Search

We demonstrate control of gelation kinetics in formation of poly(ethylene glycol) (PEG) hydrogels by enzymatic cross-linking. A predictive model for gelation kinetics based on macromer structure and composition, stoichiometric ratios of reactants, cross-linking enzyme concentration, and the underlying kinetics of enzyme reaction was developed on the basis of classical Flory−Stockmayer theory. Experiments with substrate-functionalized multiarm comb PEG showed good agreement with theory upon variation of the enzyme concentration, with a slight overprediction in the time to gelation. Experiments with a substrate-functionalized difunctional PEG in conjunction with a polypeptide where macromer concentrations were varied were also consistent with theory, but with a slight underprediction of gelation time.

Novel polymer latexes were prepared that can be applied in several ways for the control and study of cell behavior on surfaces. Acrylic latexes with glass transitions ranging from -30 to 100 degrees C were synthesized by dispersion polymerization in a water and alcohol solution using an amphiphilic comb copolymer as a stabilizing agent. The comb had a poly(methyl methacrylate) backbone and hydrophilic poly(ethylene glycol) (PEG) side chains, which served to stabilize the dispersion and create a robust hydrophilic coating on the final latex particles. The end groups of the comb stabilizer can be selectively functionalized to obtain latex particles with a controlled density of ligands tethered to their surfaces. Latexes were prepared with adhesion peptides (RGD) linked to the surface of the acrylic beads to induce attachment and spreading of cells. Coalesced films obtained from the RGD-bearing latex particles promoted attachment of WT NR6 fibroblasts, while films from unmodified latex particles were resistant to these cells. Additionally, RGD-linked beads were embedded in cell-resistant comb polymer films to create cell-interactive surfaces with discrete clustered-ligand domains. Cell attachment and morphology were seen to vary with the surface density of the RGD-bearing latex beads.

A major limitation of the widespread use of stem cells in a variety of biotechnological applications is the relatively low level of knowledge about how to maintain these cells in vitro without losing the long-term multilineage growth properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of stem cell stimulation by exogenous cytokines would thus be useful. An emerging theme from recent hematopoietic stem cell (HSC)-expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by cytokine presentation on cell surfaces. According to such a model, when the relevant ligand-receptor interaction falls below this threshold level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Taking advantage of the ability of the cytokine leukemia inhibitory factor (LIF) to maintain embryonic stem (ES) cell pluripotentiality at high concentrations, we are testing this model by investigating critical parameters in the control of ES cell responses. We have developed quantitative assays of ES cell differentiation by measuring cell-surface alkaline phosphatase activity, cell-surface stage specific embryonic antigen (SSEA)-1 expression, and the ability of ES cells to form embryoid bodies. Examination of ES cell responses over a range of LIF concentrations shows that LIF supplementation has little effect on ES cell-growth rate but significantly alters the probability of a cell undergoing a self-renewal vs. a differentiation division. In vitro culture parameters such as inoculum cell density, medium exchange, as well as cell-intrinsic processes such as autocrine secretion are shown to affect this decision. In addition to yielding new information on stem cell regulation by exogenous factors, these studies provide important clues about culture of these cells and should stimulate further investigations into the mechanistic basis of stem cell differentiation control.

Kinetic parameters have been measured for coupled nucleophilic and solvolytic reactions of 2,2,2-trifluoroethanesulfonyl (tresyl)-modified poly(ethylene glycol) based on a system of coupled differential equations implied by recently proposed elementary reaction mechanisms. Fitted kinetic parameters were found to be strong functions of pH, temperature, and steric factors. To maximize the total yield of coupled amine as well as the fraction of secondary amine linkages, our model predicts that it is desirable to run tresyl coupling reactions at low temperatures at pH approximately 8.0, depending on the amine pKa for primary, unhindered amines. For branched primary amines, our data favor room temperature at a slightly higher pH.

The ability to understand and control the morphogenesis of mammalian cells is a fundamental objective of cell and developmental biology and tissue engineering research. Numerous processes, both biochemical and biophysical in nature, have been studied in an attempt to elucidate the mechanisms underlying this behavior. We focus here on the contributions of biophysical phenomena to the morphogenetic behavior of pure and mixed cell populations on solid surfaces in vitro. These principles are illustrated using characteristic liver tissue cells as a model system. The studies discussed demonstrate that cell-substratum and cell-cell adhesive forces are critical determinants of the ultimate morphology, cytoarchitecture, and organization achieved by these cells in vitro.

ObjectiveTo evaluate the survival and function of hepatocytes (HCs) on a novel three-dimensional (3D) synthetic biodegradable polymer scaffold with an intrinsic network of interconnected channels under continuous flow conditions.Summary Background DataThe authors' laboratory has investigated HC

Cell functions are regulated by signal transduction processes triggered by binding interactions with extracellular ligands, including those coupled to insoluble matrices as well as those diffusing in free solution. Whereas receptor interactions with freely diffusible soluble ligands are quantitatively governed by the mean ligand concentration, interactions with spatially constrained substratum-bound ligands may be affected not only by the mean ligand concentration, but also by the ligand spatial microdistribution. To probe this hypothesis we have generated surfaces presenting galactose ligands at different concentrations and different capabilities for micromobility--and thus spatial microdistribution--by means of polyethylene oxide tethering and assayed for hepatocyte spreading mediated by asialoglycoprotein receptor/cytoskeleton linkages. We demonstrate that spreading is not uniquely determined by the mean galactose concentration presented by the culture substrate. Rather, the ability of primary hepatocytes to spread is additionally specified by a combination of ligand concentration and tether length. Our results suggest spreading results when monovalent ligands possess sufficient mobility to form effectively multivalent bonds with the receptor; that is, when the substratum-bound ligands to cluster in spatial microdomains.

Novel anti-neoplastic agents such as gene targeting vectors and encapsulated carriers are quite large (approximately 100–300 nm in diameter). An understanding of the functional size and physiological regulation of transvascular pathways is necessary to optimize delivery of these agents. Here we analyze the functional limits of transvascular transport and its modulation by the microenvironment. One human and five murine tumors including mammary and colorectal carcinomas, hepatoma, glioma, and sarcoma were implanted in the dorsal skin-fold chamber or cranial window, and the pore cutoff size, a functional measure of transvascular gap size, was determined. The microenvironment was modulated: ( i ) spatially, by growing tumors in subcutaneous or cranial locations and ( ii ) temporally, by inducing vascular regression in hormone-dependent tumors. Tumors grown subcutaneously exhibited a characteristic pore cutoff size ranging from 200 nm to 1.2 μm. This pore cutoff size was reduced in tumors grown in the cranium or in regressing tumors after hormone withdrawal. Vessels induced in basic fibroblast growth factor-containing gels had a pore cutoff size of 200 nm. Albumin permeability was independent of pore cutoff size. These results have three major implications for the delivery of therapeutic agents: ( i ) delivery may be less efficient in cranial tumors than in subcutaneous tumors, ( ii ) delivery may be reduced during tumor regression induced by hormonal ablation, and ( iii ) permeability to a molecule is independent of pore cutoff size as long as the diameter of the molecule is much less than the pore diameter.

