Biomaterials (v.33, #7)

The control of endothelial cell adhesion and migration by shear stress and matrix-substrate anchorage by Juliane Teichmann; Alexander Morgenstern; Jochen Seebach; Hans-Joachim Schnittler; Carsten Werner; Tilo Pompe (1959-1969).
Endothelial cells constitute the natural inner lining of blood vessels and possess anti-thrombogenic properties. This characteristic is frequently used by seeding endothelial cells on vascular prostheses. As the type of anchorage of adhesion ligands to materials surfaces is known to determine the mechanical balance of adherent cells, we investigated herein the behaviour of endothelial cells under physiological shear stress conditions. The adhesion ligand fibronectin was anchored to polymer surfaces of four physicochemical characteristics exhibiting covalent and non-covalent attachment as well as high and low hydrophobicity. The in situ analysis combined with cell tracking of shear stress-induced effects on cultured isolated cells and monolayers under venous (0.5 dyn/cm2) and arterial (12 dyn/cm2) shear stress over a time period of 24 h revealed distinct differences in their morphological and migratory features. Most pronounced, unidirectional and bimodal migration patterns of endothelial cells in or against flow direction were found in dependence on the type of substrate-matrix anchorage. Combined by an immunofluorescent analysis of the actin cytoskeleton, cell–cell junctions, cell-matrix adhesions, and matrix reorganization these results revealed a distinct balance of laminar shear stress, cell–cell contacts and substrate-matrix anchorage in affecting endothelial cell fate under flow conditions. This analysis underlines the importance of materials surface parameters as well as primary and secondary adhesion ligand anchorage in the context of artificial blood vessels for future therapeutic devices.
Keywords: Cell adhesion; ECM; Endothelial cells; Shear; Fibronectin; Migration;

Stability and biocompatibility of a library of polyester dendrimers in comparison to polyamidoamine dendrimers by Neus Feliu; Marie V. Walter; Maria I. Montañez; Andrea Kunzmann; Anders Hult; Andreas Nyström; Michael Malkoch; Bengt Fadeel (1970-1981).
Dendrimers can be designed for several biomedical applications due to their well-defined structure, functionality and dimensions. The present study focused on the in vitro biocompatibility evaluation of a library of aliphatic polyester dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) with an overall diameter of 0.5–2 nm. In addition, dendrimers with two different chemical surfaces (neutral with hydroxyl end group and anionic with carboxylic end group) and dendrons corresponding to the structural fragments of the dendrimers were evaluated. Commercial polyamidoamine dendrimers (PAMAM) with cationic (amine) or neutral (hydroxyl) end group were also included for comparison. Cell viability studies were conducted in human cervical cancer (HeLa) and acute monocytic leukemia cells (THP.1) differentiated into macrophage-like cells as well as in primary human monocyte-derived macrophages. Excellent biocompatibility was observed for the entire hydroxyl functional bis-MPA dendrimer library, whereas the cationic, but not the neutral PAMAM exerted dose-dependent cytotoxicity in cell lines and primary macrophages. Studies to evaluate material stability as a function of pH, temperature, and time, demonstrated that the stability of the 4th generation hydroxyl functional bis-MPA dendrimer increased at acidic pH. Taken together, bis-MPA dendrimers are degradable and non-cytotoxic to human cell lines and primary cells.
Keywords: Dendrimer; Biocompatibility; Cytokine secretion; Macrophage; Degradation;

The use of a dual PEDOT and RGD-functionalized alginate hydrogel coating to provide sustained drug delivery and improved cochlear implant function by Jennifer A. Chikar; Jeffrey L. Hendricks; Sarah M. Richardson-Burns; Yehoash Raphael; Bryan E. Pfingst; David C. Martin (1982-1990).
Cochlear implants provide hearing by electrically stimulating the auditory nerve. Implant function can be hindered by device design variables, including electrode size and electrode-to-nerve distance, and cochlear environment variables, including the degeneration of the auditory nerve following hair cell loss. We have developed a dual-component cochlear implant coating to improve both the electrical function of the implant and the biological stability of the inner ear, thereby facilitating the long-term perception of sound through a cochlear implant. This coating is a combination of an arginine-glycine-aspartic acid (RGD)-functionalized alginate hydrogel and the conducting polymer poly(3, 4-ethylenedioxythiophene) (PEDOT). Both in vitro and in vivo assays on the effects of these electrode coatings demonstrated improvements in device performance. We found that the coating reduced electrode impedance, improved charge delivery, and locally released significant levels of a trophic factor into cochlear fluids. This coating is non-cytotoxic, clinically relevant, and has the potential to significantly improve the cochlear implant user’s experience.
Keywords: Alginate; Drug delivery; Electroactive polymer; Growth factor; Hydrogel; Neural prosthesis;

Mechanisms underlying toxicity induced by CdTe quantum dots determined in an invertebrate model organism by Alfredo Ambrosone; Lucia Mattera; Valentina Marchesano; Alessandra Quarta; Andrei S. Susha; Angela Tino; Andrey L. Rogach; Claudia Tortiglione (1991-2000).
A systematic and thorough quantitative analysis of the in vivo effects of inorganic nanoparticles is extremely important for the design of functional nanomaterials for diagnostic and therapeutic applications, better understanding of their non-specificity toward tissues and cell types, and for assessments of their toxicity. This study was undertaken to examine the impact of CdTe quantum dots (QDs) on an invertebrate freshwater model organism, Hydra vulgaris, for assessment of long term toxicity effects. The continuous exposure of living polyps to sub-lethal doses of QDs caused time and dose dependent morphological damages more severe than Cd2+ ions at the same concentrations, impaired both reproductive and regenerative capability, activated biochemical and molecular responses. Of remarkable interest, low QD doses, apparently not effective, caused early changes in the expression of general stress responsive and apoptotic genes. The occurrence of subtle genetic variations, in the absence of morphological damages, indicates the importance of genotoxicity studies for nanoparticle risk assessment. The versatility in morphological, cellular, biochemical and molecular responses renders Hydra a perfect model system for high-throughput screening of toxicological and ecotoxicological impact of nanomaterials on human and environmental health.
Keywords: Animal model; Cytotoxicity; Genotoxicity; In vivo test; CdTe quantum dots;

Divalent cation-mediated polysaccharide interactions with zwitterionic surfaces by Luo Mi; Michelle M. Giarmarco; Qing Shao; Shaoyi Jiang (2001-2006).
One popular postulation in the design of a nonfouling surface is that a surface capable of resisting nonspecific protein adsorption should also resist bacterial adhesion and subsequent biofilm formation. Such a hypothesis, though valid in certain cases, oversimplifies complex biological systems, since they contain not only proteins but also other biomacromolecules, such as polysaccharides. This work aims to re-examine this postulation by testing the biofouling of polysaccharides onto protein-resisting zwitterionic surfaces in the presence of a multivalent cation. Our results show that Mg2+ plays an important role in mediating alginate adsorption onto zwitterionic surfaces through ion-bridged interactions from surface plasmon resonance (SPR) experiments. Three zwitterionic polymers tested in this work have clearly different responses to changes in Mg2+ concentration, indicating that such ion-bridged adsorption is strongly dependent on cation-zwitterionic polymer binding affinities and is dictated by the specific chemical structure of the polymer betaine side chain. This work underlines the necessity to go beyond current nonfouling criteria at the protein level and to take into account polysaccharides when it comes to complex environments.
Keywords: Polysaccharide; Bacterial adhesion; Surface modification; Biosensor;

Dynamics of T cells on endothelial layers aligned by nanostructured surfaces by Kwang Hoon Song; Keon Woo Kwon; Sukhyun Song; Kahp-Yang Suh; Junsang Doh (2007-2015).
In this work, well-aligned endothelial cell (EC) layers were prepared by culturing ECs on surfaces containing nanoscale ridges/grooves fabricated by UV-assisted capillary force lithography. Then, the dynamics of T cells on well-aligned ECs were compared with that on randomly oriented ECs cultured on flat surfaces. With this experimental setting, we demonstrated for the first time that EC alignment is important for the regulation of transendothelial migration (TEM) of T cells, a critical step for leukocyte infiltration; T cells preferentially underwent TEM at the junctions surrounded by more than three ECs only if ECs surrounding those junctions were poorly aligned. As a result, TEM of T cells occurred more quickly and frequently on randomly oriented ECs cultured on flat surfaces than on well-aligned ECs cultured on nanostructured surfaces. This result will suggest a new strategy for the design of synthetic small diameter vascular grafts and extend our current knowledge of leukocyte dynamics on an inflamed endothelium.
Keywords: Nanostructured surfaces; Capillary force lithography; Endothelial cell alignment; T cell dynamics; Transendothelial migration;

