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Biomaterials (v.32, #16)

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

Ultra-thin, gas permeable free-standing and composite membranes for microfluidic lung assist devices by Ramaswamy Sreenivasan; Erik K. Bassett; David M. Hoganson; Joseph P. Vacanti; Karen K. Gleason (pp. 3883-3889).
Membranes for a lung assist device must permit the exchange of gaseous O2 and CO2 while simultaneously acting as a liquid barrier, so as to prevent leakage of blood and its components from passing from one side to the other. Additionally, these membranes must be capable of being integrated into microfluidic devices possessing a vascular network. In this work, uniform, large-area, ultra-thin, polymeric free-standing membranes (FSMs) and composite membranes (CMs) are reproducibly fabricated by initiated Chemical Vapor Deposition (iCVD). The 5 μm thick FSMs remained intact during handling and exhibited a CO2 permeance that was 1.3 times that of the control membrane (8 μm thick spun-cast membrane of silicone). The CMs consisted of a dense iCVD skin layer (0.5–3 μm thick) deposited on top of a polytetrafluoroethylene (PTFE) support membrane (20 μm thick, 100 nm pores). The CMs exhibited CO2 and O2 permeance values 50–300 times that of the control membrane. The FSMs were subjected to mechanical testing to assess the impact of the absence of an underlying support structure. The CMs were subjected to liquid barrier tests to ensure that while they were permeable to gases, they acted as barriers to liquids. Both FSMs and CMs were integrated into silicone microfluidic devices and tested for bond integrity.

Keywords: Lung assist device; Permeable membrane; iCVD; Membrane oxygenator; Artificial lung


Ultra-thin, gas permeable free-standing and composite membranes for microfluidic lung assist devices by Ramaswamy Sreenivasan; Erik K. Bassett; David M. Hoganson; Joseph P. Vacanti; Karen K. Gleason (pp. 3883-3889).
Membranes for a lung assist device must permit the exchange of gaseous O2 and CO2 while simultaneously acting as a liquid barrier, so as to prevent leakage of blood and its components from passing from one side to the other. Additionally, these membranes must be capable of being integrated into microfluidic devices possessing a vascular network. In this work, uniform, large-area, ultra-thin, polymeric free-standing membranes (FSMs) and composite membranes (CMs) are reproducibly fabricated by initiated Chemical Vapor Deposition (iCVD). The 5 μm thick FSMs remained intact during handling and exhibited a CO2 permeance that was 1.3 times that of the control membrane (8 μm thick spun-cast membrane of silicone). The CMs consisted of a dense iCVD skin layer (0.5–3 μm thick) deposited on top of a polytetrafluoroethylene (PTFE) support membrane (20 μm thick, 100 nm pores). The CMs exhibited CO2 and O2 permeance values 50–300 times that of the control membrane. The FSMs were subjected to mechanical testing to assess the impact of the absence of an underlying support structure. The CMs were subjected to liquid barrier tests to ensure that while they were permeable to gases, they acted as barriers to liquids. Both FSMs and CMs were integrated into silicone microfluidic devices and tested for bond integrity.

Keywords: Lung assist device; Permeable membrane; iCVD; Membrane oxygenator; Artificial lung


Implantation of ultrathin, biofunctionalized polyimide membranes into the subretinal space of rats by Sylvie Julien; Tobias Peters; Focke Ziemssen; Blanca Arango-Gonzalez; Susanne Beck; Hagen Thielecke; Heiko Büth; Sandra Van Vlierberghe; Milada Sirova; Pavel Rossmann; Blanka Rihova; Etienne Schacht; Peter Dubruel; Eberhart Zrenner; Ulrich Schraermeyer (pp. 3890-3898).
Subretinal implants aim to replace the photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli in the subretinal space. Critical obstacles in the design of high-resolution subretinal implants include the proximity of stimulating electrodes to the target cells and enabling nutrient flow between the retina and the choroid. The present work evaluates the adhesion, migration and survival of retinal cells on an ultrathin (5 μm), highly porous (Ø 1 μm spaced 3 μm), gelatin-coated polyimide (PI) membrane. The biocompatibility was examined in mice indicating a good tolerance upon subcutaneous implantation with only a mild inflammatory response. In addition, organotypic cultures of rat retina evidenced that the porous membrane allowed the necessary nutrient flow for the retinal cell survival and maintenance. A transscleral implantation technique was applied to position the membrane into the subretinal space of rats. The effect on the obtained retinal integration was investigated in vivo using scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). In 12 out of 18 rat eyes, the implant was successfully placed subretinally. SLO and OCT demonstrated complete retinal attachment and fluorescein angiography showed no retinal vascular abnormalities over and around the implant, immediately after and up to four weeks after the implantation. Histological examination of the eyes showed a close attachment of a thin fibrocyte layer to the implant, the occlusion of the pores by living cells and the survival of some photoreceptors at the implantation site.

Keywords: Ophthalmology; Biocompatibility; Cell viability; Cell adhesion; Electron microscopy


Implantation of ultrathin, biofunctionalized polyimide membranes into the subretinal space of rats by Sylvie Julien; Tobias Peters; Focke Ziemssen; Blanca Arango-Gonzalez; Susanne Beck; Hagen Thielecke; Heiko Büth; Sandra Van Vlierberghe; Milada Sirova; Pavel Rossmann; Blanka Rihova; Etienne Schacht; Peter Dubruel; Eberhart Zrenner; Ulrich Schraermeyer (pp. 3890-3898).
Subretinal implants aim to replace the photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli in the subretinal space. Critical obstacles in the design of high-resolution subretinal implants include the proximity of stimulating electrodes to the target cells and enabling nutrient flow between the retina and the choroid. The present work evaluates the adhesion, migration and survival of retinal cells on an ultrathin (5 μm), highly porous (Ø 1 μm spaced 3 μm), gelatin-coated polyimide (PI) membrane. The biocompatibility was examined in mice indicating a good tolerance upon subcutaneous implantation with only a mild inflammatory response. In addition, organotypic cultures of rat retina evidenced that the porous membrane allowed the necessary nutrient flow for the retinal cell survival and maintenance. A transscleral implantation technique was applied to position the membrane into the subretinal space of rats. The effect on the obtained retinal integration was investigated in vivo using scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). In 12 out of 18 rat eyes, the implant was successfully placed subretinally. SLO and OCT demonstrated complete retinal attachment and fluorescein angiography showed no retinal vascular abnormalities over and around the implant, immediately after and up to four weeks after the implantation. Histological examination of the eyes showed a close attachment of a thin fibrocyte layer to the implant, the occlusion of the pores by living cells and the survival of some photoreceptors at the implantation site.

Keywords: Ophthalmology; Biocompatibility; Cell viability; Cell adhesion; Electron microscopy


The biocompatibility and biofilm resistance of implant coatings based on hydrophilic polymer brushes conjugated with antimicrobial peptides by Guangzheng Gao; Dirk Lange; Kai Hilpert; Jason Kindrachuk; Yuquan Zou; John T.J. Cheng; Mehdi Kazemzadeh-Narbat; Kai Yu; Rizhi Wang; Suzana K. Straus; Donald E. Brooks; Ben H. Chew; Robert E.W. Hancock; Jayachandran N. Kizhakkedathu (pp. 3899-3909).
Bacterial colonization on implant surfaces and subsequent infections are one of the most common reasons for the failure of many indwelling devices. Several approaches including antimicrobial and antibiotic-eluting coatings on implants have been attempted; however, none of these approaches succeed in vivo. Here we report a polymer brush based implant coating that is non-toxic, antimicrobial and biofilm resistant. These coating consists of covalently grafted hydrophilic polymer chains conjugated with an optimized series of antimicrobial peptides (AMPs). These tethered AMPs maintained excellent broad spectrum antimicrobial activity in vitro and in vivo. We found that this specially structured robust coating was extremely effective in resisting biofilm formation, and that the biofilm resistance depended on the nature of conjugated peptides. The coating had no toxicity to osteoblast-like cells and showed insignificant platelet activation and adhesion, and complement activation in human blood. Since such coatings can be applied to most currently used implant surfaces, our approach has significant potential for the development of infection-resistant implants.