Thin film miscible blends of poly(methyl methacrylate) (PMMA) and a branched random copolymer of methyl methacrylate and methoxy poly(ethylene glycol) monomethacrylate, P(MMA-r-MnG), were investigated by neutron reflectivity. The branched copolymer, which has a higher surface tension than PMMA, was nevertheless found to segregate to and completely cover both the surface and silicon substrate following annealing in 2000 A thick films with ≥2 wt % P(MMA-r-MnG). This is in contrast to linear polyethylene oxide, which was depleted at both film interfaces when blended with PMMA and annealed. The reflectivity results were confirmed by contact angle studies, which indicate that the surfaces of P(MMA-r-MnG)/PMMA blends behave like that of pure P(MMA-r-MnG), resulting in a hydrophilic surface that is stable against dissolution in water-based environments. The branched hydrophilic additive is further shown to render PMMA resistant to protein adsorption and cell adhesion.

We demonstrate formation of a hydrogel network by cross-linking functionalized poly(ethylene glycol) (PEG) and a lysine-containing polypeptide through the action of a natural tissue enzyme, transglutaminase. The enzyme reaction rate using a PEG-modified peptide substrate is the same as the reaction rate for free substrate. Both the ratio and total concentration of the two macromers determine whether gelation will occur and the nature of the gel which forms. Under suitable conditions, clear gels form and swell to give a final composition which is 90% water. Diffusion coefficients of small proteins and albumin in the gel are comparable to those in free solution. Gelation proceeds under mild conditions and thus these gels hold potential for forming highly hydrated networks around living cells.

Cultured hepatocytes typically form multicellular aggregates which are either monolayered or spheroidal in morphology. We propose that the aggregate morphology resulting from a particular cell-substratum interaction has a biophysical basis: when cell contractile forces are greater than cell-substratum adhesion forces, spheroidal aggregates form; when cell contractile forces are weaker than cell-substratum adhesion forces, cells remain essentially spread and form monolayered aggregates. We tested this hypothesis by systematically varying the morphology of hepatocellular aggregates formed on substrata coated with a series of different concentrations of Matrigel, and correlating aggregate morphology with the cell-substratum adhesion strength measured in a shear flow detachment assay. Aggregate morphology was binary-spheroidal aggregates formed at low Matrigel concentrations and monolayered aggregates formed at high Matrigel concentrations. Cell-substratum adhesion strength was similarly binary, with low adhesion strengths correlated with spheroidal aggregates and high adhesion strengths correlated with formation of monolayered aggregates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 415-426, 1997.

Objective To test the hypothesis that endometrial stromal cells (ESCs) in endometriosis exhibit increased cell motility under hypoxia. Design Prospective case-control study. Setting University research laboratory. Patient(s) Women with endometriosis (n = 18) or benign gynecological disease (n = 19). Intervention(s) Eutopic ESCs were cultured under normoxia (20% O 2 ) or hypoxia (6.5% O 2 ), and migration and invasion capacity assayed, with pathway-focused polymerase chain reaction (PCR) array and ELISAs performed. CD26/dipeptidyl peptidase IV (DPPIV) expression was determined by flow cytometric analysis and enzymatic activity assay. The ESCs supplemented with Diprotin A (CD26 inhibitor), stromal cell-derived factor-1α, or AMD3100 (C-X-C motif receptor 4; CXCR4 blocker) were assayed for their migratory potential. Main Outcome Measure(s) Endometrial stromal cell migration and invasion under hypoxia. Result(s) Endometriotic ESCs showed significantly higher migration and invasion through collagen gels under hypoxia compared with nonendometriotic ESCs. The PCR array revealed down-regulation of the migration inhibitor CD26/DPPIV and up-regulation of angiogenic factors (vascular endothelial growth factor A, C-X-C motif Ligand 6; CXCL6) in endometriotic ESCs under hypoxia. The CD26/DPPIV surface expression and activity as well as angiogenic protein secretions suggested that the molecular mechanisms underlying aberrant migratory and angiogenic behavior in endometriotic ESCs. A combinatorial treatment with diprotin A and stromal cell-derived factor-1α effectively enhanced migration and invasion preferentially in endometriotic ESCs cultured hypoxically. Conclusion(s) Loss of CD26/DPPIV under hypoxia and the subsequent increase in migratory and angiogenic factors may favor conditions for lesion development in endometriosis.

Background—Doxorubicin (DOXO) is an effective anthracycline chemotherapeutic, but its use is limited by cumulative dose-dependent cardiotoxicity. Neuregulin-1β is an ErbB receptor family ligand that is effective against DOXO-induced cardiomyopathy in experimental models but is also proneoplastic. We previously showed that an engineered bivalent neuregulin-1β (NN) has reduced proneoplastic potential in comparison with the epidermal growth factor–like domain of neuregulin-1β (NRG), an effect mediated by receptor biasing toward ErbB3 homotypic interactions uncommonly formed by native neuregulin-1β. Here, we hypothesized that a newly formulated, covalent NN would be cardioprotective with reduced proneoplastic effects in comparison with NRG. Methods and Results—NN was expressed as a maltose-binding protein fusion in Escherichia coli. As established previously, NN stimulated antineoplastic or cytostatic signaling and phenotype in cancer cells, whereas NRG stimulated proneoplastic signaling and phenotype. In neonatal rat cardiomyocytes, NN and NRG induced similar downstream signaling. NN, like NRG, attenuated the double-stranded DNA breaks associated with DOXO exposure in neonatal rat cardiomyocytes and human cardiomyocytes derived from induced pluripotent stem cells. NN treatment significantly attenuated DOXO-induced decrease in fractional shortening as measured by blinded echocardiography in mice in a chronic cardiomyopathy model (57.7±0.6% versus 50.9±2.6%, P=0.004), whereas native NRG had no significant effect (49.4±3.7% versus 50.9±2.6%, P=0.813). Conclusions—NN is a cardioprotective agent that promotes cardiomyocyte survival and improves cardiac function in DOXO-induced cardiotoxicity. Given the reduced proneoplastic potential of NN versus NRG, NN has translational potential for cardioprotection in patients with cancer receiving anthracyclines., United States. Dept. of Defense (Congressionally Directed Medical Research Programs, Breast Cancer Research Program (W81XWH-11-1-0035)), National Institutes of Health (U.S.) (grant HL112905), National Institutes of Health (U.S.) (grant AG032977), National Institutes of Health (U.S.) (grant DE019523), National Institutes of Health (U.S.) (grant DK090147), National Institutes of Health (U.S.) (grant U54-CA112967), National Institutes of Health (U.S.) (grant EB003805), United States. Dept. of Defense (Congressionally Directed Medical Research Programs, Orthopedic Research Program Career Development Award (W81XWH-11-1-0821)), Sarnoff Cardiovascular Research Foundation (fellowship), Hertz Foundation (Fellowship), Harvard Stem Cell Institute

