Amphiphilic Pentablock Copolymers Prepared from Pluronic and ε-Caprolactone by Enzymatic Ring Opening Polymerization.

Amphiphilic copolymers are appealing materials because of their interesting architecture and tunable properties. In view of their application in the biomedical field, the preparation of these materials should avoid the use of toxic compounds as catalysts. Therefore, enzymatic catalysis is a suitable alternative to common synthetic routes. Pentablock copolymers (CUC) were synthesized with high yields by ring-opening polymerization of ε-caprolactone (ε-CL) initiated by Pluronic (EPE) and catalyzed by Candida antarctica lipase B enzyme. The variables to study the structure-property relationship were EPEs' molecular weight and molar ratios between ε-CL monomer and EPE macro-initiator (M/In). The obtained copolymers were chemically characterized, the molecular weight determined, and morphologies evaluated. The results suggest an interaction between the reaction time and M/In variables. There was a correlation between the differential scanning calorimetry data with those of X-ray diffraction (WAXD). The length of the central block of CUC copolymers may have an important role in the crystal formation. WAXD analyses indicated that a micro-phase separation takes place in all the prepared copolymers. Preliminary cytotoxicity experiments on the extracts of the polymer confirmed that these materials are nontoxic.

Mass Spectrometry Reveals Molecular Structure of Polyhydroxyalkanoates Attained by Bioconversion of Oxidized Polypropylene Waste Fragments.

This study investigated the molecular structure of the polyhydroxyalkanoate (PHA) produced via a microbiological shake flask experiment utilizing oxidized polypropylene (PP) waste as an additional carbon source. The bacterial strain Cupriavidus necator H16 was selected as it is non-pathogenic, genetically stable, robust, and one of the best known producers of PHA. Making use of PHA oligomers, formed by controlled moderate-temperature degradation induced by carboxylate moieties, by examination of both the parent and fragmentation ions, the ESI-MS/MS analysis revealed the 3-hydroxybutyrate and randomly distributed 3-hydroxyvalerate as well as 3-hydroxyhexanoate repeat units. Thus, the bioconversion of PP solid waste to a value-added product such as PHA tert-polymer was demonstrated.

The Microbial Production of Polyhydroxyalkanoates from Waste Polystyrene Fragments Attained Using Oxidative Degradation.

Excessive levels of plastic waste in our oceans and landfills indicate that there is an abundance of potential carbon sources with huge economic value being neglected. These waste plastics, through biological fermentation, could offer alternatives to traditional petrol-based plastics. Polyhydroxyalkanoates (PHAs) are a group of plastics produced by some strains of bacteria that could be part of a new generation of polyester materials that are biodegradable, biocompatible, and, most importantly, non-toxic if discarded. This study introduces the use of prodegraded high impact and general polystyrene (PS0). Polystyrene is commonly used in disposable cutlery, CD cases, trays, and packaging. Despite these applications, some forms of polystyrene PS remain financially and environmentally expensive to send to landfills. The prodegraded PS0 waste plastics used were broken down at varied high temperatures while exposed to ozone. These variables produced PS flakes (PS1⁻3) and a powder (PS4) with individual acid numbers. Consequently, after fermentation, different PHAs and amounts of biomass were produced. The bacterial strain, Cupriavidus necator H16, was selected for this study due to its well-documented genetic profile, stability, robustness, and ability to produce PHAs at relatively low temperatures. The accumulation of PHAs varied from 39% for prodegraded PS0 in nitrogen rich media to 48% (w/w) of dry biomass with the treated PS. The polymers extracted from biomass were analyzed using nuclear magnetic resonance (NMR) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) to assess their molecular structure and properties. In conclusion, the PS0⁻3 specimens were shown to be the most promising carbon sources for PHA biosynthesis; with 3-hydroxybutyrate and up to 12 mol % of 3-hydroxyvalerate and 3-hydroxyhexanoate co-monomeric units generated.

Additive manufacturing of poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] scaffolds for engineered bone development.

