Sustained growth factor delivery from bioactive PNIPAM-grafted-chitosan/heparin multilayers as a tool to promote growth and migration of cells.

Delivery of growth factors (GFs) is challenging for regulation of cell proliferation and differentiation due to their rapid inactivation under physiological conditions. Here, a bioactive polyelectrolyte multilayer (PEM) is engineered by the combination of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and glycosaminoglycans to be used as reservoir for GF storage. PNIPAM-grafted-chitosan (PChi) with two degrees of substitution (DS) are synthesized, namely LMW* (DS 0.14) and HMW (DS 0.03), by grafting low (2 kDa) and high (10 kDa) molecular weight of PNIPAM on the backbone of chitosan (Chi) to be employed as polycations to form PEM with the polyanion heparin (Hep) at pH 4. Subsequently, PEMs are chemically crosslinked to improve their stability at physiological pH 7.4. Resulting surface and mechanical properties indicate that PEM containing HMW is responsive to temperature at 20 °C and 37 °C, while LMW is not. More importantly, Hep as terminal layer combined with HMW allows not only a better retention of the adhesive protein vitronectin but also a sustained release of FGF-2 at 37 °C. With the synergistic effect of vitronectin and matrix-bound FGF-2, significant promotion on adhesion, proliferation, and migration of 3T3 mouse embryonic fibroblasts is achieved on HMW-containing PEM compared to Chi-containing PEM and exogenously added FGF-2. Thus, PEM containing PNIPAM in combination with bioactive glycosaminoglycans like Hep represents a versatile approach to fabricate a GF delivery system for efficient cell culture, which can be potentially served as cell culture substrate for production of (stem) cells and bioactive wound dressing for tissue regeneration.

Fabrication of Insoluble Elastin by Enzyme-Free Cross-Linking.

Elastin is an essential extracellular matrix protein that enables tissues and organs such as arteries, lungs, and skin, which undergo continuous deformation, to stretch and recoil. Here, an approach to fabricating artificial elastin with close-to-native molecular and mechanical characteristics is described. Recombinantly produced tropoelastin are polymerized through coacervation and allysine-mediated cross-linking induced by pyrroloquinoline quinone (PQQ). A technique that allows the recovery and repeated use of PQQ for protein cross-linking by covalent attachment to magnetic Sepharose beads is developed. The produced material closely resembles natural elastin in its molecular, biochemical, and mechanical properties, enabled by the occurrence of the cross-linking amino acids desmosine, isodesmosine, and merodesmosine. It possesses elevated resistance against tryptic proteolysis, and its Young's modulus ranging between 1 and 2 MPa is similar to that of natural elastin. The approach described herein enables the engineering of mechanically resilient, elastin-like materials for biomedical applications.

3D printed neural tissues with in situ optical dopamine sensors.

Engineered neural tissues serve as models for studying neurological conditions and drug screening. Besides observing the cellular physiological properties, in situ monitoring of neurochemical concentrations with cellular spatial resolution in such neural tissues can provide additional valuable insights in models of disease and drug efficacy. In this work, we demonstrate the first three-dimensional (3D) tissue cultures with embedded optical dopamine (DA) sensors. We developed an alginate/Pluronic F127 based bio-ink for human dopaminergic brain tissue printing with tetrapodal-shaped-ZnO microparticles (t-ZnO) additive as the DA sensor. DA quenches the autofluorescence of t-ZnO in physiological environments, and the reduction of the fluorescence intensity serves as an indicator of the DA concentration. The neurons that were 3D printed with the t-ZnO showed good viability, and extensive 3D neural networks were formed within one week after printing. The t-ZnO could sense DA in the 3D printed neural network with a detection limit of 0.137 µM. The results are a first step toward integrating tissue engineering with intensiometric biosensing for advanced artificial tissue/organ monitoring.

Lipoplex-Functionalized Thin-Film Surface Coating Based on Extracellular Matrix Components as Local Gene Delivery System to Control Osteogenic Stem Cell Differentiation.