A Disintegrin and Metalloproteinases (ADAMs) are the principal enzymes for shedding receptor tyrosine kinase (RTK) ectodomains and ligands from the cell surface. Multiple layers of activity regulation, feedback, and catalytic promiscuity impede our understanding of context-dependent ADAM “sheddase” function and our ability to predictably target that function in disease. This study uses combined measurement and computational modeling to examine how various growth factor environments influence sheddase activity and cell migration in the invasive disease of endometriosis. We find that ADAM-10 and -17 dynamically integrate numerous signaling pathways to direct cell motility. Data-driven modeling reveals that induced cell migration is a quantitative function of positive feedback through EGF ligand release and negative feedback through RTK shedding. Although sheddase inhibition prevents autocrine ligand shedding and resultant EGF receptor transactivation, it also leads to an accumulation of phosphorylated receptors (HER2, HER4, and MET) on the cell surface, which subsequently enhances Jnk/p38 signaling. Jnk/p38 inhibition reduces cell migration by blocking sheddase activity while additionally preventing the compensatory signaling from accumulated RTKs. In contrast, Mek inhibition reduces ADAM-10 and -17 activities but fails to inhibit compensatory signaling from accumulated RTKs, which actually enhances cell motility in some contexts. Thus, here we present a sheddase-based mechanism of rapidly acquired resistance to Mek inhibition through reduced RTK shedding that can be overcome with rationally directed combination inhibitor treatment. We investigate the clinical relevance of these findings using targeted proteomics of peritoneal fluid from endometriosis patients and find growth-factor–driven ADAM-10 activity and MET shedding are jointly dysregulated with disease.

Multipotential stromal cells/mesenchymal stem cells (MSCs) are attractive candidates for regenerative therapy due to the ability of these cells to differentiate and positively influence neighboring cells. However, on implantation for wound reconstruction, these cells are lost as they are challenged by nonspecific inflammation signals generated in the wound environment and in response to any implanted foreign body. We have previously shown that sustained and surface-restricted epidermal growth factor receptor (EGFR) signaling by a tethered form of its prototypal ligand EGF enhances survival of MSC in the presence of death cytokines such as FasL, serum deprivation, and low oxygen in vitro. This was proposed to be due to the plasma membrane restriction of EGFR signaling. Interestingly, during wound repair, an extracellular matrix (ECM) component Tenascin-C (TNC) containing EGF-like repeats (EGFL) and fibronectin-like repeats (FNL) is upregulated. A few of the 14 EGFL on each of the 6 arms, especially the 14th, bind as low-affinity/high-avidity ligands to EGFR causing sustained surface-restricted EGFR signaling. We queried whether signaling by this physiologically relevant EGFR matrikine also protects MSCs from FasL-induced death. MSCs grown on TNC and Collagen I (as TNC by itself is antiadhesive) displayed a survival advantage in the presence of FasL. TNC neither sequestered nor neutralized FasL; rather, the effects of survival were via cell signaling. This survival was dependent on TNC activating EGFR and downstream pathways of Erk and Akt through EGFL; to a much lesser extent, the FNL of TNC also contributed to survival. Taken together, these results suggest that providing MSCs with a nonimmunogenic naturally occurring ECM moiety such as TNC enhances their survival in the presence of death factors, and this advantage occurs via signaling through EGFR primarily and integrins only to a minor extent. This matrix component is proposed to supplement MSC delivery on the scaffolds to provide a survival advantage against death upon in vivo implantation.

Background: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1, National Institutes of Health (U.S.) (Grant HL052212), National Institutes of Health (U.S.) (Grant HL066105), National Institutes of Health (U.S.) (Grant ES015241), National Institutes of Health (U.S.) (Grant GM068762)

We applied the Transport of Intensity equation for quantitative phase imaging of hydrogel microwells using a commercial bright field microscope. Reconstructions of microwell phase depths are validated computationally and experimentally.

The development of computational models capable of describing the transport of oxygen and other nutrients and resulting concentration gradients represents a critical design tool for microfluidic cell culture systems. In particular, the interplay between nutrient transport and shear forces on cultured cell populations resulting from fluid motion represents one of the most critical challenges in the design of these systems. Conventional single-chamber systems are shown to have limited flexibility in the maintaining low shear forces while sustaining sufficient nutrient transport, while two-compartment systems separated by semipermeable membranes provide a broader design space for optimization of transport and shear. This chapter addresses the development of computational models capable of investigating the tradeoffs between nutrient transport and shear forces in both single- and dual-chamber microfluidic cell culture systems.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR11) of the Conference Proceedings. Citation Format: Amanda M. Clark, Sarah E. Wheeler, Carissa L. Young, Venkateswaran C. Pillai, Donna B. Stolz, Douglas A. Lauffenburger, Raman Venkataramanan, Linda G. Griffith, Alan Wells. Elucidating candidate biomarkers of breast cancer progression and dormancy using a 3D model of metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A41.

Over the years, various liver-derived in vitro model systems have been developed to enable investigation of the potential adverse effects of chemicals and drugs. Liver tissue slices, isolated microsomes, perfused liver, immortalized cell lines, and primary hepatocytes have been used extensively. Immortalized cell lines and primary isolated liver cells are currently the most widely used in vitro models for liver toxicity testing. Limited throughput, loss of viability, and decreases in liver-specific functionality and gene expression are common shortcomings of these models. Recent developments in the field of in vitro hepatotoxicity include three-dimensional tissue constructs and bioartificial livers, co-cultures of various cell types with hepatocytes, and differentiation of stem cells into hepatic lineage-like cells. In an attempt to provide a more physiological environment for cultured liver cells, some of the novel cell culture systems incorporate fluid flow, micro-circulation, and other forms of organotypic microenvironments. Co-cultures aim to preserve liver-specific morphology and functionality beyond those provided by cultures of pure parenchymal cells. Stem cells, both embryonic- and adult tissue-derived, may provide a limitless supply of hepatocytes from multiple individuals to improve reproducibility and enable testing of the individual-specific toxicity. This review describes various traditional and novel in vitro liver models and provides a perspective on the challenges and opportunities afforded by each individual test system., National Institutes of Health (U.S.) (P42 ES005948), National Institutes of Health (U.S.) (R01 ES01524)

