Malpratice claims in cardiology and cardiac surgery: A medico-legal issue.

Medical liability has become a challenge in every physician's modern practice with the consequent loss of the physician's autonomy and an increase in "defensive medicine". From this perspective, the role of Legal Medicine in assessing medical liability has become increasingly specific and a homogenization of the methods of ascertainment is increasingly necessary, since such a process can contribute to strengthening the guarantees in professional liability procedures. Focusing on malpractice claims in the field of cardiology, the complexity of the management of cardiac pathologies and the frequency of severe adverse events implies the importance of a multi-disciplinary approach, together with the application of a shared ascertainment methodology. In particular, it is essential for the forensic pathologist to collaborate with experts in cardio-pathology, cardiology and/or cardiac surgery in cases of alleged medical liability in the cardiologic field and to follow the guidelines which have been produced to assist the expert dealing with deaths reflecting cardiac disease, in order to prevent criticism of case analysis in medico-legal environments and to promote the standardization of the structure of the juridical-legislative medical malpractice lawsuits.

Joint Measurement of the

The PROSPECT and STEREO collaborations present a combined measurement of the pure ^{235}U antineutrino spectrum, without site specific corrections or detector-dependent effects. The spectral measurements of the two highest precision experiments at research reactors are found to be compatible with χ^{2}/ndf=24.1/21, allowing a joint unfolding of the prompt energy measurements into antineutrino energy. This ν[over ¯]_{e} energy spectrum is provided to the community, and an excess of events relative to the Huber model is found in the 5-6 MeV region. When a Gaussian bump is fitted to the excess, the data-model χ^{2} value is improved, corresponding to a 2.4σ significance.

Searching for Hidden Neutrons with a Reactor Neutrino Experiment: Constraints from the STEREO Experiment.

Different extensions of the standard model of particle physics, such as braneworld or mirror matter models, predict the existence of a neutron sterile state, possibly as a dark matter candidate. This Letter reports a new experimental constraint on the probability p for neutron conversion into a hidden neutron, set by the STEREO experiment at the high flux reactor of the Institut Laue-Langevin. The limit is p<3.1×10^{-11} at 95% C.L. improving the previous limit by a factor of 13. This result demonstrates that short-baseline neutrino experiments can be used as competitive passing-through-walls neutron experiments to search for hidden neutrons.

Accurate Measurement of the Electron Antineutrino Yield of

We report a measurement of the antineutrino rate from the fission of ^{235}U with the STEREO detector using 119 days of reactor turned on. In our analysis, we perform several detailed corrections and achieve the most precise single measurement at reactors with highly enriched ^{235}U fuel. We measure an IBD cross section per fission of σ_{f}=(6.34±0.06[stat]±0.15[sys]±0.15[model])×10^{-43} cm^{2}/fission and observe a rate deficit of (5.2±0.8[stat]±2.3[sys]±2.3[model])% compared to the model, consistent with the deficit of the world average. Testing ^{235}U as the sole source of the deficit, we find a tension between the results of lowly and highly enriched ^{235}U fuel of 2.1 standard deviations.

Electrospun Polyhydroxyethyl-Aspartamide-Polylactic Acid Scaffold for Biliary Duct Repair: A Preliminary In Vivo Evaluation.

Tissue engineering has emerged as a new approach with the potential to overcome the limitations of traditional therapies. The objective of this study was to test whether our polymeric scaffold is able to resist the corrosive action of bile and to support a cell's infiltration and neoangiogenesis with the aim of using it as a biodegradable tissue substitute for serious bile duct injuries. In particular, a resorbable electrospun polyhydroxyethyl-aspartamide-polylactic acid (90 mol% PHEA, 10 mol% PLA)/polycaprolactone (50:50 w/w) plate scaffold was implanted into rabbit gallbladder to assess the in vivo effects of the lytic action of the bile on the scaffold structure and then as a tubular scaffold to create a biliary-digestive anastomosis as well. For the above evaluation, 5 animals were used and killed after 15 days and 5 animals after 3 months. At 15-day and 3-month follow-ups, the fibrillar structure was not digested by lytic action bile. The fibers of the scaffold were organized despite being in contact with bile action. A new epithelial tissue appeared on the scaffold surface suggesting the suitability of this scaffold for future studies of the repair of biliary tract injuries with the use of resorbable copolymer on biliary injuries.

