Exploiting the layer-by-layer nanoarchitectonics for the fabrication of polymer capsules: A toolbox to provide multifunctional properties to target complex pathologies.

Polymer capsules fabricated via the layer-by-layer (LbL) approach have attracted a great deal of attention for biomedical applications thanks to their tunable architecture. Compared to alternative methods, in which the precise control over the final properties of the systems is usually limited, the intrinsic versatility of the LbL approach allows the functionalization of all the constituents of the polymeric capsules following relatively simple protocols. In fact, the final properties of the capsules can be adjusted from the inner cavity to the outer layer through the polymeric shell, resulting in therapeutic, diagnostic, or theranostic (i.e., combination of therapeutic and diagnostic) agents that can be adapted to the particular characteristics of the patient and face the challenges encountered in complex pathologies. The biomedical industry demands novel biomaterials capable of targeting several mechanisms and/or cellular pathways simultaneously while being tracked by minimally invasive techniques, thus highlighting the need to shift from monofunctional to multifunctional polymer capsules. In the present review, those strategies that permit the advanced functionalization of polymer capsules are accordingly introduced. Each of the constituents of the capsule (i.e., cavity, multilayer membrane and outer layer) is thoroughly analyzed and a final overview of the combination of all the strategies toward the fabrication of multifunctional capsules is presented. Special emphasis is given to the potential biomedical applications of these multifunctional capsules, including particular examples of the performed in vitro and in vivo validation studies. Finally, the challenges in the fabrication process and the future perspective for their safe translation into the clinic are summarized.

Encapsulation of manganese dioxide nanoparticles into layer-by-layer polymer capsules for the fabrication of antioxidant microreactors.

Oxidative stress is caused by the accumulation of reactive oxygen and nitrogen species (ROS and RNS) in the cellular microenvironment. These ROS and RNS damage important cell structures leading to cell apoptosis and senescence, thus causing a detrimental effect on numerous disease pathologies such as osteoarthritis, neurodegeneration and cardiovascular diseases. For this reason, there is a growing interest in the development of antioxidant biomaterials that can eventually regulate the levels of ROS/RNS and prevent oxidative stress. The encapsulation of antioxidant enzymes (e.g., catalase or superoxide dismutase) on polymer microcapsules fabricated via the layer-by-layer (LbL) approach represents a promising strategy within this context. The diffusion of reagents and by-products through the shell of these microcapsules is timely and spatially controlled, allowing the bio-chemical reaction between ROS/RNS and the encapsulated enzyme. However, natural enzymes usually present low stability, high cost and difficult storage, which could limit their potential application in the biomedical field. Hence, nanomaterials with intrinsic enzyme-like characteristics (i.e., nanozymes) have been considered as inorganic alternatives. In the present work, manganese dioxide nanoparticles were encapsulated into LbL polymer microcapsules to yield synthetic antioxidant microreactors. These microreactors efficiently scavenged hydrogen peroxide (H2O2) from solution and protected cells from oxidative stress in an in vitro model. The versatility of the synthetic procedure presented herein allows the fabrication of capsules with either positive or negative surface charge, which has a direct impact on the cytotoxicity and cell interaction. This study represents accordingly a novel strategy to obtain antioxidant polymer microreactors based on synthetic (nano)materials for the treatment of oxidative stress.

Mechanical properties and state of miscibility in poly(racD,L-lactide-co-glycolide)/(L-lactide-co-ε-caprolactone) blends.

Polymers based on lactic acid (PLA) are a very promising category of biopolymers. As they are multi-stimuli responsive, can, in many ways, positively interact with the host, stimulating the innate reparative machinery of the human body. Since biopolymers for medical applications are subject to restrictive regulations, blending stands out as an effective method for obtaining tailored properties within a reduced time to market if compared to synthesis. Hence, in this study a set of PDLGA/PLCL blends was obtained by means of thermoplastic techniques and then further characterized. Evaluation techniques include GPC, NMR, DSC, tensile testing and SEM. Although mixtures proved to be immiscible, a full range of tensile properties was achieved. Observation of the surfaces of fracture provided visual evidence of the deformation mechanisms that occurred during the tensile tests which in the end led to failure. Interpretation of the thermal events based on molecular characterization parameters revealed phase separation, crystallization and plasticisation mechanisms that are relevant to any potential applications based on mechanical performance and shape memory behaviour.