As principal degrading enzymes of the extracellular matrix, metalloproteinases contribute to various pathologies and represent a family of promising drug targets and biomarker candidates. However, multiple proteases and endogenous inhibitors interact to govern metalloproteinase activity, often leading to highly context-dependent protease function that unfortunately has impeded associated clinical utility. We present a method for rapidly assessing the activity of multiple specific proteases in small volumes (

As a platform for investigating the individual effects of substrate stiffness, permeability, and ligand density on cellular behavior, we developed a set of hydrogels with stiffness tuned by polymer backbone rigidity, independent of cross-link density and concentration. Previous studies report that poly(propargyl-L-glutamate) (PPLG), synthesized by ring-opening polymerization of the N-carboxy anhydride of γ-propargyl-L-glutamate (γpLglu), adopts a rigid a-helix conformation: we hypothesized that a random copolymer (PPDLG) with equal amounts of γpLglu and γ-propargyl-D-glutamate (γpDglu) monomers would exhibit a more flexible random coil conformation. The resulting macromers exhibited narrow molecular weight distributions (PDI = 1.15) and were grafted with ethylene glycol groups using a highly efficient “click” azide/alkyne cycloaddition reaction with average grafting efficiency of 97% for PPLG and 85% for PPDLG. The polypeptide secondary structure, characterized via circular dichroism spectroscopy, FTIR spectroscopy, and dynamic light scattering, is indeed dependent upon monomer chirality: PPLG exhibits an α-helix conformation while PPDLG adopts a random coil conformation. Hydrogel networks produced by cross-linking either helical or random coil polypeptides with poly(ethylene glycol) (PEG) were analyzed for amount of swelling, gelation efficiency, and permeability to a model protein. In addition, the elastic modulus of helical and coil polypeptide gels was determined by AFM indentation in fluid. Importantly, we found that helical and coil polypeptide gels exhibited similar swelling and permeability but different stiffnesses, which correspond to predictions from the theory of semi-flexible chains., National Institutes of Health (U.S.) (R01 EB10246), National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems

The promise of cellular therapy lies in healing damaged tissues and organs in vivo as well as generating tissue constructs in vitro for subsequent transplantation. Postnatal stem cells are ideally suited for cellular therapies due to their pluripotency and the ease with which they can be cultured on functionalized substrates. Creating environments to control and successfully drive their differentiation toward a lineage of choice is one of the most important challenges of current cell-based engineering strategies. In recent years, a variety of biomaterials platforms have been prepared for stem cell cultures, primarily to provide efficient delivery of growth or survival factors to cells and a conductive microenvironment for their growth. Here, we demonstrate that repeating tetralayer structures composed of biocompatible poly(methacrylic acid), poly(acrylamide), and poly(ethylene oxide)-block-poly(ε-caprolactone) micelles arrayed in layer-by-layer films can serve as a payload region for dexamethasone delivery to human mesenchymal stem cells (MSCs). This architecture can induce MSC differentiation into osteoblasts in a dose-dependent manner. The amount of Dex loaded in the films is controlled by varying the deposition conditions and the film thickness. Release of Dex is tuned by changing the amount of covalent cross-linking of multilayers via thermal treatments. The multilayer architecture including payload and cell-adhesion region introduced here are well suited for extended cell culture thus affording the important and protective effect of both Dex release and immobilization. These films may find applications in the local delivery of immobilized therapeutics for biomedical applications, as they can be deposited on a wide range of substrates with different shapes, sizes, and composition., Singapore-MIT Alliance for Research and Technology, Hertz Foundation

Death Blockers 395 18.4 Conclusions and Future Directions 396 Acknowledgments 398 References 39818.1.1 Adult Stem Cells: Regenerative PotentialStem cells are found in adult tissue at low frequencies and are capable of differentiating into specic tissues based on their differentiation potential, location, stimuli, and relocation (Huh et al. 2006). Multipotent stromal cells or mesenchymal stem cells (MSCs) present in the adult bone marrow are self-renewing clonal precursors of non-hematopoietic stromal tissue and are capable of differentiating into mesenchymal lineages of bone, cartilage, fat, tendon, muscle, endothelial cells, and marrow stroma (Pittenger et al. 1999). These can be obtained by marrow aspiration. There are also stem and progenitor cells found in adipose tissue, called adipose-derived stem cells (ASCs) obtained at a higher frequency compared to bone-marrow-derived stem cells, 0.5% compared to 0.01%. These cells which differ in expression of certain cell surface markers (Fraser et al. 2006) possess similar multilineage differentiation potential though there is a predilection toward fat tissue (Brayeld et al. 2010). ASCs are called by several other names in literature including processed lipoaspirate cells (LPA) and adipose-tissue-derived stromal cells. Likewise, the muscle also holds postnatal stem cells called musclederived stem cells (MDSCs); these cells can differentiate spontaneously into cardiac and skeletal muscle and by modication ex vivo with growth factors can differentiate into the osteogenic and chondrogenic lineages (Usas and Huard 2007). There also exist stem cells with more restricted differentiation ability like neural stem cells (NSCs) found in the adult hippocampus and lateral ventricle which can differentiate into neurons and glia (astrocytes and oligodendrocytes) (Massirer et al. 2011). There are other less well clinically characterized epithelial stem cells in tissues with high homeostasis, turnover rates, and regeneration such as the skin which form hair follicles (Zhang et al. 2009), sebaceous glands (Jensen et al. 2009), and epidermal cells (reviewed [Barker et al. 2010]). Other stem cells are found in the mammary and prostate glands (Shackleton et al. 2006; Stingl et al. 2006).

Multipotential stromal cells or mesenchymal stem cells (MSCs) have been proposed as aids in regenerating bone and adipose tissues, as these cells form osteoblasts and adipocytes. A major obstacle to this use of MSC is the initial loss of cells postimplantation. This cell death in part is due to ubiquitous nonspecific inflammatory cytokines such as FasL generated in the implant site. Our group previously found that soluble epidermal growth factor (sEGF) promotes MSC expansion. Furthermore, tethering EGF (tEGF) onto a two-dimensional surface altered MSC responses, by restricting epidermal growth factor receptor (EGFR) to the cell surface, causing sustained activation of EGFR, and promoting survival from FasL-induced death. sEGF by causing internalization of EGFR does not support MSC survival. However, for tEGF to be useful in bone regeneration, it needs to allow for MSC differentiation into osteoblasts while also protecting emerging osteoblasts from apoptosis. tEGF did not block induced differentiation of MSCs into osteoblasts, or adipocytes, a common default MSC-differentiation pathway. MSC-derived preosteoblasts showed increased Fas levels and became more susceptible to FasL-induced death, which tEGF prevented. Differentiating adipocytes underwent a reduction in Fas expression and became resistant to FasL-induced death, with tEGF having no further survival effect. tEGF protected undifferentiated MSC from combined insults of FasL, serum deprivation, and physiologic hypoxia. Additionally, tEGF was dominant in the face of sEGF to protect MSC from FasL-induced death. Our results suggest that MSCs and differentiating osteoblasts need protective signals to survive in the inflammatory wound milieu and that tEGF can serve this function., National Institute of General Medical Sciences (U.S.) (GM069668), National Institute of Dental and Craniofacial Research (U.S.) (DE019523)

