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Biomaterials (v.32, #8)

Editorial board (pp. ifc).
Editorial board (pp. ifc).

Fabrication of self-assemblingd-form peptide nanofiber scaffold d-EAK16 for rapid hemostasis by Zhongli Luo; Shunkang Wang; Shuguang Zhang (pp. 2013-2020).
We previously reported a class of designer self-assembling peptides that form 3-dimensional nanofiber scaffolds using onlyl-amino acids. Here we report that usingd-amino acids, the chiral self-assembling peptide d-EAK16 also forms 3-dimensional nanofiber scaffold that is indistinguishable from its counterpart l-EAK16. These chiral peptides containing alld-amino acids, d-EAK16, self-assemble into well-ordered nanofibers. However with alternatingd- andl-amino acids, EA∗K16 and E∗AK∗16, showed poor self-assembling properties. To fully understand individual molecular building blocks and their structures, assembly properties and dynamic behaviors for rapid hemostasis, we used circular dichroism, atomic force microscopy and scanning electron microscopy to study in detail the peptides. We also used rheological measurement to study the hydrogel gelation property. Furthermore, we used an erythrocyte-agglutination test and a rabbit liver wound healing model, particularly in the transverse rabbit liver experiments, to examine rapid hemostasis. We showed that 1% d-EAK16 for the liver wound hemostasis took ∼20 s, but using 1% of E∗A∗K16 and EA∗K16 that have alternating chirald- andl-amino acids took ∼70 and ∼80 s, respectively. We here propose a plausible model not only to provide insights in understanding the chiral assembly properties for rapid hemostasis, but also to aid in further design of self-assemblingd-form peptide scaffolds for clinical applications.Display Omitted

Keywords: Chirality; Circular dichroism; d; -form amino acids; Molecular self-assembly; Scaffold hydrogel


Fabrication of self-assemblingd-form peptide nanofiber scaffold d-EAK16 for rapid hemostasis by Zhongli Luo; Shunkang Wang; Shuguang Zhang (pp. 2013-2020).
We previously reported a class of designer self-assembling peptides that form 3-dimensional nanofiber scaffolds using onlyl-amino acids. Here we report that usingd-amino acids, the chiral self-assembling peptide d-EAK16 also forms 3-dimensional nanofiber scaffold that is indistinguishable from its counterpart l-EAK16. These chiral peptides containing alld-amino acids, d-EAK16, self-assemble into well-ordered nanofibers. However with alternatingd- andl-amino acids, EA∗K16 and E∗AK∗16, showed poor self-assembling properties. To fully understand individual molecular building blocks and their structures, assembly properties and dynamic behaviors for rapid hemostasis, we used circular dichroism, atomic force microscopy and scanning electron microscopy to study in detail the peptides. We also used rheological measurement to study the hydrogel gelation property. Furthermore, we used an erythrocyte-agglutination test and a rabbit liver wound healing model, particularly in the transverse rabbit liver experiments, to examine rapid hemostasis. We showed that 1% d-EAK16 for the liver wound hemostasis took ∼20 s, but using 1% of E∗A∗K16 and EA∗K16 that have alternating chirald- andl-amino acids took ∼70 and ∼80 s, respectively. We here propose a plausible model not only to provide insights in understanding the chiral assembly properties for rapid hemostasis, but also to aid in further design of self-assemblingd-form peptide scaffolds for clinical applications.Display Omitted

Keywords: Chirality; Circular dichroism; d; -form amino acids; Molecular self-assembly; Scaffold hydrogel


The interactions of astrocytes and fibroblasts with defined pore structures in static and perfusion cultures by Tao Sun; Peter S. Donoghue; Jennifer R. Higginson; Nikolaj Gadegaard; Susan C. Barnett; Mathis O. Riehle (pp. 2021-2031).
Open pores to maintain nutrient diffusion and waste removal after cell colonization are crucial for the successful application of constructs based on assembled membranes, in our case tubular scaffolds made of ɛ-polycaprolactone (PCL), for use in tissue engineering. Due to the complex three-dimensional structure and large size of such scaffolds needed for transplantable tissues, it is difficult to investigate the cell–pore interactions in situ. Therefore miniaturized bioreactors inside Petri dishes (30 mm in diameter), containing porous PCL or poly-dimethylsiloxane (PDMS) membranes, were developed to allow the interactions of different cells with defined pores to be investigated in situ during both static and perfusion cultures. Investigation of two different cell types (fibroblasts and cortical astrocytes) and how they interact with a range of pores (100–350 μm in diameter) for up to 50 days indicated that the cells either ‘covered’ or ‘bridged’ the pores. Three distinct behaviors were observed in the way cortical astrocytes interacted with pores, while fibroblasts were able to quickly bridge the pores based on consistent “joint efforts”. Our studies demonstrate that the distinct pore sealing behaviors of both cell types were influenced by pore size, initial cell density and culture period, but not by medium perfusion within the range of shear forces investigated. These findings form important basic data about the usability of pores within scaffolds that could inform the design and fabrication of suitable scaffolds for various applications in tissue engineering.

Keywords: Scaffold porosity; Fibroblast; Astrocyte; Static culture; Perfusion culture; Tissue engineering


The interactions of astrocytes and fibroblasts with defined pore structures in static and perfusion cultures by Tao Sun; Peter S. Donoghue; Jennifer R. Higginson; Nikolaj Gadegaard; Susan C. Barnett; Mathis O. Riehle (pp. 2021-2031).
Open pores to maintain nutrient diffusion and waste removal after cell colonization are crucial for the successful application of constructs based on assembled membranes, in our case tubular scaffolds made of ɛ-polycaprolactone (PCL), for use in tissue engineering. Due to the complex three-dimensional structure and large size of such scaffolds needed for transplantable tissues, it is difficult to investigate the cell–pore interactions in situ. Therefore miniaturized bioreactors inside Petri dishes (30 mm in diameter), containing porous PCL or poly-dimethylsiloxane (PDMS) membranes, were developed to allow the interactions of different cells with defined pores to be investigated in situ during both static and perfusion cultures. Investigation of two different cell types (fibroblasts and cortical astrocytes) and how they interact with a range of pores (100–350 μm in diameter) for up to 50 days indicated that the cells either ‘covered’ or ‘bridged’ the pores. Three distinct behaviors were observed in the way cortical astrocytes interacted with pores, while fibroblasts were able to quickly bridge the pores based on consistent “joint efforts”. Our studies demonstrate that the distinct pore sealing behaviors of both cell types were influenced by pore size, initial cell density and culture period, but not by medium perfusion within the range of shear forces investigated. These findings form important basic data about the usability of pores within scaffolds that could inform the design and fabrication of suitable scaffolds for various applications in tissue engineering.

Keywords: Scaffold porosity; Fibroblast; Astrocyte; Static culture; Perfusion culture; Tissue engineering


The effect of recombinant E-cadherin substratum on the differentiation of endoderm-derived hepatocyte-like cells from embryonic stem cells by Amranul Haque; Bayar Hexig; Qingyuan Meng; Sharif Hossain; Masato Nagaoka; Toshihiro Akaike (pp. 2032-2042).
Generation of specific lineages of cells from embryonic stem (ES) cells is pre-requisite to use these cells in pre-clinical applications. Here, we developed a recombinant E-cadherin substratum for generation of hepatic progenitor populations at single cell level. This artificial acellular feeder layer supports the stepwise differentiation of ES cells to cells with characteristics of definitive endoderm, hepatic progenitor cells, and finally cells with phenotypic and functional characteristics of hepatocytes. The efficient differentiation of hepatic endoderm cells (approximately 55%) together with the absence of neuroectoderm and mesoderm markers suggests the selective induction of endoderm differentiation. The co-expression of E-cahderin and alpha-fetoprotein (approximately 98%) suggests the important role of E-cadherin as a surface marker for the enrichment of hepatic progenitor cells. With extensive expansion, approximately 92% albumin expressing cells can be achieved without any enzymatic stress and cell sorting. Furthermore, these mouse ES cell-derived hepatocyte-like cells showed higher morphological similarities to primary hepatocytes. In conclusion, we demonstrated that E-cadherin substratum can guide differentiation of ES cells into endoderm-derived hepatocyte-like cells. This recombinant extracellular matrix could be effectively used as an in vitro model for studying the mechanisms of early stages of liver development even at single cell level.

Keywords: Embryonic stem cells; E-cadherin; Endoderm; Hepatocyte differentiation


The effect of recombinant E-cadherin substratum on the differentiation of endoderm-derived hepatocyte-like cells from embryonic stem cells by Amranul Haque; Bayar Hexig; Qingyuan Meng; Sharif Hossain; Masato Nagaoka; Toshihiro Akaike (pp. 2032-2042).
Generation of specific lineages of cells from embryonic stem (ES) cells is pre-requisite to use these cells in pre-clinical applications. Here, we developed a recombinant E-cadherin substratum for generation of hepatic progenitor populations at single cell level. This artificial acellular feeder layer supports the stepwise differentiation of ES cells to cells with characteristics of definitive endoderm, hepatic progenitor cells, and finally cells with phenotypic and functional characteristics of hepatocytes. The efficient differentiation of hepatic endoderm cells (approximately 55%) together with the absence of neuroectoderm and mesoderm markers suggests the selective induction of endoderm differentiation. The co-expression of E-cahderin and alpha-fetoprotein (approximately 98%) suggests the important role of E-cadherin as a surface marker for the enrichment of hepatic progenitor cells. With extensive expansion, approximately 92% albumin expressing cells can be achieved without any enzymatic stress and cell sorting. Furthermore, these mouse ES cell-derived hepatocyte-like cells showed higher morphological similarities to primary hepatocytes. In conclusion, we demonstrated that E-cadherin substratum can guide differentiation of ES cells into endoderm-derived hepatocyte-like cells. This recombinant extracellular matrix could be effectively used as an in vitro model for studying the mechanisms of early stages of liver development even at single cell level.

