect. The results obtained suggest that new polyesters can be synthesized with desirable properties from the materials perspective but formulation with additional excipients requires careful evaluation for drug delivery applications.
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http://dx.doi.org/10.1016/j.jconrel.2016.08.003DOI Listing
December 2016

Relation between detection rate and inappropriate shocks in single versus dual chamber cardioverter-defibrillator--an analysis from the OPTION trial.

Sci Rep 2016 Feb 19;6:21748. Epub 2016 Feb 19.

Klinik für Kardiologie und Internistische Intensivmedizin, Städtisches Klinikum München-Bogenhausen, München, Germany.

The programming of implantable cardioverter-defibrillators (ICDs) influences inappropriate shock rates. The aim of the study is to analyse rates of patients with appropriate and inappropriate shocks according to detection zones in the OPTION trial. All patients received dual chamber (DC) ICDs randomly assigned to be programmed either to single chamber (SC) or to DC settings including PARAD+ algorithm. In a post-hoc analysis, rates of patients with inappropriate and appropriate shocks were calculated for shocks triggered at heart rates ≥ 170 bpm (ventricular tachycardia zone) and at rates ≥ 200 bpm (ventricular fibrillation zone). In the SC group, higher rates of patients with total and inappropriate shocks were delivered at heart rates ≥ 170 bpm than at rates ≥ 200 bpm (total shocks: 21.1% vs. 16.6%; p = 0.002; inappropriate shocks: 7.6% vs. 4.5%, p = 0.016; appropriate shocks: 15.2% vs. 13.5%; p = n.s.). No such differences were observed in the DC group (total shocks: 14.3% vs. 12.6%; p = n.s.; inappropriate shocks: 3.9% vs. 3.6%; p = n.s.; appropriate shocks: 12.2% vs. 10.4%; p = n.s.). The higher frequency of patients with total shocks with SC settings than with DC settings that benefit from PARAD+ was driven by a higher percentage of patients with inappropriate shocks in the VT zone (170-200 bpm) in the SC population.
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http://dx.doi.org/10.1038/srep21748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759595PMC
February 2016

Phosphonium Polymethacrylates for Short Interfering RNA Delivery: Effect of Polymer and RNA Structural Parameters on Polyplex Assembly and Gene Knockdown.

Biomacromolecules 2015 Nov 7;16(11):3480-90. Epub 2015 Oct 7.

School of Pharmacy, University of Nottingham , Boots Science Building, University Park, Nottingham NG7 2RD, U.K.

Synthetic polymers containing quaternary phosphonium salts are an emerging class of materials for the delivery of oligo/polynucleotides. In this work, cationic phosphonium salt-containing polymethacrylates and their corresponding ammonium analogues were synthesized by reversible addition-fragmentation chain transfer polymerization. Both the nature of the charged heteroatom (N vs P) and the length of the spacer separating the cationic units along the polymer backbone (oxyethylene vs trioxyethylene) were systematically varied. Polymers efficiently bound short interfering RNA (siRNA) at N(+)/P(-) or P(+)/P(-) ratios of 2 and above. At a 20:1 ratio, small polyplexes (Rh: 4-15 nm) suitable for cellular uptake were formed that displayed low cytotoxicity. While siRNA polyplexes from both ammonium and phosphonium polymers were efficiently internalized by green fluorescent protein (GFP)-expressing 3T3 cells, no knockdown of GFP expression was observed. However, 65% Survivin gene knockdown was observed when siRNA was replaced with novel, multimerized long interfering RNA in HeLa cells, demonstrating the importance of RNA macromolecular architecture on RNA-mediated gene silencing.
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http://dx.doi.org/10.1021/acs.biomac.5b00898DOI Listing
November 2015

Triblock Copolymer Nanovesicles for pH-Responsive Targeted Delivery and Controlled Release of siRNA to Cancer Cells.

Biomacromolecules 2015 Jul 2;16(7):1924-37. Epub 2015 Jun 2.

†Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131, Padova, Italy.