Keywords: Antimicrobial coatings; Implant-associated infection; Biofilm resistance; Blood/tissue toxicity; Antimicrobial peptides; Polymer brushes


The biocompatibility and biofilm resistance of implant coatings based on hydrophilic polymer brushes conjugated with antimicrobial peptides by Guangzheng Gao; Dirk Lange; Kai Hilpert; Jason Kindrachuk; Yuquan Zou; John T.J. Cheng; Mehdi Kazemzadeh-Narbat; Kai Yu; Rizhi Wang; Suzana K. Straus; Donald E. Brooks; Ben H. Chew; Robert E.W. Hancock; Jayachandran N. Kizhakkedathu (pp. 3899-3909).
Bacterial colonization on implant surfaces and subsequent infections are one of the most common reasons for the failure of many indwelling devices. Several approaches including antimicrobial and antibiotic-eluting coatings on implants have been attempted; however, none of these approaches succeed in vivo. Here we report a polymer brush based implant coating that is non-toxic, antimicrobial and biofilm resistant. These coating consists of covalently grafted hydrophilic polymer chains conjugated with an optimized series of antimicrobial peptides (AMPs). These tethered AMPs maintained excellent broad spectrum antimicrobial activity in vitro and in vivo. We found that this specially structured robust coating was extremely effective in resisting biofilm formation, and that the biofilm resistance depended on the nature of conjugated peptides. The coating had no toxicity to osteoblast-like cells and showed insignificant platelet activation and adhesion, and complement activation in human blood. Since such coatings can be applied to most currently used implant surfaces, our approach has significant potential for the development of infection-resistant implants.

Keywords: Antimicrobial coatings; Implant-associated infection; Biofilm resistance; Blood/tissue toxicity; Antimicrobial peptides; Polymer brushes


The promotion of cartilage defect repair using adenovirus mediated Sox9 gene transfer of rabbit bone marrow mesenchymal stem cells by Lei Cao; Fei Yang; Guangwang Liu; Degang Yu; Huiwu Li; Qiming Fan; Yaokai Gan; Tingting Tang; Kerong Dai (pp. 3910-3920).
Although Sox9 is essential for chondrogenic differentiation and matrix production, its application in cartilage tissue engineering has been rarely reported. In this study, the chondrogenic effect of Sox9 on bone marrow mesenchymal stem cells (BMSCs) in vitro and its application in articular cartilage repair in vivo were evaluated. Rabbit BMSCs were transduced with adenoviral vector containing Sox9. Toluidine blue, safranin O staining and real-time PCR were performed to check chondrogenic differentiation. The results showed that Sox9 could induce chondrogenesis of BMSCs both in monolayer and on PGA scaffold effectively. The rabbit model with full-thickness cartilage defects was established and then repaired by PGA scaffold and rabbit BMSCs with or without Sox9 transduction. HE, safranin O staining and immunohistochemistry were used to assess the repair of defects by the complex. Better repair, including more newly-formed cartilage tissue and hyaline cartilage-specific extracellular matrix and greater expression of several chondrogenesis marker genes were observed in PGA scaffold and BMSCs with Sox9 transduction, compared to that without transduction. Our findings defined the important role of Sox9 in the repair of cartilage defects in vivo and provided evidence that Sox9 had the potential and advantage in the application of tissue engineering.

Keywords: Animal model; Cartilage tissue engineering; Gene transfer; Stem cell


The promotion of cartilage defect repair using adenovirus mediated Sox9 gene transfer of rabbit bone marrow mesenchymal stem cells by Lei Cao; Fei Yang; Guangwang Liu; Degang Yu; Huiwu Li; Qiming Fan; Yaokai Gan; Tingting Tang; Kerong Dai (pp. 3910-3920).
Although Sox9 is essential for chondrogenic differentiation and matrix production, its application in cartilage tissue engineering has been rarely reported. In this study, the chondrogenic effect of Sox9 on bone marrow mesenchymal stem cells (BMSCs) in vitro and its application in articular cartilage repair in vivo were evaluated. Rabbit BMSCs were transduced with adenoviral vector containing Sox9. Toluidine blue, safranin O staining and real-time PCR were performed to check chondrogenic differentiation. The results showed that Sox9 could induce chondrogenesis of BMSCs both in monolayer and on PGA scaffold effectively. The rabbit model with full-thickness cartilage defects was established and then repaired by PGA scaffold and rabbit BMSCs with or without Sox9 transduction. HE, safranin O staining and immunohistochemistry were used to assess the repair of defects by the complex. Better repair, including more newly-formed cartilage tissue and hyaline cartilage-specific extracellular matrix and greater expression of several chondrogenesis marker genes were observed in PGA scaffold and BMSCs with Sox9 transduction, compared to that without transduction. Our findings defined the important role of Sox9 in the repair of cartilage defects in vivo and provided evidence that Sox9 had the potential and advantage in the application of tissue engineering.

Keywords: Animal model; Cartilage tissue engineering; Gene transfer; Stem cell


The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β by Jennifer S. Park; Julia S. Chu; Anchi D. Tsou; Rokhaya Diop; Zhenyu Tang; Aijun Wang; Song Li (pp. 3921-3930).
Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.

Keywords: Extracellular matrix; Cell adhesion; Mesenchymal stem cells; Smooth muscle cell; Chondrocyte; Matrix rigidity


The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β by Jennifer S. Park; Julia S. Chu; Anchi D. Tsou; Rokhaya Diop; Zhenyu Tang; Aijun Wang; Song Li (pp. 3921-3930).
Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.

Keywords: Extracellular matrix; Cell adhesion; Mesenchymal stem cells; Smooth muscle cell; Chondrocyte; Matrix rigidity


Critical areas of cell adhesion on micropatterned surfaces by Ce Yan; Jianguo Sun; Jiandong Ding (pp. 3931-3938).
The adhesive area is important to modulate cell behaviors on a substrate. This paper aims to semi-quantitatively examine the existence of the characteristic areas of cell adhesion on the level of individual cells. We prepared a series of micropatterned surfaces with adhesive microislands of various sizes on an adhesion-resistant background, and cultured cells of MC3T3-E1 (osteoblast), BMSC (bone mesenchymal stem cell) or NIH3T3 (fibroblast) on those modeled surfaces. We have defined seven characteristic areas of an adhesive microisland and confirmed that they are meaningful to describe cell adhesion behaviors. Those parameters are (1) the critical adhesion area from apoptosis to survival denoted as A or Ac1, (2) the critical area from adhesion of a single cell to adhesion of multiple cells ( Ac2), (3) the basic area for one more cell to adhere ( AΔ), (4) and (5) the characteristic areas of a microisland most probably occupied by one cell ( Apeak(1)) and two cells ( Apeak(2)), (6) and (7) the characteristic areas of a microisland occupied by one cell (A N(1)) or two cells (A N(2)) on average. Besides the introduction of those basic parameters, the present paper demonstrates how to determine them experimentally. We further discussed the relationship between those characteristic areas and the spreading area on a non-patterned adhesive surface.

