Poly-Gamma-Glutamic Acid (γ-PGA)-Based Encapsulation of Adenovirus to Evade Neutralizing Antibodies.

In recent years, there has been an increasing interest in oncolytic adenoviral vectors as an alternative anticancer therapy. The induction of an immune response can be considered as a major limitation of this kind of application. Significant research efforts have been focused on the development of biodegradable polymer poly-gamma-glutamic acid (γ-PGA)-based nanoparticles used as a vector for effective and safe anticancer therapy, owing to their controlled and sustained-release properties, low toxicity, as well as biocompatibility with tissue and cells. This study aimed to introduce a specific destructive and antibody blind polymer-coated viral vector into cancer cells using γ-PGA and chitosan (CH). Adenovirus was successfully encapsulated into the biopolymer particles with an encapsulation efficiency of 92% and particle size of 485 nm using the ionic gelation method. Therapeutic agents or nanoparticles (NPs) that carry therapeutics can be directed specifically to cancerous cells by decorating their surfaces using targeting ligands. Moreover, in vitro neutralizing antibody response against viral capsid proteins can be somewhat reduced by encapsulating adenovirus into γ-PGA-CH NPs, as only 3.1% of the encapsulated adenovirus was detected by anti-adenovirus antibodies in the presented work compared to naked adenoviruses. The results obtained and the unique characteristics of the polymer established in this research could provide a reference for the coating and controlled release of viral vectors used in anticancer therapy.

Peptide Modified Electrospun Glycopolymer Fibers.

Oligo(Glu70 -co-Leu30 ), a peptide synthesized by protease catalysis, is functionalized at the N-terminus with a 4-pentenoyl unit and grafted to polyLSL[6'Ac,6″Ac], a glycopolymer prepared by ring-opening metathesis polymerization of lactonic sophorolipid diacetate. First, polyLSL[6'Ac,6"Ac] fiber mats are fabricated by electrospinning. Oxidation of the fiber mats and subsequent reaction with cysteamine lead to thiol-functionalized fiber mats with no significant morphology changes. Grafting of the alkene-modified oligopeptide to thiol-functionalized polyLSL[6'Ac,6″Ac] fiber mats is achieved via "thiol-ene" click reaction. X-ray photoelectron spectroscopy analysis to characterize peptide grafting reveals that about 50 mol% of polyLSL[6'Ac,6''Ac] repeat units at fiber surfaces are decorated with a peptide moiety, out of which about 1/3 of the oligo(Glu70 -co-Leu30 ) units are physically adsorbed to polyLSL[6'Ac,6''Ac]. The results of this work pave the way to precise engineering of polyLSL fiber mats that can be decorated with a potentially wide range of molecules that tailor surface chemistry and biological properties.

Exploring the Solid State Properties of Enzymatic Poly(amine-co-ester) Terpolymers to Expand their Applications in Gene Transfection.

Polymers bearing amino functional groups are an important class of materials capable of serving as non-viral carriers for DNA delivery to living cells. In this work biodegradable poly(amine-co-ester) terpolymers were synthesized via ring-opening and polycondensation copolymerization of lactone (ε-caprolactone (CL), ω-dodecalactone, ω-pentadecalactone (PDL), and ω-hexadecalactone) with diethyl sebacate (DES) and N-methyldiethanolamine (MDEA) in diphenyl ether, catalyzed by Candida antarctica lipase B (CALB). All lactone-DES-MDEA terpolymers had random distributions of lactone, sebacate, MDEA repeat units in the polymer chains. PDL-DES-MDEA terpolymers were studied in the composition range from 21 mol% to 90 mol% PDL whereas the terpolymers with other lactones were investigated at a single composition (80 mol% lactone). DSC and WAXS analyses showed that all investigated terpolymers crystallize in their respective homopolylactone crystal lattice. Terpolymers with large lactones and a high lactone content melt well above room temperature and are hard solids, whereas terpolymers with small lactones (e.g. CL) or with a low lactone content melt below/around ambient temperature and are waxy/gluey materials. Given the importance of hydrophobicity in influencing gene delivery, water contact angle measurements were carried out on lactone-DES-MDEA terpolymers showing that it is possible to tune the hydrophilic-to-hydrophobic balance by varying polymer composition and size of lactone units. To demonstrate the feasibility of using solid terpolymers as nanocarriers for DNA delivery, PDL-DES-MDEA copolymers with 65-90% PDL were successfully transformed into free-standing nanoparticles with average particle size ranging from 163 to 175 nm. Our preliminary results showed that LucDNA-loaded nanoparticles of the terpolymer with 65% PDL were effective for luciferase gene transfection of HEK293 cells.