Keywords: Embryonic stem cells; E-cadherin; Endoderm; Hepatocyte differentiation


The regulation of traction force in relation to cell shape and focal adhesions by Andrew D. Rape; Wei-hui Guo; Yu-li Wang (pp. 2043-2051).
Mechanical forces provide critical inputs for proper cellular functions. The interplay between the generation of, and response to, mechanical forces regulate such cellular processes as differentiation, proliferation, and migration. We postulate that adherent cells respond to a number of physical and topographical factors, including cell size and shape, by detecting the magnitude and/or distribution of traction forces under different conditions. To address this possibility we introduce a new simple method for precise micropatterning of hydrogels, and then apply the technique to systematically investigate the relationship between cell geometry, focal adhesions, and traction forces in cells with a series of spread areas and aspect ratios. Contrary to previous findings, we find that traction force is not determined primarily by the cell spreading area but by the distance from cell center to the perimeter. This distance in turn controls traction forces by regulating the size of focal adhesions, such that constraining the size of focal adhesions by micropatterning can override the effect of geometry. We propose that the responses of traction forces to center-periphery distance, possibly through a positive feedback mechanism that regulates focal adhesions, provide the cell with the information on its own shape and size. A similar positive feedback control may allow cells to respond to a variety of physical or topographical signals via a unified mechanism.

Keywords: Microcontact printing; Polyacrylamide; Traction force; Cell shape


The regulation of traction force in relation to cell shape and focal adhesions by Andrew D. Rape; Wei-hui Guo; Yu-li Wang (pp. 2043-2051).
Mechanical forces provide critical inputs for proper cellular functions. The interplay between the generation of, and response to, mechanical forces regulate such cellular processes as differentiation, proliferation, and migration. We postulate that adherent cells respond to a number of physical and topographical factors, including cell size and shape, by detecting the magnitude and/or distribution of traction forces under different conditions. To address this possibility we introduce a new simple method for precise micropatterning of hydrogels, and then apply the technique to systematically investigate the relationship between cell geometry, focal adhesions, and traction forces in cells with a series of spread areas and aspect ratios. Contrary to previous findings, we find that traction force is not determined primarily by the cell spreading area but by the distance from cell center to the perimeter. This distance in turn controls traction forces by regulating the size of focal adhesions, such that constraining the size of focal adhesions by micropatterning can override the effect of geometry. We propose that the responses of traction forces to center-periphery distance, possibly through a positive feedback mechanism that regulates focal adhesions, provide the cell with the information on its own shape and size. A similar positive feedback control may allow cells to respond to a variety of physical or topographical signals via a unified mechanism.

Keywords: Microcontact printing; Polyacrylamide; Traction force; Cell shape


Maturational growth of self-assembled, functional menisci as a result of TGF-β1 and enzymatic chondroitinase-ABC stimulation by Daniel J. Huey; Kyriacos A. Athanasiou (pp. 2052-2058).
Replacement of the knee meniscus requires a material possessing adequate geometrical and biomechanical properties. Meniscal tissue engineering attempts have been unable to produce tissue with collagen content and biomechanical properties, particularly tensile properties, mimicking native menisci. In an effort to obtain the geometric properties and the maturational growth necessary for the recapitulation of biochemical and, thus, biomechanical properties, a scaffoldless cell-based system, the self-assembly process, was used in conjunction with the catabolic enzyme chondroitinase-ABC and TGF-β1. We show that combinations of these agents resulted in maturational growth as evidenced by synergistic enhancement of the radial tensile modulus by 5-fold and the compressive relaxation modulus by 68%, and additive increases of the compressive instantaneous modulus by 136% and Col/WW by 196%. This study shows that tissue engineering can produce a biomaterial that is on par with the biochemical and biomechanical properties of native menisci.

Keywords: Cartilage tissue engineering; Knee meniscus; TGF (transforming growth factor); C-ABC (chondroitinase-ABC); ECM; Mechanical properties


Maturational growth of self-assembled, functional menisci as a result of TGF-β1 and enzymatic chondroitinase-ABC stimulation by Daniel J. Huey; Kyriacos A. Athanasiou (pp. 2052-2058).
Replacement of the knee meniscus requires a material possessing adequate geometrical and biomechanical properties. Meniscal tissue engineering attempts have been unable to produce tissue with collagen content and biomechanical properties, particularly tensile properties, mimicking native menisci. In an effort to obtain the geometric properties and the maturational growth necessary for the recapitulation of biochemical and, thus, biomechanical properties, a scaffoldless cell-based system, the self-assembly process, was used in conjunction with the catabolic enzyme chondroitinase-ABC and TGF-β1. We show that combinations of these agents resulted in maturational growth as evidenced by synergistic enhancement of the radial tensile modulus by 5-fold and the compressive relaxation modulus by 68%, and additive increases of the compressive instantaneous modulus by 136% and Col/WW by 196%. This study shows that tissue engineering can produce a biomaterial that is on par with the biochemical and biomechanical properties of native menisci.

Keywords: Cartilage tissue engineering; Knee meniscus; TGF (transforming growth factor); C-ABC (chondroitinase-ABC); ECM; Mechanical properties


The enhancement of VEGF-mediated angiogenesis by polycaprolactone scaffolds with surface cross-linked heparin by Shivani Singh; Benjamin M. Wu; James C.Y. Dunn (pp. 2059-2069).
This study investigates the effect of surface cross-linked heparin on vascular endothelial growth factor (VEGF)-mediated angiogenesis in porous polycaprolactone (PCL) scaffolds in vivo. We tested the hypothesis that VEGF delivered by scaffolds coated with a sub-micron thick layer of immobilized heparin would accelerate angiogenesis. The bioactivity of retained VEGF was confirmed by its phosphorylation of VEGF receptor-2. After 7 and 14 days of subcutaneous implantation in mice, the heparin-PCL scaffolds loaded with VEGF displayed significantly higher infiltration of blood vessels which traversed the entire scaffold thickness (2 mm). The stability and function of the newly formed vessels were confirmed by smooth muscle cell coverage and vessel perfusability, respectively. The contribution of individual components was assessed by varying the VEGF dose and heparin thickness. Prolonging the cross-linking reaction on PCL scaffolds resulted in higher heparin content, thicker heparin layer, and higher VEGF retention. While a dose dependent angiogenic response was observed with VEGF, higher amount of cross-linked heparin did not translate into additional improvement in angiogenesis for a given dose of VEGF. The synergism of immobilized heparin and VEGF in stimulating angiogenesis was observed in vivo.

Keywords: Scaffold; Polycaprolactone; Surface modification; Heparin; Cross-linking; Angiogenesis


The enhancement of VEGF-mediated angiogenesis by polycaprolactone scaffolds with surface cross-linked heparin by Shivani Singh; Benjamin M. Wu; James C.Y. Dunn (pp. 2059-2069).
This study investigates the effect of surface cross-linked heparin on vascular endothelial growth factor (VEGF)-mediated angiogenesis in porous polycaprolactone (PCL) scaffolds in vivo. We tested the hypothesis that VEGF delivered by scaffolds coated with a sub-micron thick layer of immobilized heparin would accelerate angiogenesis. The bioactivity of retained VEGF was confirmed by its phosphorylation of VEGF receptor-2. After 7 and 14 days of subcutaneous implantation in mice, the heparin-PCL scaffolds loaded with VEGF displayed significantly higher infiltration of blood vessels which traversed the entire scaffold thickness (2 mm). The stability and function of the newly formed vessels were confirmed by smooth muscle cell coverage and vessel perfusability, respectively. The contribution of individual components was assessed by varying the VEGF dose and heparin thickness. Prolonging the cross-linking reaction on PCL scaffolds resulted in higher heparin content, thicker heparin layer, and higher VEGF retention. While a dose dependent angiogenic response was observed with VEGF, higher amount of cross-linked heparin did not translate into additional improvement in angiogenesis for a given dose of VEGF. The synergism of immobilized heparin and VEGF in stimulating angiogenesis was observed in vivo.

Keywords: Scaffold; Polycaprolactone; Surface modification; Heparin; Cross-linking; Angiogenesis


Axon guidance of rat cortical neurons by microcontact printed gradients by Rita Fricke; Peter D. Zentis; Lionel T. Rajappa; Boris Hofmann; Marko Banzet; Andreas Offenhäusser; Simone H. Meffert (pp. 2070-2076).
Substrate-bound gradients expressed in numerous spatio-temporal patterns play a crucial role during the development of complex neural circuits. A deeper understanding of the axon guidance mechanism is provided by studying the effect of a defined substrate-bound cue on a confined neural network. In this study, we constructed a discontinuous substrate-bound gradient to control neuronal cell position, the path of neurite growth, and axon directionality. A variety of gradient patterns, with slight changes in slope, width, and length were designed and fabricated by microcontact printing using laminin/poly-l-lysine (PLL) or PLL alone. The gradients were tested for neurite growth and their impact on axon guidance of embryonic rat cortical neurons. The neurite length was determined and the axon was evaluated by Tau-1 immunostaining. We found that the microgradients of laminin/PLL and PLL directed neurons’ adhesion, differentially controlled the neurite growth, and guided up to 84% of the axons. The effect of the protein micropattern on axon guidance and neurite growth depended on the protein and geometric parameters used. Our approach proved to be very successful in guiding axons of single multipolar neurons with very high efficiency. It could thereby be useful to engineer defined neural networks for analyzing signal processing of functional circuits, as well as to unravel fundamental questions of the axon guidance mechanism.

Keywords: Axon guidance; Microcontact printing; Substrate-bound gradient; Rat cortical neurons; Directed neurite growth


Axon guidance of rat cortical neurons by microcontact printed gradients by Rita Fricke; Peter D. Zentis; Lionel T. Rajappa; Boris Hofmann; Marko Banzet; Andreas Offenhäusser; Simone H. Meffert (pp. 2070-2076).
Substrate-bound gradients expressed in numerous spatio-temporal patterns play a crucial role during the development of complex neural circuits. A deeper understanding of the axon guidance mechanism is provided by studying the effect of a defined substrate-bound cue on a confined neural network. In this study, we constructed a discontinuous substrate-bound gradient to control neuronal cell position, the path of neurite growth, and axon directionality. A variety of gradient patterns, with slight changes in slope, width, and length were designed and fabricated by microcontact printing using laminin/poly-l-lysine (PLL) or PLL alone. The gradients were tested for neurite growth and their impact on axon guidance of embryonic rat cortical neurons. The neurite length was determined and the axon was evaluated by Tau-1 immunostaining. We found that the microgradients of laminin/PLL and PLL directed neurons’ adhesion, differentially controlled the neurite growth, and guided up to 84% of the axons. The effect of the protein micropattern on axon guidance and neurite growth depended on the protein and geometric parameters used. Our approach proved to be very successful in guiding axons of single multipolar neurons with very high efficiency. It could thereby be useful to engineer defined neural networks for analyzing signal processing of functional circuits, as well as to unravel fundamental questions of the axon guidance mechanism.