Keywords: Cell adhesion; Micro-patterning; Critical area; Surface modification


Critical areas of cell adhesion on micropatterned surfaces by Ce Yan; Jianguo Sun; Jiandong Ding (pp. 3931-3938).
The adhesive area is important to modulate cell behaviors on a substrate. This paper aims to semi-quantitatively examine the existence of the characteristic areas of cell adhesion on the level of individual cells. We prepared a series of micropatterned surfaces with adhesive microislands of various sizes on an adhesion-resistant background, and cultured cells of MC3T3-E1 (osteoblast), BMSC (bone mesenchymal stem cell) or NIH3T3 (fibroblast) on those modeled surfaces. We have defined seven characteristic areas of an adhesive microisland and confirmed that they are meaningful to describe cell adhesion behaviors. Those parameters are (1) the critical adhesion area from apoptosis to survival denoted as A or Ac1, (2) the critical area from adhesion of a single cell to adhesion of multiple cells ( Ac2), (3) the basic area for one more cell to adhere ( AΔ), (4) and (5) the characteristic areas of a microisland most probably occupied by one cell ( Apeak(1)) and two cells ( Apeak(2)), (6) and (7) the characteristic areas of a microisland occupied by one cell (A N(1)) or two cells (A N(2)) on average. Besides the introduction of those basic parameters, the present paper demonstrates how to determine them experimentally. We further discussed the relationship between those characteristic areas and the spreading area on a non-patterned adhesive surface.

Keywords: Cell adhesion; Micro-patterning; Critical area; Surface modification


The use of laminin modified linear ordered collagen scaffolds loaded with laminin-binding ciliary neurotrophic factor for sciatic nerve regeneration in rats by Jiani Cao; Changkai Sun; Hui Zhao; Zhifeng Xiao; Bing Chen; Jian Gao; Tiezheng Zheng; Wei Wu; Shuang Wu; Jingyu Wang; Jianwu Dai (pp. 3939-3948).
Nerve conduit provides a promising strategy for nerve injury repair in the peripheral nervous system (PNS). However, simply bridging the transected nerve with an empty conduit is hard to satisfy functional recovery. The regenerated axons may disperse during regeneration in the empty lumen, limiting the functional recovery. Our previous work had reported that linear ordered collagen scaffold (LOCS) could be used as a nerve guidance material. Here we cross-linked LOCS fibers with laminin which was a major component of the extracellular matrix in nervous system. Ciliary neurotrophic factor (CNTF) plays a critical role in peripheral nerve regeneration. But the lack of efficient CNTF delivery approach limits its clinical applications. To retain CNTF on the scaffold, a laminin binding domain (LBD) was fused to the N-terminal of CNTF. Compared with NAT-CNTF, LBD-CNTF exhibited specific laminin-binding ability and comparable neurotrophic bioactivity. We combined LBD-CNTF with the laminin modified LOCS fibers to construct a double-functional bio-scaffold. The functional scaffold was filled in silicon conduit and tested in the rat sciatic nerve transection model. Results showed that this functional biomaterial could guide the axon growth, retain more CNTF on the scaffolds and enhance the nerve regeneration as well as functional recovery.

Keywords: LOCS fibers; Laminin; LBD-CNTF; Nerve transection repair


The use of laminin modified linear ordered collagen scaffolds loaded with laminin-binding ciliary neurotrophic factor for sciatic nerve regeneration in rats by Jiani Cao; Changkai Sun; Hui Zhao; Zhifeng Xiao; Bing Chen; Jian Gao; Tiezheng Zheng; Wei Wu; Shuang Wu; Jingyu Wang; Jianwu Dai (pp. 3939-3948).
Nerve conduit provides a promising strategy for nerve injury repair in the peripheral nervous system (PNS). However, simply bridging the transected nerve with an empty conduit is hard to satisfy functional recovery. The regenerated axons may disperse during regeneration in the empty lumen, limiting the functional recovery. Our previous work had reported that linear ordered collagen scaffold (LOCS) could be used as a nerve guidance material. Here we cross-linked LOCS fibers with laminin which was a major component of the extracellular matrix in nervous system. Ciliary neurotrophic factor (CNTF) plays a critical role in peripheral nerve regeneration. But the lack of efficient CNTF delivery approach limits its clinical applications. To retain CNTF on the scaffold, a laminin binding domain (LBD) was fused to the N-terminal of CNTF. Compared with NAT-CNTF, LBD-CNTF exhibited specific laminin-binding ability and comparable neurotrophic bioactivity. We combined LBD-CNTF with the laminin modified LOCS fibers to construct a double-functional bio-scaffold. The functional scaffold was filled in silicon conduit and tested in the rat sciatic nerve transection model. Results showed that this functional biomaterial could guide the axon growth, retain more CNTF on the scaffolds and enhance the nerve regeneration as well as functional recovery.

Keywords: LOCS fibers; Laminin; LBD-CNTF; Nerve transection repair


The generation of biomolecular patterns in highly porous collagen-GAG scaffolds using direct photolithography by Teresa A. Martin; Steven R. Caliari; Paul D. Williford; Brendan A. Harley; Ryan C. Bailey (pp. 3949-3957).
The extracellular matrix (ECM) is a complex organization of structural proteins found within tissues and organs. Heterogeneous tissues with spatially and temporally modulated properties play an important role in organism physiology. Here we present a benzophenone (BP) based direct, photolithographic approach to spatially pattern solution phase biomolecules within collagen-GAG (CG) scaffolds and demonstrate creation of a wide range of patterns composed of multiple biomolecular species in a manner independent from scaffold fabrication steps. We demonstrate the ability to immobilize biomolecules at surface densities of up to 1000 ligands per square micron on the scaffold strut surface and to depths limited by the penetration depth of the excitation source into the scaffold structure. Importantly, while BP photopatterning does further crosslink the CG scaffold, evidenced by increased mechanical properties and collagen crystallinity, it does not affect scaffold microstructural or compositional properties or negatively influence cell adhesion, viability, or proliferation. We show that covalently photoimmobilized fibronectin within a CG scaffold significantly increases the speed of MC3T3-E1 cell attachment relative to the bare CG scaffold or non-specifically adsorbed fibronectin, suggesting that this approach can be used to improve scaffold bioactivity. Our findings, on the whole, establish the use of direct, BP photolithography as a methodology for covalently incorporating activity-improving biochemical cues within 3D collagen biomaterial scaffolds with spatial control over biomolecular deposition.

Keywords: Collagen; Scaffold; Micropatterning; Photolithography; Cell adhesion; Surface modification


The generation of biomolecular patterns in highly porous collagen-GAG scaffolds using direct photolithography by Teresa A. Martin; Steven R. Caliari; Paul D. Williford; Brendan A. Harley; Ryan C. Bailey (pp. 3949-3957).
The extracellular matrix (ECM) is a complex organization of structural proteins found within tissues and organs. Heterogeneous tissues with spatially and temporally modulated properties play an important role in organism physiology. Here we present a benzophenone (BP) based direct, photolithographic approach to spatially pattern solution phase biomolecules within collagen-GAG (CG) scaffolds and demonstrate creation of a wide range of patterns composed of multiple biomolecular species in a manner independent from scaffold fabrication steps. We demonstrate the ability to immobilize biomolecules at surface densities of up to 1000 ligands per square micron on the scaffold strut surface and to depths limited by the penetration depth of the excitation source into the scaffold structure. Importantly, while BP photopatterning does further crosslink the CG scaffold, evidenced by increased mechanical properties and collagen crystallinity, it does not affect scaffold microstructural or compositional properties or negatively influence cell adhesion, viability, or proliferation. We show that covalently photoimmobilized fibronectin within a CG scaffold significantly increases the speed of MC3T3-E1 cell attachment relative to the bare CG scaffold or non-specifically adsorbed fibronectin, suggesting that this approach can be used to improve scaffold bioactivity. Our findings, on the whole, establish the use of direct, BP photolithography as a methodology for covalently incorporating activity-improving biochemical cues within 3D collagen biomaterial scaffolds with spatial control over biomolecular deposition.