Recovery of amorphous polyhydroxybutyrate granules from Cupriavidus necator cells grown on used cooking oil.

Used cooking oil (UCO) was employed as the sole carbon source for the production of polyhydroxybutyrate (PHB) by cultivation in batch mode of Cupriavidus necator DSM 428. The produced biomass was used for extraction of the PHB granules with a solvent-free approach using sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and the enzyme Alcalase in an aqueous medium. The recovered PHB granules showed a degree of purity higher than 90% and no crystallization (i.e., granules were recovered in their 'native' amorphous state) as demonstrated by wide angle X-ray diffraction (WAXS). Granules were characterized according to their thermal properties and stability by differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Results show that UCO can be used as a renewable resource to produce amorphous PHB granules with excellent properties in a biocompatible manner.

Polyhydroxyalkanoates production with mixed microbial cultures: from culture selection to polymer recovery in a high-rate continuous process.

Polyhydroxyalkanoates (PHA) production with mixed microbial cultures (MMC) has been investigated by means of a sequential process involving three different stages, consisting of a lab-scale sequencing batch reactor for MMC selection, a PHA accumulation reactor and a polymer extraction reactor. All stages were performed under continuous operation for at least 4 months to check the overall process robustness as well as the related variability of polymer composition and properties. By operating both biological stages at high organic loads (8.5 and 29.1 gCOD/Ld, respectively) with a synthetic mixture of acetic and propionic acid, it was possible to continuously produce PHA at 1.43 g/Ld with stable performance (overall, the storage yield was 0.18 COD/COD). To identify the optimal operating conditions of the extraction reactor, two digestion solutions have been tested, NaOH (1m) and NaClO (5% active Cl2). The latter resulted in the best performance both in terms of yield of polymer recovery (around 100%, w/w) and purity (more than 90% of PHA content in the residual solids, on a weight basis). In spite of the stable operating conditions and performance, a large variation was observed for the HV content, ranging between 4 and 20 (%, w/w) for daily samples after accumulation and between 9 and 13 (%, w/w) for weekly average samples after extraction and lyophilization. The molecular weight of the produced polymer ranged between 3.4 × 10(5) and 5.4 × 10(5)g/mol with a large polydispersity index. By contrast, TGA and DSC analysis showed that the thermal polymer behavior did not substantially change over time, although it was strongly affected by the extraction agent used (NaClO or NaOH).

The role of 3D microenvironmental organization in MCF-7 epithelial-mesenchymal transition after 7 culture days.

We present a multi-technique study on in vitro epithelial-mesenchymal transition (EMT) in human MCF-7 cells cultured on electrospun scaffolds of poly(l-lactic acid) (PLA), with random and aligned fiber orientations. Our aim is to investigate the morphological and genetic characteristics induced by extracellular matrix in tumor cells cultured in different 3D environments, and at different time points. Cell vitality was assessed with AlamarBlue at days 1, 3, 5 and 7. Scanning electron microscopy was performed at culture days 3 and 7. Immunohistochemistry (for E-cadherin, ß-catenin, cytokeratins, nucleophosmin, tubulin, Ki-67 and vimentin), immunofluorescence (for F-actin) western blot (for E-cadherin, ß-catenin and vimentin) and transmission electron microscopy were carried out at day 7. An EMT gene array followed by PCR analysis confirmed the regulation of selected genes. At day 7, scanning electron microscopy on aligned-PLA revealed spindle-shaped cells gathered in buds and ribbon-like structures, with a higher nucleolar/nuclear ratio and a loss in E-cadherin and ß-catenin at immunohistochemistry and western blot. An up-regulation of SMAD2, TGF-ß2, TFPI2 and SOX10 was found in aligned-PLA compared to random-PLA cultured cells. The topography of the extracellular matrix has a role in tumor EMT, and a more aggressive phenotype characterizes MCF-7 cells cultured on aligned-PLA scaffold.

