Leucine-Based Pseudo-Proteins (LPPs) as Promising Biomaterials: A Study of Cell-Supporting Properties.

Scaffold-based systems have become essential in biomedical research, providing the possibility of building in vitro models that can better mimic tissue/organic physiology. A relatively new family of biomimetics-pseudo-proteins (PPs)-can therefore be considered especially promising in this context. Three different artificial leucine-based LPP films were tested in vitro as potential scaffolding materials. In vitro experiments were performed using two types of cells: primary mouse skin fibroblasts and a murine monocyte/macrophages cell line, RAW264.7. Cell adhesion and cell spreading were evaluated according to morphological parameters via scanning electron microscopy (SEM), and they were assessed according to actin cytoskeleton distribution, which was studied via confocal laser microscopy. Cell proliferation was evaluated via an MTT assay. Cell migration was studied using time-lapse microscopy. SEM images for both types of cells demonstrated prominent adhesion and perfect cell spreading on all three LPPs. Analyses of actin cytoskeleton organization revealed a high number of focal adhesions and prominent motility-associated structures. A certain stimulation of cell proliferation was detected in the cases of all three LPPs, and two of them promoted macrophage migration. Overall, our data suggest that the LPPs used in the study can be considered potential cell-friendly scaffolding materials.

Biomimetic Hybrid Systems for Tissue Engineering.

Tissue engineering approaches appear nowadays highly promising for the regeneration of injured/diseased tissues. Biomimetic scaffolds are continuously been developed to act as structural support for cell growth and proliferation as well as for the delivery of cells able to be differentiated, and also of bioactive molecules like growth factors and even signaling cues. The current research concerns materials employed to develop biological scaffolds with improved features as well as complex preparation techniques. In this work, hybrid systems based on natural polymers are discussed and the efforts focused to provide new polymers able to mimic proteins and DNA are extensively explained. Progress on the scaffold fabrication technique is mentioned, those processes based on solution and melt electrospinning or even on their combination being mainly discussed. Selection of the appropriate hybrid technology becomes vital to get optimal architecture to reasonably accomplish the final applications. Representative examples of the recent possibilities on tissue regeneration are finally given.

Biodegradable Nanoparticles Based on Pseudo-Proteins Show Promise as Carriers for Ophthalmic Drug Delivery.

Purpose: Drug delivery to treat ocular diseases still is a challenge in ophthalmology. One way to achieve drug delivery that is investigated currently is topical administration of drug-loaded polymeric nanoparticles (NPs) that are able to penetrate ocular barriers. The purpose of this study was optimal preparation of NPs made from pseudo-proteins and evaluation of their ability to penetrate ocular tissues. Methods: Biodegradable NPs of various types were prepared by nanoprecipitation of pseudo-protein composed of l-leucine (L), 1,6-hexanediol (6), and sebacic acid (8) (8L6). Arginine-based cationic polyester amides 8R6 and comb-like polyester amide containing lateral PEG-2000 chains along with 8L6 anchoring fragments in the backbones were used to construct positively charged and PEGylated NPs. They were loaded with fluorescein diacetate (FDA) or rhodamine 6G (Rh6G) as fluorescent probes. Suspensions of the NPs were given to cultivated microglial cells and retinal pigment epithelial (RPE) cells as well as topically on eyes of C57BL/6 mice. Penetration of NPs into the eyes was checked by fluorescence analysis. Results: NPs were prepared, and their properties were characterized. Cultured microglial cells and RPE cells took up the NPs. After topical administration, penetration of NPs into the cornea of the eyes was clearly seen. Small amounts of fluorescent dyes were also found in the lens, the retina, and the sclera depending on the type of NPs. Conclusions: The results showed that the new NPs penetrate ocular tissues after topical administration and are internalized by the cells. This raises confidence that the NPs may be useful carriers of therapeutic agents for ocular delivery.

A Preliminary Evaluation of the Pro-Chondrogenic Potential of 3D-Bioprinted Poly(ester Urea) Scaffolds.