A gene-activated surface coating is presented as a strategy to design smart biomaterials for bone tissue engineering. The thin-film coating is based on polyelectrolyte multilayers composed of collagen I and chondroitin sulfate, two main biopolymers of the bone extracellular matrix, which are fabricated by layer-by-layer assembly. For further functionalization, DNA/lipid-nanoparticles (lipoplexes) are incorporated into the multilayers. The polyelectrolyte multilayer fabrication and lipoplex deposition are analyzed by surface sensitive analytical methods that demonstrate successful thin-film formation, fibrillar structuring of collagen, and homogenous embedding of lipoplexes. Culture of mesenchymal stem cells on the lipoplex functionalized multilayer results in excellent attachment and growth of them, and also, their ability to take up cargo like fluorescence-labelled DNA from lipoplexes. The functionalization of the multilayer with lipoplexes encapsulating DNA encoding for transient expression of bone morphogenetic protein 2 induces osteogenic differentiation of mesenchymal stem cells, which is shown by mRNA quantification for osteogenic genes and histochemical staining. In summary, the novel gene-functionalized and extracellular matrix mimicking multilayer composed of collagen I, chondroitin sulfate, and lipoplexes, represents a smart surface functionalization that holds great promise for tissue engineering constructs and implant coatings to promote regeneration of bone and other tissues.

Metal Ion Doping of Alginate-Based Surface Coatings Induces Adipogenesis of Stem Cells.

Metal ions are important effectors of protein and cell functions. Here, polyelectrolyte multilayers (PEMs) made of chitosan (Chi) and alginate (Alg) were doped with different metal ions (Ca2+, Co2+, Cu2+, and Fe3+), which can form bonds with their functional groups. Ca2+ and Fe3+ ions can be deposited in PEM at higher quantities resulting in more positive ζ potentials and also higher water contact angles in the case of Fe3+. An interesting finding was that the exposure of PEM to metal ions decreases the elastic modulus of PEM. Fourier transformed infrared (FTIR) spectroscopy of multilayers provides evidence of interaction of metal ions with the carboxylic groups of Alg but not for hydroxyl and amino groups. The observed changes in wetting and surface potential are partly related to the increased adhesion and proliferation of multipotent C3H10T1/2 fibroblasts in contrast to plain nonadhesive [Chi/Alg] multilayers. Specifically, PEMs doped with Cu2+ and Fe3+ ions greatly promote cell attachment and adipogenic differentiation, which indicates that changes in not only surface properties but also the bioactivity of metal ions play an important role. In conclusion, metal ion-doped multilayer coatings made of alginate and chitosan can promote the differentiation of multipotent cells on implants without the use of other morphogens like growth factors.

Cerosomes as skin repairing agent: Mode of action studies with a model stratum corneum layer at liquid/air and liquid/solid interfaces.

The stratum corneum (SC) is the largest physical barrier of the human body. It protects against physical, chemical and biological damages, and avoids evaporation of water from the deepest skin layers. For its correct functioning, the homeostasis of the SC lipid matrix is fundamental. An alteration of the lipid matrix composition and in particular of its ceramide (CER) fraction can lead to the development of pathologies such as atopic dermatitis and psoriasis. Different studies showed that the direct replenishment of SC lipids on damaged skin had positive effects on the recovery of its barrier properties. In this work, cerosomes, i.e. liposomes composed of SC lipids, have been successfully prepared in order to investigate the mechanism of interaction with a model SC lipid matrix. The cerosomes contain CER[NP], D-CER[AP], stearic acid and cholesterol. In addition, hydrogenated soybean phospholipids have been added to one of the formulations leading to an increased stability at neutral pH. For the mode of action studies, monolayer models at the air-water interface and on solid support have been deployed. The results indicated that a strong interaction occurred between SC monolayers and the cerosomes. Since both systems were negatively charged, the driving force for the interaction must be based on the ability of CERs head groups to establish intermolecular hydrogen bonding networks that energetically prevailed against the electrostatic repulsion. This work proved for the first time the mode of action by which cerosomes exploit their function as skin barrier repairing agents on the SC.