Advantages of surface-initiated ATRP (SI-ATRP) for the functionalization of electrospun materials.

Surface-initiated atom transfer radical polymerization (SI-ATRP) is successfully applied to electrospun constructs of poly(L-lactide). ATRP macroinitiators are adsorbed through polyelectrolyte complexation following the introduction of negative charges on the polyester surface through its blending with a six-armed carboxy-terminated oligolactide. SI-ATRP of glycerol monomethacrylate (GMMA) or 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) allows then to grow surface films with controllable thickness, and in this way also to control the wetting and interactions of the construct.

Biocompatible two-layer tantalum/titania-polymer hybrid coating.

Using a two-step procedure, radiopaque and biocompatible coatings were obtained, consisting of a tantalum layer deposited by sputtering technique and of an upper organic-inorganic hybrid layer synthesized via sol-gel. As shown by radiographic images, tantalum confers to plastic substrates good X-ray visibility, adjustable via control of deposition time, but its adhesion to the substrate is poor and manipulation easily damages the metal layer. Polymer-titania hybrid coatings, synthesized using poly-ε-caprolactone (PCL) or carboxy-terminated polydimethylsiloxane (PDMS) as organic precursors, were applied on the metal layer as biocompatible protective coatings. Biocompatibility is demonstrated by cytotoxicity tests conducted using vascular wall resident-mesenchymal stem cells (VW-MSCs). Both coatings show very good adhesion to the substrate, showing no sign of detachment upon large substrate deformations. Under such conditions, SEM observations show that the PCL-containing hybrid forms cracks, whereas the PDMS-based hybrid does not crack, suggesting possible applications of the latter material as a protective layer of sputtered tantalum radiopaque markers for flexible medical devices.

Electrospun scaffolds of a polyhydroxyalkanoate consisting of omega-hydroxylpentadecanoate repeat units: fabrication and in vitro biocompatibility studies.

Electrospinning was used to fabricate fibrous scaffolds of lipase-catalyzed poly(omega-pentadecalactone) (PPDL). The slow resorbability of this biomaterial is expected to be valuable for tissue-engineering applications requiring long healing times. The effect of solvent systems and instrumental parameters on fiber morphology was investigated. PPDL electrospinning was optimized and defect-free fibers (diameter 410 +/- 150 nm) were obtained by using a mixed three-solvent system. Scaffolds were characterized by scanning electron microscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXS). TGA showed no residual solvent in the scaffolds. DSC and WAXS results indicated that electrospun PPDL is semicrystalline. Biocompatibility of PPDL scaffolds was evaluated through indirect cytotoxicity tests using embryonic rat cardiac H9c2 cells. The ability of PPDL electrospun mats to support cell growth was verified by culturing H9c2 cells onto the scaffold. Cell adhesion, proliferation and morphology were evaluated. The results indicated that PPDL mats are not cytotoxic and they support proliferation of H9c2 cells. The cumulative results of this study suggest further exploration of PPDL fibrous mats as scaffolds for tissue-engineered constructs.

Scaffold for tissue engineering fabricated by non-isothermal supercritical carbon dioxide foaming of a highly crystalline polyester.

Porous scaffolds of a random co-polymer of omega-pentadecalactone (PDL) and epsilon-caprolactone (CL) (poly(PDL-CL)), synthesized by biocatalysis, were fabricated by supercritical carbon dioxide (scCO(2)) foaming. The co-polymer, containing 31 mol.% CL units, is highly crystalline (T(m) = 82 degrees C, DeltaH(m) = 105 J g(-1)) thanks to the ability of the two monomer units to co-crystallize. The co-polymer can be successfully foamed upon homogeneous absorption of scCO(2) at T > T(m). The effect of soaking time, depressurization rate and cooling rate on scaffold porosity, pore size distribution and pore interconnectivity was investigated by micro X-ray computed tomography. Scaffolds with a porosity in the range 42-76% and an average pore size of 100-375 microm were successfully obtained by adjusting the main foaming parameters. Process conditions in the range investigated did not affect the degree of crystallinity of poly(PDL-CL) scaffolds. A preliminary study of the mechanical properties of the scaffolds revealed that poly(PDL-CL) foams may find application in the regeneration of cartilage tissue.