Degeneration of articular cartilage (AC) is a common healthcare issue that can result in significantly impaired function and mobility for affected patients. The avascular nature of the tissue strongly burdens its regenerative capacity contributing to the development of more serious conditions such as osteoarthritis. Recent advances in bioprinting have prompted the development of alternative tissue engineering therapies for the generation of AC. Particular interest has been dedicated to scaffold-based strategies where 3D substrates are used to guide cellular function and tissue ingrowth. Despite its extensive use in bioprinting, the application of polycaprolactone (PCL) in AC is, however, restricted by properties that inhibit pro-chondrogenic cell phenotypes. This study proposes the use of a new bioprintable poly(ester urea) (PEU) material as an alternative to PCL for the generation of an in vitro model of early chondrogenesis. The polymer was successfully printed into 3D constructs displaying adequate substrate stiffness and increased hydrophilicity compared to PCL. Human chondrocytes cultured on the scaffolds exhibited higher cell viability and improved chondrogenic phenotype with upregulation of genes associated with type II collagen and aggrecan synthesis. Bioprinted PEU scaffolds could, therefore, provide a potential platform for the fabrication of bespoke, pro-chondrogenic tissue engineering constructs.

Artificial Polymers made of α-amino Acids - Poly(Amino Acid)s, Pseudo-Poly(Amino Acid)s, Poly(Depsipeptide)s, and Pseudo-Proteins.

Degradable polymers (DPs) - "green materials" of the future, have an innumerable use in biomedicine, particularly in the fields of tissue engineering and drug delivery. Among these kind of materials naturally occurring polymers - proteins which constituted one of the most important "bricks of life" - α-amino acids (AAs) are highly suitable. A wide biomedical applicability of proteins is due to special properties such as a high affinity with tissues and releasing AAs upon biodegradation that means a nutritive potential for cells. Along with these positive characteristics proteins as biomedical materials they have some shortcomings, such as batch-to-batch variation, risk of disease transmission, and immune rejection. The last limitation is connected with the molecular architecture of proteins. Furthermore, the content of only peptide bonds in protein molecules significantly restricts their material properties. Artificial polymers with the composition of AAs are by far more promising as degradable biomaterials since they are free from the limitations of proteins retaining at the same time their positive features - a high tissue compatibility and nutritive potential. The present review deals with a brief description of different families of AA-based artificial polymers, such as poly(amino acid)s, pseudo-poly(amino acid)s, polydepsipeptides, and pseudo-proteins - relatively new and broad family of artificial AA-based DPs. Most of these polymers have a different macromolecular architecture than proteins and contain various types of chemical links along with NH-CO bonds that substantially expands properties of materials destined for sophisticated biomedical applications.

Oligoarginine Peptides, a New Family of Nicotinic Acetylcholine Receptor Inhibitors.

Many peptide ligands of nicotinic acetylcholine receptors (nAChRs) contain a large number of positively charged amino acid residues, a striking example being conotoxins RgIA and GeXIVA from marine mollusk venom, with an arginine content of >30%. To determine whether peptides built exclusively from arginine residues will interact with different nAChR subtypes or with their structural homologs such as the acetylcholine-binding protein and ligand-binding domain of the nAChR α9 subunit, we synthesized a series of R3, R6, R8, and R16 oligoarginines and investigated their activity by competition with radioiodinated α-bungarotoxin, two-electrode voltage-clamp electrophysiology, and calcium imaging. R6 and longer peptides inhibited muscle-type nAChRs, α7 nAChRs, and α3ß2 nAChRs in the micromolar range. The most efficient inhibition of ion currents was detected for muscle nAChR by R16 (IC50 = 157 nM) and for the α9α10 subtype by R8 and R16 (IC50 = 44 and 120 nM, respectively). Since the R8 affinity for other tested nAChRs was 100-fold lower, R8 appears to be a selective antagonist of α9α10 nAChR. For R8, the electrophysiological and competition experiments indicated the existence of two distinct binding sites on α9α10 nAChR. Since modified oligoarginines and other cationic molecules are widely used as cell-penetrating peptides, we studied several cationic polymers and demonstrated their nAChR inhibitory activity. SIGNIFICANT STATEMENT: By using radioligand analysis, electrophysiology, and calcium imaging, we found that oligoarginine peptides are a new group of inhibitors for muscle nicotinic acetylcholine receptors (nAChRs) and some neuronal nAChRs, the most active being those with 16 and 8 Arg residues. Such compounds and other cationic polymers are cell-penetrating tools for drug delivery, and we also demonstrated the inhibition of nAChRs for several of the latter. Possible positive and negative consequences of such an action should be taken into account.

Perspectives of using of "aseptic" drains for abdominal drainage.

