Zwitterionic polymer ligands: An ideal surface coating to totally suppress protein-nanoparticle corona formation?

International audienceIn the last few years, zwitterionic polymers have been developed as antifouling surface coatings. However, their ability to completely suppress protein adsorption at the surface of nanoparticles in complex biological media remains undemonstrated. Here we investigate the formation of hard (irreversible) and soft (reversible) protein corona around model nanoparticles (NPs) coated with sulfobetaine (SB), phosphorylcholine (PC) and carboxybetaine (CB) polymer ligands in model albumin solutions and in whole serum. We show for the first time a complete absence of protein corona around SB-coated NPs, while PC-and CB-coated NPs undergo reversible adsorption or partial aggregation. These dramatic differences cannot be described by naïve hard/soft acid/base electrostatic interactions. Single NP tracking in the cytoplasm of live cells corroborate these in vitro observations. Finally, while modification of SB polymers with additional charged groups lead to consequent protein adsorption, addition of small neutral targeting moieties preserves antifouling and enable efficient intracellular targeting

Novel Zwitterionic Copolymers to Enhance Hydrophilicity of PVDF Membranes: A Comprehensive Computational Study

Membrane technology covers all the engineering approaches with a key growth for large-scale industrial applications, including biotechnology, biomedical applications, food industry, and water and wastewater treatment. Poly (vinylidene fluoride) (PVDF) membrane has been gained remarkable attentions in recent years due to its excellent advantages in terms of thermal stability, chemical resistance, and high mechanical strength for water treatment. Despite its outstanding advantages, the performances of PVDF membranes are substantially limited by fouling problems. In this research study, we designed novel zwitterionic (ZW)-PVDF membranes with high hydrophilicity by employing a set of comprehensive computational methods. To achieve our goal, we first investigated the interactions occurring between water molecules and the fragments of hydrophobic and hydrophilic membrane models at the molecular level using the pair interaction energy decomposition analysis (PIEDA) as part of the fragment molecular orbital (FMO) method’s framework. This research direction is critical, since a research study of the reasons behind the interactions between water molecules and membrane materials would help design ground-breaking membranes with superior hydrophilicity. The computational studies and experimental analyses of PVDF and Polyacrylonitrile (PAN) membranes were considered as the models for hydrophobic and hydrophilic membranes, respectively. Density-functional theory (DFT), based on B3LYP functional and split-valance 6-311+G (d, p) basis sets, was used in order to optimize the geometry of PAN, PVDF, and their complexes with different numbers of water molecules. Furthermore, the functional groups of membrane surfaces were experimentally evaluated through Fourier-transform infrared spectroscopy (FTIR- ATR), 13C cross polarization magic angle spinning (13C CP MAS) Solid State Nuclear magnetic resonance SSNMR, and Fourier transform Raman (FT-Raman) spectroscopies. The confocal microscopic was also employed to interrogate water transport and the interactions between fluorescence particles through the membrane matrices. The non-covalent interactions in terms of electrostatic, exchange-repulsion, and charge-transfer parameters were comprehensively investigated for the designed ZW-PVDF copolymers. The performance of ZW moieties was derived from three different anionic groups in the ZW head, specifically, carboxylate, sulfonate, and phosphate. This approach was used in addition to the inclusion of a linker between the ZW head and the PVDF backbone, such as trimethyl ammonium groups and hydroxyl group, for an improvement of PVDF hydrophilicity. The quantum chemical calculations were conducted to examine the hydration structure of moieties. The interactions between the ZW moieties, with water molecules confirmed that it depended on the charged groups in addition to the chemical functional groups between charged groups. Furthermore, the types of anionic groups, the polar functional groups between charged groups, and the hydrophilic group, as a linker between charged groups of the ZW to the PVDF polymer backbone are the key reason for membrane hydrophilicity and the membrane water uptake. The double Zwitterionic PMAL®-C8-CB-OH-SB-PVDF was designed through the addition of protonated carboxyl group on a backbone of copolymer PMAL®-C8, and the protonated nitrogen atom of the amide group. This double zwitterion showed strong electrostatic interactions between individual water molecules and the secondary ammonium and the Oxygen of carboxybetaine, compared to PMAL®-C8-OH-SB-PVDF model. Our designed hydrophilic ZW-PVDF membranes, and especially the double zwitterion membrane, are an exciting development that can be applied in a broad range of water applications