Surface-initiated ATRP modification of tissue culture substrates: poly(glycerol monomethacrylate) as an antifouling surface.

Surface-initiated atom transfer radical polymerization (SI-ATRP) can be used to produce conformal coatings of controlled thickness on virtually any surface, providing to it specific physico-chemical and biological properties. Here we have tackled the problem of modulating cell adhesion on typical culture substrates; tissue culture polystyrene (TCPS) offers a number of favorable properties (optical transparency, chemical stability, sterilizability, availability in a wide variety of shapes) but somehow limited biological function. A fine tuning of cell adhesion can, on the contrary, allow better control cell phenotype during cell expansion or, by using responsive polymers, allow attachment/detachment cycles with reduced cell damage. Here we have optimized a procedure of TCPS surface oxidation to allow the adsorption of cationic macroinitiators and the successive growth of surface-born polymer chains, producing films with controlled thickness. We have specifically focused our attention on the preparation of films containing poly(glycerol monomethacrylate) (PGMMA), showing that PGMMA is nontoxic but nonadhesive to cells, possibly providing "stealth" surfaces. Cell adhesion can be reinstated by copolymerizing GMMA with other monomers: films containing N,N-dimethylamino ethyl methacrylate (DMAEMA; in the surface-grown films this monomer is substantially hydrophobic at physiological pH) together with GMMA provided cell attachment and spreading to comparable to TCPS. Last, cell circularity was here shown to be a valid reporter for the assessment of cell spreading.

Organic-inorganic hybrids as transparent coatings for UV and X-ray shielding.

This work reports the synthesis of new polymer-titania hybrids, where the organic phase is constituted by polyesters such as poly(-caprolactone), poly(d,l-lactic acid), and poly(l-lactic acid). In these hybrids, very strong interaction between the organic and inorganic domains is obtained through transesterification of ester moieties by titanium atoms, which leads to organotitanium esters. The influence of the structure of the polyester on the mode of its interaction with titania is also investigated. Hybrids with a range of solid-state properties, which depend on the starting polymer characteristics and on the inorganic-phase content, are obtained. Hybrids are applied as coatings on a variety of substrates. They show intrinsic optical transparency and the ability to completely block UV radiation in the range UVB and UV-A2. In particular, when applied to different textiles, the hybrids impart radiopacity to the fabrics, opening new perspectives in the field of personal protective clothing and equipment.

Shape memory behavior of novel (L-lactide-glycolide-trimethylene carbonate) terpolymers.

Bioresorbable new terpolymers of L-lactide, glycolide, and trimethylene carbonate with different compositions were synthesized via ring-opening polymerization reaction of the cyclic monomers using low-toxicity zirconium(IV) acetylacetonate as initiator. The thermal and mechanical properties were investigated by means of thermogravimetry, differential scanning calorimetry, stress-strain measurements, and dynamical mechanical analysis. The glass transition temperature of the terpolymers changes with composition from 12 to 42 degrees C in a predictable manner. All terpolymers display shape memory properties and, after undergoing 100% deformation, they recover the permanent shape in a time frame of seconds. Terpolymers with high L-lactide content show a glass transition in the range of 38-42 degrees C, recovery temperature close to body temperature, and good recovery ratio (>0.89). Low-toxicity bioresorbable terpolymers with shape memory properties are promising new materials for biomedical applications.

Lipase-catalyzed copolymerization of omega-pentadecalactone with p-dioxanone and characterization of copolymer thermal and crystalline properties.