AIM: Aim of the study was to evaluate the effectiveness of using different types of drain tubes to prevent and reduce the drain-associated infection rate of abdominal drainage procedures. MATERIALS AND METHODS: 80 cases of used so called "standard", "coladerm" and "chlorhexidine" drain tubes for abdominal drainage were analysed. "Standard" drain tubes were used 35 times and "coladerm" and "chlorhexidine" tubes - 20 and 25 times respectively. There were adopted in different elective and emergency so called "clean", "potentially contaminated" and "contaminated" abdominal surgical procedures. The drain tubes were removed between 2 to 14 days after the operations followed by the bacteriological study in search of bacteria growth on the surface of drainage tubes were examined. RESULTS: Of all 35 cases of used "standard" drain tubes the bacterial growth was found in 23 cases, that means 65,7%; of 20 cases of drains covered by "coladerm" polymer the bacterial growth was found in 6 cases (30%) and only in 3 cases of 25 cases of drain tubes covered by polymer and "chlorhexidine" were positive, that means 12%. The most interesting data were obtained considering the so called "clean" and "contaminated" operations. After the so called "clean" operations the bacterial growth using "standard" drain tubes was found almost in 50% of cases and in 8,3% of cases using "chlorhexidine" drain tubes. After the "potentially contaminated" and "contaminated" operations the bacterial growth was found in 68,2% using "standard" tubes, and using "coladerm" and "chlorhexidine" drain tubes - in 50% and 16,7% respectively. CONCLUSIONS: In our limited experience using of new antimicrobial polymeric composites as coatings mean the adhesion of bacteria and formation of biofilm at drainage tubes is prevented, which can significantly reduce the drain-associated infection rate. KEY WORDS: Abdominal drainage, Bacterial growth, Infection rate.

Generation of cortical neurons from human induced-pluripotent stem cells by biodegradable polymeric microspheres loaded with priming factors.

Ischemic stroke is often associated with loss of cortical neurons leading to various neurological deficits. A cell replacement based on stem cell transplantation to repair the damaged brain requires the generation of specific neuronal subtypes. Recently, induced pluripotent stem cells have been used to generate various subtypes of neurons in vitro for transplantation in stroke-damaged brains. However, whether these cells can be primed as neuronal precursors to become cortical projection neurons by means of biomaterials releasing differentiation factors is not known. Here, we report that microspheres of biodegradable poly(ester-amide) composed of adipic acid, L-phenyl-alanine and 1,4-butanediol, loaded with differentiation factors, can be used to fate human induced pluripotent stem cell-derived long-term expandable neuroepithelial-like stem cells to cortical projection neurons. The three factors, Wnt3A, BMP4 and cyclopamine, were released from loaded microspheres over at least one month following biphasic dynamic time course, promoting cortical differentiation of the cells in vitro. Microspheres did not evoke significant inflammatory response after transplantation into intact rodent brain. Our study shows the potential of biodegradable polymer microspheres to promote neuronal differentiation by continuous release of factors, thereby creating the appropriate microenvironment. This new strategy may improve the efficacy of stem cell-based therapeutic approaches.

New poly(ester urea) derived from L-leucine: electrospun scaffolds loaded with antibacterial drugs and enzymes.

Electrospun scaffolds from an amino acid containing poly(ester urea) (PEU) were developed as promising materials in the biomedical field and specifically in tissue engineering applications. The selected poly(ester urea) was obtained with a high yield and molecular weight by reaction of phosgene with a bis(α-aminoacyl)-α,ω-diol-diester monomer. The polymer having L-leucine, 1,6-hexanediol and carbonic acid units had a semicrystalline character and relatively high glass transition and melting temperatures. Furthermore it was highly soluble in most organic solvents, an interesting feature that facilitated the electrospinning process and the effective incorporation of drugs with bactericidal activity (e.g. biguanide derivatives such as clorhexidine and polyhexamethylenebiguanide) and enzymes (e.g. α-chymotrypsin) that accelerated the degradation process. Continuous micro/nanofibers were obtained under a wide range of processing conditions, being diameters of electrospun fibers dependent on the drug and solvent used. Poly(ester urea) samples were degradable in media containing lipases and proteinases but the degradation rate was highly dependent on the surface area, being specifically greater for scaffolds with respect to films. The high hydrophobicity of new scaffolds had repercussions on enzymatic degradability since different weight loss rates were found depending on how samples were exposed to the medium (e.g. forced or non-forced immersion). New scaffolds were biocompatible, as demonstrated by adhesion and proliferation assays performed with fibroblast and epithelial cells.

Improving the safety of Staphylococcus aureus polyvalent phages by their production on a Staphylococcus xylosus strain.