EFFECT OF CHEMICAL IDENTITY AND MORPHOLOGY ON AMPHIPHILIC-ZWITTERIONIC BLOCK COPOLYMER MEMBRANES

Amphiphilic block copolymers have gained a broad research interest attributed to their self-assembly properties over a range of pH, temperature, and ionic strength. Polyzwitterions have attracted special attention due to their hydrophilicity, charge sensitivity and coulombic attraction of the opposite charges over a range of environments making them a popular material of study in the field of stimuli responsive systems, for example in self-healing hydrogels, and water transport membranes. Combining the stimuli responsiveness and higher hydrophilicity of zwitterionic polymers with self-assembly behavior of amphiphilic block copolymers created an interest to study the effect of composition and identity of the zwitterionic block on the morphology of novel amphiphilic- zwitterionic block copolymer systems. A novel chemistry to synthesis a series of amphiphilic-zwitterionic block copolymer was developed. As a first step, a parent block copolymer precursors of poly (dimethyl amino ethyl methacrylate)-b-poly (n butyl acrylate-ran-allyl methacrylate) (PDMAEMA-b- P(nBA-ran-AMA)) with different copolymer volume fractions were synthesized using controlled radical polymerization. The advantage of using this technique is the ability to synthesize a polymer chain with predetermined molecular weight and well-defined chemical composition while also maintaining a narrow dispersity. Variation of the zwitterionic groups; sulfobetaine, carboxybetaine or cholinephosphate were investigated through post-polymerization modification of the PDMAEMA block using nucleophilic ring-opening reactions of 1,3-propane sultone , β-propiolactone or n-butyl substituted phospholane. Proton NMR spectroscopy data were analyzed to calculate degree of polymerization (DP) from the distinct peak of each repeating unit of pendent groups in parent neutral block copolymers and its modified amphiphilic-zwitterionic counterpart. These DP of the neutral block copolymer were then used to calculate the relative volume fraction of each block and aid the future calculation of post-polymerization modification on the PDMAEMA block and crosslinking chemistry on the poly allyl methacrylate (PAMA) block. Thermal stability of these zwitterionic systems was investigated by using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Mechanically robust free standing amphiphilic zwitterionic copolymer poly (n butyl acrylate-ran-allyl methacrylate)-b-polybetaine methacrylate membrane were tailored using thiol-ene click chemistry on the pendent double bond of the allyl methacrylate repeating unit with a dithiol crosslinking agent under UV irradiation. The analysis of the effect of composition, and identity of the zwitterionic block on the resultant films ix morphologies was probed. The systematic variations in volume fractions of each block were targeted to generate different morphologies. The impact of the copolymer composition on the morphology were analyzed using small angle X-ray scattering (SAXS). Various relative humidity sweeps and temperature variation SAXS were performed through collaboration with Argonne National Laboratory to investigate the effect of different environmental conditions of the morphologies. In addition to SAXS, transmission electron microscopy (TEM) was performed to study real space imaging of these structures. With the results from these characterization methods, it was possible to perform a structure-property relationship study of these novel Amphiphilic-Zwitterionic block copolymers. Looking forward, results from this study have the potential to guide future applications in the field of water transport filtration membranes. The effect on morphology, zwitterionic content and block copolymer composition on water uptake and salt uptake was evaluated using gravimetric analysis, and dead-end filtration performance studies showing promising initial results

Interfacial properties of grafted zwitterionic polymers and interaction with proteins at the nanoscale

