Preparation, Properties, and Bioapplications of Block Copolymer Nanopatterns.

Block copolymer (BCP) self-assembly has emerged as a feasible method for large-scale fabrication with remarkable precision - features that are not common for most of the nanofabrication techniques. In this review, recent advancements in the molecular design of BCP along with state-of-the-art processing methodologies based on microphase separation alone or its combination with different lithography methods are presented. Furthermore, the bioapplications of the generated nanopatterns in the development of protein arrays, cell-selective surfaces, and antibacterial coatings are explored. Finally, the current challenges in the field are outlined and the potential breakthroughs that can be achieved by adopting BCP approaches already applied in the fabrication of electronic devices are discussed.

End-on PEGylation of heparin: Effect on anticoagulant activity and complexation with protamine.

Heparin is the most common anticoagulant used in clinical practice but shows some downsides such as short half-life (for the high molecular weight heparin) and secondary effects. On the other hand, its low molecular weight analogue cannot be neutralized with protamine, and therefore cannot be used in some treatments. To address these issues, we conjugated polyethylene glycol (PEG) to heparin reducing end (end-on) via oxime ligation and studied the interactions of the conjugate (Hep-b-PEG) with antithrombin III (AT) and protamine. Isothermal titration calorimetry showed that Hep-b-PEG maintains the affinity to AT. Dynamic light scattering demonstrated that the Hep-b-PEG formed colloidal stable nanocomplexes with protamine instead of large multi-molecular aggregates, associated with heparin side effects. The in vitro (human plasma) and in vivo experiments (Sprague Dawley rats) evidenced an extended half-life and higher anticoagulant activity of the conjugate when compared to unmodified heparin.

Antibacterial nanopatterned coatings for dental implants.

Dental implants, usually made of titanium, are exposed to hostile oral microflora that facilitate bacterial infections and subsequent inflammation. To mitigate these processes, we coated titanium substrates with block copolymer nanopatterns and investigated the bactericidal effect of these coatings against Gram-positive and Gram-negative bacteria. We found that the bactericidal efficacy of the coatings depends on their morphology and surface chemistry as well as on the bacterial strain: an optimal combination can lead to significant bacterial death for a short time, i.e. 90% for 90 min. Human gingival fibroblasts in contact with the nanopatterned coatings showed similar cell attachment and morphology as on bare Ti. Immunostaining assays showed similar levels of CCR7 and CD206 in macrophages cultured over the nanopatterns and bare Ti, demonstrating adequate properties for tissue integration. The nanopatterns induced a small increase in macrophage aspect ratio, which might indicate early states of M2 polarization, given the absence of CD206.

Hyaluronan Brush-like Copolymers Promote CD44 Declustering in Breast Cancer Cells.

We report on the synthesis of hyaluronan (HA) brush-like copolymers and their application as antagonists of tumorigenic CD44-HA interactions. HA (4.8 kDa, ca. 24 saccharides) was grafted on 2-hydrohyethyl methacrylate (HEMA) by end-on oxime ligation. The obtained copolymers were compared with low and high molecular weight HA in terms of hydrolysis kinetics in the presence of hyaluronidase (isothermal titration calorimetry) and interactions with CD44 (surface plasmon resonance). The results evidenced that the high molecular weight HA and HA-g-HEMA have a much higher affinity to CD44 than low molecular weight HA. Additionally, slower enzymatic degradation was observed for the copolymer, making it an excellent candidate for active targeting of tumorigenic CD44-HA interactions. We, therefore, investigated the effect of the copolymer on cancer cell lines with different expression of CD44 and observed an efficient declustering of CD44 that is usually associated with reduction of metastasis and drug resistance.

Unveiling an NMR-Invisible Fraction of Polymers in Solution by Saturation Transfer Difference.

