Cell-Taxi: Mesenchymal Cells Carry and Transport Clusters of Cancer Cells.

Cell clusters that collectively migrate from primary tumors appear to be far more potent in forming distant metastases than single cancer cells. A better understanding of the collective cell migration phenomenon and the involvement of various cell types during this process is needed. Here, an in vitro platform based on inverted-pyramidal microwells to follow and quantify the collective migration of hundreds of tumor cell clusters at once is developed. These results indicate that mesenchymal stromal cells (MSCs) or cancer-associated fibroblasts (CAFs) in the heterotypic tumor cell clusters may facilitate metastatic dissemination by transporting low-motile cancer cells in a Rac-dependent manner and that extracellular vesicles secreted by mesenchymal cells only play a minor role in this process. Furthermore, in vivo studies show that cancer cell spheroids containing MSCs or CAFs have faster spreading rates. These findings highlight the active role of co-traveling stromal cells in the collective migration of tumor cell clusters and may help in developing better-targeted therapies.

Biomass-Derived Poly(ether-amide)s Incorporating Hydroxycinnamates.

Lignin-derived chemicals have great potential as feedstock to produce polymeric materials, due to the low cost and high abundance of lignin biomass. Lignin is one of the few nonpetroleum sources of aromatic carbon, a desirable moiety in high-performance polymers. Herein we describe the synthesis and characterization of a series of 21 poly(ether-amide)s that incorporate hydroxycinnamates derived from lignin. Three different hydroxycinnamates (coumaric acid, ferulic acid, sinapinic acid) were incorporated into dimers, and then copolymerized with a series of seven aliphatic and aromatic diamines via interfacial polymerization. The resultant polymers exhibited poor solubility in standard organic solvents (excluding DMF), but exhibited moderate glass transition temperatures and moderate thermal stabilities. Additionally, the polymers exhibit excellent resistance to hydrolysis. The modularity of this synthetic approach could be used to rapidly generate diverse polymers with a broad range of well-tuned properties.

Strategies for the Conversion of Lignin to High-Value Polymeric Materials: Review and Perspective.

The majority of commodity plastics and materials are derived from petroleum-based chemicals, illustrating the strong dependence on products derived from non-renewable energy sources. As the most accessible, renewable form of carbon (in comparison to CO2), lignocellulosic biomass (defined as organic matter available on a renewable basis) has been acknowledged as the most logical carbon-based feedstock for a variety of materials such as biofuels and chemicals. This Review focuses on methods developed to synthesize polymers derived from lignin, monolignols, and lignin-derived chemicals. Major topics include the structure and processing of lignocellulosic biomass to lignin, polymers utilizing lignin as a macromonomer, synthesis of monomers and polymers from monolignols, and polymers from lignin-derived chemicals, such as vanillin.

Designing Hybrid Antibiotic Peptide Conjugates To Cross Bacterial Membranes.

We design hybrid antibiotic peptide conjugates that can permeate membranes. Integration of multiple components with different functions into a single molecule is often problematic, due to competing chemical requirements for different functions and to mutual interference. By examining the structure of antimicrobial peptides (AMPs), we show that it is possible to design and synthesize membrane active antibiotic peptide conjugates (MAAPCs) that synergistically combine multiple forms of antimicrobial activity, resulting in unusually strong activity against persistent bacterial strains.

Poly(methyl 6-acryloyl-ß-d-glucosaminoside) as a Cationic Glycomimetic of Chitosan.

Chitosan, a cationic polysaccharide derived from one of the most abundant natural polymers, chitin, has been investigated extensively for its antimicrobial properties. However, it suffers from the inherent drawbacks of natural products such as batch-to-batch variability, limited supply, contamination, and potential adverse reaction. Additionally, its solubility depends on the degree of deacetylation and pH, as it is only soluble under acidic conditions. As an alternative to chitosan, we synthesized the protected cationic glycomimetic monomer methyl N-Fmoc-6-acryloyl-ß-d-glucosaminoside from glucosamine. This monomer retains structural features critical to recapitulating the properties of the chitosan repeat unit, namely, the pKa of the protonated amine. We optimized the free radical polymerization of methyl N-Fmoc-6-acryloyl-ß-d-glucosaminoside and fractionated the resultant poly(methyl N-Fmoc-6-acryloyl-ß-d-glucosaminoside) to obtain a range of molecular weights. Following Fmoc deprotection, the cationic glycopolymers retained 95% of their expected amine content by mass and exhibited a pKa of 6.61. Poly(methyl 6-acryloyl-ß-d-glucosaminoside) mimicked the molecular weight-dependent bacterial inhibitory property of chitosan in acidic solutions. Importantly, poly(methyl 6-acryloyl-ß-d-glucosaminoside) remained soluble at elevated pH (conditions under which chitosan is insoluble) and maintained its antibacterial activity. Mammalian cell viability in the presence of poly(methyl 6-acryloyl-ß-d-glucosaminoside) at acidic pH is good, although somewhat lower than viability in the presence of chitosan. No cytotoxic effect was observed at neutral pH. These results demonstrate that poly(methyl 6-acryloyl-ß-d-glucosaminoside) is not only a suitable biomimetic for chitosan, but that it can be utilized as an antibacterial agent in a broader range of biologically relevant pHs.