Primary rat liver sinusoidal endothelial cells (LSEC) are difficult to maintain in a differentiated state in culture for scientific studies or technological applications. Relatively little is known about molecular regulatory processes that affect LSEC differentiation because of this inability to maintain cellular viability and proper phenotypic characteristics for extended times in vitro, given that LSEC typically undergo death and detachment around 48–72 h even when treated with VEGF. We demonstrate that particular lipid supplements added to serum-free, VEGF-containing medium increase primary rat liver LSEC viability and maintain differentiation. Addition of a defined lipid combination, or even oleic acid (OA) alone, promotes LSEC survival beyond 72 h and proliferation to confluency. Moreover, assessment of LSEC cultures for endocytic function, CD32b surface expression, and exhibition of fenestrae showed that these differentiation characteristics were maintained when lipids were included in the medium. With respect to the underlying regulatory pathways, we found lipid supplement-enhanced phosphatidylinositol 3-kinase and MAPK signaling to be critical for ensuring LSEC function in a temporally dependent manner. Inhibition of Akt activity before 72 h prevents growth of SEC, whereas MEK inhibition past 72 h prevents survival and proliferation. Our findings indicate that OA and lipids modulate Akt/PKB signaling early in culture to mediate survival, followed by a switch to a dependence on ERK signaling pathways to maintain viability and induce proliferation after 72 h. We conclude that free fatty acids can support maintenance of liver LSEC cultures in vitro; key regulatory pathways involved include early Akt signaling followed by ERK signaling., National Science Foundation (U.S.) (Grant EFRI-0735997), National Institutes of Health (U.S.) (Grant R01 GM069668)

An insiduously terrifying aspect of breast cancer is its propensity to recur in metastatic sites even over a decade after all evidence of cancer has passed. It is obvious that these cells had escaped very early from the primary tumor as this occurs even in small, node-negative and in situ primary lesions, and that these micrometastases survival chemotherapeutic regimens that shrink and extirpate the primary carcinomas. Thus this mortal turn of events leads to three key questions how do the cells escape early?, how do they survive over extended periods?, and what causes these dormant lesions to become aggressive at these late dates? The area of metastatic dissemination of primary cells has received a great level of inspection with an understanding of underlying molecular mechanisms, even if we do not yet have therapies. While chemotherapy survival in ectopic sites has been studies more recently, this is usually done in the context of a growing lesion. The truly under-appreciated and under-studied aspect is the last, that of re-emergence from dormancy. Understanding what triggers dormant breast cancer cells to emerge and form frank and mortal metastases would allow the development not only of rationale therapeutics but of prevention and possibly lifestyle avoidance. Herein, these issues are addressed using a novel organotypic bioreactor in which tumor cells can be followed for weeks to months, the process of seeding, dormancy and emergence can be followed.

Prodomains of A disintegrin and metalloproteinase (ADAM) metallopeptidases can act as highly specific intra- and intermolecular inhibitors of ADAM catalytic activity. The mouse ADAM9 prodomain (proA9; amino acids 24–204), expressed and characterized from Escherichia coli, is a competitive inhibitor of human ADAM9 catalytic/disintegrin domain with an overall inhibition constant of 280 ± 34 nm and high specificity toward ADAM9. In SY5Y neuroblastoma cells overexpressing amyloid precursor protein, proA9 treatment reduces the amount of endogenous ADAM10 enzyme in the medium while increasing membrane-bound ADAM10, as shown both by Western and activity assays with selective fluorescent peptide substrates using proteolytic activity matrix analysis. An increase in membrane-bound ADAM10 generates higher levels of soluble amyloid precursor protein α in the medium, whereas soluble amyloid precursor protein β levels are decreased, demonstrating that inhibition of ADAM9 increases α-secretase activity on the cell membrane. Quantification of physiological ADAM10 substrates by a proteomic approach revealed that substrates, such as epidermal growth factor (EGF), HER2, osteoactivin, and CD40-ligand, are increased in the medium of BT474 breast tumor cells that were incubated with proA9, demonstrating that the regulation of ADAM10 by ADAM9 applies for many ADAM10 substrates. Taken together, our results demonstrate that ADAM10 activity is regulated by inhibition of ADAM9, and this regulation may be used to control shedding of amyloid precursor protein by enhancing α-secretase activity, a key regulatory step in the etiology of Alzheimer disease., National Institutes of Health (U.S.) (Grant R01EB010246), National Institutes of Health (U.S.) (Grant R01GM081336), Heptagon Fund (London, England), Cancer Research UK, Whitehead Foundation, Duke University. School of Medicine (Bridge Funding Program), Germany. Bundesministerium für Bildung und Forschung, China (National Fellowship from the Chinese Scholarship Council), Florida State University

We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator, which enhances the limited sensitivity of low-abundance enzyme assays by concentrating biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultralow levels of matrix metalloproteinases (MMPs) from diluted cellular supernatant and showed that it significantly (~10-fold) reduced the time required to complete the assay and the sample volume used., National Institutes of Health (U.S.) (Grant GM68762), National Institutes of Health (U.S.) (Grant U54-CA112967), National Institutes of Health (U.S.) (Grant R01-EB010246), National Institutes of Health (U.S.) (Grant R01-GM081336), National Science Foundation (U.S.) (Graduate Fellowship), United States. Defense Advanced Research Projects Agency (Cipher Program)

Protein kinases catalyze protein phosphorylation and thereby control the flow of information through signaling cascades. Currently available methods for concomitant assessment of the enzymatic activities of multiple kinases in complex biological samples rely upon indirect proxies for enzymatic activity, such as posttranslational modifications to protein kinases. Our laboratories have recently described a method for directly quantifying the enzymatic activity of kinases in unfractionated cell lysates using substrates containing a phosphorylation-sensitive unnatural amino acid termed CSox, which can be monitored using fluorescence. Herein, we demonstrate the utility of this methodology using a probe set encompassing p38α, MK2, ERK1/2, Akt, and PKA. This panel of chemosensors provides activity measurements of individual kinases in a model of skeletal muscle differentiation and can be readily used to generate individualized kinase activity profiles for tissue samples from clinical cancer patients.