Keywords: Axon guidance; Microcontact printing; Substrate-bound gradient; Rat cortical neurons; Directed neurite growth


Chimeric fibronectin matrix mimetic as a functional growth- and migration-promoting adhesive substrate by Daniel C. Roy; Susan J. Wilke-Mounts; Denise C. Hocking (pp. 2077-2087).
Therapeutic protein engineering combines genetic, biochemical, and functional information to improve existing proteins or invent new protein technologies. Using these principles, we developed an approach to deliver extracellular matrix (ECM) fibronectin-specific signals to cells. Fibronectin matrix assembly is a cell-dependent process that converts the inactive, soluble form of fibronectin into biologically-active ECM fibrils. ECM fibronectin stimulates cell functions required for normal tissue regeneration, including cell growth, spreading, migration, and collagen reorganization. We have developed recombinant fibronectin fragments that mimic the effects of ECM fibronectin on cell function by coupling the cryptic heparin-binding fragment of fibronectin’s first type III repeat (FNIII1H) to the integrin-binding domain (FNIII8–10). GST/III1H,8–10 supports cell adhesion and spreading and stimulates cell proliferation to a greater extent than plasma fibronectin. Deletion and site-specific mutant constructs were generated to identify the active regions in GST/III1H,8–10 and reduce construct size. A chimeric construct in which the integrin-binding, RGDS loop was inserted into the analogous site in FNIII8 (GST/III1H,8RGD), supported cell adhesion and migration, and enhanced cell proliferation and collagen gel contraction. GST/III1H,8RGD was expressed in bacteria and purified from soluble lysate fractions by affinity chromatography. Fibronectin matrix assembly is normally up-regulated in response to tissue injury. Decreased levels of ECM fibronectin are associated with non-healing wounds. Engineering fibronectin matrix mimetics that bypass the need for cell-dependent fibronectin matrix assembly in chronic wounds is a novel approach to stimulating cellular activities critical for tissue repair.

Keywords: Fibronectin; Recombinant protein; Extracellular matrix; Cell proliferation; Cell adhesionAbbreviations; ECM; Extracellular matrix; FNIII; Fibronectin type III repeat; FN-null MEFs; Fibronectin-null mouse embryonic fibroblasts; GST; Glutathione-S-transferase


Chimeric fibronectin matrix mimetic as a functional growth- and migration-promoting adhesive substrate by Daniel C. Roy; Susan J. Wilke-Mounts; Denise C. Hocking (pp. 2077-2087).
Therapeutic protein engineering combines genetic, biochemical, and functional information to improve existing proteins or invent new protein technologies. Using these principles, we developed an approach to deliver extracellular matrix (ECM) fibronectin-specific signals to cells. Fibronectin matrix assembly is a cell-dependent process that converts the inactive, soluble form of fibronectin into biologically-active ECM fibrils. ECM fibronectin stimulates cell functions required for normal tissue regeneration, including cell growth, spreading, migration, and collagen reorganization. We have developed recombinant fibronectin fragments that mimic the effects of ECM fibronectin on cell function by coupling the cryptic heparin-binding fragment of fibronectin’s first type III repeat (FNIII1H) to the integrin-binding domain (FNIII8–10). GST/III1H,8–10 supports cell adhesion and spreading and stimulates cell proliferation to a greater extent than plasma fibronectin. Deletion and site-specific mutant constructs were generated to identify the active regions in GST/III1H,8–10 and reduce construct size. A chimeric construct in which the integrin-binding, RGDS loop was inserted into the analogous site in FNIII8 (GST/III1H,8RGD), supported cell adhesion and migration, and enhanced cell proliferation and collagen gel contraction. GST/III1H,8RGD was expressed in bacteria and purified from soluble lysate fractions by affinity chromatography. Fibronectin matrix assembly is normally up-regulated in response to tissue injury. Decreased levels of ECM fibronectin are associated with non-healing wounds. Engineering fibronectin matrix mimetics that bypass the need for cell-dependent fibronectin matrix assembly in chronic wounds is a novel approach to stimulating cellular activities critical for tissue repair.

Keywords: Fibronectin; Recombinant protein; Extracellular matrix; Cell proliferation; Cell adhesionAbbreviations; ECM; Extracellular matrix; FNIII; Fibronectin type III repeat; FN-null MEFs; Fibronectin-null mouse embryonic fibroblasts; GST; Glutathione-S-transferase


Micropatterning neural cell cultures in 3D with a multi-layered scaffold by Anja Kunze; Michele Giugliano; Ana Valero; Philippe Renaud (pp. 2088-2098).
Cortical neurons, in their native state, are organized in six different cell layers; and the thickness of the cell layer ranges from 0.12 mm to 0.4 mm. The structure of cell layers plays an important role in neurodegenerative diseases or corticogenesis. We developed a 3D microfluidic device for creating physiologically realistic, micrometer scaled neural cell layers. Using this device, we demonstrated that (1) agarose–alginate mixture can be gelled thermally, thus an excellent candidate for forming multi-layered scaffolds for micropatterning embedded cells; (2) primary cortical neurons were cultured successfully for up to three weeks in the micropatterned multi-layered scaffold; (3) B27 concentration gradient enhanced neurite outgrowth. In addition, this device is compatible with optical microscopy, the dynamic process of neural growth can be imaged, and density and number of neurites can be quantified. This device can potentially be used for drug development, as well as research in basic neural biology.

Keywords: Micropatterning; Agarose-alginate hydrogel; Multi-layered scaffold; Primary neural cell culture; Neurite outgrowth


Micropatterning neural cell cultures in 3D with a multi-layered scaffold by Anja Kunze; Michele Giugliano; Ana Valero; Philippe Renaud (pp. 2088-2098).
Cortical neurons, in their native state, are organized in six different cell layers; and the thickness of the cell layer ranges from 0.12 mm to 0.4 mm. The structure of cell layers plays an important role in neurodegenerative diseases or corticogenesis. We developed a 3D microfluidic device for creating physiologically realistic, micrometer scaled neural cell layers. Using this device, we demonstrated that (1) agarose–alginate mixture can be gelled thermally, thus an excellent candidate for forming multi-layered scaffolds for micropatterning embedded cells; (2) primary cortical neurons were cultured successfully for up to three weeks in the micropatterned multi-layered scaffold; (3) B27 concentration gradient enhanced neurite outgrowth. In addition, this device is compatible with optical microscopy, the dynamic process of neural growth can be imaged, and density and number of neurites can be quantified. This device can potentially be used for drug development, as well as research in basic neural biology.

Keywords: Micropatterning; Agarose-alginate hydrogel; Multi-layered scaffold; Primary neural cell culture; Neurite outgrowth


Role of material-driven fibronectin fibrillogenesis in cell differentiation by Manuel Salmerón-Sánchez; Patricia Rico; David Moratal; Ted T. Lee; Jean E. Schwarzbauer; Andrés J. García (pp. 2099-2105).
Fibronectin (FN) is a ubiquitous extracellular matrix protein (ECM) protein that is organized into fibrillar networks by cells through an integrin-mediated process that involves contractile forces. This assembly allows for the unfolding of the FN molecule, exposing cryptic domains that are not available in the native globular FN structure and activating intracellular signalling complexes. However, organization of FN into a physiological fibrillar network upon adsorption on a material surface has not been observed. Here we demonstrate cell-free, material-induced FN fibrillogenesis into a biological matrix with enhanced cellular activities. We found that simple FN adsorption onto poly(ethyl acrylate) surfaces, but not control polymers, triggered FN organization into a fibrillar network via interactions in the amino-terminal 70 kDa fragment, which is involved in the formation of cell-mediated FN fibrils. Moreover, the material-driven FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules and even type I collagen. Our results demonstrate that molecular assembly of FN can take place at the material interface, giving rise to a physiological protein network similar to fibrillar matrices assembled by cells. This research identifies material surfaces that trigger the organization of extracellular matrix proteins into biological active fibrils and establishes a new paradigm to engineer ECM-mimetic biomaterials.

Keywords: Fibronectin; Cell adhesion; Integrin; Interface


Role of material-driven fibronectin fibrillogenesis in cell differentiation by Manuel Salmerón-Sánchez; Patricia Rico; David Moratal; Ted T. Lee; Jean E. Schwarzbauer; Andrés J. García (pp. 2099-2105).
Fibronectin (FN) is a ubiquitous extracellular matrix protein (ECM) protein that is organized into fibrillar networks by cells through an integrin-mediated process that involves contractile forces. This assembly allows for the unfolding of the FN molecule, exposing cryptic domains that are not available in the native globular FN structure and activating intracellular signalling complexes. However, organization of FN into a physiological fibrillar network upon adsorption on a material surface has not been observed. Here we demonstrate cell-free, material-induced FN fibrillogenesis into a biological matrix with enhanced cellular activities. We found that simple FN adsorption onto poly(ethyl acrylate) surfaces, but not control polymers, triggered FN organization into a fibrillar network via interactions in the amino-terminal 70 kDa fragment, which is involved in the formation of cell-mediated FN fibrils. Moreover, the material-driven FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules and even type I collagen. Our results demonstrate that molecular assembly of FN can take place at the material interface, giving rise to a physiological protein network similar to fibrillar matrices assembled by cells. This research identifies material surfaces that trigger the organization of extracellular matrix proteins into biological active fibrils and establishes a new paradigm to engineer ECM-mimetic biomaterials.

Keywords: Fibronectin; Cell adhesion; Integrin; Interface


The potential of combinations of drug-loaded nanoparticle systems and adult stem cells for glioma therapy by Mathilde Roger; Anne Clavreul; Marie-Claire Venier-Julienne; Catherine Passirani; Claudia Montero-Menei; Philippe Menei (pp. 2106-2116).
The prognosis of patients with malignant glioma remains extremely poor, despite surgery and improvements in radio- and chemo-therapies. Nanotechnologies hold great promise in glioma therapy as they protect the therapeutic agent and allow its sustained release. However, new paradigms permitting tumor-specific targeting and extensive intratumoral distribution must be developed to efficiently deliver nanoparticles. Modifications and functionalizations of nanoparticles have been developed to specifically track tumor cells. However, these nanoparticles have yielded few clinical results due to intra-patient heterogeneity and inter-patient variability. Stem cells with a specific tropism for brain tumors could be used as delivery vehicles for nanoparticles. Indeed, these cells have a natural tendency to migrate and distribute within the tumor mass and they can also incorporate nanoparticles. Stem cell therapy combined with nanotechnology could be a promising tool to efficiently deliver drugs to brain tumors.