A wide range of poly(hydroxyalkanoate)s (PHAs), a class of biodegradable polyesters produced by various bacteria grown under unbalanced conditions, have been proposed for the fabrication of tissue-engineering scaffolds. In this study, the manufacture of poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (or PHBHHx) scaffolds, by means of an additive manufacturing technique based on a computer-controlled wet-spinning system, was investigated. By optimizing the processing parameters, three-dimensional scaffolds with different internal architectures were fabricated, based on a layer-by-layer approach. The resulting scaffolds were characterized by scanning electron microscopy, which showed good control over the fibre alignment and a fully interconnected porous network, with porosity in the range 79-88%, fibre diameter 47-76 µm and pore size 123-789 µm. Moreover, the resulting fibres presented an internal porosity connected to the external fibre surface as a consequence of the phase-inversion process governing the solidification of the polymer solution. Scaffold compressive modulus and yield stress and strain could be varied in a certain range by changing the architectural parameters. Cell-culture experiments employing the MC3T3-E1 murine pre-osteoblast cell line showed good cell proliferation after 21 days of culture. The PHBHHx scaffolds demonstrated promising results in terms of cell differentiation towards an osteoblast phenotype. Copyright © 2014 John Wiley & Sons, Ltd.

Additive manufacturing techniques for the production of tissue engineering constructs.

'Additive manufacturing' (AM) refers to a class of manufacturing processes based on the building of a solid object from three-dimensional (3D) model data by joining materials, usually layer upon layer. Among the vast array of techniques developed for the production of tissue-engineering (TE) scaffolds, AM techniques are gaining great interest for their suitability in achieving complex shapes and microstructures with a high degree of automation, good accuracy and reproducibility. In addition, the possibility of rapidly producing tissue-engineered constructs meeting patient's specific requirements, in terms of tissue defect size and geometry as well as autologous biological features, makes them a powerful way of enhancing clinical routine procedures. This paper gives an extensive overview of different AM techniques classes (i.e. stereolithography, selective laser sintering, 3D printing, melt-extrusion-based techniques, solution/slurry extrusion-based techniques, and tissue and organ printing) employed for the development of tissue-engineered constructs made of different materials (i.e. polymeric, ceramic and composite, alone or in combination with bioactive agents), by highlighting their principles and technological solutions.

Surface decorated poly(ester-ether-urethane)s nanoparticles: a versatile approach towards clinical translation.

Poly(ester-ether-urethane)s copolymers are a resourceful class of biopolymers for the preparation of nanocarriers for drug delivery applications. However, a simple clinical translation for this synthetic material with biological and quality features is still needed. In this view, poly(ε-caprolactone)-co-poly(ethylene glycol) copolymers were synthesized as semi-bulk pilot (Kg) scale under mild conditions in absence of catalyst, bearing functional termini such as fluorescein tag and anticancer targeting moieties. The obtained materials were processed into surface decorated paclitaxel (PTX) loaded nanoparticles (NPs). The NPs were fully characterized in vitro and in vivo biodistribution in healthy mice evidenced no sign of toxicity and lower levels of PTX in lung and spleen, compared to clinically applied PTX dosage form.

Nano/microfibrous polymeric constructs loaded with bioactive agents and designed for tissue engineering applications: a review.

Nano/microfibrous polymeric constructs present various inherent advantages, such as highly porous architecture and high surface to volume ratio, making them attractive for tissue engineering purposes. Electrospinning is the most preferred technique for the fabrication of polymeric nanofibrous assemblies that can mimic the physical functions of native extracellular matrix greatly favoring cells attachment and thus influencing their morphology and activities. Different approaches have been developed to apply polymeric microfiber fabrication techniques (e.g. wet-spinning) for the obtainment of scaffolds with a three-dimensional network of micropores suitable for effective cells migration. Progress in additive manufacturing technology has led to the development of complex scaffold's shapes and microfibrous structures with a high degree of automation, good accuracy and reproducibility. Various loading methods, such as direct blending, coaxial electrospinning and microparticles incorporation, are enabling to develop customized strategies for the biofunctionalization of nano/microfibrous scaffolds with a tailored kinetics of release of different bioactive agents, ranging from small molecules, such as antibiotics, to protein drugs, such as growth factors, and even cells. Recent activities on the combination of different processing techniques and loading methods for the obtainment of biofunctionalized polymeric constructs with a complex multiscale structure open new possibilities for the development of biomimetic scaffolds endowed with a hierarchical architecture and a sophisticated release kinetics of different bioactive agents. This review is aimed at summarizing current advances in technologies and methods for manufacturing nano/microfibrous polymeric constructs suitable as tissue engineering scaffolds, and for their combination with different bioactive agents to promote tissue regeneration and therapeutic effects.

Silk microgels formed by proteolytic enzyme activity.