Computational Bench Testing to Evaluate the Short-Term Mechanical Performance of a Polymeric Stent.

Over the last decade, there has been a significant volume of research focussed on the utilization of biodegradable polymers such as poly-L-lactide-acid (PLLA) for applications associated with cardiovascular disease. More specifically, there has been an emphasis on upgrading current clinical shortfalls experienced with conventional bare metal stents and drug eluting stents. One such approach, the adaption of fully formed polymeric stents has led to a small number of products being commercialized. Unfortunately, these products are still in their market infancy, meaning there is a clear non-occurrence of long term data which can support their mechanical performance in vivo. Moreover, the load carry capacity and other mechanical properties essential to a fully optimized polymeric stent are difficult, timely and costly to establish. With the aim of compiling rapid and representative performance data for specific stent geometries, materials and designs, in addition to reducing experimental timeframes, Computational bench testing via finite element analysis (FEA) offers itself as a very powerful tool. On this basis, the research presented in this paper is concentrated on the finite element simulation of the mechanical performance of PLLA, which is a fully biodegradable polymer, in the stent application, using a non-linear viscous material model. Three physical stent geometries, typically used for fully polymeric stents, are selected, and a comparative study is performed in relation to their short-term mechanical performance, with the aid of experimental data. From the simulated output results, an informed understanding can be established in relation to radial strength, flexibility and longitudinal resistance, that can be compared with conventional permanent metal stent functionality, and the results show that it is indeed possible to generate a PLLA stent with comparable and sufficient mechanical performance. The paper also demonstrates the attractiveness of FEA as a tool for establishing fundamental mechanical characteristics of polymeric stent performance.

Pyrene-end-functionalized poly(L-lactide) as an efficient carbon nanotube dispersing agent in poly(L-lactide): mechanical performance and biocompatibility study.

In order to improve the mechanical properties of poly(L-lactide) (PLLA) based implants, a study was made of how far well dispersed multi-walled carbon nanotubes (MWCNTs) within a PLLA matrix were able to positively affect these properties. To this end, pyrene-end-functionalized poly(L-lactide) (py-end-PLLA) was evaluated as a dispersing agent. Transmission electron microscopy (TEM) analyses and mechanical tests of MWCNTs-based materials demonstrated an enhancement of MWCNT dispersion in the PLLA matrix and improved Young's modulus (E) when 4 wt% of py-end-PLLA was used as the dispersing agent. Subsequently, the bioacceptance of PLLA/py-end-PLLA/MWCNTs nanocomposites was evaluated using human bone marrow stromal cells (HBMC) in vitro. The inclusion of py-end-PLLA and MWCNTs supported HBMC adhesion and proliferation. The expression levels of the bone-specific markers indicated that the cells kept their potential to undergo osteogenic differentiation. The results of this study indicate that the addition of MWCNT combined with py-end-PLLA in PLLA/py-end-PLLA/MWCNTs nanocomposites may widen the range of applications of PLLA within the field of bone tissue engineering thanks to their mechanical strength and cytocompatibility.

Supramolecular structure, phase behavior and thermo-rheological properties of a poly (L-lactide-co-ε-caprolactone) statistical copolymer.

PLAcoCL samples, both unaged, termed PLAcoCLu, and aged over time, PLAcoCLa, were prepared and analyzed to study the phase structure, morphology, and their evolution under non-quiescent conditions. X- ray diffraction, Differential Scanning Calorimetry and Atomic Force Microscopy were complemented with thermo-rheological measurements to reveal that PLAcoCL evolves over time from a single amorphous metastable state to a 3 phase system, made up of two compositionally different amorphous phases and a crystalline phase. The supramolecular arrangements developed during aging lead to a rheological complex behavior in the PLAcoCLa copolymer: Around Tt=131 °C thermo-rheological complexity and a peculiar chain mobility reduction were observed, but at T>Tt the thermo-rheological response of a homogeneous system was recorded. In comparison with the latter, the PLLA/PCL 70:30 physical blend counterpart showed double amorphous phase behavior at all temperatures, supporting the hypothesis that phase separation in the PLAcoCLa copolymer is caused by the crystallization of polylactide segment blocks during aging.