Zwitterionic polymers are considered to be great candidate for surface modifications of biosensors and implantable materials because of their super hydrophilicity and ability to prevent non-specific protein adsorption or ‘fouling’. They also provide steric and electrostatic stabilization for colloid or nanoparticles in electrolytes, protein/drug stabilization and prevent marine fouling. Their functional performance as biocompatible, non-fouling coatings in different environment such as salt concentration in medium, types of salt, temperature etc. affect their solubility, swelling behavior and molecular level surface properties. In this thesis, the design parameters of grafted polysulfobetaine thin films- one of the commonly known polyzwitterions, are identified to be- grafted chain densities, polymer molecular weight, and/or film thickness and demonstrated how these parameters tune protein adsorption and surface forces at varying ionic strength of the surrounding medium and at different grafting densities. The first part of this study address the research question- why do grafted zwitterionic polymers display excellent non-fouling properties but directly interact with proteins in solution? The results of the study reported that proteins do adsorb on this so-called non-fouling polysulfobetaine grafted chains. The amount of adsorbed proteins follow a bell shaped curve, with the maximum adsorption happening at low (non-overlapping mushrooms) grafting densities and a low adsorption high (dense brush) grafting densities. This adsorption profile is a signature of ternary adsorption of proteins on weakly attractive grafted polymer chains is well described by theory, and it allows us to both test our hypothesis that the polysulfobetaines form segment-protein attractive interactions by overcoming the osmotic repulsion of insertion into the grafted chain layer as well as identify design parameters to tune protein adsorption on such zwitterionic thin films. In the second part of the study, the surface forces between grafted polysulfobetaine chains and mica were measured as a function of distance in order to investigate the influence of ionic strength, polymer molecular weight and surface density on the range of interactions and the amplitude of interfacial forces. The repulsive forces generated by thin films were quantified with sub nanometer resolution in distance by using a Surface Force Apparatus (SFA). SFA, based on multiple beam interferometry, can directly inform us of the efficacy of polysulfobetaines as entropic barriers in colloid or nanoparticle stabilization in salt solutions. The results from this study highlighted the potential for using sparsely grafted chains for developing non-fouling coatings and/or particle stabilization, whereas previous reports only focused on densely grafted brushes of polysulfobetaines. Next, surface force measurements were performed between a statistical copolymer consisting of non-ionic oligoethylene glycol and zwitterionic polysulfobetaine polymers at high and low grafting densities and testing surface mica at varying zwitterionic composition. Here, we tested the hypothesis that although the monomer constituents are chemical structurally different, they are well-mixed and non-interacting and thus, their influence on steric repulsive forces depend on the zwitterionic content in the copolymer chains. The ionic strength dependence of the chain extension and repulsive forces increased proportionally with the sulfobetaine content, reflecting the increasing influence of charged monomers and their interactions with ions in solution. These results suggested that ethyene glycol and sulfobetaine behave as non-interacting, miscible monomers that contribute independently to the polymer extension and chain interactions with ions. These findings have important practical implication in stabilizing proteins/drugs by differential interactions of zwitterionic and non-ionic counterparts in the copolymer chains. Prior to beginning my research project in surface science, I had investigated protein mediated binding kinetics by using Micropipette Aspiration Assay (MPA), the results of which are included in the fifth chapter of this thesis. Finally in the last chapter of my thesis, I discussed future directions of the project. Here I elaborated on using thin films of polyzwitterions to tune surface interactions with proteins for direct, in-situ measurements of protein folding dynamics of immobilized, high value proteins or drugs at the interface

Structure-Property Relationships of Polymer Films and Hydrogels to Control Bacterial Adhesion

The emergence and spread of antibiotic resistance across microbial species necessitates the need for alternative approaches to mitigate the risk of infection without relying on commercial antibiotics. Biofilm-related infections are a class of notoriously difficult to treat healthcare-associated infections that frequently develop on the surface of implanted medical devices. As biofilm formation is a surface-associated phenomenon, understanding how the intrinsic properties of materials affect bacterial adhesion enables the development of structure-property relationships that can guide the future design of infection-resistant materials. Despite lacking visual, auditory, and olfactory perception, bacteria still manage to sense and attach to surfaces. Previously, it has been reported that bacteria can detect and differentiate the surface chemistry and topography of surfaces; however, the influence of the stiffness and thickness on bacterial-surface interactions remains unknown. In this thesis, the effect that the fundamental material properties of polymer films and hydrogels (stiffness, thickness, and chemistry) have on the adhesion and surface-associated transport of bacteria was investigated. By decoupling the effect of the hydrogel’s stiffness and thickness from their chemistry, we suggest a key takeaway design rule: to optimize fouling-resistance, hydrogel coatings should be thick and soft. Two chemically distinct hydrogels, poly(ethylene glycol) and agar, were synthesized over a 1-1000 kPa range of Young’s modulus. Static adhesion experiments, conducted on 150 µm thick hydrogels, determined that Escherichia coli and Staphylococcus aureus colony surface coverage correlated positively with an increase in Young’s modulus. Notably, a substantial increase in adhesion occurred for both bacteria when the thickness of the hydrogels was reduced to 10 µm. The stiffness of poly(ethylene glycol) brushes and hydrogels was also found to influence the length and frequency of Staphylococcus aureus surface-associated transport via dynamic shear flow experiments. Furthermore, a universal hydrogel functionalization platform was developed for instances where mechanical properties of hydrogels are not adjustable. The incorporation of the fouling-resistant polymer zwitterion, poly(2-methacryloyloxyethyl phosphorylcholine), enhanced resistance to bacterial adhesion without altering the mechanical properties of covalently or physically crosslinked hydrogels. This thesis demonstrates that by combining structure-property relationships with fouling-resistant zwitterionic chemistry, the adhesion of proteins and microorganisms to polymer hydrogels is reduced