Electrospun PHEA-PLA/PCL Scaffold for Vascular Regeneration: A Preliminary in Vivo Evaluation.

BACKGROUND: There is increasing interest in the development of vessel substitutes, and many studies are currently focusing on the development of biodegradable scaffolds capable of fostering vascular regeneration. We tested a new biocompatible and biodegradable material with mechanical properties similar to those of blood vessels. METHODS: The material used comprises a mixture of α,ß-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) and polylactic acid (PLA), combined with polycaprolactone (PCL) by means of electrospinning technique. Low-molecular-weight heparin was also linked to the copolymer. A tubular PHEA-PLA/PCL sample was used to create an arteriovenous fistula in a pig model with the use of the external iliac vessels. The flow was assessed by means of Doppler ultrasound examination weekly, and 1 month after the implantation we removed the scaffold for histopathologic evaluation. RESULTS: The implants showed a perfect leak-proof seal and adequate elastic tension to blood pressure. About ∼3 weeks after the implantation, Doppler examination revealed thrombosis of the graft, so we proceeded to its removal. Histologic examination showed chronic inflammation, with the presence of foreign body cells and marked neovascularization. The material had been largely absorbed, leaving some isolated spot residues. CONCLUSIONS: The biocompatibility of PHEA-PLA/PCL and its physical properties make it suitable for the replacement of vessels. In the future, the possibility of functionalizing the material with a variety of molecules, to modulate the inflammatory and coagulative responses, will allow obtaining devices suitable for the replacement of native vessels.

Smart Inulin-Based Polycationic Nanodevices for siRNA Delivery.

The advances of short interfering RNA (siRNA) mediated therapy provide a powerful option for the treatment of many diseases by silencing the expression of targeted genes including cancer development and progression. Inulin is a very simple and biocompatible polysaccharide proposed by our groups to produce interesting delivery systems for Nucleic Acid Based Drugs (NABDs), such as siRNA, either as polycations able to give polyplexes and polymeric coatings for nanosystems having a metallic core. In this research field, different functionalizing groups were linked to the inulin backbone with specific aims including oligoamine such as Ethylendiammine (EDA), Diethylediamine (DETA), Spermine, (SPM) etc. In this contribution the main Inulin-based nanodevices for the delivery of siRNA have been reported, analysed and compared with particular reference to their chemical design and structure, biocompatibility, siRNA complexing ability, silencing ability.

Development of a simple, biocompatible and cost-effective Inulin-Diethylenetriamine based siRNA delivery system.

Small interfering RNAs (siRNAs) have the potential to be of therapeutic value for many human diseases. So far, however, a serious obstacle to their therapeutic use is represented by the absence of appropriate delivery systems able to protect them from degradation and to allow an efficient cellular uptake. In this work we developed a siRNA delivery system based on inulin (Inu), an abundant and natural polysaccharide. Inu was functionalized via the conjugation with diethylenetriamine (DETA) residues to form the complex Inu-DETA. We studied the size, surface charge and the shape of the Inu-DETA/siRNA complexes; additionally, the cytotoxicity, the silencing efficacy and the cell uptake-mechanisms were studied in the human bronchial epithelial cells (16HBE) and in the hepatocellular carcinoma derived cells (JHH6). The results presented here indicate that Inu-DETA copolymers can effectively bind siRNAs, are highly cytocompatible and, in JHH6, can effectively deliver functional siRNAs. Optimal delivery is observed using a weight ratio Inu-DETA/siRNA of 4 that corresponds to polyplexes with an average size of 600nm and a slightly negative surface charge. Moreover, the uptake and trafficking mechanisms, mainly based on micropinocytosis and clatrin mediated endocytosis, allow the homogeneous diffusion of siRNA within the cytoplasm of JHH6. Notably, in 16 HBE where the trafficking mechanism (caveolae mediated endocytosis) does not allow an even distribution of siRNA within the cell cytoplasm, no significant siRNA activity is observed. In conclusion, we developed a novel inulin-based siRNA delivery system able to efficiently release siRNA in JHH6 with negligible cytotoxicity thus opening the way for further testing in more complex in vivo models.

