Biomaterials (v.32, #26)

The impact of hyperglycemia and the presence of encapsulated islets on oxygenation within a bioartificial pancreas in the presence of mesenchymal stem cells in a diabetic Wistar rat model by Sophie Vériter; Najima Aouassar; Pierre-Yves Adnet; Marie-Sophie Paridaens; Charlotte Stuckman; Bénédicte Jordan; Oussama Karroum; Bernard Gallez; Pierre Gianello; Denis Dufrane (5945-5956).
This study investigates the potential of bone marrow (BM-MSCs) versus adipose mesenchymal stem cells (AMSCs) to potentiate the oxygenation of encapsulated islets in a subcutaneous bioartificial pancreas. Oxygen pressures (inside subcutaneous implants) were followed in vivo (by electronic paramagnetic resonance) in non-diabetic/diabetic rats transplanted with encapsulated porcine islets or empty implants up to 4 weeks post-transplantation. After graft explantation, neoangiogenesis surrounding the implants was assessed by histomorphometry. Angiogenic properties of BM-MSCs and AMSCs were first assessed in vitro by incubation of the cells in hypoxia chambers, under normoxic/hypoxic and hypo-/hyperglycemic conditions, followed by quantification of vascular endothelial growth factor (VEGF) release. Second, the in vivo aspect was studied by subcutaneous transplantation of encapsulated BM-MSCs and AMSCs in diabetic rats and assessment of the cells’ angiogenic properties as described above. Diabetic state and islet encapsulation induced a significant decrease of oxygenation of the subcutaneous implant and an increased number of cells expressing VEGF. AMSCs demonstrated a significantly higher VEGF secretion than BM-MSCs in vitro. In vivo, AMSCs improved the implant’s oxygenation and vascularization. Diabetes and islet encapsulation significantly reduced the oxygenation of a subcutaneous bioartificial pancreas. AMSCs can improve oxygenation by VEGF release in hypoxia and hyperglycemia states.
Keywords: Islet; Transplantation; Adipose tissue engineering; Stem cells; Oxygenation; Diabetes;

The hemocompatibility of a nitric oxide generating polymer that catalyzes S-nitrosothiol decomposition in an extracorporeal circulation model by Terry C. Major; David O. Brant; Charles P. Burney; Kagya A. Amoako; Gail M. Annich; Mark E. Meyerhoff; Hitesh Handa; Robert H. Bartlett (5957-5969).
Nitric oxide (NO) generating (NOGen) materials have been shown previously to create localized increases in NO concentration by the catalytic decomposition of blood S-nitrosothiols (RSNO) via copper (Cu)-containing polymer coatings and may improve extracorporeal circulation (ECC) hemocompatibility. In this work, a NOGen polymeric coating composed of a Cuo-nanoparticle (80 nm)-containing hydrophilic polyurethane (SP-60D-60) combined with the intravenous infusion of an RSNO, S- nitroso-N-acetylpenicillamine (SNAP), is evaluated in a 4 h rabbit thrombogenicity model and the anti-thrombotic mechanism is investigated. Polymer films containing 10 wt.% Cuo-nanoparticles coated on the inner walls of ECC circuits are employed concomitantly with systemic SNAP administration (0.1182 μmol/kg/min) to yield significantly reduced ECC thrombus formation compared to polymer control + systemic SNAP or 10 wt.% Cu NOGen + systemic saline after 4 h blood exposure (0.4 ± 0.2 NOGen/SNAP vs 4.9 ± 0.5 control/SNAP or 3.2 ± 0.2 pixels/cm2 NOGen/saline). Platelet count (3.9 ± 0.7 NOGen/SNAP vs 1.8 ± 0.1 control/SNAP or 3.0 ± 0.2 × 108/ml NOGen/saline) and plasma fibrinogen levels were preserved after 4 h blood exposure with the NOGen/SNAP combination vs either the control/SNAP or the NOGen/saline groups. Platelet function as measured by aggregometry (51 ± 9 NOGen/SNAP vs 49 ± 3% NOGen/saline) significantly decreased in both the NOGen/SNAP and NOGen/saline groups while platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry was not decreased after 4 h on ECC to ex vivo collagen stimulation (26 ± 2 NOGen/SNAP vs 29 ± 1 MFI baseline). Western blotting showed that fibrinogen activation as assessed by Aγ dimer expression was reduced after 4 h on ECC with NOGen/SNAP (68 ± 7 vs 83 ± 3% control/SNAP). These results suggest that the NOGen polymer coating combined with SNAP infusion preserves platelets in blood exposure to ECCs by attenuating activated fibrinogen and preventing platelet aggregation. These NO-mediated platelet changes were shown to improve thromboresistance of the NOGen polymer-coated ECCs when adequate levels of RSNOs are present.
Keywords: Nitric oxide; Copper nanoparticles; Monocyte CD11b; Platelet P-selectin; Hemocompatible polymer coating; S-Nitroso acetylpenicillamine (SNAP);

Determinants of the thrombogenic potential of multiwalled carbon nanotubes by Andrew R. Burke; Ravi N. Singh; David L. Carroll; John D. Owen; Nancy D. Kock; Ralph D’Agostino; Frank M. Torti; Suzy V. Torti (5970-5978).
Multiwalled carbon nanotubes (MWCNTs) are cylindrical tubes of graphitic carbon with unique physical and electrical properties. MWCNTs are being explored for a variety of diagnostic and therapeutic applications. Successful biomedical application of MWCNTs will require compatibility with normal circulatory components, including constituents of the hemostatic cascades. In this manuscript, we compare the thrombotic activity of MWCNTs in vitro and in vivo. We also assess the influence of functionalization of MWCNTs on thrombotic activity. In vitro, MWCNT activate the intrinsic pathway of coagulation as measured by activated partial thromboplastin time (aPTT) assays. Functionalization by amidation or carboxylation enhances this procoagulant activity. Mechanistic studies demonstrate that MWCNTs enhance propagation of the intrinsic pathway via a non-classical mechanism strongly dependent on factor IX. MWCNTs preferentially associate with factor IXa and may provide a platform that enhances its enzymatic activity. In addition to their effects on the coagulation cascade, MWCNTs activate platelets in vitro, with amidated MWCNTs exhibiting greater platelet activation than carboxylated or pristine MWCNTs. However, contrasting trends are obtained in vivo, where functionalization tends to diminish rather than enhance procoagulant activity. Thus, following systemic injection of MWCNTs in mice, pristine MWCNTs decreased platelet counts, increased vWF, and increased D-dimers. In contrast, carboxylated MWCNTS exhibited little procoagulant tendency in vivo, eliciting only a mild and transient decrease in platelets. Amidated MWCNTs elicited no statistically significant change in platelet count. Further, neither carboxylated nor amidated MWCNTs increased vWF or D-dimers in mouse plasma. We conclude that the procoagulant tendencies of MWCNTs observed in vitro are not necessarily recapitulated in vivo. Further, functionalization can markedly attenuate the procoagulant activity of MWCNTs in vivo. This work will inform the rational development of biocompatible MWCNTs for systemic delivery.
Keywords: Blood; Blood compatibility; Clotting; Nanoparticle; Platelet activation; Thrombosis;

The influence of substrate creep on mesenchymal stem cell behaviour and phenotype by Andrew. R. Cameron; Jessica. E. Frith; Justin. J. Cooper-White (5979-5993).
Human mesenchymal stem cells (hMSCs) are capable of probing and responding to the mechanical properties of their substrate. Although most biological and synthetic matrices are viscoelastic materials, previous studies have primarily focused upon substrate compressive modulus (rigidity), neglecting the relative contributions that the storage (elastic) and loss (viscous) moduli make to the summed compressive modulus. In this study we aimed to isolate and identify the effects of the viscous component of a substrate on hMSC behaviour. Using a polyacrlyamide gel system with constant compressive modulus and varying loss modulus we determined that changes to substrate loss modulus substantially affected hMSC morphology, proliferation and differentiation potential. In addition, we showed that the effect of substrate loss modulus on hMSC behaviour is due to a reduction in both passive and actively generated isometric cytoskeletal tension caused by the inherent creep of substrates with a high loss modulus. These findings highlight substrate creep, or more explicitly substrate loss modulus, as an important mechanical property of a biomaterial system that can be tailored to encourage the growth and differentiation of specific cell types.
Keywords: Mesenchymal stem cell; Mechanical properties; Creep; Viscoelasticity; Tension;