The observation of signals in solution NMR requires nuclei with sufficiently large transverse relaxation times (T2). Otherwise, broad signals embedded in the baseline afford an invisible fraction of nuclei (IF). Based on the STD (saturation transfer difference) sequence, IF-STD is presented as a quick tool to unveil IF in the 1H NMR spectra of polymers. The saturation of a polymer in a region of the NMR spectrum with IF (very short 1H T2) results in an efficient propagation of the magnetization by spin diffusion through the network of protons to a visible-invisible interphase with larger 1H T2 (STDon). Subtracting this spectrum from one recorded without saturation (STDoff) produces a difference spectrum (STDoff-on), with the nuclei at the visible-invisible interphase, that confirms the presence of an IF. Analysis of a wide collection of polymers by IF-STD reveals IF more common than previously thought, with relevant IF figures when STD > 0.4% at 750 MHz. A fundamental property of the IF-STD experiment is that the signal is generated within a single state comprising polymer domains with different dynamics, as opposed to several states in exchange with different degrees of aggregation. Contrary to a reductionist visible-invisible dichotomy, our results confirm a continuous distribution of nuclei with diverse dynamics. Since nuclei observed (edited) by IF-STD at the visible-invisible interphase are in close spatial proximity to the IF (tunable with the saturation time), they emerge as a privileged platform from which gaining an insight into the IF itself.

Isolation and Characterization of Polysaccharides from the Ascidian Styela clava.

Styela clava is an edible sea squirt farmed in Korea that has gradually invaded other seas, negatively impacting the ecology and economy of coastal areas. Extracts from S. clava have shown wide bioactivities, and ascidians have the unique capability among animals of biosynthesizing cellulose. Thus, S. clava is a relevant candidate for valorization. Herein, we aimed at surveying and characterizing polysaccharides in both tunic and flesh of this ascidian. To this end, we enzymatically hydrolyzed both tissues, recovering crystalline cellulose from the tunic with high aspect ratios, based on results from microscopy, X-ray diffraction, and infrared spectroscopy analyses. Alkaline hydroalcoholic precipitation was applied to isolate the polysaccharide fraction that was characterized by gel permeation chromatography (with light scattering detection) and NMR. These techniques allowed the identification of glycogen in the flesh with an estimated Mw of 7 MDa. Tunic polysaccharides consisted of two fractions of different Mw. Application of Diffusion-Ordered NMR allowed spectroscopically separating the low-molecular-weight fraction to analyze the major component of an estimated Mw of 40-66 kDa. We identified six different sugar residues, although its complexity prevented the determination of the complete structure and connectivities of the residues. The two more abundant residues were N-acetylated and possibly components of the glycosaminoglycan-like (GAG-like) family, showing the remaining similarities to sulfated galactans. Therefore, Styela clava appears as a source of nanocrystalline cellulose and GAG-like polysaccharides.

Hyaluronic Acid Oligomer Immobilization as an Angiogenic Trigger for the Neovascularization of TE Constructs.

Tissue engineered (TE) substitutes of clinically relevant sizes need an adequate vascular system to ensure function and proper tissue integration after implantation. However, the predictable vascularization of TE substitutes is yet to be achieved. Molecular weight variations in hyaluronic acid (HA) have been pointed to trigger angiogenesis. Thus, this study investigates HA oligomer immobilization as a promoter for TE construct vascularization. As a proof-of-concept, the surface of methacrylated gelatin (GelMA) hydrogels were functionalized with high molecular weight (HMW; 1.5 to 1.8 MDa) and low molecular weight (LMW; < 10 kDa) HA, previously modified with aldehyde groups to enable the immobilization through Schiff's base formation. The ability of A-HA to bind amine-presenting surfaces was confirmed by Surface Plasmon Resonance (SPR). Human Umbilical Vein Endothelial Cells (HUVECs) seeded over hydrogels functionalized with LMW HA showed higher proliferation and expression of angiogenic markers (KDR and CD31), than those grown in HMW HA conjugated- or plain surfaces, in line with the activation of HA ERK1/2 mediated downstream signaling. Moreover, when cocultured with human dental pulp cells (hDPCs) encapsulated into the GelMA, an increase in endothelial cell migration was observed for the LMW HA functionalized formulations. Overall LMW HA functionalization enhanced endothelial cell response showing potential as an angiogenesis inducer for TE applications.