Elastic fibers: formation, function, and fate during aging and disease.

Elastic fibers are extracellular components of higher vertebrates and confer elasticity and resilience to numerous tissues and organs such as large blood vessels, lungs, and skin. Their formation and maturation take place in a complex multistage process called elastogenesis. It requires interactions between very different proteins but also other molecules and leads to the deposition and crosslinking of elastin's precursor on a scaffold of fibrillin-rich microfibrils. Mature fibers are exceptionally resistant to most influences and, under healthy conditions, retain their biomechanical function over the life of the organism. However, due to their longevity, they accumulate damages during aging. These are caused by proteolytic degradation, formation of advanced glycation end products, calcification, oxidative damage, aspartic acid racemization, lipid accumulation, carbamylation, and mechanical fatigue. The resulting changes can lead to diminution or complete loss of elastic fiber function and ultimately affect morbidity and mortality. Particularly, the production of elastokines has been clearly shown to influence several life-threatening diseases. Moreover, the structure, distribution, and abundance of elastic fibers are directly or indirectly influenced by a variety of inherited pathological conditions, which mainly affect organs and tissues such as skin, lungs, or the cardiovascular system. A distinction can be made between microfibril-related inherited diseases that are the result of mutations in diverse microfibril genes and indirectly affect elastogenesis, and elastinopathies that are linked to changes in the elastin gene. This review gives an overview on the formation, structure, and function of elastic fibers and their fate over the human lifespan in health and disease.

Effect of metal ions on the physical properties of multilayers from hyaluronan and chitosan, and the adhesion, growth and adipogenic differentiation of multipotent mouse fibroblasts.

Polyelectrolyte multilayers (PEMs) consisting of the polysaccharides hyaluronic acid (HA) as the polyanion and chitosan (Chi) as the polycation were prepared with layer-by-layer technique (LbL). The [Chi/HA]5 multilayers were exposed to solutions of metal ions (Ca2+, Co2+, Cu2+ and Fe3+). Binding of metal ions to [Chi/HA]5 multilayers by the formation of complexes with functional groups of polysaccharides modulates their physical properties and the bioactivity of PEMs with regard to the adhesion and function of multipotent murine C3H10T1/2 embryonic fibroblasts. Characterization of multilayer formation and surface properties using different analytical methods demonstrates changes in the wetting, surface potential and mechanical properties of multilayers depending on the concentration and type of metal ion. Most interestingly, it is observed that Fe3+ metal ions greatly promote adhesion and spreading of C3H10T1/2 cells on the low adhesive [Chi/HA]5 PEM system. The application of intermediate concentrations of Cu2+, Ca2+ and Co2+ as well as low concentrations of Fe3+ to PEMs also results in increased cell spreading. Moreover, it can be shown that complex formation of PEMs with Cu2+ and Fe3+ ions leads to increased metabolic activity in cells after 24 h and induces cell differentiation towards adipocytes in the absence of any additional adipogenic media supplements. Overall, complex formation of [Chi/HA]5 PEM with metal ions like Cu2+ and Fe3+ represents an interesting and cheap alternative to the use of growth factors for making cell-adhesive coatings and guiding stem cell differentiation on implants and scaffolds to regenerate connective-type of tissues.

A guide to the composition and functions of the extracellular matrix.

Extracellular matrix (ECM) is a dynamic 3-dimensional network of macromolecules that provides structural support for the cells and tissues. Accumulated knowledge clearly demonstrated over the last decade that ECM plays key regulatory roles since it orchestrates cell signaling, functions, properties and morphology. Extracellularly secreted as well as cell-bound factors are among the major members of the ECM family. Proteins/glycoproteins, such as collagens, elastin, laminins and tenascins, proteoglycans and glycosaminoglycans, hyaluronan, and their cell receptors such as CD44 and integrins, responsible for cell adhesion, comprise a well-organized functional network with significant roles in health and disease. On the other hand, enzymes such as matrix metalloproteinases and specific glycosidases including heparanase and hyaluronidases contribute to matrix remodeling and affect human health. Several cell processes and functions, among them cell proliferation and survival, migration, differentiation, autophagy, angiogenesis, and immunity regulation are affected by certain matrix components. Structural alterations have been also well associated with disease progression. This guide on the composition and functions of the ECM gives a broad overview of the matrisome, the major ECM macromolecules, and their interaction networks within the ECM and with the cell surface, summarizes their main structural features and their roles in tissue organization and cell functions, and emphasizes the importance of specific ECM constituents in disease development and progression as well as the advances in molecular targeting of ECM to design new therapeutic strategies.

