Biomaterials (v.32, #11)

The prevention of biofilm colonization by multidrug-resistant pathogens that cause ventilator-associated pneumonia with antimicrobial-coated endotracheal tubes by Issam I. Raad; Jamal A. Mohamed; Ruth A. Reitzel; Ying Jiang; Tanya L. Dvorak; Mahmoud A. Ghannoum; Ray Y. Hachem; Anne-Marie Chaftari (2689-2694).
Ventilator-associated pneumonia (VAP) continues to be the nosocomial infection associated with the highest mortality in critically ill patients. Since silver-coated endotracheal tubes (ETT) was shown in a multicenter prospective randomized trials to decrease the risk of VAP, we compared the efficacy of two antiseptic agents such as gardine- and gendine-coated ETTs with that of silver-coated ETTs in preventing biofilm. The ETTs were tested for their ability to prevent the biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, and Candida albicans. Scanning electron microscopy studies revealed a heavy biofilm on uncoated and silver-coated ETT but not on the gardine-coated ETT. The gardine and gendine ETTs completely inhibited the formation of biofilms by all organisms tested and were more effective in preventing biofilm growth than the silver ETTs (p < 0.001). The gardine- and gendine-coated ETTs were more durable against MRSA than either the silver-coated or uncoated ETTs for up to 2 weeks (p < 0.0001). We have therefore shown that gardine- and gendine-coated ETTs are superior to silver-coated ETTs in preventing biofilm. Future animal and clinical studies are warranted to determine whether the gardine- and gendine-coated ETTs can significantly reduce the risk of VAP.
Keywords: Endotracheal tube; Biofilm; Pneumonia; Silver; Gardine; Gendine;

A mineralized polymeric matrix has been extensively studied to understand biomineralization processes and to further regulate phenotypic functions of various cells involved in osteogenesis and physiological homeostasis. It has been often proposed that several matrix variables including charge density, hydrophobicity, and pore size play vital roles in modulating composition and morphology of minerals formed within a three dimensional (3D) matrix. However, the aspects have not yet been systematically examined because a tool enabling the independent control of the matrix variables is lacking. This study presents an advanced integrative strategy to control morphology and composition of biominerals with matrix properties, by using a hydrogel formulated to independently control charge density, hydrophobicity, and porosity. The hydrogel consists of poly(ethylene glycol) monomethacrylate (PEGmM), poly(propylene glycol) monomethacrylate (PPGmM), and methacrylic alginate (MA), so the charge density and hydrophobicity of the hydrogel can be separately controlled with mass fractions of MA and PPGmM. Also, hydrogels which present only nano-sized pores, termed nanoporous hydrogels, are lyophilized and rehydrated to prepare the hydrogels containing micro-sized pores, termed microporous hydrogels. We find that increasing the mass fractions of MA and PPGmM of the microporous hydrogel promotes the growth of apatite layers because of the increases in the charge density, hydrophobicity and pore size. In contrast, increasing mass fractions of MA and PPGmM of the nanoporous hydrogel enhances the formation of calcium carbonate minerals. The dependency of the mineralization on hydrogel variables is related to the change in supersaturation of mineral ions. Overall, the results of this study will be highly useful to better understand the interplay of matrix variables in biomineralization and to design a wide array of mineralized matrix potentially used in cell therapies and tissue engineering.
Keywords: Biomineralization; Hydrogel; Hydrophobicity; Charge density; Pore size;

A photopolymerized antimicrobial hydrogel coating derived from epsilon-poly-l-lysine by Chuncai Zhou; Peng Li; Xiaobao Qi; Abdul Rahim Mohamed Sharif; Yin Fun Poon; Ye Cao; Matthew W. Chang; Susanna Su Jan Leong; Mary B. Chan-Park (2704-2712).
Hydrogels made from epsilon-poly-l-lysine-graft-methacrylamide (EPL-MA) have been found to have impressive wide spectrum antimicrobial activity against both bacteria (specifically Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus) and fungi (specifically Candida albicans and Fusarium solani). The EPL-MA hydrogel also possesses in vitro biocompatibility and EPL-MA solution is relatively non-hemolytic: the concentration needed for onset of human red blood cell (hRBC) hemolysis is 12,500 μg/mL so that the selectivity for the pathogenic microorganisms over hRBCs is 230–1560. Further, EPL-MA hydrogel can be conveniently ultraviolet-immobilized onto plasma-treated plastic surfaces to form thin highly adherent antimicrobial hydrogel coatings for medical devices and implants.
Keywords: Antimicrobial peptide; Contact-active coating; Hydrogel; Hemolysis; Epsilon-poly-l-lysine; Graft;

Systemic distribution, nuclear entry and cytotoxicity of amorphous nanosilica following topical application by Hiromi Nabeshi; Tomoaki Yoshikawa; Keigo Matsuyama; Yasutaro Nakazato; Kazuhiko Matsuo; Akihiro Arimori; Masaaki Isobe; Saeko Tochigi; Sayuri Kondoh; Toshiro Hirai; Takanori Akase; Takuya Yamashita; Kohei Yamashita; Tokuyuki Yoshida; Kazuya Nagano; Yasuhiro Abe; Yasuo Yoshioka; Haruhiko Kamada; Takayoshi Imazawa; Norio Itoh; Shinsaku Nakagawa; Tadanori Mayumi; Shin-ichi Tsunoda; Yasuo Tsutsumi (2713-2724).
Currently, nanomaterials (NMs) with particle sizes below 100 nm have been successfully employed in various industrial applications in medicine, cosmetics and foods. On the other hand, NMs can also be problematic in terms of eliciting a toxicological effect by their small size. However, biological and/or cellular responses to NMs are often inconsistent and even contradictory. In addition, relationships among NMs physicochemical properties, absorbency, localization and biological responses are not yet well understood. In order to open new frontiers in medical, cosmetics and foods fields by the safer NMs, it is necessary to collect the information of the detailed properties of NMs and then, build the prediction system of NMs safety. The present study was designed to examine the skin penetration, cellular localization, and cytotoxic effects of the well-dispersed amorphous silica particles of diameters ranging from 70 nm to 1000 nm. Our results suggested that the well-dispersed amorphous nanosilica of particle size 70 nm (nSP70) penetrated the skin barrier and caused systemic exposure in mouse, and induced mutagenic activity in vitro. Our information indicated that further studies of relation between physicochemical properties and biological responses are needed for the development and the safer form of NMs.
Keywords: Nanoparticles; Silica; Transdermal penetration; Cytotoxicity; Mutagenicity;

Directional cell migration induced by a mechanical gradient on a substrate surface toward a harder region, so-called mechanotaxis or durotaxis, has recently drawn attention not only in the field of mechanobiology but also for possible cell manipulation in biomedical engineering. Before we can use mechanotaxis to control cell migration on a biomaterial surface, quantitative design criteria for a microelasticity gradient should be established. To clarify the conditions required to induce mechanotaxis, the effects of a microelasticity boundary on cell culture hydrogels have been systematically assessed with regard to fibroblast migration based on a custom-built reduction projection-type photolithographic microelasticity patterning system with elasticity-tunable photocurable styrenated gelatins, which is a thoroughly-improved system of our previous simple photomasking method . As a result, the conditions required to induce mechanotaxis were found to include a certain threshold jump in elasticity (30–40 kPa) and a sufficiently narrow width of the elasticity boundary (50 μm) comparable to a single cell’s adhered area, i.e., a sufficiently high gradient strength (30–40 kPa/50 μm in our gelatinous gel system). A significant asymmetric distribution of the number and size of focal adhesions across the elasticity boundary was confirmed to be one of the driving factors of mechanotaxis by indirect immunofluorescence microscopy, and mechanistic considerations in the design criteria are discussed.
Keywords: Mechanotaxis; Durotaxis; Cell migration; Styrenated gelatin; Microelasticity patterning;