Novel amphiphilic chitosan micelles as carriers for hydrophobic anticancer drugs.

Chitosan was grafted with O-methyl-O'-succinylpolyethylene glycol and oleic acid after a two-step carbodiimide coupling. The structural and physicochemical characterization of the compounds confirmed the successful conjugation of the hydrophilic and hydrophobic moieties to the chitosan backbone. The amphiphilic chitosan derivative obtained allowed the formation of polymeric micelles with an average size of 140 nm, a polydispersity index <0.234, and a positive superficial charge. Camptothecin, used as a model hydrophobic drug, was successfully carried into the polymeric micelles with an encapsulation efficiency of 78%. The in vitro drug release was evaluated in simulated gastrointestinal fluids, exhibiting a low release of camptothecin in gastric media and a controlled release in intestinal fluids. Furthermore, it was demonstrated that chitosan micelles were able to stabilize camptothecin, protecting up to 75% of the drug from hydrolysis, preserving its active lactone form. This new chitosan amphiphilic system exhibits great potential to load hydrophobic drugs, acting as a promising delivery system.

Structure, rheology, and copper-complexation of a hyaluronan-like exopolysaccharide from Vibrio.

MO245 exopolysaccharide (EPS) was produced in laboratory conditions from Vibrio genus microorganism isolated from bacterial mats found in Moorea Island. Its structure consists of a linear tetrasaccharide repeating unit →4)-ß-D-GlcpA-(1→4)-α-D-GalpNAc-(1→3)-ß-D-GlcpNAc-(1→4)-ß-D-GlcpA-(1→ containing covalently-linked 5% of glucose, galactose, and rhamnose, determined by methylation analyses and NMR spectroscopy. The molecular weight, radius of gyration (Rg) and intrinsic viscosity, [η], determined by gel permeation chromatography with light scattering and viscosity detection, were 513 ± 4 kDa (PDI, 1.42 ± 0.01), 6.7 ± 0.3 dl/g and 56 ± 0.3 nm respectively. The chelation of the EPS with copper divalent ions leads to the instantaneous formation of gels. The structural similitude proposed, based in an equal ratio of GlcA to N-acetylated sugars and in the same type of glyosidic linkages present in the repeating unit (alternated 1→3 and 1→4 linkages), is translated into analogous physicochemical properties: MO245 EPS is a flexible polyelectrolyte, with scaling exponents similar to that described for HA. This similitude opens opportunities in future drug delivery, tissue engineering, and cosmetic applications.

Minimalistic supramolecular proteoglycan mimics by co-assembly of aromatic peptide and carbohydrate amphiphiles.

We report the co-assembly of aromatic carbohydrate and dipeptide amphiphiles under physiological conditions as a strategy to generate minimalistic proteoglycan mimics. The resulting nanofibers present a structural, fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) core and a functional carbohydrate (Fmoc-glucosamine-6-sulfate or -phosphate) shell. The size, degree of bundling and mechanical properties of the assembled structures depend on the chemical nature of the carbohydrate amphiphile used. In cell culture medium, these nanofibers can further organize into supramolecular hydrogels. We demonstrate that, similar to proteoglycans, the assembled gels prolong the stability of growth factors and preserve the viability of cultured cells. Our results demonstrate that this approach can be applied to the design of extracellular matrix (ECM) substitutes for future regenerative therapies.

Optimal isolation and characterisation of chondroitin sulfate from rabbit fish (Chimaera monstrosa).

