Microgels with controlled network topologies by photocrosslinking-assisted continuous precipitation polymerization.

In this study, we present a new synthesis methodology based on photo-crosslinking-assisted continuous precipitation polymerization which allows controlling the distribution of crosslinks in microgels. In our approach we substituted conventional crosslinking agent by a comonomer carrying photo-crosslinkable 4-oxocyclopent-2-en-1-yl group. Microgel size, morphology, distribution of crosslinks and packing density of the polymer chains are studied as a function of retention time (Rt) in the flow reactor. Dynamic and static light scattering (DLS and SLS) as well as small angle X-ray scattering (SAXS) proved an excellent level of control over the distribution of crosslinks in microgels during the polymerization process. These results were confirmed by atomic force microscopy (AFM), indicating a difference in microgel stiffness and arrangement of the polymer network as resulting from increased Rt.

Microgels with Immobilized Glycosyltransferases for Enzymatic Glycan Synthesis.

Glycans, composed of linked monosaccharides, play crucial roles in biology and find diverse applications. Enhancing their enzymatic synthesis can be achieved by immobilizing enzymes on materials such as microgels. Here, we present microgels with immobilized glycosyltransferases, synthesized through droplet microfluidics, immobilizing enzymes either via encapsulation or postattachment. SpyTag-SpyCatcher interaction was used for enzyme binding, among others. Fluorescamine and permeability assays confirmed enzyme immobilization and microgel porosity, while enzymatic activities were determined using HPLC. The potential application of microgels in cascade reactions involving multiple enzymes was demonstrated by combining ß4GalT and α3GalT in an enzymatic reaction with high yields. Moreover, a cascade of ß4GalT and ß3GlcNAcT was successfully implemented. These results pave the way toward a modular membrane bioreactor for automated glycan synthesis containing the presented biocatalytic microgels.

Adsorption of Mono- and Divalent Ions onto Dendritic Polyglycerol Sulfate (dPGS) as Studied Using Isothermal Titration Calorimetry.

The effective charge of highly charged polyelectrolytes is significantly lowered by a condensation of counterions. This effect is more pronounced for divalent ions. Here we present a study of the counterion condensation to dendritic polyglycerol sulfate (dPGS) that consists of a hydrophilic dendritic scaffold onto which sulfate groups are appended. The interactions between the dPGS and divalent ions (Mg2+ and Ca2+) were analyzed using isothermal titration calorimetry (ITC) and showed no ion specificity upon binding, but clear competition between the monovalent and divalent ions. Our findings, in line with the latest theoretical studies, demonstrate that a large fraction of the monovalent ions is sequentially replaced with the divalent ions.

Multicompartment polymeric colloids from functional precursor Microgel: Synthesis in continuous process.

Raspberry-like poly(oligoethylene methacrylate-b-N-vinylcaprolactam)/polystyrene (POEGMA-b-PVCL/PS) patchy particles (PPs) and complex colloidal particle clusters (CCPCs) were fabricated in two-, and one-step (cascade) flow process. Surfactant-free, photo-initiated reversible addition-fragmentation transfer (RAFT) precipitation polymerization (Photo-RPP) was used to develop internally cross-linked POEGMA-b-PVCL microgels with narrow size distribution. Resulting microgel particles were then used to stabilize styrene seed droplets in water, producing raspberry-like PPs. In the cascade process, different hydrophobicity between microgel and PS induced the self-assembly of the first formed raspberry particles that then polymerized continuously in a Pickering emulsion to form the CCPCs. The internal structure as well as the surface morphology of PPs and CCPCs were studied as a function of polymerization conditions such as flow rate/retention time (Rt), temperature and the amount of used cross-linker. By performing Photo-RPP in tubular flow reactor we were able to gained advantages over heat dissipation and homogeneous light distribution in relation to thermally-, and photo-initiated bulk polymerizations. Tubular reactor also enabled detailed studies over morphological evolution of formed particles as a function of flow rate/Rt.