The aim of this study was to test the hypotheses that retroviral gene transfer of SOX trio enhances the in vitro chondrogenic differentiation of ASCs, and that SOX trio-co-transduced ASCs in fibrin gel promote the healing of osteochondral defects, and arrest the progression of surgically-induced osteoarthritis in a rat model. ASCs isolated from inguinal fat in rats were transduced with SOX trio genes using retrovirus, and further cultured in vitro in pellets for 21 days, then analyzed for gene and protein expression of SOX trio and chondrogenic markers. SOX trio-co-transduced ASCs in fibrin gel were implanted on the osteochondral defect created in the patellar groove of the distal femur, and also injected into the knee joints of rats with surgically-induced osteoarthritis. Rats were sacrificed after 8 weeks, and analyzed grossly and microscopically. After 21 days, ASCs transduced with SOX-5, -6, or -9 had hundreds-fold greater gene expression of each gene compared with the control with the SOX protein expression matching gene expression. SOX trio-co-transduction significantly increased GAG contents as well as type II collagen gene and protein expression. ASCs co-transduced with SOX trio significantly promoted the in vivo cartilage healing in osteochondral defect model, and prevented the progression of degenerative changes in surgically-induced osteoarthritis.
Keywords: Retroviral gene transfer; SOX trio; Chondrogenic differentiation; Fibrin gel; Osteochondral defect; Surgically-induced osteoarthritis;

The disruption of balance between osteogenesis and adipogenesis of mesenchymal stem cells (MSCs) leads to the disorders such as osteoporosis. Controlling the balance of these processes during MSC differentiation is important to maintain bone homeostasis. The extracellular microenvironment, especially the extracellular matrix (ECM), plays an important role in regulating MSC differentiation. Here, we investigated the role of ECM in controlling the balance between osteogenesis and adipogenesis of MSCs with matrices that mimic the stepwise tissue development of ECM during osteogenesis and adipogenesis. The osteogenesis of MSCs was enhanced by matrices with upregulated RUNX2 expression and suppressed PPARG expression, which mimic the attributes of the ECM during the early stages of osteogenesis. MSC adipogenesis was enhanced by matrices with suppressed expression of RUNX2, MSX2, and TAZ, which mimics the characteristics of ECM during the early stages of adipogenesis. These results showed that ECM may regulate the expression of various transcription factors to control the balance of osteogenesis and adipogenesis of MSCs. Tissue- and stage-specific ECM are required to control differentiation of MSCs into a specific cell types.
Keywords: ECM (extracellular matrix); Biomimetic material; Mesenchymal stem cell; Osteogenesis; Adipogenesis;

The effect of vitronectin on the differentiation of embryonic stem cells in a 3D culture system by Sepideh Heydarkhan-Hagvall; Jessica M. Gluck; Connor Delman; Monica Jung; Nazanin Ehsani; Sean Full; Richard J. Shemin (2032-2040).
While stem cell niches in vivo are complex three-dimensional (3D) microenvironments, the relationship between the dimensionality of the niche to its function is unknown. We have created a 3D microenvironment through electrospinning to study the impact of geometry and different extracellular proteins on the development of cardiac progenitor cells (Flk-1+) from resident stem cells and their differentiation into functional cardiovascular cells. We have investigated the effect of collagen IV, fibronectin, laminin and vitronectin on the adhesion and proliferation of murine ES cells as well as the effects of these proteins on the number of Flk-1+ cells cultured in 2D conditions compared to 3D system in a feeder free condition. We found that the number of Flk-1+ cells was significantly higher in 3D scaffolds coated with laminin or vitronectin compared to colIV-coated scaffolds. Our results show the importance of defined culture systems in vitro for studying the guided differentiation of pluripotent embryonic stem cells in the field of cardiovascular tissue engineering and regenerative medicine.
Keywords: Extracellular matrix; Niche; Cardiovascular tissue engineering; Stem cell; Scaffold;

Modulation of Wnt/β-catenin signaling in human embryonic stem cells using a 3-D microwell array by Samira M. Azarin; Xiaojun Lian; Elise A. Larson; Heidi M. Popelka; Juan J. de Pablo; Sean P. Palecek (2041-2049).
Intercellular interactions in the cell microenvironment play a critical role in determining cell fate, but the effects of these interactions on pathways governing human embryonic stem cell (hESC) behavior have not been fully elucidated. We and others have previously reported that 3-D culture of hESCs affects cell fates, including self-renewal and differentiation to a variety of lineages. Here we have used a microwell culture system that produces 3-D colonies of uniform size and shape to provide insight into the effect of modulating cell–cell contact on canonical Wnt/β-catenin signaling in hESCs. Canonical Wnt signaling has been implicated in both self-renewal and differentiation of hESCs, and competition for β-catenin between the Wnt pathway and cadherin-mediated cell–cell interactions impacts various developmental processes, including the epithelial–mesenchymal transition. Our results showed that hESCs cultured in 3-D microwells exhibited higher E-cadherin expression than cells on 2-D substrates. The increase in E-cadherin expression in microwells was accompanied by a downregulation of Wnt signaling, as evidenced by the lack of nuclear β-catenin and downregulation of Wnt target genes. Despite this reduction in Wnt signaling in microwell cultures, embryoid bodies (EBs) formed from hESCs cultured in microwells exhibited higher levels of Wnt signaling than EBs from hESCs cultured on 2-D substrates. Furthermore, the Wnt-positive cells within EBs showed upregulation of genes associated with cardiogenesis. These results demonstrate that modulation of intercellular interactions impacts Wnt/β-catenin signaling in hESCs.
Keywords: Stem cell; Micropatterning; Cell signaling; Cell adhesion;

The reparative response to cross-linked collagen-based scaffolds in a rat spinal cord gap model by Rahmatullah H. Cholas; Hu-Ping Hsu; Myron Spector (2050-2059).
Prior work demonstrated the improvement of peripheral nerve regeneration in gaps implanted with collagen scaffold-filled collagen tubes, compared with nerve autografts, and the promise of such implants for treating gaps in spinal cord injury (SCI) in rats. The objective of this study was to investigate collagen implants alone and incorporating select therapeutic agents in a 5-mm full-resection gap model in the rat spinal cord. Two studies were performed, one with a 6-week time point and one with a 2-week time point. For the 6-week study the groups included: (1) untreated control, (2) dehydrothermally (DHT)-cross-linked collagen scaffold, (3) DHT-cross-linked collagen scaffold seeded with adult rat neural stem cells (NSCs), and (4) DHT-cross-linked collagen scaffold incorporating plasmid encoding glial cell line-derived neurotropic factor (pGDNF). The 2-week study groups were: (1) nontreated control, (2) DHT-cross-linked collagen scaffold; (3) DHT-cross-linked collagen scaffold containing laminin; and (4) carbodiimide-cross-linked collagen scaffold containing laminin. The tissue filling the defect of all groups at 6 weeks was largely composed of fibrous scar; however, the tissue was generally more favorably aligned with the long axis of the spinal cord in all of the treatment groups, but not in the control group. Quantification of the percentage of animals per group containing cystic cavities in the defect showed a trend toward fewer rats with cysts in the groups in which the scaffolds were implanted compared to control. All of the collagen implants were clearly visible and mostly intact after 2 weeks. A band of fibrous tissue filling the control gaps was not seen in the collagen implant groups. In all of the groups there was a narrowing of the spinal canal within the gap as a result of surrounding soft tissue collapse into the defect. The narrowing of the spinal canal occurred to a greater extent in the control and DHT scaffold alone groups compared to the DHT scaffold/laminin and EDAC scaffold/laminin groups. Collagen biomaterials can be useful in the treatment of SCI to: favorably align the reparative tissue with the long axis of the spinal cord; potentially reduce the formation of fluid-filled cysts; serve as a delivery vehicle for NSCs and the gene for GDNF; and impede the collapse of musculature and connective tissue into the defect.
Keywords: Collagen; Scaffold; Nerve tissue engineering; Nerve guide; Stem cells; Growth factors;

