Biomaterials (v.32, #21)

The use of multifunctional magnetic mesoporous core/shell heteronanostructures in a biomolecule separation system by Zhen Liu; Meng Li; Xinjian Yang; Meili Yin; Jinsong Ren; Xiaogang Qu (4683-4690).
A multifunctional magnetic mesoporous core/shell heteronanostructure (designated as Fe3O4@NiSiO3) has been designed and constructed that combined the capacity of effective protein purification from protein mixture and selective low molecule weight (MW) biomolecule enrichment. The nanoparticle is composed by magnetite nanoparticle with immobilized metal ion surface and solid porous shell which presents a number of important features, such as controllable shell thickness, uniform pore size and excellent magnetism. By taking advantages of the high affinity of Ni2+ on the shell surface toward His-tagged proteins and the fast response toward an assistant magnet, the heteronanoparticles can be applied to selectively bind to and magnetically separate of His-tagged proteins from a cell lysate of E. coli. Additionally, owing to the homogeneous 3D mesopores on the nickel silicate shell, the heteronanoparticles can selectively capture low MW biomolecules from complex mixture. Significantly, it is expected that this approach can be extended to other biomolecule separation and enrichment systems by changing the immobilized surface and the pore size.
Keywords: Heteronanostructure; Mesoporous material; Protein separation; Peptide enrichment; Bionanotechnology;

The effect of coimmobilizing heparin and fibronectin on titanium on hemocompatibility and endothelialization by Guicai Li; Ping Yang; Wei Qin; Manfred F. Maitz; Shuo Zhou; Nan Huang (4691-4703).
Currently available cardiovascular implants, such as heart valves and stents, exhibit suboptimal biocompatibility because of the incomplete endothelialization and sequential thrombosis formation especially after a long-term implantation. To improve the blood compatibility and endothelialization simultaneously and ensure the long-term effect of the cardiovascular implants, a technique of combining electrostatic interaction and coimmobilization was developed to form heparin and fibronectin (Hep/Fn) films on aminosilanized titanium (Ti) surfaces. The Hep/Fn coimmobilized films were stable after immersion in PBS for five days, probed by wettability studies and by the release kinetics of heparin and fibronectin. Blood compatibility tests showed that the coimmobilized Hep/Fn films displayed lower hemolysis rate, prolonged blood coagulation time, higher AT III binding density, less platelets activation and aggregation, and less fibrinogen conformational change compared with Ti surface. Endothelial cells (ECs) seeding and fibronectin bioactivity results showed more attached and proliferated ECs and exposed cell-binding sites on the Hep/Fn immobilized samples than that on Ti surfaces. Thus, the Hep/Fn coimmobilized films kept excellent bioactivity even after immersion in PBS for five days. Systemic evaluation suggests that the coimmobilization of Hep/Fn complex improves the blood compatibility and promotes the endothelialization simultaneously. We envisage that this method will provide a potential and effective selection for biomaterials surface modification of cardiovascular implants.
Keywords: Coimmobilization; Heparin; Fibronectin; Stability; Blood compatibility; Endothelialization;

The targeted antibacterial and antifungal properties of magnetic nanocomposite of iron oxide and silver nanoparticles by Robert Prucek; Jiří Tuček; Martina Kilianová; Aleš Panáček; Libor Kvítek; Jan Filip; Milan Kolář; Kateřina Tománková; Radek Zbořil (4704-4713).
Two types of magnetic binary nanocomposites, Ag@Fe3O4 and γ-Fe2O3@Ag, were synthesized and characterized and their antibacterial activities were tested. As a magnetic component, Fe3O4 (magnetite) nanoparticles with an average size of about 70 nm and monodisperse γ-Fe2O3 (maghemite) nanoparticles with an average size of 5 nm were used. Nanocomposites were prepared via in situ chemical reduction of silver ions by maltose in the presence of particular magnetic phase and molecules of polyacrylate serving as a spacer among iron oxide and silver nanoparticles. In the case of the Ag@Fe3O4 nanocomposite, silver nanoparticles, caught at the surfaces of Fe3O4 nanocrystals, were around 5 nm in a size. On the contrary, in the case of the γ-Fe2O3@Ag nanocomposite, ultrafine γ-Fe2O3 nanoparticles surrounded silver nanoparticles ranging in a size between 20 and 40 nm. In addition, the molecules of polyacrylate in this nanocomposite type suppress considerably interparticle magnetic interactions as proved by magnetization measurements. Both synthesized nanocomposites exhibited very significant antibacterial and antifungal activities against ten tested bacterial strains (minimum inhibition concentrations (MIC) from 15.6 mg/L to 125 mg/L) and four candida species (MIC from 1.9 mg/L to 31.3 mg/L). Moreover, acute nanocomposite cytotoxicity against mice embryonal fibroblasts was observed at concentrations of higher than 430 mg/L (Ag@Fe3O4) and 292 mg/L (γ-Fe2O3@Ag). With respect to the non-cytotoxic nature of the polyacrylate linker, both kinds of silver nanocomposites are well applicable for a targeted magnetic delivery of silver nanoparticles in medicinal and disinfection applications.
Keywords: Nanocomposite; Nanoparticles; Silver; Magnetism; Iron oxides; Antimicrobial agent; Cytotoxicity;

In this work, polydimethylsiloxane was activated with oxygen plasma and treated with silanes bearing ethylene imine units. Hyaluronic acid was then grafted covalently onto the aminated surfaces. The influence of silane structure on surface amination was assessed and the influence of the modification on surface physiochemical properties and protein adsorption of modified polydimethylsiloxane were investigated. Collagen type I was conjugated onto the modified polydimethylsiloxane to improve its cyto-compatibility for neural applications. In vitro cultivation of rat pheochromocytoma cells on the bioactive polydimethylsiloxane showed a significant increase in cell growth and differentiation. The potential applications of the bio-functionalized polydimethylsiloxane in cochlear implant electrode arrays were discussed.
Keywords: Polydimethylsiloxane; Surface modification; Hyaluronic acid; Neural cells; Differentiation; Neural prosthesis;