Angiogenesis in ischemic tissue produced by spheroid grafting of human adipose-derived stromal cells by Suk Ho Bhang; Seung-Woo Cho; Wan-Geun La; Tae-Jin Lee; Hee Seok Yang; Ah-Young Sun; Sang-Hong Baek; Jong-Won Rhie; Byung-Soo Kim (2734-2747).
Stem cells offer significant therapeutic promise for the treatment of ischemic disease. However, stem cells transplanted into ischemic tissue exhibit limited therapeutic efficacy due to poor engraftment in vivo. Several strategies for improving the survival and engraftment of stem cells in ischemic tissue have been developed including transplantation in combination with growth factor delivery, genetic modification of stem cells, and the use of cell-transplantation scaffolds. Here, we demonstrate that human adipose-derived stromal cells (hADSCs) cultured and grafted as spheroids exhibit improved therapeutic efficacy for ischemia treatment. hADSCs were cultured in monolayer or spheroids. Spheroid cultures were more effective in preconditioning hADSCs to a hypoxic environment, upregulating hypoxia-adaptive signals (i.e., stromal cell-derived factor-1α and hypoxia-inducible factor-1α), inhibiting apoptosis, and enhancing secretion of both angiogenic and anti-apoptotic factors (i.e., hepatocyte growth factor, vascular endothelial growth factor, and fibroblast growth factor 2) compared to monolayer cultures. Moreover, cell harvesting following spheroid cultures avoided damage to extracellular matrices due to harsh proteolytic enzyme treatment, thereby preventing anoikis (apoptosis induced by a lack of cell–matrix interaction). Following intramuscular transplantation to ischemic hindlimbs of athymic mice, hADSC spheroids showed improved cell survival, angiogenic factor secretion, neovascularization, and limb survival as compared to hADSCs grafted as dissociated cells. Taken together, spheroid cultures precondition hADSCs to a hypoxic environment, and grafting hADSCs as spheroids to ischemic limbs improves therapeutic efficacy for ischemia treatment due to enhanced cell survival and paracrine effects. Spheroid-based cell delivery could be a simple and effective strategy for improving stem cell therapy for ischemic diseases, eliminating the need for growth factor delivery, biomaterial scaffolds or genetic modification.
Keywords: Human adipose stromal cells; Spheroid; Therapeutic angiogenesis;

Recapitulation of the embryonic cardiovascular progenitor cell niche by Katja Schenke-Layland; Ali Nsair; Ben Van Handel; Ekaterini Angelis; Jessica M. Gluck; Miriam Votteler; Joshua I. Goldhaber; Hanna K. Mikkola; Michael Kahn; William R. MacLellan (2748-2756).
Stem or progenitor cell populations are often established in unique niche microenvironments that regulate cell fate decisions. Although niches have been shown to be critical for the normal development of several tissues, their role in the cardiovascular system is poorly understood. In this study, we characterized the cardiovascular progenitor cell (CPC) niche in developing human and mouse hearts, identifying signaling pathways and extracellular matrix (ECM) proteins that are crucial for CPC maintenance and expansion. We demonstrate that collagen IV (ColIV) and β-catenin-dependent signaling are essential for maintaining and expanding undifferentiated CPCs. Since niches are three-dimensional (3D) structures, we investigated the impact of a 3D microenvironment that mimics the in vivo niche ECM. Employing electrospinning technologies, 3D in vitro niche substrates were bioengineered to serve as culture inserts. The three-dimensionality of these structures increased mouse embryonic stem cell differentiation into CPCs when compared to 2D control cultures, which was further enhanced by incorporation of ColIV into the substrates. Inhibiting p300-dependent β-catenin signals with the small molecule IQ1 facilitated further expansion of CPCs. Our study represents an innovative approach to bioengineer cardiac niches that can serve as unique 3D in vitro systems to facilitate CPC expansion and study CPC biology.
Keywords: Extracellular matrix; Cardiac tissue engineering; Stem cell; Scaffold; Heart;

A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics by Alexander Hoppe; Nusret S. Güldal; Aldo R. Boccaccini (2757-2774).
Several inorganic materials such as special compositions of silicate glasses, glass-ceramics and calcium phosphates have been shown to be bioactive and resorbable and to exhibit appropriate mechanical properties which make them suitable for bone tissue engineering applications. However, the exact mechanism of interaction between the ionic dissolution products of such inorganic materials and human cells are not fully understood, which has prompted considerable research work in the biomaterials community during the last decade. This review comprehensively covers literature reports which have investigated specifically the effect of dissolution products of silicate bioactive glasses and glass-ceramics in relation to osteogenesis and angiogenesis. Particularly, recent advances made in fabricating dense biomaterials and scaffolds doped with trace elements (e.g. Zn, Sr, Mg, and Cu) and investigations on the effect of these elements on the scaffold biological performance are summarized and discussed in detail. Clearly, the biological response to artificial materials depends on many parameters such as chemical composition, topography, porosity and grain size. This review, however, focuses only on the ion release kinetics of the materials and the specific effect of the released ionic dissolution products on human cell behaviour, providing also a scope for future investigations and identifying specific research needs to advance the field. The biological performance of pure and doped silicate glasses, phosphate based glasses with novel specific compositions as well as several other silicate based compounds are discussed in detail. Cells investigated in the reviewed articles include human osteoblastic and osteoclastic cells as well as endothelial cells and stem cells.
Keywords: Bone tissue engineering; Bioactive glasses; Bioactivity; Metal ion release; Cell proliferation; Osteoblasts;

Spatially directed guidance of stem cell population migration by immobilized patterns of growth factors by Eric D. Miller; Kang Li; Takeo Kanade; Lee E. Weiss; Lynn M. Walker; Phil G. Campbell (2775-2785).
We investigated how engineered gradients of exogenous growth factors, immobilized to an extracellular matrix material, influence collective guidance of stem cell populations over extended time (>1 day) and length (>1 mm) scales in vitro. Patterns of low-to-high, high-to-low, and uniform concentrations of heparin-binding epidermal growth factor-like growth factor were inkjet printed at precise locations on fibrin substrates. Proliferation and migration responses of mesenchymal stem cells seeded at pattern origins were observed with time-lapse video microscopy and analyzed using both manual and automated computer vision-based cell tracking techniques. Based on results of established chemotaxis studies, we expected that the low-to-high gradient would most effectively direct cell guidance away from the cell source. All printed patterns, however, were found to direct net collective cell guidance with comparable responses. Our analysis revealed that collective “cell diffusion” down a cell-to-cell confinement gradient originating at the cell starting lines and not the net sum of directed individual cell migration up a growth factor concentration gradient is the principal driving force for directing mesenchymal stem cell population outgrowth from a cell source. These results suggest that simple uniform distributions of growth factors immobilized to an extracellular matrix material may be as effective in directing cell migration into a wound site as more complex patterns with concentration gradients.
Keywords: Growth factors; Scaffold; Stem cells; Migration; Bioprinting;

Self-organization, interfacial interaction and photophysical properties of gold nanoparticle complexes derived from resilin-mimetic fluorescent protein rec1-resilin by Sundar Mayavan; Naba K. Dutta; Namita R. Choudhury; Misook Kim; Christopher M. Elvin; Anita J. Hill (2786-2796).
In this investigation we report the synthesis of optically coupled hybrid architectures based on a new biomimetic fluorescent protein rec1-resilin and nanometer-scale gold nanoparticles (AuNPs) in a one-step method using a non-covalent mode of binding protocol. The presence of uniformly distributed fluorophore sequences, –Ser(Thr)-Tyr-Gly- along the molecular structure of rec1-resilin provides signnificant opportunity to synthesize fluorophore-modified AuNPs bioconjugates with unique photophysical properties. The detailed analyses of the AuNP-bioconjugates, synthesized under different experimental conditions using spectroscopic, microscopic and scattering techniques demonstrate the organizational pathways and the electronic and photophysical properties of the developed AuNP-rec1-resilin bioconjugates. The calculation of the bimolecular quenching constant using the Stern–Volmer equation confirms that the dominant mechanism involved in quenching of fluorescence of rec1-resilin in the presence of AuNP is static. Photoacoustic infrared spectroscopy was employed to understand the nature of the interfacial interaction between the AuNP and rec1-resilin and its evolution with pH. In such bioconjugates the quenched emission of fluorescence by AuNP on the fluorophore moiety of rec1-resilin in the immediate vicinity of the AuNP has significant potential for fluorescence-based detection schemes, sensors and also can be incorporated into nanoparticle-based devices.
Keywords: Biomimetic protein; Self-assembly; Gold-protein bioconjugates; Gold nanoparticles; Photophysical properties of bioconjugates;

The objective of this study is to evaluate the activity of gelatin hydrogels incorporating combined stromal cell-derived factor-1 (SDF-1) and bone morphogenetic protein-2 (BMP-2) on the in vivo bone regeneration at an ulna critical-sized defect and subcutaneous site of rats, and compared with that of those incorporating either SDF-1 or BMP-2. The similar release profile of SDF-1 and BMP-2 from the hydrogels was observed with or without the combination of BMP-2 and SDF-1, respectively. An enhanced bone regeneration by the hydrogels incorporating combined SDF-1 and BMP-2 was observed. In addition, the implantation of hydrogels incorporating combined SDF-1 and BMP-2 enhanced the expression level of CXC chemokine cell-surface receptor-4 (Cxcr4), Runt-related factor-2 (Runx2), and Osteocalcin genes. The experiments with green fluorescent protein (GFP)-positive Chimeric mice revealed that the recruitment of bone marrow-derived cells was promoted and a vascular-like structure together with strong accumulation of CD31- and CD34-positive cells was observed at the site of hydrogels incorporating combined SDF-1 and BMP-2 implanted. In addition, a large fraction of CD29- and CD44-positive non-hematopoietic cells was detected. It is concluded that the combined release of SDF-1 and BMP-2 enhanced the recruitment of osteogenic cells and angiogenesis, resulting in the synergistic effect on bone regeneration.
Keywords: Stromal cell-derived factor-1 (SDF-1); Bone morphogenetic protein-2 (BMP-2); Gelatin hydrogels; Combined release; Bone regeneration;

Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds by Sarindr Bhumiratana; Warren L. Grayson; Andrea Castaneda; Danielle N. Rockwood; Eun S. Gil; David L. Kaplan; Gordana Vunjak-Novakovic (2812-2820).
We describe a composite hydroxyapatite (HA)–silk fibroin scaffold designed to induce and support the formation of mineralized bone matrix by human mesenchymal stem cells (hMSCs) in the absence of osteogenic growth factors. Porous three-dimensional silk scaffolds were extensively used in our previous work for bone tissue engineering and showed excellent biodegradability and biocompatibility. However, silk is not an osteogenic material and has a compressive stiffness significantly lower than that of native bone. In the present study, we explored the incorporation of silk sponge matrices with HA (bone mineral) micro-particles to generate highly osteogenic composite scaffolds capable of inducing the in vitro formation of tissue-engineered bone. Different amounts of HA were embedded in silk sponges at volume fractions of 0%, 1.6%, 3.1% and 4.6% to enhance the osteoconductive activity and mechanical properties of the scaffolds. The cultivation of hMSCs in the silk/HA composite scaffolds under perfusion conditions resulted in the formation of bone-like structures and an increase in the equilibrium Young’s modulus (up to 4-fold or 8-fold over 5 or 10 weeks of cultivation, respectively) in a manner that correlated with the initial HA content. The enhancement in mechanical properties was associated with the development of the structural connectivity of engineered bone matrix. Collectively, the data suggest two mechanisms by which the incorporated HA enhanced the formation of tissue engineered bone: through osteoconductivity of the material leading to increased bone matrix production, and by providing nucleation sites for new mineral resulting in the connectivity of trabecular-like architecture.
Keywords: Tissue engineering; Bone; Scaffold; Silk; Hydroxyapatite; Mineralization;

Osteoblast function on electrically conductive electrospun PLA/MWCNTs nanofibers by Shijun Shao; Shaobing Zhou; Long Li; Jinrong Li; Chao Luo; Jianxin Wang; Xiaohong Li; Jie Weng (2821-2833).
The electrospinning process was utilized successfully to fabricate the random oriented and aligned electrically conductive nanofibers of biodegradable poly-DL-lactide (PLA) in which multiwalled carbon nanotubes (MWCNTs) were embedded. The topographical features of the composite nanofibers were characterized by SEM. The dispersion and alignment of MWCNTs in nanofiber matrix were observed by TEM. The in vitro degradation was characterized in terms of the morphological change, the mass loss and the reduction of polymer molecular weight as well as the decrease of pH value of degradation media. In particular, these conductive nanofiber meshes offered a unique system to study the synergistic effect of topographic cues and electrical stimulation on osteoblasts outgrowth as a way of exploring their potential application in bone tissue engineering. The results of obsteoblasts assay unstimulated showed that the aligned nanofibers as topographic cues could enhance the extension and direct the outgrowth of obsteoblasts better than random fibers. In the presence of direct current (DC) of 100 μA, the obsteoblasts on all samples grew along the electrical current direction. The cellular elongation and proliferation were mainly dependent on the electrical stimulation whereas the topographical features played a minor role in them. Therefore, electrical stimulation with an appropriate DC value imparted on conductive substrate had great potential in application of bone tissue engineering.
Keywords: Biodegradable; Carbon nanotube; Electrospinning; Conductive; Nanofiber;

The mechanical coupling of adult marrow stromal stem cells during cardiac regeneration assessed in a 2-D co-culture model by Mani T. Valarmathi; John W. Fuseler; Richard L. Goodwin; Jeffrey M. Davis; Jay D. Potts (2834-2850).
Postnatal cardiomyocytes undergo terminal differentiation and a restricted number of human cardiomyocytes retain the ability to divide and regenerate in response to ischemic injury. However, whether these neo-cardiomyocytes are derived from endogenous population of resident cardiac stem cells or from the exogenous double assurance population of resident bone marrow-derived stem cells that populate the damaged myocardium is unresolved and under intense investigation. The vital challenge is to ameliorate and/or regenerate the damaged myocardium. This can be achieved by stimulating proliferation of native quiescent cardiomyocytes and/or cardiac stem cell, or by recruiting exogenous autologous or allogeneic cells such as fetal or embryonic cardiomyocyte progenitors or bone marrow-derived stromal stem cells. The prerequisites are that these neo-cardiomyocytes must have the ability to integrate well within the native myocardium and must exhibit functional synchronization. Adult bone marrow stromal cells (BMSCs) have been shown to differentiate into cardiomyocyte-like cells both in vitro and in vivo. As a result, BMSCs may potentially play an essential role in cardiac repair and regeneration, but this concept requires further validation. In this report, we have provided compelling evidence that functioning cardiac tissue can be generated by the interaction of multipotent BMSCs with embryonic cardiac myocytes (ECMs) in two-dimensional (2-D) co-cultures. The differentiating BMSCs were induced to undergo cardiomyogenic differentiation pathway and were able to express unequivocal electromechanical coupling and functional synchronization with ECMs. Our 2-D co-culture system provides a useful in vitro model to elucidate various molecular mechanisms underpinning the integration and orderly maturation and differentiation of BMSCs into neo-cardiomyocytes during myocardial repair and regeneration.
Keywords: Bone marrow stromal cells; Mesenchymal stem cells; Embryonic cardiac myocytes; Dedifferentiation; Myocardial regeneration; Cardiac tissue engineering;

Osteoblasts proliferate slowly on the surface of calcium phosphate apatite which is widely used as a substrate biomaterial in bone regeneration. Owing to poor adhesion signaling in the cells grown on the calcium phosphate surface, inadequate growth factor signaling is generated to trigger cell cycle progression. The present study investigated an intracellular signal transduction pathway involved in the slow cell proliferation in osteoblasts grown on the calcium phosphate surface. Small GTPase RhoA and phosphatase and tensin homolog (PTEN) were more activated in cells grown on the surface of calcium phosphate apatite than on tissue culture plate. Specific inhibition of RhoA and PTEN induced the cells on calcium phosphate apatite surface to proliferate at a similar rate as cells on tissue culture plate surface. Specific inhibition of ROCK, which is a downstream effector of RhoA and an upstream activator of PTEN also increased proliferation of these osteoblasts. Present results indicate that physical property of calcium phosphate crystals that impede cell proliferation may be surmounted by the inhibition of the RhoA/ROCK/PTEN pathway to rescue delayed proliferation of osteoblasts on the calcium phosphate apatite surface. In addition, specific inhibition of ROCK promoted cell migration and osteoblast differentiation. Inhibition of the RhoA/ROCK/PTEN intracellular signaling pathway is expected to enhance cell activity to promote and accelerate bone regeneration on the calcium phosphate apatite surface.
Keywords: Cell adhesion; Calcium phosphate; Cell proliferation; Cell signaling;

An injectable vehicle for nucleus pulposus cell-based therapy by Estelle C. Collin; Sibylle Grad; Dimitrios I. Zeugolis; Claire S. Vinatier; Johann R. Clouet; Jérôme J. Guicheux; Pierre Weiss; Mauro Alini; Abhay S. Pandit (2862-2870).
An injectable hydrogel, acting as a reservoir for cell delivery and mimicking the native environment, offers promise for nucleus pulposus (NP) repair and regeneration. Herein, the potential of a stabilised type II collagen hydrogel using poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG) cross-linker, enriched with hyaluronic acid (HA) was investigated. The optimally stabilised type II collagen hydrogel was determined by assessing free amine groups, resistance to enzymatic degradation, gel point. The potential toxicity of the cross-linker was initially assessed against adipose-derived stem cells (ADSCs). After addition of HA (molar ratio type II collagen:HA 9:0, 9:1, 9:4.5, 9:9) within the hydrogel, the behaviour of the encapsulated NP cells was evaluated using cell proliferation assay, gene expression analysis, cell distribution and cell morphology. A significant decrease (p < 0.05) in the free amine groups of collagen was observed, confirming successful cross-linking. Gelation was independent of the concentration of 4S-StarPEG (8 min at 37 °C). The 1 mm cross-linked hydrogel yielded the most stable after enzymatic degradation (p < 0.05). No toxicity of the 4S-StarPEG was noted for the ADSCs. NP cell viability was high regardless of the concentration of HA (>80%). A cell proliferation was not seen after 14 days in its presence. At a gene expression level, HA did not influence NP cells phenotype after seven days in culture. After seven days in culture, the type I collagen mRNA expression was maintained (p > 0.05). The optimally stabilised and functionalised type II collagen/HA hydrogel system developed in this study shows promise as an injectable reservoir system for intervertebral disc regeneration.
Keywords: Injectable hydrogel; Intervertebral disc; Type II collagen; Hyaluronan;