Candida antarctica Lipase B (CALB), a metal-free enzyme, was successfully employed as catalyst for ring-opening copolymerization of omega-pentadecalactone (PDL) with p-dioxanone (DO) under mild reaction conditions (<80 degrees C, atmospheric pressure). Poly(PDL-co-DO) with high molecular weight (Mw > 30 000) and a wide range of comonomer contents was synthesized using various PDL/DO feed ratios. During the copolymerization reaction, large ring PDL was found to be more reactive than its smaller counterpart DO, resulting in higher PDL/DO unit ratios in polymer chains than the corresponding PDL/DO monomer ratios in the feed. The copolymers were typically isolated in 50-90 wt % yields as the monomer conversion was limited by the equilibrium between monomers and copolymer. 1H and 13C NMR analysis on poly(PDL-co-DO) formed by CALB showed that the copolymers contain nearly random sequences of PDL and DO units with a slight tendency toward alternating arrangements. Copolymerization with PDL was found to remarkably enhance PDO thermal stability. Differential scanning calorimetry (DSC) and wide-angle X-ray scattering (WAXS) results demonstrate high crystallinity in all copolymers over the whole range of compositions. Depending on copolymer composition, the crystal lattice of either PDO or PPDL hosts units of the other comonomer, a behavior typical of an isodimorphic system. In poly(PDL-co-DO), both melting temperature and melting enthalpy display a minimum at 70 mol % DO, that is, at the pseudoeutectic composition. WAXS diffractograms show one crystal phase (that of either PPDL or PDO) on either side of the pseudoeutectic and coexistence of PPDL and PDO crystals at the pseudoeutectic.

Carboxylate-induced degradation of poly(3-hydroxybutyrate)s.

This communication shows that thermal degradation of poly(3-hydroxybutyrate)s (PHBs) is induced by carboxylate groups via a newly proposed E1cB mechanism. In PHBs with end groups in the form of carboxylic acid salts with Na+, K+, and Bu4N+ counterions, the proposed mechanism explains the dependence of thermal stability on the size of the counterion. The degradation via intermolecular alpha-deprotonation by carboxylate is suggested to be the main PHB decomposition pathway at moderate temperatures. The results of the present study show the ability to control the degradation and stability of poly(3-hydroxybutyrate)s as well as of their blends via chemical structure and concentration of the carboxylate polymer end groups.

Radiopaque organic-inorganic hybrids based on poly(D,L-lactide).

Hybrid organic-inorganic nanocomposites were prepared starting from alpha,omega-triethoxysilane-terminated poly(d,l-lactic acid) (PDLLA) to be used as potential radiopaque biocompatible coatings for medical devices. The synthesis of the organic phase precursors of given chain length was achieved via anionic polymerization of d,l-lactide using a bifunctional initiator and subsequent triethoxysilane functionalization of the end groups. PDLLA-based ceramers (ceramic polymers) were then synthesized by the sol-gel process at room temperature (rt) in the presence of different amounts of tetraethoxysilane. The rt-synthesized hybrids were then cured (at 80 or 130 degrees C), and their thermal and viscoelastic properties were investigated. All obtained hybrids were optically transparent, due to the nanometric dimension of the silica particles, and yielded clearly contrasted radiographic images.

Cocrystallization of random copolymers of omega-pentadecalactone and epsilon-caprolactone synthesized by lipase catalysis.

Random copolymers were prepared by Candida antarctica lipase B (Novozyme-435) catalyzed copolymerization of omega-pentadecalactone (PDL) with epsilon-caprolactone (CL). Over the whole composition range PDL-CL copolymers are highly crystalline (melting enthalpy by differential scanning calorimetry, above 100 J/g; crystallinity degree by wide-angle X-ray scattering, WAXS, 60-70%). The copolymers melt at temperatures that linearly decrease with composition from that of poly(omega-pentadecalactone) (PPDL; 97 degrees C) to that of poly(epsilon-caprolactone) (PCL; 59 degrees C). The WAXS profiles of PCL and PPDL homopolymers are very similar, except for the presence in PPDL of the (001) reflection at 2theta = 4.58 degrees that corresponds to a 19.3 angstroms periodicity in the chain direction. In PDL-CL copolymers the intensity of this reflection decreases with increasing content of CL units and vanishes at 50 mol % CL, as a result of randomization of the ester group alignment and loss of chain periodicity. PDL-CL copolymers crystallize in a lattice that gradually changes from that of one homopolymer to that of the other, owing to comonomer isomorphous substitution. Cocrystallization of comonomer units is also shown by a random PDL-CL copolymer obtained in a polymerization/transesterification reaction catalyzed by C. antarctica lipase B (Novozyme-435) starting from preformed PCL and PDL monomer.