The proteolytic enzyme α-chymotrypsin selectively cleaves the amorphous regions of silk fibroin protein (SFP) and allows the crystalline regions to self-assemble into silk microgels (SMGs) at physiological temperature. These microgels consist of lamellar crystals in the micrometer scale, in contrast to the nanometer-scaled crystals in native silkworm fibers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zeta potential results demonstrated that α-chymotrypsin utilized only the non-amorphous domains or segments of the heavy chain of SFP to form negatively charged SMGs. The SMGs were characterized in terms of size, charge, structure, morphology, crystallinity, swelling kinetics, water content and thermal properties. The results suggest that the present technique of preparing SMGs by α-chymotrypsin is simple and efficient, and that the prepared SMGs have useful features for studies related to biomaterial and pharmaceutical needs. This process is also an easy way to obtain the amorphous peptide chains for further study.

Extraction of short-chain-length poly-[(R)-hydroxyalkanoates] (scl-PHA) by the "anti-solvent" acetone under elevated temperature and pressure.

A novel method was developed for extraction of short-chain-length poly(hydroxyalkanoates) (scl-PHA) from microbial biomass by the well-known "scl-PHA anti-solvent" acetone at elevated temperature and pressure in a closed system combining components for extraction, filtration, and product work-up. Recovery of scl-PHA using this new approach was compared with established methods using chloroform at ambient pressure. The new method performs similar regarding product purity (98.4 vs. 97.7%) and extraction yield (96.8% by both methods), and is by far faster than established chloroform extraction (20 min vs. 12 h). Separation of the polymer from acetone is simply achieved by cooling down the acetone solution of scl-PHA, thus allows for a nearly quantitative recovery of the solvent that conveniently can be reused. Characterization of scl-PHA extracted by both methods does not reveal any significant difference in terms of molar mass and thermo analytical parameters.

Polyhydroxyalkanoate biosynthesis by Hydrogenophaga pseudoflava DSM1034 from structurally unrelated carbon sources.

In the present paper we report the exclusive microbial preparation of polyhydroxyalkanoates (PHA) containing 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB) as comonomers through the use of unexpensive carbon sources such as whey from dairy industry. Polymers were produced by growing H. pseudoflava DSM 1034 in minimal medium supplemented with sucrose, lactose or whey without any co-substrate added. The chemical and physical properties of the polymers were fully characterized by GPC, DSC, TGA analyses and the composition by GC and (1)H NMR examinations to especially confirm the content of different monomeric units. The presence of 4HB units into PHA samples is particularly aimed in thermoplastic applications where greater flexibility is required and conventional rigid PHAs tend to fail. Usually the insertion of 4HB into chain backbone consisting of 3-hydroxyalkanoates requires expensive carbon sources mostly of petrochemical origin. According to our study the production of P(3HB-co-3HV-co-4HB) terpolymer can be obtained directly by the use of lactose or waste raw materials such as cheese whey as carbon sources. Although the amount of 4HB in the produced terpolymers was usually low and not exceeding 10% of the total molar composition, a PHA containing 18.4% of 4HB units was produced in 1 step fermentation process from this structurally unrelated carbon sources. The crystallinity of the terpolymer is basically to be markedly affected with respect to that of conventional PHAs, thus obtaining a comparatively less rigid material and easier to be processed.

Cationic polymers and their therapeutic potential.

The last decade has witnessed enormous research focused on cationic polymers. Cationic polymers are the subject of intense research as non-viral gene delivery systems, due to their flexible properties, facile synthesis, robustness and proven gene delivery efficiency. Here, we review the most recent scientific advances in cationic polymers and their derivatives not only for gene delivery purposes but also for various alternative therapeutic applications. An overview of the synthesis and preparation of cationic polymers is provided along with their inherent bioactive and intrinsic therapeutic potential. In addition, cationic polymer based biomedical materials are covered. Major progress in the fields of drug and gene delivery as well as tissue engineering applications is summarized in the present review.

Identification of selective ligands for human fibrin recognition using high-throughput docking.

The ultimate aim of this study is to identify new molecules that are able to recognize polymerized fibrin, which is the main component of a thrombus. These selective ligands can be exposed on the surface of particular nanoparticles used for the targeted delivery of fibrinolytic drugs. The targeted delivery of these drugs is expected to help to keep under control the severe side effects which can occur if the drugs are administered systemically. The study focuses on the application of high-throughput docking methods used to screen a library of thousands of commercial compounds. The aim was to identify molecules that are potentially capable of interacting with the human fibrin γ(312-324) epitope. The best scoring compounds were purchased and tested through fluorimetric assays in order to estimate their affinity toward fibrin. The results show that the protocol proposed here for identifying new compounds of interest may provide a valuable contribution to the discovery of lead molecules for human fibrin recognition.