Carboxybetaine Ester Feature as a Platform for Switchable Surface Properties

A lot of strategies for smart approaches on surfaces were applied such as hydrogel layer, polymer brushes or self-assembly monolayers (SAM). [1] Nowadays switchable zwitterionic materials consisting of molecules with internally balanced charge between positive ammonium and negative carboxy group are promising candidates for this application. [2] They can combine antifouling properties of their zwitterion state and complexation or sticky character in their pre-zwitterionic carboxybetaine ester form. Zwitterionic forms possess antibiofouling properties due to electrostatic interaction between charged moieties, highly hydration capability and overall neutral charge in material as well as biomimetic character because zwitterions are structural similarity to biomembranes. We showed that modifications of surface by zwitterionic based self-assemble monolayer allow enhance detection limit of biosensors down to 10–15 M for analyte, [3,4] or improve electrorheological response. [5] Carboxybetaine esters have cationic character and permit complexation with polyanionic bioabsorbents as well as character of counter ion can adjust wettability and interaction with biomolecules. These studies will present on the utilization of pre-zwiterionic molecules: carboxybetaine based derivates formed from lipoic acid precursor in order to modify surface for construction of impedimetric lectin biosensors and for tuning wettability and interaction with DNA and other charged (bio)molecules. Novel pre-zwitterionic carboxybetaine ester (hydrolysable and photolysable) derivates were synthetized by protocol consists of several synthetic steps and fully characterized. Subsequently, modification of a gold surface was performed by a self-assembled monolayer deposited from a solution containing prezwitterion molecules. Self-assembly monolayer, formed from derivates, was characterized by set instrumentation as atomic force microscopy, quartz crystal microbalance XPS, contact angle etc. Hydrolysable carboxybetaine derivate was able to from complex with polycationic DNA molecules to preconcentrate and release at pH dependent manner. During course of hydrolysis carboxybetaine ester is transferred to carboxybetaine zwitterionic form to promote DNA release due to formation of carboxylate negative charge. Additionally, gradient in wettability can be observed within progress of hydrolysis and present of long perfluorinated or aliphatic types of counter ions. For example switch in wettability can be achieved only by simple and rapid couterion exchange between superhydrophilic (contact angle (CA) below 10° (to very high hydrophobilic (CA over 140°) on rough gold surface. After completed hydrolyses zwitterionic surface can be utilized as a platform for biosensor surface with nonfouling properties. Carboxylic functionality allows immobilizing sensing molecules as lectins for electrochemical impedance spectroscopy by means of EDC/NHS chemistry. This methodology provides opportunity for ultrasensitive detection up to 10–15 M of lectins which may result of a biomarker discovery on several diseases in whole media. Moreover utilization of photolabile ester of carboxybetaine derivates allowing spatially control wettability and pattering with photomask was performed. Photolabile 2-nitrophenyl methyl ester group was introduced to pre-zwitterionic molecule and after irradiation of prepared surface with light at 365 nm was transformed from carboxybetaine ester group to zwitterionic carboxybetaine. Progress of photolysis can be observed by change of surface zeta potential, quartz crystal microbalance and contact angle measurement. This irreversible switch along with different interaction of biological species before and after photolysis will be discussed in this contribution as well. This contribution was made possible by NPRP grant 6-381-1-078 from the Qatar National Research Fund (a member of the Qatar Foundation). The statements contained are entirely the responsibility of the authors.qscienc

Bacteria-Resistant, Transparent, Free-standing Films Prepared from Complex Coacervates

We report the fabrication, properties, and bacteria-resistance of polyelectrolyte complex (PEC) coatings and free-standing films. Poly(4-styrenesulfonic acid), poly(diallyldimethylammonium chloride), and salt were spin-coated into PEC films. After thermal annealing in a humid environment, highly transparent, mechanically strong, and chemically robust films were formed. Notably, we demonstrate that PEC coatings significantly reduce the attachment of Escherichia coli K12 without killing the microorganisms. We suggest that forming bacteria-resistant surface coatings from commercially available polymers holds the potential for use across a wide range of applications, including high-touch surfaces in medical settings