Team1 (vB_SauM_Team1) is a polyvalent staphylococcal phage belonging to the Myoviridae family. Phage Team1 was propagated on a Staphylococcus aureus strain and a non-pathogenic Staphylococcus xylosus strain used in industrial meat fermentation. The two Team1 preparations were compared with respect to their microbiological and genomic properties. The burst sizes, latent periods, and host ranges of the two derivatives were identical as were their genome sequences. Phage Team1 has 140,903 bp of double stranded DNA encoding for 217 open reading frames and 4 tRNAs. Comparative genomic analysis revealed similarities to staphylococcal phages ISP (97%) and G1 (97%). The host range of Team1 was compared to the well-known polyvalent staphylococcal phages phi812 and K using a panel of 57 S. aureus strains collected from various sources. These bacterial strains were found to represent 18 sequence types (MLST) and 14 clonal complexes (eBURST). Altogether, the three phages propagated on S. xylosus lysed 52 out of 57 distinct strains of S. aureus. The identification of phage-insensitive strains underlines the importance of designing phage cocktails with broadly varying and overlapping host ranges. Taken altogether, our study suggests that some staphylococcal phages can be propagated on food-grade bacteria for biocontrol and safety purposes.

Arginine-based biodegradable ether-ester polymers with low cytotoxicity as potential gene carriers.

The success of gene therapy depends on safe and effective gene carriers. Despite being widely used, synthetic vectors based on poly(ethylenimine) (PEI), poly(l-lysine) (PLL), or poly(l-arginine) (poly-Arg) are not yet fully satisfactory. Thus, both improvement of established carriers and creation of new synthetic vectors are necessary. A series of biodegradable arginine-based ether-ester polycations was developed, which consists of three main classes: amides, urethanes, and ureas. Compared to that of PEI, PLL, and poly-Arg, much lower cytotoxicity was achieved for the new cationic arginine-based ether-ester polymers. Even at polycation concentrations up to 2 mg/mL, no significant negative effect on cell viability was observed upon exposure of several cell lines (murine mammary carcinoma, human cervical adenocarcinoma, murine melanoma, and mouse fibroblast) to the new polymers. Interaction with plasmid DNA yielded compact and stable complexes. The results demonstrate the potential of arginine-based ether-ester polycations as nonviral carriers for gene therapy applications.

Synthesis, properties and applications of biodegradable polymers derived from diols and dicarboxylic acids: from polyesters to poly(ester amide)s.

Poly(alkylene dicarboxylate)s constitute a family of biodegradable polymers with increasing interest for both commodity and speciality applications. Most of these polymers can be prepared from biobased diols and dicarboxylic acids such as 1,4-butanediol, succinic acid and carbohydrates. This review provides a current status report concerning synthesis, biodegradation and applications of a series of polymers that cover a wide range of properties, namely, materials from elastomeric to rigid characteristics that are suitable for applications such as hydrogels, soft tissue engineering, drug delivery systems and liquid crystals. Finally, the incorporation of aromatic units and α-amino acids is considered since stiffness of molecular chains and intermolecular interactions can be drastically changed. In fact, poly(ester amide)s derived from naturally occurring amino acids offer great possibilities as biodegradable materials for biomedical applications which are also extensively discussed.

Cell compatible arginine containing cationic polymer: one-pot synthesis and preliminary biological assessment.

Synthetic cationic polymers are of interest as both nonviral vectors for intracellular gene delivery and antimicrobial agents. For both applications synthetic polymers containing guanidine groups are of special interest since such kind of organic compounds/polymers show a high transfection potential along with antibacterial activity. It is important that the delocalization of the positive charge of the cationic group in guanidine significantly decreases the toxicity compared to the ammonium functionality. One of the most convenient ways for incorporating guanidine groups is the synthesis of polymers composed of the amino acid arginine (Arg) via either application of Arg-based monomers or chemical modification of polymers with derivatives of Arg. It is also important to have biodegradable cationic polymers that will be cleared from the body after their function as transfection or antimicrobial agent is fulfilled. This chapter deals with a two-step/one-pot synthesis of a new biodegradable cationic polymer-poly(ethylene malamide) containing L-arginine methyl ester covalently attached to the macrochains in ß-position of the malamide residue via the α-amino group. The goal cationic polymer was synthesized by in situ interaction of arginine methyl ester dihydrochloride with intermediary poly(ethylene epoxy succinimide) formed by polycondensation of di-p-nitrophenyl-trans-epoxy succinate with ethylenediamine. The cell compatibility study with Chinese hamster ovary (CHO) and insect Schneider 2 cells (S2) within the concentration range of 0.02-500 mg/mL revealed that the new polymer is not cytotoxic. It formed nanocomplexes with pDNA (120-180 nm in size) at low polymer/DNA weight ratios (WR = 5-10). A preliminarily transfection efficiency of the Arg-containing new cationic polymer was assessed using CHO, S2, H5, and Sf9 cells.