In recent years, microfluidic devices have emerged as a platform in which to culture tissue for various applications such as drug discovery, toxicity testing, and fundamental investigations of cell-cell interactions. We examine the transport phenomena associated with gradients of soluble factors and oxygen in a microfluidic device for co-culture. This work focuses on emulating conditions known to be important in sustaining a viable culture of cells. Critical parameters include the flow and the resulting shear stresses, the transport of various soluble factors throughout the flow media, and the mechanical arrangement of the cells in the device. Using analytical models derived from first principles, we investigate interactions between flow conditions and transport in a microfluidic device. A particular device of interest is a bilayer configuration in which critical solutes such as oxygen are delivered through the media into one channel, transported across a nanoporous membrane, and consumed by cells cultured in another. The ability to control the flow conditions in this membrane bilayer device to achieve sufficient oxygenation without shear damage is shown to be superior to the case present in a single channel system. Using the results of these analyses, a set of criteria that characterize the geometric and transport properties of a robust microfluidic device are provided.

Author Manuscript 2012 May 21., Design of 3D scaffolds that can facilitate proper survival, proliferation, and differentiation of progenitor cells is a challenge for clinical applications involving large connective tissue defects. Cell migration within such scaffolds is a critical process governing tissue integration. Here, we examine effects of scaffold pore diameter, in concert with matrix stiffness and adhesivity, as independently tunable parameters that govern marrow-derived stem cell motility. We adopted an “inverse opal” processing technique to create synthetic scaffolds by crosslinking poly(ethylene glycol) at different densities (controlling matrix elastic moduli or stiffness) and small doses of a heterobifunctional monomer (controlling matrix adhesivity) around templating beads of different radii. As pore diameter was varied from 7 to 17 µm (i.e., from significantly smaller than the spherical cell diameter to approximately cell diameter), it displayed a profound effect on migration of these stem cells—including the degree to which motility was sensitive to changes in matrix stiffness and adhesivity. Surprisingly, the highest probability for substantive cell movement through pores was observed for an intermediate pore diameter, rather than the largest pore diameter, which exceeded cell diameter. The relationships between migration speed, displacement, and total path length were found to depend strongly on pore diameter. We attribute this dependence to convolution of pore diameter and void chamber diameter, yielding different geometric environments experienced by the cells within. Bioeng. 2011; 108:1181–1193, (National Institute of General Medical Sciences (U.S.) (NRSA Fellowship GM083472), National Institutes of Health (U.S.) (National Institute of General Medical Sciences (U.S.) Cell Migration Consortium Grant GM064346), National Science Foundation (U.S.) (CAREER CBET-0644846)

Multipotential stromal cells (MSCs) have been touted to provide an alternative to conservative procedures of therapy, be it heart transplants, bone reconstruction, kidney grafts, or skin, neuronal and cartilage repair. A wide gap exists, however, between the number of MSCs that can be obtained from the donor site and the number of MSCs needed for implantation to regenerate tissue. Standard methods of MSC expansion being followed in laboratories are not fully suitable due to time and age-related constraints for autologous therapies, and transplant issues leave questions for allogenic therapies. Beyond these issues of sufficient numbers, there also exists a problem of MSC survival at the graft. Experiments in small animals have shown that MSCs do not persist well in the graft environment. Either there is no incorporation into the host tissue, or, if there is incorporation, most of the cells are lost within a month. The use of growth and other trophic factors may be helpful in counteracting these twin issues of MSC expansion and death. Growth factors are known to influence cell proliferation, motility, survival and morphogenesis. In the case of MSCs, it would be beneficial that the growth factor does not induce differentiation at an early stage since the number of early-differentiating progenitors would be very low. The present review looks at the effect of and downstream signaling of various growth factors on proliferation and survival in MSCs.

Refer to Web version on PubMed Central for supplementary material., Idiosyncratic drug hepatotoxicity is a major problem in pharmaceutical development due to poor prediction capability of standard preclinical toxicity assessments and limited knowledge of its underlying mechanisms. Findings in animal models have shown that adverse effects of numerous drugs with idiosyncratic hepatotoxicity in humans can be reproduced in the presence of coincident inflammatory cytokine signaling. Following these observations, we have recently developed an in vitro drug/inflammatory cytokine co-treatment approach that can reproduce clinical drug hepatotoxicity signatures—particularly for idiosyncratic drugs—in cultured primary human hepatocytes. These observations have suggested that drug-induced stresses may interact with cytokine signaling to induce hepatic cytotoxicity, but the hepatocyte signaling mechanisms governing these interactions are poorly understood. Here, we collect high-throughput phosphoprotein signaling and cytotoxicity measurements in cultured hepatocytes, from multiple human donors, treated with combinations of hepatotoxic drugs (e.g. trovafloxacin, clarithromycin) and cytokines (tumor necrosis factor-α, interferon-γ, interleukin-1α, and interleukin-6). We demonstrate, through orthogonal partial least-squares regression (OPLSR) modeling of these signal-response data, that drug/cytokine hepatic cytotoxicity is integratively controlled by four key signaling pathways: Akt, p70 S6 kinase, MEK–ERK, and p38–HSP27. This modeling predicted, and experimental studies confirmed, that the MEK–ERK and p38–HSP27 pathways contribute pro-death signaling influences in drug/cytokine hepatic cytotoxicity synergy. Further, our four-pathway OPLSR model produced successful prediction of drug/cytokine hepatic cytotoxicities across different human donors, even though signaling and cytotoxicity responses were both highly donor-specific. Our findings highlight the critical role of kinase signaling in drug/cytokine hepatic cytotoxicity synergies and reveal that hepatic cytotoxicity responses are governed by multi-pathway signaling network balance., Pfizer Inc., Institute for Collaborative Biotechnologies, MIT Center for Cell Decision Processes, National Institute of Mental Health (U.S.) (grant P50-GM68762), Massachusetts Institute of Technology. Biotechnology Process Engineering Center, Massachusetts Institute of Technology. Center for Environmental Health Sciences, National Institute of Mental Health (U.S.) (grant U19ES011399), Whitaker Foundation

A fluorescence-based imaging system has been developed for real-time, in vivo tumor margin assessment with microscopic sensitivity. Intra-operative imaging results have matched post-surgical histo-pahtological margin assessment in all the sarcoma mice cases tested so far.