Bisphosphonate-polyaspartamide conjugates as bone targeted drug delivery systems.

Poly-hydroxy-aspartamide was used as a backbone to synthesize bisphosphonate derivatives thus achieving macromolecular carriers to be potentially used as targeting agents for bone drug delivery. Molecules bearing bisphosphonate groups, such as aminobisphosphonate (ABP) and neridronate (NRD), have been conjugated to polyaspartamide (α,ß-poly(N-2-hydroxyethyl)-dl-aspartamide, PHEA), with or without a spacer (succinic acid or 6-aminocaproic acid) thus obtaining PHEA-succinate-ABP and PHEA-caproylcarbamate-ABP and PHEA-ABP and PHEA-NRD, respectively. Bisphosphonate-polymer conjugates were physico-chemically characterized using size exclusion chromatography and 1H-NMR; and their in vitro and ex vivo affinity for bone tissue has been further tested using the hydroxylapatite and rabbit bone binding assays, respectively. In vivo studies were carried out using rats to evaluate the biodistribution features of bisphosphonate-polymer conjugates in comparison with the starting PHEA. In vivo findings evidenced a suitable selectivity of bisphosphonate-polymer conjugates toward the bone tissues also as a function of time.

Inulin-based polymer coated SPIONs as potential drug delivery systems for targeted cancer therapy.

This paper deal with the synthesis and characterization of PEGylated squalene-grafted-inulin amphiphile capable of self-assembling and self-organizing into nanocarriers once placed in aqueous media. It was exploited as coating agent for obtaining doxorubicin loaded superparamagnetic iron oxide nanoparticles (SPIONs) endowed with stealth like behavior and excellent physicochemical stability. Inulin was firstly modified in the side chain with primary amine groups, followed in turn by conjugation with squalenoyl derivatives through common amidic coupling agents and PEGylation by imine linkage. Polymer coated SPIONs were so obtained by spontaneous self-assembling of inulin copolymer onto magnetite surface involving hydrophobic-hydrophobic interactions between the metallic core and the squalene moieties. The system was characterized in terms of hydrodynamic radius, zeta potential, shape and drug loading capacity. On the whole, the stealth-like shell stabilized the suspension in aqueous media, though allowing the release of the doxorubicin loaded in therapeutic range. The cytotoxicity profile on cancer (HCT116) cell line and in vitro drug uptake were evaluated both with and without an external magnetic field used as targeting agent and uptake promoter, displaying that magnetic targeting implies advantageous therapeutic effects, that is amplified drug uptake and increased anticancer activity throughout the tumor mass.

Synthesis and characterization of polyaspartamide copolymers obtained by ATRP for nucleic acid delivery.