Enhancement of neurite outgrowth in PC12 cells by iron oxide nanoparticles by Jeong Ah Kim; Nohyun Lee; Byung Hyo Kim; Won Jong Rhee; Sungjun Yoon; Taeghwan Hyeon; Tai Hyun Park (2871-2877).
Despite the many potential therapeutic applications of iron oxide nanoparticle such as its use as an imaging and targeting tool, its biological effects have not yet been extensively characterized. Herein, we report that iron oxide nanoparticles taken up by PC12 cells can enhance neurite outgrowth. PC12 cells exposed to both iron oxide nanoparticles and nerve growth factor (NGF) synergistically increased the efficiency of neurite outgrowth in a dose-dependent manner. This may have resulted from the activation of cell adhesion molecules that are associated with cell–matrix interactions through iron. Immunoblotting assays also revealed that both neural specific marker protein and cell adhesion protein expression were upregulated by iron oxide nanoparticles compared with non-treated cells via activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Our findings point to the possibility that iron oxide nanoparticles can affect cell–substrate interactions and regulate cell behaviors, which provides clinical insights into potential neurologic and therapeutic applications of iron oxide nanoparticles.
Keywords: Neurite outgrowth; Iron oxide nanoparticle; Neuronal differentiation; Extracellular matrix; Cell adhesion;

The influence of the scaffold design on the distribution of adhering cells after perfusion cell seeding by Ferry P.W. Melchels; Beatrice Tonnarelli; Andy L. Olivares; Ivan Martin; Damien Lacroix; Jan Feijen; David J. Wendt; Dirk W. Grijpma (2878-2884).
In natural tissues, the extracellular matrix composition, cell density and physiological properties are often non-homogeneous. Here we describe a model system, in which the distribution of cells throughout tissue engineering scaffolds after perfusion seeding can be influenced by the pore architecture of the scaffold. Two scaffold types, both with gyroid pore architectures, were designed and built by stereolithography: one with isotropic pore size (412 ± 13 μm) and porosity (62 ± 1%), and another with a gradient in pore size (250–500 μm) and porosity (35%–85%). Computational fluid flow modelling showed a uniform distribution of flow velocities and wall shear rates (15–24 s-1) for the isotropic architecture, and a gradient in the distribution of flow velocities and wall shear rates (12–38 s-1) for the other architecture. The distribution of cells throughout perfusion-seeded scaffolds was visualised by confocal microscopy. The highest densities of cells correlated with regions of the scaffolds where the pores were larger, and the fluid velocities and wall shear rates were the highest. Under the applied perfusion conditions, cell deposition is mainly determined by local wall shear stress, which, in turn, is strongly influenced by the architecture of the pore network of the scaffold.
Keywords: Scaffolds; Microstructure; Cell adhesion; Confocal microscopy; Image analysis; Computational fluid dynamics;

Differences in valvular and vascular cell responses to strain in osteogenic media by Zannatul Ferdous; Hanjoong Jo; Robert M. Nerem (2885-2893).
Calcification is the primary cause of failure of bioprosthetic and tissue-engineered vascular and valvular grafts. We used tissue-engineered collagen gels containing human aortic smooth muscle cells (HASMC) and human aortic valvular interstitial cells (HAVIC) as a model to investigate cell-mediated differences in early markers of calcification. The HASMCs and HAVICs were isolated from non-sclerotic human tissues. After 21 days of culture in either regular or osteogenic media with or without 10% cyclic strain at 1 Hz, the collagen gels were assessed for DNA content, collagen I, matrix metalloproteinase (MMP)-2 and glycosaminoglycan (GAG) content. The collagen gels containing HASMCs contained significantly greater amounts of collagen I and GAG compared to HAVICs. Although strain increased MMP-2 activity for both cell types, this trend was significant (p ≤ 0.05) only for HAVICs. Cultured gels were also assessed for osteogenic markers calcium content, alkaline phosphatase (ALP), and Runx2 and were present at greater amounts in gels containing HASMCs than HAVICs. Calcium content, Runx2 expression, and ALP activity were also modulated by mechanical strain. The results indicate that cell-mediated differences exist between the vascular and valvular calcification processes. Further investigation is necessary for improved understanding and to detect biomarkers for early detection or prevention of these diseases.
Keywords: Calcification; Tissue engineering; Cyclic strain; Aortic valve; Collagen gels;

Multifunctional Pluronic P123/F127 mixed polymeric micelles loaded with paclitaxel for the treatment of multidrug resistant tumors by Wei Zhang; Yuan Shi; Yanzuo Chen; Jiang Ye; Xianyi Sha; Xiaoling Fang (2894-2906).
The aim of this study was to exploit the possibility of combination of active targeting function of folic acid by folate receptor-mediated endocytosis and overcoming multidrug resistance (MDR) by Pluronic block copolymers to promote drug delivery to MDR tumor following intravenous administration with paclitaxel (PTX) as model drug. Folic acid functionalized Pluronic P123/F127 mixed micelles encapsulating PTX (FPF-PTX) was firstly developed and tested in vitro and in vivo, while PTX-loaded Pluronic P123/F127 mixed micelles (PF-PTX) and Taxol were used as control. FPF-PTX was about 20 nm in diameter with spherical shape and high encapsulation efficiency. Cellular uptake of FPF-PTX was found to be higher than that of PF-PTX due to the folate receptor-mediated endocytosis effect. In vitro cytotoxicity, cell apoptosis and cell cycle arrest studies also revealed that FPF-PTX was more potent than those of PF-PTX and Taxol. In vivo pharmacokinetic study in rats showed that the polymeric micelles significantly enhanced the bioavailability of PTX (∼3 fold) than Taxol. Moreover, in BALB/c mice bearing KBv MDR tumor xenografts, stronger antitumor efficacy was shown in FPF-PTX group, with good correlation between in vitro and in vivo. In conclusion, folate-conjugated Pluronic micelles could be a potential vehicle for delivering hydrophobic chemotherapeutic drugs to MDR tumors.
Keywords: Tumor targeting; Paclitaxel; Polymeric micelles; Folic acid; Multidrug resistance;

Patterns of cancer invasion revealed by QDs-based quantitative multiplexed imaging of tumor microenvironment by Chun-Wei Peng; Xiu-Li Liu; Chuang Chen; Xiong Liu; Xue-Qin Yang; Dai-Wen Pang; Xiao-Bo Zhu; Yan Li (2907-2917).
Tumor growth and progression depends on their microenvironment, which undergoes constant co-evolution because of the dynamic tumor–stormal interactions. Such co-evolution has long been under appreciated due to the lack of appropriate technology platforms to simultaneously reveal these complex interactions. Here we report on a quantum dots based multiplexed imaging and spectrum analysis technology to simultaneously study major components of tumor stroma, including type IV collagen, tumor angiogenesis, macrophages infiltration and tissue destructive proteolytic enzyme matrix metalloproteinase 9. The new technology revealed a panoramic picture of the tempo-spatial co-evolution of tumor cells and their stroma at the architecture level. Four patterns of tumor invasion with distinctive co-evolution features were identified as Washing pattern, Ameba-like pattern, Polarity pattern and Linear pattern. This quantum dots based multiplexed technology could help gain new insight into the complex process of tumor invasion, and formulate new anti-cancer strategies.
Keywords: Quantum dots; Tumor microenvironment; Cancer invasion; Multiplexed imaging; Nanomedicine;

Combined cytotoxic and anti-invasive properties of redox-active nanoparticles in tumor–stroma interactions by Lirija Alili; Maren Sack; Ajay S. Karakoti; Sarah Teuber; Katharina Puschmann; Suzanne M. Hirst; Christopher M. Reilly; Klaus Zanger; Wilhelm Stahl; Soumen Das; Sudipta Seal; Peter Brenneisen (2918-2929).
Tumor–stroma interaction plays an important role in tumor progression. Myofibroblasts, pivotal for tumor progression, populate the microecosystem of reactive stroma. The formation of myofibroblasts is mediated by tumor derived transforming growth factor β1 (TGFβ1) which initiates a reactive oxygen species cell type dependent expression of alpha-smooth muscle actin, a biomarker for myofibroblastic cells. Myofibroblasts express and secrete proinvasive factors significantly increasing the invasive capacity of tumor cells via paracrine mechanisms. Although antioxidants prevent myofibroblast formation, the same antioxidants increase the aggressive behavior of the tumor cells. In this study, the question was addressed of whether redox-active polymer-coated cerium oxide nanoparticles (CNP, nanoceria) affect myofibroblast formation, cell toxicity, and tumor invasion. Herein, nanoceria downregulate both the expression of alpha-smooth muscle actin positive myofibroblastic cells and the invasion of tumor cells. Furthermore, concentrations of nanoceria being non-toxic for normal (stromal) cells show a cytotoxic effect on squamous tumor cells. The treatment with redox-active CNP may form the basis for protection of stromal cells from the dominating influence of tumor cells in tumor–stroma interaction, thus being a promising strategy for chemoprevention of tumor invasion.
Keywords: Antioxidant; Carcinogenesis; Cell culture; Tumor-stroma interactions; Fibroblast; Nanoparticle;