Zwitterionic ceramics for biomedical applications

Bioceramics for bone tissue regeneration, local drug delivery and nanomedicine, are receiving growing attention by the biomaterials scientific community. The design of bioceramics with improved surface properties able to overcome clinical issues is a great scientific challenge. Zwitterionization of surfaces has arisen as a powerful alternative in the design of biocompatible bioceramics capable to inhibit bacterial and non-specific protein adsorption, which opens up new insights into the biomedical applications of these materials. This manuscript reviews the different approaches reported up to date for the synthesis and characterization of zwitterionic bioceramics with potential clinical applications. Statement of Significance Zwitterionic bioceramics are receiving growing attention by the biomaterials scientific community due to their great potential in bone tissue regeneration, local drug delivery and nanomedicines. Herein, the different strategies developed so far to synthesize and characterize zwitterionic bioceramics with potential clinical applications are summarized. (C) 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Zwitterionic microscale hydrogels for protein delivery, stabilization, and immobilization

Proteins are incredibly useful in medicine and industrial chemistry. Many of the most recent breakthroughs in cancer therapy are based on monoclonal antibody treatments. Yet, there are major difficulties that can act as deterrents in developments of such therapies. Sustained subcutaneous, oral or pulmonary deliveries of such therapeutics are limited by the poor stability, short half-life, and non-specific interactions between the therapeutic biomolecules (e.g. antibody) and the delivery vehicle. Similarly, usage of proteins as enzymes in processes is limited by poor stability, short half-life, and difficulties with reusability. With growing usage of proteins as pharmaceuticals and biocatalysts, and apparent shortcomings in both fields, there is a growing need to design materials that are protein compatible and can improve protein stability. The key to successfully utilizing proteins as therapeutics, biocatalysts or biosensors is to maintain their conformation and function. There is emerging evidence that biomimetic, biocompatible zwitterionic polymers can prevent non-specific interactions within protein systems and increase protein stability. In this work, zwitterionic microscale hydrogels of two different zwitterionic moieties (carboxybetaine and sulfobetaine), were synthesized. For the purpose of protein delivery, a biodegradable zwitterionic poly(carboxybetaine), pCB, based microscale hydrogel (microgel) covalently crosslinked with tetra(ethylene glycol) diacrylate was synthesized for antibody encapsulation. The resulting microgels were characterized via FTIR, diffusion NMR, SANS, and cell culture studies. The microgels were found to contain up to 97.5% water content and showed excellent degradability that can be tuned with crosslinking density. Cell compatibility of the microgel was studied by assessing the toxicity and immunogenicity in vitro. Cells exposed to the microgel showed complete viability and no pro-inflammatory secretion of interleukin 6 (IL6) or tumor necrosis factor-alpha (TNFa). The microgel was loaded with Immunoglobulin G (as a model antibody), using a post-fabrication loading technique, and antibody sustained release from microgels of varying crosslinking densities was studied. The released antibodies (especially from the high crosslinked microgels) proved to be completely active and able to bind with antibody receptors. Furthermore, for the purpose of protein immobilization, a reaction scheme was developed and studied for covalent immobilization of the protein (a-chymotrypsin) (ChT) within the zwitterionic microscale hydrogels. Confocal laser microscopy studies showed that immobilized ChT (i-ChT) was distributed within the hydrogel. The enzyme-immobilized microgels showed excellent reusability (72% of its initial activity after 10 uses) and could undergo several freezing/drying/rehydration cycles while retaining enzymatic activity. The i-ChT activity, half-life, and conformational stability were studied at varying pH and temperatures with results compared to free ChT in buffer. ChT immobilized within pCB hydrogel showed increased enzymatic stability as observed by a 13 degrees C increase in the temperature at which i-ChT loses activity compared to free ChT. Furthermore, enzyme half-life increased up to seven-fold for the pCB immobilized ChT, and the increased stability resulted in higher activity at elevated pH. The i-ChT was most active at pH of 8.5 and was partially active up to the pH of 10.2. This research paves the way for designing protein delivery vectors as well as fabrication of enzyme immobilized materials with extended enzyme lifetime and activity