Tissue response to biphasic calcium phosphate covalently modified with either heparin or hyaluronic acid in a mouse subcutaneous implantation model.

Biphasic calcium phosphate (BCP) materials are widely employed as bone substitute materials due to their resorption/degradation properties. Inflammation after implantation of such materials represents a prerequisite for bone tissue repair and regeneration but can be also problematic if it is not only transient and if it is followed by fibrosis and scarring. Here, we modified BCP covalently with hyaluronan (HA) and heparin (Hep), glycosaminoglycans that possess anti-inflammatory properties. Beside the characterization of particle surface properties, the focus was on in vivo tissue response after subcutaneous implantation in mice. Histological analysis revealed a decrease in signs of inflammatory response to BCP when modified with either HA or Hep. Reduced vascularization after 30 days was noticed when BCP was modified with either HA or Hep with greater cellularity in all examined time points. Compared to plain BCP, expression of endothelial-related genes Flt1 and Vcam1 was higher in BCP-HA and BCP-Hep group at day 30. Expression of osteogenesis-related genes Sp7 and Bglap after 30 days was the highest in BCP group, followed by BCP-Hep, while the lowest expression was in BCP-HA group which correlates with collagen amount. Hence, coating of BCP particles with HA seems to suppress inflammatory response together with formation of new bone-like tissue, while the presence of Hep delays the onset of inflammatory response but permits osteogenesis in this subcutaneous bone-forming model. Transferring the results of this study to other coated materials intended for biomedical application may also pave the way to reduction of inflammation after their implantation.

Towards the Therapeutic Use of Thrombospondin 1/CD47 Targeting TAX2 Peptide as an Antithrombotic Agent.

OBJECTIVE: TSP-1 (thrombospondin 1) is one of the most expressed proteins in platelet α-granules and plays an important role in the regulation of hemostasis and thrombosis. Interaction of released TSP-1 with CD47 membrane receptor has been shown to regulate major events leading to thrombus formation, such as, platelet adhesion to vascular endothelium, nitric oxide/cGMP (cyclic guanosine monophosphate) signaling, platelet activation as well as aggregation. Therefore, targeting TSP-1:CD47 axis may represent a promising antithrombotic strategy. Approach and Results: A CD47-derived cyclic peptide was engineered, namely TAX2, that targets TSP-1 and selectively prevents TSP-1:CD47 interaction. Here, we demonstrate for the first time that TAX2 peptide strongly decreases platelet aggregation and interaction with collagen under arterial shear conditions. TAX2 also delays time for complete thrombotic occlusion in 2 mouse models of arterial thrombosis following chemical injury, while Thbs1-/- mice recapitulate TAX2 effects. Importantly, TAX2 administration is not associated with increased bleeding risk or modification of hematologic parameters. CONCLUSIONS: Overall, this study sheds light on the major contribution of TSP-1:CD47 interaction in platelet activation and thrombus formation while putting forward TAX2 as an innovative antithrombotic agent with high added-value.

Extracellular Matrix Stiffness and Composition Regulate the Myofibroblast Differentiation of Vaginal Fibroblasts.