Crystallization and its effect on the mechanical properties of a medium chain length polyhydroxyalkanoate.

Medium chain length polyhydroxyalkanoates (mcl-PHAs) could play a role in the growing demand for highly elastic and biodegradable materials in the medical field. In this study, a poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate) (P(3HO-co-3HH)) was first fully characterized in terms of molecular weight, microstructural chain parameters and chemical structure by means of gel permeation chromatography (GPC), nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR). As determined by NMR, the synthesized polymer contained 94.3% and 5.7% molar content of 3-hydroxyoctanoate and 3-hydroxyhexanoate, respectively. Since mechanical properties are closely related to thermal history, the effect of crystallization on tensile properties was also investigated in the present study. Three crystallization temperatures were selected (0, 23 and 37°C), the conclusion reached is that the maximum crystallization rate for this copolymer was achieved at 0°C. On the other hand, evolution of tensile properties of P(3HO-co-3HH) films stored at room temperature demonstrated that, as crystallization occurred toward the equilibrium state, the polymer underwent a stiffening process. In this sense, secant modulus and tensile strength increased respectively from 8.3 ± 1.0 MPa and 6.4 ± 0.8 MPa after 1 day stored at room temperature to 36.2 ± 3.3 MPa and 16.3 ± 2.1 MPa after 16 weeks.

A study of the mechanical properties and cytocompatibility of lactide and caprolactone based scaffolds filled with inorganic bioactive particles.

The mechanical properties of highly porous (90% porosity) poly(l-lactide) (PLLA), poly(ε-caprolactone) (PCL) and poly(l-lactide/ε-caprolactone) (PLCL) were investigated. Young's modulus of non-porous PLLA, PCL and PLCL dropped from 2263.4, 183.7 and 5.7 MPa to 16.8, 1.0 and 1.0 MPa, respectively, for their ~90% porous counterparts. Elongation at break of PCL decreased noticeably with porosity fraction while PLCL maintained a highly elastomeric character and strain recovery capacity even in the presence of pores. Inorganic bioactive particles (hydroxyapatite or bioglass) were added to confer bioactivity to the aforementioned synthetic bioresorbable polymers, and their effect on the mechanical properties was also investigated. Addition of 15 vol.% of inorganic bioactive particles increased the Young's modulus of highly porous PLLA from 16.2 to ~30 MPa. On the contrary, the difference between Young's modulus of filled and unfilled PCL and PLCL scaffolds was not statistically significant. Finally, an in vitro study of the cytocompatibility and adhesion of adipose derived stem cells (ADSCs) was conducted. The observed viability and excellent adhesion of these cells to both porous and non-porous templates indicate that the employed materials can be good candidates for application in tissue engineering.

Anhydric maleic functionalization and polyethylene glycol grafting of lactide-co-trimethylene carbonate copolymers.

Lactide and trimethylene carbonate copolymers were successfully grafted with polyethylene glycol via previous functionalization with maleic anhydride and using N,N'-diisopropylcarbodiimide as condensing agent. Maleinization led to moderate polymer degradation. Specifically, the weight average molecular weight decreased from 36,200 to 30,200 g/mol for the copolymer having 20 mol% of trimethylene carbonate units. Copolymers were characterized by differential scanning calorimetry, thermogravimetry and X-ray diffraction. Morphology of spherulites and lamellar crystals was evaluated with optical and atomic force microscopies, respectively. The studied copolymers were able to crystallize despite the randomness caused by the trimethylene carbonate units and the lateral groups. Contact angle measurements indicated that PEG grafted copolymers were more hydrophilic than parent copolymers. This feature justified that enzymatic degradation in lipase medium and proliferation of both epithelial-like and fibroblast-like cells were enhanced. Grafted copolymers were appropriate to prepare regular drug loaded microspheres by the oil-in-water emulsion method. Triclosan release from loaded microspheres was evaluated in two media.

