Zwitterionic polymer-dexamethasone conjugates penetrate and protect cartilage from inflammation.

Improving the pharmacokinetics of intra-articularly injected therapeutics is a major challenge in treating joint disease. Small molecules and biologics are often cleared from the joint within hours, which greatly reduces their therapeutic efficacy. Furthermore, they are often injected at high doses, which can lead to local cytotoxicity and systemic side effects. In this study, we present modular polymer-drug conjugates of zwitterionic poly(carboxybetaine acrylamide) (pCBAA) and the anti-inflammatory glucocorticoid dexamethasone (DEX) to create cartilage-targeted carriers with slow-release kinetics. pCBAA polymers showed excellent cartilage penetration (full thickness in 1 h) and retention (>50 % after 2 weeks of washing). DEX was loaded onto the pCBAA polymer by employing two different DEX-bearing comonomers to produce pCBAA-co-DEX conjugates with different release kinetics. The slow-releasing conjugate showed zero-order release kinetics in PBS over 70 days. The conjugates elicited no oxidative stress on chondrocytes compared to dose-matched free DEX and protected bovine cartilage explants from the inflammatory response after treatment with IL-1ß. By combining cartilage targeting and sustained drug release properties, the pCBAA-co-DEX conjugates solve many issues of today's intra-articular therapeutics, which could ultimately enable better long-term clinical outcomes with fewer side effects.

Ionically annealed zwitterionic microgels for bioprinting of cartilaginous constructs.

Foreign body response (FBR) is a pervasive problem for biomaterials used in tissue engineering. Zwitterionic hydrogels have emerged as an effective solution to this problem, due to their ultra-low fouling properties, which enable them to effectively inhibit FBRin vivo. However, no versatile zwitterionic bioink that allows for high resolution extrusion bioprinting of tissue implants has thus far been reported. In this work, we introduce a simple, novel method for producing zwitterionic microgel bioink, using alginate methacrylate (AlgMA) as crosslinker and mechanical fragmentation as a microgel fabrication method. Photocrosslinked hydrogels made of zwitterionic carboxybetaine acrylamide (CBAA) and sulfobetaine methacrylate (SBMA) are mechanically fragmented through meshes with aperture diameters of 50 and 90µm to produce microgel bioink. The bioinks made with both microgel sizes showed excellent rheological properties and were used for high-resolution printing of objects with overhanging features without requiring a support structure or support bath. The AlgMA crosslinker has a dual role, allowing for both primary photocrosslinking of the bulk hydrogel as well as secondary ionic crosslinking of produced microgels, to quickly stabilize the printed construct in a calcium bath and to produce a microporous scaffold. Scaffolds showed ∼20% porosity, and they supported viability and chondrogenesis of encapsulated human primary chondrocytes. Finally, a meniscus model was bioprinted, to demonstrate the bioink's versatility at printing large, cell-laden constructs which are stable for furtherin vitroculture to promote cartilaginous tissue production. This easy and scalable strategy of producing zwitterionic microgel bioink for high resolution extrusion bioprinting allows for direct cell encapsulation in a microporous scaffold and has potential forin vivobiocompatibility due to the zwitterionic nature of the bioink.

Zwitterionic Granular Hydrogel for Cartilage Tissue Engineering.

Zwitterionic hydrogels have high potential for cartilage tissue engineering due to their ultra-hydrophilicity, nonimmunogenicity, and superior antifouling properties. However, their application in this field has been limited so far, due to the lack of injectable zwitterionic hydrogels that allow for encapsulation of cells in a biocompatible manner. Herein, a novel strategy is developed to engineer cartilage employing zwitterionic granular hydrogels that are injectable, self-healing, in situ crosslinkable and allow for direct encapsulation of cells with biocompatibility. The granular hydrogel is produced by mechanical fragmentation of bulk photocrosslinked hydrogels made of zwitterionic carboxybetaine acrylamide (CBAA), or a mixture of CBAA and zwitterionic sulfobetaine methacrylate (SBMA). The produced microgels are enzymatically crosslinkable using horseradish peroxidase, to quickly stabilize the construct, resulting in a microporous hydrogel. Encapsulated human primary chondrocytes are highly viable and able to proliferate, migrate, and produce cartilaginous extracellular matrix (ECM) in the zwitterionic granular hydrogel. It is also shown that by increasing hydrogel porosity and incorporation of SBMA, cell proliferation and ECM secretion are further improved. This strategy is a simple and scalable method, which has high potential for expanding the versatility and application of zwitterionic hydrogels for diverse tissue engineering applications.