Biodegradable and thermoreversible PCLA–PEG–PCLA hydrogel as a barrier for prevention of post-operative adhesion by Zheng Zhang; Jian Ni; Liang Chen; Lin Yu; Jianwei Xu; Jiandong Ding (4725-4736).
Biodegradable polymers can serve as barriers to prevent the post-operative intestinal adhesion. Herein, we synthesized a biodegradable triblock copolymer poly(ɛ-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ɛ-caprolactone-co-lactide) (PCLA–PEG–PCLA). The concentrated polymeric aqueous solution was injectable, and a hydrogel could be rapidly formed due to percolation of a self-assembled micelle network at the body temperature without requirement of any chemical reactions. This physical hydrogel retained its integrity in vivo for a bit more than 6 weeks and was eventually degraded due to hydrolysis. The synthesized polymer exhibited little cytotoxicity and hemolysis; the acute inflammatory response after implanting the hydrogel was acceptable, and the degradation products were less acidic than those of other polyester-containing materials. A rabbit model of sidewall defect-bowel abrasion was employed, and a significant reduction of post-operative peritoneal adhesion has been found in the group of in situ formed PCLA–PEG–PCLA hydrogels.
Keywords: Post-operative adhesion; Thermoreversible hydrogel; Adhesion prevention; Biocompatibility; Block copolymer;

Enhanced proliferation of neural stem cells in a collagen hydrogel incorporating engineered epidermal growth factor by Edgar Y. Egawa; Koichi Kato; Makiko Hiraoka; Tadashi Nakaji-Hirabayashi; Hiroo Iwata (4737-4743).
Neural stem cells (NSCs) have received much attention in cell-transplantation therapy for central nervous disorders such as Parkinson’s disease. However, poor engraftment of transplanted cells limits the efficacy of the treatments. To overcome this problem, collagen-based hydrogels were designed in this study to provide microenvironments for embedded cells to survive and proliferate. Our approach was to incorporate epidermal growth factor (EGF), known as a mitogen for NSCs, into a collagen hydrogel. For the stable binding of EGF with collagen under mild conditions, EGF was fused with a collagen-binding polypeptide domain by recombinant DNA technology. A cell population containing NSCs was derived from the fetal rat brain and cultured in the composite hydrogels for 7 d followed by analysis for cell proliferation. It was shown that the number of living cells was significantly higher in hydrogels incorporating collagen-binding EGF. This effect is largely owing to the collagen-binding domain that serves to sustain presentation of EGF toward cells within the hydrogel. It is further revealed by gene expression analysis that cells proliferated in the EGF-incorporating collagen hydrogel contained subpopulations expressing the marker of stem cells, neurons, astrocytes, or oligodendrocytes.
Keywords: Collagen; ECM (extracellular matrix); Growth factors; Hydrogel; Neural cell; Stem cell;

Controlled growth and differentiation of MSCs on grooved films assembled from monodisperse biological nanofibers with genetically tunable surface chemistries by Haibao Zhu; Binrui Cao; Zipeng Zhen; Ayyagari A. Laxmi; Dong Li; Shaorong Liu; Chuanbin Mao (4744-4752).
The search for a cell-supporting scaffold with controlled topography and surface chemistry is a constant topic within tissue engineering. Here we have employed M13 phages, which are genetically modifiable biological nanofibers (∼880 nm long and ∼6.6 nm wide) non-toxic to human beings, to form films for supporting the growth of mesencymal stem cells (MSCs). Films were built from nearly parallel phage bundles separated by grooves. The bundles can guide the elongation and alignment of MSCs along themselves. Phage with peptides displayed on the surface exhibited different control over the fine morphologies and differentiation of the MSCs. When an osteogenic peptide was displayed on the surface of phage, the proliferation and differentiation of MSCs into osteoblasts were significantly accelerated. The use of the grooved phage films allows us to control the proliferation and differentiation of MSCs by simply controlling the concentrations of phages as well as the peptides displayed on the surface of the phages. This work will advance our understanding on the interaction between stem cells and proteins.
Keywords: M13 phage; Mesenchymal stem cells; Differentiation; Topography; Surface chemistry;

The use of dynamic surface chemistries to control msc isolation and function by J.M. Curran; F. Pu; R. Chen; J.A. Hunt (4753-4760).
Material modifications can be used to induce cell responses, in particular–CH3 and –NH2 have shown potential in enhancing the ability of a material to support mesenchymal stem cell (MSC) adhesion and differentiation. Currently this process is variable, due to the lack of definition of controlled contextual presentation of the chemical group of interest across the surface. This paper defines the potential of –CH3 modified surfaces, with optimised dynamic surface chemistry, to manipulate initial MSC adhesive events, integrin binding, and subsequent cell function. An array of –CH3 silane modified glass substrates was produced using different –CH3 chain lengths and mechanisms of bonding to the base substrate. We show that changing the chain length affects the ability of the surfaces to support viable adult MSC adhesion, directly related to induced FGF release, and expression of STRO-1, CD29, 73, 90 and 105. Chlorodimethyloctylsilane (ODMCS) modified surfaces resulted in significant increases of associated adult MSC markers compared to all other –CH3 modified and control substrates. In contrast Dichlorodimethylsilane (DMDCS) modified surfaces did not support adult MSC adhesion due to high levels of early FGF release, which had an inhibitory effect on adult MSC culture, but enhanced the efficiency and cell selective properties of the substrate in isolation of multi-potent progenitor/MSC from adult human whole blood. Incorporation of optimised –CH3 groups is a cost effective route for producing substrates that significantly enhance MSC isolation and expansion, highlighting the potential of the optimised substrates to replace RGD and fibronectin modifications in selected applications.
Keywords: Cell adhesion; Stem cell; Surface modification; Integrin;