Improvement of toughness by stereocomplex crystal formation in optically pure polylactides of high molecular weight.

A solution casting method followed by thermal homogenization was performed for the preparation of 1:1 blends and non-blended films from poly(d-lactide) (PDLA) and poly(l-lactide) (PLLA) of three different molecular weights, and their thermal and mechanical properties were determined via differential scanning calorimetry (DSC) and tensile tests. According to the literature, when Mw is below 1.0×10(5)g/mol only stereocomplex crystallization takes place, and when it is higher, both homocrystallites and stereocomplex crystallites co-exist. In order to promote crystallization as a homocrystal in neat polylactides and to promote the stereoselective crystallization as stereocomplex in the case of non-blended films, and in turn, to achieve different degrees of crystallinity, several thermal treatments of annealing were carried out in this work. Highly stereocomplexed blends were found by the stereospecific thermal treatments. As a consequence, the toughness of 1:1 blends was found significantly enhanced over those of non-blended films, irrespective of molecular weight. For instance, in B2-5050 stereocomplexed blend having poly(l-lactide) and poly(d-lactide) of Mw=1.2×10(5)g/mol, tensile strength increased from 44.0±2.1MPa to 65.1±6.1MPa, and the elongation at break from 10.8±2.5% to 33.1±8.1% with respect to its non-blended poly(l-lactide) counterpart crystallized as homocrystal. This improvement in mechanical properties in stereocomplexed blends is not attributed to the inherent properties of the type of crystal polymorph but to the presence of a higher density of intercrystalline connections through a mobile amorphous phase, i.e. tie chains in the stereocomplexed supramolecular spherulitic entities that provide in the stereocomplexed samples enhanced strength and elongation at break at the same time.

Tensile behavior and dynamic mechanical analysis of novel poly(lactide/δ-valerolactone) statistical copolymers.

Lactide-co-δ-valerolactone copolymers (PLVL) have not attracted as much research interest as the more popular poly(lactide-co-ε-caprolactone) (PLCL) elastomeric materials. In this work the study of the mechanical performance is focused on the former with the aim of identifying the potential advantages of these thermoplastic elastomers for their application in the biomedical field. Mechanical testing (at 21°C and at 37°C) of at least 5 specimens and dynamic mechanical analysis (DMA) in duplicate were carried out on various PLVL, which include a moderately blocky l-lactide/δ-valerolactone copolymer (~70% of l-LA and R=0.68) and several that showed a random distribution of sequences (R~1): some terpolymers based on l-lactide, d-lactide and δ-valerolactone (with a lactone content of ~25 and ~14%) and a series of copolymers of l-LA and δ-VL having l-LA molar contents ranging from 69 to 74%. In view of the results, it can be concluded that noteworthy improvements in stiffness and strength were achieved by adding δ-VL to the reaction mix instead of ε-CL, although both monomers have analogous chemical properties. For example, a PLVL with a 75:25M composition of l-LA/δ-VL at 21°C presented a secant modulus of 213.7±36.5MPa and σu=14.7±1.4MPa whereas a previously studied PLCL of equal composition had a secant modulus and an ultimate stress value of 19.4±1.3MPa and 3.2±0.6MPa, respectively. At 37°C, the differences in the mechanical properties between the different PLVLs of this work were far less relevant, with most of them showing a fully elastomeric behavior. Referring to the DMA measurements, the reduction in the peak of tan δ (from ~2.5 to 0.5) through the glass transition was a clear indicator that crystalline domains formed during hydrolytic degradation in some of the polymers. However, the more amorphous PLVLs with short l-LA average sequence lengths (ll-LA<2.91) did not undergo changes in the storage modulus and tan δ curves after two weeks submerged in PBS at 37°C.

Ultra-fast laser microprocessing of medical polymers for cell engineering applications.