Keywords: Glioma; Nanoparticles; Adult stem cells; Targeting


The potential of combinations of drug-loaded nanoparticle systems and adult stem cells for glioma therapy by Mathilde Roger; Anne Clavreul; Marie-Claire Venier-Julienne; Catherine Passirani; Claudia Montero-Menei; Philippe Menei (pp. 2106-2116).
The prognosis of patients with malignant glioma remains extremely poor, despite surgery and improvements in radio- and chemo-therapies. Nanotechnologies hold great promise in glioma therapy as they protect the therapeutic agent and allow its sustained release. However, new paradigms permitting tumor-specific targeting and extensive intratumoral distribution must be developed to efficiently deliver nanoparticles. Modifications and functionalizations of nanoparticles have been developed to specifically track tumor cells. However, these nanoparticles have yielded few clinical results due to intra-patient heterogeneity and inter-patient variability. Stem cells with a specific tropism for brain tumors could be used as delivery vehicles for nanoparticles. Indeed, these cells have a natural tendency to migrate and distribute within the tumor mass and they can also incorporate nanoparticles. Stem cell therapy combined with nanotechnology could be a promising tool to efficiently deliver drugs to brain tumors.

Keywords: Glioma; Nanoparticles; Adult stem cells; Targeting


Ultrasensitive electrochemical immunoassay for BRCA1 using BMIM·BF4-coated SBA-15 as labels and functionalized graphene as enhancer by Yanyan Cai; He Li; Bin Du; Minghui Yang; Yan Li; Dan Wu; Yanfang Zhao; Yuxue Dai; Qin Wei (pp. 2117-2123).
BRCAl is an anti-oncogene in women, who are genetically predisposed to breast and ovary cancer. The detection of BRCA1 can offer an opportunity to characterize the function of genetic features in breast and ovarian cancer and to screen breast or ovarian cancer patients. In this study, we designed a new label and fabricated a novel sandwich-type electrochemical immunoassay for the ultrasensitive detection of BRCAl. Horseradish peroxidase (HRP) was entrapped in the pores of amino-group functionalized SBA-15 and the secondary antibody (Ab2) combined with SBA-15 by covalent bond. Ionic liquid (IL) was added into the mixed solution of SBA-15/HRP/Ab2 and application of IL increased the electrochemical activity of HRP and promoted electron transport. The synergistic effect between IL, SBA-15, Ab2 and HRP could retain the bioactivity of HRP and Ab2. The sensitivity of the sandwich-type immunosensor using SBA-15/HRP/Ab2/BMIM·BF4 as labels for BRCA1 detection was much higher than that using either SBA-15/HRP/Ab2 or SBA-15/Ab2 as labels. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.01 to 15 ng/mL with a detection limit of 4.86 pg/mL BRCA1. The precision, reproducibility, and stability of the immunoassay were acceptable.

Keywords: SBA-15; Horseradish peroxidase; Amperometric immunosensor; Ionic liquid; Graphene nanocomposites; Breast cancer susceptibility gene


Ultrasensitive electrochemical immunoassay for BRCA1 using BMIM·BF4-coated SBA-15 as labels and functionalized graphene as enhancer by Yanyan Cai; He Li; Bin Du; Minghui Yang; Yan Li; Dan Wu; Yanfang Zhao; Yuxue Dai; Qin Wei (pp. 2117-2123).
BRCAl is an anti-oncogene in women, who are genetically predisposed to breast and ovary cancer. The detection of BRCA1 can offer an opportunity to characterize the function of genetic features in breast and ovarian cancer and to screen breast or ovarian cancer patients. In this study, we designed a new label and fabricated a novel sandwich-type electrochemical immunoassay for the ultrasensitive detection of BRCAl. Horseradish peroxidase (HRP) was entrapped in the pores of amino-group functionalized SBA-15 and the secondary antibody (Ab2) combined with SBA-15 by covalent bond. Ionic liquid (IL) was added into the mixed solution of SBA-15/HRP/Ab2 and application of IL increased the electrochemical activity of HRP and promoted electron transport. The synergistic effect between IL, SBA-15, Ab2 and HRP could retain the bioactivity of HRP and Ab2. The sensitivity of the sandwich-type immunosensor using SBA-15/HRP/Ab2/BMIM·BF4 as labels for BRCA1 detection was much higher than that using either SBA-15/HRP/Ab2 or SBA-15/Ab2 as labels. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.01 to 15 ng/mL with a detection limit of 4.86 pg/mL BRCA1. The precision, reproducibility, and stability of the immunoassay were acceptable.

Keywords: SBA-15; Horseradish peroxidase; Amperometric immunosensor; Ionic liquid; Graphene nanocomposites; Breast cancer susceptibility gene


Dual-aptamer-based delivery vehicle of doxorubicin to both PSMA (+) and PSMA (−) prostate cancers by Kyoungin Min; Hunho Jo; Kyungmi Song; Minseon Cho; Yang-Sook Chun; Sangyong Jon; Won Jong Kim; Changill Ban (pp. 2124-2132).
We have designed a dual-aptamer complex specific to both prostate-specific membrane antigens (PSMA) (+) and (−) prostate cancer cells. In the complex, an A10 RNA aptamer targeting PSMA (+) cells and a DUP-1 peptide aptamer specific to PSMA (−) cells were conjugated through streptavidin. Doxorubicin-loaded onto the stem region of the A10 aptamer was delivered not only to PSMA (+) cells but to PSMA (−) cells, and eventually induced apoptosis in both types of prostate cancer cells. Cell death was monitored by measuring guanine concentration in cells using differential pulse voltammetry (DPV), a simple and rapid electrochemical method, and was further confirmed by directly observing cell morphologies cultured on the transparent indium tin oxide (ITO) glass electrode and checking their viabilities using a trypan blue assay. To investigate the in vivo application of the dual-aptamer system, both A10 and DUP-1 aptamers were immobilized on the surface of thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION). Selective cell uptakes and effective drug delivery action of these probes were verified by Prussian blue staining and trypan blue staining, respectively.

Keywords: Drug delivery; PSMA (+) and PSMA (−) prostate cancer cells; A10 RNA aptamer; DUP-1 peptide aptamer; Differential pulse voltammetry; Superparamagnetic iron oxide nanoparticle


Dual-aptamer-based delivery vehicle of doxorubicin to both PSMA (+) and PSMA (−) prostate cancers by Kyoungin Min; Hunho Jo; Kyungmi Song; Minseon Cho; Yang-Sook Chun; Sangyong Jon; Won Jong Kim; Changill Ban (pp. 2124-2132).
We have designed a dual-aptamer complex specific to both prostate-specific membrane antigens (PSMA) (+) and (−) prostate cancer cells. In the complex, an A10 RNA aptamer targeting PSMA (+) cells and a DUP-1 peptide aptamer specific to PSMA (−) cells were conjugated through streptavidin. Doxorubicin-loaded onto the stem region of the A10 aptamer was delivered not only to PSMA (+) cells but to PSMA (−) cells, and eventually induced apoptosis in both types of prostate cancer cells. Cell death was monitored by measuring guanine concentration in cells using differential pulse voltammetry (DPV), a simple and rapid electrochemical method, and was further confirmed by directly observing cell morphologies cultured on the transparent indium tin oxide (ITO) glass electrode and checking their viabilities using a trypan blue assay. To investigate the in vivo application of the dual-aptamer system, both A10 and DUP-1 aptamers were immobilized on the surface of thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION). Selective cell uptakes and effective drug delivery action of these probes were verified by Prussian blue staining and trypan blue staining, respectively.

Keywords: Drug delivery; PSMA (+) and PSMA (−) prostate cancer cells; A10 RNA aptamer; DUP-1 peptide aptamer; Differential pulse voltammetry; Superparamagnetic iron oxide nanoparticle


Ultra-photostable, non-cytotoxic, and highly fluorescent quantum nanospheres for long-term, high-specificity cell imaging by Yao He; Haoting Lu; Yuanyuan Su; Liman Sai; Mei Hu; Chunhai Fan; Lianhui Wang (pp. 2133-2140).
A new class of fluorescent quantum nanospheres (QNs) is directly achieved in aqueous phase through a facile one-pot microwave irradiation (MWI) strategy. Multi-color QNs with maximum emission wavelengths ranging from 525 to 610 nm and PLQY of 30–60% are facilely prepared through this new MWI strategy. In addition to strong fluorescence, these QNs possess excellent photostability, preserving ∼90% of the original intensity after 70 min high-power UV irradiation (100 W Xeon lamp). In sharp contrast, the fluorescence of CdTe/CdS/ZnS core-shell-shell quantum dots (QDs), recognized as established fluorescent probes with robust photostability, decrease to ∼50% under the same conditions. Besides, cytotoxicity assessment demonstrates that the prepared QNs exhibit favorable cytocompatibility to K562 cells with high concentration (3 μmol) and long-time incubation (24 h). Furthermore, cellular imaging results demonstrate that the as-prepared QNs are remarkably efficacious for long-term and high-specificity immunofluorescent cellular labeling, and multi-color cell imaging. Our systematical investigation clearly shows that these high-performance QNs may serve as practical and powerful tools for various biological researches, such as in vivo and in vitro imaging.