Real time responses of fibroblasts to plastically compressed fibrillar collagen hydrogels by Chiara E. Ghezzi; Naser Muja; Benedetto Marelli; Showan N. Nazhat (4761-4772).
In vitro reconstituted type I collagen hydrogels are widely utilized for tissue engineering studies. However, highly hydrated collagen (HHC) gels exhibit insufficient mechanical strength and unstable geometrical properties, thereby limiting their therapeutic application. Plastic compression (PC) is a simple and reproducible approach for the immediate production of dense fibrillar collagen (DC) scaffolds which demonstrate multiple improvements for tissue engineered constructs including extracellular matrix (ECM)-like meso scale characteristics, increased mechanical properties (modulus and strength), enhanced cell growth and differentiation, and reduced long-term scaffold deformation. In order to determine at which stage these benefits become apparent, and the underlying mechanisms involved, the immediate response of NIH/3T3 fibroblasts to PC as well as longer-term cell growth within DC scaffolds were examined herein. The real time three-dimensional (3D) distribution of fluorescently labelled cells during PC was sequentially monitored using confocal laser scanning microscopy (CLSM), observing excellent cell retention and negligible numbers of expelled cells. Relative to cells grown in HHC gels, a significant improvement in cell survival within DC scaffolds was evident as early as day 1. Cell growth and metabolic activity within DC gels were significantly increased over the course of one week. While cells within DC scaffolds reached confluency, an inhomogeneous distribution of cells was present in HHC gels, as detected using x-ray computed micro-tomography analysis of phosphotungstic acid labelled cells and CLSM, which both showed a significant cell loss within the HHC core. Therefore, PC generates a DC gel scaffold without detrimental effects towards seeded cells, surpassing HHC gels as a 3D scaffold for tissue engineering.
Keywords: Dense collagen gels; Plastic compression; Extracellular matrix; In vitro tissue models; Soft tissue x-ray micro-tomography;

The role of bFGF in down-regulating α-SMA expression of chondrogenically induced BMSCs and preventing the shrinkage of BMSC engineered cartilage by Qiong Li; Tianyi Liu; Lu Zhang; Yu Liu; Wenjie Zhang; Wei Liu; Yilin Cao; Guangdong Zhou (4773-4781).
Bone marrow stromal cells (BMSCs) have proved to be an ideal cell source for cartilage regeneration. Our previous studies demonstrated that a three-dimensional (3D) cartilage could be constructed successfully in vitro using BMSCs and biodegradable scaffolds. However, an obvious shrinkage and deformation was observed during in vitro chondrogenic induction. According to the literatures, it can be speculated that the up-regulation of smooth muscle actin-alpha (α-SMA) caused by transforming growth factor beta (TGFβ) is one of the leading reasons and that basic fibroblast growth factor (bFGF) could antagonize the role of TGFβ to down-regulate α-SMA expression and prevent the shrinkage of BMSC engineered cartilage. This study testified these speculations by adding bFGF to chondrogenic media. According to the current results, chondrogenic induction significantly up-regulated α-SMA expression of BMSCs at both cell and tissue levels, and the engineered tissue only retained 12.4% of original size after 6 weeks of chondrogenic induction. However, the supplement of bFGF in chondrogenic media efficiently down-regulated α-SMA expression and the engineered tissue still retained over 60% of original size after 6 weeks of culture. Moreover, bFGF showed a beneficial influence on 3D cartilage formation of BMSCs in terms of gene expression and deposition of cartilage specific matrices. All these results suggested that bFGF could repress α-SMA expression caused by chondrogenic induction, efficiently prevent shrinkage of BMSC engineered tissue, and have a positive influence on cartilage formation, which provides a clue for both shape control and quality improvement of BMSC engineered 3D cartilage.
Keywords: Bone marrow stromal cells (BMSCs); Chondrogenesis; Shrinkage; Smooth muscle actin-alpha (α-SMA); Basic fibroblast growth factor (bFGF);

A denatured collagen microfiber scaffold seeded with human fibroblasts and keratinocytes for skin grafting by Margit Kempf; Yuki Miyamura; Pei-Yun Liu; Alice C.-H. Chen; Hideki Nakamura; Hiroshi Shimizu; Yasuhiko Tabata; Roy M. Kimble; James R. McMillan (4782-4792).
Biomaterial scaffolds are categorized into artificial or natural polymers, or combinations of the two. Artificial polymers often undergo serum protein adsorption, elicit foreign body and encapsulation immune responses post-implantation. Large pore bovine electrospun collagen I was therefore screened as a candidate for human keratinocyte and fibroblast cell scaffolds. Human HaCaT keratinocyte and dermal fibroblasts were seeded on electrospun denatured collagen I microfiber (DCM) scaffolds and after 72 h Livedead® assays performed to determine adhesive cell, survival and scaffold penetration. Both keratinocytes and fibroblasts attached to and survived on DCM scaffolds, however only fibroblasts migrated over and into this biomaterial. HaCaT keratinocytes remained largely stationary on the scaffold surface in discrete islands of monolayered cells. For this reason, normal human epidermal keratinocyte (NHEK) scaffold interactions were assessed using scanning and transmission electron microscopy (EM) that demonstrated DCM scaffolds comprised networks of interlocking and protruding collagen fibers with a mean diameter of 2–5 μm, with a mean inter-fiber pore size of 6.7 μm (range 3–10 μm) and scaffold thickness 50–70 μm. After 72 h the keratinocytes and fibroblasts on DCM scaffolds had attached, flattened and spread over the entire scaffold with assembly of lamellapodia and focal adhesion (FA)-like junctions. Using transmission EM, NHEKs and HaCaT keratinocytes assembled desmosomes, lamellapodia and FA junctions, however, neither hemidesmosomes nor basal lamina were present. In long term (21 day) co-culture fibroblasts migrated throughout the scaffold and primary keratinocytes (and to a lesser extend HaCaTs) stratified on the scaffold surface forming a human skin equivalent (HSE). In vivo testing of these HSEs on immunocompetent (BalbC) and immunodeficient (SCID) excisionally wounded model mice demonstrated scaffold wound biocompatibility and ability to deliver human cells after scaffold biodegradation.
Keywords: Collagen; Dermis; Scaffold; Keratinocyte; Fibroblast; Surface graft;

Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds by Jiangning Chen; Huan Chen; Pei Li; Huajia Diao; Shiyu Zhu; Lei Dong; Rui Wang; Ting Guo; Jianning Zhao; Junfeng Zhang (4793-4805).
Engineering complex tissues is important but difficult to achieve in tissue regeneration. Osteochondral tissue engineering for the repair of osteochondral defect, involving simultaneous regeneration of bone and cartilage, has attracted considerable attention and also serves as an optimal model system for developing effective strategies aimed at regenerating complex tissues. In the present study, we formulated a bilayered gene-activated osteochondral scaffold consisting of plasmid TGF-β1-activated chitosan-gelatin scaffold for chondrogenic layer and plasmid BMP-2-activated hydroxyapatite/chitosan-gelatin scaffold for osteogenic layer. Mesenchymal stem cells seeded in each layer of the bilayered gene- activated osteochondral scaffold showed significant cell proliferation, high expression of TGF-β1 protein and BMP-2 protein respectively. The results showed that spatially controlled and localized gene delivery system in the bilayered integrated scaffolds could induce the mesenchymal stem cells in different layers to differentiate into chondrocytes and osteoblasts in vitro, respectively, and simultaneously support the articular cartilage and subchondral bone regeneration in the rabbit knee ostochondral defect model. This study gives the evidence that multi-tissue regeneration through the combination of biomimetic and multi-phasic scaffold design, spatially controlled and localized gene delivery system and multi-lineage differentiation of a single stem cell population represents a promising strategy for facilitating the development of complex tissue or organ systems.
Keywords: Bilayered scaffold; Local gene delivery; Mesenchymal stem cells; Osteochondral tissue;