Chondroitin sulfate (CS) is a glycosaminoglycan widely explored for cartilage regeneration. Its bioactivity is influenced by sulfation degree and pattern, and distinct sulfation in marine CS may open new therapeutic possibilities. In this context, we studied for the first time the isolation and characterisation of CS from Rabbit Fish (Chimaera monstrosa). We propose an efficient process starting with enzymatic hydrolysis, followed by chemical treatments and ending in membrane purification. All steps were optimised by response surface methodology. Chemical treatment by alkaline-hydroalcoholic precipitation led to 99% purity CS suitable for biomedical and pharmaceutical applications, and treatment by alkaline hydrolysis yielded CS adequate for nutraceutical formulations (89% purity). Molecular weight and sulfation profiles were similar for both materials. Gel permeation chromatography analyses resulted in molecular weights (Mn) of 51-55 kDa. NMR and SAX-HPLC revealed dominant 6S-GalNAc sulfation (4S/6S ratio of 0.4), 17% of GlcA 2S-GalNAc 6S and minor quantities of other disaccharides.

A multifunctional drug nanocarrier for efficient anticancer therapy.

So far, the success of anticancer nanomedicines has been moderate due to their lack of adequate targeting properties and/or to their difficulties for penetrating tumors. Here we report a multifunctional drug nanocarrier consisting of hyaluronic acid nanocapsules conjugated with the tumor homing peptide tLyp1, which exhibits both, dual targeting properties (to the tumor and to the lymphatics), and enhanced tumor penetration. Data from a 3D co-culture in vitro model showed the capacity of these nanocapsules to interact with the NRP1 receptors over-expressed in cancer cells. The targeting capacity of the nanocapsules was evidenced in orthotopic lung cancer-bearing mice, using docetaxel as a standard drug. The results showed a dramatic accumulation of docetaxel in the tumor (37-fold the one achieved with Taxotere®). This biodistribution profile correlated with the high efficacy shown in terms of tumor growth regression and drastic reduction of metastasis in the lymphatics. When efficacy was validated in a pancreatic patient-derived tumor, the nanocapsule's activity was comparable to that of a dose ten times higher of Abraxane®. Multi-functionality was found to be the key to the success of this new therapy.

Star-Like Glycosaminoglycans with Superior Bioactivity Assemble with Proteins into Microfibers.

Here it is shown that glycosaminoglycans (GAGs) with high molecular weight can be grafted via their reducing end on hyperbranched synthetic cores by oxime condensation without the need of any previous functionalisation of the polysaccharide. The versatility of this reaction is demonstrated by the use of hyaluronan, chondroitin sulfate and heparin with up to 60 sugar units. The isothermal calorimetry analysis demonstrated that the generated star-like glycopolymers have superior bioactivity. Moreover, when mixed with positively charged proteins (e.g., fibroblast growth factor-2, FGF-2) they form microfiber structures instead of the spherical nanocomplexes described for linear GAGs. The results suggest that the described star-like GAG are closer mimics of the proteoglycans at the structural and functional level and therefore have huge potential in the development of tissue engineering platforms and therapeutics by modulating the activity and presentation of various proteins such as growth factors.

Redox-Responsive Micellar Nanoparticles from Glycosaminoglycans for CD44 Targeted Drug Delivery.

Cancer progression is associated with overexpression of various receptors at the cell surface. Among these, CD44 is known to recognize and bind specifically hyaluronan (HA) and interact with less affinity to other glycosaminoglycans (GAGs), such as chondroitin sulfate (CS). In this study, we describe a simple method to obtain micellar nanoparticles with a GAG shell (HA or CS) as potential drug delivery systems that target cancer cells overexpressing CD44. Alkanethiol was conjugated at the reducing end of the respective GAG using highly efficient oxime chemistry. The alkane moiety confers amphiphilic behavior to the obtained conjugates and triggers their self-assembly into micellar nanoparticles, while the thiol group adds redox-responsiveness to the system. The properties of the particles depend on the used GAG: HA amphiphiles form more dense, smaller assemblies that are redox sensitive. Both systems allow encapsulation of either hydrophobic or hydrophilic cargos with high efficiency. We demonstrate that the GAGs exposed on the surface of the nanoparticles are with preserved bioactivity and recognized by the cellular receptors: the particles were internalized via CD44 dependent pathways.

Design of protein delivery systems by mimicking extracellular mechanisms for protection of growth factors.