Guiding Lights: Tissue Bioprinting Using Photoactivated Materials.

Photoactivated materials have found widespread use in biological and medical applications and are playing an increasingly important role in the nascent field of three-dimensional (3D) bioprinting. Light can be used as a trigger to drive the formation or the degradation of chemical bonds, leading to unprecedented spatiotemporal control over a material's chemical, physical, and biological properties. With resolution and construct size ranging from nanometers to centimeters, light-mediated biofabrication allows multicellular and multimaterial approaches. It promises to be a powerful tool to mimic the complex multiscale organization of living tissues including skin, bone, cartilage, muscle, vessels, heart, and liver, among others, with increasing organotypic functionality. With this review, we comprehensively discuss photochemical reactions, photoactivated materials, and their use in state-of-the-art deposition-based (extrusion and droplet) and vat polymerization-based (one- and two-photon) bioprinting. By offering an up-to-date view on these techniques, we identify emerging trends, focusing on both the chemistry and instrument aspects, thereby allowing the readers to select the best-suited approach. Starting with photochemical reactions and photoactivated materials, we then discuss principles, applications, and limitations of each technique. With a critical eye to the most recent achievements, the reader is guided through this exciting, emerging field, with special emphasis on cell-laden hydrogel constructs.

Modulation of Living Cell Behavior with Ultra-Low Fouling Polymer Brush Interfaces.

Ultra-low fouling and functionalizable coatings represent emerging surface platforms for various analytical and biomedical applications such as those involving examination of cellular interactions in their native environments. Ultra-low fouling surface platforms as advanced interfaces enabling modulation of behavior of living cells via tuning surface physicochemical properties are presented and studied. The state-of-art ultra-low fouling surface-grafted polymer brushes of zwitterionic poly(carboxybetaine acrylamide), nonionic poly(N-(2-hydroxypropyl)methacrylamide), and random copolymers of carboxybetaine methacrylamide (CBMAA) and HPMAA [p(CBMAA-co-HPMAA)] with tunable molar contents of CBMAA and HPMAA are employed. Using a model Huh7 cell line, a systematic study of surface wettability, swelling, and charge effects on the cell growth, shape, and cytoskeleton distribution is performed. This study reveals that ultra-low fouling interfaces with a high content of zwitterionic moieties (>65 mol%) modulate cell behavior in a distinctly different way compared to coatings with a high content of nonionic HPMAA. These differences are attributed mostly to the surface hydration capabilities. The results demonstrate a high potential of carboxybetaine-rich ultra-low fouling surfaces with high hydration capabilities and minimum background signal interferences to create next-generation bioresponsive interfaces for advanced studies of living objects.

Nanoparticle-Cell Interactions: Surface Chemistry Effects on the Cellular Uptake of Biocompatible Block Copolymer Assemblies.

The development of nanovehicles for intracellular drug delivery is strongly bound to the understating and control of nanoparticles cellular uptake process, which in turn is governed by surface chemistry. In this study, we explored the synthesis, characterization, and cellular uptake of block copolymer assemblies consisting of a pH-responsive poly[2-(diisopropylamino)ethyl methacrylate] (PDPA) core stabilized by three different biocompatible hydrophilic shells (a zwitterionic type poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) layer, a highly hydrated poly(ethylene oxide) (PEO) layer with stealth effect, and an also proven nontoxic and nonimmunogenic poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) layer). All particles had a spherical core-shell structure. The largest particles with the thickest hydrophilic stabilizing shell obtained from PMPC40-b-PDPA70 were internalized to a higher level than those smaller in size and stabilized by PEO or PHPMA and produced from PEO122-b-PDPA43 or PHPMA64-b-PDPA72, respectively. Such a behavior was confirmed among different cell lines, with assemblies being internalized to a higher degree in cancer (HeLa) as compared to healthy (Telo-RF) cells. This fact was mainly attributed to the stronger binding of PMPC to cell membranes. Therefore, cellular uptake of nanoparticles at the sub-100 nm size range may be chiefly governed by the chemical nature of the stabilizing layer rather than particles size and/or shell thickness.