Healing parameters in a rabbit partial tendon defect following tenocyte/biomaterial implantation by Christiane Stoll; Thilo John; Claudia Conrad; Anke Lohan; Sylvia Hondke; Wolfgang Ertel; Christian Kaps; Michaela Endres; Michael Sittinger; Jochen Ringe; G. Schulze-Tanzil (4806-4815).
Although rabbits are commonly used as tendon repair model, interpretative tools are divergent and comprehensive scoring systems are lacking. Hence, the aim was to develop a multifaceted scoring system to characterize healing in a partial Achilles tendon defect model. A 3 mm diameter defect was created in the midsubstance of the medial M. gastrocnemius tendon, which remained untreated or was filled with a polyglycolic-acid (PGA) scaffold + fibrin and either left cell-free or seeded with Achilles tenocytes. After 6 and 12 weeks, tendon repair was assessed macroscopically and histologically using self-constructed scores. Macroscopical scoring revealed superior results in the tenocyte seeded PGA + fibrin group compared with the controls at both time points. Histology of all operated tendons after 6 weeks proved extracellular matrix (ECM) disorganization, hypercellularity and occurrence of irregular running elastic fibres with no significance between the groups. Some inflammation was associated with PGA implantation and increased sulphated proteoglycan deposition predominantly with the empty defects. After 12 weeks defect areas became hard to recognize and differences between groups, except for the increased sulphated proteoglycans content in the empty defects, were almost nullified. We describe a partial Achilles tendon defect model and versatile scoring tools applicable for characterizing biomaterial-supported tendon healing.
Keywords: Tendon; Polyglycolic-acid; Fibrin; Implantation;

There are few synthetic elastomeric biomaterials that simultaneously provide the required biological conditioning and the ability to translate biomechanical stimuli to vascular smooth muscle cells (VSMCs). Biomechanical stresses are important physiological elements that regulate VSMC function, and polyurethane elastomers are a class of materials capable of facilitating the translation of stress induced biomechanics. In this study, human coronary artery smooth muscle cells (hCASMCs), which were seeded into a porous degradable polar/hydrophobic/ionic (D-PHI) polyurethane scaffold, were subjected to uniaxial cyclic mechanical strain (CMS) over a span of four weeks using a customized bioreactor. The distribution, proliferation and contractile protein expression of hCASMCs in the scaffold were then analyzed and compared to those grown under static conditions. Four weeks of CMS, applied to the elastomeric scaffold, resulted in statistically greater DNA mass, more cell area coverage and a better distribution of cells deeper within the scaffold construct. Furthermore, CMS samples demonstrated improved tensile mechanical properties following four weeks of culture, suggesting the generation of more extracellular matrix within the polyurethane constructs. The expression of smooth muscle α-actin, calponin and smooth muscle myosin heavy chain and the absence of Ki-67+ cells in both static and CMS cultures, throughout the 4 weeks, suggest that hCASMCs retained their contractile character on these biomaterials. The study highlights the importance of implementing physiologically-relevant biomechanical stimuli in the development of synthetic elastomeric tissue engineering scaffolds.
Keywords: Smooth muscle cell; Polyurethane; Scaffold; Arterial tissue engineering; Cell proliferation; Soft tissue biomechanics;

The promotion of axon extension in vitro using polymer-templated fibrin scaffolds by John B. Scott; Mehdi Afshari; Richard Kotek; Justin M. Saul (4830-4839).
Biomaterial nerve cuffs are a clinical alternative to autografts and allografts as a means to repair segmental peripheral nerve defects. However, existing clinical biomaterial constructs lack true incorporation of physical guidance cues into their design. In both two- and three-dimensional systems, it is known that substrate geometry directly affects rates of axon migration. However, the ability to incorporate these cues into biomaterial scaffolds of sufficient porosity to promote robust nerve regeneration in three-dimensional systems is a challenge. We have developed fibrin constructs fabricated by a sacrificial templating approach, yielding scaffolds with multiple 10–250 μm diameter conduits depending on the diameter of the template fibers. The resulting scaffolds contained numerous, highly aligned conduits, had porosity of ∼ 80%, and showed mechanical properties comparable to native nerve (150–300 kPa Young’s modulus). We studied the effects of the conduit diameters on the rate of axon migration through the scaffold to investigate if manipulation of this geometry could be used to ultimately promote more rapid bridging of the scaffold. All diameters studied led to axon migration, but in contrast to effects of fiber diameters in other systems, the rate of axon migration was independent of conduit diameter in these templated scaffolds. However, aligned conduits did support more rapid axon migration than non-aligned, tortuous controls.
Keywords: Fibrin; Micropatterning; Nerve guide; Nerve tissue engineering; Scaffold;

Hyaluronan-grafted particle clusters loaded with Mitomycin C as selective nanovectors for primary head and neck cancers by Gideon Bachar; Keren Cohen; Roy Hod; Raphael Feinmesser; Aviram Mizrachi; Thomas Shpitzer; Odelia Katz; Dan Peer (4840-4848).
CD44, a well-documented cell surface receptor, is involved in cell proliferation, migration, signaling, adhesion, differentiation and angiogenesis, which are important properties for normal and cancerous cell function. We recently developed particle clusters coated with hyaluronan (termed gagomers; GAG), and showed that they can deliver the insoluble drug paclitaxel directly into CD44-over-expressing tumors in a mouse tumor model. Here, we tested primary head and neck cancers (HNC) and normal cells taken from the same patient, and found that although CD44 expression in both types of cells was high, GAGs bind only to the cancerous cells in a selective manner. We next formulated the anti cancer agent mitomycin C (MMC) in the GAGs. MMC-based chemoradiation is a potential treatment for HNC, however, due to patient’s toxicity, MMC is not part of the standard treatment of HNC. MMC encapsulation efficiency was about 70% with a half-life drug efflux of 1.2 ± 0.3 days. The Ex vivo study of the targeted MMC-GAG showed significant increase in the therapeutic effect on HNC cells (compared to free MMC), while it had no effect on normal cells taken from the same patient. These results demonstrate the specificity of the nanovectors towards head and neck cancers, which might be applicable as future therapy to many CD44-expressing tumors.
Keywords: Hyaluronan; CD44; Nanomedicine; Head and neck cancers;