The beneficial effects of deferred delivery on the efficiency of hydrogel therapy post myocardial infarction by Karen Kadner; Stephan Dobner; Thomas Franz; Deon Bezuidenhout; Mazin S. Sirry; Peter Zilla; Neil H. Davies (2060-2066).
Biomaterials are increasingly being investigated as a means of reducing stress within the ventricular wall of infarcted hearts and thus attenuating pathological remodelling and loss of function. In this context, we have examined the influence of timing of delivery on the efficacy of a polyethylene glycol hydrogel polymerised with an enzymatically degradable peptide sequence. Delivery of the hydrogel immediately after infarct induction resulted in no observable improvements, but a delay of one week in delivery resulted in significant increases in scar thickness and fractional shortening, as well as reduction in end-systolic diameter against saline controls and immediately injected hydrogel at both 2 and 4 weeks post-infarction (p < 0.05). Hydrogels injected at one week were degraded significantly slower than those injected immediately and this may have played a role in the differing outcomes. The hydrogel assumed markedly different morphologies at the two time points having either a fibrillar or bulky appearance after injection immediately or one week post-infarction respectively. We argue that the different morphologies result from infarction induced changes in the cardiac structure and influence the degradability of the injectates. The results indicate that timing of delivery is important and that very early time points may not be beneficial.
Keywords: Heart; Hydrogel; Infarction; Remodelling;

Functional improvement and neurogenesis after collagen-GAG matrix implantation into surgical brain trauma by Kuo-Feng Huang; Wei-Cherng Hsu; Wen-Ta Chiu; Jia-Yi Wang (2067-2075).
Surgical or traumatic brain injury often leads to loss of cerebral parenchyma but there is as yet no clinically effective strategy for neural regeneration. Collagen glycosaminoglycan (collagen-GAG, CG) scaffolds have previously been used in many tissues in vivo but have never been utilized in the brain. Using an animal model, we investigated the effects of the implantation of CG scaffold matrix following surgical brain trauma. Results indicated that implantation of CG scaffold could significantly promote functional recovery following surgical brain trauma. The CG scaffold was found to facilitate proliferation, differentiation and migration of endogenous neural precursor cells (NPCs) both in the intra-matrix zone (IMZ) and lesion boundary zone (LBZ). The tissue concentration of brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF) in the cortex demonstrated a sustained increase after implantation of CG scaffold following surgical brain trauma. These results suggest that the utilization of CG scaffolds can be considered as a potential clinical strategy for tissue regeneration and functional recovery after brain injury.
Keywords: Brain; Cell proliferation; Collagen-GAG; Neurotrophic factor; Scaffold; Functional recovery;

Hypoxia-mimicking mesoporous bioactive glass scaffolds with controllable cobalt ion release for bone tissue engineering by Chengtie Wu; Yinghong Zhou; Wei Fan; Pingping Han; Jiang Chang; Jones Yuen; Meili Zhang; Yin Xiao (2076-2085).
Low oxygen pressure (hypoxia) plays an important role in stimulating angiogenesis; there are, however, few studies to prepare hypoxia-mimicking tissue engineering scaffolds. Mesoporous bioactive glass (MBG) has been developed as scaffolds with excellent osteogenic properties for bone regeneration. Ionic cobalt (Co) is established as a chemical inducer of hypoxia-inducible factor (HIF)-1α, which induces hypoxia-like response. The aim of this study was to develop hypoxia-mimicking MBG scaffolds by incorporating ionic Co2+ into MBG scaffolds and investigate if the addition of Co2+ ions would induce a cellular hypoxic response in such a tissue engineering scaffold system. The composition, microstructure and mesopore properties (specific surface area, nano-pore volume and nano-pore distribution) of Co-containing MBG (Co-MBG) scaffolds were characterized and the cellular effects of Co on the proliferation, differentiation, vascular endothelial growth factor (VEGF) secretion, HIF-1α expression and bone-related gene expression of human bone marrow stromal cells (BMSCs) in MBG scaffolds were systematically investigated. The results showed that low amounts of Co (<5%) incorporated into MBG scaffolds had no significant cytotoxicity and that their incorporation significantly enhanced VEGF protein secretion, HIF-1α expression, and bone-related gene expression in BMSCs, and also that the Co-MBG scaffolds support BMSC attachment and proliferation. The scaffolds maintain a well-ordered mesopore channel structure and high specific surface area and have the capacity to efficiently deliver antibiotics drugs; in fact, the sustained released of ampicillin by Co-MBG scaffolds gives them excellent anti-bacterial properties. Our results indicate that incorporating cobalt ions into MBG scaffolds is a viable option for preparing hypoxia-mimicking tissue engineering scaffolds and significantly enhanced hypoxia function. The hypoxia-mimicking MBG scaffolds have great potential for bone tissue engineering applications by combining enhanced angiogenesis with already existing osteogenic properties.
Keywords: Hypoxia; Mesoporous bioactive glass; Bone tissue engineering; VEGF secretion; HIF-1α expression;

The role of bFGF on the ability of MSC to activate endogenous regenerative mechanisms in an ectopic bone formation model by Roberta Tasso; Massimiliano Gaetani; Erica Molino; Angela Cattaneo; Massimiliano Monticone; Angela Bachi; Ranieri Cancedda (2086-2096).
The view depicting bone marrow (BM)-derived mesenchymal stem cells (MSC) as a uniform population differentiating into new-tissue builder cells is evolving toward the concept of a heterogeneous population of stem/progenitor cells secreting bioactive molecules, and contributing to establish an on-site regenerative microenvironment. We report that in an ectopic bone formation model the intrinsic MSC capability to activate endogenous regenerative mechanisms is critically dependent on the commitment level of implanted MSC. We demonstrate that the presence of bFGF in the culture medium during mouse MSC expansion in vitro is the key factor for the selection of subpopulations inducing host regenerative responses. We developed a novel strategy combining SILAC-LC-MS/MS quantitative proteomics of conditioned culture media and gene expression profiling to disentangle the major role of MSC in modulating the microenvironment toward the damage resolution. The correspondence between results provided by the applied techniques proved that the most statistically significant biological processes favored by the bFGF treatment were carried out by secreted factors. In particular, the immune response, the inflammatory response, the response to wounding and chemotaxis were all upregulated in bFGF-selected MSC. We propose these processes as majorly involved in activating the endogenous responses triggered by trophic effects of implanted bFGF-selected MSC.
Keywords: Bone regeneration; Cell signaling; Mesenchymal stem cells; Fibroblast growth factor;

Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs by Duohong Zou; Zhiyuan Zhang; Jiacai He; Kai Zhang; Dongxia Ye; Wei Han; Jian Zhou; Yuanyin Wang; Quanli Li; Xin Liu; Xin Zhang; Shaoyi Wang; Jingzhou Hu; Chao Zhu; Wenjie Zhang; Yong zhou; Honghai Fu; Yuanliang Huang; Xinquan Jiang (2097-2108).
The successful clinical outcome of the implanted tissue-engineered bone is dependent on the establishment of a functional vascular network. A gene-enhanced tissue engineering represents a promising approach for vascularization. Our previous study indicated that hypoxia-inducible factor-1α (HIF-1α) can up-regulate the expression of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1) in bone mesenchymal stem cells (BMSCs). The angiogenesis is a co-ordinated process that requires the participation of multiple angiogenic factors. To further explore the angiogenic effect of HIF-1α mediated stem cells, in this study, we systematically evaluated the function of HIF-1α in enhancing BMSCs angiogenesis in vitro and in vivo. A constitutively active form of HIF-1α (CA5) was inserted into a lentivirus vector and transduced into BMSCs, and its effect on vascularization and vascular remodeling was further evaluated in a rat critical-sized calvarial defects model with a gelatin sponge (GS) scaffold. The expression of the key angiogenic factors including VEGF, SDF-1, basic fibroblast growth factor (bFGF), placental growth factor (PLGF), angiopoietin 1 (ANGPT1), and stem cell factor (SCF) at both mRNAs and proteins levels in BMSCs were significantly enhanced by HIF-1α overexpression compared to the in vitro control group. In addition, HIF-1α-over expressing BMSCs showed dramatically improved blood vessel formation in the tissue-engineered bone as analyzed by photography of specimen, micro-CT, and histology. These data confirm the important role of HIF-1α in angiogenesis in tissue-engineered bone. Improved understanding of the mechanisms of angiogenesis may offer exciting therapeutic opportunities for vascularization, vascular remodeling, and bone defect repair using tissue engineering strategies in the future.
Keywords: HIF-1a; Angiogenesis; The tissue-engineered bone; Gene therapy;