Nucleic acid molecules such as small interfering RNAs (siRNAs) and plasmidic DNAs (pDNAs) have been shown to have the potential to be of therapeutic value in different human diseases. Their practical use is however compromised by the lack of appropriate release systems. Delivered as naked molecules, siRNAs/pDNAs are rapidly degraded by extracellular nucleases thus considerably reducing the amount of molecule which can reach the target cells. Additionally, the anionic charge of the phosphate groups present on the siRNAs/pDNAs backbone, disfavors the interaction with the negatively charged surface of the cell membrane. In this paper we describe the generation of a novel polymer able to deliver both siRNAs and pDNAs. The combined release of these molecules is used in many different experimental settings such as the evaluation of the silencing efficiency of a given siRNA targeted against a given RNA, encoded by the pDNA. The possibility to use the same delivery system is very convenient from the technical point of view and it allows minimizing possible artifacts introduced by the use of different delivery agents for siRNAs and pDNA. The copolymer described here is based on α,ß-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) bearing positively chargeable side oligochains, with diethylamino ethyl methacrylate (DEAEMA) as monomer. Monomer polymerization has been obtained by atom transfer radical polymerization (ATRP), a technique which allows the precise polymerization of the monomer. In addition to the chemical-physical characterization of the polymer, we provide evidences of the polymer ability to delivery both siRNAs and pDNA to cultured cells. Whereas additional investigations are necessary to study the delivery mechanisms of this polyplex, the polymer generated represents a novel and convenient device for the delivery of both siRNAs and pDNA.

Amphiphilic inulin graft co-polymers as self-assembling micelles for doxorubicin delivery.

This paper reports the synthesis and characterization of a new amphiphilic inulin graft copolymer able to self-assemble in water into a micelle type structure and to deliver the anticancer model drug doxorubicin. For this aim, inulin was chemically modified in the side chain with primary amine groups (INU-EDA) and these were used as reactive moieties for the conjugation of poly ethylene glycol 2000 and succinyl-ceramide. The CMC of obtained amphiphilic inulin derivatives (INU-ceramide and INU-ceramide-PEG2000) was measured by means of fluorescence analysis using pyrene as the fluorescent probe. The obtained micelles were characterized by DLS and AFM analysis and the ability to release the loaded doxorubicin was studied in different media. Finally the cytotoxicity profile on both cancer (HCT116) and normal (16 HBE) cell lines and in vitro ability to deliver the drug into cancer cells were evaluated.

Amphiphilic polyaspartamide copolymer-based micelles for rivastigmine delivery to neuronal cells.

A novel polysorbate-80 (PS(80))-attached amphiphilic copolymer comprising a hydrophilic α,ß-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) backbone and hydrophobic squalenyl-C(17) (Sq(17)) portions was synthesized and characterized; the formation of polymeric micelles was also evaluated. Rivastigmine free-base (Riv), a hydrophobic drug employed to treat Alzheimer's disease, was chosen as model drug to investigate micelle's ability to incorporate hydrophobic molecules and target them to neuronal cells. Micelle formation was studied through analyses including fluorescence spectroscopy and 2D (1)H-NMR NOESY experiments. Finally, the capacity of Riv-loaded micelles, versus free drug, to penetrate mouse neuroblastoma cells (Neuro2a) was evaluated. 2D (1)H-NMR NOESY experiments demonstrated that the PHEA-EDA-Sq(17)-PS(80) copolymer self-assembles into micelle structures in water, with a micelle core formed by hydrophobic interaction between Sq(17) alkyl chains. Fluorescence probe studies revealed the CAC of PHEA-EDA-Sq(17)-PS(80) micelles, which was 0.25 mg mL(-1). The micelles obtained had a nanometric hydrodynamic diameter with narrow size distribution and negative surface charge. The PHEA-EDA-Sq(17)-PS(80) micelles incorporated a large amount of Riv, and the system maintained the stability of Riv after incubation in human plasma. An in vitro biological assay evidenced no cytotoxic effects of either empty or loaded micelles on the neuronal cell lines tested. Moreover, the micelles are internalized by neuroblastoma cell lines with drug uptake depending on the micelles concentration.

New pegylated polyaspartamide-based polyplexes as gene delivery vectors.