New pH-responsive polymersomes for active anticancer oligonucleotide delivery were prepared from triblock copolymers. The delivery systems were formed by two terminal hydrophilic blocks, PEG and polyglycerolmethacrylate (poly-GMA), and a central weakly basic block, polyimidazole-hexyl methacrylate (poly-ImHeMA), which can complex with oligonucleotides and control vesicle formation/disassembly via pH variations. Targeted polymersomes were prepared by mixing folate-derivatized and underivatized copolymers. At pH 5, ds-DNA was found to complex with the pH-responsive copolymers at a N/P molar ratio above ∼2:1, which assisted the encapsulation of ds-DNA in the polymersomes, while low association was observed at pH 7.4. Cytotoxicity studies performed on folate receptor overexpressing KB and B16-F10 cells and low folate receptor expressing MCF-7 cells showed high tolerance of the polymersomes at up to 3 mg/mL concentration. Studies performed with red blood cells showed that at pH 5.0 the polymersomes have endosomolytic properties. Cytofluorimetric studies showed a 5.5-fold higher uptake of ds-DNA loaded folate-functional polymersomes in KB cells compared to nontargeted polymersomes. In addition, ds-DNA was found to be localized both in the nucleus and in the cytosol. The incubation of luciferase transfected B16-F10 cells with targeted polymersomes loaded with luciferase and Hsp90 expression silencing siRNAs yielded 31 and 23% knockdown in target protein expression, respectively.
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http://dx.doi.org/10.1021/acs.biomac.5b00286DOI Listing
July 2015

Reduced risk for inappropriate implantable cardioverter-defibrillator shocks with dual-chamber therapy compared with single-chamber therapy: results of the randomized OPTION study.

JACC Heart Fail 2014 Dec 1;2(6):611-9. Epub 2014 Oct 1.

University Hospital, Tours, France.

Objectives: The OPTION (Optimal Anti-Tachycardia Therapy in Implantable Cardioverter-Defibrillator Patients Without Pacing Indications) trial sought to compare long-term rates of inappropriate shocks, mortality, and morbidity between dual-chamber and single-chamber settings in implantable cardioverter-defibrillators (ICDs) patients.

Background: The use of dual-chamber ICDs potentially allows better discrimination of supraventricular arrhythmias and thereby reduces inappropriate shocks. However, it may lead to detrimental ventricular pacing.

Methods: This prospective multicenter, single-blinded trial enrolled 462 patients with de novo primary or secondary prevention indications for ICD placement and with left ventricular ejection fractions ≤40% despite optimal tolerated pharmacotherapy. All patients received atrial leads and dual-chamber defibrillators that were randomized to be programmed either with dual-chamber or single-chamber settings. In the dual-chamber setting arm, the PARAD+ algorithm, which differentiates supraventricular from ventricular arrhythmias, and SafeR mode, to minimize ventricular pacing, were activated. In the single-chamber setting arm, the acceleration, stability, and long cycle search discrimination criteria were activated, and pacing was set to VVI 40 beats/min. Ventricular tachycardia detection was required at rates between 170 and 200 beats/min, and ventricular fibrillation detection was activated above 200 beats/min.

Results: During a follow-up period of 27 months, the time to the first inappropriate shock was significantly longer in the dual-chamber setting arm (p = 0.012, log-rank test), and 4.3% of patients in the dual-chamber setting group compared with 10.3% in the single-chamber setting group experienced inappropriate shocks (p = 0.015). Rates of all-cause death or cardiovascular hospitalization were 20% for the dual-chamber setting group and 22.4% for the single-chamber setting group and satisfied the pre-defined margin for equivalence (p < 0.001).

Conclusions: Therapy with dual-chamber settings for ICD discrimination combined with algorithms for minimizing ventricular pacing was associated with reduced risk for inappropriate shock compared with single-chamber settings, without increases in mortality and morbidity. (Optimal Anti-Tachycardia Therapy in Implantable Cardioverter-Defibrillator [ICD] Patients Without Pacing Indications [OPTION]; NCT00729703).
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http://dx.doi.org/10.1016/j.jchf.2014.05.015DOI Listing
December 2014

Bacteria-instructed synthesis of polymers for self-selective microbial binding and labelling.

Nat Mater 2014 Jul 11;13(7):748-55. Epub 2014 May 11.

School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK.

