Intramolecular Cross-linking Research Articles

Radiation synthesis of poly(acrylic acid) nanogels for drug delivery applications – post-synthesis product colloidal stability

Abstract Synthesis of polymer nanogels (NGs) for biomedical applications is considered to be a very promising application in radiation engineering. Under high-dose pulse irradiation of dilute aqueous polymer solution, reactive species generated by water radiolysis can create multiple radicals on each macromolecule and consequently induce intramolecular cross-linking of polymer chains, resulting in NG formation. The obtained products are free from harmful monomers, initiators, and cross-linking agents, which makes them potentially applicable for drug delivery applications. One of the biggest challenges in handling and use of nanoparticles, however, is the colloidal stability, when aqueous suspensions are stored for prolonged periods. Therefore, development of the best protocols for the particular nanocarrier storage is key. To address this need, we have performed the prospective study in which we systematically assessed the influence of various processing and storage scenarios feasible in our lab, on the colloidal stability of the radiation-synthesized poly(acrylic acid) (PAA) NG particles in suspension. This allowed us to choose the optimal way of handling the product after its synthesis. We confirmed that none of the strategies we used and tested are substantially detrimental to our product. Filtration with 0.2-μm filters was proven sufficient for sample purification and prolonged storage in aqueous suspension did not exert a negative effect on the colloidal stability of particles suspension. We have also demonstrated that lyoprotectant-free lyophilization was suitable for our polymer nanoparticles. This is an important fact for further application of particles as nanocarriers for biologically active compounds such as targeting ligands or therapeutic moieties.

Cytosolic Delivery of Single-Chain Polymer Nanoparticles.

Cytosolic delivery of therapeutic agents is key to improving their efficacy, as the therapeutics are primarily active in specific organelles. Single-chain polymer nanoparticles (SCNPs) are a promising nanocarrier platform in biomedical applications due to their unique size range of 5-20 nm, modularity, and ease of functionalization. However, cytosolic delivery of SCNPs remains challenging. Here, we report the synthesis of active ester-functional SCNPs of approximately 10 nm via intramolecular thiol-Michael addition cross-linking and their functionalization with increasing amounts of tertiary amines 0 to 60 mol % to obtain SCNPs with increasing positive surface charges. No significant cytotoxicity was detected in bEND.3 cells for the SCNPs, except when SCNPs with high amounts of tertiary amines were incubated over prolonged periods of time at high concentrations. Cellular uptake of the SCNPs was analyzed, presenting different uptake behavior depending on the degree of functionalization. Confocal microscopy revealed successful cytosolic delivery of SCNPs with high degrees of functionalization (45%, 60%), while SCNPs with low amounts (0% to 30%) of tertiary amines showed high degrees of colocalization with lysosomes. This work presents a strategy to direct the intracellular location of SCNPs by controlled surface modification to improve intracellular targeting for biomedical applications.

Intramolecular Crosslinking of Polyvinylidene Fluoride by Homogeneous Solution Irradiation

Intramolecular Crosslinking of Polyvinylidene Fluoride by Homogeneous Solution Irradiation

On the Mechanism and Kinetics of Synthesizing Polymer Nanogels by Ionizing Radiation-Induced Intramolecular Crosslinking of Macromolecules.

Nanogels—internally crosslinked macromolecules—have a growing palette of potential applications, including as drug, gene or radioisotope nanocarriers and as in vivo signaling molecules in modern diagnostics and therapy. This has triggered considerable interest in developing new methods for their synthesis. The procedure based on intramolecular crosslinking of polymer radicals generated by pulses of ionizing radiation has many advantages. The substrates needed are usually simple biocompatible polymers and water. This eliminates the use of monomers, chemical crosslinking agents, initiators, surfactants, etc., thus limiting potential problems with the biocompatibility of products. This review summarizes the basics of this method, providing background information on relevant aspects of polymer solution thermodynamics, radiolysis of aqueous solutions, generation and reactions of polymer radicals, and the non-trivial kinetics and mechanism of crosslinking, focusing on the main factors influencing the outcomes of the radiation synthesis of nanogels: molecular weight of the starting polymer, its concentration, irradiation mode, absorbed dose of ionizing radiation and temperature. The most important techniques used to perform the synthesis, to study the kinetics and mechanism of the involved reactions, and to assess the physicochemical properties of the formed nanogels are presented. Two select important cases, the synthesis of nanogels based on polyvinylpyrrolidone (PVP) and/or poly(acrylic acid) (PAA), are discussed in more detail. Examples of recent application studies on radiation-synthesized PVP and PAA nanogels in transporting drugs across the blood–brain barrier and as targeted radioisotope carriers in nanoradiotherapy are briefly described.