Picosecond laser micromachining technology (PLM) has been employed as a tool for the fabrication of 3D structured substrates. These substrates have been used as supports in the in vitro study of the effect of substrate topography on cell behavior. Different micropatterns were PLM-generated on polystyrene (PS) and poly-L-lactide (PLLA) and employed to study cellular proliferation and morphology of breast cancer cells. The laser-induced microstructures included parallel lines of comparable width to that of a single cell (which in this case is roughly 20µm), and the fabrication of square-like compartments of a much larger area than a single cell (250,000µm(2)). The results obtained from this in vitro study showed that though the laser treatment altered substrate roughness, it did not noticeably affect the adhesion and proliferation of the breast cancer cells. However, pattern direction directly affected cell proliferation, leading to a guided growth of cell clusters along the pattern direction. When cultured in square-like compartments, cells remained confined inside these for eleven incubation days. According to these results, laser micromachining with ultra-short laser pulses is a suitable method to directly modify the cell microenvironment in order to induce a predefined cellular behavior and to study the effect of the physical microenvironment on cell proliferation.

Novel hydrogels of chitosan and poly(vinyl alcohol)-g-glycolic acid copolymer with enhanced rheological properties.

Poly(vinyl alcohol) (PVA) has been grafted with glycolic acid (GL), a biodegradable hydroxyl acid to yield modified poly(vinyl alcohol) (PVAGL). The formation of hydrogels at pH = 6.8 and physiological temperature through blending chitosan (CS) and PVAGL at different concentrations has been investigated. FTIR, DOSY NMR and oscillatory rheology measurements have been carried out on CS/PVAGL hydrogels and the results have been compared to those obtained for CS/PVA hydrogels prepared under the same conditions. The experimental results point to an increase in the number of interactions between chitosan and PVAGL in polymer hydrogels prepared with modified PVA. The resulting materials with enhanced elastic properties and thixotropic behavior are potential candidates to be employed as injectable materials for biomedical applications.

A new generation of poly(lactide/ε-caprolactone) polymeric biomaterials for application in the medical field.

Thermoplastic biodegradable polymers displaying an elastomeric behavior are greatly valued for the regeneration of soft tissues and for various medical devices. In this work, terpolymers composed of ε-caprolactone (CL), D-lactide (D-LA), and L-lactide (L-LA) were synthesized. These poly(lactide-ε-caprolactone) (PLCLs) presented an elevated randomness character (R∼1), glass transition temperatures (Tg ) higher than 20°C and adjusted L-LA content. In this way, the L-LA average sequence length (/L-LA ) was reduced to below 3.62 and showed little or no crystallization capability during in vitro degradation. As a result, the obtained materials underwent homogenous degradation exhibiting KMw ranging from 0.030 to 0.066 d(-1) and without generation of crystalline remnants in advanced stages of degradation. Mechanical performance was maintained over a period of 21 days for a rac-lactide-ε-caprolactone copolymer composed of ∼85% D,L-LA and ∼15% CL and also for a terpolymer composed of ∼72% L-LA, ∼12% D-LA and ∼16% CL. Terpolymers having L-LA content from ∼60 to 70% and CL content from ∼10 to 27% were also studied. In view of the results, those materials having CL and D-LA units disrupting the microstructural arrangement of the L-LA crystallizable chains, an L-LA content <72% and a random distribution of sequences, may display proper and tunable mechanical behavior and degradation performance for a large number of medical applications. Those with a CL content from 15 to 30% will fulfill the demand of elastomeric materials of Tg higher than 20°C whereas those with a CL content from 5 to 15% might be applied as ductile stiff materials.

[Biodegradable catheters for fistula prevention in hypospadias. Experimental preliminary study]. / Sondas biodegradables para la prevención de las fístulas de hipospadias. Estudio preliminar experimental.