Multivalent 3D Display of Glycopolymer Chains for Enhanced Lectin Interaction.

Synthetic glycoprotein conjugates were synthesized through the polymerization of glycomonomers (mannose and/or galactose acrylate) directly from a protein macroinitiator. This design combines the multivalency of polymer structures with 3D display of saccharides randomly arranged around a central protein structure. The conjugates were tested for their interaction with mannose binding lectin (MBL), a key protein of immune complement. Increasing mannose number (controlled through polymer chain length) and density (controlled through comonomer feed ratio of mannose versus galactose) result in greater interaction with MBL. Most significantly, mannose glycopolymers displayed in a multivalent and 3D configuration from the protein exhibit dramatically enhanced interaction with MBL compared to linear glycopolymer chains with similar total valency but lacking 3D display. These findings demonstrate the importance of the 3D presentation of ligand structures for designing biomimetic materials.

Synthesis, characterization, and biological interaction of glyconanoparticles with controlled branching.

Branched amphiphilic copolymers were synthesized through the reversible addition-fragmentation chain transfer (RAFT) chain extension of a poly(methyl acrylate) macro-chain transfer agent using a protected galactose monomer and a polymerizable chain transfer agent branching unit. After galactose deprotection, the copolymers were self-assembled via nanoprecipitation. The resultant nanoparticles were analyzed for their size, shape, and biological interaction with a galactose binding lectin. Using light scattering, the nanoparticles were determined to be solid spheres. Nanoparticles containing branched glycoblocks bound significantly more lectin than those containing comparable linear blocks. By adjusting the molecular weight and branching of the copolymer, the size of the self-assembled nanoparticle and the saccharide density on its surface can be varied.

Tandem mass spectrometry and ion mobility mass spectrometry for the analysis of molecular sequence and architecture of hyperbranched glycopolymers.

Multidimensional mass spectrometry techniques, combining matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) with tandem mass spectrometry (MS(2)), multistage mass spectrometry (MS(n)) or ion mobility mass spectrometry (IM-MS), have been employed to gain precise structural insight on the compositions, sequences and architectures of small oligomers of a hyperbranched glycopolymer, prepared by atom transfer radical copolymerization of an acrylate monomer (A) and an acrylate inimer (B), both carrying mannose ester pendants. The MS data confirmed the incorporation of multiple inimer repeat units, which ultimately lead to the hyperbranched material. The various possible structures of n-mers with the same composition were subsequently elucidated based on MS(2) and MS(n) studies. The characteristic elimination of bromomethane molecule provided definitive information about the comonomer connectivity in the copolymeric AB2 trimer and A2B2 tetramer, identifying as present only one of the three possible trimeric isomers (viz. sequence BBA) and only two of the six possible tetrameric isomers (viz. sequences BBA2 and BABA). Complementary IM-MS studies confirmed that only one of the tetrameric structures is formed. Comparison of the experimentally determined collision cross-section of the detected isomer with those predicted by molecular simulations for the two possible sequences ascertained BBA2 as the predominant tetrameric architecture. The multidimensional MS approaches presented provide connectivity information at the atomic level without requiring high product purity (due to the dispersive nature of MS) and, hence, should be particularly useful for the microstructure characterization of novel glycopolymers and other types of complex copolymers.

Nontoxic Initiator Alternatives to TEMED for Redox Hydrogel Polymerization.