In-Line Characterization of the Temperature-Responsive Behavior of Surface-Bound Microgel Coatings by QCM-D: A Novel Strategy for Protein Repellence Evaluation.

In this work, quartz crystal microbalance with dissipation monitoring (QCM-D) was used to develop a new method to evaluate the protein repellency of microgel coatings. Compared to traditional protocols for surface analysis, QCM has the advantage of a real-time quantitative approach with high sensitivity, allowing us to describe variations of the adsorbed mass with unprecedented accuracy. To enable the detectability of the film throughout the whole operational temperature interval, a poly(N-isopropylacrylamide-co-glycidyl methacrylate) p(NIPAm-co-GMA) microgel monolayer with defined thickness and rigidity was designed. Covalent adhesion of the film to the silica surface was achieved by epoxy-thiol click chemistry and tested for repeated temperature cycles, showing substantial reproducibility. Further functionalization of microgel surfaces by grafting polyzwitterionic chains remarkably improved the protein repellence leaving the strong surface adhesion unaltered. Before and after exposure to fluorescein-tagged bovine serum albumin (FITC-BSA), the coatings showed identical responsive behavior, proving the absence of protein deposition. In nonrepellent coatings, QCM monitoring instead displayed a characteristic shift in the volume phase transition (VPT), pointing out the effect of adsorbed proteins on the swelling behavior of pNIPAm. The combination of QCM-D and UV-visible (UV-vis) was used to evaluate the effect of increasing surface coverage, enabling to distinguish between the protein deposition occurring over the coated and the uncoated portion of the sensor.

Interaction of Linear Polyelectrolytes with Proteins: Role of Specific Charge-Charge Interaction and Ionic Strength.

We present a thermodynamic study of the interaction of synthetic, linear polyelectrolytes with bovine serum albumin (BSA). All polyelectrolytes are based on poly(allyl glycidyl ether) which has been modified by polymer-analogous reaction with anionic (-SO3Na), cationic (-NH3Cl or -NHMe2Cl) or zwitterionic groups (-NMe2(CH2)3SO3). While the anionic polymer shows a very weak interaction, the zwitterionic polymer exhibits no interaction with BSA (pI = 4.7) under the applied pH = 7.4, ionic strength (I = 23-80 mM) and temperature conditions (T = 20-37 °C). A strong binding, however, was observed for the polycations bearing primary amino or tertiary dimethyl amino groups, which could be analysed in detail by isothermal titration calorimetry (ITC). The analysis was done using an expression which describes the free energy of binding, ΔGb, as the function of the two decisive variables, temperature, T, and salt concentration, cs. The underlying model splits ΔGb into a term related to counterion release and a term related to water release. While the number of released counter ions is similar for both systems, the release of bound water is more important for the primary amine compared to the tertiary N,N-dimethyl amine presenting polymer. This finding is further traced back to a closer contact of the polymers' protonated primary amino groups in the complex with oppositely charged moieties of BSA as compared to the bulkier protonated tertiary amine groups. We thus present an investigation that quantifies both driving forces for electrostatic binding, namely counterion release and change of hydration, which contribute to a deeper understanding with direct impact on future advancements in the biomedical field.

Interaction of Polyelectrolytes with Proteins: Quantifying the Role of Water.

A theoretical model is presented for the free energy ΔGb of complex formation between a highly charged polyelectrolyte and a protein. The model introduced here comprises both the effect of released counterions and the uptake or release of water molecules during complex formation. The resulting expression for ΔGb is hence capable of describing the dependence of ΔGb on temperature as well as on the concentration of salt in the system: An increase of the salt concentration in the solution increases the activity of the ions and counterion release becomes less effective for binding. On the other hand, an increased salt concentration leads to the decrease of the activity of water in bulk. Hence, release of water molecules during complex formation will be more advantageous and lead to an increase of the magnitude of ΔGb and the binding constant. It is furthermore demonstrated that the release or uptake of water molecules is the origin of the marked enthalpy-entropy cancellation observed during complex formation of polyelectrolytes with proteins. The comparison with experimental data on complex formation between a synthetic (sulfated dendritic polyglycerol) and natural polyelectrolytes (DNA; heparin) with proteins shows full agreement with theory.