Here we report an electrochemical sensor that can realize label-free cancer cell detection using the first clinical trial II used aptamer AS1411 and functionalized graphene. By taking advantages of AS1411 high binding affinity and specificity to the overexpressed nucleolin on the cancer cell surface, our developed electrochemical aptasensor can distinguish cancer cells and normal ones and detect as low as one thousand cells. With DNA hybridization technique, this E-DNA sensor can be regenerated and reusable for cancer cell detection. Our work gives a good example for label-free cancer cell detection based on aptamer and graphene-modified electrode.
Keywords: Graphene; AS1411; G-quadruplex; Aptamer; Cancer cell detection; Reusable sensor;

Dimercaptosuccinic acid-coated magnetite nanoparticles for magnetically guided in vivo delivery of interferon gamma for cancer immunotherapy by Raquel Mejías; Sonia Pérez-Yagüe; Lucía Gutiérrez; Lourdes I. Cabrera; Roberto Spada; Pilar Acedo; Carlos J. Serna; Francisco J. Lázaro; Ángeles Villanueva; María del Puerto Morales; Domingo F. Barber (2938-2952).
As radio- and chemotherapy-based cancer treatments affect both tumors and healthy tissue, cancer immunotherapy attempts to specifically enhance the natural immune response to tumor cells. In mouse models of cancer, we tested uniform dimercaptosuccinic acid (DMSA)-coated monodisperse magnetic nanoparticles as a delivery system for the anti-tumorigenic cytokine IFN-γ. IFN-γ-adsorbed DMSA-coated magnetic nanoparticles were targeted to the tumor site by application of an external magnetic field. We analyzed nanoparticle biodistribution before and after IFN-γ conjugation, as well as the efficiency of nanoparticle accumulation in tumors, IFN-γ release in the area of interest, and the effects of both on tumor development. At the tumor site, we observed a high degree of nanoparticle accumulation and of cytokine delivery, which led to increased T cell and macrophage infiltration and promoted an anti-angiogenic effect. The combined action led to a notable reduction in tumor size. Our findings indicate that IFN-γ-adsorbed DMSA-coated magnetite nanoparticles can be used as an efficient in vivo drug delivery system for tumor immunotherapy.
Keywords: Immunemodulation; Nanoparticle; Carcinogenesis; Cytokine; Magnetism;

Endosomolytic polymers can aid in the endosomal release of therapeutics to improve intracellular drug delivery. pH-responsive biomimetic pseudo-peptides were synthesised by grafting l-phenylalanine onto the pendant carboxylic acids of a polyamide, poly(l-lysine isophthalamide). PP-75 (stoichiometric l-phenylalanine grafting of 75 mol%) was determined to have the best endosomolytic property. The mean hydrodynamic size of PP-75 decreased with lower pH as the polymers adopted a more compact conformation due to protonation of acidic groups and increase in hydrophobicity. PP-75 was demonstrated to deliver model drugs effectively in three dimensional (3D) magnetic HeLa multicellular spheroids used as in vitro tumour models. These spheroids can be isolated easily and quickly by magnetic separation. Due to its relatively small size, PP-75 was able to penetrate from the exterior to the interior of these spheroids and was internalised by the cells in the spheroids. It could retain its pH-mediated membrane-lytic capability in 3D drug delivery by releasing internalised calcein from intracellular endosomes in the tumour models. Furthermore, cell viability results suggest that PP-75 showed no significant cytotoxicity towards cells in the spheroids. The pH-responsive PP-75 can potentially enhance the extracellular and intracellular delivery of therapeutics in tumours.
Keywords: Drug delivery; pH-responsive polymer; l-phenylalanine; Pseudo-peptide; Multicellular spheroids; Magnetic separation;

High-quality water-soluble and surface-functionalized upconversion nanocrystals as luminescent probes for bioimaging by Tianye Cao; Yang Yang; Yuan Gao; Jing Zhou; Zhengquan Li; Fuyou Li (2959-2968).
Rare-earth upconversion nanophosphors (UCNPs) have great potential to become excellent biological luminescent labels for fluorescence bioimaging. However, it is still difficult to directly synthesize high-quality water-soluble UCNPs bearing appropriate functional groups using a one-step synthetic strategy. Herein, we report a one-step synthetic strategy for high-quality water-soluble and surface-functionalized UCNPs using a hydrothermal reaction assisted by binary cooperative ligands (HR-BCL). In this system, 6-aminohexanoic acid and oleate were introduced to control nuclear generation and crystal growth of small nanoparticles. The UCNPs synthesized here showed high crystalline and intense upconversion luminescence emission. Fourier-transform infrared and nuclear magnetic resonance spectroscopy indicated that 6-aminohexanoic acid and oleate cooperate as surface ligands to co-control the surface of UCNPs. Thus, the water-solubility of the as-prepared UCNPs can be tuned by changing the molar ratio of 6-aminohexanoic acid to oleate. The AA-modified UCNPs provided a free amine content of (6.0 ± 0.2) × 10−5 mol/g, which renders them dispersible in aqueous solution and allows further conjugation with folic acid (FA) for targeted bioimaging. Furthermore, the amine-functionalized UCNPs show intense near-infrared upconversion luminescence and were successfully applied in the lymphatic capillary bioimaging of small animals with a high signal-to-noise ratio, suggesting that these surface-functionalized UCNPs are promising candidates for luminescent biolabels.
Keywords: Upconversion luminescence; Nanophosphors; One-step synthesis; Lymph bioimaging;

Multimodal tumor imaging by iron oxides and quantum dots formulated in poly (lactic acid)-d-alpha-tocopheryl polyethylene glycol 1000 succinate nanoparticles by Yang Fei Tan; Prashant Chandrasekharan; Dipak Maity; Cai Xian Yong; Kai-Hsiang Chuang; Ying Zhao; Shu Wang; Jun Ding; Si-Shen Feng (2969-2978).
This work developed a multimodal imaging system by co-encapsulating superparamagnetic iron oxides (IOs) and quantum dots (QDs) in the nanoparticles of poly (lactic acid) - d-α-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS) for concurrent imaging of the magnetic resonance imaging (MRI) and the fluorescence imaging to combine their advantages and to overcome their disadvantages as well as to promote a sustained and controlled imaging with passive targeting effects to the diseased cells. The QDs and IOs-loaded PLA-TPGS NPs were prepared by a modified nanoprecipitation method, which were then characterized for their size and size distribution, zeta potential and the imaging agent encapsulation efficiency. The transmission electron microscopy (TEM) images showed direct evidence for the well-dispersed distribution of the QDs and IOs within the PLA-TPGS NPs. The cellular uptake and the cytotoxicity of the PLA-TPGS NPs formulation of QDs and IOs were investigated in vitro with MCF-7 breast cancer cells, which were conducted in close comparison with the free QDs and IOs at the same agent dose. The Xenograft model was also conducted for biodistribution of the QDs and IOs-loaded PLA-TPGS NPs among the various organs, which showed greatly enhanced tumor imaging due to the passively targeting effects of the NPs to the tumor. Images of tumors were acquired in vivo by a 7T MRI scanner. Further ex vivo images of the tumors were obtained by confocal laser scanning microscopy. Such a multimodal imaging system shows great advantages of both contrast agents making the resultant probe highly sensitive with good depth penetration, which confirms the diagnosis obtained from each individual imaging. With therapeutics co-encapsulation and ligand conjugation, such nanoparticles system can realize a multi-functional system for medical diagnosis and treatment.
Keywords: Biodegradable polymers; Cancer nanotechnology; Magnetic resonance imaging (MRI); Molecular imaging; Multifunctional nanoparticles; Nanomedicine;

Computed tomography imaging of cancer cells using acetylated dendrimer-entrapped gold nanoparticles by Han Wang; Linfeng Zheng; Chen Peng; Rui Guo; Mingwu Shen; Xiangyang Shi; Guixiang Zhang (2979-2988).
We report a new use of acetylated dendrimer-entrapped gold nanoparticles (Au DENPs) for in vitro and in vivo computed tomography (CT) imaging of cancer cells. In this study, Au DENPs prepared using amine-terminated generation 5 poly(amidoamine) dendrimers were subjected to an acetylation reaction to neutralize the positive surface potential. The acetylated Au DENPs were used for both in vitro and in vivo CT imaging of a human lung adencarcinoma cell line (SPC-A1 cells). Micro-CT images show that SPC-A1 cells can be detected under X-ray after incubation with the acetylated Au DENPs in vitro and the xenograft tumor model can be imaged after both intratumoral and intraperitoneal administration of the particles. Transmission electron microscopy data further confirm that the acetylated Au DENPs are able to be uptaken dominantly in the lysosomes of the cells. Combined morphological observation of cells after hematoxylin and eosin staining, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay of cell viability, and flow cytometric analysis of cell cycle show that the acetylated Au DENPs do not appreciably affect the cell morphology, viability, and cell cycle, indicating their good biocompatibility at the given concentration range. Findings from this study suggest that the developed acetylated Au DENPs have a great potential to be used for CT imaging of cancer cells.
Keywords: Dendrimers; Gold nanoparticles; Molecular imaging; Computed tomography; Cancer cells;