Regeneration of dental pulp following pulpectomy by fractionated stem/progenitor cells from bone marrow and adipose tissue by Ryo Ishizaka; Koichiro Iohara; Masashi Murakami; Osamu Fukuta; Misako Nakashima (2109-2118).
Pulp stem/progenitor cells can induce complete pulp regeneration. However, due to the limited availability of pulp tissue with age, there is a need to examine other sources for fractions of side population (SP) cells. In the present investigation bone marrow and adipose tissues of the same individual were evaluated as alternate sources. Pulp CD31 SP cells have higher migration activity and higher expression of angiogenic/neurotrophic factors than bone marrow and adipose CD31 SP cells. Adipose tissue CD31 SP cell transplantation yielded the same amount of regenerated tissue as pulp derived cells. However, bone marrow CD31 SP cell transplantation yielded significantly less regenerated tissue in pulpectomized root canals in dogs. The rate of matrix formation was much higher in adipose CD31 SP cell transplantation compared to pulp CD31 SP cell transplantation on day 28. Microarray analysis demonstrated similar qualitative and quantitative patterns of mRNA expression characteristic of pulp in the regenerated tissues from all three cell sources. Expression of many angiogenic/neurotrophic factors in the transplanted cells demonstrated trophic effects. Our results demonstrate that bone marrow and adipose CD31 SP cells might be suitable alternative cell sources for pulp regeneration.
Keywords: Dental pulp stem cells (DPSC); Adipose-derived stem cells (ADSC); Bone marrow-derived stem cells (BMSC); Transplantation; Pulp regeneration; Side population (SP) cells;

The focus on creating tissue engineered constructs of clinically relevant sizes requires new approaches for monitoring construct health during tissue development. A few key requirements are that the technology be in situ, non-invasive, and provide temporal and spatial information. In this work, we demonstrate that optical coherence microscopy (OCM) can be used to assess cell viability without the addition of exogenous probes in three-dimensional (3D) tissue scaffolds maintained under standard culture conditions. This is done by collecting time-lapse images of speckle generated by sub-cellular features. Image cross-correlation is used to calculate the number of features the final image has in common with the initial image. If the cells are live, the number of common features is low. The number of common features approaches 100% if the cells are dead. In control experiments, cell viability is verified by the addition of a two-photon fluorescence channel to the OCM. Green fluorescent protein transfected human bone marrow stromal cells cultured in a transparent poly(ethylene glycol) tetramethacrylate hydrogel scaffold is used as the control system. Then, the utility of this approach is demonstrated by determining L929 fibroblast cell viability in a more challenging matrix, collagen, an optical scatterer. These results demonstrate a new technique for in situ mapping of single cell viability without any exogenous probes that is capable of providing continuous monitoring of construct health.
Keywords: Cell viability; Hydrogel; Image analysis; Scaffold; Stem cell;

Vascularization and restoration of heart function in rat myocardial infarction using transplantation of human cbMSC/HUVEC core-shell bodies by Wen-Yu Lee; Hao-Ji Wei; Jiun-Jie Wang; Kun-Ju Lin; Wei-Wen Lin; Ding-Yuan Chen; Chieh-Cheng Huang; Ting-Yin Lee; Hsiang-Yang Ma; Shiaw-Min Hwang; Yen Chang; Hsing-Wen Sung (2127-2136).
Cell transplantation is a promising strategy for therapeutic treatment of ischemic heart diseases. In this study, cord blood mesenchymal stem cells (cbMSCs) and human umbilical vein endothelial cells (HUVECs) in the form of core-shell bodies (cbMSC/HUVEC bodies) were prepared to promote vascularization and restore heart functions in an experimentally-created myocardial infarction (MI) rat model. Saline, cbMSC bodies and HUVEC bodies were used as controls. In vitro results indicated that cbMSC/HUVEC bodies possessed the capability of heterotypic assembly of cbMSCs and HUVECs into robust and durable tubular networks on Matrigel. The up-regulated gene expressions of VEGF and IGF-1 reflected the robust expansion of tubular networks; in addition, the augmented levels of SMA and SM22 suggested smooth muscle differentiation of cbMSCs, possibly helping to improve the durability of networks. Moreover, according to the in vivo echocardiographic, magnetic resonance and computed-tomographic results, transplantation of cbMSC/HUVEC bodies benefited post-MI dysfunction. Furthermore, the vascularization analyses demonstrated the robust vasculogenic potential of cbMSC/HUVEC bodies in vivo, thus contributing to the greater viable myocardium and the less scar region, and ultimately restoring the cardiac function. The concept of core-shell bodies composed of perivascular cells and endothelial cells may serve as an attractive cell delivery vehicle for vasculogenesis, thus improving the cardiac function significantly.
Keywords: Thermo-responsive hydrogel; Vasculogenesis; Blood perfusion; Cell therapy; Cardiac regeneration; Thermo-responsive hydrogel;

Tenogenic differentiation of human MSCs induced by the topography of electrochemically aligned collagen threads by Vipuil Kishore; Whitney Bullock; Xuanhao Sun; William Scott Van Dyke; Ozan Akkus (2137-2144).
Topographical cues from the extracellular microenvironment can influence cellular activity including proliferation and differentiation. Information on the effects of material topography on tenogenic differentiation of human mesenchymal stem cells (human MSCs) is limited. A methodology using the principles of isoelectric focusing has previously been developed in our laboratory to synthesize electrochemically aligned collagen (ELAC) threads that mimics the packing density, alignment and strength of collagen dense connective tissues. In the current study, human MSCs were cultured on ELAC and randomly oriented collagen threads and the effect of collagen orientation on cell morphology, proliferation and tenogenic differentiation was investigated. The results indicate that higher rates of proliferation were observed on randomly oriented collagen threads compared to ELAC threads. On the other hand, tendon specific markers such as scleraxis and tenomodulin, were significantly increased on ELAC threads compared to randomly oriented collagen threads. Additionally, osteocalcin, a specific marker of bone differentiation was suppressed on ELAC threads. Previous studies have reported that BMP-12 is a key growth factor to induce tenogenic differentiation of MSCs. To evaluate the synergistic effect of BMP-12 and collagen orientation, human MSCs were cultured on ELAC threads in culture medium supplemented with and without BMP-12. The results revealed that BMP-12 did not have an additional effect on the tenogenic differentiation of human MSCs on ELAC threads. Together, these results suggest that ELAC induces tenogenic differentiation of human MSCs by presenting an aligned and dense collagen substrate, akin to the tendon itself. In conclusion, ELAC has a significant potential to be used as a tendon replacement and in the development of an osteotendinous construct towards the regeneration of bone–tendon interfaces.
Keywords: Collagen; Topography; Mesenchymal stem cells; Differentiation; Tenogenic; Tendon;

Injectable shear-thinning hydrogels engineered with a self-assembling Dock-and-Lock mechanism by Hoang D. Lu; Manoj B. Charati; Iris L. Kim; Jason A. Burdick (2145-2153).
Injected therapeutics, such as cells or biological molecules, may have enhanced efficiency when delivered within a scaffold carrier. Here, we describe a dual-component Dock-and-Lock (DnL) self-assembly mechanism that can be used to construct shear-thinning, self-healing, and injectable hydrogels. One component is derived from the RIIα subunit of cAMP-dependent kinase A and is engineered as a telechelic protein with end groups that dimerize (docking step). The second component is derived from the anchoring domain of A-kinase anchoring protein (AD) and is attached to multi-arm crosslinker polymers and binds to the docked proteins (locking step). When mixed, these two DnL components form robust physical hydrogels instantaneously and under physiological conditions. Mechanical properties and erosion rates of DnL gels can be tuned through the AD peptide sequence, the concentration and ratio of each component, and the number of peptides on the cross-linking polymer. DnL gels immediately self-recover after deformation, are resistant to yield at strains as high as 400%, and completely self-heal irrespective of prior mechanical disruption. Mesenchymal stem cells mixed in DnL gels and injected through a fine needle remain highly viable (>90%) during the encapsulation and delivery process, and encapsulated large molecules are released with profiles that correspond to gel erosion. Thus, we have used molecular engineering strategies to develop cytocompatible and injectable hydrogels that have the potential to support cell and drug therapies.
Keywords: Self assembly; Hydrogel; Cell encapsulation; Mesenchymal stem cell; Drug delivery; Shear;