Thermodynamic Analysis of the Interaction of Heparin with Lysozyme.

Glycosaminoglycan (GAG)-protein binding governs critically important signaling events in living matter. Aiming at a quantitative analysis of the involved processes, we herein present a thermodynamic study of the interaction of the model GAG heparin and lysozyme in aqueous solution. Heparin is a highly charged linear polyelectrolyte with a charge parameter of 2.9 (37 °C). The binding constant Kb was determined by ITC as a function of the temperature and ionic strength adjusted through the concentration cs of added salt. The dependence on salt concentration cs was used to determine the net number of released counterions. Moreover, the binding constant at a reference salt concentration of 1 M Kb(1 M) was determined by extrapolation. The dependence on temperature of Kb was used to dissect the binding free energy ΔGb into the respective enthalpies ΔHb and entropies ΔSb together with the specific heat Δcp. A strong enthalpy-entropy cancelation was found similar to the results for many other systems. The binding free energy ΔGb could furthermore be split up into a part ΔGci due to counterion release and a residual part ΔGres. The latter quantity reflects specific contributions as, e.g., salt bridges, van der Waals interactions, or hydrogen bonds. The entire analysis shows that heparin-lysozyme interactions are mainly caused by counterion release; that is, ca. three counterions are being released upon binding one lysozyme molecule. Our reported approach of quantifying interactions between glycosaminoglycans and proteins is generally applicable and suitable to provide new insights in the physical modulation of biomolecular signals.

Interaction of Proteins with a Planar Poly(acrylic acid) Brush: Analysis by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D).

We describe the preparation of a poly(acrylic acid) (PAA) brush, polymerized by atom transfer radical polymerization (ATRP) of tert-butyl acrylate (tBA) and subsequent acid hydrolysis, on the flat gold surfaces of quartz-crystal microbalance (QCM) crystals. The PAA brushes were characterized by Fourier transform infrared (FT-IR) spectroscopy, ellipsometry and water contact angle analysis. The interaction of the PAA brushes with human serum albumin (HSA) was studied for a range of ionic strengths and pH conditions by quartz-crystal microbalance with dissipation monitoring (QCM-D). The quantitative analysis showed a strong adsorption of protein molecules onto the PAA brush. By increasing the ionic strength, we were able to release a fraction of the initially bound HSA molecules. This finding highlights the importance of counterions in the polyelectrolyte-mediated protein adsorption/desorption. A comparison with recent calorimetric studies related to the binding of HSA to polyelectrolytes allowed us to fully analyze the QCM data based on the results of the thermodynamic analysis of the binding process.

Thermodynamic Analysis of the Uptake of a Protein in a Spherical Polyelectrolyte Brush.

A thermodynamic study of the adsorption of Human Serum Albumin (HSA) onto spherical polyelectrolyte brushes (SPBs) by isothermal titration calorimetry (ITC) is presented. The SPBs are composed of a solid polystyrene core bearing long chains of poly(acrylic acid). ITC measurements done at different temperatures and ionic strengths lead to a full set of thermodynamicbinding constants together with the enthalpies and entropies of binding. The adsorption of HSA onto SPBs is described with a two-step model. The free energy of binding ΔGb depends only weakly on temperature because of a marked compensation of enthalpy by entropy. Studies of the adsorbed HSA by Fourier transform infrared spectroscopy (FT-IR) demonstrate no significant disturbance in the secondary structure of the protein. The quantitative analysis demonstrates that counterion release is the major driving force for adsorption in a process where proteins become multivalent counterions of the polyelectrolyte chains upon adsorption. A comparison with the analysis of other sets of data related to the binding of HSA to polyelectrolytes demonstrates that the cancellation of enthalpy and entropy is a general phenomenon that always accompanies the binding of proteins to polyelectrolytes dominated by counterion release.