A cationic prodrug/therapeutic gene nanocomplex for the synergistic treatment of tumors by Xiao Lu; Qing-Qing Wang; Fu-Jian Xu; Gu-Ping Tang; Wan-Tai Yang (4849-4856).
The combination of gene therapy and chemotherapy may increase the therapeutic efficacy in the treatment of patients. In this work, the cationic polymer prodrug/plasmid nanocomplexes were designed to in vivo synergistically treat drug-resistant breast tumors. Cationic β-cyclodextrin-polyethylenimine-Dox (PC-Dox) conjugates were prepared for carrying wt p53 plasmid in the form of PC-Dox/p53 nanocomplexes to achieve synergistic cancer therapeutic effects of drug and gene therapies. Such PC-Dox/p53 nanocomplexes ensure that both drug and gene can be delivered to the same cancer cells. The physicochemical properties and Dox release profiles of the PC-Dox conjugates, as well as their antitumor activities in vitro and in vivo, were determined. mRNA expression and western blot experiments also proved that co-delivery of Dox with wt p53 plasmid from PC-Dox/wt p53 complexes could promote wt p53 gene expression largely. By investigating anticancer efficacy via multi-drug resistant MCF-7/Adr breast cancer cells, it was found that PC-Dox/wt p53 complexes promoted the inhibition of tumor growth in vivo and prolonged the survival time of tumor-bearing mice. With the efficient ability to co-deliver drug and gene, such multifunctional PC-Dox/pDNA complexes should have great potential applications in cancer therapy.
Keywords: DNA; Gene therapy; Vectors; Transfection; Wild-type p53; Doxorubicin;

In spite of tremendous demand for the development and implementation of effective therapeutic strategies, limitations are still associated with doxorubicin-induced cardiotoxicity. Arjunolic acid (AA) has been shown to possess a multitude of biological functions. The purpose of the present study was to explore whether AA plays any protective role against doxorubicin-induced cardiotoxicity; and if so, what molecular mechanism it utilizes for its protective action. In rat cardiomyocytes, doxorubicin administration activated the proapoptotic p53, p38 and JNK MAPKs, Bax translocation, disrupted mitochondrial membrane potential, precipitated mitochondrion mediated caspase-dependent apoptotic signalling and reduced viability of cardiomyocytes. Doxorubicin exposure increases dichlorofluorescein (DCF) intensity corresponding to the intracellular H2O2 generation in myocytes; catalase (CAT) treatment, however, reduced this intensity and preserves cell viability. Intracellular H2O2 thus produced now activates the p38-JNK and p53-mediated pathways. CAT treatment also markedly decreased the doxorubicin-mediated activation of p38 and JNK, suggesting that H2O2 is involved in the activation of MAPKs. Blockage of p53 and p38-JNK by pharmacological inhibitors also suppressed the doxorubicin-induced apoptosis with the concomitant inhibition of anti-apoptotic Bcl-2 family proteins. AA treatment ameliorates nearly all of these apoptotic actions of doxorubicin and preserves cell viability. Similarly, rats treated with doxorubicin displayed retarded growth of body and heart as well as elevated apoptotic indices in heart tissue, whereas AA treatment effectively neutralised all these doxorubicin-induced cardiac-abnormalities. Combining all, our results suggest that doxorubicin induces cardiac apoptosis via the activation of JNK-p38 and p53-mediated signalling pathways, where H2O2 acts as the mediators of these pathways. AA can effectively and extensively counteract this action of doxorubicin, and may potentially protect the heart and cardiomyocytes from the severe doxorubicin-induced cardiovascular burden.
Keywords: Doxorubicin; Cardiac oxidative stress; p53; Apoptosis; Arjunolic acid; Cardioprotection;

The behavior after intravenous injection in mice of multiwalled carbon nanotube / Fe3O4 hybrid MRI contrast agents by Huixia Wu; Gang Liu; Yeming Zhuang; Dongmei Wu; Haoqiang Zhang; Hong Yang; He Hu; Shiping Yang (4867-4876).
Fe3O4 nanoparticles were in situ loaded on the surface of multiwalled carbon nanotubes (MWCNTs) by a solvothermal method using diethylene glycol and diethanolamine as solvents and complexing agents. The as-prepared MWCNT/Fe3O4 hybrids exhibited excellent hydrophilicity, superparamagnetic property at room temperature, and a high T 2 relaxivity of 175.5 mM−1 s−1 in aqueous solutions. In vitro experiments revealed that MWCNT/Fe3O4 had an excellent magnetic resonance imaging (MRI) enhancement effect on cancer cells, and importantly, they displayed low cytotoxicity and neglectable hemolytic activity. After intravenous administration, the T 2-weighted MRI signal in the liver and spleen of mice decreased significantly, suggesting the potential application of the hybrids as MRI contrast agents. The organ biodistribution studies, histological analyses and elimination investigations showed that the hybrids were uptaken by the liver, lung and spleen after intravenous injection, and could be excreted from the liver and kidney.
Keywords: Carbon nanotubes; Magnetic nanoparticles; Magnetic resonance imaging; Hemolysis; Biodistribution; Elimination;

Surface-enhanced Raman scattering-based signal detection and molecular identification faces the lack of reproducibility and reliability thus hampers its practical applications. Here, we demonstrate a facile particle mediated aggregation protocol to synthesize highly roughened mesosuperstructure - silver polyhedral mesocages. The individual silver octahedral mesocage, owing to highly-roughed surface topography, anisotropic growth as well as intraparticle effect, creates homogenously distributed multiple effective hot spots on the surface of single mesoparticle, hereby exhibits a high reproducibility and an unusual SERS enhancement, i.e., ∼108–109 magnitude. As such, the current protocol opens avenues for the fabrication of structurally reproducible mesosuperstructure-based SERS sensors.
Keywords: Single particle SERS; Polyhedral Ag mesocages; Biosensor; Ag2O template;