Biodegradable polyesters reinforced with surface-modified vegetable fibers.

Flax fibers are investigated as reinforcing agents for biodegradable polyesters (Bionolle and poly(lactic acid) plasticized with 15 wt.-% of acetyltributyl citrate, p-PLLA). The composites are obtained either by high temperature compression molding fiber mats sandwiched between polymer films, or by batch mixing fibers with the molten polymer. Fibers in composites obtained by the latter method are much shorter (140-200 microm) than those of the mats (5,000 microm). Flax fibers are found to reinforce both p-PLLA and Bionolle (i.e. tensile modulus and strength increase) when composites based on fiber mats are investigated. Conversely, analogous composites obtained by batch mixing show poor mechanical properties. The observed behavior is attributed to the combined effect of fiber length and fiber-matrix adhesion. If flax fibers with a modified surface chemistry are used, the strength of short fiber composites is seen to improve significantly because the interface strengthens and load is more efficiently transferred. Appropriate surface modifications are performed by heterogeneous acylation reactions or by grafting poly(ethylene glycol) chains (PEG, molecular weight 350 and 750). The highest tensile strength of p-PLLA composites is reached when PEG-grafted flax fibers are used, whereas in the case of Bionolle the best performance is observed with acylated fibers.

Biodegradation of chemically modified flax fibers in soil and in vitro with selected bacteria.

The extent and rate of degradation of flax (Linum usitatissimum) fibers, both in the native state and after surface chemical modification (acetylation or poly(ethylene glycol), PEG, grafting), was investigated under laboratory conditions in two different biodegrading environments. Degradation of the fibers under aerobic conditions by the action of the microorganisms present in soil is assessed with the ASTM 5988-96 method by monitoring carbon dioxide evolution. In vitro biodegradation experiments were carried out by exposing the fibers to a pure culture of Cellvibrio fibrovorans bacteria and measuring the mass loss as a function of time. Despite the complexity of the system, the results of degradation in soil were satisfactorily reproducible, although the absolute rates were found to change in different experiments using the same soil. The degradation rate of acetylated fibers in soil nearly equals that of unmodified fibers, whereas in the pure culture, acetylated fibers biodegrade slower than native fibers. The opposite happens with the PEG-grafted fibers, which degrade slower than unmodified flax in soil and at a comparable rate upon in vitro exposure to the bacterial culture. The different biodegradation kinetics observed in the two biodegrading environments were attributed to differences of biocenoses, abiotic factors, and biodegradation assessing methods. Nevertheless, the final extent of biodegradation was the same for modified and unmodified fibers both in soil and in the pure culture, showing that the surface chemical modifications applied do not significantly affect biodegradability of the flax fibers.

Structural study of poly(L-lactic acid) spherulites.

Spherulites of poly(L-lactic acid) (PLLA) and of its blends with atactic poly(3-hydroxybutyrate) (a-PHB, from 10 to 75 wt %) were investigated by microfocus X-ray diffraction using synchrotron radiation. Radial scans in 5 microm steps with 3 microm beam diameter were performed. In PLLA, tens of identical diffraction images were collected. The unit cell a-axis was radially oriented, and the other axes lacked any specific orientation. In contrast, all PLLA/a-PHB blends showed a periodic change of diffraction pattern with increasing distance from the spherulite center. In all cases, the a-axis lay along the radius, while the b- and c-axes rotated about a with a defined periodicity. The unit cell twisting frequency increased with a-PHB content and closely matched the band spacing observed by polarized optical microscopy, which changed from 250 to 60 microm when the amount of a-PHB increased from 10 to 75 wt %. Concomitantly, a gradual broadening of all X-ray reflections was observed.