Modulating physicochemical properties of collagen films by cross-linking with glutaraldehyde at varied pH values

Collagen films cross-linked with glutaraldehyde (GTA) at different pH values were prepared to explore the influence of pH on their physicochemical properties. The films were characterized using FTIR spectroscopy, XRD analysis, SDS-PAGE, shrinkage temperature, fluorescence analysis and SEM. The results indicated that cross-linking degree and reaction position were different at different pH values. The optimal cross-linking degree in collagen films was obtained under neutral and alkaline conditions. The main reaction under acidic conditions was intramolecular cross-linking and this gradually shifted to intermolecular cross-linking with an increase in pH. Cross-linked films had improved mechanical properties and thermal stability as well as higher b* values compared to films without cross-linking. These improvements correlated with the degree and position of cross-linking reactions. Controlling the reaction pH can be used to modulate the physicochemical properties of the collagen films to match specific application requirements.

Single-Chain Nanoparticle-Based Coatings with Improved Bactericidal Activity and Antifouling Properties.

Dual-function antibacterial surfaces have exhibited promising potential in addressing implant-associated infections. However, both bactericidal and antifouling properties need to be further improved prior to practical uses. Herein, we report the preparation and properties of a linear block copolymer coating (LP-KF) and a single-chain nanoparticle coating (NP-KF) with poly(ethylene glycol) (PEG) and cationic polypeptide segments. NP-KF with cyclic PEG segments and densely charged polypeptide segments was expected to display improved bactericidal and antifouling properties. LP-KF was prepared by the combination of ring-opening polymerization of N-carboxyanhydride (NCA) monomers and subsequent deprotection. NP-KF was prepared by intramolecular cross-linking of LP-KF in diluted solutions. Both LP-KF- and NP-KF-coated PDMS surfaces were prepared by dipping with polydopamine-coated surfaces. They showed superior in vitro bactericidal activity against both Staphylococcus aureus and Escherichia coli with >99.9% killing efficacy, excellent protein adsorption resistance, antibacterial adhesion, and low cytotoxicity. The NP-KF coating showed higher bactericidal activity and antifouling properties than its linear counterpart. It also showed significant anti-infective property and histocompatibility in vivo, which makes it a good candidate for implants and biomedical device applications.

Stapled β-Hairpins Featuring 4-Mercaptoproline.

Peptides constrained by intramolecular cross-links, especially stapled α-helices, have emerged as versatile scaffolds for drug development. However, there are fewer examples of similarly constrained scaffolds for other secondary structures. Here, we used a novel computational strategy to identify an optimal staple for antiparallel β-strands, and then we incorporated that staple within a β-hairpin peptide. The hairpin uses 4-mercaptoproline as a novel staple component, which contributes to a unique, kinked structure. The stapled hairpins show a high degree of structure in aqueous solution, excellent resistance to degradation in cell lysates, and cytosolic penetration at micromolar concentrations. They also overlay with a unique subset of kinked hairpin motifs at protein-protein interaction interfaces. Thus, these scaffolds represent promising starting points for developing inhibitors of cellular protein-protein interactions.