Keywords: Quantum nanospheres; Biocompatibility; Cell imaging; Biological probes


Ultra-photostable, non-cytotoxic, and highly fluorescent quantum nanospheres for long-term, high-specificity cell imaging by Yao He; Haoting Lu; Yuanyuan Su; Liman Sai; Mei Hu; Chunhai Fan; Lianhui Wang (pp. 2133-2140).
A new class of fluorescent quantum nanospheres (QNs) is directly achieved in aqueous phase through a facile one-pot microwave irradiation (MWI) strategy. Multi-color QNs with maximum emission wavelengths ranging from 525 to 610 nm and PLQY of 30–60% are facilely prepared through this new MWI strategy. In addition to strong fluorescence, these QNs possess excellent photostability, preserving ∼90% of the original intensity after 70 min high-power UV irradiation (100 W Xeon lamp). In sharp contrast, the fluorescence of CdTe/CdS/ZnS core-shell-shell quantum dots (QDs), recognized as established fluorescent probes with robust photostability, decrease to ∼50% under the same conditions. Besides, cytotoxicity assessment demonstrates that the prepared QNs exhibit favorable cytocompatibility to K562 cells with high concentration (3 μmol) and long-time incubation (24 h). Furthermore, cellular imaging results demonstrate that the as-prepared QNs are remarkably efficacious for long-term and high-specificity immunofluorescent cellular labeling, and multi-color cell imaging. Our systematical investigation clearly shows that these high-performance QNs may serve as practical and powerful tools for various biological researches, such as in vivo and in vitro imaging.

Keywords: Quantum nanospheres; Biocompatibility; Cell imaging; Biological probes


Affibody-based nanoprobes for HER2-expressing cell and tumor imaging by Jinhao Gao; Kai Chen; Zheng Miao; Gang Ren; Xiaoyuan Chen; Sanjiv S. Gambhir; Zhen Cheng (pp. 2141-2148).
This article reports the affibody-based nanoprobes specifically target and image human epidermal growth factor receptor type 2 (HER2)-expressing cells and tumors. The affibody molecules are a promising class of targeting ligands with simple, robust, and precise structure and high affinity. Using near-infrared (NIR) quantum dots (QDs) and iron oxide (IO) nanoparticles as two representative nanomaterials, we designed anti–HER2 affibody molecules with a N-terminus cysteine residue (Cysteine-ZHER2:342) and precisely conjugated with maleimide-functionalized nanoparticles to make nanoparticle-affibody conjugates. The in vitro and in vivo study showed the conjugates are highly specific to target and image HER2-expressing cells and tumors. This work indicated the nanoparticle-affibody conjugates may be excellent candidates as targeting probes for molecular imaging and diagnosis.

Keywords: Affibody; Bioconjugation; Nanoprobes; HER2; Molecular imaging


Affibody-based nanoprobes for HER2-expressing cell and tumor imaging by Jinhao Gao; Kai Chen; Zheng Miao; Gang Ren; Xiaoyuan Chen; Sanjiv S. Gambhir; Zhen Cheng (pp. 2141-2148).
This article reports the affibody-based nanoprobes specifically target and image human epidermal growth factor receptor type 2 (HER2)-expressing cells and tumors. The affibody molecules are a promising class of targeting ligands with simple, robust, and precise structure and high affinity. Using near-infrared (NIR) quantum dots (QDs) and iron oxide (IO) nanoparticles as two representative nanomaterials, we designed anti–HER2 affibody molecules with a N-terminus cysteine residue (Cysteine-ZHER2:342) and precisely conjugated with maleimide-functionalized nanoparticles to make nanoparticle-affibody conjugates. The in vitro and in vivo study showed the conjugates are highly specific to target and image HER2-expressing cells and tumors. This work indicated the nanoparticle-affibody conjugates may be excellent candidates as targeting probes for molecular imaging and diagnosis.

Keywords: Affibody; Bioconjugation; Nanoprobes; HER2; Molecular imaging


Engineered silk fibroin protein 3D matrices for in vitro tumor model by Sarmistha Talukdar; Mahitosh Mandal; Dietmar W. Hutmacher; Pamela J. Russell; Carolina Soekmadji; Subhas C. Kundu (pp. 2149-2159).
3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs.

Keywords: Silk; Fibroin; 3D; Cell culture; Tumour


Engineered silk fibroin protein 3D matrices for in vitro tumor model by Sarmistha Talukdar; Mahitosh Mandal; Dietmar W. Hutmacher; Pamela J. Russell; Carolina Soekmadji; Subhas C. Kundu (pp. 2149-2159).
3D in vitro model systems that are able to mimic the in vivo microenvironment are now highly sought after in cancer research. Antheraea mylitta silk fibroin protein matrices were investigated as potential biomaterial for in vitro tumor modeling. We compared the characteristics of MDA-MB-231 cells on A. mylitta, Bombyx mori silk matrices, Matrigel, and tissue culture plates. The attachment and morphology of the MDA-MB-231 cell line on A. mylitta silk matrices was found to be better than on B. mori matrices and comparable to Matrigel and tissue culture plates. The cells grown in all 3D cultures showed more MMP-9 activity, indicating a more invasive potential. In comparison to B. mori fibroin, A. mylitta fibroin not only provided better cell adhesion, but also improved cell viability and proliferation. Yield coefficient of glucose consumed to lactate produced by cells on 3D A. mylitta fibroin was found to be similar to that of cancer cells in vivo. LNCaP prostate cancer cells were also cultured on 3D A. mylitta fibroin and they grew as clumps in long term culture. The results indicate that A. mylitta fibroin scaffold can provide an easily manipulated microenvironment system to investigate individual factors such as growth factors and signaling peptides, as well as evaluation of anticancer drugs.

Keywords: Silk; Fibroin; 3D; Cell culture; Tumour


A small MRI contrast agent library of gadolinium(III)-encapsulated supramolecular nanoparticles for improved relaxivity and sensitivity by Kuan-Ju Chen; Stephanie M. Wolahan; Hao Wang; Chao-Hsiung Hsu; Hsing-Wei Chang; Armando Durazo; Lian-Pin Hwang; Mitch A. Garcia; Ziyue K. Jiang; Lily Wu; Yung-Ya Lin; Hsian-Rong Tseng /. (pp. 2160-2165).
We introduce a new category of nanoparticle-based T1 MRI contrast agents (CAs) by encapsulating paramagnetic chelated gadolinium(III), i.e., Gd3+·DOTA, through supramolecular assembly of molecular building blocks that carry complementary molecular recognition motifs, including adamantane (Ad) and β-cyclodextrin (CD). A small library of Gd3+·DOTA-encapsulated supramolecular nanoparticles (Gd3+·DOTA⊂SNPs) was produced by systematically altering the molecular building block mixing ratios. A broad spectrum of relaxation rates was correlated to the resulting Gd3+·DOTA⊂SNP library. Consequently, an optimal synthetic formulation of Gd3+·DOTA⊂SNPs with an r1 of 17.3 s−1 mM−1 (ca. 4-fold higher than clinical Gd3+ chelated complexes at high field strengths) was identified. T1-weighted imaging of Gd3+·DOTA⊂SNPs exhibits an enhanced sensitivity with a contrast-to-noise ratio (C/N ratio) ca. 3.6 times greater than that observed for free Gd3+·DTPA. A Gd3+·DOTA⊂SNPs solution was injected into foot pads of mice, and MRI was employed to monitor dynamic lymphatic drainage of the Gd3+·DOTA⊂SNPs-based CA. We observe an increase in signal intensity of the brachial lymph node in T1-weighted imaging after injecting Gd3+·DOTA⊂SNPs but not after injecting Gd3+·DTPA. The MRI results are supported by ICP-MS analysis ex vivo. These results show that Gd3+·DOTA⊂SNPs not only exhibits enhanced relaxivity and high sensitivity but also can serve as a potential tool for diagnosis of cancer metastasis.

Keywords: Supramolecular Chemistry; Cyclodextrin; T1-weighted Contrast Agent; MRI; Gadolinium; Dynamic imaging


A small MRI contrast agent library of gadolinium(III)-encapsulated supramolecular nanoparticles for improved relaxivity and sensitivity by Kuan-Ju Chen; Stephanie M. Wolahan; Hao Wang; Chao-Hsiung Hsu; Hsing-Wei Chang; Armando Durazo; Lian-Pin Hwang; Mitch A. Garcia; Ziyue K. Jiang; Lily Wu; Yung-Ya Lin; Hsian-Rong Tseng /. (pp. 2160-2165).
We introduce a new category of nanoparticle-based T1 MRI contrast agents (CAs) by encapsulating paramagnetic chelated gadolinium(III), i.e., Gd3+·DOTA, through supramolecular assembly of molecular building blocks that carry complementary molecular recognition motifs, including adamantane (Ad) and β-cyclodextrin (CD). A small library of Gd3+·DOTA-encapsulated supramolecular nanoparticles (Gd3+·DOTA⊂SNPs) was produced by systematically altering the molecular building block mixing ratios. A broad spectrum of relaxation rates was correlated to the resulting Gd3+·DOTA⊂SNP library. Consequently, an optimal synthetic formulation of Gd3+·DOTA⊂SNPs with an r1 of 17.3 s−1 mM−1 (ca. 4-fold higher than clinical Gd3+ chelated complexes at high field strengths) was identified. T1-weighted imaging of Gd3+·DOTA⊂SNPs exhibits an enhanced sensitivity with a contrast-to-noise ratio (C/N ratio) ca. 3.6 times greater than that observed for free Gd3+·DTPA. A Gd3+·DOTA⊂SNPs solution was injected into foot pads of mice, and MRI was employed to monitor dynamic lymphatic drainage of the Gd3+·DOTA⊂SNPs-based CA. We observe an increase in signal intensity of the brachial lymph node in T1-weighted imaging after injecting Gd3+·DOTA⊂SNPs but not after injecting Gd3+·DTPA. The MRI results are supported by ICP-MS analysis ex vivo. These results show that Gd3+·DOTA⊂SNPs not only exhibits enhanced relaxivity and high sensitivity but also can serve as a potential tool for diagnosis of cancer metastasis.

Keywords: Supramolecular Chemistry; Cyclodextrin; T1-weighted Contrast Agent; MRI; Gadolinium; Dynamic imaging


Multifunctional polyglycerol-grafted Fe3O4@SiO2 nanoparticles for targeting ovarian cancer cells by Liang Wang; Koon Gee Neoh; En-Tang Kang; Borys Shuter (pp. 2166-2173).
Ligand-mediated magnetic resonance (MR) contrast agents would be highly desirable for cancer diagnosis. In the present study, nanoparticles of Fe3O4 core with fluorescent SiO2 shell were synthesized and grafted with hyperbranched polyglycerol (HPG-grafted Fe3O4@SiO2 nanoparticles). These nanoparticles have a hydrodynamic diameter of 47.0 ± 4.0 nm, and are very stable in aqueous solution as well as in cell culture medium. Numerous surface hydroxyl groups of these nanoparticles were conjugated with folic acid by a thiol ‘click’ reaction. The successful covalent attachment of folic acid on the nanoparticles was confirmed by FTIR and XPS analyses. Both MR imaging and fluorescence microscopy show significant preferential uptake of the folic acid-conjugated polyglycerol-grafted Fe3O4@SiO2 (FA-HPG-grafted Fe3O4@SiO2) nanoparticles by human ovarian carcinoma cells (SKOV-3) as compared to macrophages and fibroblasts. Such nanoparticles can potentially be used to provide real-time imaging in ovarian cancer resection.