Interactions of vimentin- or desmin-expressing liver cells with N-acetylglucosamine-bearing polymers by Sun-Jung Kim; Hirohiko Ise; Mitsuaki Goto; Toshihiro Akaike (2154-2164).
It is necessary to develop highly functionalized liver cell culture systems for liver tissue engineering such as bioartificial livers and liver cell chips. To maintain a high level of hepatocyte function, well-organized patterning culture systems of hepatocytes and nonparenchymal cells would be advantageous. To design the patterning culture system using these cells, cell-recognizable polymers should be useful to regulate not only the hepatocytes, but also the nonparenchymal cells. Here, we report that N-acetylglucosamine (GlcNAc)-bearing polymers are useful as nonparenchymal cell-recognizable polymers. It has previously been reported that mesenchymal cells adhered to GlcNAc-bearing polymer-coated dishes through surface vimentin. It was also observed that nonparenchymal cells expressing vimentin or desmin specifically adhered to GlcNAc-bearing polymer-coated dishes. Especially, in hepatic stellate cells (HSCs) cultured on GlcNAc-bearing polymer-coated dishes, the expression of α-smooth muscle actin as an activated HSCs marker was suppressed in long-term. Therefore, HSCs were shown to maintain a quiescent state on PVGlcNAc-coated dishes during a long-term culture. These results demonstrated that GlcNAc-bearing polymers could be beneficial to culture nonparenchymal cells such as HSCs. Our findings suggest that galactose- and GlcNAc-bearing polymers can regulate the culture of all liver cells and may be useful tools for the establishment of liver tissue engineering.
Keywords: Cell adhesion; Hepatocyte; Liver tissue engineering; Nonparenchymal cells; Hepatic stellate cells;

Tethered spheroids as an in vitro hepatocyte model for drug safety screening by Lei Xia; Rashidah Binte Sakban; Yinghua Qu; Xin Hong; Wenxia Zhang; Bramasta Nugraha; Wen Hao Tong; Abhishek Ananthanarayanan; Baixue Zheng; Ian Yin-Yan Chau; Ruirui Jia; Michael McMillian; Jose Silva; Shannon Dallas; Hanry Yu (2165-2176).
Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 μm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.
Keywords: 3D hepatocyte model; Xenobiotics; RGD; Galactose; CYP450 enzymes; Drug safety testing;

Monitoring transplanted adipose tissue-derived stem cells combined with heparin in the liver by fluorescence imaging using quantum dots by Hiroshi Yukawa; Masaki Watanabe; Noritada Kaji; Yukihiro Okamoto; Manabu Tokeshi; Yoshitaka Miyamoto; Hirofumi Noguchi; Yoshinobu Baba; Shuji Hayashi (2177-2186).
Adipose tissue-derived stem cell (ASC) transplantation, when used in combination with heparin, has proven to be an effective treatment for acute liver failure in mice. However, the behavior and organ-specific accumulation of transplanted ASCs alone or in combination with heparin is poorly understood. In this paper, we investigated whether quantum dots (QDs) labeling using octa-arginine peptide (R8) for ASCs could be applied for in vivo fluorescence imaging in mice with acute liver failure, and analyzed the behavior and organ-specific accumulation of ASCs that were transplanted alone or in combination with heparin using an IVIS® Spectrum analysis. Almost all of the transplanted ASCs were observed to accumulate in the lungs within 10 min without heparin. However, when heparin was used in combination with the ASCs, the accumulation of the transplanted ASCs was found not only in the lungs but also in the liver. The region of interest (ROI) analysis of ex vivo fluorescence imaging showed that the accumulation rate of transplanted ASCs in the liver increased to about 30%. In the time course analysis, the accumulation rate of ASCs in the liver was about 10% in 1 day and was maintained at that level for at least 2 day. We observed that heparin was effective for increasing the accumulation of transplanted ASCs in the liver using fluorescence imaging technology. We suggest that fluorescence imaging by means of QDs labeling using R8 can be useful for tracing the transplanted cells.
Keywords: Cell transplantation; Adipose tissue-derived stem cells (ASCs); Fluorescence imaging; Quantum dots (QDs); Octa-arginine peptide (R8); Liver failure;

Doxorubicin-loaded glycyrrhetinic acid-modified alginate nanoparticles for liver tumor chemotherapy by Chuangnian Zhang; Wei Wang; Tong Liu; Yukun Wu; Hua Guo; Ping Wang; Qin Tian; Yongming Wang; Zhi Yuan (2187-2196).
Doxorubicin (DOX)-loaded glycyrrhetinic acid (GA)-modified alginate (ALG) nanoparticles (DOX/GA-ALG NPs) were prepared for targeting therapy of liver cancer. This study focused on the biodistribution of DOX/GA-ALG NPs in Kunming mice as well as their antitumor activity against liver tumors in situ and side effects. The biodistribution data showed that the concentration of DOX in the liver reached 67.8 ± 4.9 μg/g after intravenous administration of DOX/GA-ALG NPs, which was 2.8-fold and 4.7-fold higher compared to non-GA-modified nanoparticles (DOX/CHO-ALG NPs) and DOX·HCl, respectively. The concentration of DOX in the heart of mice treated with DOX/GA-ALG NPs at any sampling time was relatively lower than that of mice treated with DOX·HCl. The liver tumor growth inhibition rate (IR) in situ was about 52.6% and the mortality was 33% in DOX·HCl group. In contrast, the IR was 76.6% and no mice died in the DOX/GA-ALG NPs group. Histological examination showed tumor necrosis in both experimental groups. Most importantly, the heart cells and the liver cells surrounding the tumor were not affected by administration of DOX/GA-ALG NPs, whereas myocardial necrosis and apparent liver cell swelling were observed after DOX·HCl administration.
Keywords: Glycyrrhetinic acid; Liver targeting; Biodistribution; Antitumor; Liver tumors in situ;

Activation of inflammasomes by tumor cell death mediated by gold nanoshells by Hai T. Nguyen; Kenny K. Tran; Bingbing Sun; Hong Shen (2197-2205).
Gold nanoshell-enabled photothermal therapy (NEPTT) utilizes the efficient thermal conversion of near infrared (NIR) light for the ablation of cancer cells. Cancer therapies that combine cell killing with the induction of a strong immune response against the dying tumor cells have been shown to increase therapeutic efficacy in the clearance and regression of cancers. In this study, we assessed the ability of dying cells generated by in vitro NEPTT to activate inflammasome complexes. We quantified levels of major danger-associated molecular patterns (DAMPs), including adenosine triphosphate (ATP), adenosine diphosphate (ADP), and uric acid, released from tumor cells treated by NEPTT. The amount of DAMPs released was dependent on the dose of nanoshells internalized by cells. However, under all the employed conditions, the levels of generated DAMPs were insufficient to activate inflammasome complexes and to induce the production of pro-inflammatory cytokines (i.e. IL-1β). The results from this study provide insights into the development of nanoplasmonics for combining both photothermal therapy and immunotherapy to eradicate cancers.
Keywords: Gold nanoshells; nanoplasmonics; Apoptosis; Necrosis; NALP3 inflammasome; DAMPs;

Photothermal therapy as a physical treatment approach to destruct cancer has emerged as an alternative of currently used cancer therapies. Previously we have shown that polyethylene glycol (PEG) functionalized nano-graphene oxide (nGO-PEG) with strong optical absorption in the near-infrared (NIR) region was a powerful photothermal agent for in vivo cancer treatment. In this work, by using ultra-small reduced graphene oxide (nRGO) with non-covalent PEG coating, we study how sizes and surface chemistry affect the in vivo behaviors of graphene, and remarkably improve the performance of graphene-based in vivo photothermal cancer treatment. Owing to the enhanced NIR absorbance and highly efficient tumor passive targeting of nRGO-PEG, excellent in vivo treatment efficacy with 100% of tumor elimination is observed after intravenous injection of nRGO-PEG and the followed 808 nm laser irradiation, the power density (0.15 W/cm2, 5 min) of which is an order of magnitude lower than that usually applied for in vivo tumor ablation using many other nanomaterials. All mice after treatment survive over a period of 100 days without a single death or any obvious sign of side effect. Our results highlight that both surface chemistry and sizes are critical to the in vivo performance of graphene, and show the promise of using optimized nano-graphene for ultra-effective photothermal treatment, which may potentially be combined with other therapeutic approaches to assist our fight against cancer.
Keywords: Nano-reduced graphene oxide; Non-covalent functionalization; Size effect; Photothermal therapy; Ultra-low laser power; Cancer treatment;