Reprogramming induced pluripotent stem cells in the absence of c-Myc for differentiation into hepatocyte-like cells by Hsin-Yang Li; Yueh Chien; Yi-Jen Chen; Szu-Fu Chen; Yuh-Lih Chang; Chih-Hung Chiang; Shaw-Yeu Jeng; Chia-Ming Chang; Mong-Lien Wang; Liang-Kung Chen; Shuen-Iu Hung; Teh-Ia Huo; Shou-Dong Lee; Shih-Hwa Chiou (5994-6005).
Induced pluripotent stem cells (iPSCs) with four reprogramming factors (Oct-4/Sox2/Klf-4/c-Myc) have been shown to differentiate into hepatic lineages. However, it was unclear whether obviation of the c-Myc oncogene in iPSCs affected hepatic differentiation or inhibited in vivo tumor formation. In this study, we demonstrated that iPSCs without c-Myc had the capacity to differentiate into hepatocyte-like cells (iPSC-Heps) with biological functions. As detected using planar-radionuclide imaging and Hoechst labeling assays, these iPSCs and iPSC-Heps tended to mobilize to the injured liver area in thioacetamide (TAA)-treated mice. Intravenous transplantation of both iPSCs and iPSC-Heps but not mouse embryonic fibroblasts (MEFs) reduced the hepatic necrotic area, improved liver functions, and rescued TAA-treated mice from lethal acute hepatic failure (AHF). In addition, microarray-based bioinformatics and quantitative RT-PCR showed high expression of antioxidant genes in iPSCs and iPSC-Heps compared to MEFs. In vivo and in vitro studies of NAC pretreatment confirmed that iPSCs and iPSC-Heps potentially suppressed ROS production and activated antioxidant enzymes in TAA-injured livers. Six months after transplantation in TAA-treated mice, tumor formation was not seen in non-c-Myc iPSC grafts. Therefore, reprogramming adult somatic cells without c-Myc may prevent oxidative stress-induced damage and provide a safer alternative for hepatic regeneration in AHF.
Keywords: Acute hepatic failure; Induced pluripotent stem cell; Reactive oxygen species; Hepatocytes;

Large-scale production of murine embryonic stem cell-derived osteoblasts and chondrocytes on microcarriers in serum-free media by Roz Alfred; Jaymi T. Taiani; Roman J. Krawetz; Akihiro Yamashita; Derrick E. Rancourt; Michael S. Kallos (6006-6016).
The generation of tissue-engineered constructs from stem cells for the treatment of musculoskeletal diseases may have immense impact in regenerative medicine, but there are difficulties associated with stem cell culture and differentiation, including the use of serum. Here we present serum-free protocols for the successful production of murine embryonic stem cell (mESC) derived osteoblasts and chondrocytes on CultiSpher S macroporous microcarriers in stirred suspension bioreactors. Various inoculum forms and agitation rates were investigated. Produced osteogenic cells were implanted ectopically into SCID mice and orthotopically into a murine burr-hole fracture model. Osterix, osteocalcin and collagen type I were upregulated in osteogenic cultures, while aggrecan and collagen type II were upregulated in chondrogenic cultures. Histological analysis using alizarin red S, von Kossa and alcian blue staining confirmed the presence of osteoblasts and chondrocytes, respectively in cultured microcarriers and excised tissue. Finally, implantation of derived cells into a mouse fracture model revealed cellular integration without any tumor formation. Overall, microcarriers may provide a supportive scaffold for ESC expansion and differentiation in a serum-free bioprocess for in vivo implantation. These findings lay the groundwork for the development of clinical therapies for musculoskeletal injuries and diseases using hESCs and iPS cells.
Keywords: Osteoblasts; Chondrocytes; Stirred suspension bioreactors; Microcarriers; Serum-free cultivation;

Engineered implants derived from neonatal rabbit chondrocytes and collagen type I hydrogel, were loaded in dialyzer pockets and implanted in muscle and articular cavity of rabbits to simulate different stress and tissue fluid micro-environments. After 4 and 12 weeks, the expressions of main histocompatibility complex (MHC) molecules as well as the mixed lymphocyte chondrocytes reactions (MLChR) levels of the seeded cells were detected. The results indicated that with stress and synovial fluid microenvironment, the formation of chondroid tissue was prominently promoted in articular cavity. It gave the seeded chondrocytes lower and gradually decreasing levels of allogeneic lymphocytes activation, however, with the higher cell mortality, the MHC molecules expression, especially MHC-I were up-regulated obviously in early stage. These results are very different to those seen in muscle and prove that stress and tissue fluid micro-environments can greatly impact the differentiation and immunological properties of the engineered cartilage. From the perspective of avoiding severe rejection, to promote the formation of the matrix as fast and select scaffold with higher “isolation” ability may be meaningful. Furthermore, the suitably treated dialyzer pockets model can be used for the study of the differentiation and immunological properties of the tissue engineered cartilage.
Keywords: Cartilage tissue engineering; Immunological properties; Chondrocytes; MHC; Mixed lymphocytes chondrocytes reaction;

Biological and mechanical implications of PEGylating proteins into hydrogel biomaterials by Maya Gonen-Wadmany; Revital Goldshmid; Dror Seliktar (6025-6033).
Protein PEGylation has been successfully applied in pharmaceuticals and more recently in biomaterials development for making bioactive and structurally versatile hydrogels. Despite many advantages in this regard, PEGylation of proteins is also known to alter biological activity and modify biophysical characteristics in ways that may be detrimental to cells. The aim of this study was to evaluate the relative loss of biological compatibility associated with PEGylating a fibrinogen precursor into a hydrogel scaffold, in comparison to thrombin cross-linked fibrin hydrogels. Specifically, we investigated the consequences of conjugating fibrinogen with linear polyethtylene glycol (PEG) polymer chains (10 kDa) on the ability to cultivate neonatal human foreskin fibroblasts (HFFs) in 3-D. For this purpose, thrombin cross-linked fibrin (TCL-Fib) and PEGylated fibrinogen (PEG-Fib) gels were prepared with HFFs and cultured for up to seven days. The benchmark biological compatibility test was based on a combined assessment of cellular morphology, proliferation, actin expression, and matrix metalloproteinase (MMP) expression in the 3-D culture systems. The results showed correlations between modulus and proteolytic biodegradation in both materials, but no correlation between the mechanical properties and the ability of HFFs to remodel the microenvironment. A slight reduction of actin, MMPs, and spindled morphology of the cells in the PEG-Fib hydrogels indicated that the PEGylation process altered the biological compatibility of the fibrin. Nevertheless, the overall benchmark performance of the two materials demonstrated that PEGylated fibrinogen hydrogels still retains much to the inherent biofunctionality of the fibrin precursor when used as a scaffold for 3-D cell cultivation.
Keywords: Fibrinogen; Polymer; Scaffold; Hydrogel; Fibroblast;

Biodegradation and in vivo biocompatibility of a degradable, polar/hydrophobic/ionic polyurethane for tissue engineering applications by Joanne E. McBane; Soroor Sharifpoor; Kuihua Cai; Rosalind S. Labow; J. Paul Santerre (6034-6044).
A degradable, polar/hydrophobic/ionic polyurethane (D-PHI) scaffold was optimized in in vitro studies to yield mechanical properties appropriate to replicate vascular graft tissue while eliciting a more wound-healing phenotype macrophage when compared to established materials. The objectives of this study were to characterize the biodegradation (in vitro and in vivo) and assess the in vivo biocompatibility of D-PHI, comparing it to a well-established, commercially-available scaffold biomaterial, polylactic glycolic acid (PLGA), recognized as being degradable, non-cytotoxic, and showing good biocompatibility. PLGA and D-PHI were formed into 6 mm diameter disk-shaped scaffolds (2 mm thick) of similar porosity (∼82%) and implanted subcutaneously in rats. Both PLGA and D-PHI scaffolds were well-tolerated at the 7 d time point in vivo. In vitro D-PHI scaffolds degraded slowly (only 12 wt% in PBS in vitro after 120 d at 37 °C). In vivo, D-PHI scaffolds degraded at a more controlled rate (7 wt% loss over the acute 7 d implant phase and subsequently a linear profile of degradation leading to a 21 wt% mass loss by 100 d (chronic period)) than PLGA scaffolds which showed an initial more rapid degradation (14 wt% over 7 d), followed by minimal change between 7 and 30 d, and then a very rapid breakdown of the scaffold over the next 60 d. Histological examination of D-PHI scaffolds showed tissue ingrowth into the pores increased with time whereas PLGA scaffolds excluded cells/tissue from its porous structure as it degraded. The results of this study suggest that D-PHI has promising qualities for use as an elastomeric scaffold material for soft TE applications yielding well integrated tissue within the scaffold and a controlled rate of degradation stabilizing the form and shape of the implant.
Keywords: Polyurethane; Polylactic glycolic acid; Biodegradation; Biocompatibility; Rat subcutaneous implant; Porous scaffold;