Polymeric hydrogels are versatile biomaterials, offering unique advantages in tunability and biocompatibility that make them well-suited to a range of applications. Cross-linking, a fundamental step in hydrogel fabrication, is often initiated using a toxic redox system, ammonium persulfate (APS), and tetramethylethylenediamine (TEMED), which hinders hydrogel utility in direct contact with cells (e.g., wound dressings). To overcome this limitation, we developed alternative redox gelation systems that serve as nontoxic replacements for TEMED. The alternate initiators were either synthetic or bioinspired amine-containing polymers, Glycofect and polyethylenimine (PEI). Used with APS, these initiator candidates produced hydrogels with short gelation time and comparable moduli to TEMED-based gels and underwent further mechanical testing and biocompatibility characterization. While achieving mechanical properties similar to those of the control, the gels based on Glycofect and PEI outperformed TEMED-based gels in two cell viability studies, with Glycofect-initiated gels displaying significantly higher cytocompatibility. Taken together, these results indicate that Glycofect may serve as a drop-in replacement for TEMED to fabricate hydrogels with improved biocompatibility.

Effect of branching density on avidity of hyperbranched glycomimetics for mannose binding lectin.

Hyperbranched glycopolymers containing mannose units in the branch point were synthesized through the copolymerization of a mannose inimer and mannose acrylate via atom transfer radical polymerization (ATRP). Incorporating a saccharide residue at the branch point results in a closer analogue to natural branched polysaccharides. Gel permeation chromatography characterization of the polymers qualitatively indicates branching in samples from polymerizations utilizing the mannose inimer. Deprotection of the acetate protecting groups from the hyperbranched mannose polymers yields water-soluble polymers that interact with mannose binding lectin (MBL), a key protein of the innate immunity complement system. MBL interaction increases with increasing polymer molecular weight and increasing branching density. Notably, incorporating mannose into the branching repeat unit also increases the interaction of the glycopolymers with MBL compared with glycopolymers with the same branching density but with no mannose at the branch point.

Steric effects in peptide and protein exchange with activated disulfides.

Disulfide exchange is an important bioconjugation tool, enabling chemical modification of peptides and proteins containing free cysteines. We previously reported the synthesis of a macromer bearing an activated disulfide and its incorporation into hydrogels. Despite their ability to diffuse freely into hydrogels, larger proteins were unable to undergo in-gel disulfide exchange. In order to understand this phenomenon, we synthesized four different activated disulfide-bearing model compounds (Mn = 300 Da to 10 kDa) and quantified their rate of disulfide exchange with a small peptide (glutathione), a moderate-sized protein (ß-lactoglobulin), and a large protein (bovine serum albumin) in four different pH solutions (6.0, 7.0, 7.4, and 8.0) to mimic biological systems. Rate constants of exchange depend significantly on the size and accessibility of the thiolate. pH also significantly affects the rate of reaction, with the faster reactions occurring at higher pH. Surprisingly, little difference in exchange rates is seen between macromolecular disulfides of varying size (Mn = 2 kDa - 10 kDa), although all undergo exchange more slowly than their small molecule analogue (MW = 300 g/mol). The maximum exchange efficiencies (% disulfides exchanged after 24 h) are not siginificantly affected by thiol size or pH, but somewhat affected by disulfide size. Therefore, while all three factors investigated (pH, disulfide size, and thiolate size) can influence the exchange kinetics and extent of reaction, the size of the thiolate and its accessibility plays the most significant role.

Synthesis of photodegradable macromers for conjugation and release of bioactive molecules.

Hydrogel scaffolds are used in biomedicine to study cell differentiation and tissue evolution, where it is critical to control the delivery of chemical cues both spatially and temporally. While large molecules can be physically entrapped in a hydrogel, moderate molecular weight therapeutics must be tethered to the hydrogel network through a labile linkage to allow controlled release. We synthesized and characterized a library of polymerizable ortho-nitrobenzyl (o-NB) macromers with different functionalities at the benzylic position (alcohol, amine, BOC-amine, halide, acrylate, carboxylic acid, activated disulfide, N-hydroxysuccinyl ester, biotin). This library of polymerizable macromers containing o-NB groups should allow direct conjugation of nearly any type of therapeutic agent and its subsequent controlled photorelease from a hydrogel network. As proof-of-concept, we incorporated the N-hydroxysuccinyl ester macromer into hydrogels and then reacted phenylalanine with the NHS ester. Upon exposure to light (λ = 365 nm; 10 mW/cm(2), 10 min), 81.3% of the phenylalanine was released from the gel. Utilizing the photodegradable macromer incorporating an activated disulfide, we conjugated a cell-adhesive peptide (GCGYGRGDSPG), a protein that exhibits enzymatic activity (bovine serum albumin (BSA)), and a growth factor (transforming growth factor-ß1 (TGF-ß1)) into hydrogels, controlled their release with light (λ = 365 nm; 10 mW/cm(2), 0-20 min), and verified the bioactivity of the photoreleased molecules. The photoreleasable peptide allows real-time control over cell adhesion. BSA maintains full enzymatic activity upon sequestration and release from the hydrogel. Photoreleased TGF-ß1 is able to induce chondrogenic differentiation of human mesenchymal stem cells comparable to native TGF-ß1. Through this approach, we have demonstrated that photodegradable tethers can be used to sequester peptides and proteins into hydrogel depots and release them in an externally controlled, predictable manner without compromising biological function.