Peptide-conjugated polyamidoamine dendrimer as a nanoscale tumor-targeted T1 magnetic resonance imaging contrast agent by Liang Han; Jianfeng Li; Shixian Huang; Rongqin Huang; Shuhuan Liu; Xing Hu; Peiwei Yi; Dai Shan; Xuxia Wang; Hao Lei; Chen Jiang (2989-2998).
A tumor-targeting carrier, peptide HAIYPRH (T7)-conjugated polyethylene glycol-modified polyamidoamine dendrimer (PAMAM-PEG-T7) was explored to deliver magnetic resonance imaging (MRI) contrast agents targeting to the tumor cells specifically. Two different types of tumors, liver cancer and early brain glioma model (involved with the blood–brain barrier), were chosen to evaluate the imaging capacity of this contrast agent. PAMAM-PEG-T7 was synthesized, conjugated with diethylene triamine pentaacetic acid (DTPA) and further chelated gadolinium (Gd), yielding GdDTPA-PAMAM-PEG-T7. The result of ICP-AES showed that about 92 Gd ions could be loaded per PAMAM molecule. The calculated longitudinal relaxivity R1 of the GdDTPA-PAMAM-PEG-T7 was 10.7 mm −1 S−1 per Gd (984.4 mm −1 S−1 per PAMAM), while that of GdDTPA was only 4.8 mm −1 S−1. PAMAM-PEG-T7 had better targeting capacity to the liver cancer cells in vitro and in vivo, compared with PAMAM-PEG. The accumulation of PAMAM-PEG-T7 was 162.5% times that of PAMAM-PEG. But for glioma cells, PAMAM-PEG-T7 did not show its specificity. Furthermore, GdDTPA-PAMAM-PEG-T7 could improve the diagnostic efficiency of liver cancer with the enhanced signal (187%), compared to 130% for PAMAM-PEG and 121% for GdDTPA. GdDTPA-PAMAM-PEG-T7 could selectively identify liver cancer but not early glioma. This nanoscaled MRI contrast agent GdDTPA-PAMAM-PEG-T7 might allow for selective and efficient diagnosis of tumors without the natural barrier including liver cancer.
Keywords: Receptor-mediated endocytosis; Transferrin receptor; Peptide; GdDTPA; Magnetic resonance imaging; Contrast agent;

Fluorine-18 labeled rare-earth nanoparticles for positron emission tomography (PET) imaging of sentinel lymph node by Yun Sun; Mengxiao Yu; Sheng Liang; Yingjian Zhang; Chenguang Li; Tiantian Mou; Wenjiang Yang; Xianzhong Zhang; Biao Li; Chunhui Huang; Fuyou Li (2999-3007).
Rare-earth-based nanoparticles have attracted increasing attention for their unique optical and magnetic properties. However, their application in bioimaging has been limited to photoluminescence bioimaging and magnetic resonance imaging. To facilitate their use in other bioimaging techniques, we developed a simple, rapid, efficient and general synthesis strategy for 18F-labeled rare-earth nanoparticles through a facile inorganic reaction between rare-earth cations and fluoride ions. The 18F-labeling process based on rare-earth elements was achieved efficiently in water at room temperature with an 18F-labeling yield of >90% and completed within 5 min, with only simple purification by aqueous washing and centrifugation, and without the use of organic agents. The effectiveness of 18F-labeled rare-earth nanoparticles was further evaluated by positron emission tomography (PET) imaging of their in vivo distribution and application in lymph monitoring. In addition, this strategy is proposed for the creation of a dual-model bioimaging technique, combining upconversion luminescence bioimaging and PET imaging.
Keywords: Rare-earth; Upconversion luminescence; Fluorine-18; Positron emission tomography; Lymphatic imaging;

Challenges for the effective molecular imprinting of proteins by Ellen Verheyen; Joris P. Schillemans; Martin van Wijk; Marie-Astrid Demeniex; Wim E. Hennink; Cornelus F. van Nostrum (3008-3020).
Molecular imprinting is a technique that is used to create artificial receptors by the formation of a polymer network around a template molecule. This technique has proven to be particularly effective for molecules with low molecular weight (<1500 Da), and during the past five years the number of research articles on the imprinting of larger (bio)templates is increasing considerably. However, expanding the methodology toward imprinted materials for selective recognition of proteins, DNA, viruses and bacteria appears to be extremely challenging. This paper presents a critical analysis of data presented by several authors and our own experiments, showing that the molecular imprinting of proteins still faces some fundamental challenges. The main topics of concern are proper monomer selection, washing method/template removal, quantification of the rebinding and reproducibility. Use of charged monomers can lead to strong electrostatic interactions between monomers and template but also to undesired high aspecific binding. Up till now, it has not been convincingly shown that electrostatic interactions lead to better imprinting results. The combination of a detergent (SDS) and AcOH, commonly used for template removal, can lead to experimental artifacts, and should ideally be avoided. In many cases template rebinding is unreliably quantified, results are not evaluated critically and lack statistical analysis. Therefore, it can be argued that presently, in numerous publications the scientific evidence of molecular imprinting of proteins is not convincing.
Keywords: Molecular imprinting; Protein imprinting; Crosslinked polymers; Functional monomers; Template removal; Protein–polymer interaction;

Reduction of oxidative stress by p-hydroxybenzyl alcohol-containing biodegradable polyoxalate nanoparticulate antioxidant by Soojin Kim; Hyunjin Park; Yiseul Song; Donghyun Hong; Onyou Kim; Eunhye Jo; Gilson Khang; Dongwon Lee (3021-3029).
The large production of reactive oxygen species (ROS) leads to the oxidative stress and the subsequent functional decline of organ systems. p-Hydroxybenzyl alcohol (HBA) is known to play a pivotal protective role against oxidative stress-related diseases. We have developed biodegradable antioxidant copolyoxalate, in which HBA is chemically incorporated into its backbone for the treatment of oxidative stress-related diseases. HBA-incorporated copolyoxalate (HPOX) was designed to possess aromatic peroxalate ester linkages in its backbone and release HBA during its hydrolytic degradation. Peroxalate ester linkages in the backbone reacted with and scavenged hydrogen peroxide, leading the release of HBA in vitro. HBA released from HPOX exerted excellent antioxidant activity, such as inhibition of nitric oxide (NO) production by suppressing iNOS (inducible nitric oxide synthases) expression in lipopolysaccharide (LPS)-activated RAW 264.7 cells. HPOX nanoparticles delivered intranasally significantly reduced pulmonary inflammation and suppressed the iNOS expression. Given their excellent antioxidant and anti-inflammatory activities, we anticipate that HPOX nanoparticles are highly potent for the treatment of oxidative damage-related diseases, such as asthma.
Keywords: Inflammation; Oxidative stress; Antioxidant; Polymer; Nanoparticles;

Folate receptor mediated gene targeting provides several advantages such as delivery of high concentration of gene at specific tumor sites including brain, lung, ovary, uterus and kidney where folate receptors are over expressed. In the present study for both systemic stability and tumor targeting ability, poly (ethylene glycol)–folic acid (PEG–FA) conjugate was coupled with an arginine modified oligo (alkylaminosiloxane) graft poly (ethyleneimine) having enhanced transfection efficiency compared to poly (ethyleneimine). The resultant polymer P(SiDAAr)5FP2 complexed pDNA effectively and showed protection against nuclease degradation. The PEG group provided improved blood compatibility and cell viability. Uniformly oriented arginine moiety helped to enhance cellular and nuclear localisation, which led to improved transfection. The polymer was capable of releasing pDNA at the nucleus and being cleared from the cell after its purpose. Transfection in presence of cellular uptake inhibitors showed multiple pathways for cellular uptake of the targeted polymer, out of which folate receptor mediated uptake was more prominent. The folate mediated cellular uptake of P(SiDAAr)5FP2 was then confirmed by flow cytometric evaluation. The high accumulation of targeted polymers in the tumor tissues of tumor bearing mice from 2nd hour onwards proved the active targeting effect of the polymer. Besides tumor accumulation, the material showed capability to diffuse through the vascular endothelium. This property is expected to be beneficial for brain targeting experiments.
Keywords: Folate mediated; Folic acid; Tumour targeting; Poly(ethyleneimine); Poly(ethylene glycol);