Non-covalent nano-adducts of co-poly(ester amide) and poly(ethylene glycol): preparation, characterization and model drug-release studies.

Biodegradable, biocompatible poly(ester amide)s (co-PEAs), composed of amino acids, fatty diols and carboxylic acids, have been synthesized. To improve the performance of co-PEAs in Federal Drug Administration-approved solvents such as water and ethanol, these polymers were complexed with poly(ethylene glycol) (PEG) of 10 kDa molecular mass have been prepared by solution blending. The non-covalent adducts were purified by precipitation into hexanes. Co-PEAs are soluble in organic solvents but are insoluble in water and ethanol; however, the co-PEA/PEG (0.8:1, w/w) adducts are soluble in ethanol and slightly soluble in water. 2D-NOESY NMR spectroscopy suggests that the non-covalent adducts are held together by multiple non-covalent interactions between the -CH2- groups of the two polymers (co-PEA and PEG). Differential scanning calorimetry studies indicate that the two polymers are interacting in the non-covalent adducts; the thermal properties of the adducts are different from those of the pure polymers. The solid-state adduct structures have been determined by atomic force microscopy (AFM). By one sample preparation method, nanoscale pancake-like structures were observed with an average diameter of 260 nm and an average height of 16 nm. Films of co-PEAs and (co-PEA)/PEG adducts containing Rhodamine B Base (RhBB), a model hydrophobic drug, were prepared. From the adduct/RhBB film containing 3% RhBB, 20% of the total RhBB was released within the first 2 h. Film and adduct composition may be varied to obtain different release profiles. The studies reported here demonstrate that non-covalent conjugation is a relatively easy and effective approach in developing new materials for application as biomaterials.

In-vivo biocompatibility evaluation of stents coated with a new biodegradable elastomeric and functional polymer.

BACKGROUND: In-stent restenosis may be prevented by impregnating an antiproliferative agent in a polymer from a stent platform. This approach requires both an antiproliferative agent effective in small doses and a biocompatible polymer. METHODS: A series of new biodegradable elastomeric poly(ester-amide)(co-PEA) polymers having functional carboxyl groups for drug conjugation were synthesized from non-toxic building blocks. The in-vivo biocompatibility was tested in porcine coronary arteries, by comparing the polymer-coated stents with bare metal stents in 10 pigs. RESULTS: All animals survived until sacrifice 28 days later and follow-up angiography prior to sacrifice revealed identical diameter stenosis (21 +/- 23%) in both groups. Histology confirmed similar injury scores (0.34 +/- 0.34 compared with 0.34 +/- 0.32), inflammatory reaction (1.18 +/- 0.38 compared with 1.11 +/- 0.32) and area stenosis (26 +/- 17% compared with 28 +/- 22%). CONCLUSIONS: This study suggests that the newly developed copoly(ester-amide) elastomers may be suitable for stent-based local drug delivery.

A novel sustained-release matrix based on biodegradable poly(ester amide)s and impregnated with bacteriophages and an antibiotic shows promise in management of infected venous stasis ulcers and other poorly healing wounds.

Healing of poorly vascularized and venous stasis ulcers is often refractory to therapy, particularly when they are infected. Systemic antibiotic therapy may be of little benefit in this setting because of poor penetration of the antibiotic into the wound and the frequent associated emergence of bacterial strains resistant to common antimicrobial agents. Given the clinical significance of these problems, there is a need to explore alternative management approaches for these difficult-to-treat wounds. PhagoBioDerm is a novel wound-healing preparation consisting of a biodegradable polymer impregnated with an antibiotic and lytic bacteriophages, which was recently licensed for sale in the Republic of Georgia (one of the former Soviet Union republics). In 1999-2000, in Tbilisi, Georgia, 107 patients who had ulcers that had failed to respond to conventional therapy were treated with PhagoBioDerm alone or in combination with other interventions. The wounds/ulcers healed completely in 67 (70%) of 96 patients for whom follow-up data were available. In 22 cases in which microbiologic data were available, healing was associated with the concomitant elimination of, or a reduction in, specific pathogenic bacteria in the ulcers. Our findings suggest that this slow-release biopolymer is safe and of possible benefit in the management of refractory wounds, and they support the apparent utility of bacteriophages in this setting. Further studies, including carefully designed clinical trials, will be required to rigorously evaluate the efficacy of this novel wound dressing preparation.