Continuous production of poly([R]-3-hydroxybutyrate) by Cupriavidus necator in a multistage bioreactor cascade.

Poly(hydroxyalkanoates) (PHAs) constitute biodegradable polyesters and are considered among the most promising candidates to replace common petrochemical plastics in various applications. To date, all commercial processes for PHA production employ microbial discontinuous fed-batch fermentations. These processes feature drawbacks such as varying product quality and the inevitable periods of downtime for preparation and post-treatment of the bioreactor equipment. An unprecedented approach to PHA production was chosen in the presented work using a multistage system consisting of five continuous stirred tank reactors in series (5-SCR), which can be considered as a process engineering substitute of a continuous tubular plug flow reactor. The first stage of the reactor cascade is the site of balanced bacterial growth; thereafter, the fermentation broth is continuously fed from the first into the subsequent reactors, where PHA accumulation takes place under nitrogen-limiting conditions. Cupriavidus necator was used as production strain. The focus of the experimental work was devoted to the development of a PHA production process characterized by high productivity and high intracellular polymer content. The results of the experimental work with the reactor cascade demonstrated its potential in terms of volumetric and specific productivity (1.85 g L⁻¹ h⁻¹ and 0.100 g g⁻¹ h⁻¹, respectively), polymer content (77%, w/w) and polymer properties (M (w) = 665 kg/mol, PDI = 2.6). Thus, implementing the technology for 5-SCR production of PHB results in an economically viable process. The study compares the outcome of the work with literature data from continuous two-stage PHA production and industrial PHA production in fed-batch mode.

Optimized electro- and wet-spinning techniques for the production of polymeric fibrous scaffolds loaded with bisphosphonate and hydroxyapatite.

This research activity was aimed at the development of composite bioactive scaffolds made of biodegradable three-arm branched-star poly(ε-caprolactone) (*PCL), hydroxyapatite nanoparticles (HNPs) and clodronate (CD), a bisphosphonate that has demonstrated efficacy in the treatment of various bone diseases and as an anti-inflammatory drug. During the experimental work, the processing conditions for the fabrication of fibrous meshes, by either electrospinning or wet-spinning, were optimized. Stemming from a previous research activity on electrospinning of *PCL, *PCL/HNPs 3D meshes were developed, evaluating the influence of fabrication parameters on the fibres' morphology. By exploiting the binding affinity of bisphosphonates for hydroxyapatite, a methodology was set up for obtaining a physical linkage between CD and HNPs, with the aim of having a dual bioactive system loaded into *PCL fibrous mats. Fibres loaded with either CD or CD-HNP particles were thus produced and analysed by scanning electron microscopy for their morphology and by energy dispersive X-ray spectroscopy for their elemental composition.

Three-dimensional models of the oligomeric human asialoglycoprotein receptor (ASGP-R).

The work presented here is aimed at suggesting plausible hypotheses for functional oligomeric forms of the human asialoglycoprotein receptor (ASGP-R), by applying a combination of different computational techniques. The functional ASGP-R is a hetero-oligomer, that comprises of several subunits of two different kinds (H1 and H2), which are highly homologous. Its stoichiometry is still unknown. An articulated step-wise modeling protocol was used in order to build the receptor model in a minimal oligomeric form, necessary for it to bind multi-antennary carbohydrate ligands. The ultimate target of the study is to contribute to increasing the knowledge of interactions between the human ASGP-R and carbohydrate ligands, at the molecular level, pertinent to applications in the field of hepatic tissue engineering.

Development of electrospun three-arm star poly(ε-caprolactone) meshes for tissue engineering applications.

We have developed three-dimensional electrospun microfibrous meshes of a novel star branched three-arm poly(ε-caprolactone) (*PCL) as potential scaffolds for tissue engineering applications. The processing conditions required to obtain uniform fibers were optimized by studying their influence on fiber morphology and size. Polymer molecular weight and solution feed rate influenced both the mesh microstructure and the tensile properties of the developed mats. Electrospun samples were also tested for their mechanical properties in wet conditions, showing higher yield strength and strain in comparison to that observed in dry conditions. Cell culture experiments employing MC3T3-E1 osteoblast like cells showed good cell viability adhesion and collagen production on the *PCL scaffolds.