The intrinsic redox reactions of polyamic acid derivatives and their application in hydrogen peroxide sensor by Mu-Yi Hua; Hsiao-Chien Chen; Cheng-Keng Chuang; Rung-Ywan Tsai; Jyh-Long Jeng; Hung-Wei Yang; Yaw-Terng Chern (4885-4895).
Polyamic acids (PAAs) containing benzothiazole (BT) and benzoxazole (BO) pendent groups (PAA-BT and PAA-BO, respectively) which possessed electroactivity were synthesized successfully. The addition of H2O2 chemically oxidized the intrinsic carboxylic acid groups of PAA to form peroxy acid groups, and the peroxy acid further oxidized the electroactive sites of BT and BO to form N-oxides. The N-oxides could be reverted to their original form by electrochemical reduction, thus increasing the electrochemical reductive current. Based on this mechanism, enzyme-free hydrogen peroxide (H2O2) biosensors were prepared by modifying gold electrodes with the PAA derivatives (PAA-BT/Au and PAA-BO/Au, respectively). These biosensors had rapid response times (3.9–5.2 s) and high selectivity and sensitivity (280.6–311.2 μA/mM-cm2). A comparison of the PAA-BT/Au and PAA-BO/Au electrodes with electrodes prepared using polyamide-BT or polyamide-BO (i.e., lacking the carboxylic acid groups) confirmed the mechanism by which PAA derivatives detect H2O2. Modifying the surface morphology of the electrode from a planar to a three-dimensional (3D) configuration enhanced the performance of the PAA-BO/Au electrode. The sensitivity of the 3D-PAA-BO/Au electrode was 1394.9 μA/mM-cm2, ∼4.5 times higher than that of the planar electrode. The detection limit was also enhanced from 5.0 to 1.43 μM. The biosensor was used analytically to detect and measure H2O2 in urine samples collected from healthy individuals and patients suffering from noninvasive bladder cancer. The results were promising and comparable to that measured by a classical HPLC method, which verified the developed biosensor had a potential to provide a usefully analytical approach for bladder cancer.
Keywords: Polyamic acid; Hydrogen peroxide; Enzyme-free; Biosensor; Benzothiazole; Benzoxazole;

Control of gene transfer on a DNA–fibronectin–apatite composite layer by the incorporation of carbonate and fluoride ions by Yushin Yazaki; Ayako Oyane; Yu Sogo; Atsuo Ito; Atsushi Yamazaki; Hideo Tsurushima (4896-4902).
Gene transfer techniques are useful tools for controlling cell behavior, such as proliferation and differentiation. We have recently developed an efficient area-specific gene transfer system using a DNA–fibronectin–apatite composite layer (DF-Ap layer). In this system, partial dissolution of the composite layer is likely to be a crucial step for gene transfer. In the present study, layer solubility was adjusted by incorporating various contents of carbonate or fluoride ions into the DF-Ap layer via ionic substitution for the apatite crystals. Carbonate ion incorporation increased the solubility of the DF-Ap layer, thereby increasing the efficiency of gene transfer on the layer. In contrast, the incorporation of fluoride ions decreased the solubility of the DF-Ap layer, thereby decreasing the efficiency and delaying the timing of gene transfer on the layer dose-dependently. The present gene transfer system with controllable efficiency and timing would be useful in tissue engineering applications because cell differentiation can be induced effectively by regulating appropriate gene expression with suitable timing.
Keywords: Gene transfer; Fibronectin; Hydroxyapatite composite; Hydroxyapatite coating; Controlled drug release;

Spatial and temporal concentration gradients of chemoattractants direct many biological processes, especially the guidance of immune cells to tissue sites during homeostasis and responses to infection. Such gradients are ultimately generated by secretion of attractant proteins from single cells or collections of cells. Here we describe cell-sized chemoattractant-releasing polysaccharide microspheres, capable of mimicking chemokine secretion by host cells and generating sustained bioactive chemokine gradients in their local microenvironment. Exploiting the common characteristic of net cationic charge and reversible glycosaminoglycan binding exhibited by many chemokines, we synthesized alginate hydrogel microspheres that could be loaded with several different chemokines (including CCL21, CCL19, CXCL12, and CXCL10) by electrostatic adsorption. These polysaccharide microspheres subsequently released the attractants over periods ranging from a few hours to at least 1 day when placed in serum-containing medium or collagen gels. The generated gradients were able to attract cells more than hundreds of microns away to make contact with individual microspheres. This versatile system for chemoattractant delivery could find applications in immunotherapy, vaccines and fundamental chemotaxis studies in vivo and in vitro.
Keywords: Alginate; Hydrogel; Microsphere; Chemokine; Chemotaxis; Leukocyte;

Cationic polymers are potential intracellular carriers for small interfering RNA (siRNA). The short and rigid nature of an siRNA chain often results in larger and more loosely packed particles compared to plasmid DNA (pDNA) after complexing with carrier polycations, and in turn, poor silencing effects are seen against the target mRNAs. A helper polyanion, pDNA, was incorporated along with siRNA to form compact nanosized polyplexes. At C/A (cation/anion) ratios of 2 and 5, poly(l-lysine) (PLL)/siRNA-pGFP and PLL/siRNA-pGFP-OSDZ (oligomeric sulfadiazine (OSDZ) for endosomolysis) complexes produced particles 90–150 nm in size with a 15–45 mV surface charge, while PLL/siRNA complexes yielded particles 1–2 μm in size at the same C/A ratios. The PLL/siRNA-pGFP (C/A 2) complexes showed significantly higher specific gene silencing (50–90% vs. 10–25%) than the complexes formed at C/A 5. PLL/siRNA-pGFP-OSDZ (C/A 2) complexes improved the specific gene silencing (90%) more dramatically than PLL/siRNA-pGFP (C/A 2) complexes (50%), demonstrating a potential role for OSDZ. PLL/siRNA-pGFP-OSDZ (C/A 2) complexes sustained higher specific gene silencing compared with PLL/siRNA-pGFP (C/A 2) complexes. Other oligomeric sulfonamides (OSA) with varying pKa used in PLL/siRNA-pGFP-OSA complexes also caused effective gene silencing. The pGFP in the PLL/siRNA-pGFP complexes successfully expressed GFP protein without interfering with the siRNA. In conclusion, this study demonstrates that long pDNA helps effectively form nanosized siRNA particles and that OSA enhances specific gene silencing. In a single nucleic acid carrier formulation, co-delivery of siRNA and pDNA is feasible to maximize therapeutic effects or to include therapeutic or diagnostic functionalities.
Keywords: Co-delivery; Oligomeric sulfonamide; pDNA delivery; Polymer gene delivery; siRNA delivery;