Plasmonic Hepatitis B Biosensor for the Analysis of Clinical Saliva.

A biosensor for the detection of hepatitis B antibodies in clinical saliva was developed. Compared to conventional analysis of blood serum, it offers the advantage of noninvasive collection of samples. Detection of biomarkers in saliva imposes two major challenges associated with the low analyte concentration and increased surface fouling. The detection of minute amounts of hepatitis B antibodies was performed by plasmonically amplified fluorescence sandwich immunoassay. To have access to specific detection, we prevented the nonspecific adsorption of biomolecules present in saliva by brushes of poly[(N-(2-hydroxypropyl) methacrylamide)-co-(carboxybetaine methacrylamide)] grafted from the gold sensor surface and post modified with hepatitis B surface antigen. Obtained results were validated against the response measured with ELISA at a certified laboratory using serum from the same patients.

Ultralow-Fouling Behavior of Biorecognition Coatings Based on Carboxy-Functional Brushes of Zwitterionic Homo- and Copolymers in Blood Plasma: Functionalization Matters.

Fouling from complex biological fluids such as blood plasma to biorecognition element (BRE)-functionalized coatings hampers the use of affinity biosensor technologies in medical diagnostics. Here, we report the effects the molecular mechanisms involved in functionalization of low-fouling carboxy-functional coatings have on the BRE capacity and resistance to fouling from blood plasma. The specific mechanisms of EDC/NHS activation of carboxy groups, BRE attachment, and deactivation of residual activated groups on recently developed ultra-low-fouling carboxybetaine polymer and copolymer brushes (pCB) as well as conventional carboxy-terminated oligo(ethylene glycol)-based alkanethiolate self-assembled monolayers (OEG-SAMs) are studied using the polarization modulation infrared reflection/absorption spectroscopy, X-ray photoelectron spectroscopy, and surface plasmon resonance methods. It is shown that the fouling resistance of BRE-functionalized pCB coatings is strongly influenced by a deactivation method affecting the ultra-low-fouling molecular structure of the brush and surface charges. It is revealed that, in contrast to free carboxy-group-terminated OEG-SAMs, only a partial deactivation of EDC/NHS-activated zwitterionic carboxy groups by spontaneous hydrolysis is possible in the pCB brushes. The fouling resistance of activated/BRE-functionalized pCB is shown to be recovered only by covalent attachment of amino acid deactivation agents to residual activated carboxy groups of pCB. The developed deactivation procedure is further combined with ultra-low-fouling brushes of random copolymer carboxybetaine methacrylamide (CBMAA) and N-(2-hydroxypropyl) methacrylamide (HPMAA) with optimized CBMAA content (15%) providing a BRE-functionalized coating with superior fouling resistance over various carboxy-functional low-fouling coatings including homopolymer pCB brushes and OEG-SAMs. The biorecognition capabilities of pHPMAA-CBMAA(15%) are demonstrated via the sensitive label-free detection of a microRNA cancer biomarker (miR-16) in blood plasma.

Copolymer Brush-Based Ultralow-Fouling Biorecognition Surface Platform for Food Safety.

Functional polymer coatings that combine the ability to resist nonspecific fouling from complex media with high biorecognition element (BRE) immobilization capacity represent an emerging class of new functional materials for a number of bioanalytical and biosensor technologies for medical diagnostics, security, and food safety. Here, we report on a random copolymer brush surface - poly(CBMAA-ran-HPMAA) - providing high BRE immobilization capacity while simultaneously exhibiting ultralow-fouling behavior in complex food media. We demonstrate that both the functionalization and fouling resistance capabilities of such copolymer brushes can be tuned by changing the surface contents of the two monomer units: nonionic N-(2-hydroxypropyl) methacrylamide (HPMAA) and carboxy-functional zwitterionic carboxybetaine methacrylamide (CBMAA). It is demonstrated that the resistance to fouling decreases with the surface content of CBMAA; poly(CBMAA-ran-HPMAA) brushes with CBMAA molar content up to 15 mol % maintain excellent resistance to fouling from a variety of homogenized foods (hamburger, cucumber, milk, and lettuce) even after covalent attachment of BREs to carboxy groups of CBMAA. The poly(CBMAA 15 mol %-ran-HPMAA) brushes functionalized with antibodies are demonstrated to exhibit fouling resistance from food samples by up to 3 orders of magnitude better when compared with the widely used low-fouling carboxy-functional oligo(ethylene glycol) (OEG)-based alkanethiolate self-assembled monolayers (AT SAMs) and, furthermore, by up to 2 orders of magnitude better when compared with the most successful ultralow-fouling biorecognition coatings - poly(carboxybetaine acrylamide), poly(CBAA). When model SPR detections of food-borne bacterial pathogens in homogenized foods are used, it is also demonstrated that the antibody-functionalized poly(CBMAA 15 mol %-ran-HPMAA) brush exhibits superior biorecognition properties over the poly(CBAA).