Influence of pH on radical reactions between kynurenic acid and amino acids tryptophan and tyrosine. Part II. Amino acids within the protein globule of lysozyme

An acidosis, a decrease of pH within a living tissue, may alter yields of radical reactions if participating radicals undergo partial or complete protonation. One of photosensitizers found in the human eye lens, kynurenic acid (KNA−), possesses pKa 5.5 for its radical form that is close to physiological pH 6.89 for a healthy lens. In this work we studied the influence of pH on mechanisms and products of photoinduced radical reactions between KNA− and amino acids tryptophan (Trp) and tyrosine (Tyr) within a globule of model protein, Hen White Egg Lysozyme (HEWL). Our results show that the rate constant of back electron transfer from kynurenyl to HEWL• radicals with the restoration of initial reagents – the major decay pathway for these radicals – does not change in the pH 3–7. The quantum yield of HEWL degradation is also pH independent, however a shift of pH from 7 to 5 completely changes the outcome of photoinduced damage to HEWL from intermolecular cross-linking to oxygenation. HPLC-MS analysis has shown that four of six Trp and all Tyr residues of HEWL are modified in different extents at all pH, but the lowering of pH from 7 to 5 significantly changes the direction of main photodamage from Trp62 to Trp108 located at the entrance and bottom of enzymatic center, respectively. A decrease of intermolecular cross-links via Trp62 is followed by an increase in quantities of intramolecular cross-links Tyr20-Tyr23 and Tyr23-Tyr53. The obtained results point out the competence of cross-linking and oxygenation reactions for Trp and Tyr radicals within a protein globule and significant increase of oxygenation to the total damage of protein in the case of cross-linking deceleration by coulombic repulsion of positively charged protein globules.

Supercooled melt structure and dynamics of single-chain nanoparticles: A computer simulation study.

By using coarse-grained molecular dynamics simulations, we have investigated the structure and dynamics of supercooled single-chain cross-linked nanoparticle (SCNP) melts having a range of cross-linking degrees ϕ. We find a nearly linear increase in glass-transition temperature (Tg) with increasing ϕ. Correspondingly, we have also experimentally synthesized a series of polystyrene-based SCNPs and have found that the measured Tg estimated from differential scanning calorimetry is qualitatively consistent with the trend predicted by our simulation estimates. Experimentally, an increase in Tg as large as ΔTg = 61K for ϕ = 0.36 is found compared with their linear chain counterparts, indicating that the changes in dynamics with cross-links are quite appreciable. We attribute the increase in Tg to the enlarged effective hard-core volume and the corresponding reduction in the free volume of the polymer segments. Topological constraints evidently frustrate the local packing. In addition, the introduction of intra-molecular cross-linking bonds slows down the structural relaxation and simultaneously enhances the local coupling motion on the length scales within SCNPs. Consequently, a more pronounced dynamical heterogeneity (DH) is observed for larger ϕ, as quantified by measuring the dynamical correlation length through the four-point susceptibility parameter, χ4. The increase in DH is directly related to the enhanced local cooperative motion derived from intra-molecular cross-linking bonds and structural heterogeneity derived from the cross-linking process. These results shed new light on the influence of intra-molecular topological constraints on the segmental dynamics of polymer melts.

Inhibition Effect of Ultrasound on the Formation of Lysinoalanine in Rapeseed Protein Isolates during pH Shift Treatment.

pH shift is an effective technique for modifying functional properties of food proteins. However, it can increase lysinoalanine (LAL) content under alkali conditions, thus limiting the use of proteins. This study investigated the inhibition effect of ultrasonic parameters on LAL formation in rapeseed protein isolates (RPI) during pH shift treatment (pH-ST). Results showed that the content of LAL decreased by 49.5% and 74.1%, following the use of ultrasound (28 kHz, 40 W/L, 40 °C, and 30 min) under alkali and acidic treatment, respectively. Structural analysis showed that after ultrasonic irradiation, increased sulfhydryl groups and amino acids reduced the dehydroalanine and, thus, decreased LAL content. Particle size, secondary structure, and microstructure (SEM, AFM) analyses showed relative dispersion in protein distribution, reducing intermolecular or intramolecular cross-linking, thereby lowering the LAL content. Thus, ultrasonic-aided pH-ST may be an operational technique toward minimizing LAL formation in RPI.