Keywords: Collagen; Scaffold; Micropatterning; Photolithography; Cell adhesion; Surface modification


Role of fibronectin in topographical guidance of neurite extension on electrospun fibers by Vivek J. Mukhatyar; Manuel Salmerón-Sánchez; Soumon Rudra; Shoumit Mukhopadaya; Thomas H. Barker; Andrés J. García; Ravi V. Bellamkonda (pp. 3958-3968).
Bridging of long peripheral nerve gaps remains a significant clinical challenge. Electrospun nanofibers have been used to direct and enhance neurite extension in vitro and in vivo. While it is well established that oriented fibers influence neurite outgrowth and Schwann cell migration, the mechanisms by which they influence these cells are still unclear. In this study, thin films consisting of aligned poly-acrylonitrile methylacrylate (PAN-MA) fibers or solvent casted smooth, PAN-MA films were fabricated to investigate the potential role of differential protein adsorption on topography-dependent neural cell responses. Aligned nanofiber films promoted enhanced adsorption of fibronectin compared to smooth films. Studies employing function-blocking antibodies against cell adhesion motifs suggest that fibronectin plays an important role in modulating Schwann cell migration and neurite outgrowth from dorsal root ganglion (DRG) cultures. Atomic Force Microscopy demonstrated that aligned PAN-MA fibers influenced fibronectin distribution, and promoted aligned fibronectin network formation compared to smooth PAN-MA films. In the presence of topographical cues, Schwann cell-generated fibronectin matrix was also organized in a topographically sensitive manner. Together these results suggest that fibronectin adsorption mediated the ability of topographical cues to influence Schwann cell migration and neurite outgrowth. These insights are significant to the development of rational approaches to scaffold designs to bridge long peripheral nerve gaps.

Keywords: Neural tissue engineering; Fibronectin; Schwann cell migration; Protein distribution; Electrospun nanofibers; Peripheral nerve regeneration


Role of fibronectin in topographical guidance of neurite extension on electrospun fibers by Vivek J. Mukhatyar; Manuel Salmerón-Sánchez; Soumon Rudra; Shoumit Mukhopadaya; Thomas H. Barker; Andrés J. García; Ravi V. Bellamkonda (pp. 3958-3968).
Bridging of long peripheral nerve gaps remains a significant clinical challenge. Electrospun nanofibers have been used to direct and enhance neurite extension in vitro and in vivo. While it is well established that oriented fibers influence neurite outgrowth and Schwann cell migration, the mechanisms by which they influence these cells are still unclear. In this study, thin films consisting of aligned poly-acrylonitrile methylacrylate (PAN-MA) fibers or solvent casted smooth, PAN-MA films were fabricated to investigate the potential role of differential protein adsorption on topography-dependent neural cell responses. Aligned nanofiber films promoted enhanced adsorption of fibronectin compared to smooth films. Studies employing function-blocking antibodies against cell adhesion motifs suggest that fibronectin plays an important role in modulating Schwann cell migration and neurite outgrowth from dorsal root ganglion (DRG) cultures. Atomic Force Microscopy demonstrated that aligned PAN-MA fibers influenced fibronectin distribution, and promoted aligned fibronectin network formation compared to smooth PAN-MA films. In the presence of topographical cues, Schwann cell-generated fibronectin matrix was also organized in a topographically sensitive manner. Together these results suggest that fibronectin adsorption mediated the ability of topographical cues to influence Schwann cell migration and neurite outgrowth. These insights are significant to the development of rational approaches to scaffold designs to bridge long peripheral nerve gaps.

Keywords: Neural tissue engineering; Fibronectin; Schwann cell migration; Protein distribution; Electrospun nanofibers; Peripheral nerve regeneration


A collagen-poly(lactic acid-co-ɛ-caprolactone) hybrid scaffold for bladder tissue regeneration by Eva-Maria Engelhardt; Lionel A. Micol; Stephanie Houis; Florian M. Wurm; Jöns Hilborn; Jeffrey A. Hubbell; Peter Frey (pp. 3969-3976).
Scaffold materials should favor cell attachment and proliferation, and provide designable 3D structures with appropriate mechanical strength. Collagen matrices have proven to be beneficial scaffolds for tissue regeneration. However, apart from small intestinal submucosa, they offer a limited mechanical strength even if crosslinking can enhance their mechanical properties. A more cell-friendly way to increase material strength is to combine synthetic polymer meshes with plastic compressed collagen gels. This work describes the potential of plastic compressed collagen–poly(lactic acid-co-ɛ-caprolactone) (PLAC) hybrids as scaffolds for bladder tissue regeneration. Human bladder smooth muscle and urothelial cells were cultured on and inside collagen–PLAC hybrids in vitro. Scaffolds were analyzed by electron microscopy, histology, immunohistochemistry, and AlamarBlue assay. Both cell types proliferated in and on the hybrid, forming dense cell layers on top after two weeks. Furthermore, hybrids were implanted subcutaneously in the backs of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed. Hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. Collagen–PLAC hybrids have potential for bladder tissue regeneration, as they show efficient cell seeding, proliferation, and good mechanical properties.

Keywords: Bladder tissue engineering; Scaffold; Collagen; Copolymer; In vitro; test; In vivo; test


A collagen-poly(lactic acid-co-ɛ-caprolactone) hybrid scaffold for bladder tissue regeneration by Eva-Maria Engelhardt; Lionel A. Micol; Stephanie Houis; Florian M. Wurm; Jöns Hilborn; Jeffrey A. Hubbell; Peter Frey (pp. 3969-3976).
Scaffold materials should favor cell attachment and proliferation, and provide designable 3D structures with appropriate mechanical strength. Collagen matrices have proven to be beneficial scaffolds for tissue regeneration. However, apart from small intestinal submucosa, they offer a limited mechanical strength even if crosslinking can enhance their mechanical properties. A more cell-friendly way to increase material strength is to combine synthetic polymer meshes with plastic compressed collagen gels. This work describes the potential of plastic compressed collagen–poly(lactic acid-co-ɛ-caprolactone) (PLAC) hybrids as scaffolds for bladder tissue regeneration. Human bladder smooth muscle and urothelial cells were cultured on and inside collagen–PLAC hybrids in vitro. Scaffolds were analyzed by electron microscopy, histology, immunohistochemistry, and AlamarBlue assay. Both cell types proliferated in and on the hybrid, forming dense cell layers on top after two weeks. Furthermore, hybrids were implanted subcutaneously in the backs of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed. Hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. Collagen–PLAC hybrids have potential for bladder tissue regeneration, as they show efficient cell seeding, proliferation, and good mechanical properties.

Keywords: Bladder tissue engineering; Scaffold; Collagen; Copolymer; In vitro; test; In vivo; test


Multinucleated giant cells from fibroblast cultures by Dolly J. Holt; David W. Grainger (pp. 3977-3987).
Many multinucleated giant cells are well-known to form from macrophage origin. Those formed from other cell types are less described, but may be as prevalent in pathological tissue. Giant multinucleated cells derived from secondary and primary fibroblast sources in various cultures with similar characteristics to foreign body giant cells are reported. Secondary-transformed NIH 3T3 fibroblasts rapidly fuse within 24 h in contact co-cultures with RAW 264.7 immortalized macrophages, while 3T3 monocultures, non-contact (transwell) co-cultures, and macrophage-conditioned media-treated 3T3 monocultures all do not fuse. Primary-derived murine fibroblasts also form multinucleated cells, both in the presence or absence of co-cultured macrophages that increase during long-term culture (5–30 days). In contrast to 3T3 fusion, this primary cell phenomenon is not due to fibroblast fusion, but rather to nuclear division without cytokinesis. That these multinucleated fibroblasts can originate via different mechanisms may influence and distinguish their behaviors in conditions under which they may arise, including various in vitro culture assays, and in certain fibroblastic pathologies such as the foreign body response, fibrosis, cancer and aged tissue.