Keywords: Polyglycerol; Iron oxide nanoparticles; Silica shell; Folic acid; fluorescence; MRI


Multifunctional polyglycerol-grafted Fe3O4@SiO2 nanoparticles for targeting ovarian cancer cells by Liang Wang; Koon Gee Neoh; En-Tang Kang; Borys Shuter (pp. 2166-2173).
Ligand-mediated magnetic resonance (MR) contrast agents would be highly desirable for cancer diagnosis. In the present study, nanoparticles of Fe3O4 core with fluorescent SiO2 shell were synthesized and grafted with hyperbranched polyglycerol (HPG-grafted Fe3O4@SiO2 nanoparticles). These nanoparticles have a hydrodynamic diameter of 47.0 ± 4.0 nm, and are very stable in aqueous solution as well as in cell culture medium. Numerous surface hydroxyl groups of these nanoparticles were conjugated with folic acid by a thiol ‘click’ reaction. The successful covalent attachment of folic acid on the nanoparticles was confirmed by FTIR and XPS analyses. Both MR imaging and fluorescence microscopy show significant preferential uptake of the folic acid-conjugated polyglycerol-grafted Fe3O4@SiO2 (FA-HPG-grafted Fe3O4@SiO2) nanoparticles by human ovarian carcinoma cells (SKOV-3) as compared to macrophages and fibroblasts. Such nanoparticles can potentially be used to provide real-time imaging in ovarian cancer resection.

Keywords: Polyglycerol; Iron oxide nanoparticles; Silica shell; Folic acid; fluorescence; MRI


Alginate-folic acid-modified chitosan nanoparticles for photodynamic detection of intestinal neoplasms by Shu-Jyuan Yang; Feng-Huei Lin; Han-Min Tsai; Chia-Fu Lin; Han-Chiang Chin; Jau-Min Wong; Ming-Jium Shieh (pp. 2174-2182).
Colorectal cancer is one of the leading causes of cancer death and often goes undetected with current colonoscopy practices. Improved methods of detecting dysplasia and tumors during colonoscopy could significantly improve mortality. Herein, we report a high-performance nanoparticle for photodynamic detection of colorectal cancer, where alginate is physically complexed with folic acid-modified chitosan to form nanoparticles with improved drug release in the cellular lysosome. The incorporated alginate molecules could complex stably with chitosan via electrostatic attraction, and the z-average diameter and zeta-potential of the prepared nanoparticles (fCAN) was 115 nm and 22 mV, respectively, enough to keep the nanoparticles stable in aqueous suspension without aggregation. When loaded with 5-aminolevulinic acid (5-ALA; 27% loading efficiency), the nanoparticles (fCANA) displayed no differences in particle size or zeta-potential compared to fCAN. Moreover, the fCANA nanoparticles were readily taken up by colorectal cancer cells via folate receptor-mediated endocytosis. Subsequently, the loaded 5-ALA was release in the lysosome, and this was promoted by the reduced attraction intensity between chitosan and 5-ALA via the deprotonated alginate, resulting in a higher intracellular PpIX accumulation for the photodynamic detection. These studies demonstrate that the alginate incorporated and folic acid-conjugated chitosan nanoparticles are excellent vectors for colorectal-specific delivery of 5-ALA for fluorescent endoscopic detection.

Keywords: Chitosan; Alginate; Colorectal cancer; 5-Aminolevulinic acid; Nanoparticle


Alginate-folic acid-modified chitosan nanoparticles for photodynamic detection of intestinal neoplasms by Shu-Jyuan Yang; Feng-Huei Lin; Han-Min Tsai; Chia-Fu Lin; Han-Chiang Chin; Jau-Min Wong; Ming-Jium Shieh (pp. 2174-2182).
Colorectal cancer is one of the leading causes of cancer death and often goes undetected with current colonoscopy practices. Improved methods of detecting dysplasia and tumors during colonoscopy could significantly improve mortality. Herein, we report a high-performance nanoparticle for photodynamic detection of colorectal cancer, where alginate is physically complexed with folic acid-modified chitosan to form nanoparticles with improved drug release in the cellular lysosome. The incorporated alginate molecules could complex stably with chitosan via electrostatic attraction, and the z-average diameter and zeta-potential of the prepared nanoparticles (fCAN) was 115 nm and 22 mV, respectively, enough to keep the nanoparticles stable in aqueous suspension without aggregation. When loaded with 5-aminolevulinic acid (5-ALA; 27% loading efficiency), the nanoparticles (fCANA) displayed no differences in particle size or zeta-potential compared to fCAN. Moreover, the fCANA nanoparticles were readily taken up by colorectal cancer cells via folate receptor-mediated endocytosis. Subsequently, the loaded 5-ALA was release in the lysosome, and this was promoted by the reduced attraction intensity between chitosan and 5-ALA via the deprotonated alginate, resulting in a higher intracellular PpIX accumulation for the photodynamic detection. These studies demonstrate that the alginate incorporated and folic acid-conjugated chitosan nanoparticles are excellent vectors for colorectal-specific delivery of 5-ALA for fluorescent endoscopic detection.

Keywords: Chitosan; Alginate; Colorectal cancer; 5-Aminolevulinic acid; Nanoparticle


Polyethylene glycol modified, cross-linked starch-coated iron oxide nanoparticles for enhanced magnetic tumor targeting by Adam J. Cole; Allan E. David; Jianxin Wang; Craig J. Galbán; Hannah L. Hill; Victor C. Yang (pp. 2183-2193).
While successful magnetic tumor targeting of iron oxide nanoparticles has been achieved in a number of models, the rapid blood clearance of magnetically suitable particles by the reticuloendothelial system (RES) limits their availability for targeting. This work aimed to develop a long-circulating magnetic iron oxide nanoparticle (MNP) platform capable of sustained tumor exposure via the circulation and, thus, potentially enhanced magnetic tumor targeting. Aminated, cross-linked starch (DN) and aminosilane (A) coated MNPs were successfully modified with 5 kDa (A5, D5) or 20 kDa (A20, D20) polyethylene glycol (PEG) chains using simple N-Hydroxysuccinimide (NHS) chemistry and characterized. Identical PEG-weight analogues between platforms (A5 & D5, A20 & D20) were similar in size (140–190 nm) and relative PEG labeling (1.5% of surface amines – A5/D5, 0.4% – A20/D20), with all PEG–MNPs possessing magnetization properties suitable for magnetic targeting. Candidate PEG–MNPs were studied in RES simulations in vitro to predict long-circulating character. D5 and D20 performed best showing sustained size stability in cell culture medium at 37 °C and 7 (D20) to 10 (D5) fold less uptake in RAW264.7 macrophages when compared to previously targeted, unmodified starch MNPs (D). Observations in vitro were validated in vivo, with D5 (7.29 h) and D20 (11.75 h) showing much longer half-lives than D (0.12 h). Improved plasma stability enhanced tumor MNP exposure 100 (D5) to 150 (D20) fold as measured by plasma AUC0–∞. Sustained tumor exposure over 24 h was visually confirmed in a 9L-glioma rat model (12 mg Fe/kg) using magnetic resonance imaging (MRI). Findings indicate that a polyethylene glycol modified, cross-linked starch-coated MNP is a promising platform for enhanced magnetic tumor targeting, warranting further study in tumor models.

Keywords: Iron oxide nanoparticles; Magnetic nanoparticles; Magnetic targeting; Polyethylene glycol (PEG); Pharmacokinetics; Reticuloendothelial system (RES)


Polyethylene glycol modified, cross-linked starch-coated iron oxide nanoparticles for enhanced magnetic tumor targeting by Adam J. Cole; Allan E. David; Jianxin Wang; Craig J. Galbán; Hannah L. Hill; Victor C. Yang (pp. 2183-2193).
While successful magnetic tumor targeting of iron oxide nanoparticles has been achieved in a number of models, the rapid blood clearance of magnetically suitable particles by the reticuloendothelial system (RES) limits their availability for targeting. This work aimed to develop a long-circulating magnetic iron oxide nanoparticle (MNP) platform capable of sustained tumor exposure via the circulation and, thus, potentially enhanced magnetic tumor targeting. Aminated, cross-linked starch (DN) and aminosilane (A) coated MNPs were successfully modified with 5 kDa (A5, D5) or 20 kDa (A20, D20) polyethylene glycol (PEG) chains using simple N-Hydroxysuccinimide (NHS) chemistry and characterized. Identical PEG-weight analogues between platforms (A5 & D5, A20 & D20) were similar in size (140–190 nm) and relative PEG labeling (1.5% of surface amines – A5/D5, 0.4% – A20/D20), with all PEG–MNPs possessing magnetization properties suitable for magnetic targeting. Candidate PEG–MNPs were studied in RES simulations in vitro to predict long-circulating character. D5 and D20 performed best showing sustained size stability in cell culture medium at 37 °C and 7 (D20) to 10 (D5) fold less uptake in RAW264.7 macrophages when compared to previously targeted, unmodified starch MNPs (D). Observations in vitro were validated in vivo, with D5 (7.29 h) and D20 (11.75 h) showing much longer half-lives than D (0.12 h). Improved plasma stability enhanced tumor MNP exposure 100 (D5) to 150 (D20) fold as measured by plasma AUC0–∞. Sustained tumor exposure over 24 h was visually confirmed in a 9L-glioma rat model (12 mg Fe/kg) using magnetic resonance imaging (MRI). Findings indicate that a polyethylene glycol modified, cross-linked starch-coated MNP is a promising platform for enhanced magnetic tumor targeting, warranting further study in tumor models.