The role of pore size on vascularization and tissue remodeling in PEG hydrogels by Yu-Chieh Chiu; Ming-Huei Cheng; Holger Engel; Shu-Wei Kao; Jeffery C. Larson; Shreya Gupta; Eric M. Brey (6045-6051).
Vascularization is influenced by the physical architecture of a biomaterial. The relationship between pore size and vascularization has been examined for hydrophobic polymer foams, but there has been little research on tissue response in porous hydrogels. The goal of this study was to examine the role of pore size on vessel invasion in porous poly(ethylene glycol) (PEG) hydrogels. Vascularized tissue ingrowth was examined using three-dimensional cell culture and rodent models. In culture, all porous gels supported vascular invasion with the rate increasing with pore size. Following subfascia implantation, porous gels rapidly absorbed wound fluid, which promoted tissue ingrowth even in the absence of exogenous growth factors. Pore size influenced neovascularization, within the scaffolds and also the overall tissue response. Cell and vessel invasion into gels with pores 25–50 μm in size was limited to the external surface, while gels with pores larger pores (50–100 and 100–150 μm) permitted mature vascularized tissue formation throughout the entire material volume. A thin layer of inflammatory tissue was present at all PEG-tissue interfaces, effectively reducing the area available for tissue growth. These results show that porous PEG hydrogels can support extensive vascularized tissue formation, but the nature of the response depends on the pore size.
Keywords: Porous; Poly (ethylene glycol); Vascularization; Hydrogels;

The use of nanoimprinted scaffolds as 3D culture models to facilitate spontaneous tumor cell migration and well-regulated spheroid formation by Yukie Yoshii; Atsuo Waki; Kaori Yoshida; Anna Kakezuka; Maki Kobayashi; Hideo Namiki; Yusei Kuroda; Yasushi Kiyono; Hiroshi Yoshii; Takako Furukawa; Tatsuya Asai; Hidehiko Okazawa; Juri G. Gelovani; Yasuhisa Fujibayashi (6052-6058).
Two-dimensional (2D) cell cultures are essential for drug development and tumor research. However, the limitations of 2D cultures are widely recognized, and a better technique is needed. Recent studies have indicated that a strong physical contact between cells and 2D substrates induces cellular characteristics that differ from those of tumors growing in vivo. 3D cell cultures using various substrates are then developing; nevertheless, conventional approaches have failed in maintenance of cellular proliferation and viability, uniformity, reproducibility, and/or simplicity of these assays. Here, we developed a 3D culture system with inorganic nanoscale scaffolding using nanoimprinting technology (nano-culture plates), which reproduced the characteristics of tumor cells growing in vivo. Diminished cell-to-substrate physical contact facilitated spontaneous tumor cell migration, intercellular adhesion, and multi-cellular 3D-spheroid formation while maintaining cellular proliferation and viability. The resulting multi-cellular spheroids formed hypoxic core regions similar to tumors growing in vivo. This technology allows creating uniform and highly-reproducible 3D cultures, which is easily applicable for microscopic and spectrophotometric assays, which can be used for high-throughput/high-content screening of anticancer drugs and should accelerate discovery of more effective anticancer therapies.
Keywords: Hypoxia; Multi-cellular spheroid; Nanoimprinting technology; Tumor cell migration; 3D cell culture;

Establishment of self-organization system in rapidly formed multicellular heterospheroids by Nobuhiko Kojima; Shoji Takeuchi; Yasuyuki Sakai (6059-6067).
Multicellular heterospheroids including two or more cell types have some tissue/organ properties and can be used in cell-to-cell interaction studies. However, the spheroid formation is difficult to control because the adhesion efficacy is different in each cell type. To solve this, we applied a rapid cell-to-cell adhesion method, avidin–biotin (AB) binding, to spheroid formation. Introduction of avidin or biotin molecules to the cell surfaces of Mile Sven 1 (MS1) cells promoted formation of spheroid in minutes. This method allowed the construction of heterospheroids having homogenous distributions of different cell types. Interestingly, cells showed self-organization and MS1 cells formed networks with Hep G2 cells. NIH3T3 cells also remodeled when mixed with Hep G2 cells. In contrast, a combination of MS1 and NIH3T3 cells failed to show pattern formation, indicating that self-organization was based on the composition of cell types. Actin polymerization not cell proliferation was the dominant factor in remodeling of heterospheroids in the first 24 h. We also demonstrated the self-organization of spheroids comprising three different cell types. The new technology to assemble cells is important not only to study cell-to-cell interaction but also to make three-dimensional complicated tissues.
Keywords: Avidin–biotin binding system; Heterospheroid; Self-organization; Cell migration; Remodeling;

Robust CNS regeneration after complete spinal cord transection using aligned poly-l-lactic acid microfibers by Andres Hurtado; Jared M. Cregg; Han B. Wang; Dane F. Wendell; Martin Oudega; Ryan J. Gilbert; John W. McDonald (6068-6079).
Following spinal cord injury, axons fail to regenerate without exogenous intervention. In this study we report that aligned microfiber-based grafts foster robust regeneration of vascularized CNS tissue. Film, random, and aligned microfiber-based conduits were grafted into a 3 mm thoracic rat spinal cord gap created by complete transection. Over the course of 4 weeks, microtopography presented by aligned or random poly-l-lactic acid microfibers facilitated infiltration of host tissue, and the initial 3 mm gap was closed by endogenous cell populations. This bulk tissue response was composed of regenerating axons accompanied by morphologically aligned astrocytes. Aligned fibers promoted long distance (2055 ± 150 μm), rostrocaudal axonal regeneration, significantly greater than random fiber (1162 ± 87 μm) and film (413 ± 199 μm) controls. Retrograde tracing indicated that regenerating axons originated from propriospinal neurons of the rostral spinal cord, and supraspinal neurons of the reticular formation, red nucleus, raphe and vestibular nuclei. Our findings outline a form of regeneration within the central nervous system that holds important implications for regeneration biology.
Keywords: Spinal cord injury; Axonal regeneration; Axon guidance; Polylactic acid; Electrospinning; Aligned microfibers;

The enhancement of mature vessel formation and cardiac function in infarcted hearts using dual growth factor delivery with self-assembling peptides by Ji Hyun Kim; Youngmee Jung; Sang-Heon Kim; Kyung Sun; Jaesoon Choi; Hee Chan Kim; Yongdoo Park; Soo Hyun Kim (6080-6088).
For successful treatment of myocardial infarction (MI), it is important to prevent cardiac fibrosis and maintain cardiac function by protecting cardiomyocytes and inducing angiogenesis. To establish functional and stable vessels, various growth factors, ones stimulating both endothelial cells (EC) and vascular smooth muscle cells (VSMC), are required. Self-assembling peptides form fibers (<10 nm) and provide 3-dimensional microenvironments that can recruit EC and VSMC to promote vascularization and long-term delivery of growth factors. Here we demonstrate myocardial protection of infarcted heart using dual growth factor delivery with self-assembling peptides. After coronary artery ligation in rats, growth factors (PDGF-BB and FGF-2) with self-assembling peptides were injected. There were 6 rats in each group. Hearts were harvested at 4 and 8 weeks for functional and histological analysis. Infarct size and cardiomyocyte apoptosis in dual growth factors along with self-assembling peptides group were dramatically reduced compared to sham. The capillary and arterial density of this group recovered with angiogenic synergism and cardiac functions had almost recovered. In conclusion, dual growth factors along with self-assembling peptides lead to myocardial protection, stable vessel formation, and improvement in cardiac function.
Keywords: Myocardial infarction; Self-assembling peptides; Dual growth factor delivery; Angiogenesis; Cardiac tissue engineering;