Multifunctional nanoparticles for upconversion luminescence/MR multimodal imaging and magnetically targeted photothermal therapy by Liang Cheng; Kai Yang; Yonggang Li; Xiao Zeng; Mingwang Shao; Shuit-Tong Lee; Zhuang Liu (2215-2222).
Theranostics, the combination of diagnostics and therapies, has become a new concept in the battles with various major diseases such as cancer. Herein, we develop multifunctional nanoparticles (MFNPs) with highly integrated functionalities including upconversion luminescence, superparamagnetism, and strong optical absorption in the near-infrared (NIR) region with high photostability. In vivo dual modal optical/magnetic resonance imaging of mice uncovers that by placing a magnet nearby the tumor, MFNPs tend to migrate toward the tumor after intravenous injection and show high tumor accumulation, which is ∼8 folds higher than that without magnetic targeting. NIR laser irradiation is then applied to the tumors grown on MFNP-injected mice under magnetic tumor-targeting, obtaining an outstanding photothermal therapeutic efficacy with 100% of tumor elimination in a murine breast cancer model. We present here a strategy for multimodal imaging-guided, magnetically targeted physical cancer therapy and highlight the promise of using multifunctional nanostructures for cancer theranostics.
Keywords: Multifunctional nanoparticles; Multimodal imaging; Magnetic tumor targeting; In vivo cancer treatment; Photothermal therapy;

Drug delivery to solid tumours remains a challenge because both tumour physiology and drug solubility are unfavourable. Engineered materials can provide the basis for drug reformulation, incorporating active compounds and modulating their pharmacokinetic and biodistribution behaviour. To this end, we encapsulated docetaxel, a poorly soluble taxane drug, in a self-assembled polymeric nanoparticle micelle of poly(2-methyl-2-carboxytrimethylene carbonate-co- d,l -lactide)-graft-poly(ethylene glycol) (poly(TMCC-co-LA)-g-PEG). This formulation was compared with its conventional ethanolic polysorbate 80 formulation in terms of plasma circulation and biodistribution in an orthotopic mouse model of breast cancer. Notably, the polymeric nanoparticle formulation achieved greater tumour retention, resulting in prolonged exposure of cancer cells to the active drug. This behaviour was unique to the tumour tissue. The active drug was eliminated at equal or greater rates in all other tissues assayed when delivered in the polymeric nanoparticles vs. the free drug formulation. Thus, these polymeric nanoparticles are promising vehicles for solid tumour drug delivery applications, offering greater tumour exposure while eliminating the need for toxic solvents and surfactants in the dosing formulation.
Keywords: Polymeric nanoparticles; In vivo evaluation; Micelle; Self-assembly; Chemotherapy; Drug delivery;

A NIR heptamethine dye with intrinsic cancer targeting, imaging and photosensitizing properties by Xu Tan; Shenglin Luo; Dechun Wang; Yongping Su; Tianmin Cheng; Chunmeng Shi (2230-2239).
The future personalized oncology significantly relies on the development of multifunctional agents to integrate tumor targeting, imaging, and cytotoxic activities. In our recent study, we have recently identified a near infrared fluorescent heptamethine dye, IR-780, with unexpected preferential accumulation in a broad spectra of tumor cells for in vivo tumor targeting and imaging. On the basis of this foregoing work, in this study, we describe here the chemical synthesis and biological characterization of an analog of IR-780, termed as IR-808, which not only possesses similar tumor targeting and imaging properties of IR-780, but also has unique photodependent cytotoxic activity. In addition, IR-808 also exhibits favorable optical and pharmacokinetic properties, as well as good biocompatibility. This dye may hold promise as a candidate multifunctional theranostic agent for future tumor targeted imaging and photodynamic therapy.
Keywords: Cancer targeting; Molecular imaging; Photodynamic therapy; Multifunctional theranostic agent;

An oligopeptide ligand-mediated therapeutic gene nanocomplex for liver cancer-targeted therapy by M. Liu; Z.H. Li; F.J. Xu; L.H. Lai; Q.Q. Wang; G.P. Tang; W.T. Yang (2240-2250).
The epidermal growth factor receptor (EGFR) is over-expressed in a wide variety of epithelial-derived cancer cells. In this study, EGFR-targeted gene carriers were designed to complex the therapeutic acetylcholinesterase gene (AChE gene), which suppresses cell proliferation via inactivating mitogen-activated protein kinase and PI3K/Akt pathways in cells, for treatment of EGFR-positive liver cancers. Different amounts of target ligand YC21 (an oligopeptide composed of 21 amino acid units) were coupled with the PEI600-CD (PC) vectors composed of β-cyclodextrin (β-CD) and low-molecular-weight polyethylenimine (PEI, Mw 600) to form the EGFR-targeted gene vectors (termed as YPCs). The YPC vectors possessed the highly efficient gene delivery ability to the EGFR-positive liver cancer cells. YPCs could effectively promote AChE gene expression. The YPC/AChE complexes produced excellent gene transfection abilities in EGFR-positive liver cancer cells in vitro and in vivo.
Keywords: Tumor; Gene therapy; Vector; Target; YC21; AChE;

Long-term theranostic hydrogel system for solid tumors by Jang Il Kim; Beom Suk Lee; ChangJu Chun; Jung-Kyo Cho; Sang-Yoon Kim; Soo-Chang Song (2251-2259).
The long-term theranostic hydrogel system for solid tumors was prepared via simple physical mixing, which consisted of three major parts: the thermosensitive/biodegradable poly(organophosphazene) hydrogel, PEGylated cobalt ferrite nanoparticles, and paclitaxel (PTX). The PEGylated cobalt ferrite nanoparticles showed extremely low cytotoxicity due to the surface modification using PEG chains. The long-term theranostic hydrogel system showed adequate properties to be used for long-term MR theragnosis. In particular, the theranostic hydrogel gradually degraded over 28 days, and the PTX was sustainedly released out from the theranostic hydrogel over the same period in vitro. Furthermore, the in vivo efficacy of long-term MR theragnosis using the theranostic hydrogel system was estimated successfully over 3 weeks by using high field (4.7 T) animal MRI and solid tumor-bearing mice. Based on our results, we expect that this system can supply multiple data regarding a) the progress of therapy and b) the treatment processes via one- or two-time i.t. administration for cases in which surgical approaches are difficult to apply. Meanwhile, cancer patients can be free from the pain of multiple surgical treatments and have the advantage of therapy through a simple i.t. administration.
Keywords: Phosphazene; Long-term theragnosis; Contrast agents; Sustained release; Drug delivery system;

Treatment of prostate carcinoma with (Galectin-3)-targeted HPMA copolymer-(G3-C12)-5-Fluorouracil conjugates by Yang Yang; Zhou Zhou; Shuang He; Tingting Fan; Yun Jin; Xi Zhu; Chunhui Chen; Zhi-rong Zhang; Yuan Huang (2260-2271).
Galectin-3 (Gal-3), over-expressed on a variety of human tumor cells, is a potential binding site for targeted metastatic prostate cancer therapy. The aim of this study was to develop a G3-C12-mediated drug delivery system based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers targeting to Gal-3-expressed human PC-3 prostate carcinoma cells. 5-Fluorouracil (5-Fu), an anti-tumor agent, was selected as a model drug. G3-C12, a binding peptide, which specifically binds to the carbohydrate-recognition domain (CRD) of Gal-3, was attached to HPMA copolymers as a targeting moiety. Compared with non-targeted conjugates (P-Fu), Gal-3-targeted HPMA copolymer-(G3-C12)-5-Fu conjugates (P-(G3-C12)-Fu) displayed a superior intracellular internalization followed by enhanced cytotoxicity and apoptosis-induction. Subsequently, the in vitro migration study on PC-3 cells indicated that P-(G3-C12)-Fu was able to efficiently inhibit the cell migration ability after wounding. On PC-3 tumor-bearing mice model, G3-C12-modified copolymers showed a higher tumor accumulation coupled with a faster clearance from blood circulation than non-modified ones. Finally, Gal-3-targeted conjugates significantly improved the anti-tumor activity of 5-Fu in nude mice bearing PC-3 tumor xenografts. Consequently, G3-C12 would be a promising targeting moiety for cell-specific prostate cancer therapy in future.
Keywords: G3-C12 peptide; Human PC-3 prostate carcinoma cells; 5-Fluorouracil; Gal-3-targeted HPMA copolymer-(G3-C12)-5-Fu conjugates;