Multifunctional silica nanotubes for dual-modality gene delivery and MR imaging by Ran Namgung; Yuhai Zhang; Qi Ling Fang; Kaushik Singha; Hwa Jeong Lee; Il Keun Kwon; Yong Yeon Jeong; In-Kyu Park; Sang Jun Son; Won Jong Kim (3042-3052).
This work demonstrated the development of multifunctional silica nanotubes (SNT) by functionalization of their inner void and outer surface with magnetic-fluorescence nanocomposites and cationic polymers, respectively. The successful construction of BPEI-SNT was established by electron energy loss spectroscopy in conjugation with standard analytical tools. The mean fluorescence intensity in a FACS assay, a luciferase gene expression assay and a confocal fluorescence study demonstrated the efficacy of BPEI-SNT as a gene delivery vector. Endocytotic uptake was also demonstrated by the colocalization of LysoTracker Red® and green fluorescent quantum dots. Moreover, enhanced magnetic resonance imaging revealed the potential of the BPEI-SNT nanocomposite to act as a dual-modality nano-device.
Keywords: Gene delivery; MR imaging; Silica nanotubes; Polyethylenimine; Iron oxide; Quantum dot;

Cellular and extracellular programming of cell fate through engineered intracrine-, paracrine-, and endocrine-like mechanisms by Debanjan Sarkar; James A. Ankrum; Grace S.L. Teo; Christopher V. Carman; Jeffrey M. Karp (3053-3061).
A cell’s fate is tightly controlled by its microenvironment. Key factors contributing to this microenvironment include physical contacts with the extracellular matrix and neighboring cells, in addition to soluble factors produced locally or distally. Alterations to these cues can drive homeostatic processes, such as tissue regeneration/wound healing, or may lead to pathologic tissue dysfunction. In vitro models of cell and tissue microenvironments are desirable for enhanced understanding of the biology and ultimately for improved treatment. However, mechanisms to exert specific control over cellular microenvironments remains a significant challenge. Genetic modification has been used but is limited to products that can be manufactured by cells and release kinetics of therapeutics cannot easily be controlled. Herein we describe a non-genetic approach to engineer cells with an intracellular depot of phenotype altering agent/s that can be used for altering cell fate via intracrine-, paracrine-, and endocrine-like mechanisms. Specifically, we show that human mesenchymal stem cells (MSCs) can be engineered with poly lactide-co-glycolic acid (PLGA) particles containing dexamethasone, which acts on cytoplasmic receptors. The controlled release properties of these particles allowed for sustained intracellular and extracellular delivery of agent to promote differentiation of particle-carrying cells, as well as neighboring cells and distant cells that do not contain particles.
Keywords: Mesenchymal stem cell; Controlled drug release; Cell signaling; Microsphere; Osteogenesis;

Spatial control of gene expression within a scaffold by localized inducer release by Priya R. Baraniak; Devin M. Nelson; Cory E. Leeson; Anand K. Katakam; Jennifer L. Friz; Dean E. Cress; Yi Hong; Jianjun Guan; William R. Wagner (3062-3071).
Gene expression can be controlled in genetically modified cells by employing an inducer/promoter system where presence of the inducer molecule regulates the timing and level of gene expression. By applying the principles of controlled release, it should be possible to control gene expression on a biomaterial surface by the presence or absence of inducer release from the underlying material matrix, thus avoiding alternative techniques that rely upon uptake of relatively labile DNA from material surfaces. To evaluate this concept, a modified ecdysone-responsive gene expression system was transfected into B16 murine cells and the ability of an inducer ligand, which was released from elastomeric poly(ester urethane) urea (PEUU), to initiate gene expression was studied. The synthetic inducer ligand was first loaded into PEUU to demonstrate extended release of the bioactive molecule at various loading densities over a one year period in vitro. Patterning films of PEUU variably-loaded with inducer resulted in spatially controlled cell expression of the gene product (green fluorescent protein, GFP). In porous scaffolds made from PEUU by salt leaching, where the central region was exclusively loaded with inducer, cells expressed GFP predominately in the loaded central regions whereas expression was minimal in outer regions where ligand was omitted. This scaffold system may ultimately provide a means to precisely control progenitor cell commitment in a spatially-defined manner in vivo for soft tissue repair and regeneration.
Keywords: Gene expression; Controlled release; Spatial control; Elastomer; Tissue engineering;

Flotillin-dependent endocytosis and a phagocytosis-like mechanism for cellular internalization of disulfide-based poly(amido amine)/DNA polyplexes by Dries Vercauteren; Martin Piest; Leonardus J. van der Aa; Monerah Al Soraj; Arwyn T. Jones; Johan F.J. Engbersen; Stefaan C. De Smedt; Kevin Braeckmans (3072-3084).
Extensive research is currently performed on designing safe and efficient non-viral carriers for gene delivery. To increase their efficiency, it is essential to have a thorough understanding of the mechanisms involved in cellular attachment, internalization and intracellular processing in target cells. In this work, we studied in vitro the cellular dynamics of polyplexes, composed of a newly developed bioreducible poly(amido amine) carrier, formed by polyaddition of N,N-cystamine bisacrylamide and 1-amino-4-butanol (p(CBA-ABOL)) on retinal pigment epithelium (RPE) cells, which are attractive targets for ocular gene therapy. We show that these net cationic p(CBA-ABOL)/DNA polyplexes require a charge-mediated attachment to the sulfate groups of cell surface heparan sulfate proteoglycans in order to be efficiently internalized. Secondly, we assessed the involvement of defined endocytic pathways in the internalization of the polyplexes in ARPE-19 cells by using a combination of endocytic inhibitors, RNAi depletion of endocytic proteins and live cell fluorescence colocalization microscopy. We found that the p(CBA-ABOL) polyplexes enter RPE cells both via flotillin-dependent endocytosis and a PAK1 dependent phagocytosis-like mechanism. The capacity of polyplexes to transfect cells was, however, primarily dependent on a flotillin-1-dependent endocytosis pathway.
Keywords: Non-viral gene delivery; Endocytosis; Retinal pigment epithelium; Polyplexes;

Intracellular trafficking and gene expression of pH-sensitive, artificially enveloped adenoviruses in vitro and in vivo by Jeroen Van den Bossche; Wafa’ T. Al-Jamal; Açelya Yilmazer; Elisabetta Bizzarri; Bowen Tian; Kostas Kostarelos (3085-3093).
Recombinant adenovirus (Ad) has shown great promise in gene therapy. Artificial envelopment of adenovirus within lipid bilayers has previously been shown to decrease the immunogenicity and hepatic affinity of naked Ad in vivo. Unfortunately, this also resulted in a significant reduction of gene expression, which we attributed to poor endosomal release of the Ad from its artificial lipid envelope. In this work, we explored the artificial envelopment of Ad within pH-sensitive DOPE:CHEMS bilayers and characterized this vector by TEM, AFM, dot blot, dynamic light scattering and zeta potential measurements. The artificially enveloped viral vectors exhibited good stability at physiological pH but immediately collapsed and released naked Ad virions at pH 5.5. Intracellular trafficking using confocal laser scanning microscopy (CLSM) revealed that Cy3-labelled Ad enveloped in DOPE:CHEMS bilayers exhibited the characteristic Ad distribution within the cytoplasm that led to virion accumulation around the nuclear membrane, indicating endosomal release of Ad. We obtained equivalent levels of gene expression as those of naked Ad in a series of CAR-positive (CAR+) and CAR-negative (CAR-) cell lines. This suggested that the mechanism of infection for the artificially enveloped Ad remained dependent on the presence of CAR receptors. Finally, the pH-sensitive enveloped Ad were injected intratumorally in human cervical carcinoma xenograft-bearing nude mice, also illustrating their capacity for efficient in vivo marker gene expression. This study is a step forward toward the engineering of functional, artificially enveloped adenovirus vectors for gene transfer applications.
Keywords: Gene therapy; Liposome; Virus envelope; Nanotechnology; Viral; Non-viral;

The impact of nanoparticle ligand density on dendritic-cell targeted vaccines by Arunima Bandyopadhyay; Rebecca L. Fine; Stacey Demento; Linda K. Bockenstedt; Tarek M. Fahmy (3094-3105).
Dendritic-cell (DC) targeted antigen delivery systems hold promise for enhancing vaccine efficacy and delivery of therapeutics. However, it is not known how the number and density of targeting ligands on such systems may affect DC function and subsequent T cell response. We modified the surface of biodegradable nanoparticles loaded with antigen with different densities of the mAb to the DC lectin DEC-205 receptor and assessed changes in the cytokine response of DCs and T cells. DEC-205 targeted nanoparticles unexpectedly induced a differential cytokine response that depended on the density of ligands on the surface. Strikingly, nanoparticle surface density of DEC-205 mAb increased the amount of anti-inflammatory, IL-10, produced by DCs and T cells. Boosting mice with DEC-205 targeted OVA-nanoparticles after immunization with an antigen in CFA induced a similar pattern of IL-10 response. The correlation between DC production of IL-10 as a function of the density of anti-DEC-205 is shown to be due to cross-linking of the DEC-205 receptor. Cross-linking also increased DC expression of the scavenger receptor CD36, and blockade of CD36 largely abrogated the IL-10 response. Our studies highlight the importance of target ligand density in the design of vaccine delivery systems.
Keywords: Dendritic cells; C-type lectin; Nanoparticles; Cytokines; Vaccines;