Pro-osteogenic trophic effects by PKA activation in human mesenchymal stromal cells by Joyce Doorn; Jeroen van de Peppel; Johannes P.T.M. van Leeuwen; Nathalie Groen; Clemens A. van Blitterswijk; Jan de Boer (6089-6098).
Human mesenchymal stromal cells (hMSCs) are able to differentiate into a wide variety of cell types, which makes them an interesting source for tissue engineering applications. On the other hand, these cells also secrete a broad panel of growth factors and cytokines that can exert trophic effects on surrounding tissues. In bone tissue engineering applications, the general assumption is that direct differentiation of hMSCs into osteoblasts accounts for newly observed bone formation in vivo. However, the secretion of bone-specific growth factors, but also pro-angiogenic factors, could also contribute to this process. We recently demonstrated that secretion of bone specific growth factors can be enhanced by treatment of hMSCs with the small molecule db-cAMP (cAMP) and here we investigate the biological activity of these secreted factors. We demonstrate that conditioned medium contains a variety of secreted growth factors, with differences between medium from basic-treated and cAMP-treated hMSCs. We show that conditioned medium from cAMP-treated hMSCs increases proliferation of various cell types and also induces osteogenic differentiation, whereas it has differential effects on migration. Microarray analysis on hMSCs exposed to conditioned medium confirmed upregulation of pathways involved in proliferation as well as osteogenic differentiation. Our data suggests that trophic factors secreted by hMSCs can be tuned for specific applications and that a good balance between differentiation on the one hand and secretion of bone trophic factors on the other, could potentially enhance bone formation for bone tissue engineering applications.
Keywords: hMSCs; Bone tissue engineering; Trophic effect; Insulin-like growth factor-1; Bone-morphogenetic protein-2;

Functional fibrils derived from the peptide TTR1-cycloRGDfK that target cell adhesion and spreading by Marie N. Bongiovanni; Denis B. Scanlon; Sally L. Gras (6099-6110).
Peptide self-assembly offers a route for the production of fibrous nanomaterials with advanced bioactive properties that promote specific cell interactions. In this study the peptide TTR1-cycloRGDfK was designed to form amyloid-like fibrils that display the functional cyclic RGDfK pentapeptide ligand to target mammalian cell surface αVβ3 integrin receptors. The TTR105–115 (or TTR1) sequence was used as the self-assembling domain. Once assembled, TTR1-cycloRGDfK fibrils display a characteristic cross-β core structure by X-ray fibre diffraction that was preserved following dehydration. Thin films of fibrils were characterised by infrared synchrotron mapping, scanning electron microscopy and atomic force microscopy. Cell adhesion and spreading were promoted on thin films of TTR1-cycloRGDfK fibrils via specific interactions with the cyclic RGDfK ligand. Low levels of non-specific interactions were also observed between cells and non-functionalised fibrils. TTR1-cycloRGDfK fibrils are an advance on bioactive fibrils previously designed to interact with a range of RGD binding integrins and our findings show that the assembly of amyloid-like fibrils based on the TTR1 sequence is robust and can be directed to form materials with specific properties.
Keywords: Self assembly; Bioactivity; RGD peptide; Nanotopography; Adhesion; Cell spreading;

An aligned engineered microvascular network is critical to the culture of thick or highly metabolic tissue in vitro due to the need for inlet and outlet sides for perfusion of the network. Contact guidance may be a way to achieve aligned networks, but the relationship between the alignment of endothelial sprouts and the alignment of extracellular matrix fibers has yet to be fully elucidated. The data presented here show that sprouts from human blood outgrowth endothelial cell spheroids align with fibrin fibrils, and that the extent to which the sprouts align depends upon the strength of the fibril alignment. This was true for both magnetically-aligned fibrin and fibrin aligned via cell-induced gel compaction, although magnetically-aligned fibrin was more effective over the same culture period. The data also demonstrate that longer sprouts are grown when the fibrils, and thus the sprouts, are more strongly aligned. The formation of aligned endothelial sprouts using these methods can be an essential step in the generation of aligned microvascular networks.
Keywords: Angiogenesis; Endothelial cell; Fibrin; Co-culture;

Extracellular matrix (ECM) comprises a rich meshwork of proteins and proteoglycans, which not only contains biological cues for cell behavior, but is also a reservoir for binding growth factors and controlling their release. Here we aimed to create a suitable bony microenvironment with cell-derived ECM and biodegradable β-tricalcium phosphate (β-TCP). More specifically, we investigated whether the ECM produced by bone marrow-derived mesenchymal stem cells (hBMSC) on a β-TCP scaffold can bind bone morphogenetic protein-2 (BMP-2) and control its release in a sustained manner, and further examined the effect of ECM and the BMP-2 released from ECM on cell behaviors. The ECM was obtained through culturing the hBMSC on a β-TCP porous scaffold and performing decellularization and sterilization. SEM, XPS, FTIR, and immunofluorescent staining results indicated the presence of ECM on the β-TCP and the amount of ECM increased with the incubation time. BMP-2 was loaded onto the β-TCP with and without ECM by immersing the scaffolds in the BMP-2 solution. The loading and release kinetics of the BMP-2 on the β-TCP/ECM were significantly slower than those on the β-TCP. The β-TCP/ECM exhibited a sustained release profile of the BMP-2, which was also affected by the amount of ECM. This is probably because the β-TCP/ECM has different binding mechanisms with BMP-2. The β-TCP/ECM promoted cell proliferation. Furthermore, the BMP-2-loaded β-TCP/ECM stimulated reorganization of the actin cytoskeleton, increased expression of alkaline phosphatase and calcium deposition by the cells compared to those without BMP-2 loading and the β-TCP with BMP-2 loading.
Keywords: ECM (extracellular matrix); BMP-2 (bone morphogenetic protein-2); Beta-tricalcium phosphate (β-TCP); Bone tissue engineering; Scaffold;

Three-dimensional porous silk tumor constructs in the approximation of in vivo osteosarcoma physiology by Pamela H.S. Tan; K.Z. Aung; S.L. Toh; James C.H. Goh; S.S. Nathan (6131-6137).
The lack of good preclinical models has hampered anticancer drug discovery. Standard preclinical protocols require the growth of cells in high throughput two-dimensional (2D) culture systems. However, such in vitro drug testing methods yield drug efficacy results that differ greatly from animal models. Conversely, it is much more difficult and expensive to use animal models for large-scale molecular biology research. It is conceivable that three-dimensional (3D) growth may be responsible for some of these changes. Porous silk sponges were fabricated through freeze drying and seeded with 143.98.2 osteosarcoma cells. Molecular profiles were obtained by carrying out real-time polymerase chain reaction for angiogenic growth factors and proliferation markers for osteosarcoma cells grown under 2D, 3D, and SCID mouse xenograft conditions. The angiogenic factor expression profiles for cells grown in 2D differed greatly from the 3D silk scaffold model (P < 0.05 for bFGF, HIF-1α, IL-8, and VEGF-A), whereas 3D tumor model profiles were found to be able to approximate that for the in vivo tumor better with no statistically different expression of HIF-1α and VEGF-A between the two. Immunohistochemistry staining for HIF-1α, VEGF-A, and VEGF receptor on osteosarcoma cells grown on the scaffolds validated the results obtained with the gene expression profiles. The results suggest that 3D tumor models could be used to bridge the gap between in vitro and in vivo tumor studies, and aid in the study of mechanisms activated during tumorigenesis for the development of novel targeted chemotherapy.
Keywords: Scaffold; Angiogenesis; Silk; Cell signalling; Cell culture;

Chemically amplified photoresist for high resolution autoradiography in targeted radiotherapy by Nadia Falzone; Roger Nathan; Sverre Myhra; Radka Chakalova; Thomas Altebaeumer; Katherine Vallis (6138-6144).
Evaluation of the intracellular distribution of radionuclides used for targeted radiotherapy (tRT) is essential for accurate dosimetry. Therefore, a direct and quantitative method for subcellular micro-autoradiography using radiation sensitive polymers (PMMA, UV1116 and AZ40XT) was developed. The electron exposure dose in radio-labelled cells due to Auger and internal conversion (IC) electron emissions of indium (111In), a radionuclide currently used for tRT, was calculated using Monte Carlo (MC) simulation. Electron beam lithography using pre-defined exposure doses was used to calibrate the resist response. The topography of the exposed and developed resists was analysed with atomic force microscopy (AFM) and the resulting pattern depth was related to a specific exposure dose. UV1116 exhibited the best contrast as compared to AZ40XT and PMMA, while AZ40XT exhibited the highest sensitivity at low doses (<10 μC/cm2). AFM analysis of the exposure pattern from radio-labelled cells and nuclei in UV1116 revealed a non-uniform distribution of 111In-EGF in the cell and nucleus, consistent with less well-resolved data from confocal microscopy and micro-autoradiography.
Keywords: Auger electrons; Targeted radiotherapy; Intracellular distribution; Microdosimetry; Chemically amplified photoresists;