Although immunosuppressive agents play a pivotal role in the success of organ transplantation, chronic toxicity has been a major issue for long-term treatment. The development of therapies that induce donor-specific immunological tolerance remains an important clinical challenge. In the present study, we investigated the underlying mechanisms and applications of prostaglandin (PG) E2 for the induction of immunological tolerance in mice with concanavalin A(Con A)-induced immune-mediated liver injury. The immunological tolerogenic effect of 16,16 dimethyl PGE2 (dmPGE2) pretreatment in C57B/6 male mice with Con A-induced liver injury was observed, and it was revealed that its response was partially associated with the expression of interleukin (IL)-10, an anti-inflammatory cytokine, in Kupffer cells. To apply native eicosanoids of PGE2 for tolerance induction in vivo, PGE2 was incorporated into l-lactic acid oligomer-grafted pullulan of an amphiphilic polymer to form a nano-sized hydrogel (PGE2-nanogel). Pharmacokinetics studies revealed that nanogel incorporation enabled PGE2 to have a prolonged life-time in circulating blood, and a tolerogenic effect was also observed in Con A-induced liver injury, the same as with dmPGE2 pretreatment. Nanogel-based prostaglandin administration might be developed as a therapeutic agent to induce immunological tolerance, which is necessary in allogenic organ and cell transplantation.
Keywords: Liver; Immunological tolerance; PGE2; Nanoparticle (nanogel);

In vitro and in vivo complement activation and related anaphylactic effects associated with polyethylenimine and polyethylenimine-graft-poly(ethylene glycol) block copolymers by Olivia M. Merkel; Rudolf Urbanics; Peter Bedőcs; Zoltán Rozsnyay; László Rosivall; Miklós Toth; Thomas Kissel; Janos Szebeni (4936-4942).
Complement activation by polymeric gene and drug delivery systems has been overlooked in the past. As more reports appear in the literature concerning immunogenicity of polymers and their impact on gene expression patterns, it is important to address possible immune side effects of polymers, namely complement activation. Therefore, in this study the activity of low and high molecular weight poly(ethylene imine) and two PEGylated derivatives to induce complement activation were investigated in human serum. These in vitro results revealed that PEI 25 kDa caused significant and concentration dependent complement activation, whereas none of the other polymers induced such effects at their IC50 concentrations determined by MTT-assays. To verify these in vitro results, additionally, studies were carried out in a swine model after intravenous administration, showing complement activation-related pseudoallergy (CARPA), reflected in symptoms of transient cardiopulmonary distress. Injections of PEI 25 kDa or PEI(25k)–PEG(2k)10 at a dose of 0.05 and 0.1 mg/kg caused strong reactivity, while PEI 5 kDa and with PEI(25k)–PEG(20k)1 were also reactogenic at 0.1 mg/kg. It was found that PEI 25 kDa caused both self- and cross-tolerance, whereas the PEG–PEIs were neither self- nor cross-reactively tachyphylactic. As a result of this study, it was shown that PEGylation of polycations with PEG of 20 kDa or higher molecular weight may be favorable. However, potential safety concerns in the development of PEI-based polymeric carriers for drugs and nucleic acids and their translation from bench to bedside need to be taken into consideration for human application.
Keywords: PEI; PEGylation; Complement activation; CARPA; Swine model; Cardiopulmonary distress;

Targeting the brain with PEG–PLGA nanoparticles modified with phage-displayed peptides by Jingwei Li; Liang Feng; Li Fan; Yuan Zha; Liangran Guo; Qizhi Zhang; Jun Chen; Zhiqing Pang; Yuchen Wang; Xinguo Jiang; Victor C. Yang; Longping Wen (4943-4950).
The relative impermeability of the blood-brain barrier (BBB) results from tight junctions and efflux transport systems limits drug delivery to the central nervous system (CNS), and thus severely restricts the therapy of many central nervous system diseases. In order to enhance the brain-specific drug delivery, we employed a 12-mer phage display peptide library to isolate peptides that could target the drug delivery system to the brain. A 12-amino-acid-peptide (denoted as Pep TGN) which was displayed by bacteriophage Clone 12-2 was finally selected by rounds of in vivo screening. Pep TGN was covalently conjugated onto the surface of poly (ethyleneglycol)-poly (lactic-co-glycolic acid) (PEG–PLGA) based nanoparticles (NPs). The cellular uptake of Pep TGN decorated nanoparticles was significantly higher than that of unmodified nanoparticles when incubated with bEnd.3 cells. Enhanced brain accumulation efficiency together with lower accumulation in liver and spleen was observed in the nude mice intravenously injected with Pep TGN conjugated nanoparticles compared with those injected with plain nanoparticles, showing powerful brain selectivity of Pep TGN. Coumarin 6 was used as a fluorescent probe for the evaluation of brain delivery properties. The brain Drug Targeting Index (DTI) of coumarin 6 incorporated in targeted nanoparticles was significantly higher than that of coumarin 6 incorporated in plain nanoparticles. In conclusion, the Pep TGN is a motif never been reported before and Pep TGN modified nanoparticles showed great potential in targeted drug delivery across the blood brain barrier.
Keywords: Phage display; Blood-brain barrier (BBB); Brain delivery; Biodegradable nanoparticle;