Keywords: Iron oxide nanoparticles; Magnetic nanoparticles; Magnetic targeting; Polyethylene glycol (PEG); Pharmacokinetics; Reticuloendothelial system (RES)


Engineering complement activation on polypropylene sulfide vaccine nanoparticles by Susan N. Thomas; André J. van der Vlies; Conlin P. O’Neil; Sai T. Reddy; Shann S. Yu; Todd D. Giorgio; Melody A. Swartz; Jeffrey A. Hubbell (pp. 2194-2203).
The complement system is an important regulator of both adaptive and innate immunity, implicating complement as a potential target for immunotherapeutics. We have recently presented lymph node-targeting, complement-activating nanoparticles (NPs) as a vaccine platform. Here we explore modulation of surface chemistry as a means to control complement deposition, in active or inactive forms, on polypropylene sulfide core, block copolymer Pluronic corona NPs. We found that nucleophile-containing NP surfaces activated complement and became functionalized in situ with C3 upon serum exposure via the alternative pathway. Carboxylated NPs displayed a higher degree of C3b deposition and retention relative to hydroxylated NPs, upon which deposited C3b was more substantially inactivated to iC3b. This in situ functionalization correlated with in vivo antigen-specific immune responses, including antibody production as well as T cell proliferation and IFN-γ cytokine production upon antigen restimulation. Interestingly, inactivation of C3b to iC3b on the NP surface did not correlate with NP affinity to factor H, a cofactor for protease factor I that degrades C3b into iC3b, indicating that control of complement protein C3 stability depends on architectural details in addition to factor H affinity. These data show that design of NP surface chemistry can be used to control biomaterials-associated complement activation for immunotherapeutic materials.

Keywords: Complement; Nanoparticle; Immunomodulation; Protein adsorption


Engineering complement activation on polypropylene sulfide vaccine nanoparticles by Susan N. Thomas; André J. van der Vlies; Conlin P. O’Neil; Sai T. Reddy; Shann S. Yu; Todd D. Giorgio; Melody A. Swartz; Jeffrey A. Hubbell (pp. 2194-2203).
The complement system is an important regulator of both adaptive and innate immunity, implicating complement as a potential target for immunotherapeutics. We have recently presented lymph node-targeting, complement-activating nanoparticles (NPs) as a vaccine platform. Here we explore modulation of surface chemistry as a means to control complement deposition, in active or inactive forms, on polypropylene sulfide core, block copolymer Pluronic corona NPs. We found that nucleophile-containing NP surfaces activated complement and became functionalized in situ with C3 upon serum exposure via the alternative pathway. Carboxylated NPs displayed a higher degree of C3b deposition and retention relative to hydroxylated NPs, upon which deposited C3b was more substantially inactivated to iC3b. This in situ functionalization correlated with in vivo antigen-specific immune responses, including antibody production as well as T cell proliferation and IFN-γ cytokine production upon antigen restimulation. Interestingly, inactivation of C3b to iC3b on the NP surface did not correlate with NP affinity to factor H, a cofactor for protease factor I that degrades C3b into iC3b, indicating that control of complement protein C3 stability depends on architectural details in addition to factor H affinity. These data show that design of NP surface chemistry can be used to control biomaterials-associated complement activation for immunotherapeutic materials.

Keywords: Complement; Nanoparticle; Immunomodulation; Protein adsorption


Synthetic cationic amphiphilic α-helical peptides as antimicrobial agents by Nikken Wiradharma; Ulung Khoe; Charlotte A.E. Hauser; See Voon Seow; Shuguang Zhang; Yi-Yan Yang (pp. 2204-2212).
Antimicrobial peptides (AMPs) secreted by the innate immune system are prevalent as the effective first-line of defense to overcome recurring microbial invasions. They have been widely accepted as the blueprints for the development of new antimicrobial agents for the treatment of drug resistant infections. However, there is also a growing concern that AMPs with a sequence that is too close to the host organism’s AMP may inevitably compromise its own natural defense. In this study, we design a series of synthetic (non-natural) short α-helical AMPs to expand the arsenal of the AMP families and to gain further insights on their antimicrobial activities. These cationic and amphiphilic peptides have a general sequence of (XXYY)n (X: hydrophobic residue, Y: cationic residue, and n: the number of repeat units), and are designed to mimic the folding behavior of the naturally-occurring α-helical AMPs. The synthetic α-helical AMPs with 3 repeat units, (FFRR)3, (LLRR)3, and (LLKK)3, are found to be more selective towards microbial cells than rat red blood cells, with minimum inhibitory concentration (MIC) values that are more than 10 times lower than their 50% hemolytic concentrations (HC50). They are effective against Gram-positive B. subtilis and yeast C. albicans; and the studies using scanning electron microscopy (SEM) have elucidated that these peptides possess membrane-lytic activities against microbial cells. Furthermore, non-specific immune stimulation assays of a typical peptide shows negligible IFN-α, IFN-γ, and TNF-α inductions in human peripheral blood mononuclear cells, which implies additional safety aspects of the peptide for both systemic and topical use. Therefore, the peptides designed in this study can be promising antimicrobial agents against the frequently-encountered Gram-positive bacteria- or yeast-induced infections.

Keywords: Antimicrobial peptides (AMP); α-Helix; Minimum inhibitory concentration (MIC); Membrane lysis; Hemolysis


Synthetic cationic amphiphilic α-helical peptides as antimicrobial agents by Nikken Wiradharma; Ulung Khoe; Charlotte A.E. Hauser; See Voon Seow; Shuguang Zhang; Yi-Yan Yang (pp. 2204-2212).
Antimicrobial peptides (AMPs) secreted by the innate immune system are prevalent as the effective first-line of defense to overcome recurring microbial invasions. They have been widely accepted as the blueprints for the development of new antimicrobial agents for the treatment of drug resistant infections. However, there is also a growing concern that AMPs with a sequence that is too close to the host organism’s AMP may inevitably compromise its own natural defense. In this study, we design a series of synthetic (non-natural) short α-helical AMPs to expand the arsenal of the AMP families and to gain further insights on their antimicrobial activities. These cationic and amphiphilic peptides have a general sequence of (XXYY)n (X: hydrophobic residue, Y: cationic residue, and n: the number of repeat units), and are designed to mimic the folding behavior of the naturally-occurring α-helical AMPs. The synthetic α-helical AMPs with 3 repeat units, (FFRR)3, (LLRR)3, and (LLKK)3, are found to be more selective towards microbial cells than rat red blood cells, with minimum inhibitory concentration (MIC) values that are more than 10 times lower than their 50% hemolytic concentrations (HC50). They are effective against Gram-positive B. subtilis and yeast C. albicans; and the studies using scanning electron microscopy (SEM) have elucidated that these peptides possess membrane-lytic activities against microbial cells. Furthermore, non-specific immune stimulation assays of a typical peptide shows negligible IFN-α, IFN-γ, and TNF-α inductions in human peripheral blood mononuclear cells, which implies additional safety aspects of the peptide for both systemic and topical use. Therefore, the peptides designed in this study can be promising antimicrobial agents against the frequently-encountered Gram-positive bacteria- or yeast-induced infections.

Keywords: Antimicrobial peptides (AMP); α-Helix; Minimum inhibitory concentration (MIC); Membrane lysis; Hemolysis


Multifunctional hollow nanoparticles based on graft-diblock copolymers for doxorubicin delivery by Pei-Lin Lu; Yi-Chun Chen; Ta-Wei Ou; Hung-Hao Chen; Hsieh-Chih Tsai; Chih-Jen Wen; Chun-Liang Lo; Shiaw-Pyng Wey; Kun-Ju Lin; Tzu-Chen Yen; Ging-Ho Hsiue (pp. 2213-2221).
This article reports a flexible hollow nanoparticles, self-assembling from poly(N-vinylimidazole-co-N-vinylpyrrolidone)-g-poly(d,l-lactide) graft copolymers and methoxyl/functionalized-PEG-PLA diblock copolymers, as an anticancer drug doxorubicin (Dox) carrier for cancer targeting, imaging, and cancer therapy. This multifunctional hollow nanoparticle exhibited a specific on-off switch drug release behavior, owning to the pH-sensitive structure of imidazole, to release Dox in acidic surroundings (intracellular endosomes) and to capsulate Dox in neutral surroundings (blood circulation or extracellular matrix). Imaging by SPECT/CT shows that nanoparticle conjugated with folic acids ensures a high intratumoral accumulation due to the folate-binding protein (FBP)-binding effect. In vivo tumor growth inhibition shows that nanoparticles exhibited excellent antitumor activity and a high rate of apoptosis in cancer cells. After 80-day treatment course of nanoparticles, it did not appreciably cause heart, liver and kidney damage by inactive Dox or polymeric materials. The results indicate that the flexible carriers with an on-off switched drug release may be allowed to accurately deliver to targeted tumors for cancer therapy.

Keywords: Multifunctional hollow nanoparticles; Intracellular drug delivery; Cancer targeting; SPECT/CT imaging; Cancer therapy


Multifunctional hollow nanoparticles based on graft-diblock copolymers for doxorubicin delivery by Pei-Lin Lu; Yi-Chun Chen; Ta-Wei Ou; Hung-Hao Chen; Hsieh-Chih Tsai; Chih-Jen Wen; Chun-Liang Lo; Shiaw-Pyng Wey; Kun-Ju Lin; Tzu-Chen Yen; Ging-Ho Hsiue (pp. 2213-2221).
This article reports a flexible hollow nanoparticles, self-assembling from poly(N-vinylimidazole-co-N-vinylpyrrolidone)-g-poly(d,l-lactide) graft copolymers and methoxyl/functionalized-PEG-PLA diblock copolymers, as an anticancer drug doxorubicin (Dox) carrier for cancer targeting, imaging, and cancer therapy. This multifunctional hollow nanoparticle exhibited a specific on-off switch drug release behavior, owning to the pH-sensitive structure of imidazole, to release Dox in acidic surroundings (intracellular endosomes) and to capsulate Dox in neutral surroundings (blood circulation or extracellular matrix). Imaging by SPECT/CT shows that nanoparticle conjugated with folic acids ensures a high intratumoral accumulation due to the folate-binding protein (FBP)-binding effect. In vivo tumor growth inhibition shows that nanoparticles exhibited excellent antitumor activity and a high rate of apoptosis in cancer cells. After 80-day treatment course of nanoparticles, it did not appreciably cause heart, liver and kidney damage by inactive Dox or polymeric materials. The results indicate that the flexible carriers with an on-off switched drug release may be allowed to accurately deliver to targeted tumors for cancer therapy.