The detection and inactivation of pathogenic strains of bacteria continues to be an important therapeutic goal. Hence, there is a need for materials that can bind selectively to specific microorganisms for diagnostic or anti-infective applications, but that can be formed from simple and inexpensive building blocks. Here, we exploit bacterial redox systems to induce a copper-mediated radical polymerization of synthetic monomers at cell surfaces, generating polymers in situ that bind strongly to the microorganisms that produced them. This 'bacteria-instructed synthesis' can be carried out with a variety of microbial strains, and we show that the polymers produced are self-selective binding agents for the 'instructing' cell types. We further expand on the bacterial redox chemistries to 'click' fluorescent reporters onto polymers directly at the surfaces of a range of clinical isolate strains, allowing rapid, facile and simultaneous binding and visualization of pathogens.
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http://dx.doi.org/10.1038/nmat3949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286827PMC
July 2014

Novel pH-responsive nanovectors for controlled release of ionisable drugs.

J Mater Chem B 2013 Oct 14;1(39):5335-5346. Epub 2013 Jun 14.

School of Pharmacy, University of Nottingham, Nottingham, UK.

A family of novel 'smart' drug nanovectors based on 2-(methacryloyloxy)ethyl-3-chloro-4-hydroxybenzoate (MCH), a functional pH-responsive monomer, and poly(methoxy-ethylene glycol) methacrylate (PEGMA) block copolymers is presented. Modification of the monomers' relative ratios allowed facile switching from micellar (PEGMA-b-MCH) to polymersome-based (PEGMA-b-MCH) drug delivery nanocarriers. The ability of the latter to incorporate model anticancer drugs - tamoxifen, paclitaxel and doxorubicin hydrochloride - was investigated. High drug loading - up to 18 w/w% - was observed for tamoxifen, a hydrophobic drug which bears an amino group able to form ion pairs with MCH acidic functionalities. Non-loaded nanovectors were characterized (CAC, DLS and TEM), and were found to be very stable under physiological conditions (PBS pH 7.4, 37 °C), even in the presence of 10% plasma proteins for at least 48 h. Tamoxifen loaded nanocarriers showed slow drug release at pH 7.4 and faster release after exposure to weakly acidic environments, due to the loss of polymer/drug ionic interactions. Importantly, at pH 7.4 tamoxifen-loaded PEGMA-b-MCH micelles were found to be less cytotoxic than free tamoxifen against MCF-7 cells, while under more acidic conditions, at pH 6.8, the opposite behaviour was observed, with a 10-fold increase in cytotoxicity for the micellar nanocarriers. The empty nanocarriers were found to be non-toxic in 48 h incubation time experiments. Pharmacokinetic studies proved the increased half-life and the slower clearance of tamoxifen after encapsulation in the micelles. These PEGMA-b-MCH based nanoassemblies could represent a novel promising delivery platform for low molecular weight ionisable drugs.
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http://dx.doi.org/10.1039/c3tb20360cDOI Listing
October 2013

Direct peptide bioconjugation/PEGylation at tyrosine with linear and branched polymeric diazonium salts.

J Am Chem Soc 2012 May 21;134(17):7406-13. Epub 2012 Apr 21.

Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.

Direct polymer conjugation at peptide tyrosine residues is described. In this study Tyr residues of both leucine enkephalin and salmon calcitonin (sCT) were targeted using appropriate diazonium salt-terminated linear monomethoxy poly(ethylene glycol)s (mPEGs) and poly(mPEG) methacrylate prepared by atom transfer radical polymerization. Judicious choice of the reaction conditions-pH, stoichiometry, and chemical structure of diazonium salt-led to a high degree of site-specificity in the conjugation reaction, even in the presence of competitive peptide amino acid targets such as histidine, lysines, and N-terminal amine. In vitro studies showed that conjugation of mPEG(2000) to sCT did not affect the peptide's ability to increase intracellular cAMP induced in T47D human breast cancer cells bearing sCT receptors. Preliminary in vivo investigation showed preserved ability to reduce [Ca(2+)] plasma levels by mPEG(2000)-sCT conjugate in rat animal models.
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http://dx.doi.org/10.1021/ja211855qDOI Listing
May 2012

Relationship between the affinity of PEO-PPO-PEO block copolymers for biological membranes and their cellular effects.

Pharm Res 2012 Jul 3;29(7):1908-18. Epub 2012 Mar 3.

Division of Drug Delivery and Tissue Engineering, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

Purpose: The interactions of poly(ethylene oxide)-co-poly(propylene oxide) tri-block copolymers (PEO-PPO-PEO block copolymers, Pluronics®, Synperonics®, Poloxamers) of differing chemical composition with cell membranes were systematically investigated in order to clarify the mechanisms behind their previously reported various cellular responses.