AIMS: To synthesize novel polyhydroxyethylaspartamide (PHEA) copolymers containing spermine (Spm) and polyethylene glycol (PEG) moieties in high yields, with the expectation that this material would show stealth properties and the ability to complex DNA by electrostatic interactions. MATERIALS & METHODS: PHEA-PEG-Spm copolymer was prepared with a two-step reaction. Chemical, physicochemical and biological characterizations of PHEA-PEG-Spm copolymers and their obtained polyplexes with pDNA were performed. RESULTS: The introduction of spermine in PHEA structure allows to obtain a copolymer bearing in the side chains polyamine moieties capable to interact with DNA. On the other hand, the introduction of PEG in polymeric structure increased the DNA condensing ability of PHEA-PEG-Spm copolymer in comparison with the derivatives without PEG (PHEA-Spm), and improved its characteristics of biocompatibility. CONCLUSIONS: PHEA-PEG-Spm copolymer shows excellent ability to complex and condense plasmidic DNA giving interpolyelectrolyte complexes to act as gene delivery systems. Moreover, PEGylation confers to the obtained interpolyelectrolyte complexes stealth properties.

Polysaccharide/polyaminoacid composite scaffolds for modified DNA release.

In this work composite polymeric films or sponges, based on hyaluronic acid (HA) covalently crosslinked with alpha,beta-poly(N-2-hydroxyethyl)(2-aminoethylcarbamate)-D,L-aspartamide (PE), have been prepared and characterized as local gene delivery systems. In particular, HA/PE scaffolds have been loaded with PE/DNA interpolyelectrolyte complexes, employing PE as a macromolecular crosslinker for HA and as a non-viral vector for DNA. In vitro studies showed that HA/PE films and sponges have high compatibility with human dermal fibroblasts and they give a sustained DNA release, whose trend can be easily tailored by varying the crosslinking ratio between HA and PE. Electrophoresis analysis and transfection studies on B16-F10 cells revealed that DNA is released as a complex with PE and it retains its bioactivity.

Polyhydroxyethylaspartamide-based micelles for ocular drug delivery.

In this paper three copolymers of polyhydroxyethylaspartamide (PHEA), bearing in the side chains polyethylene glycol (PEG) and/or hexadecylamine (C(16)) (PHEA-PEG, PHEA-PEG-C(16) and PHEA-C(16) respectively) have been studied as potential colloidal drug carriers for ocular drug delivery. The physical characterization of all three PHEA derivatives, using the Langmuir trough (LT) and micellar affinity capillary electrophoresis (MACE) techniques allowed to assume that whereas alone PHEA backbone is an inert polymer with respect to the interactions with lipid membranes and drug complexation, when PHEA chains are grafted with long alkyl chains like C(16) or in combination C(16) chains and hydrophilic chains like PEG, copolymers with lipid membrane interaction ability and drug complexation capability are obtained. In vitro permeability studies performed on primary cultured rabbit conjunctival and corneal epithelia cells, using PHEA-C(16) and PHEA-PEG-C(16) as micelle carriers for netilmicin sulphate, dexamethasone alcohol and dexamethasone phosphate, demonstrated that in all cases drug loaded PHEA-C(16) and PHEA-PEG-C(16) micelles provide a drug permeation across ocular epithelia greater than simple drug solutions or suspensions. In particular PHEA-PEG-C(16) acts as the best permeability enhancer in our experimental model. In vivo bioavailability studies conducted with PHEA-PEG-C(16) micelles loaded with dexamethasone alcohol, confirmed that this system also provides a drug bioavailability greater in comparison with that obtained with water suspension of the same drug after ocular administration to rabbits.

Microwave-assisted synthesis of PHEA-oligoamine copolymers as potential gene delivery systems.