Synergistic effects of Akt1 shRNA and paclitaxel-incorporated conjugated linoleic acid-coupled poloxamer thermosensitive hydrogel on breast cancer by Ding-Ding Guo; Seong-Ho Hong; Hu-Lin Jiang; Ji-Hye Kim; Arash Minai-Tehrani; Ji-Eun Kim; Ji-Young Shin; Tao Jiang; You-Kyoung Kim; Yun-Jaie Choi; Chong-Su Cho; Myung-Haing Cho (2272-2281).
The phosphoinositide 3-kinase/Akt1 signaling pathway has emerged as a target for cancer therapy. In this study, we aimed to develop a strategy to enhance Akt-targeted cancer therapy. We hypothesized that combination of Akt1-targeted therapy with conventional chemotherapy using paclitaxel-incorporated conjugated linoleic acid-coupled poloxamer thermosensitive hydrogel may have synergistic effects in cancer therapeutic efficiency compared with chemotherapy alone. In this study, we found that the combination of shAkt1 with paclitaxel exerted synergistic anti-cancer effects, thus, inhibiting the growth of human breast cancer cells, and breast cancer xenografts in mice as well. The combination therapy demonstrated enhanced anti-cancer effects through inhibiting Akt1 signaling and inducing apoptosis. Our results suggest that the presented strategy of combination of shAkt1 with paclitaxel may have a potential for treatment of breast cancer.
Keywords: Combination therapy; Akt1; Paclitaxel; Breast cancer; Poloxamer hydrogel; Conjugated linoleic acid;

A pyrene-imidazolium derivative that selectively Recognizes G-Quadruplex DNA by Ha Na Kim; Eun-Hae Lee; Zhaochao Xu; Hee-Eun Kim; Hee-Seung Lee; Joon-Hwa Lee; Juyoung Yoon (2282-2288).
G-quadruplexes, formed of four stranded guanine bases stabilized by monovalent cations, serve important role in cancer cell growth and control gene expression in telomere. Since there are various types of quadruplex structures, rapid and simple screening methods with high selectivity, sensitivity and nontoxicity are required for understanding about the biological roles of quadruplex DNA as well as in designing therapeutics. Herein, we report a pyrene-imidazolium derivative, JY-1, which can with high selectivity recognize G-quadruplex using fluorescence and NMR spectroscopy. This is the first example based on the imidazolium derivative, which can detect the G-quadruplex directly to utilize the excimer/monomer emission change in pyrene fluorophore. The selectivity of strong binding to a specific sequence can allow for quadruplex sensing and the detection method presented here is very simple, using fluorescence and NMR study. Also, the groove binding characteristic of JY-1 to the G-quadruplex has a relatively low nonspecific toxicity and the structure-specific differences in fluorescent character between DNA duplex and G-quadruplex may offer more discovery and application in biological study.
Keywords: G-quadruplex; NMR; Fluorescent sensor; Imidazolium; Pyrene; Excimer emission;

Ophthalmic drug delivery through eye drops is inefficient because of low corneal bioavailability and short residence time in tears. Contact lenses are ideally suited for extended and targeted drug delivery to cornea, but commercial contact lenses release ophthalmic drugs for only 1–2 h. This study focuses on dispersing timolol encapsulating highly crosslinked nanoparticles in contact lenses to increase the duration of drug release from 1 to 2 h to about 2–4 weeks. The highly crosslinked particles were prepared from monomers with multivinyl functionalities such as EGDMA (ethylene glycol dimethacrylate) and PGT (propoxylated glyceryl triacylate). The nanoparticles were about 3.5 nm in size and encapsulated 48–66% of the drug depending on the composition. Drug release studies in a diffusion cell showed that the particles released the drug for a period of about 4 weeks. The drug loaded particles were dispersed in hydroxy methyl methacrylate (HEMA) gels, which are common contact lens materials. The particle loaded gels release timolol in phosphate buffered saline (PBS) for 2–4 weeks at therapeutic dose, which is promising for extended drug release applications. The proposed mechanism of drug transport is hydrolysis of ester bonds that link timolol to the particle matrix which form during the particle formation process. The drug release profiles can be described by a first order reaction model with a temperature dependent rate constant. The rate constant of ester hydrolysis was significantly smaller than that in previous studies on timolol esters possibly due to steric effects and the low water content of the highly crosslinked hydrophobic particles. The results of this study provide evidences that contact lenses loaded with nanoparticles could be very useful for extended delivery of ophthalmic drugs.
Keywords: Contact lenses; Drug delivery; Nanoparticles; Glaucoma; Timolol;

Diblock copolymers with tunable pH transitions for gene delivery by Matthew J. Manganiello; Connie Cheng; Anthony J. Convertine; James D. Bryers; Patrick S. Stayton (2301-2309).
A series of diblock copolymers containing an endosomal-releasing segment composed of diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The materials were designed to condense plasmid DNA (pDNA) through electrostatic interactions with a cationic poly(N,N-dimethylaminoethyl methacrylate) (DMAEMA) first block. The pDMAEMA was employed as a macro chain transfer agent (macroCTA) for the synthesis of a series in which the relative feed ratios of DEAEMA and BMA were systematically varied from 20% to 70% BMA. The resultant diblock copolymers exhibited low polydispersity (PDI ≤ 1.06) with similar molecular weights (Mn  = 19.3–23.1 kDa). Dynamic light scattering (DLS) measurements in combination with 1H NMR D2O studies demonstrated that the free copolymers assemble into core-shell micelles at physiological pH. Reduction of the solution pH to values representative of endosomal/lysosomal compartments induced an increase in the net cationic charge of the core through protonation of the DEAEMA residues. This protonation promotes micelle destabilization and exposure of the hydrophobic BMA residues that destabilize biological membranes. The pH value at which this micelle-to-unimer transition occurred was dependent on the hydrophobic content of the copolymer, with higher BMA-containing copolymer compositions exhibiting pH-induced transitions to the membrane-destabilizing state at successively lower pH values. The ability of the diblock copolymers to deliver pDNA was subsequently investigated using a GFP expression vector in two monocyte cell lines. High levels of DNA transfection were observed for the copolymer compositions exhibiting the sharpest pH transitions and membrane destabilizing activities, demonstrating the importance of tuning the endosomal-releasing segment composition.
Keywords: Copolymer; Cell culture; DNA; Drug delivery; Gene therapy; Micelle;

Redox-sensitive micelles self-assembled from amphiphilic hyaluronic acid-deoxycholic acid conjugates for targeted intracellular delivery of paclitaxel by Jing Li; Meirong Huo; Jing Wang; Jianping Zhou; Jumah M. Mohammad; Yinlong Zhang; Qinnv Zhu; Ayman Y. Waddad; Qiang Zhang (2310-2320).
A targeted intracellular delivery system of paclitaxel (PTX) was successfully developed based on redox-sensitive hyaluronic acid-deoxycholic acid (HA-ss-DOCA) conjugates. The conjugates self-assembled into nano-size micelles in aqueous media and exhibited excellent drug-loading capacities (34.1%) and entrapment efficiency (93.2%) for PTX. HA-ss-DOCA micelles were sufficiently stable at simulated normal physiologic condition but fast disassembled in the presence of 20 mm reducing agent, glutathione. In vitro drug release studies showed that the PTX-loaded HA-ss-DOCA micelles accomplished rapid drug release under reducing condition. Intracellular release of fluorescent probe nile red indicated that HA-ss-DOCA micelles provide an effective approach for rapid transport of cargo into the cytoplasm. Enhanced cytotoxicity of PTX-loaded HA-ss-DOCA micelles further confirmed that the sensitive micelles are more potent for intracellular drug delivery as compared to the insensitive control. Based on flow cytometry and confocal microscopic analyses, observations revealed that HA-ss-DOCA micelles were taken up to human breast adenocarcinoma cells (MDA-MB-231) via HA-receptor mediated endocytosis. In vivo investigation of micelles in tumor-bearing mice confirmed that HA-ss-DOCA micelles possessed much higher tumor targeting capacity than the insensitive control. These results suggest that redox-sensitive HA-ss-DOCA micelles hold great potential as targeted intracellular delivery carriers of lipophilic anticancer drugs.
Keywords: Drug delivery; Redox-sensitive; Hyaluronic acid; Glutathione; Polymeric micelle; Paclitaxel;