Methods: Relationships between the structural components of a defined series of PEO-PPO-PEO block copolymers and i) their interactions with biological membranes; ii) their cytotoxic potential were probed using a combination of haemolysis studies and cytotoxicity assays in the Caco-2 and HMEC-1 cell lines.

Results: The length of the PPO block as well as the PEO/PPO ratio were determinants of their membrane binding constant and cytotoxicity endpoints measured in the MTS and LDH assays. Similar 2D parabolic relationships were found between polymer composition and their affinity for membranes or their cytotoxicity potential. Cytotoxicity was related to the ability of the copolymers to form ion transversable pores within the cell membrane.

Conclusions: The data suggest a link between the affinity of certain Pluronics for biological membranes and their cellular adverse effects. This first cell-based investigation of the interactions of Pluronics with biological membranes is an important step towards unravelling the complex mechanisms which govern the biological effects of widely used amphiphilic materials.
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http://dx.doi.org/10.1007/s11095-012-0716-6DOI Listing
July 2012

Polymer control of ligand display on gold nanoparticles for multimodal switchable cell targeting.

Chem Commun (Camb) 2011 Sep 2;47(35):9846-8. Epub 2011 Aug 2.

Department of Pharmaceutical Sciences, University of Padua, Padova, Italy.

The function of cell-specific ligands on gold nanoparticles can be selectively gated by the action of co-grafted thermosensitive polymers. Below the LCST the responsive chain-extended polymers prevent cell-surface receptors from accessing the affinity ligands while above the LCST, the polymers collapse exposing the ligands and allowing binding to receptors, which in turn promotes cell internalisation.
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http://dx.doi.org/10.1039/c1cc12654gDOI Listing
September 2011

Effect of PEGylation on the toxicity and permeability enhancement of chitosan.

Biomacromolecules 2010 Nov 27;11(11):2854-65. Epub 2010 Sep 27.

Drug Delivery and Tissue Engineering Division, School of Pharmacy, and School of Chemistry, University of Nottingham, Nottingham, NG7 2RD, United Kingdom and Critical Pharmaceuticals Limited, BioCity, Pennyfoot Street, Nottingham, NG1 1GF, United Kingdom.

The aim of the present work is to investigate if conditions can be devised where PEGylation of chitosan would reduce its toxicity toward the nasal mucosa while maintaining its ability to open the cellular tight junctions and, consequently, produce an enhancement of macromolecular permeability. A series of mPEG-g-chitosan copolymers with varying levels of mPEG substitution, mPEG molecular weight, and chitosan molecular weight were synthesized by grafting carboxylic acid-terminated mPEGs (Mw 1.9 and 5.0 × 10(3) g mol(-1)) to chitosans (Mw 28.9 and 82.0 × 10(3) g mol(-1)) using a NHS/EDC coupling system. The synthesized mPEG-g-chitosans were fully characterized using a number of techniques, including FT-IR, (1)H NMR, and SEC-MALLS and their physicochemical properties were analyzed by TGA and DSC. Thereafter, the conjugates were tested for their cytotoxicity and tight junction modulating property in a relevant cell model, a mucus producing Calu-3 monolayer. mPEG-g-chitosan conjugates exhibited reduced toxicity toward cells, as compared to unmodified chitosan counterparts. Furthermore, the conjugates demonstrated a dramatic effect on cell monolayer transepithelial electrical resistance (TEER) and enhancement of permeability of model macromolecules. TEER and permeability-enhancing effects, as measurable indicators of tight junction modulation, were found to be pH-dependent and were notably more pronounced than those exhibited by unmodified chitosans. This work therefore demonstrates that conditions can be contrived where PEGylation improves the toxicity profile of chitosan, while preserving its effect on epithelial tight junctions in the nose.
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http://dx.doi.org/10.1021/bm100522cDOI Listing
November 2010

Rationale and design of the OPTION study: optimal antitachycardia therapy in ICD patients without pacing indications.

Pacing Clin Electrophysiol 2010 Sep;33(9):1141-8

Deutsches Herzzentrum und 1, Faculty of Medicine, Technische Universität München, Munich, Germany.