Gel Formation in Reversibly Cross-Linking Polymers

By means of Langevin dynamics simulations, we investigate the gel formation of randomly functionalized polymers in solution, with the ability to form both intra- and intermolecular reversible bonds. Under highly dilute conditions, these polymers form soft nano-objects (so-called single-chain nanoparticles, SCNPs), resulting from the purely intramolecular cross-linking of the reactive functional groups. Here we show that the competition between intra- and intermolecular bonds at finite concentration is governed by a delicate balance of various entropic contributions and leads to a density dependent effective valence. System-spanning networks are formed at relatively low monomer densities and their stability is mediated by just a small number of intermolecular connections per chain. The formation of intermolecular bonds furthermore can induce a non-monotonic dependence of the polymer size on the density for long bond lifetimes. Concomitantly, the polymers in the percolating cluster adopt an intramolecular structure characteristic for self-avoiding chains, which constitutes a strong contrast to the fractal globular behavior of irreversible SCNPs in crowded solutions with purely topological interactions (no intermolecular bonds). Finally, we study the dynamics of the system, which displays signatures expected for reversible gel-forming systems. An interesting behavior emerges in the reorganization dynamics of the percolating cluster. The relaxation is mostly mediated by the diffusion over long distances, through breaking and formation of bonds, of chains that do not leave the percolating cluster. Regarding the few chains that are transiently free, the time they spend until they reattach to the cluster is solely governed by the bond strength.

Protein Macrocyclization for Tertiary Structure Stabilization.

Proteins possess unique molecular recognition capabilities and enzymatic activities, features that are usually tied to a particular tertiary structure. To make use of proteins for biotechnological and biomedical purposes, it is often required to enforce their tertiary structure in order to ensure sufficient stability under the conditions inherent to the application of interest. The introduction of intramolecular crosslinks has proven efficient in stabilizing native protein folds. Herein, we give an overview of methods that allow the macrocyclization of expressed proteins, discussing involved reaction mechanisms and structural implications.

Synthesis of Photo, Oxidation, Reduction Triple‐Stimuli‐Responsive Interface‐Cross‐Linked Polymer Micelles as Nanocarriers for Controlled Release

AbstractStimuli‐responsive chemical bonds have already been widely applied to construct nanomaterials, however, such materials rarely have great amounts of responsive groups and stable structures. Herein, a triple‐stimuli‐responsive interface‐cross‐linked (ICL) copolymer which is synthesized via Cu(I)‐catalyzed azide‐alkyne cycloaddition click polymerization and atom transfer radical polymerization (ATRP), is reported. N, N′‐bis(bromoacetyl) cystamine is used for intramolecular crosslinking of the linear polymer chains. The ο‐nitrobenzyl groups, peroxalate ester bonds, disulfide bonds, and triazole units are regularly and repeatedly arranged in the hydrophobic blocks, therefore enabling the polymer a large amount of responsive chemical bonds. The polymer self‐assembles into spherical micelles with uniform size in deionized water. Using hydrophobic dye Nile red (NR) as a model drug to evaluate the effect of release in vitro, the multi‐stimuli‐sensitive release properties of the ICL micelles are investigated in detail by means of UV–vis, transmission electron microscopy, dynamic light scattering, and fluorescence spectrophotometry, and the results indicate the ICL micelles can respond to the photo, oxidation, or reduction responsiveness either independently or synergistically. Furthermore, the cross‐linked structure enhances the stability of the micelles and rarely impacts the total amount of drug release. The research may have applications in the field of drug delivery systems for cancer therapy.