Upconversion nanoparticles (UCNPs) that emit high-energy photons upon excitation by the low-energy near-infrared (NIR) light are emerging as new optical nano-probes useful in biomedicine. Herein, we load Chlorin e6 (Ce6), a photosensitizer, on polymer-coated UCNPs, forming a UCNP-Ce6 supramolecular complex that produces singlet oxygen to kill cancer cells under NIR light. Excellent photodynamic therapy (PDT) efficacy is achieved in tumor-bearing mice upon intratumoral injection of UCNP-Ce6 and the followed NIR light exposure. It is further uncovered that UCNPs after PDT treatment are gradually cleared out from mouse organs, without rendering appreciable toxicity to the treated animals. Moreover, we demonstrate that the NIR-induced PDT based on UCNP-Ce6 exhibits a remarkably increased tissue penetration depth compared to the traditional PDT using visible excitation light, offering significantly improved treatment efficacy for tumors blocked by thick biological tissues. Our work demonstrates NIR light-induced in vivo PDT treatment of cancer in animals, and highlights the promise of UCNPs for multifunctional in vivo cancer treatment and imaging.
Keywords: Unconversion nanoparticles; Cancer; In vivo photodynamic therapy; Near-infrared light;

Iron oxide nanoparticle-containing microbubble composites as contrast agents for MR and ultrasound dual-modality imaging by Zhe Liu; Twan Lammers; Josef Ehling; Stanley Fokong; Jörg Bornemann; Fabian Kiessling; Jessica Gätjens (6155-6163).
Magnetic resonance (MR) and ultrasound (US) imaging are widely used diagnostic modalities for various experimental and clinical applications. In this study, iron oxide nanoparticle-embedded polymeric microbubbles were designed as multi-modal contrast agents for hybrid MR–US imaging. These magnetic nano-in-micro imaging probes were prepared via a one-pot emulsion polymerization to form poly(butyl cyanoacrylate) microbubbles, along with the oil-in-water (O/W) encapsulation of iron oxide nanoparticles in the bubble shell. The nano-in-micro embedding strategy was validated using NMR and electron microscopy. These hybrid imaging agents exhibited strong contrast in US and an increased transversal relaxation rate in MR. Moreover, a significant increase in longitudinal and transversal relaxivities was observed after US-induced bubble destruction, which demonstrated triggerable MR imaging properties. Proof-of-principle in vivo experiments confirmed that these nanoparticle-embedded microbubble composites are suitable contrast agents for both MR and US imaging. In summary, these magnetic nano-in-micro hybrid materials are highly interesting systems for bimodal MR–US imaging, and their enhanced relaxivities upon US-induced destruction recommend them as potential vehicles for MR-guided US-mediated drug and gene delivery.
Keywords: Nanoparticle; MRI (magnetic resonance imaging); Contrast agent; Polymerization; Molecular imaging;

Mechanism and consequence of chitosan-mediated reversible epithelial tight junction opening by Tzyy-Harn Yeh; Li-Wen Hsu; Michael T. Tseng; Pei-Ling Lee; Kiran Sonjae; Yi-Cheng Ho; Hsing-Wen Sung (6164-6173).
In order to increase the absorption of hydrophilic macromolecules in the small intestine, permeation enhancers such as chitosan (CS) and its derivatives have been evaluated. The aim of the current work was to investigate, on molecular levels, the effect of CS on tight junction (TJ) integrity in Caco-2 cells. The observed changes in transepithelial-electrical-resistance measurements and the staining patterns of the monolayer Caco-2 cells demonstrate that CS can transiently and reversibly open the TJs between cells, thus enhancing the paracellular permeability. TJ ultra-structures examined by transmission electron microscopy support the concept that CS did induce transient opening of TJs. We then assessed TJ disruption at the gene and protein expression levels. Our data indicate that exposure to CS followed by recovery resulted in a significant increase in claudin-4 (Cldn4) gene transcription. Additionally, CS treatment induced redistribution of the TJ protein CLDN4 intracellularly following by its degradation in lysosomes, which represented an important contributing factor in TJ weakening, leading to the opening of TJs. The recovery of TJ after CS disruption required CLDN4 protein synthesis. These results suggest that CS regulates TJs by inducing changes in transmembrane CLDN4 protein. Understanding the mechanism of interaction between CS and epithelial cells is of paramount importance and needs to be established to aid further development in the use of CS to mediate the trans-epithelial drug delivery.
Keywords: Epithelial cell; Tight junction; Chitosan; Claudin-4; Paracellular transport;

Ambidextrous magnetic nanovectors for synchronous gene transfection and labeling of human MSCs by Jaemoon Yang; Eun-Sook Lee; Min-Young Noh; Seong-Ho Koh; Eun-Kyung Lim; A-Rum Yoo; Kwangyeol Lee; Jin-Suck Suh; Seung Hyun Kim; Seungjoo Haam; Yong-Min Huh (6174-6182).
The synchronization of gene expression and cell trafficking in transfected stem cells is crucial for augmentation of stem cell functions (differentiation and neurotropic factor secretion) and real time in vivo monitoring. We report a magnetic nanoparticle-based gene delivery system that can ensure simultaneous gene delivery and in vivo cell trafficking by high resolution MR imaging. The polar aprotic solvent soluble MnFe2O4 nanoparticles were enveloped using cationic polymers (branched polyethyleneimine, PEI) by the solvent shifting method for a gene loading. Using our magnetic nanovector system (PEI-coated MnFe2O4 nanoparticles), thus, we synchronized stem cell migration and its gene expression in a rat stroke model.
Keywords: Magnetic; Nanovectors; Gene delivery; Stem cell; Solvent shifting;

Tunable dual growth factor delivery from polyelectrolyte multilayer films by Nisarg J. Shah; Mara L. Macdonald; Yvette M. Beben; Robert F. Padera; Raymond E. Samuel; Paula T. Hammond (6183-6193).
A promising strategy to accelerate joint implant integration and reduce recovery time and failure rates is to deliver a combination of certain growth factors to the integration site. There is a need to control the quantity of growth factors delivered at different times during the healing process to maximize efficacy. Polyelectrolyte multilayer (PEM) films, built using the layer-by-layer (LbL) technique, are attractive for releasing controlled amounts of potent growth factors over a sustained period. Here, we present PEM films that sequester physiological amounts of osteogenic rhBMP-2 (recombinant human bone morphogenetic protein - 2) and angiogenic rhVEGF165 (recombinant human vascular endothelial growth factor) in different ratios in a degradable [poly(β-amino ester)/polyanion/growth factor/polyanion] LbL tetralayer repeat architecture where the biologic load scaled linearly with the number of tetralayers. No burst release of either growth factor was observed as the films degraded. The release of rhBMP-2 was sustained over a period of 2 weeks, while rhVEGF165 eluted from the film over the first 8 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. rhBMP-2 initiated a dose dependent differentiation cascade in MC3T3-E1S4 pre-osteoblasts while rhVEGF165 upregulated HUVEC proliferation, and accelerated closure of a scratch in HUVEC cell cultures in a dose dependent manner. In vivo, the mineral density of ectopic bone formed de novo by rhBMP-2/rhVEGF165 PEM films was approximately 33% higher than when only rhBMP-2 was introduced, with a higher trabecular thickness, which would indicate a decrease in the risk of osteoporotic fracture. Bone formed throughout the scaffold when both growth factors were released, which suggests more complete remodeling due to an increased local vascular network. This study demonstrates a promising approach to delivering precise doses of multiple growth factors for a variety of implant applications where control over spatial and temporal release profile of the biologic is desired.
Keywords: Controlled drug release; BMP; VEGF; Bone; Layer-by-layer;

We have developed a polymer nanoparticle-based siRNA delivery system that exploits a cell surface binding synergism between targeting ligands and cell-penetrating peptides. Nanoparticles were coated with folate and penetratin via a PEGylated phospholipid linker (DSPE-PEG): the combination of both of these ligands represents a strategy for enhancing intracellular delivery of attached polymer nanoparticles. Nanoparticles were characterized for size, morphology, density of surface modification, and ligand association and retention. The surface coverage achieved on DSPE-PEG-coated nanoparticles is as high as (or higher than) obtained with other ligand-modified nano-scale particulate systems (∼0.5–5 pmol ligand/cm2). Additionally, these nanoparticles were loaded with a high density of siRNA (∼130–140 pmol siRNA/mg nanoparticles), which is slowly released upon incubation in water. Synergies between the activity of surface binding and cell internalizing ligands on these siRNA-loaded nanoparticles impart delivery enhancements that improve their gene silencing efficacy both in culture and in tumor models. Traditionally, targeting ligands function by binding to cell surface receptors, while cell-penetrating peptides function by nonspecifically transporting across cell membranes. Interestingly, we have observed that improved delivery of these dual-functionalized nanoparticles was in part, a result of increased cell surface avidity afforded by both ligands. This siRNA delivery system presents an approach to surface modification of nanovehicles, in which multiple ligands function in parallel to enhance cell binding and uptake.
Keywords: Nanoparticle; Copolymer; Gene therapy; Phospholipid; Drug delivery; Controlled drug release;