Target specific systemic delivery of TGF-β siRNA/(PEI-SS)-g-HA complex for the treatment of liver cirrhosis by Kitae Park; Sung Woo Hong; Wonhee Hur; Min-Young Lee; Jeong-A. Yang; Sung Woo Kim; Seung Kew Yoon; Sei Kwang Hahn (4951-4958).
A target specific systemic delivery system of siRNA therapeutics was successfully developed using reducible polyethyleneimine grafted hyaluronic acid [(PEI-SS)-g-HA] conjugates. The PEI-SS was synthesized by Michael addition of low molecular weight PEI (MW = 2000) with cystaminebisacrylamide (CBA), and grafted to carboxyl groups of HA via amide bond formation after activation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole monohydrate (HOBt). The confocal microscopic and fluorometric analyses confirmed the effective cellular uptake of siRNA/(PEI-SS)-g-HA complex by HA receptor mediated endocytosis. In vitro gene silencing efficiency was ca. 80% in the presence of 10 vol% serum and ca. 50% in the presence of 50 vol% serum in B16F1 melanoma cells and activated hepatic stellate cells (HSCs). Furthermore, target specific systemic delivery of apolipoprotein B (ApoB) siRNA/(PEI-SS)-g-HA complex resulted in a drastically reduced ApoB mRNA level down to ca. 20% in a dose-dependent manner. Finally, TGF-β siRNA/(PEI-SS)-g-HA complex showed a feasible therapeutic effect on liver cirrhosis with a significantly reduced nodule formation, collagen content, and HSC number. The siRNA/(PEI-SS)-g-HA complex can be exploited for the target specific systemic treatment of various liver diseases.
Keywords: Hyaluronic acid; Polyethyleneimine; siRNA; Targeted systemic delivery; Liver cirrhosis;

Physicochemical properties, such as particle size, shape, molecular weight, surface charge and composition, play a key role in the cellular uptake of polymeric nanoparticles. Antigen-encapsulated biodegradable nanoparticles have considerable potential for use in vaccine delivery systems. Although it is accepted that particle size is important for the induction of antigen-specific immune responses in vivo, little is known about how their size affects their intracellular fate. Here, we demonstrate that the size effects on the cellular uptake, intracellular degradation and distribution of protein-encapsulated nanoparticles. We prepared size-regulated ovalbumin (OVA)-encapsulated nanoparticles composed of hydrophobically modified poly(γ-glutamic acid) (γ-PGA). These nanoparticles were efficiently taken up by macrophages, and also delivered encapsulated OVA from the endosomes to the cytoplasm. Comparing 40–200 nm-sized nanoparticles, there was no significant difference in their intracellular distribution. Interestingly, the size of the nanoparticles affected the intracellular degradation of the encapsulated OVA. The uptake of OVA alone by macrophages resulted in early degradation of the OVA. In contrast, the degradation of OVA encapsulated into the nanoparticles was attenuated as compared to free OVA. A difference in OVA degradation kinetics was observed between the particle sizes, the degradation of small nanoparticles was slower than for the larger ones. These results indicate that particle size is an important factor for the intracellular degradation of encapsulated proteins and nanoparticles. These results will provide a rational design of nanoparticle-based vaccines to control immune responses.
Keywords: Nanoparticles; Intracellular degradation; Particle size; Vaccine; Macrophages;

A brain-targeted rabies virus glycoprotein-disulfide linked PEI nanocarrier for delivery of neurogenic microRNA by Do Won Hwang; Sejin Son; Jaeho Jang; Hyewon Youn; Song Lee; Duhwan Lee; Yun-Sang Lee; Jae Min Jeong; Won Jong Kim; Dong Soo Lee (4968-4975).
Recent advances in efficient microRNA (miRNA) delivery techniques using brain-targeted nanoparticles offer critical information for understanding the functional role of miRNAs in vivo, and for supporting targeted gene therapy in terms of treating miRNA-associated neurological diseases. Here, we report the rabies virus glycoprotein (RVG)-labeled non-toxic SSPEI nanomaterials capable of neuron-specific miR-124a delivery to neuron in vivo. The RVG-labeled BPEI-SS (RVG-SSPEI) nanocarrier showed less toxicity in acetylcholine receptor-positive Neuro2a cells, and electrostatic interaction of RVG-SSPEI with miR-124a exhibited optimal transfection efficacy. The RVG-SSPEI polymer specifically targeted Neuro2a using cy5.5-miR-124a mixed with RVG-SSPEI. The functional action of miR-124a oligomers released from polyplexes in the cytoplasmic region was evaluated by a reporter vector containing a miR-124a -binding sequence, and showed a significantly reduced reporter signal in a dose-dependent manner. Cy5.5-miR-124a/RVG-SSPEI- injected into mice via tail veins displayed the enhanced accumulation of miR-124a in the isolated brain. Hindrance of the efficient penetration of neuronal cells by size limitation of the miR-124a/RVG-SSPEI improved with the help of mannitol through blood-brain barrier disruption. These findings indicated that the RVG peptide combined with mannitol infusion using SSPEI polymer for neuron-specific targeting in vivo is sufficient to deliver neurogenic microRNA into the brain.
Keywords: MicroRNA; RVG peptide; PEI polymer; Brain-targeting agent; Polyplex;

Hollow chitosan–silica nanospheres as pH-sensitive targeted delivery carriers in breast cancer therapy by Ziwei Deng; Zipeng Zhen; Xiaoxi Hu; Shuilin Wu; Zushun Xu; Paul K. Chu (4976-4986).
Promising drug nanocarriers consisting of mono-dispersed and pH sensitive chitosan–silica hollow nanospheres (CS–SiO2 HNPs) suitable for breast cancer therapy are produced and investigated. The SiO2 HNPs are fabricated using a one-step, one-medium process which obviates the need for post-treatment to remove the templates, additional dissolution, or calcination. Taking advantage of the cross-linking reaction with (3-Glycidyloxypropyl) trimethoxysilane (GTPMS), cationic polysaccharide-chitosan decorates the surface and produces pH sensitive CS–SiO2 HNPs. The materials enable controlled release of loaded drugs in pericellular and interstitial environments. In particular, the antibody molecule (to ErbB 2) can be conjugated onto the surface of the CS–SiO2 HNPs thereby allowing the hollow nanospheres to serve as a targeted delivery agent to breast cancer cells. TNF-α are delivered to MCF-7 breast cancer cells under both in vitro and in vivo conditions to suppress the growth of cancerous cells and even kill them with high therapeutic efficacy. Owing to their hollow inner cavity and porous structures, the CS–SiO2 HNPs are excellent pH-responsive targeted nanocarriers.
Keywords: Silica; Chitosan; Hollow nanospheres; pH responsive; Drug release; Breast cancer therapy;