AIMS: To prepare new copolymers, useful for gene delivery, based on alpha, beta-poly-(N-2-hydroxyethyl)-D, L-aspartamide (PHEA) as a polymeric backbone and bearing an oligoamine such as diethylenetriamine in the side chain. Moreover, in order to reduce solvent volume and make the reaction faster, microwave-assisted heating was used. MATERIALS & METHODS: PHEA copolymers bearing different amounts of diethylenetriamine were prepared using bis(4-nitrophenyl) carbonate as a condensing agent with the use of microwaves. Chemical, physico-chemical and biological characterization of PHEA-diethylenetriamine copolymers and their complexes obtained with DNA were performed. RESULTS: Copolymers showed good DNA complexing and condensing abilities depending on the oligoamine derivatization degree and good hemocompatibility. Moreover, plasmid DNA/copolymer polyplexes showed very good cytocompatibility on B16F10 and N2A cell lines. CONCLUSION: Results support the use of these copolymers as gene delivery systems in the future. Finally, the use of microwaves makes the proposed synthetic method advantageous as time and solvents are saved.

Polyhydroxyethylaspartamide-spermine copolymers: efficient vectors for gene delivery.

Aim of this paper was that to prepare biocompatible, polyaspartamide based copolymers containing spermine or spermine/hydrophobic side chains able to condense nucleic acids and to transfect mammalian cells. Copolymers were prepared starting from alpha,beta-poly-(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) and exploiting the reactive hydroxyl groups in the polymeric side chains by subsequent activation reactions to obtain PHEA-Spermine (PHEA-Spm) and PHEA-Spermine-Butyramide (PHEA-Spm-C(4)). Molecular, physico-chemical and biological characterization of copolymers and interpolyelectrolyte complexes with plasmid DNA was performed. Experimental results evidenced that these copolymers are able to form complexes with plasmid DNA already at low polycation/DNA weight ratio ranging from 0.75/1 to 2/1. Interpolyelectrolyte complexes with decreased size were obtained when increasing the polycation/DNA weight ratio, until nanosized dimensions were reached. Copolymers as well as complexes were not haemolytic and non toxic in vitro. In vitro cell transfection with PHEA derivatives showed good biocompatibility and high transfection efficiency (luciferase) in cancer cells in comparison with commercially available, but toxic transfection agents.

Polyaspartylhydrazide copolymer-based supramolecular vesicular aggregates as delivery devices for anticancer drugs.

In this paper we report on three different hydrophilic copolymers based on alpha,beta-polyaspartylhydrazide (PAHy) bearing butyric groups in the side chain (C 4) (PAHy-C 4) or a combination of butyric groups and positive charged residues ((carboxypropyl)trimethylammonium chloride, CPTACl) (PAHy-C 4-CPTA) that were synthesized and used for the preparation of new supramolecular vesicular aggregates (SVAs) containing gemcitabine as an antitumor drug. Gemcitabine-loaded SVAs containing synthesized PAHy derivatives were characterized from the physicochemical and technological point of view and the in vitro toxicity and anticancer activity on two different human cancer cell lines, i.e., CaCo-2 (human colon carcinoma) and ARO (human anaplastic thyroid carcinoma) cells, were also evaluated. Moreover, considering that carrier-cell interaction is an important factor to achieve an improvement of anticancer drug activity, confocal laser scanning microscopy and flow cytometric experiments were carried out on the two different cancer cell lines.

Novel cationic copolymers of a polyasparthylhydrazide: synthesis and characterization.

Alpha,beta-poly(asparthylhydrazide) (PAHy), a water soluble synthetic polymer, was functionalized by using EDCI chemistry with 3-(carboxypropyl)trimethyl-ammonium chloride (CPTACl) obtaining carboxypropyltrimethyl ammonium copolymers (PAHy-CPTA). Three PAHy-CPTA copolymers at increasing derivatization degrees (38%, 48%, 58%) were chosen for subsequent investigations. The capability of these copolymers to bind, neutralize, and protect DNA against degradation by DNase II was evalued by gel retardation assay and DNA degradation test at pH 5.5. Zeta potential measurements show that all studied polymers are able to neutralize the anionic charge of DNA at polymer/DNA weight ratio in the range of 0.8/1-5/1. Polyplex dimensional distribution analyses in bistilled water, saline solution NaCl 0.9%, and HEPES pH 7 show that polyplex size is strongly affected by both presence and type of electrolyte and with time incubation.