Heparin sulfate proteoglycans (HSPGs) are responsible for the storage and stabilization of numerous growth factors in the extracellular matrix. In this complex native environment, the efficient binding of the growth factors is determined by multivalent, specific and reversible electrostatic interactions between the sulfate groups of HSPGs and the positively charged amino acids of the growth factor. Inspired by this naturally occurring stabilization process, we propose the use of diblock copolymers of heparin and polyethylene glycol (Hep-b-PEG) for protection and delivery of FGF-2. We describe the encapsulation of FGF-2 into spontaneously assembling polyelectrolyte complexes (PECs) with Hep-b-PEG in which the Hep block ensures the formation of the PECs, while the PEG moiety confers stability of the generated complex by a stealth corona. Our results demonstrate that by this method we can generate homogeneous complexes (ca. 400nm diameter, PDI 0.29±0.07) with a very high encapsulation efficiency (about 99% encapsulated FGF-2). The release of the growth factor in response to different stimuli such as pH, ionic strength or presence of heparinase was also studied. We report a sustained release of up to 80% during 28days which is not influenced by the presence of heparinase - a result that clearly demonstrates the protective effect of the stealth corona. We also show that FGF-2 remains bioactive as it influences the morphology of bone marrow mesenchymal stem cells. STATEMENT OF SIGNIFICANCE: We describe a biopolymer that uses the way the cells shield a type of proteins (growth factors) to simultaneously assemble, slowly deliver and shield the protein in a "nanocarrier". Growth factors are essential for the regeneration of cartilage, bones by stem cell therapies but have a short life time as when added directly to tissues. Our design makes use of the heparin bioactivity towards such proteins in combination with a polyethylene glycol moiety (PEG) that makes a protecting shell. PEG, is biocompatible and used in approved medicines and countless cosmetic products. The highest novelty is the reaction (oxime click) used to bound these molecules that does not require modification of heparin and allows preservation of its bioactivity.

Optimization of high purity chitin and chitosan production from Illex argentinus pens by a combination of enzymatic and chemical processes.

The present report illustrates the optimisation of the experimental conditions for the chemical and enzymatic production of chitin and chitosan from Illex argentinus pen by-products. Optima conditions for chitin isolation were established at 0.82M NaOH/36.4°C, 57.5°C/pH=9.29, 59.6°C/pH=9.30 and 49.6°C/pH=5.91 for chemical, alcalase, esperase and neutrase deproteinization, respectively. Chitin samples were subsequently deacetylated by alkaline treatment reaching the highest degrees of deacetylation (DD>93%) at 61.0-63.7% of NaOH and 14.9-16.4h of hydrolysis depending on the type of process previously performed to the squid pens. Molecular weight (as number average molecular weight, Mn) of chitosan produced in the experimental designs ranged from 143kDa (PDI 2.37) to 339kDa (PDI 2.38).

Glycosaminoglycans from marine sources as therapeutic agents.

Glycosaminoglycans (GAGs) in marine animals are different to those of terrestrial organisms, mainly in terms of molecular weight and sulfation. The therapeutic properties of GAGs are related to their ability to interact with proteins, which is very much influenced by sulfation position and patterns. Since currently GAGs cannot be chemically synthesized, they are sourced from natural products, with high intra- but also inter-species variability, in terms of chain length, disaccharide composition and sulfation pattern. Consequently, sulfated GAGs are the most interesting molecules in the marine environment and constitute the focus of the present review. In particular, chondroitin sulfate (CS) appears as the most promising compound. CS-E chains [GlcA-GalNAc(4S,6S)] extracted from squid possess antiviral and anti-metastatic activities and seem to impart signalling properties and improve the mechanical performance of cartilage engineering constructs; Squid CS-E and octopus CS-K [GlcA(3S)-GalNAc(4S)], dermatan sulfate (DS) from sea squirts [-iK units, IdoA(3S)-GalNAc(4S)] and sea urchins [-iE units, IdoA-GalNAc(4S,6S)] and hybrids CS/DS from sharks (-B/iB [GlcA/IdoA(2S)-GalNAc(4S)], -D/iD [GlcA/IdoA(2S)-GalNAc(6S)] and -E/iE units [GlcA/IdoA-GalNAc(4S,6S)]) promote neurite outgrowth and could be valuable materials for nerve regeneration. Also displaying antiviral and anti-metastatic properties, a rare CS with fucosylated branches isolated from sea cucumbers is an anticoagulant and anti-inflammatory agent. In this same line, marine heparin extracted from shrimp and sea squirt has proven anti-inflammatory properties, with the added advantage of decreased risk of bleeding because of its low anticoagulant activity.