Age-related type I collagen modifications reveal tissue-defining differences between ligament and tendon

Tendons and ligaments tend to be pooled into a single category as dense elastic bands of collagenous connective tissue. They do have many similar properties, for example both tissues are flexible cords of fibrous tissue that join bone to either muscle or bone. Tendons and ligaments are both prone to degenerate and rupture with only limited capacity to heal, although tendons tend to heal faster than ligaments. Type I collagen constitutes about 80% of the dry weight of tendons and ligaments and is principally responsible for the core strength of each tissue. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including proline and lysine hydroxylation, hydroxylysine glycosylation and covalent cross-linking. The chemistry, placement and quantity of intramolecular and intermolecular cross-links are believed to be key contributors to the tissue-specific variations in material strength and biological properties of collagens. As tendons and ligaments grow and develop, the collagen cross-links are known to chemically mature, strengthen and change in profile. Accordingly, changes in cross-linking and other post-translational modifications are likely associated with tissue development and degeneration. Using mass spectrometry, we have compared tendon and ligaments from fetal and adult bovine knee joints to investigate changes in collagen post-translational properties. Although hydroxylation levels at the type I collagen helical cross-linking lysine residues were similar in all adult tissues, ligaments had significantly higher levels of glycosylation at these sites compared to tendon. Differences in lysine hydroxylation were also found between the tissues at the telopeptide cross-linking sites. Total collagen cross-linking analysis, including mature trivalent cross-links and immature divalent cross-links, revealed unique cross-linking profiles between tendon and ligament tissues. Tendons were found to have a significantly higher frequency of smaller diameter collagen fibrils compared with ligament, which we suspect is functionally associated with the unique cross-linking profile of each tissue. Understanding the specific molecular characteristics that define and distinguish these specialized tissues will be important to improving the design of orthopedic treatment approaches.

RESEARCH OF THERMAL PROPERTIES OF UHMWPE BASED COMPOSITES AFTER EXPLOSIVE PRESSING

The thermal characteristics of the initial UHMWPE and its composites with QGP and MDF, as well as those obtained by explosive pressing (VP), have been investigated. It has been established that explosive pressing leads to hindering the processes of melting and thermal destruction, which may be associated with chemical interaction, the formation of inter- and intramolecular cross-links characteristic of this polymer. UHMWPE and its compositions with 3% CHP after VP have higher melting points of the crystalline phase, thermal oxidation and thermal destruction in comparison with the starting materials of similar composition. In the UHMWPE composition with 10% MDF, no increase in thermal properties after VP was observed, similar to the original fluoroplastic.

RESEARCH OF THERMAL PROPERTIES OF UHMWPE BASED COMPOSITES AFTER EXPLOSIVE PRESSING

The thermal characteristics of the initial UHMWPE and its composites with QGP and MDF, as well as those obtained by explosive pressing (VP), have been investigated. It has been established that explosive pressing leads to hindering the processes of melting and thermal destruction, which may be associated with chemical interaction, the formation of inter- and intramolecular cross-links characteristic of this polymer. UHMWPE and its compositions with 3% CHP after VP have higher melting points of the crystalline phase, thermal oxidation and thermal destruction in comparison with the starting materials of similar composition. In the UHMWPE composition with 10% MDF, no increase in thermal properties after VP was observed, similar to the original fluoroplastic.

S-Layer Ultrafiltration Membranes.

Monomolecular arrays of protein subunits forming surface layers (S-layers) are the most common outermost cell envelope components of prokaryotic organisms (bacteria and archaea). Since S-layers are periodic structures, they exhibit identical physicochemical properties for each constituent molecular unit down to the sub-nanometer level. Pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes. The functional groups on the surface and in the pores of the S-layer protein lattice are accessible for chemical modifications and for binding functional molecules in very precise fashion. S-layer ultrafiltration membranes (SUMs) can be produced by depositing S-layer fragments as a coherent (multi)layer on microfiltration membranes. After inter- and intramolecular crosslinking of the composite structure, the chemical and thermal resistance of these membranes was shown to be comparable to polyamide membranes. Chemical modification and/or specific binding of differently sized molecules allow the tuning of the surface properties and molecular sieving characteristics of SUMs. SUMs can be utilized as matrices for the controlled immobilization of functional biomolecules (e.g., ligands, enzymes, antibodies, and antigens) as required for many applications (e.g., biosensors, diagnostics, enzyme- and affinity-membranes). Finally, SUM represent unique supporting structures for stabilizing functional lipid membranes at meso- and macroscopic scale.