Keywords: Fibroblast; Macrophage; Co-culture; Foreign body response; Animal model; Cell culture


Multinucleated giant cells from fibroblast cultures by Dolly J. Holt; David W. Grainger (pp. 3977-3987).
Many multinucleated giant cells are well-known to form from macrophage origin. Those formed from other cell types are less described, but may be as prevalent in pathological tissue. Giant multinucleated cells derived from secondary and primary fibroblast sources in various cultures with similar characteristics to foreign body giant cells are reported. Secondary-transformed NIH 3T3 fibroblasts rapidly fuse within 24 h in contact co-cultures with RAW 264.7 immortalized macrophages, while 3T3 monocultures, non-contact (transwell) co-cultures, and macrophage-conditioned media-treated 3T3 monocultures all do not fuse. Primary-derived murine fibroblasts also form multinucleated cells, both in the presence or absence of co-cultured macrophages that increase during long-term culture (5–30 days). In contrast to 3T3 fusion, this primary cell phenomenon is not due to fibroblast fusion, but rather to nuclear division without cytokinesis. That these multinucleated fibroblasts can originate via different mechanisms may influence and distinguish their behaviors in conditions under which they may arise, including various in vitro culture assays, and in certain fibroblastic pathologies such as the foreign body response, fibrosis, cancer and aged tissue.

Keywords: Fibroblast; Macrophage; Co-culture; Foreign body response; Animal model; Cell culture


Long term in vivo biotransformation of iron oxide nanoparticles by Michael Levy; Nathalie Luciani; Damien Alloyeau; Dan Elgrabli; Vanessa Deveaux; Christine Pechoux; Sophie Chat; Guillaume Wang; Nidhi Vats; François Gendron; Cécile Factor; Sophie Lotersztajn; Alain Luciani; Claire Wilhelm; Florence Gazeau (pp. 3988-3999).
The long term outcome of nanoparticles in the organism is one of the most important concerns raised by the development of nanotechnology and nanomedicine. Little is known on the way taken by cells to process and degrade nanoparticles over time. In this context, iron oxide superparamagnetic nanoparticles benefit from a privileged status, because they show a very good tolerance profile, allowing their clinical use for MRI diagnosis. It is generally assumed that the specialized metabolism which regulates iron in the organism can also handle iron oxide nanoparticles. However the biotransformation of iron oxide nanoparticles is still not elucidated. Here we propose a multiscale approach to study the fate of nanomagnets in the organism. Ferromagnetic resonance and SQUID magnetization measurements are used to quantify iron oxide nanoparticles and follow the evolution of their magnetic properties. A nanoscale structural analysis by electron microscopy complements the magnetic follow-up of nanoparticles injected to mice. We evidence the biotransformation of superparamagnetic maghemite nanoparticles into poorly-magnetic iron species probably stored into ferritin proteins over a period of three months. A putative mechanism is proposed for the biotransformation of iron-oxide nanoparticles.

Keywords: Nanoparticles; Nanomagnetism; Nanotoxicity; Biodegradation; Iron oxide; Contrast agent


Long term in vivo biotransformation of iron oxide nanoparticles by Michael Levy; Nathalie Luciani; Damien Alloyeau; Dan Elgrabli; Vanessa Deveaux; Christine Pechoux; Sophie Chat; Guillaume Wang; Nidhi Vats; François Gendron; Cécile Factor; Sophie Lotersztajn; Alain Luciani; Claire Wilhelm; Florence Gazeau (pp. 3988-3999).
The long term outcome of nanoparticles in the organism is one of the most important concerns raised by the development of nanotechnology and nanomedicine. Little is known on the way taken by cells to process and degrade nanoparticles over time. In this context, iron oxide superparamagnetic nanoparticles benefit from a privileged status, because they show a very good tolerance profile, allowing their clinical use for MRI diagnosis. It is generally assumed that the specialized metabolism which regulates iron in the organism can also handle iron oxide nanoparticles. However the biotransformation of iron oxide nanoparticles is still not elucidated. Here we propose a multiscale approach to study the fate of nanomagnets in the organism. Ferromagnetic resonance and SQUID magnetization measurements are used to quantify iron oxide nanoparticles and follow the evolution of their magnetic properties. A nanoscale structural analysis by electron microscopy complements the magnetic follow-up of nanoparticles injected to mice. We evidence the biotransformation of superparamagnetic maghemite nanoparticles into poorly-magnetic iron species probably stored into ferritin proteins over a period of three months. A putative mechanism is proposed for the biotransformation of iron-oxide nanoparticles.

Keywords: Nanoparticles; Nanomagnetism; Nanotoxicity; Biodegradation; Iron oxide; Contrast agent


The potential of nanoscale combinations of self-assembling peptides and amino acids of the Src tyrosine kinase inhibitor in acute lung injury therapy by Shan-Yu Fung; Takeshi Oyaizu; Hong Yang; Yongfang Yuan; Bing Han; Shaf Keshavjee; Mingyao Liu (pp. 4000-4008).
Many newly discovered therapeutic agents require a delivery platform in order to translate them into clinical applications. For this purpose, a nanoscale formulation strategy was developed for the Src tyrosine kinase inhibitor PP2. The formulation utilizes the combination of the self-assembling peptides (EAK16-II) and amino acids to minimize the use of the toxic organic solvent DMSO; hence, the biocompatibility of the PP2 nanoformulations was significantly improved. They were found to be non-hemolytic and safe for intravenous and intratracheal administration; the formulations did not alter PP2 activity in Src inhibition on cultured cells. The PP2 nanoformulation was further evaluated on a lipopolysaccharide (LPS)-induced acute lung injury mouse model. Results revealed that the pretreatment of PP2 nanoformulation could decrease the inflammatory cell infiltration and the pro-inflammatory cytokine TNF-α production in the bronchoalveolar lavage fluid after LPS stimulation. The promising therapeutic efficacy and the formulation strategy developed in this work may help further translate PP2 and other hydrophobic therapeutic agents into clinical applications.

Keywords: Drug delivery; Self-assembling peptide; Acute inflammatory response; Biocompatibility; Nanoformulation


The potential of nanoscale combinations of self-assembling peptides and amino acids of the Src tyrosine kinase inhibitor in acute lung injury therapy by Shan-Yu Fung; Takeshi Oyaizu; Hong Yang; Yongfang Yuan; Bing Han; Shaf Keshavjee; Mingyao Liu (pp. 4000-4008).
Many newly discovered therapeutic agents require a delivery platform in order to translate them into clinical applications. For this purpose, a nanoscale formulation strategy was developed for the Src tyrosine kinase inhibitor PP2. The formulation utilizes the combination of the self-assembling peptides (EAK16-II) and amino acids to minimize the use of the toxic organic solvent DMSO; hence, the biocompatibility of the PP2 nanoformulations was significantly improved. They were found to be non-hemolytic and safe for intravenous and intratracheal administration; the formulations did not alter PP2 activity in Src inhibition on cultured cells. The PP2 nanoformulation was further evaluated on a lipopolysaccharide (LPS)-induced acute lung injury mouse model. Results revealed that the pretreatment of PP2 nanoformulation could decrease the inflammatory cell infiltration and the pro-inflammatory cytokine TNF-α production in the bronchoalveolar lavage fluid after LPS stimulation. The promising therapeutic efficacy and the formulation strategy developed in this work may help further translate PP2 and other hydrophobic therapeutic agents into clinical applications.