The Key Role of Sulfation and Branching on Fucoidan Antitumor Activity.

There is an urgent need for antitumor bioactive agents with minimal or no side effects over normal adjacent cells. Fucoidan is a marine-origin polymer with known antitumor activity. However, there are still some concerns about its application due to the inconsistent experimental results, specifically its toxicity over normal cells and the mechanism behind its action. Herein, three fucoidan extracts (FEs) have been tested over normal and breast cancer cell lines. From cytotoxicity results, only one of the extracts shows selective antitumor behavior (at 0.2 mg mL-1 ), despite similarities in sulfation degree and carbohydrates composition. Although the three FEs present different molecular weights, depolymerization of selected samples discarded Mw as the key factor in the antitumor activity. Significant differences in sulfates position and branching are observed, presenting FE 2 the higher branching degree. Based on all these experimental data, it is believed that these last two properties are the ones that influence the cytotoxic effects of fucoidan extracts.

By-products of Scyliorhinus canicula, Prionace glauca and Raja clavata: A valuable source of predominantly 6S sulfated chondroitin sulfate.

Chondroitin sulfate (CS) was isolated from Scyliorhinus canicula (fin, head and skeleton), Prionace glauca (head), and Raja clavata (skeleton) by-products from fish processing industry using environmentally friendly processes. The molecular weight was determined by gel permeation chromatography and the sugar composition and sulfation position by NMR and SAX-HPLC after enzymatic digestion. The CSs showed a prevalent 6S GalNAc sulfation for the 3 species (4S/6S ratio lower than 1). A higher 6S sulfation was observed for P. glauca head and R. clavata skeleton (4S/6S ratio below 0.20) than for S. canicula (4S/6S ratio ca. 0.6). The existence of CS samples with such low 4S/6S ratio has only been observed before in a rare species of shark (Mitsukutina owatoni, globin shark). The good extraction yields achieved make S. canicula, P. glauca and R. clavata fish industry by-products a useful source of 6-sulfated chondroitin sulfate.

Neovascularization Induced by the Hyaluronic Acid-Based Spongy-Like Hydrogels Degradation Products.

Neovascularization has been a major challenge in many tissue regeneration strategies. Hyaluronic acid (HA) of 3-25 disaccharides is known to be angiogenic due to its interaction with endothelial cell receptors. This effect has been explored with HA-based structures but a transitory response is observed due to HA burst biodegradation. Herein we developed gellan gum (GG)-HA spongy-like hydrogels from semi-interpenetrating network hydrogels with different HA amounts. Enzymatic degradation was more evident in the GG-HA with high HA amount due to their lower mechanical stability, also resulting from the degradation itself, which facilitated the access of the enzyme to the HA in the bulk. GG-HA spongy-like hydrogels hyaluronidase-mediated degradation lead to the release of HA oligosaccharides of different amounts and sizes in a HA content-dependent manner which promoted in vitro proliferation of human umbilical cord vein endothelial cells (HUVECs) but not their migration. Although no effect was observed in human dermal microvascular endothelial cells (hDMECs) in vitro, the implantation of GG-HA spongy-like hydrogels in an ischemic hind limb mice model promoted neovascularization in a material-dependent manner, consistent with the in vitro degradation profile. Overall, GG-HA spongy-like hydrogels with a sustained release of HA oligomers are valuable options to improve tissue vascularization, a critical issue in several applications in the tissue engineering and regenerative medicine field.