Engineered macromolecular Toll-like receptor agents and assemblies.

Macromolecular Toll-like receptor (TLR) agents have been utilized as agonists and inhibitors in preclinical and clinical settings. These agents interface with the TLR class of innate immune receptors which recognize macromolecular ligands that are characteristic of pathogenic material. As such, many agents that have been historically investigated are derived from the natural macromolecules which activate or inhibit TLRs. This review covers recent research and clinically available TLR agents that are macromolecular or polymeric. Synthetic materials that have been found to interface with TLRs are also discussed. Assemblies of these materials are investigated in the context of improving stability or efficacy of ligands. Attention is given to strategies which modify or enhance the current agents and to future outlooks on the development of these agents.

Photodegradable macromers and hydrogels for live cell encapsulation and release.

Hydrogel scaffolds are commonly used as 3D carriers for cells because their properties can be tailored to match natural extracellular matrix. Hydrogels may be used in tissue engineering and regenerative medicine to deliver therapeutic cells to injured or diseased tissue through controlled degradation. Hydrolysis and enzymolysis are the two most common mechanisms employed for hydrogel degradation, but neither allows sequential or staged release of cells. In contrast, photodegradation allows external real-time spatial and temporal control over hydrogel degradation, and allows for staged and sequential release of cells. We synthesized and characterized a series of macromers incorporating photodegradbale ortho-nitrobenzyl (o-NB) groups in the macromer backbone. We formed hydrogels from these macromers via redox polymerization and quantified the apparent rate constants of degradation (kapp) of each via photorheology at 370 nm, 10 mW/cm(2). Decreasing the number of aryl ethers on the o-NB group increases kapp, and changing the functionality from primary to seconday at the benzylic site dramatically increases kapp. Human mesenchymal stem cells (hMSCs) survive encapsulation in the hydrogels (90% viability postencapsulation). By exploiting the differences in reactivity of two different o-NB linkers, we quantitatively demonstrate the biased release of one stem cell population (green-fluoroescent protein expressing hMSCs) over another (red-fluorescent protein expressing hMSCs).

Photodegradable Polyacrylamide Gels for Dynamic Control of Cell Functions.

Cross-linked polyacrylamide hydrogels are commonly used in biotechnology and cell culture applications due to advantageous properties, such as the precise control of material stiffness and the attachment of cell adhesive ligands. However, the chemical and physical properties of polyacrylamide gels cannot be altered once fabricated. Here, we develop a photodegradable polyacrylamide gel system that allows for a dynamic control of polyacrylamide gel stiffness with exposure to light. Photodegradable polyacrylamide hydrogel networks are produced by copolymerizing acrylamide and a photocleavable ortho-nitrobenzyl (o-NB) bis-acrylate cross-linker. When the hydrogels are exposed to light, the o-NB cross-links cleave and the stiffness of the photodegradable polyacrylamide gels decreases. Further examination of the effect of dynamic stiffness changes on cell behavior reveals that in situ softening of the culture substrate leads to changes in cell behavior that are not observed when cells are cultured on presoftened gels, indicating that both dynamic and static mechanical environments influence cell fate. Notably, we observe significant changes in nuclear localization of YAP and cytoskeletal organization after in situ softening; these changes further depend on the type and concentration of cell adhesive proteins attached to the gel surface. By incorporating the simplicity and well-established protocols of standard polyacrylamide gel fabrication with the dynamic control of photodegradable systems, we can enhance the capability of polyacrylamide gels, thereby enabling cell biologists and engineers to study more complex cellular behaviors that were previously inaccessible using regular polyacrylamide gels.