Keywords: Drug delivery; Self-assembling peptide; Acute inflammatory response; Biocompatibility; Nanoformulation


Dual mode polyspermine with tunable degradability for plasmid DNA and siRNA delivery by Min Suk Shim; Young Jik Kwon (pp. 4009-4020).
Stimuli-responsive degradability is an indispensable design component for polymeric gene carriers. In order to obtain enhanced, non-cytotoxic, and molecularly tunable nonviral gene delivery, spermine, a bioavailable small cationic molecule, was polymerized with diacrylate cross-linkers with or without acid-degradable ketal linkages for controlled dual mode-degradability (i.e., differential degradations in the endosome and the cytosol). The effects of ketal to ester ratios in the polymeric backbone on degradation rate, condensation of both plasmid DNA and siRNA, cellular uptake, intracellular disassembly, and consequent DNA transfection and RNA interference efficiency in vitro and in vivo were investigated. Limited nucleic acid complexation and cellular uptake but efficient intracellular release of nucleic acids were obtained with poly(spermine ketal ester) (PSKE), the most acid-degradable polyspermine. In contrast, poly(spermine ester) (PSE), which is not acid-degradable, demonstrated efficient nucleic acid complexation and cellular uptake but inefficient intracellular release of nucleic acids. The highest in vitro DNA transfection was obtained by the random co-polymer of PSKE and PSE at an equal ratio (PSKE-PSE), attributed to its balanced DNA complexation and acid-responsive release efficiency, while efficient siRNA unpackaging by PSKE resulted in the highest gene silencing efficiency. Preliminary in vivo studies demonstrated that the highest DNA transfection was obtained by using PSE, while both PSKE and PSE silenced GFP expression at the similar level. In conclusion, dual mode-degradable polyspermine is a non-cytotoxic nonviral gene carrier, and its acid-degradability can be molecularly tuned for differentially controlled transfection and gene silencing in vitro and in vivo.

Keywords: Polyspermine; Nonviral gene delivery; Biodegradability; DNA and siRNA delivery; In vitro; and; in vivo; transfection; Intracellular release


Dual mode polyspermine with tunable degradability for plasmid DNA and siRNA delivery by Min Suk Shim; Young Jik Kwon (pp. 4009-4020).
Stimuli-responsive degradability is an indispensable design component for polymeric gene carriers. In order to obtain enhanced, non-cytotoxic, and molecularly tunable nonviral gene delivery, spermine, a bioavailable small cationic molecule, was polymerized with diacrylate cross-linkers with or without acid-degradable ketal linkages for controlled dual mode-degradability (i.e., differential degradations in the endosome and the cytosol). The effects of ketal to ester ratios in the polymeric backbone on degradation rate, condensation of both plasmid DNA and siRNA, cellular uptake, intracellular disassembly, and consequent DNA transfection and RNA interference efficiency in vitro and in vivo were investigated. Limited nucleic acid complexation and cellular uptake but efficient intracellular release of nucleic acids were obtained with poly(spermine ketal ester) (PSKE), the most acid-degradable polyspermine. In contrast, poly(spermine ester) (PSE), which is not acid-degradable, demonstrated efficient nucleic acid complexation and cellular uptake but inefficient intracellular release of nucleic acids. The highest in vitro DNA transfection was obtained by the random co-polymer of PSKE and PSE at an equal ratio (PSKE-PSE), attributed to its balanced DNA complexation and acid-responsive release efficiency, while efficient siRNA unpackaging by PSKE resulted in the highest gene silencing efficiency. Preliminary in vivo studies demonstrated that the highest DNA transfection was obtained by using PSE, while both PSKE and PSE silenced GFP expression at the similar level. In conclusion, dual mode-degradable polyspermine is a non-cytotoxic nonviral gene carrier, and its acid-degradability can be molecularly tuned for differentially controlled transfection and gene silencing in vitro and in vivo.

Keywords: Polyspermine; Nonviral gene delivery; Biodegradability; DNA and siRNA delivery; In vitro; and; in vivo; transfection; Intracellular release


Tumor-homing photosensitizer-conjugated glycol chitosan nanoparticles for synchronous photodynamic imaging and therapy based on cellular on/off system by So Jin Lee; Heebeom Koo; Dong-Eun Lee; Solki Min; Seulki Lee; Xiaoyuan Chen; Yongseok Choi; James F. Leary; Kinam Park; Seo Young Jeong; Ick Chan Kwon; Kwangmeyung Kim; Kuiwon Choi (pp. 4021-4029).
Herein, we developed the photosensitizer, protoporphyrin IX (PpIX), conjugated glycol chitosan (GC) nanoparticles (PpIX–GC–NPs) as tumor-homing drug carriers with cellular on/off system for photodynamic imaging and therapy, simultaneously. In order to prepare PpIX–GC–NPs, hydrophobic PpIXs were chemically conjugated to GC polymer and the amphiphilic PpIX–GC conjugates formed a stable nanoparticle structure in aqueous condition, wherein conjugated PpIX molecules formed hydrophobic inner-cores and they were covered by the hydrophilic GC polymer shell. Based on the nanoparticle structure, PpIX–GC–NPs showed the self-quenching effect that is ‘off’ state with no fluorescence signal and phototoxicity with light exposure. It is due to the compact crystallized PpIX molecules in the nanoparticles as confirmed by dynamic light scattering and X-ray diffraction methods. However, after cellular uptake, compact nanoparticle structure gradually decreased to generate strong fluorescence signal and singlet oxygen generation when irradiated. Importantly, PpIX–GC–NPs-treated mice presented prolonged blood circulation, enhanced tumor targeting ability, and improved in vivo therapeutic efficiency in tumor-bearing mice, compared to that of free PpIX-treated mice. These results proved that this tumor-homing cellular ‘on/off’ nanoparticle system of PpIX–GC–NPs has a great potential for synchronous photodynamic imaging and therapy in cancer treatment.

Keywords: Photosensitizer; Nanoparticle; Photodynamic therapy; Drug delivery; Glycol chitosan; Cellular on-off system


Tumor-homing photosensitizer-conjugated glycol chitosan nanoparticles for synchronous photodynamic imaging and therapy based on cellular on/off system by So Jin Lee; Heebeom Koo; Dong-Eun Lee; Solki Min; Seulki Lee; Xiaoyuan Chen; Yongseok Choi; James F. Leary; Kinam Park; Seo Young Jeong; Ick Chan Kwon; Kwangmeyung Kim; Kuiwon Choi (pp. 4021-4029).
Herein, we developed the photosensitizer, protoporphyrin IX (PpIX), conjugated glycol chitosan (GC) nanoparticles (PpIX–GC–NPs) as tumor-homing drug carriers with cellular on/off system for photodynamic imaging and therapy, simultaneously. In order to prepare PpIX–GC–NPs, hydrophobic PpIXs were chemically conjugated to GC polymer and the amphiphilic PpIX–GC conjugates formed a stable nanoparticle structure in aqueous condition, wherein conjugated PpIX molecules formed hydrophobic inner-cores and they were covered by the hydrophilic GC polymer shell. Based on the nanoparticle structure, PpIX–GC–NPs showed the self-quenching effect that is ‘off’ state with no fluorescence signal and phototoxicity with light exposure. It is due to the compact crystallized PpIX molecules in the nanoparticles as confirmed by dynamic light scattering and X-ray diffraction methods. However, after cellular uptake, compact nanoparticle structure gradually decreased to generate strong fluorescence signal and singlet oxygen generation when irradiated. Importantly, PpIX–GC–NPs-treated mice presented prolonged blood circulation, enhanced tumor targeting ability, and improved in vivo therapeutic efficiency in tumor-bearing mice, compared to that of free PpIX-treated mice. These results proved that this tumor-homing cellular ‘on/off’ nanoparticle system of PpIX–GC–NPs has a great potential for synchronous photodynamic imaging and therapy in cancer treatment.