Fibroblast to myofibroblast differentiation is a key feature of wound-healing in soft tissues, including the vagina. Vaginal fibroblasts maintain the integrity of the vaginal wall tissues, essential to keep pelvic organs in place and avoid pelvic organ prolapse (POP). The micro-environment of vaginal tissues in POP patients is stiffer and has different extracellular matrix (ECM) composition than healthy vaginal tissues. In this study, we employed a series of matrices with known stiffnesses, as well as vaginal ECMs, in combination with vaginal fibroblasts from POP and healthy tissues to investigate how matrix stiffness and composition regulate myofibroblast differentiation in vaginal fibroblasts. Stiffness was positively correlated to production of α-smooth muscle actin (α-SMA). Vaginal ECMs induced myofibroblast differentiation as both α-SMA and collagen gene expressions were increased. This differentiation was more pronounced in cells seeded on POP-ECMs that were stiffer than those derived from healthy tissues and had higher collagen and elastin protein content. We showed that stiffness and ECM content regulate vaginal myofibroblast differentiation. We provide preliminary evidence that vaginal fibroblasts might recognize POP-ECMs as scar tissues that need to be remodeled. This is fundamentally important for tissue repair, and provides a rational basis for POP disease modelling and therapeutic innovations in vaginal reconstruction.

Direct three-dimensional imaging for morphological analysis of electrospun fibers with laboratory-based Zernike X-ray phase-contrast computed tomography.

Electrospinning is a well-established and widely used method for the production of protein-based fibrous biomaterials. The visualization of the morphology and the characterization of sample features related to the three-dimensional (3D) structure, like the porosity and fibers thickness, is crucial for the design and fabrication of tailor-made and application-optimized materials. Here, we evaluated the benefits of using 3D X-ray imaging in a laboratory setup with a resolution in the sub-micrometer range for the characterization of electrospun gelatin fibrous mats. We used phase-contrast X-ray computed tomography at the nanoscale (nano-CT) for the evaluation of the time-course morphological changes of the mats induced by chemical cross-linking of the gelatin fibers. We present an image processing protocol that enables the segmentation of the fibers and quantification of the mats porosity, the analysis of the shape and size of the pores, and of the fibers thickness and orientation. We compared the results obtained from the processed nano-CT data with those obtained with the conventional methods used for the characterization of electrospun fibrous materials, and we discuss the advantages and limitations of each method when applied to gelatin electrospun samples. Our results reveal that the use of phase-contrast nano-CT provides quick additional and relevant information for the characterization of fibrous mats and, thus, provides beneficial insights for the design and fabrication of novel fibrous materials.

Expression of elastolytic cathepsins in human skin and their involvement in age-dependent elastin degradation.

BACKGROUND: Skin ageing is associated with structure-functional changes in the extracellular matrix, which is in part caused by proteolytic degradation. Since cysteine cathepsins are major matrix protein-degrading proteases, we investigated the age-dependent expression of elastolytic cathepsins K, S, and V in human skin, their in vitro impact on the integrity of the elastic fibre network, their cleavage specificities, and the release of bioactive peptides. METHODS: Cathepsin-mediated degradation of human skin elastin samples was assessed from young to very old human donors using immunohistochemical and biochemical assays, scanning electron microscopy, and mass spectrometry. RESULTS: Elastin samples derived from patients between 10 and 86 years of age were analysed and showed an age-dependent deterioration of the fibre structure from a dense network of thinner fibrils into a beaded and porous mesh. Reduced levels of cathepsins K, S, and V were observed in aged skin with a predominant epidermal expression. Cathepsin V was the most potent elastase followed by cathepsin K and S. Biomechanical analysis of degraded elastin fibres corroborated the destructive activity of cathepsins. Mass spectrometric determination of the cleavage sites in elastin revealed that all three cathepsins predominantly cleaved in hydrophobic domains. The degradation of elastin was efficiently inhibited by an ectosteric inhibitor. Furthermore, the degradation of elastin fibres resulted in the release of bioactive peptides, which have previously been associated with various pathologies. CONCLUSION: Cathepsins are powerful elastin-degrading enzymes and capable of generating a multitude of elastokines. They may represent a viable target for intervention strategies to reduce skin ageing.

Unique molecular networks: Formation and role of elastin cross-links.