Functionalized PEG hydrogels through reactive dip-coating for the formation of immunoactive barriers by Patrick S. Hume; Christopher N. Bowman; Kristi S. Anseth (6204-6212).
Influencing the host immune system via implantable cell-delivery devices has the potential to reduce inflammation at the transplant site and increase the likelihood of tissue acceptance. Towards this goal, an enzymatically-initiated, dip-coating technique is adapted to fabricate conformal hydrogel layers and to create immunoactive polymer coatings on cell-laden poly(ethylene glycol) (PEG) hydrogels. Glucose oxidase (GOx)-initiated dip coatings enable the rapid formation of uniform, PEG-based coatings on the surfaces of PEG hydrogels, with thicknesses up to 500 μm where the thickness is proportional to the reaction time. Biofunctional coatings were fabricated by thiolating biomolecules that were subsequently covalently incorporated into the coating layer via thiol-acrylate copolymerization. The presence of these proteins was verified via fluorescent confocal microscopy and a modified ELISA, which indicated IgG concentrations as high as 13 ± 1 ng/coated cm2 were achievable. Anti-Fas antibody, known to induce T cell apoptosis, was incorporated into coatings, with or without the addition of ICAM-1 to promote T cell interaction with the functionalized coating. Jurkat T cells were seeded atop functionalized coatings and the induction of apoptosis was measured as an indicator of coating bioactivity. After 48 h of interaction with the functionalized coatings, 61 ± 9% of all cells were either apoptotic or dead, compared to only 18 ± 5% of T cells on non-functionalized coatings. Finally, the cytocompatibility of the surface-initiated GOx coating process was confirmed by modifying gels with either encapsulated β-cells or 3T3 fibroblasts within a gel that contained a PEG methacrylate coating.
Keywords: Apoptosis; Cell Encapsulation; Immunomodulation; Lymphocyte; Surface modification;

Polysorbate 80 (P80) anchored poly(propyleneimine) (PPI) dendritic nanoconjugate was developed and evaluated for targeting anti-cancer drug, docetaxel (DTX) to the brain tumor. In vitro cytotoxicity studies of free DTX, DTX–PPI and DTX–P80-PPI dendrimers were carried out using U87MG human glioblastoma cell line. The in vivo anti-cancer activity in brain tumor bearing rats revealed that DTX loaded P80 conjugated dendrimers reduced the tumor volume extremely significantly (p < 0.0001; more than 50%). The median survival time for brain tumor bearing rats treated with DTX–P80-PPI dendrimers (42 days) was extended very significantly as compared to DTX–PPI (23 days; p < 0.001), receptor blocked group (15 days; p < 0.001) and free DTX (18 days; p < 0.001). Gamma scintigraphy and biodistribution studies further confirmed the targeting efficiency and higher biodistribution of ligand conjugated dendrimer into the brain. The results concluded that the developed nanoconjugate has potential to deliver significantly higher amount of drug to brain tumor for improved therapeutic outcome.
Keywords: Animal model; Apolipoprotein; Brain; Dendrimer; Drug delivery;

Effects of ligands with different water solubilities on self-assembly and properties of targeted nanoparticles by Pedro M. Valencia; Mikhail H. Hanewich-Hollatz; Weiwei Gao; Fawziya Karim; Robert Langer; Rohit Karnik; Omid C. Farokhzad (6226-6233).
The engineering of drug-encapsulated targeted nanoparticles (NPs) has the potential to revolutionize drug therapy. A major challenge for the smooth translation of targeted NPs to the clinic has been developing methods for the prediction and optimization of the NP surface composition, especially when targeting ligands (TL) of different chemical properties are involved in the NP self-assembly process. Here we investigated the self-assembly and properties of two different targeted NPs decorated with two widely used TLs that have different water solubilities, and developed methods to characterize and optimize NP surface composition. We synthesized two different biofunctional polymers composed of poly(lactide-co-glycolide)-b-polyethyleneglycol-RGD (PLGA-PEG-RGD, high water solubility TL) and PLGA-PEG-Folate (low water solubility TL). Targeted NPs with different ligand densities were prepared by mixing TL-conjugated polymers with non-conjugated PLGA-PEG at different ratios through nanoprecipitation. The NP surface composition was quantified and the results revealed two distinct nanoparticle assembly behaviors: for the case of PLGA-PEG-RGD, nearly all RGD molecules conjugated to the polymer were found to be on the surface of the NPs. In contrast, only ∼20% of the folate from PLGA-PEG-Folate was present on the NP surface while the rest remained presumably buried in the PLGA NP core due to hydrophobic interactions of PLGA and folate. Finally, in vitro phagocytosis and cell targeting of NPs were investigated, from which a window of NP formulations exhibiting minimum uptake by macrophages and maximum uptake by targeted cells was determined. These results underscore the impact that the ligand chemical properties have on the targeting capabilities of self-assembled targeted nanoparticles and provide an engineering strategy for improving their targeting specificity.
Keywords: Nanoparticles; Targeting ligand; Surface ligand density; RGD; Folate;

Surfactant-assisted controlled release of hydrophobic drugs using anionic surfactant templated mesoporous silica nanoparticles by Chih-Hsiang Tsai; Juan L. Vivero-Escoto; Igor I. Slowing; I-Ju Fang; Brian G. Trewyn; Victor S.-Y. Lin (6234-6244).
A series of mesoporous silica nanoparticles (MSNs) were synthesized using the co-structure directing method. A non-cytotoxic anionic surfactant, undec-1-en-11-yltetra(ethylene glycol) phosphate monoester surfactant (PMES), was used as a structure directing agent (SDA) together with aminopropyltrimethoxysilane that functioned as a co-structure directing agent (CSDA). The morphology and mesoporous structure of these materials were tuned by changing the molar ratio of CSDA and SDA. These mesoporous nanomaterials containing PMES inside the pores showed excellent biocompatibility in vitro. The cellular internalization and endosome escape of PMES–MSNs in cervical cancer cells (HeLa) was demonstrated by flow cytometry and confocal microscopy, respectively. The PMES–MSNs were used as drug delivery carriers for resveratrol, a low water solubility drug, by taking advantage of the hydrophobic environment created by the PMES micelle inside the pores. This surfactant-assisted delivery strategy was tested under physiological conditions showing an increase of the drug loading compared to the material without surfactant and steady release of resveratrol. Finally, the therapeutic properties of resveratrol-loaded PMES–MSNs were evaluated in vitro using HeLa and Chinese hamster ovarian cells. We envision that this surfactant-assisted drug delivery method using MSNs as nanovehicles would lead to a new generation of carrier materials for intracellular delivery of a variety of hydrophobic therapeutic agents.
Keywords: Mesoporous silica nanoparticles; Anionic surfactant; Drug delivery system; Surfactant-assisted drug release; Non-cytotoxic nanomaterials; Intracellular drug delivery;

The delivery of bioactive proteins to tumors is associated with many difficulties that have impeded clinical translation of these promising therapeutics. Herein we present an approach, including (1) use of magnetically-responsive and MRI-visible nanoparticles as drug carriers, (2) topography-optimized intra-arterial magnetic targeting, (3) MRI-guided subject alignment within the magnetic field, and (4) surface modification of the protein drug with membrane-permeable polyethyleneimine (PEI), to prevail over the obstacles in protein delivery. Applying these methodologies, we demonstrated the delivery of a significant quantity of β-Galactosidase selectively into brain tumors of glioma-bearing rats, while limiting the exposure of normal brain regions. Clinical viability of the technologies utilized, and the ability to deliver proteins at high nanomolar-range tumor concentrations, sufficient to completely eradicate a tumor lesion with existing picomolar-potency protein toxins, renders the prospect of enabling protein-based cancer therapy extremely promising.
Keywords: Drug delivery; Protein delivery; Magnetic nanoparticles; Magnetic targeting; MR imaging; Brain tumor targeting;