Keywords: Photosensitizer; Nanoparticle; Photodynamic therapy; Drug delivery; Glycol chitosan; Cellular on-off system


The inhibition of death receptor mediated apoptosis through lysosome stabilization following internalization of carboxyfullerene nanoparticles by Wei Li; Lina Zhao; Taotao Wei; Yuliang Zhao; Chunying Chen (pp. 4030-4041).
Cells undergo apoptosis through two major pathways, the extrinsic pathway (death receptor pathway) and the intrinsic pathway (the mitochondrial pathway). It is well known that nanomaterials of water- soluble fullerene derivatives are potent antioxidants and help to prevent the overproduction of mitochondrial reactive oxygen species (ROS). However, whether their interaction with cells via the death receptor pathway is direct or indirect remains poorly understood. Here, we show that a bis-adduct malonic acid derivative of fullerene, C60(C(COOH)2)2, inhibits tumor necrosis factor alpha-initiated cellular apoptosis via stabilizing lysosomes. Data presented here demonstrate that nano-sized aggregates of this water-soluble fullerene derivative are endocytosed into cells and enriched in the lysosomes. During the internalization of C60(C(COOH)2)2, the expression of Hsp 70 is significantly upregulated, promoting cell survival by inhibiting the permeabilization of lysosomal membranes. In addition, the acidic environment inside lysosomes has a marked but temporary effect on the size distribution of fullerenic nanoparticles, and may disperse the aggregated C60(C(COOH)2)2 nanoparticles into single molecules or smaller aggregates. These single molecules or smaller aggregates may insert into the lysosomal membranes, further stabilizing them and decreasing the release of cathepsins from lysosomes, leading to the inhibition of tumor necrosis factor-induced apoptosis. C60(C(COOH)2)2 nanoparticles can thus protect cells by stabilizing lysosomal membranes via both upregulated expression of Hsp 70 and by their interactions with lysosomal membranes.

Keywords: Fullerene derivative; Endocytosis; Lysosomal membrane; Apoptosis; Hsp 70


The inhibition of death receptor mediated apoptosis through lysosome stabilization following internalization of carboxyfullerene nanoparticles by Wei Li; Lina Zhao; Taotao Wei; Yuliang Zhao; Chunying Chen (pp. 4030-4041).
Cells undergo apoptosis through two major pathways, the extrinsic pathway (death receptor pathway) and the intrinsic pathway (the mitochondrial pathway). It is well known that nanomaterials of water- soluble fullerene derivatives are potent antioxidants and help to prevent the overproduction of mitochondrial reactive oxygen species (ROS). However, whether their interaction with cells via the death receptor pathway is direct or indirect remains poorly understood. Here, we show that a bis-adduct malonic acid derivative of fullerene, C60(C(COOH)2)2, inhibits tumor necrosis factor alpha-initiated cellular apoptosis via stabilizing lysosomes. Data presented here demonstrate that nano-sized aggregates of this water-soluble fullerene derivative are endocytosed into cells and enriched in the lysosomes. During the internalization of C60(C(COOH)2)2, the expression of Hsp 70 is significantly upregulated, promoting cell survival by inhibiting the permeabilization of lysosomal membranes. In addition, the acidic environment inside lysosomes has a marked but temporary effect on the size distribution of fullerenic nanoparticles, and may disperse the aggregated C60(C(COOH)2)2 nanoparticles into single molecules or smaller aggregates. These single molecules or smaller aggregates may insert into the lysosomal membranes, further stabilizing them and decreasing the release of cathepsins from lysosomes, leading to the inhibition of tumor necrosis factor-induced apoptosis. C60(C(COOH)2)2 nanoparticles can thus protect cells by stabilizing lysosomal membranes via both upregulated expression of Hsp 70 and by their interactions with lysosomal membranes.

Keywords: Fullerene derivative; Endocytosis; Lysosomal membrane; Apoptosis; Hsp 70


Multivalent artificial opsonin for the recognition and phagocytosis of Gram-positive bacteria by human phagocytes by Kristy N. Katzenmeyer; James D. Bryers (pp. 4042-4051).
Hospital-acquired infections (HAIs) remain a leading cause of death in the United States. Unfortunately, treatment of HAIs is complicated by the emergence of antibiotic-resistant bacterial strains. In an effort to enhance the body’s natural immune response to infection, we have developed an artificial opsonin to promote the recognition, phagocytosis, and destruction of pathogenic bacteria by human phagocytes. The artificial opsonin is constructed from multivalent conjugates of poly(l-lysine)- graft-poly(ethylene glycol) with vancomycin and human IgG-Fc. Our approach utilizes vancomycin’s inherent ability to bind tod-Ala-d-Ala terminated peptides present in the cell wall of Gram-positive bacteria. Here, we show that conjugation of vancomycin to PLL- g-PEG prevents its action as an antibiotic and allows vancomycin to function solely as a recognition molecule. Human IgG-Fc antibody fragment serves as a phagocyte recognition molecule and is recognized by the Fcγ cell surface receptors expressed on professional human phagocytes. Using flow cytometry, we found that a polysaccharide-encapsulated, methicillin-resistant strain of Staphylococcus epidermidis is efficiently recognized by the artificial opsonin (nearly 100% of cells were opsonized) and that opsonin binding is specific since it can be inhibited by the soluble cell wall peptide analog acetyl-Lys-d-Ala-d-Ala. Opsonization of S. epidermidis resulted in an approximate 2-fold increase in phagocytosis by a human neutrophil cell line. Notably, Enterococcus faecalis VanB, a bacterial strain with inducible vancomycin resistance, was used to show that the artificial opsonin does not unintentionally induce antibiotic resistance mechanisms.

Keywords: Bacteria; Flow cytometry; Immune response; Immunostimulation; Infection; Neutrophil


Multivalent artificial opsonin for the recognition and phagocytosis of Gram-positive bacteria by human phagocytes by Kristy N. Katzenmeyer; James D. Bryers (pp. 4042-4051).
Hospital-acquired infections (HAIs) remain a leading cause of death in the United States. Unfortunately, treatment of HAIs is complicated by the emergence of antibiotic-resistant bacterial strains. In an effort to enhance the body’s natural immune response to infection, we have developed an artificial opsonin to promote the recognition, phagocytosis, and destruction of pathogenic bacteria by human phagocytes. The artificial opsonin is constructed from multivalent conjugates of poly(l-lysine)- graft-poly(ethylene glycol) with vancomycin and human IgG-Fc. Our approach utilizes vancomycin’s inherent ability to bind tod-Ala-d-Ala terminated peptides present in the cell wall of Gram-positive bacteria. Here, we show that conjugation of vancomycin to PLL- g-PEG prevents its action as an antibiotic and allows vancomycin to function solely as a recognition molecule. Human IgG-Fc antibody fragment serves as a phagocyte recognition molecule and is recognized by the Fcγ cell surface receptors expressed on professional human phagocytes. Using flow cytometry, we found that a polysaccharide-encapsulated, methicillin-resistant strain of Staphylococcus epidermidis is efficiently recognized by the artificial opsonin (nearly 100% of cells were opsonized) and that opsonin binding is specific since it can be inhibited by the soluble cell wall peptide analog acetyl-Lys-d-Ala-d-Ala. Opsonization of S. epidermidis resulted in an approximate 2-fold increase in phagocytosis by a human neutrophil cell line. Notably, Enterococcus faecalis VanB, a bacterial strain with inducible vancomycin resistance, was used to show that the artificial opsonin does not unintentionally induce antibiotic resistance mechanisms.