Elastic fibers are essential assemblies of vertebrates and confer elasticity and resilience to various organs including blood vessels, lungs, skin, and ligaments. Mature fibers, which comprise a dense and insoluble elastin core and a microfibrillar mantle, are extremely resistant toward intrinsic and extrinsic influences and maintain elastic function over the human lifespan in healthy conditions. The oxidative deamination of peptidyl lysine to peptidyl allysine in elastin's precursor tropoelastin is a crucial posttranslational step in their formation. The modification is catalyzed by members of the family of lysyl oxidases and the starting point for subsequent manifold condensation reactions that eventually lead to the highly cross-linked elastomer. This review summarizes the current understanding of the formation of cross-links within and between the monomer molecules, the molecular sites, and cross-link types involved and the pathological consequences of abnormalities in the cross-linking process.

Risperidone-Loaded PLGA-Lipid Particles with Improved Release Kinetics: Manufacturing and Detailed Characterization by Electron Microscopy and Nano-CT.

For parenteral controlled drug release, the desired zero order release profile with no lag time is often difficult to achieve. To overcome the undesired lag time of the current commercial risperidone controlled release formulation, we developed PLGA-lipid microcapsules (MCs) and PLGA-lipid microgels (MGs). The lipid phase was composed of middle chain triglycerides (MCT) or isopropylmyristate (IPM). Hydroxystearic acid was used as an oleogelator. The three-dimensional inner structure of Risperidone-loaded MCs and MGs was assessed by using the invasive method of electron microscopy with focused ion beam cutting (FIB-SEM) and the noninvasive method of high-resolution nanoscale X-ray computed tomography (nano-CT). FIB-SEM and nano-CT measurements revealed the presence of highly dispersed spherical structures around two micrometres in size. Drug release kinetics did strongly depend on the used lipid phase and the presence or absence of hydroxystearic acid. We achieved a nearly zero order release without a lag time over 60 days with the MC-MCT formulation. In conclusion, the developed lipid-PLGA microparticles are attractive alternatives to pure PLGA-based particles. The advantages include improved release profiles, which can be easily tuned by the lipid composition.

Effect of Different Crosslinking Strategies on Physical Properties and Biocompatibility of Freestanding Multilayer Films Made of Alginate and Chitosan.

Freestanding multilayer films prepared by layer-by-layer technique have attracted interest as promising materials for wound dressings. The goal is to fabricate freestanding films using chitosan (CHI) and alginate (ALG) including subsequent crosslinking to improve the mechanical properties of films while maintaining their biocompatibility. Three crosslinking strategies are investigated, namely use of calcium ions for crosslinking ALG, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide combined with N-hydroxysuccinimide for crosslinking ALG with CHI, and Genipin for crosslinking chitosan inside the films. Different characteristics, such as surface morphology, wettability, swelling, roughness, and mechanical properties are investigated showing that films became thinner, exhibited rougher surfaces, had lower water uptake, and increased mechanical strength after crosslinking. Changes of wettability are moderate and dependent on the crosslinking method. In vitro cytotoxicity and cell attachment studies with human dermal fibroblasts show that freestanding CHI-ALG films represent a poorly adhesive substratum for fibroblasts, while studies using incubation of plastic-adherent fibroblast beneath floating films show no signs of cytotoxicity in a time frame of 7 days. Results from cell experiments combined with film characteristics after crosslinking, indicate that crosslinked freestanding films made of ALG and CHI may be interesting candidates for wound dressings.

MMP-14 degrades tropoelastin and elastin.