Simultaneous in vivo tracking of dendritic cells and priming of an antigen-specific immune response by Young-Woock Noh; Yong-Suk Jang; Kook-Jin Ahn; Yong Taik Lim; Bong Hyun Chung (6254-6263).
We report the fabrication of a one-pot antigen system that delivers antigen to dendritic cells (DCs) and tracks their in vivo migration after injection. Multifunctional polymer nanoparticles containing ovalbumin protein, magnetic resonance imaging contrast agents (iron oxide nanoparticles), and near-infrared fluorophores (indocyanine green, ICG), MPN-OVA, were prepared using a double emulsion method. The MPN-OVA was efficiently taken up by the dendritic cells and subsequently localized in the lysosome. Flow cytometry analysis revealed an increase in the uptake of OVA antigen by MPN-OVA at 37 °C, when compared with soluble OVA protein. We found that MPN-OVA had no effect on DC surface expression of MHC class I, costimulatory (CD80, CD86) or adhesion (CD54) molecules or the ability of DCs to mature in response to LPS. Following the uptake of MPN-OVA, exogenous OVA antigen was delivered to the cytoplasm, and OVA peptides were presented on MHC class I molecules, which enhanced OVA antigen-specific cross-presentation to OT-1 T cells and CD8OVA1.3 T cell hybridoma in vitro. The immunization of mice with MPN-OVA-treated DCs induced OVA-specific CTL activity in draining lymph nodes. The presence of MPN allowed us to monitor the migration of DCs via lymphatic drainage using NIR fluorescence imaging, and the homing of DCs into the lymph nodes was imaged using MRI. This system has potential for use as a delivery system to induce T cell priming and to image DC-based immunotherapies.
Keywords: Polymer nanoparticles; Dendritic cells; Dual-modality imaging; Antigen delivery; Immunity;

A gene delivery system for human cells mediated by both a cell-penetrating peptide and a piggyBac transposase by Cheng-Yi Lee; Jheng-Fong Li; Ji-Sing Liou; Yuh-Chyang Charng; Yue-Wern Huang; Han-Jung Lee (6264-6276).
The piggyBac (PB) transposable element has recently accumulated enormous attention as a tool for the transgenesis in various eukaryotic organisms. Arginine-rich cell-penetrating peptides (CPPs) are protein transduction domains containing a large amount of basic amino acids that were found to be capable of delivering biologically active macromolecules into living cells. In this study, we demonstrate a strategy, which we called “transposoduction”, which is a one-plasmid gene delivery system mediated by the nontoxic CPP-piggyBac transposase (CPP-PBase) fusion protein to accomplish both protein transduction and transposition. CPPs were proven to be able to synchronously deliver covalently linked PBase and noncovalently linked a cis plasmid into human cells. The expression of promoterless reporter genes coding for red (dTomato) and yellow (mOrange) fluorescent proteins (RFP and YFP) with PB elements could be detected in cells treated with the PBase-expressing plasmid after 3 days indicating transposition of coding regions to downstream of endogenous promoter sequences. An enhanced green fluorescent protein (EGFP) plasmid-based excision assay further confirmed the efficiency of the bifunctional CPP-PBase fusion protein. In conclusion, this strategy representing a combinational concept of both protein transduction and mobile transposition may provide tremendous potential for safe and efficient cell line transformation, gene therapy and functional genomics.
Keywords: Cell-penetrating peptide (CPP); Gene delivery; Polyarginine; piggyBac (PB); Transposon;

Controlled release of DNA from poly(vinylpyrrolidone) capsules using cleavable linkers by Sher Leen Ng; Georgina K. Such; Angus P.R. Johnston; Gema Antequera-García; Frank Caruso (6277-6284).
The design of polymer carriers with tunable degradation and cargo release is fundamental for applications in drug and gene delivery. In this study, we report low-fouling poly(N-vinyl pyrrolidone) (PVPON) capsules assembled via hydrogen bonding and stabilized using covalent cross-linking. We first investigated the effects of pH and ionic strength to optimize the assembly conditions. A model therapeutic cargo (plasmid DNA) was then loaded in the capsules and used for encapsulation and release studies. Two bisazide cross-linkers that contain a disulfide bond, termed PEG8 (poly(ethylene glycol)) and PEG16, were employed to stabilize the multilayer films, and used to tune the degradation and cargo release behavior of the capsules in simulated cytoplasmic conditions. The results suggest that PEG8-stabilized capsules were more efficiently cross-linked, and hence displayed higher plasmid encapsulation. Consequently, the capsules cross-linked with PEG8 also showed a two-fold reduction in degradation rate. This ability to achieve controlled carrier degradation and cargo release makes these capsules of potential interest for drug and gene delivery.
Keywords: Controlled drug release; Click chemistry; Degradation; DNA; Drug delivery; Microcapsule;

Drug carrier nanoparticles that penetrate human chronic rhinosinusitis mucus by Samuel K. Lai; Jung Soo Suk; Amanda Pace; Ying-Ying Wang; Ming Yang; Olcay Mert; Jeane Chen; Jean Kim; Justin Hanes (6285-6290).
No effective therapies currently exist for chronic rhinosinusitis (CRS), a persistent inflammatory condition characterized by the accumulation of highly viscoelastic mucus (CRSM) in the sinuses. Nanoparticle therapeutics offer promise for localized therapies for CRS, but must penetrate CRSM in order to avoid washout during sinus cleansing and to reach underlying epithelial cells. Prior research has not established whether nanoparticles can penetrate the tenacious CRSM barrier, or instead become trapped. Here, we first measured the diffusion rates of polystyrene nanoparticles and the same nanoparticles modified with muco-inert polyethylene glycol (PEG) coatings in fresh, minimally perturbed CRSM collected during endoscopic sinus surgery from CRS patients with and without nasal polyp. We found that uncoated polystyrene particles, previously shown to be mucoadhesive in a number of human mucus secretions, were immobilized in all CRSM samples tested. In contrast, densely PEGylated particles as large as 200 nm were able to readily penetrate all CRSM samples from patients with CRS alone, and nearly half of CRSM samples from patients with nasal polyp. Based on the mobility of different sized PEGylated particles, we estimated the average pore size of fresh CRSM to be at least 150 ± 50 nm. Guided by these studies, we formulated mucus-penetrating particles composed of poly(lactide-co-glycolide) (PLGA) and Pluronics, two materials with a long history of safety and use in humans. We showed that these biodegradable particles are capable of rapidly penetrating CRSM at average speeds up to only 20-fold slower than their theoretical speeds in water. Our findings strongly support the development of mucus-penetrating nanomedicines for the treatment of CRS.
Keywords: Drug delivery; Sinusitis; Polymer; PEG; Mucus-penetrating particles; Particle tracking;

Magnetic brain tumor targeting and biodistribution of long-circulating PEG-modified, cross-linked starch-coated iron oxide nanoparticles by Adam J. Cole; Allan E. David; Jianxin Wang; Craig J. Galbán; Victor C. Yang (6291-6301).
Magnetic iron oxide nanoparticles (MNPs) have been studied to circumvent the limitations of status-quo brain tumor therapy and can be targeted by applying an external magnetic field to lesions. To address the pharmacokinetic shortcomings of MNPs that can limit targeting efficiency, we recently reported a long-circulating polyethylene glycol modified, cross-linked starch MNP (PEG-MNP) suitable for magnetic targeting. Using a rat model, this work explores the biodistribution patterns of PEG-MNPs in organs of elimination (liver, spleen, lung, and kidney) and shows proof-of-concept that enhanced magnetic brain tumor targeting can be achieved due to the relatively long circulation lifetime of the nanoparticles. Reductions in liver (∼12-fold) and spleen (∼2.5-fold) PEG-MNP concentrations at 1 h compared to parent starch-coated MNPs (D) confirm plasma pharmacokinetics observed previously. While liver concentrations of PEG-MNPs remained considerably lower than those observed for D at 1 h through 60 h, spleen values continue to increase and are markedly higher at later time points – a trend also observed with histology. Limited to no distribution of PEG-MNPs was visualized in lung or kidney throughout the 60 h course evaluated. Enhanced, selective magnetic brain tumor targeting (t = 1 h) of PEG-MNPs (12 mg Fe/kg) was confirmed in 9L-glioma tumors, with up to 1.0% injected dose/g tissue nanoparticle delivery achieved – a 15-fold improvement over targeted D (0.07% injected dose/g tissue). MRI and histological analyses visually confirmed enhanced targeting and also suggest a limited contribution of passive mechanisms to tissue retention of nanoparticles. Our results are exciting and justify both further development of PEG-MNP as a drug delivery platform and concurrent optimization of the magnetic brain tumor targeting strategy utilized.
Keywords: Magnetic nanoparticles; Magnetic targeting; Polyethylene glycol (PEG); Pharmacokinetics; Drug delivery; Brain tumor;