Keywords: Bacteria; Flow cytometry; Immune response; Immunostimulation; Infection; Neutrophil


In vivo evidence of oral vaccination with PLGA nanoparticles containing the immunostimulant monophosphoryl lipid A by Federica Sarti; Glen Perera; Fabian Hintzen; Katerina Kotti; Vassilis Karageorgiou; Olga Kammona; Costas Kiparissides; Andreas Bernkop-Schnürch (pp. 4052-4057).
Although oral vaccination has numerous advantages over the commonly used parenteral route, degradation of vaccine and its low uptake in the lymphoid tissue of the gastrointestinal (GI) tract still impede their development. In this study, the model antigen ovalbumin (OVA) and the immunostimulant monophosphoryl lipid A (MPLA) were incorporated in polymeric nanoparticles based on poly(d,l-lactide-co-glycolide) (PLGA). These polymeric carriers were orally administered to BALB/c mice (Bagg albino, inbred strain of mouse) and the resulting time-dependent systemic and mucosal immune responses towards OVA were assessed by measuring the OVA-specific IgG and IgA titers using an enzyme-linked immunosorbent assay (ELISA). PLGA nanoparticles were spherical in shape, around 320 nm in size, negatively charged (around −20 mV) and had an OVA and MPLA payload of 9.6% and 0.86%, respectively. A single immunization with formulation containing (OVA + MPLA) incorporated in PLGA nanoparticles induced a stronger IgG immune response than that induced by OVA in PBS solution or OVA incorporated into PLGA nanoparticles. Moreover, significantly higher IgA titers were generated by administration of (OVA + MPLA)/PLGA nanoparticles compared to IgA stimulated by control formulations, proving the capability of inducing a mucosal immunity. These findings demonstrate that co-delivery of OVA and MPLA in PLGA nanoparticles promotes both systemic and mucosal immune responses and represents therefore a suitable strategy for oral vaccination.

Keywords: Ovalbumin; Mucosa; Poly(; dl; -lactide-co-glycolide) nanoparticles; Vaccination; Oral delivery; Immune response


In vivo evidence of oral vaccination with PLGA nanoparticles containing the immunostimulant monophosphoryl lipid A by Federica Sarti; Glen Perera; Fabian Hintzen; Katerina Kotti; Vassilis Karageorgiou; Olga Kammona; Costas Kiparissides; Andreas Bernkop-Schnürch (pp. 4052-4057).
Although oral vaccination has numerous advantages over the commonly used parenteral route, degradation of vaccine and its low uptake in the lymphoid tissue of the gastrointestinal (GI) tract still impede their development. In this study, the model antigen ovalbumin (OVA) and the immunostimulant monophosphoryl lipid A (MPLA) were incorporated in polymeric nanoparticles based on poly(d,l-lactide-co-glycolide) (PLGA). These polymeric carriers were orally administered to BALB/c mice (Bagg albino, inbred strain of mouse) and the resulting time-dependent systemic and mucosal immune responses towards OVA were assessed by measuring the OVA-specific IgG and IgA titers using an enzyme-linked immunosorbent assay (ELISA). PLGA nanoparticles were spherical in shape, around 320 nm in size, negatively charged (around −20 mV) and had an OVA and MPLA payload of 9.6% and 0.86%, respectively. A single immunization with formulation containing (OVA + MPLA) incorporated in PLGA nanoparticles induced a stronger IgG immune response than that induced by OVA in PBS solution or OVA incorporated into PLGA nanoparticles. Moreover, significantly higher IgA titers were generated by administration of (OVA + MPLA)/PLGA nanoparticles compared to IgA stimulated by control formulations, proving the capability of inducing a mucosal immunity. These findings demonstrate that co-delivery of OVA and MPLA in PLGA nanoparticles promotes both systemic and mucosal immune responses and represents therefore a suitable strategy for oral vaccination.

Keywords: Ovalbumin; Mucosa; Poly(; dl; -lactide-co-glycolide) nanoparticles; Vaccination; Oral delivery; Immune response


Formulation of Docetaxel by folic acid-conjugatedd-α-tocopheryl polyethylene glycol succinate 2000 (Vitamin E TPGS2k) micelles for targeted and synergistic chemotherapy by Yu Mi; Yutao Liu; Si-Shen Feng (pp. 4058-4066).
Although high efficacy has been showed, Paclitaxel and Docetaxel cause serious side effects due to the adjuvant used in their clinical formulation Taxol® and Taxotere®. We developed a micelle system with a newly synthesized TPGS2k polymer, which shows lower CMC of 0.0219 mg/ml compared with 0.2 mg/ml for traditional micelles with TPGS involved, to achieve sustained and controlled drug delivery with Docetaxel used as a model anti-cancer drug. The TPGS2k micelles were further conjugated to folic acid (FA) for targeted drug delivery. The Docetaxel-loaded TPGS2k micelles with and without FA conjugation were found of desired size and size distribution, high drug encapsulation efficiency and favorable drug release. In vitro studies using MCF-7 cancer cells demonstrated significantly the higher cellular uptake of the formulated drug for TPGS2k micelle formulation than that for Taxotere®. The targeting effects for the FA conjugated TPGS2k micelles are also demonstrated. The IC50 value, which is the drug concentration needed for 50% cell viability in the designated time period, is 103.4, 1.280 and 0.1480 μg/ml for MCF-7 cancer cells after 24, 48, and 72 h treatment respectively, which is greatly decreased to be 0.526, 0.251 and 0.233 μg/ml, i.e. a 99.5%, 80.4% decrease and 57.5% increase for the TPGS2k micelle formulation, and further decreased to be 0.1780, 0.1520 and 0.1140 μg/ml, i.e. a 99.8%, 88.1% and 23.0% decrease for the folic acid conjugated micelles, respectively. A synergistic effect between TPGS2k and Docetaxel is also achieved. The present work represents a new concept in the design of drug delivery systems – the carrier materials of the drug delivery system can also have therapeutic effects, which either modulate the side effects of, or promote a synergistic interaction with the formulated drug.

Keywords: Biodegradable polymers; Cancer nanotechnology; Chemotherapeutic engineering; Drug formulation; Nanomedicine; Pharmaceutical nanotechnology


Formulation of Docetaxel by folic acid-conjugatedd-α-tocopheryl polyethylene glycol succinate 2000 (Vitamin E TPGS2k) micelles for targeted and synergistic chemotherapy by Yu Mi; Yutao Liu; Si-Shen Feng (pp. 4058-4066).
Although high efficacy has been showed, Paclitaxel and Docetaxel cause serious side effects due to the adjuvant used in their clinical formulation Taxol® and Taxotere®. We developed a micelle system with a newly synthesized TPGS2k polymer, which shows lower CMC of 0.0219 mg/ml compared with 0.2 mg/ml for traditional micelles with TPGS involved, to achieve sustained and controlled drug delivery with Docetaxel used as a model anti-cancer drug. The TPGS2k micelles were further conjugated to folic acid (FA) for targeted drug delivery. The Docetaxel-loaded TPGS2k micelles with and without FA conjugation were found of desired size and size distribution, high drug encapsulation efficiency and favorable drug release. In vitro studies using MCF-7 cancer cells demonstrated significantly the higher cellular uptake of the formulated drug for TPGS2k micelle formulation than that for Taxotere®. The targeting effects for the FA conjugated TPGS2k micelles are also demonstrated. The IC50 value, which is the drug concentration needed for 50% cell viability in the designated time period, is 103.4, 1.280 and 0.1480 μg/ml for MCF-7 cancer cells after 24, 48, and 72 h treatment respectively, which is greatly decreased to be 0.526, 0.251 and 0.233 μg/ml, i.e. a 99.5%, 80.4% decrease and 57.5% increase for the TPGS2k micelle formulation, and further decreased to be 0.1780, 0.1520 and 0.1140 μg/ml, i.e. a 99.8%, 88.1% and 23.0% decrease for the folic acid conjugated micelles, respectively. A synergistic effect between TPGS2k and Docetaxel is also achieved. The present work represents a new concept in the design of drug delivery systems – the carrier materials of the drug delivery system can also have therapeutic effects, which either modulate the side effects of, or promote a synergistic interaction with the formulated drug.

Keywords: Biodegradable polymers; Cancer nanotechnology; Chemotherapeutic engineering; Drug formulation; Nanomedicine; Pharmaceutical nanotechnology

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