Background: Implantable cardioverter-defibrillators (ICDs) represent the treatment of choice for primary and secondary prevention of sudden cardiac death but ICD therapy is also plagued by inappropriate shocks due to supraventricular tachyarrhythmias. Dual-chamber (DC) ICDs are considered to exhibit an enhanced discrimination performance in comparison to single-chamber (SC) ICDs, which results in reduction of inappropriate detections in a short- to mid-term follow-up. Comparative data on long-term follow-up and especially on inappropriate shocks are limited.

Methods: The aim of the OPTION study is to assess whether an optimized treatment with DC ICDs improves patient outcome and decreases the rate of inappropriate shocks in comparison to SC ICDs. DC ICD therapy optimization is achieved by optimal customizing of antitachycardia therapy parameters, activation of discrimination algorithms, antitachycardia pacing in the slow ventricular tachycardia zone, and avoidance of right ventricular pacing with the SafeR algorithm mode. The OPTION study, a prospective, multicenter, randomized, single-blinded, parallel study, will randomize 450 patients on a 1:1 allocation to either an SC arm with backup pacing at VVI 40 beats per minute (bpm) or to the DC arm with SafeR pacing at 60 bpm. Patients will be followed for 27 months. Primary outcome measure is the time to first occurrence of inappropriate shock and a combined endpoint of cardiovascular morbidity and all-cause mortality.

Conclusion: The study will evaluate the relative performance of DC in comparison to SC ICDs in terms of inappropriate shock reduction and patient outcome.
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http://dx.doi.org/10.1111/j.1540-8159.2010.02790.xDOI Listing
September 2010

Antibacterial effects of poly(2-(dimethylamino ethyl)methacrylate) against selected gram-positive and gram-negative bacteria.

Biomacromolecules 2010 Feb;11(2):443-53

UCD Conway Institute, University College Dublin, Ireland.

Antimicrobial coatings can reduce the occurrence of medical device-related bacterial infections. Poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) is one such polymer that is being researched in this regard. The aims of this study were to (1) elucidate pDMAEMA's antimicrobial activity against a range of Gram-positive and Gram-negative bacteria and (2) to investigate its antimicrobial mode of action. The methods used include determination of minimum inhibitory concentration (MIC) values against various bacteria and the effect of pH and temperature on antimicrobial activity. The ability of pDMAEMA to permeabilise bacterial membranes was determined using the dyes 1-N-phenyl-naphthylamine and calcein-AM. Flow cytometry was used to investigate pDMAEMA's capacity to be internalized by bacteria and to determine effects on bacterial cell cycling. pDMAEMA was bacteriostatic against Gram-negative bacteria with MIC values between 0.1-1 mg/mL. MIC values against Gram-positive bacteria were variable. pDMAEMA was active against Gram-positive bacteria around its pK(a) and at lower pH values, while it was active against Gram-negative bacteria around its pK(a) and at higher pH values. pDMAEMA inhibited bacterial growth by binding to the outside of the bacteria, permeabilizing the outer membrane and disrupting the cytoplasmic membrane. By incorporating pDMAEMA with erythromycin, it was found that the efficacy of the latter was increased against Gram-negative bacteria. Together, the results illustrate that pDMAEMA acts in a similar fashion to other cationic biocides.
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http://dx.doi.org/10.1021/bm901166yDOI Listing
February 2010

Phosphine-mediated one-pot thiol-ene "click" approach to polymer-protein conjugates.

Chem Commun (Camb) 2009 Sep 21(35):5272-4. Epub 2009 Jul 21.

Department of Chemistry, University of Warwick, Coventry, UKCV4 7AL.

We employ water-soluble organic phosphines as key reagents in a one-pot synthetic protocol where a (poly)peptide disulfide bridge is first reduced followed by subsequent reaction of the two thiols in situ with poly(monomethoxy ethylene glycol)(meth)acrylates (p(mPEG)(M)A); we use salmon calcitonin (sCT) as a disulfide bridge-containing peptide, which contains a disulfide bridge-Cys1-Cys7-that can be reduced to give two sulfhydryl units available for thiol functionalisation; bioactivity is retained.
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http://dx.doi.org/10.1039/b906865aDOI Listing
September 2009

Sonographic features of acute colonic diverticulitis.

J Clin Ultrasound 2009 Oct;37(8):457-63

Urgency/Emergency, General Surgery and Transplantation Department, Radiology Unit, S. Orsola-Malpighi, University Hospital, Bologna, Italy.