Idiosyncratic drug hepatotoxicity is a hepatotoxicity subset that occurs in a very small fraction of human patients, is poorly predicted by standard preclinical models and in clinical trials, and frequently leads to post-approval drug failure. Animal models utilizing bacterial LPS co-administration to induce an inflammatory background and hepatocyte cell culture models utilizing cytokine mix co-treatment have successfully reproduced idiosyncratic hepatotoxicity signatures for certain drugs, but the hepatocyte signaling mechanisms governing these drug-cytokine toxicity synergizes are largely unclear. Here, we summarize our efforts to computationally model the signaling mechanisms regulating inflammatory cytokine-associated idiosyncratic drug hepatotoxicity. We collected a “cue-signal-response” (CSR) data compendium in cultured primary human hepatocytes treated with many combinations of idiosyncratic hepatotoxic drugs and inflammatory cytokine mixes (“cues”) and subjected this compendium to orthogonal partial-least squares regression (OPLSR) to computationally relate the measured intracellular phosphoprotein signals and hepatocellular death responses. This OPLSR model suggested that hepatocytes specify their cell death responses to toxic drug/cytokine conditions by integrating signals from four key pathways -- Akt, p70 S6K, ERK, and p38. An OPLSR model focused on data from these four signaling pathways demonstrated accurate predictions of idiosyncratic drug- and cytokine-induced hepatotoxicities in a second human hepatocyte donor, suggesting that hepatocytes from different individuals have shared network control mechanisms governing toxicity responses to diverse combinations of idiosyncratic hepatotoxicants and inflammatory cytokines.

In vitro models that capture the complexity of in vivo tissue and organ behaviors in a scalable and easy-to-use format are desirable for drug discovery. To address this, we have developed a bioreactor that fosters maintenance of 3D tissue cultures under constant perfusion and we have integrated multiple bioreactors into an array in a multiwell plate format. All bioreactors are fluidically isolated from each other. Each bioreactor in the array contains a scaffold that supports formation of hundreds of 3D microscale tissue units. The tissue units are perfused with cell culture medium circulated within the bioreactor by integrated pneumatic diaphragm micropumps. Electronic controls for the pumps are kept outside the incubator and connected to the perfused multiwell by pneumatic lines. The docking design and open-well bioreactor layout make handling perfused multiwell plates similar to using standard multiwell tissue culture plates. A model of oxygen consumption and transport in the circulating culture medium was used to predict appropriate operating parameters for primary liver cultures. Oxygen concentrations at key locations in the system were then measured as a function of flow rate and time after initiation of culture to determine oxygen consumption rates. After seven days of culture, tissue formed from cells seeded in the perfused multiwell reactor remained functionally viable as assessed by immunostaining for hepatocyte and liver sinusoidal endothelial cell (LSEC) phenotypic markers., National Institute of Environmental Health Sciences (grant number 5P30ES002109-30), National Institutes of Health (U.S.) (NIH grant number 5R01ES015241), DuPont MIT Alliance, Pfizer Inc., National Science Foundation (U.S.) (NSF grant number EEC-9843342)

Increasing interest has focused on capturing the complexity of tissues and organs in vitro as models of human pathophysiological processes. In particular, a need exists for a model that can investigate the interactions in three dimensions (3D) between epithelial tissues and a microvascular network since vascularization is vital for reconstructing functional tissues in vitro. Here, we implement a microfluidic platform to analyze angiogenesis in 3D cultures of rat primary hepatocytes and rat/human microvascular endothelial cells (rMVECs/hMVECs). Liver and vascular cells were cultured on each sidewall of a collagen gel scaffold between two microfluidic channels under static or flow conditions. Morphogenesis of 3D hepatocyte cultures was found to depend on diffusion and convection across the nascent tissue. Furthermore, rMVECs formed 3D capillary-like structures that extended across an intervening gel to the hepatocyte tissues in hepatocyte-rMVEC coculture while they formed 2D sheet-like structures in rMVEC monoculture. In addition, diffusion of fluorescent dextran across the gel scaffold was analyzed, demonstrating that secreted proteins from the hepatocytes and MVECs can be exchanged across the gel scaffold by diffusional transport. The experimental approach described here is useful more generally for investigating microvascular networks within 3D engineered tissues with multiple cell types in vitro.—Sudo, R., Chung, S., Zervantonakis, I. K., Vickerman, V., Toshimitsu, Y., Griffith, L. G., Kamm, R. D. Transport-mediated angiogenesis in 3D epithelial coculture.

Author Manuscript 2011 April 29., Epidermal growth factor receptor (EGFR)-mediated signaling helps regulate bone development and healing through its effects on osteogenic cells. Here, we show how EGFR activity and osteogenic differentiation responses in primary human bone marrow-derived multipotent stromal cells (MSCs) are influenced by presenting covalently tethered epidermal growth factor (tEGF) on the culture substratum, a presentation mode that reduces EGFR internalization and restricts signaling to the cell surface. In both absence and presence of tEGF, MSCs increase expression levels of EGFR and its heterodimerization partner HER2 during the course of osteogenic differentiation. tEGF substrata increased levels of phosphorylated EGFR and phosphorylated extracellular regulated kinase (ERK) compared to control substrata, and these elevations were associated with a twofold enhancement of MSC alkaline phosphatase activity at day 7 and matrix mineralization at day 21. Surprisingly, addition of soluble EGF (sEGF) to cells cultured on tEGF substrata reduces osteogenic differentiation, even though EGFR signaling is more strongly activated in acute, short-term manner by sEGF treatment than by tEGF treatment. A striking concomitant result of the sEGF effects is near-complete downregulation of EGFR and HER2, demonstrating that the tEGF/EGFR interaction is dynamically reversible even though temporally sustained. Taken together, our results show that enhanced MSC osteogenic differentiation corresponds to a sustained combination of receptor expression and ligand presentation, both of which are maintained by tEGF., National Institutes of Health (U.S.) (Grant R01-GM059870-07), National Institutes of Health (U.S.) (Grant R01-DE 019523-10), National Institutes of Health (U.S.) (Grant P40RR017447), United Negro College Fund (Merck Postdoctoral Fellowship), Georgia Institute of Technology (FACES Fellowship), National Center for Research Resources (U.S.) (Grant P40RR017447)

Multitarget compounds that act on a diverse set of regulatory pathways are emerging as a therapeutic approach for a variety of cancers. Toward a more specified use of this approach, we hypothesize that the desired efficacy can be recreated in terms of a particular combination of relatively more specific (i.e., ostensibly single target) compounds. We test this hypothesis for the geldanamycin analogue 17-Allylamino-17-demethoxygeldanamycin (17AAG) in hepatocellular carcinoma cells, measuring critical phosphorylation levels that indicate the kinase pathway effects correlating with apoptotic responsiveness of the Hep3B cell line in contrast to the apoptotic resistance of the Huh7 cell line. A principal components analysis (PCA) constructed from time course measurements of seven phosphoprotein signaling levels identified modulation of the AKT, IκB kinase, and signal transducer and activator of transcription 3 pathways by 17AAG treatment as most important for distinguishing these cell-specific death responses. The analysis correctly suggested from 17AAG-induced effects on these phosphoprotein levels that the FOCUS cell line would show apoptotic responsiveness similarly to Hep3B. The PCA also guided the inhibition of three critical pathways and rendered Huh7 cells responsive to 17AAG. Strikingly, in all three hepatocellular carcinoma lines, the three-inhibitor combination alone exhibited similar or greater efficacy to 17AAG. We conclude that (a) the PCA captures and clusters the multipathway phosphoprotein time courses with respect to their 17AAG-induced apoptotic responsiveness and (b) we can recreate, in a more specified manner, the cellular responses of a prospective multitarget cancer therapeutic., National Institute of General Medical Sciences (U.S.). Cell Decision Processes Center, National Cancer Institute (U.S.). Integrative Cancer Biology Program, Massachusetts Institute of Technology. Presidential Fellowship, National Institutes of Health (U.S.)