Enhanced endosomal escape of siRNA-incorporating hybrid nanoparticles from calcium phosphate and PEG-block charge-conversional polymer for efficient gene knockdown with negligible cytotoxicity by Frederico Pittella; Mingzhen Zhang; Yan Lee; Hyun J. Kim; Theofilus Tockary; Kensuke Osada; Takehiko Ishii; Kanjiro Miyata; Nobuhiro Nishiyama; Kazunori Kataoka (3106-3114).
Development of safe and efficient short interfering RNA (siRNA) delivery system for RNA interference (RNAi)-based therapeutics is a current critical challenge in drug delivery field. The major barriers in siRNA delivery into the target cytoplasm are the fragility of siRNA in the body, the inefficient cellular uptake, and the acidic endosomal entrapment. To overcome these barriers, this study is presenting a hybrid nanocarrier system composed of calcium phosphate comprising the block copolymer of poly(ethylene glycol) (PEG) and charge-conversional polymer (CCP) as a siRNA vehicle. In these nanoparticles, the calcium phosphate forms a stable core to incorporate polyanions, siRNA and PEG–CCP. The synthesized PEG–CCP is a non-toxic endosomal escaping unit, which induces endosomal membrane destabilization by the produced polycation through degradation of the flanking cis-aconitylamide of CCP in acidic endosomes. The nanoparticles prepared by mixing of each component was confirmed to possess excellent siRNA-loading efficiency (∼80% of dose), and to present relatively homogenous spherical shape with small size. With negligible cytotoxicity, the nanoparticles efficiently induced vascular endothelial growth factor (VEGF) mRNA knockdown (∼80%) in pancreatic cancer cells (PanC-1). Confocal laser scanning microscopic observation revealed rapid endosomal escape of siRNA with the nanoparticles for the excellent mRNA knockdown. The results obtained demonstrate our hybrid nanoparticle as a promising candidate to develop siRNA therapy.
Keywords: Calcium phosphate nanoparticles; Endosomal escape; Vascular endothelial growth factor (VEGF); siRNA; Charge-conversional polymer (CCP); Poly(ethylene glycol) (PEG);

Anti-Flt1 peptide – Hyaluronate conjugate for the treatment of retinal neovascularization and diabetic retinopathy by Eun Ju Oh; Jun-Sub Choi; Hyemin Kim; Choun-Ki Joo; Sei Kwang Hahn (3115-3123).
Anti-angiogenic therapeutics has been investigated extensively for the treatment of retinal and choroidal vascular diseases, and diabetic retinopathy. Anti-Flt1 peptide of GNQWFI is an antagonistic peptide for vascular endothelial growth factor receptor 1 (VEGFR1 or Flt1) inhibiting VEGFR1-mediated endothelial cell migration and tube formation. In this work, anti-Flt1 peptide (GGNQWFI) was chemically conjugated to tetra-n-butyl ammonium modified hyaluronate (HA-TBA) via amide bond formation in dimethyl sulfoxide (DMSO) using benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). The resulting HA – GGNQWFI conjugate self-assembled to form micelle-like nanoparticles in aqueous solution, as confirmed and characterized by transmission electron microscopy (TEM). According to in vitro biological activity tests, HA – GGNQWFI conjugate exhibited a dose-dependent inhibition effect on the binding of Flt1-Fc to VEGF165 coated on the well. Furthermore, anti-Flt1 peptide – HA conjugate effectively inhibited retinal choroidal neovascularization (CNV) in laser induced CNV model rats. The retinal vascular permeability and the deformation of retinal vascular structure were also significantly reduced in diabetic retinopathy model rats after treatment with anti-Flt1 peptide – HA conjugate. Pharmacokinetic analysis confirmed the increased mean residence time of anti-Flt1 peptide after conjugation to HA longer than 2 weeks.
Keywords: Hyaluronate; Anti-Flt1 peptide; Drug delivery; Choroidal neovascularization; Diabetic retinopathy;

A biodegradable amphiphilic and cationic triblock copolymer for the delivery of siRNA targeting the acid ceramidase gene for cancer therapy by Cheng-Qiong Mao; Jin-Zhi Du; Tian-Meng Sun; Yan-Dan Yao; Pei-Zhuo Zhang; Er-Wei Song; Jun Wang (3124-3133).
One of the key challenges in the development of RNA interference-based cancer therapy is the lack of an efficient delivery system for synthetic small interfering RNAs (siRNAs) that would enable efficient uptake by tumor cells and allow for significant knockdown of a target transcript in vivo. Here, we describe a micelleplex system based on an amphiphilic and cationic triblock copolymer, which can systemically deliver siRNA targeting the acid ceramidase (AC) gene for cancer therapy. This triblock copolymer, consisting of monomethoxy poly(ethylene glycol), poly(ε-caprolactone) and poly(2-aminoethyl ethylene phosphate), self-assembles into micellar nanoparticles (MNPs) in aqueous solution with an average diameter of 60 nm and a zeta potential of approximately 48 mV. The resulting micelleplex, formed by the interaction of MNPs and siRNA, was effectively internalized by BT474 breast cancer cells and siRNA was subsequently released, resulting in significant gene knockdown. This effect was demonstrated by significant down-regulation of luciferase expression in BT474-luciferase cells which stably express luciferase, and suppression of AC expression in BT474 cells at both the transcriptional and protein level, following delivery of specific siRNAs by the micelleplex. Furthermore, a micelleplex carrying siRNA targeting the AC (micelleplexsiAC ) gene was found to induce remarkable apoptosis and reduce the proliferation of cancer cells. Systemic delivery of micelleplexsiAC significantly inhibited tumor growth in a BT474 xenograft murine model, with depressed expression of AC and no positive activation of the innate immune response, suggesting therapeutic promise for micelleplex siRNA delivery in cancer therapy.
Keywords: siRNA delivery; Cancer therapy; Acid ceramidase; Micelleplex; Polyphosphoester;

Dissolving microneedles, three-dimensional polymer structures with microscale cross-sectional dimensions, have been introduced as a means of safe transdermal drug delivery. Most dissolving microneedles have been fabricated using a traditional micro-casting method that cures biopolymers within three-dimensional mold, nevertheless, repeated molding process may cause damage to encapsulated drugs, a critical hurdle for clinical application. Here, we describe the stepwise controlled drawing technique that can directly fabricate dissolving microneedle from maltose by precise controlling the drawing time and the viscosity of the maltose. Controlled drawing shaped the particular sharp-conical microneedles of 1200 μm length with tip diameter of 60 μm, and dissolved within 20 min in-vivo after inserting to the skin. This technique surpasses the limitations of micro-casting for dissolving microneedle. Furthermore, transdermal delivery of impermeable hydrophilic molecules such as ascorbic acid-2-glucoside and niacinamide was confirmed as inhibition of cutaneous hypermelanosis. We anticipate that controlled drawing technique will be suitable to design dissolving microneedles for use in minimally invasive transcutaneous drug delivery to patients.
Keywords: Controlled drawing lithography; Dissolving microneedle; Transdermal drug delivery; Cutaneous depigmentation;

The suppression of prion propagation using poly-l-lysine by targeting plasminogen that stimulates prion protein conversion by Chongsuk Ryou; William B. Titlow; Charles E. Mays; Younsoo Bae; Sehun Kim (3141-3149).
Poly-l-lysine (PLL), a homopolymer of amino acid l-lysine (LL), has been frequently used for drug delivery. Here, we report that PLL is an effective agent to inhibit propagation of prions that cause fatal and incurable neurologic disorders in humans and animals, termed prion diseases. In our recent investigation on prion propagation facilitated by conversion of the cellular prion protein (PrP) to the misfolded, disease-associated PrP (PrPSc), we demonstrated that plasminogen stimulates PrP conversion as a cellular cofactor. In the current study, we targeted plasminogen using PLL and assessed its anti-prion efficacy. The results showed that PLL strongly inhibited PrPSc propagation in the cell-free, cell culture, and mouse models of prion disease. These results confirm the role of plasminogen in PrPSc propagation, validates plasminogen as a therapeutic target to combat prion disease, and suggests PLL as a potential anti-prion agent. Therefore, our study represents a proof-of-concept that targeting plasminogen, a cofactor for PrP conversion, using PLL results in suppression of prion propagation, which represents a successful translation of our understanding on details of prion propagation into a potential therapeutic strategy for prion diseases.
Keywords: Poly-l-lysine; Prion conversion; Cofactor; Plasminogen; Therapeutic target; Translational research;