Receptor-targeted liposome-peptide nanocomplexes for siRNA delivery by Aristides D. Tagalakis; Lin He; Luisa Saraiva; Kenth T. Gustafsson; Stephen L. Hart (6302-6315).
RNA interference induced by double-stranded, small interfering RNA (siRNA) molecules has attracted great attention as a genetic therapeutic approach. Despite major advances in this field, new nanoparticle formulations are required for in vivo delivery of siRNA, particularly for tissue-specific delivery of siRNA reagents. We have developed and optimized LYR nanocomplex formulations for siRNA delivery that consist of a liposome (DOTMA/DOPE; L) and a targeting peptide (K16GACYGLPHKFCG; Y) which self-assemble on mixing at optimal ratios with siRNA (R). Biophysical measurements indicated that LYR nanocomplexes were strongly cationic, mainly spherical particles of less than 100 nm. These formulations packaged and protected siRNA on incubation with RNAseA with >90% intact siRNA recovery. In addition, intact siRNA was recovered from LYRs upon heparin treatment. A critical synergy was observed between the lipid and peptide components for LYR particle stability and transfection efficiency. To evaluate targeting, transfections were compared with non-targeted formulations containing K16 with no targeting ligand. Gene knockdown efficiencies with targeted formulations were more than two-fold better in all cell lines tested (p < 0.01). LYR formulations with liposomes containing DOTMA, which has an 18-carbon (C18) alkyl tail, were significantly better in silencing than formulations containing cationic lipids with shorter alkyl tails. LYRs with siRNA against endogenous luciferase and GAPDH were successful in silencing these genes in 3 cell lines (1HAEo- human airway epithelial, B104 rat neuroblastoma, Neuro2A-Luc mouse neuroblastoma) in vitro with 80% efficiency, similar in efficiency to Lipofectamine 2000. Confocal microscopy analysis with LYRs containing fluorescently labelled siRNA (Cy3) showed that the siRNA was located in the perinuclear region of the cytoplasm, where the RNA-induced silencing complex (RISC) is likely to be found. The LYR formulations may have applications for the further development of siRNA-based therapeutics.
Keywords: Nanoparticle; Targeting; siRNA delivery; RNA interference; Liposomes; Gene silencing;

Carbon nanotube nanoreservior for controlled release of anti-inflammatory dexamethasone by Xiliang Luo; Christopher Matranga; Susheng Tan; Nicolas Alba; Xinyan T. Cui (6316-6323).
On demand release of anti-inflammatory drug or neurotropic factors have great promise for maintaining a stable chronic neural interface. Here we report the development of an electrically controlled drug release system based on conducting polymer and carbon nanotubes. Drug delivery research using carbon nanotubes (CNTs) has taken advantage of the ability of CNTs to load large amounts of drug molecules on their outer surface. However, the utility of the inner cavity of CNTs, which can increase the drug loading capacity, has not yet been explored. In this paper, the use of multi-wall CNTs as nanoreserviors for drug loading and controlled release is demonstrated. The CNTs are pretreated with acid sonication to open their ends and make their outer and inner surfaces more hydrophilic. When dispersed and sonicated in a solution containing the anti-inflammatory drug dexamethasone, experiments show that the pretreated CNTs are filled with the drug solution. To prevent the unwanted release of the drug, the open ends of the drug-filled CNTs are then sealed with polypyrrole (PPy) films formed through electropolymerization. The prepared electrode coating significantly reduced the electrode impedance, which is desired for neural recording and stimulation. More importantly, the coating can effectively store drug molecules and release the bioactive drug in a controlled manner using electrical stimulation. The dexamethasone released from the PPy/CNT film was able to reduce lipopolysaccharide induced microglia activation to the same degree as the added dexamethasone.
Keywords: Carbon nanotubes; Drug nanoreservior; Conducting polymer; Drug release; Dexamethasone;

Targeted delivery of non-viral vectors to cartilage in vivo using a chondrocyte-homing peptide identified by phage display by Yanbin Pi; Xin Zhang; Junjun Shi; Jinxian Zhu; Wenqing Chen; Chenguang Zhang; Weiwei Gao; Chunyan Zhou; Yingfang Ao (6324-6332).
Gene therapy is a promising method for osteoarthritis and cartilage injury. However, specifically delivering target genes into chondrocytes is a great challenge because of their non-vascularity and the dense extracellular matrix of cartilage. In our study, we identified a chondrocyte-affinity peptide (CAP, DWRVIIPPRPSA) by phage display technology. Subsequent analysis suggests that the peptide can efficiently interact specifically with chondrocytes without any species specificity. Polyethylenimine (PEI) was covalently modified with CAP to construct a non-viral vector for cartilage-targeted therapy. To investigate the cartilage-targeting property of the CAP-modified vector, FITC-labeled CAP conjugated PEI/DNA particles were injected into rabbit knee joints, and visualized under confocal microscope. Higher concentrations of CAP-modified vector were detected in the cartilage and specifically taken up by chondrocytes compared with a randomly scrambled peptide (SP)-modified vector. To evaluate cartilage-targeting transfection efficiency, the GFP and luciferase genes were delivered into knee joints using CAP- and SP-modified PEI. Cartilage transfections mediated by CAP-modified PEI were much more efficient and specific than those by SP-modified PEI. This result suggests that CAP-modified PEI could be used as a specific cartilage-targeting vector for cartilage disorders.
Keywords: Phage display; Cartilage-targeting; Gene therapy; Peptide-modified polyethylenimine;

Pluronic–lysozyme conjugates as anti-adhesive and antibacterial bifunctional polymers for surface coating by Agnieszka K. Muszanska; Henk J. Busscher; Andreas Herrmann; Henny C. van der Mei; Willem Norde (6333-6341).
This paper describes the preparation and characterization of polymer–protein conjugates composed of a synthetic triblock copolymer with a central polypropylene oxide (PPO) block and two terminal polyethylene oxide (PEO) segments, Pluronic F-127, and the antibacterial enzyme lysozyme attached to the telechelic groups of the PEO chains. Covalent conjugation of lysozyme proceeded via reductive amination of aldehyde functionalized PEO blocks (CHO-Pluronic) and the amine groups of the lysine residues in the protein. SDS-PAGE gel electrophoresis together with MALDI-TOF mass spectrometry analysis revealed formation of conjugates of one or two lysozyme molecules per Pluronic polymer chain. The conjugated lysozyme showed antibacterial activity towards Bacillus subtilis. Analysis with a quartz crystal microbalance with dissipation revealed that Pluronic–lysozyme conjugates adsorb in a brush conformation on a hydrophobic gold-coated quartz surface. X-ray photoelectron spectroscopy indicated surface coverage of 32% by lysozyme when adsorbed from a mixture of unconjugated Pluronic and Pluronic–lysozyme conjugate (ratio 99:1) and of 47% after adsorption of 100% Pluronic–lysozyme conjugates. Thus, bifunctional brushes were created, possessing both anti-adhesive activity due to the polymer brush, combined with the antibacterial activity of lysozyme. The coating having a lower degree of lysozyme coverage proved to be more bactericidal.
Keywords: Block copolymers; Protein–polymer conjugates; Functional coatings; Medical applications;

KALA-modified multi-layered nanoparticles as gene carriers for MHC class-I mediated antigen presentation for a DNA vaccine by Sharif M. Shaheen; Hidetaka Akita; Takashi Nakamura; Shota Takayama; Shiroh Futaki; Atsushi Yamashita; Ryo Katoono; Nobuhiko Yui; Hideyoshi Harashima (6342-6350).
DNA vaccines are a new-generation vaccines that elicit an immunological response against a wide-variety of antigens with frequent mutations. However, an effective non-viral vector for genetically engineered DNA to dendritic cells is yet to be developed. We previously reported that an octaarginine (R8)-modified tetra-lamellar multi-functional envelope-type nano device (R8-T-MEND) increases transfection efficiency in dendritic cell cultures (JAWS II). The critical structural elements of the R8-T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes, and two endosome-fusogenic outer envelopes. While the gene expression was drastically enhanced by R8-T-MEND, antigen presentation using an epitope-encoding plasmid DNA remains an obstacle for future non-viral vectors in DNA vaccinations. In the present study, we upgraded the function of R8-T-MEND by improving the membrane-fusion processes with endosome- and nuclear membranes by incorporating the KALA peptide, and by reducing the charge ratio (+/−), in an attempt to accelerate intra-nuclear decondensation. The resulting KALA-modified T-MEND (R8/KALA-T-MEND) showed an approximately 20-fold higher transgene expression compared with the conventional R8-T-MEND in JAWS II, and exceeded that of Lipofectamine PLUS, a commercially available transfection reagent. Furthermore, significant antigen presentation of a specific epitope (SIINFEKL) was observed for the R8/KALA-T-MEND but was not detected for the conventional T-MEND or Lipofectamine PLUS when an ovalbumin (OVA)-encoding plasmid DNA was transfected. It thus appears that the R8/KALA-T-MEND has the potential for use as a vector in DNA vaccinations.
Keywords: Stearylated KALA; Antigen presentation; Transfection; Dendritic cells; Non-viral vectors;