Strategies to combine aspirated marrow cells with scaffolds to treat connective tissue defects are gaining increasing clinical attention and use. In situations such as large defects where initial survival and proliferation of transplanted connective tissue progenitors (CTPs) are limiting, therapeutic outcomes might be improved by using the scaffold to deliver growth factors that promote the early stages of cell function in the graft. Signaling by the epidermal growth factor receptor (EGFR) plays a role in cell survival and has been implicated in bone development and homeostasis. Providing epidermal growth factor (EGF) in a scaffold-tethered format may sustain local delivery and shift EGFR signaling to pro-survival modes compared to soluble ligand. We therefore examined the effect of tethered EGF on osteogenic colony formation from human bone marrow aspirates in the context of three different adhesion environments using a total of 39 donors. We found that tethered EGF, but not soluble EGF, increased the numbers of colonies formed regardless of adhesion background, and that tethered EGF did not impair early stages of osteogenic differentiation., National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AR42997), National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AG024980), National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 GM59870), National Institute of General Medical Sciences (U.S.) (Grant NIH DE019523)

The confinement of polymer chains to spaces smaller than their bulk dimensions is realized in a variety of systems of scientific and commercial interest1-8. Under these circumstances, the conformations9 and dynamics10 of individual polymer chains may differ greatly from their bulk state. Strategies exploiting such confinement effects have been proposed for creating nanolithographic templates1, separating polyelectrolytes by molecular weight2, and controlling the spatial distribution of surface-bound peptides in order to enhance their bioactivity3-5. Conformations of polymer chains confined to two dimensions are predicted to depend strongly on polymer chain concentration. Swollen polymer coils in two dimensions have been modeled as self-avoiding random walks, for which the radius of gyration (Rg) scales with the number of segments (N) as Rg∼Nv where ν=0.75.11 With increasing chain concentration, screening effects cause a reduction in chain dimensions such that for a 2D monodisperse melt, chains exhibit ideal behavior with ν=0.5.9,12-14 These scaling predictions have been verified through Monte Carlo (MC) simulations.14,15 Three-dimensional chains exhibit identical scaling in the melt (ν=0.5), while ν=0.6 in dilute solution.9 There have been few experimental reports of the determination of ν for polymers in 2D confinement.16-23 Jones et al.16,17 performed small-angle neutron scattering studies on thin polystyrene films incorporating a deuterated polystyrene fraction and found that the scattering intensity scaled with wavevector as I(k)∼k−2 for films of sub-Rg thickness, consistent with ideal chain statistics (ν=0.5). Maier and Radler investigated the conformations of micron-length strands of fluorescently labeled DNA confined in 2D via adsorption to mobile cationic lipid bilayers. For isolated chains they found that Rg scaled with number of DNA base pairs as Rg∼N0.79, while for concentrated systems chain collapse was observed, consistent with theoretical predictions, though no scaling exponent was reported.18,19 Wang and Foltz20 conducted atomic force microscopy (AFM) studies on dense 2D films of nanoropes (worm-like micelles) formed from polystyrene-block-polybutadiene copolymers and found that the lateral dimension scaled with contour length as R∼L0.63. For dilute surface concentrations, however, surface tension effects resulted in more collapsed configurations with R∼L0.51. Sukhishvili et al.21 performed fluctuation correlation spectroscopy on isolated fluorescently-labeled polyethylene oxide (PEO) chains adsorbed onto a self-assembled monolayer on silica and obtained a diffusion coefficient scaling of D∼N−3/2, implying Rg∼N−3/4. From Langmuir trough experiments on polymers confined at the air/water interface, Vilanove and Rondelez22 extracted ν values of 0.56 and 0.79 for poly(methyl methacrylate) and poly(vinyl acetate), respectively, using the semidilute scaling relation Π∼c2ν/(2ν–1), where Π and c denote the surface pressure and concentration, respectively. More recently, a similar analysis by Gavranovic et al.23 on poly(tert-butyl methacrylate) yielded ν=0.53. Akin to the surface-confined DNA and PEO systems described above, amphiphilic comb copolymers composed of a hydrophobic poly(methyl methacrylate) (PMMA) backbone and short, hydrophilic PEO side chains, PMMA-g-PEO, have been proposed to self-organize at the polymer/water interface, resulting in the effective confinement of the backbone to two dimensions3,24,25 for chains at the immediate surface of a PMMA-g-PEO film (Figure 1). To directly probe such 2D conformations, in this study combs modified with maleimide groups on the PEO chain ends were blended at 0.5-10 wt% with unmodified PMMA-g-PEO and cast into films of thickness ∼3Rg. Films were immersed in aqueous solution to induce orientation of surface molecules, and maleimide-functionalized chains at the film/water interface were labeled with 1.4 nm dia. Au nanoparticles. Transmission electron microscopy (TEM) was then used to trace the trajectories of individual nanoparticle-decorated chains. The distribution of observed 2D chain lengths was compared to the molecular weight distribution obtained by gel permeation chromatography. The scaling exponent ν for the 2D radius of gyration was calculated, and compared to that obtained from Monte Carlo simulation of a 2D melt having a chain length distribution fitted to that of our experimental system. The results suggest that polydispersity significantly influences 2D melt conformations. Figure 1 (a) Schematic illustration of amphiphilic comb copolymers confined in two dimensions at the polymer film/water interface. Nanoparticles attached to side chains of maleimide-modified comb polymers create nanoparticle clusters at the surface. By contrast, ...

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials.

On p. 2836, Daniel G. Anderson and co-workers report the development of end-modified poly(β-amino ester)s that are able to deliver DNA to primary human umbilical vein endothelial cells at levels comparable to adenovirus at a Multiplicity of Infection between 100 and 500, and two orders of magnitude better than the commonly used non-viral polymeric vector, poly(ethylene imine). Small structural changes were found to have dramatic effects on multiple steps of gene delivery including the DNA binding affinity, nanoparticle size, intracellular DNA uptake, and final protein expression. In vivo, these polymer modifications enhance DNA delivery to ovarian tumors.