Surface segregation assessment in poly(epsilon-caprolactone)-poly(ethylene glycol) multiblock copolymer films.

The ability to predict the in vivo performance of multiblock-copolymer-based biomaterials is crucial for their applicability in the biomedical field. In this work, XPS analysis of PCL-PEG copolymers was carried out, as well as morphological and wettability evaluations by SEM and CA measurements, respectively. XPS analysis on films equilibrated in PBS demonstrated a further enrichment in the PEG component on the surface. Copolymer films obtained by casting using different solvents showed a dependence in segregation according to the solvent employed. Cell adhesion tests demonstrated the importance of copolymer segregation and rearrangement in a wet environment, with a dependence of these phenomena on the copolymer molecular weight.

The surface-segregated nanostructure of fluorinated copolymer-poly(dimethylsiloxane) blend films.

Two fluorinated/siloxane copolymers, O5/19 and D5/3, carrying 6 and 8 CF(2) groups in the fluoroalkyl tail, respectively, were used as the surface-active components of cured poly(dimethylsiloxane) (PDMS) blends at different loadings (0.3-5.0 wt % with respect to PDMS). The surface chemical composition was determined by angle-resolved X-ray photoelectron spectroscopy at the takeoff angles theta of 0 degrees, 60 degrees, and 75 degrees. It was found that the fluorinated copolymer was surface-segregated, and in-depth segregation (approximately 5 nm) depended upon the chemical structure of the copolymer. The surface fluorine atomic percentage of the blends with D5/3 was up to 3 orders of magnitude higher than the theoretical value expected for ideal homogeneous samples. Moreover, small amounts of the copolymer in the blends were sufficient to saturate the outermost surface in fluorine content. The chemical composition of the surface-segregated nanostructure of the films was also proven to be affected by external environment, namely, exposure to water.

More fluorous surface modifier makes it less oleophobic: fluorinated siloxane copolymer/PDMS coatings.

A copolyacrylate with semifluorinated and polydimethylsiloxane side chains (D5-3) was used as a surface modifier for a condensation-cured PDMS coating. The decyl fluorous group is represented by "D"; "5" is a 5 kDa silicone, and "3" is the mole ratio of fluorous to silicone side chains. Wetting behavior was assessed by dynamic contact angle (DCA) analysis using isopropanol, which differentiates silicone and fluorous wetting behavior. Interestingly, a maximum in surface oleophobicity was found at low D5-3 concentration (0.4 wt %). Higher concentrations result in decreased oleophobicity, as reflected in decreased contact angles. To understand this unexpected observation, dynamic light scattering (DLS) studies were initiated on a model system consisting of hydroxyl-terminated PDMS (18 kDa) containing varying amounts of D5-3. DLS revealed D5-3 aggregation to be a function of temperature and concentration. A model is proposed by which D5-3 surface concentration is depleted via phase separation favoring D5-3 aggregation at concentrations >0.4 wt %, that is, the cmc. This model suggests increasing aggregate/micelle concentrations at increased D5-3 concentration. Bulk morphologies studied by scanning electron microscopy (SEM) and atomic force microscopy (AFM) support this model by showing increased aggregate concentrations with increased D5-3 > 0.4 wt %.

Poly(hydroxyalkanoates)-based polymeric nanoparticles for drug delivery.

Poly (hydroxyalkanoates) (PHAs) have recently attracted a great deal of academic and industrial interest for their biodegradability and biocompatibility making them suitable for environmental and biomedical applications. Poly(3-hydroxybutyrate-) (PHB-) and Poly(DL-lactide-co-glycolide) (PLGA-) based nanoparticles were prepared using the dialysis method as yet unreported for the preparation of nanoparticles based on PHB. Processing conditions were varied in order to evaluate their influence on morphology, drug encapsulation, and size of nanoparticles. The relevant results obtained give a theoretical understanding of the phenomenon occurring during colloidal formation. The adopted procedure allows for a relatively small diameter and homogeneity in size distribution of the PHB nanoparticles to be obtained compared to other methods like the one based on solvent evaporation which leads to particles on microscale. The biocompatibility of PHB and relative nanoparticles was investigated and both exhibited very good cytocompatibility.