Molecular Transformations in DNA under the Influence of UV-radiation

Background: The processes of destruction and crosslinking of macromolecules occur simultaneously under the influence of ultraviolet (UV) radiation in synthetic polymers, dry DNA and their concentrated solutions. Objective: The effect of UV radiation on calf thymus DNA in dilute solutions subjected to UV- irradiation was studied in this work. Methods: The calf thymus DNA was studied in dilute solutions using viscometry, absorption spectroscopy and electrophoresis. Results: It was shown that at a low concentration of DNA in the buffer solution ([DNA] = 85 μg / ml) under the influence of UV radiation, the processes of destruction of macromolecules and an increase in their flexibility predominate, which are accompanied by a gradual decrease in the viscosity of their solution. In addition, due to the low concentration of the solution, intramolecular crosslinking of macromolecules predominates, which also reduces their size and, consequently, the viscosity of the solution. Conclusion: It was concluded that in dilute DNA solutions, due to the predominance of the processes of intramolecular crosslinking of macromolecules over intermolecular, only constant processes of decreasing the sizes of DNA macromolecules occur. As a result, their solubility remains virtually unchanged during UV irradiation. The described explanations are also confirmed by the results of absorption spectroscopy and electrophoresis.

Improvement of gel properties of low salt surimi using low-dose l-arginine combined with oxidized caffeic acid

The combined effects of l-arginine (Arg) and oxidized caffeic acid (OCF) treatment under different salt concentrations on gelling properties of bighead carp (Aristichthys nobilis) surimi gels were investigated. The results showed that the improvement of gel strength and water-holding capacity (WHC) of low-salt (0.5%) surimi gels could be obtained by adding Arg at various levels (0.1–0.4%) rather than OCF (0.1–0.9%, based on protein content). However, the gel strength and WHC of the low-salt surimi gel added with low-dose (0.1%) Arg alone were still far lower than that of regular-salt (3.0%) control. Whereas the significant improvements in low-salt surimi gels could be achieved when 0.1% Arg was combined with various levels of OCF, especially in combination with 0.5% OCF (LC-A-O). The water distribution in LC-A-O was markedly improved and its β-sheet structure content was significantly increased compared with the low-salt control (P < 0.05). Moreover, a denser and more uniform network structure of LC-A-O was obtained, which was probably due to much stronger and more intra-molecular cross-linking of proteins, via the formation of new adducts between OCF and surimi proteins. Hence, the combined treatment of Arg and OCF showed a potential for improving the gel properties of low-salt surimi.

An experimental study comparing the stability of colloidal dispersion gels with normal polymeric solutions for enhanced‐oil‐recovery purposes

AbstractWaterflooding in oil reservoirs is associated with several drawbacks. Recently, colloidal dispersion gels (CDG) have been proposed as a new method for conformance control to address these drawbacks. CDGs are prepared by mixing crosslinkers and polymers in very low concentrations as a result of intramolecular crosslinking. In this research, a comprehensive study was conducted on the stability of sulfonated polyacrylamide (SPAM) solutions and CDGs prepared using the same SPAM in harsh conditions. The effects of high temperature, shear rate, salts, and high mechanical degrading on the viscosity of both polymeric solution types were studied using a rheometer. The results showed that, in similar conditions, CDGs are less susceptible to salts than are normal SPAM solutions. Moreover, both solutions have nearly the same sensitivity to temperature. The effect of extremely high shear rates was also studied, and the results suggested that, in similar conditions, CDG is less susceptible to high shear rates than normal SPAM solutions. Being treated at 2500 1/s for 2 hours, the CDG solution lost almost 0% of its original viscosity, while the SPAM solution lost 0.97% of its original viscosity. Given that CDG is more stable in harsh reservoir conditions, it may be a suitable substitute to the highly sensitive polymeric solutions for improving the volumetric sweep efficiency of waterfloods.