Three-dimensionally printable shear-thinning triblock copolypeptide hydrogels with antimicrobial potency.

Through rational design, block sequence controlled triblock copolypeptides comprising cysteine and tyrosine as well as a lysine or glutamic acid central block are devised. In these copolypeptides, each block contributes a specific property to the hydrogels to render them extrusion printable and antimicrobial. Three-dimensional (3D) printing of complex hydrogel structures with high shape retention is demonstrated. Moreover, composition dependent potent antimicrobial activity in contact-killing assays is elucidated.

Photodegradation as a mechanism for controlled drug delivery.

A drug-releasing model compound based on photosensitive acrylated ortho-nitrobenzylether (o-NBE) moiety and fluorescein was synthesized to demonstrate photolysis as a mechanism for drug release. Release of this model compound from a hydrogel network can be controlled with light intensity (5-20 mW/cm(2)), exposure duration (0-20 min) and wavelength (365, 405, 436 nm). Due to the high molar absorptivity of the compound (5,984 M(-1) cm(-1)), light attenuation is significant in this system. Light attenuation can be used to self-limit the dosing from a hydrogel, and allow subsequent release from the drug reservoir after equilibration, or attenuation can be utilized to create a chemical gradient within the hydrogel. A model of photodegradation that uses an integrated form of Beer-Lambert's law quantitatively predicts release from hydrophilic hydrogels with low crosslink density, but fails to quantitatively predict release from more hydrophobic systems, presumably due to partitioning of the hydrophobic model compound in the hydrogel. In contrast to other mechanisms of release (enzymolysis, hydrolysis), photolysis provides real-time on demand control over drug release along with the unique ability to create chemical gradients within the hydrogel.

Selective Promotion of Adhesion of Shewanella oneidensis on Mannose-Decorated Glycopolymer Surfaces.

Using glycopolymer surfaces, we have stimulated Shewanella oneidensis bacterial colonization and induced where the bacteria attach on a molecular pattern. When adherent bacteria were rinsed with methyl α-d-mannopyranoside, the glycopolymer-functionalized surfaces retained more cells than self-assembled monolayers terminated by a single mannose unit. These results suggest that the three-dimensional multivalency of the glycopolymers both promotes and retains bacterial attachment. When the methyl α-d-mannopyranoside competitor was codeposited with the cell culture, however, the mannose-based polymer was not significantly different from bare gold surfaces. The necessity for equilibration between methyl α-d-mannopyranoside and the cell culture to remove the enhancement suggests that the retention of cells on glycopolymer surfaces is kinetically controlled and is not a thermodynamic result of the cluster glycoside effect. The MshA lectin appears to facilitate the improved adhesion observed. Our findings that the surfaces studied here can induce stable initial attachment and influence the ratio of bacterial strains on the surface may be applied to harness useful microbial communities.

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or   l-tryptophan were selectively recognized by previously identified dopamine or l-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though, slightly greater than the previously determined solution dissociation constant. Using prefunctionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with l-3,4-dihydroxyphenylalanine, l- threo-dihydroxyphenylserine, and l-5-hydroxytryptophan enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons and future identification and characterization of novel aptamers targeting neurotransmitters or other important small molecules.

Small-Molecule Patterning via Prefunctionalized Alkanethiols.

Interactions between small molecules and biomolecules are important physiologically and for biosensing, diagnostic, and therapeutic applications. To investigate these interactions, small molecules can be tethered to substrates through standard coupling chemistries. While convenient, these approaches co-opt one or more of the few small-molecule functional groups needed for biorecognition. Moreover, for multiplexing, individual probes require different surface functionalization chemistries, conditions, and/or protection/deprotection strategies. Thus, when placing multiple small-molecules on surfaces, orthogonal chemistries are needed that preserve all functional groups and are sequentially compatible. Here, we approach high-fidelity small-molecule patterning by coupling small-molecule neurotransmitter precursors, as examples, to monodisperse asymmetric oligo(ethylene glycol)alkanethiols during synthesis and prior to self-assembly on Au substrates. We use chemical lift-off lithography to singly and doubly pattern substrates. Selective antibody recognition of pre-functionalized thiols was comparable to or better than recognition of small molecules functionalized to alkanethiols after surface assembly. These findings demonstrate that synthesis and patterning approaches that circumvent sequential surface conjugation chemistries enable biomolecule recognition and afford gateways to multiplexed small-molecule functionalized substrates.