Keywords: Multifunctional hollow nanoparticles; Intracellular drug delivery; Cancer targeting; SPECT/CT imaging; Cancer therapy


The synergistic effect of hierarchical assemblies of siRNA and chemotherapeutic drugs co-delivered into hepatic cancer cells by Nuo Cao; Du Cheng; Seyin Zou; Hua Ai; Jinming Gao; Xintao Shuai (pp. 2222-2232).
Diblock copolymers (PEI-PCL) of poly(ε-caprolactone) (PCL) and linear poly(ethylene imine) (PEI) were synthesized and assembled to biodegradable nano-carriers for co-delivery of BCL-2 siRNA and doxorubicin (DOX). Folic acid as a tumor-targeting ligand was conjugated to the polyanion, poly(ethylene glycol)-block-poly(glutamic acid) (FA-PEG-PGA). Driven by the electrostatic interaction, FA-PEG-PGA was coated onto the surface of the cationic PEI-PCL nanoparticles pre-loaded with siRNA and DOX, potentiating a ligand-directed delivery to human hepatic cancer cells Bel-7402. At certain N/P and C/N ratios (N/P: PEI-PCL nitrogen to siRNA phosphate; C/N: FA-PEG-PGA carboxyl to PEI-PCL amine), the nanoparticles exhibited not only high transfection efficiency but also ideally controlled release of drug. Compared to non-specific delivery, the folate-targeted delivery of BCL-2 siRNA resulted in more significant gene suppression at both the BCL-2 mRNA and protein expression levels, inducing cancer cell apoptosis and improving the therapeutic efficacy of the co-administered DOX. Herein we demonstrated that co-loading siRNA and small molecular drug in a multifunctional hierarchical nano-assembly enabled simultaneously delivering siRNA and drug into the same cancer cells, yielding synergistic effect of RNA interference and chemotherapy in cancer.

Keywords: Co-delivery of siRNA and drug; tumor-targeting; Electrostatic assembly; Gene silencing; Apoptosis; ChemotherapyAbbreviations; SCR; scrambled siRNA; BCL-2 siRNA; siRNA targeting BCL-2 gene; N/P ratio; molar ratio of PEI-PCL nitrogen to siRNA phosphorus; C/N ratio; molar ratio of FA-PEG-PGA or PEG-PGA carboxyl to PEI-PCL amino group; D-PCE or B-PCE; DOX-loaded or blank PEI-PCL micelles; D-PCE/BCL-2 or B-PCE/BCL-2; DOX-loaded or blank PEI-PCL micelles complexing with BCL-2 siRNA; D-PCE/FITC or B-PCE/FITC; DOX-loaded or blank PEI-PCL micelles complexing with FITC-labeled SCR; D-PCE/SCR or B-PCE/SCR; DOX-loaded or blank PEI-PCL micelles complexing with SCR; D-PCE/BCL-2/FA or B-PCE/BCL-2/FA; DOX-loaded or blank PEI-PCL micelles complexing with BCL-2 siRNA and then with FA-PEG-PGA; D-PCE/BCL-2/NFA or B-PCE/BCL-2/NFA; DOX-loaded or blank PEI-PCL micelles complexing with BCL-2 siRNA and then with PEG-PGA; D-PCE/FITC/FA or B-PCE/FITC/FA; DOX-loaded or blank PEI-PCL micelles complexing with FITC-labeled SCR and then with FA-PEG-PGA; D-PCE/FITC/NFA or B-PCE/FITC/NFA; DOX-loaded or blank PEI-PCL micelles complexing with FITC-labeled SCR and then with PEG-PGA; D-PCE/SCR/FA or B-PCE/SCR/FA; DOX-loaded or blank PEI-PCL micelles complexing with SCR and then with FA-PEG-PGA; D-PCE/SCR/NFA or B-PCE/SCR/NFA; DOX-loaded or blank PEI-PCL micelles complexing with SCR and then with PEG-PGA; Real-time PCR; real-time polymerase chain reaction; TUNEL; terminal deoxynucleotidyltransferase-mediated UTP end labeling


The synergistic effect of hierarchical assemblies of siRNA and chemotherapeutic drugs co-delivered into hepatic cancer cells by Nuo Cao; Du Cheng; Seyin Zou; Hua Ai; Jinming Gao; Xintao Shuai (pp. 2222-2232).
Diblock copolymers (PEI-PCL) of poly(ε-caprolactone) (PCL) and linear poly(ethylene imine) (PEI) were synthesized and assembled to biodegradable nano-carriers for co-delivery of BCL-2 siRNA and doxorubicin (DOX). Folic acid as a tumor-targeting ligand was conjugated to the polyanion, poly(ethylene glycol)-block-poly(glutamic acid) (FA-PEG-PGA). Driven by the electrostatic interaction, FA-PEG-PGA was coated onto the surface of the cationic PEI-PCL nanoparticles pre-loaded with siRNA and DOX, potentiating a ligand-directed delivery to human hepatic cancer cells Bel-7402. At certain N/P and C/N ratios (N/P: PEI-PCL nitrogen to siRNA phosphate; C/N: FA-PEG-PGA carboxyl to PEI-PCL amine), the nanoparticles exhibited not only high transfection efficiency but also ideally controlled release of drug. Compared to non-specific delivery, the folate-targeted delivery of BCL-2 siRNA resulted in more significant gene suppression at both the BCL-2 mRNA and protein expression levels, inducing cancer cell apoptosis and improving the therapeutic efficacy of the co-administered DOX. Herein we demonstrated that co-loading siRNA and small molecular drug in a multifunctional hierarchical nano-assembly enabled simultaneously delivering siRNA and drug into the same cancer cells, yielding synergistic effect of RNA interference and chemotherapy in cancer.

Keywords: Co-delivery of siRNA and drug; tumor-targeting; Electrostatic assembly; Gene silencing; Apoptosis; ChemotherapyAbbreviations; SCR; scrambled siRNA; BCL-2 siRNA; siRNA targeting BCL-2 gene; N/P ratio; molar ratio of PEI-PCL nitrogen to siRNA phosphorus; C/N ratio; molar ratio of FA-PEG-PGA or PEG-PGA carboxyl to PEI-PCL amino group; D-PCE or B-PCE; DOX-loaded or blank PEI-PCL micelles; D-PCE/BCL-2 or B-PCE/BCL-2; DOX-loaded or blank PEI-PCL micelles complexing with BCL-2 siRNA; D-PCE/FITC or B-PCE/FITC; DOX-loaded or blank PEI-PCL micelles complexing with FITC-labeled SCR; D-PCE/SCR or B-PCE/SCR; DOX-loaded or blank PEI-PCL micelles complexing with SCR; D-PCE/BCL-2/FA or B-PCE/BCL-2/FA; DOX-loaded or blank PEI-PCL micelles complexing with BCL-2 siRNA and then with FA-PEG-PGA; D-PCE/BCL-2/NFA or B-PCE/BCL-2/NFA; DOX-loaded or blank PEI-PCL micelles complexing with BCL-2 siRNA and then with PEG-PGA; D-PCE/FITC/FA or B-PCE/FITC/FA; DOX-loaded or blank PEI-PCL micelles complexing with FITC-labeled SCR and then with FA-PEG-PGA; D-PCE/FITC/NFA or B-PCE/FITC/NFA; DOX-loaded or blank PEI-PCL micelles complexing with FITC-labeled SCR and then with PEG-PGA; D-PCE/SCR/FA or B-PCE/SCR/FA; DOX-loaded or blank PEI-PCL micelles complexing with SCR and then with FA-PEG-PGA; D-PCE/SCR/NFA or B-PCE/SCR/NFA; DOX-loaded or blank PEI-PCL micelles complexing with SCR and then with PEG-PGA; Real-time PCR; real-time polymerase chain reaction; TUNEL; terminal deoxynucleotidyltransferase-mediated UTP end labeling


Recombinant spider silk particles as drug delivery vehicles by Andreas Lammel; Martin Schwab; Markus Hofer; Gerhard Winter; Thomas Scheibel ∗ (pp. 2233-2240).
Spider silk has been in the focus of research mainly due to the superior mechanical characteristics of silk fibers. However, it has been previously shown that spider silk proteins can also adopt other morphologies such as submicroparticles. This study examines the applicability of such particles as drug carriers. Particle characterization revealed that particles made of the engineered spider silk protein eADF4(C16) are colloidally stable in solution. Here, it is shown that small molecules with positive net-charge can diffuse into the negatively charged spider silk protein matrix driven by electrostatic interactions. The loading efficiencies correlate with the distribution coefficient (log D) of small molecules of weak alkaline nature. Interestingly, constant release rates can be realized for a period of two weeks at physiological conditions in vitro, with accelerated release rates within acidic environments. Enzymatic degradation studies of eADF4(C16) particles indicated that the silk proteins degrade slowly and the particles decrease in size. Along with their all-aqueous and easy preparation, drug loaded eADF4(C16) particles provide a high potential for diverse applications in which controlled release from biodegradable carriers is desired.

Keywords: Biomimetics; Biodegradation; Controlled release; Drug loading; Protein


Recombinant spider silk particles as drug delivery vehicles by Andreas Lammel; Martin Schwab; Markus Hofer; Gerhard Winter; Thomas Scheibel ∗ (pp. 2233-2240).
Spider silk has been in the focus of research mainly due to the superior mechanical characteristics of silk fibers. However, it has been previously shown that spider silk proteins can also adopt other morphologies such as submicroparticles. This study examines the applicability of such particles as drug carriers. Particle characterization revealed that particles made of the engineered spider silk protein eADF4(C16) are colloidally stable in solution. Here, it is shown that small molecules with positive net-charge can diffuse into the negatively charged spider silk protein matrix driven by electrostatic interactions. The loading efficiencies correlate with the distribution coefficient (log D) of small molecules of weak alkaline nature. Interestingly, constant release rates can be realized for a period of two weeks at physiological conditions in vitro, with accelerated release rates within acidic environments. Enzymatic degradation studies of eADF4(C16) particles indicated that the silk proteins degrade slowly and the particles decrease in size. Along with their all-aqueous and easy preparation, drug loaded eADF4(C16) particles provide a high potential for diverse applications in which controlled release from biodegradable carriers is desired.

Keywords: Biomimetics; Biodegradation; Controlled release; Drug loading; Protein

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