Matrix metalloproteinases are a class of enzymes, which degrade extracellular matrix components such as collagens, elastin, laminin or fibronectin. So far, four matrix metalloproteinases have been shown to degrade elastin and its precursor tropoelastin, namely matrix metalloproteinase-2, -7, -9 and -12. This study focuses on investigating the elastinolytic capability of membrane-type 1 matrix metalloproteinase, also known as matrix metalloproteinase-14. We digested recombinant human tropoelastin and human skin elastin with matrix metalloproteinase-14 and analyzed the peptide mixtures using complementary mass spectrometric techniques and bioinformatics tools. The results and additional molecular docking studies show that matrix metalloproteinase-14 cleaves tropoelastin as well as elastin. While tropoelastin was well degraded, fewer cleavages occurred in the highly cross-linked mature elastin. The study also provides insights into the cleavage preferences of the enzyme. Similar to cleavage preferences of matrix metalloproteinases-2, -7, -9 and -12, matrix metalloproteinase-14 prefers small and medium-sized hydrophobic residues including Gly, Ala, Leu and Val at cleavage site P1'. Pro, Gly and Ala were preferably found at P1-P4 and P2'-P4' in both tropoelastin and elastin. Cleavage of mature skin elastin by matrix metalloproteinase-14 released a variety of bioactive elastin peptides, which indicates that the enzyme may play a role in the development and progression of cardiovascular diseases that go along with elastin breakdown.

A comprehensive map of human elastin cross-linking during elastogenesis.

Elastin is an essential structural protein in the extracellular matrix of vertebrates. It is the core component of elastic fibers, which enable connective tissues such as those of the skin, lungs or blood vessels to stretch and recoil. This function is provided by elastin's exceptional properties, which mainly derive from a unique covalent cross-linking between hydrophilic lysine-rich motifs of units of the monomeric precursor tropoelastin. To date, elastin's cross-linking is poorly investigated. Here, we purified elastin from human tissue and cleaved it into soluble peptides using proteases with different specificities. We then analyzed elastin's molecular structure by identifying unmodified residues, post-translational modifications and cross-linked peptides by high-resolution mass spectrometry and amino acid analysis. The data revealed the presence of multiple isoforms in parallel and a complex and heterogeneous molecular interconnection. We discovered that the same lysine residues in different monomers were simultaneously involved in various cross-link types or remained unmodified. Furthermore, both types of cross-linking domains, Lys-Pro and Lys-Ala domains, participate not only in bifunctional inter- but also in intra-domain cross-links. We elucidated the sequences of several desmosine-containing peptides and the contribution of distinct domains such as 6, 14 and 25. In contrast to earlier assumptions proposing that desmosine cross-links are formed solely between two domains, we elucidated the structure of a peptide that proves a desmosine formation with participation of three Lys-Ala domains. In summary, these results provide new and detailed insights into the cross-linking process, which takes place within and between human tropoelastin units in a stochastic manner.

Ligand-Binding Cooperativity Effects in Polymer-Protein Conjugation.

We present an electron paramagnetic resonance (EPR) spectroscopic characterization of structural and dynamic effects that stem from post-translational modifications of bovine serum albumin (BSA), an established model system for polymer-protein conjugation. Beyond the typical drug delivery and biocompatibility aspect of such systems, we illustrate the causes that alter internal dynamics and therefore functionality in terms of ligand-binding to the BSA protein core. Uptake of the paramagnetic fatty acid derivative 16-doxyl stearic acid by several BSA-based squaric acid macroinitiators and polymer-protein conjugates was studied by EPR spectroscopy, aided by dynamic light scattering (DLS) and zeta potential measurements. The conjugates were grafted from oligo(ethylene glycol) methyl ether methacrylate (OEGMA), forming an overall core-shell-like structure. It is found that ligand-binding and associated parameters such as binding affinity, cooperativity, and the number of binding sites of BSA change drastically with the extent of surface modification. In the course of processing BSA, the ligands also change their preference for individual binding sites, as observed from a comparative view of their spatial alignments in double electron electron resonance (DEER) experiments. The protein-attached polymers constitute a diffusion barrier that significantly hamper ligand uptake. Moreover, zeta potentials (ζ) decrease linearly with the degree of surface modification in protein macroinitiators and an effective dielectric constant can be estimated for the polymer layer in the conjugates. All this suggests that ligand uptake characteristics in BSA can be fine-tuned by the extent and nature of such post-translational modifications (PTMs). We show that EPR spectroscopy is suitable for quantifying these subtle PTM-based functional effects from self-assembly of substrate and ligand.