INTRODUCTION: Continuous technical innovations are not enough to resolve the high incidence of fistula after hypospadias repair. A urethral catheter-tutor made of reabsorbable polymeric biomaterial (RPB) which could be left in situ long enough could reduce the complications. TARGET: To investigate in an animal model differents RPB to be used in urology. METHODOLOGY: CRL Wistar rats, males, divided into 5 equal groups according to the used polymers: polylactide; lactic-coprolactone copolymer; lactic-glycolic copolymer; simulated; control silicones. Three individuals were sacrificed per group at 4th, 10th and 16th week. In all animals (exceptuating the simulated group), biomaterial was fixed to the bladder wall bylaparotomy. Animals remained in individual housing and kept under daily control of hematuria during the first 15 days and weekly weight and urine control for pH and lactate. After being slaughtered, remaining polymer was collected for chemical analysis and bladder tissue for hystologic study. RESULTS: There was no mortality, hematuria nor other clinical signs. The bladder wall showed a mild foreign body reaction. The values of lactate and pH in urine did not reach toxic levels. Lactic-glycolic was totally reabsorbed by the 10th week and had the lowest degree of calcification. Polylactide and lactic-coprolactone remained intact. CONCLUSION: The model of urinary bladder has proven useful for studying the degradation of bioresorbable polymers. The analyzed polymers have spent long time to be reabsorbed, so we will have to study new others.

Crystallinity assessment and in vitro cytotoxicity of polylactide scaffolds for biomedical applications.

Bioresorbable polylactides are one of the most important materials for tissue engineering applications. In this work we have prepared scaffolds based on the two optically pure stereoisomers: poly(L: -lactide) (PLLA) and poly(D: -lactide) (PDLA). The crystalline structure and morphology were evaluated by DSC, AFM and X-ray diffraction. PLLA and PDLA crystallized in the α form and the equimolar PLLA/PDLA blend, crystallized in the stereocomplex form, were analyzed by a proliferation assay in contact with mouse L-929 and human fibroblasts and neonatal keratinocytes for in vitro cytotoxicity evaluation. SEM analysis was conducted to determine the cell morphology, spreading and adhesion when in contact with the different polymer surfaces. The preserved proliferation rate showed in MTT tests and the high colonization on the surface of polylactides observed by SEM denote that PLLA, PDLA and the equimolar PLLA/PDLA are useful biodegradable materials in which the crystalline characteristics can be tuned for specific biomedical applications.

Conformational behavior of poly(L-lactide) studied by infrared spectroscopy.

This paper reports the analysis of the C=O stretching region of poly(L-lactide). This spectral band splits into up to four components, a phenomenon that a priori can be explained in terms of carbonyl-carbonyl coupling or specific interactions (such as C-H...O hydrogen bonding or dipole-dipole). Hydrogen bonding can be discarded from the analysis of the C-H stretching spectral region. In addition, low molecular weight dicarbonyl compounds of chemical structure similar to that of PLLA, such as diacyl peroxides, show a remarkable splitting of the carbonyl band attributed to intramolecular carbonyl-carbonyl coupling. Several mechanisms can be responsible for this behavior, such as mechanical coupling, electronic effects, or through-space intramolecular TDC (transition dipole coupling) interactions. Intermolecular dipole-dipole interactions (possible in the form of interchain TDC interactions) are proven to be of minor relevance taking into account the spatial structure of the PLLA conformers. The Simply Coupled Oscilator (SCO) model, which only accounts for mechanical coupling, has been found to predict adequately the relative intensity of the symmetric and asymmetric bands of dicarbonyl compounds. The dispersion curves predicted for PLLA by the SCO model also match those given by more general treatments, such as Miyazawa's first-order perturbation theory. Hence, the SCO model is adopted here as an adequate yet simple tool for the interpretation of band splitting caused by intramolecular coupling of polylactide. The four components observed in the C=O stretching band of semicrystalline PLLA are attributed to the four possible conformers: gt, gg, tt, and tg. The narrow bands observed for the interlamellar material are attributed to highly ordered chains, indicating the absence of a truly amorphous phase in the crystalline polymer. The interphase seems to extend over the whole interlamellar region, showing the features of a semiordered metastable phase. In amorphous PLLA, bands corresponding to gt, gg, and tt conformers also can be resolved by second derivative techniques, and curve-fitting results provide information about the conformational population at different temperatures.