Acute colonic diverticulitis is a common cause of acute abdominal symptoms, especially in elderly patients. Sonography is frequently used as the initial imaging modality because of its ready availability. This pictorial essay aims to provide an overview of the sonographic features of acute colonic diverticulitis and of the more common differential diagnosis.
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http://dx.doi.org/10.1002/jcu.20619DOI Listing
October 2009

Dexamethasone-pDMAEMA polymeric conjugates reduce inflammatory biomarkers in human intestinal epithelial monolayers.

J Control Release 2009 Apr 6;135(1):35-43. Epub 2008 Dec 6.

School of Agriculture, Food Science and Veterinary Medicine, and UCD Conway Institute, University College Dublin, Dublin 4, Ireland.

The mucoadhesive polymer, poly(dimethylamino)ethyl methacrylate, (pDMAEMA), was synthesised by living radical polymerisation and subsequently conjugated by quaternisation reaction to a functionalised anti-inflammatory corticosteroid dexamethasone, to separately yield two conjugates with either 9:1 or 18:1 molar ratios of dexamethasone:polymer respectively. The hypothesis was to test whether the active agent maintained in vitro bioactivity when exposed to the apical side of human intestinal epithelial monolayers, Caco-2 and mucus-covered HT29-MTX-E12 (E12). HPLC analysis indicated high conjugate purity. Similar to pDMAEMA, fluorescently-labelled dexamethasone-pDMAEMA conjugates were bioadhesive to Caco-2 and mucoadhesive to E12. Apical addition of conjugates suppressed mRNA expression of the inflammatory markers, NURR1 and ICAM-1 in E12 following stimulation by PGE(2) and TNF-alpha, respectively. Conjugates also suppressed TNF-alpha stimulated cytokine secretion to the basolateral side of Caco-2 monolayers. Measurement of dexamethasone permeability from conjugates across monolayers suggested that conjugation reduced permeability compared to free dexamethasone. LDH assay indicated that conjugates were not cytotoxic to monolayers. Anti-inflammatory agents can therefore be successfully conjugated to polymers and they retain adhesion and bioactivity and have potential to be formulated for topical administration.
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http://dx.doi.org/10.1016/j.jconrel.2008.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047761PMC
April 2009

Advances in PEGylation of important biotech molecules: delivery aspects.

Expert Opin Drug Deliv 2008 Apr;5(4):371-83

University College Dublin, School of Agriculture, Food Science and Veterinary Medicine, Veterinary Sciences Building, Room 214, Belfield, Dublin 4, Ireland.

Background: Although various injected peptide and protein therapeutics have been developed successfully over the past 25 years, several pharmacokinetic and immunological challenges are still encountered that can limit the efficacy of both novel and established biotech molecules.

Objective And Method: PEGylation is a popular technique to address such properties. PEGylated drugs exhibit prolonged half-life, higher stability, water solubility, lower immunogenicity and antigenicity, as well as potential for specific cell targeting. Although PEGylated drug conjugates have been on the market for many years, the technology has steadily developed in respect of site-specific chemistry, chain length, molecular weights and purity of conjugate. These developments have occurred in parallel to improvements in physicochemical methods of characterization.

Conclusion: This review will discuss recent achievements in PEGylation processes with an emphasis on novel PEG-drugs constructs, the unrealized potential of PEGylation for non-injected routes of delivery, and also on PEGylated versions of polymeric nanoparticles, including dendrimers and liposomes.
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http://dx.doi.org/10.1517/17425247.5.4.371DOI Listing
April 2008

Simultaneous copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and living radical polymerization.

Angew Chem Int Ed Engl 2008 ;47(22):4180-3

Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.

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http://dx.doi.org/10.1002/anie.200800179DOI Listing
July 2008

Tunable thermoresponsive water-dispersed multiwalled carbon nanotubes.

Chem Commun (Camb) 2008 Mar 1(9):1097-9. Epub 2008 Feb 1.

Department of Chemistry, The University of Warwick, Coventry, UK.

Polymers containing poly(ethylene glycol) methacrylate and 2-(2-methoxyethoxy)ethyl methacrylate have been synthesized by Cu(0)-mediated radical polymerisation for use as thermoresponsive water-dispersants for carbon nanotubes.
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http://dx.doi.org/10.1039/b718112dDOI Listing
March 2008