Hepatitis B plasmonic biosensor for the analysis of clinical serum samples.

A plasmonic biosensor for rapid detection of protein biomarkers in complex media is reported. Clinical serum samples were analyzed by using a novel biointerface architecture based on poly[(N-(2-hydroxypropyl) methacrylamide)-co-(carboxybetaine methacrylamide)] brushes functionalized with bioreceptors. This biointerface provided an excellent resistance to fouling even after the functionalization and allowed for the first time the direct detection of antibodies against hepatitis B surface antigen (anti-HBs) in clinical serum samples using surface plasmon resonance (SPR). The fabricated SPR biosensor allowed discrimination of anti-HBs positive and negative clinical samples in 10min. Results are validated by enzyme-linked immunoassays of the sera in a certified laboratory. The sensor could be regenerated by simple treatment with glycine buffer.

Polymer brushes interfacing blood as a route toward high performance blood contacting devices.

In the current study, well-defined polymer brushes are shown as an effective surface modification to resist the adhesion of whole blood and its components. Poly[oligo(ethylene glycol)methylether methacrylate] (poly(MeOEGMA)), poly(hydroxyethyl methacrylate) (poly(HEMA)), poly[N-(2-hydroxypropyl) methacrylamide] (poly(HPMA)), and poly(carboxybetaine acrylamide) (poly(CBAA)) brushes were grown by surface initiated atom transfer radical polymerization (SI-ATRP) and subsequently characterized by Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), dynamic contact angle measurements, atomic force microscopy (AFM), and surface plasmon resonance (SPR) spectroscopy. All brushes decreased the fouling from blood plasma over 95% and prevented the adhesion of platelets, erythrocytes, and leukocytes as evidenced by SPR and SEM measurements.

Self-organization of 1-methylnaphthalene on the surface of artificial snow grains: a combined experimental-computational approach.

A combined experimental-computational approach was used to study the self-organization and microenvironment of 1-methylnaphthalene (1MN) deposited on the surface of artificial snow grains from vapors at 238 K. The specific surface area of this snow (1.1 × 10(4) cm(2) g(-1)), produced by spraying very fine droplets of pure water from a nebulizer into liquid nitrogen, was determined using valerophenone photochemistry to estimate the surface coverage by 1MN. Fluorescence spectroscopy at 77 K, in combination with molecular dynamics simulations, and density functional theory (DFT) and second-order coupled cluster (CC2) calculations, provided evidence for the occurrence of ground- and excited-state complexes (excimers) and other associates of 1MN on the snow grains' surface. Only weak excimer fluorescence was observed for a loading of 5 × 10(-6) mol kg(-1), which is ∼2-3 orders of magnitude below monolayer coverage. However, the results indicate that the formation of excimers is favored at higher surface loadings (>5 × 10(-5) mol kg(-1)), albeit still being below monolayer coverage. The calculations of excited states of monomer and associated moieties suggested that a parallel-displaced arrangement is responsible for the excimer emission observed experimentally, although some other associations, such as T-shape dimer structures, which do not provide excimer emission, can still be relatively abundant at this surface concentration. The hydrophobic 1MN molecules, deposited on the ice surface, which is covered by a relatively flexible quasi-liquid layer at 238 K, are then assumed to be capable of dynamic motion resulting in the formation of energetically preferred associations to some extent. The environmental implications of organic compounds' deposition on snow grains and ice are discussed.