Synergistic effects of conjugating cell penetrating peptides and thiomers on non-viral transfection efficiency by Deni Rahmat; Mohammad I. Khan; Gul Shahnaz; Duangkamon Sakloetsakun; Glen Perera; Andreas Bernkop-Schnürch (2321-2326).
Nanoparticles generated by complex coacervation of plasmid DNA (pDNA) and modified chitosans namely chitosan–thioglycolic acid (TGA) conjugate and chitosan–HIV-1 Tat peptide conjugate were evaluated as gene delivery systems. In order to optimize transfection efficiency, chitosan–HIV-1 Tat peptide conjugate was combined with chitosan–TGA before its complexation with pDNA. Particle size and zeta potential measurements were performed to characterize the generated nanoparticles. The nanoparticles transfection efficiencies were assessed by exploitation of the green fluorescent protein (GFP) reporter gene. HEK293 cells were incubated for 24 h with the nanoparticles and the GFP positive cells were observed by fluorescence microscopy. The nanoparticles in the size range of 200–300 nm could transfect HEK293 cells as a model cell line with different transfection efficiencies. Unlike chitosan–TGA, chitosan–HIV-1 Tat peptide led to increased zeta potential of nanoparticles as compared to unmodified chitosan. The transfection efficiency of the nanoparticles generated by combination of chitosan–HIV-1 Tat peptide with chitosan–TGA was comparatively higher than that of the nanoparticles generated by either chitosan–TGA or the combination of chitosan–HIV-1 Tat peptide with unmodified chitosan. After 72 h of incubation, the combination of chitosan–HIV-1 Tat peptide with chitosan–TGA was found to be 7.12- and 67.37 times more efficient than unmodified chitosan and pDNA alone, respectively and showed a synergistic effect in transfection of pDNA into the cells. Moreover, none of the nanoparticles showed any severe cytotoxicity. Accordingly, this strategy might result in a potent carrier for gene delivery.
Keywords: Nanoparticles; Thioglycolic acid; Tat peptide; Transfection; Gene delivery;

The molecular mechanism of action of bactericidal gold nanoparticles on Escherichia coli by Yan Cui; Yuyun Zhao; Yue Tian; Wei Zhang; Xiaoying Lü; Xingyu Jiang (2327-2333).
This work examines the molecular mechanism of action of a class of bactericidal gold nanoparticles (NPs) which show potent antibacterial activities against multidrug-resistant Gram-negative bacteria by transcriptomic and proteomic approaches. Gold NPs exert their antibacterial activities mainly by two ways: one is to collapse membrane potential, inhibiting ATPase activities to decrease the ATP level; the other is to inhibit the subunit of ribosome from binding tRNA. Gold NPs enhance chemotaxis in the early-phase reaction. The action of gold NPs did not include reactive oxygen species (ROS)-related mechanism, the cause for cellular death induced by most bactericidal antibiotics and nanomaterials. Our investigation would allow the development of antibacterial agents that target the energy-metabolism and transcription of bacteria without triggering the ROS reaction, which may be at the same time harmful for the host when killing bacteria.
Keywords: Gold nanoparticle; Molecular mechanism; Transcriptomic/proteomic analysis; ROS;

Mono-methoxy-poly(3-hydroxybutyrate-co-4-hydroxybutyrate)-graft-hyper-branched polyethylenimine copolymers for siRNA delivery by Li Zhou; Zhifei Chen; Weilin Chi; Xiuqun Yang; Wei Wang; Biliang Zhang (2334-2344).
A class of non-viral siRNA vectors consisting of biodegradable poly(hydroxyalkanoates) (PHA) grafted onto branched poly(ethyleneimine) (bPEI, 25 kDa) was synthesized and evaluated for siRNA delivery. The mPHA-g-bPEI copolymers were synthesized through Michael addition between acrylated mono-methoxy-poly(hydroxyalkanoates) (mPHA-acrylated) and bPEI with various block length poly(hydroxyalkanoates) from 1300 to 2900 Da. Our research showed that mPHA-g-bPEI copolymers could effectively bind siRNA, protect it from degradation by nucleases and efficiently release the complexed siRNA in the presence of low concentrations of polyanionic heparin. The particle size of mPHA-g-bPEI/siRNA complexes was <200 nm with ζ-potential between 33 and 43 mV. mPHA-g-bPEI copolymers displayed low cytotoxicity compared to unmodified bPEI and efficient cellular uptake of Cy3-siRNA in A549 cells by flow cytometry and confocal microscopy. siRNA delivery efficiency of the copolymers was assessed by siRNA against luciferase in cultured A549-Luc and MCF-7-Luc cells. Those mPHA-g-bPEI copolymers revealed a higher transfection efficiency and lower cytotoxicity than bPEI in two cell lines. Furthermore, a remarkable knockdown of luciferase expression of mPHA-g-bPEI (mAP2) complex (up to 85%) in vitro was found to be equivalent to that of commercially available transfection agent Lipofectamine™ 2000.
Keywords: siRNA delivery; Poly(hydroxyalkanoates); Poly(ethyleneimine); Non-viral vector;

Off-resonance plasmonic enhanced femtosecond laser optoporation and transfection of cancer cells by Judith Baumgart; Laure Humbert; Étienne Boulais; Rémi Lachaine; Jean-Jaques Lebrun; Michel Meunier (2345-2350).
A femtosecond laser based transfection method using off-resonance plasmonic gold nanoparticles is described. For human cancer melanoma cells, the treatment leads to a very high perforation rate of 70%, transfection efficiency three times higher than for conventional lipofection, and very low toxicity (<1%). Off-resonance laser excitation inhibited the fracture of the nanoparticles into possibly toxic DNA intercalating particles. This efficient and low toxicity method is a promising alternative to viral transfection for skin cancer treatment.
Keywords: Optoperforation; Transfection; Gold nanoparticle; Plasmonic; Femtosecond laser; Cancer cell;

Thermal-sensitive hydrogel as adjuvant-free vaccine delivery system for H5N1 intranasal immunization by Youbin Wu; Wei Wei; Meng Zhou; Yueqi Wang; Jie Wu; Guanghui Ma; Zhiguo Su (2351-2360).
For H5N1 influenza immunization, we developed a thermal-sensitive hydrogel as intranasal vaccine delivery system, which was formulated with N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) and α, β-glycerophosphate (α, β-GP). The flowing solution of HTCC/GP under room temperature could gelate rapidly at body temperature, which significantly prolonged the H5N1 split antigen residence time in nasal cavity. This system also enhanced the transepithelial transport via the paracellular routes due to the disorganization of ZO-1 protein in nasal epithelial tissue. In comparison to naked H5N1 split antigen and MF59 adjuvanted antigen, as designed hydrogel/H5N1 vaccine induced greater antigen-specific systemic immune responses and mucosal IgA immunity without adjuvants. Furthermore, a boosted cellular and humoral response was also obtained by examination of IFN-γ and IL-4 cytokines, respectively. In addition, hydrogel based formulation promoted the antigen-specific CD8+ T cell immune memory as determined by the proportion of central and effector memory CD8+ T cells in nasal associated lymphoid tissue (NALT). These results demonstrate that the HTCC hydrogel has potential as an adjuvant-free platform for H5N1 split antigen intranasal vaccination.
Keywords: Thermal-sensitive hydrogel; Intranasal immunization; Vaccine delivery system; Adjuvant-free; H5N1 split antigen;

Enhancement of airway gene transfer by DNA nanoparticles using a pH-responsive block copolymer of polyethylene glycol and poly-l-lysine by Nicholas J. Boylan; Anthony J. Kim; Jung Soo Suk; Pichet Adstamongkonkul; Brian W. Simons; Samuel K. Lai; Mark J. Cooper; Justin Hanes (2361-2371).
Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of polyethylene glycol and poly-l-lysine (PEG-CK30), have shown considerable promise in human gene therapy clinical trials in the nares, but may be less capable of transfecting cells that lack surface nucleolin. To address this potential shortcoming, we formulated pH-responsive DNA nanoparticles that mediate gene transfer via a nucleolin-independent pathway. Poly-l-histidine was inserted between PEG and poly-l-lysine to form a triblock copolymer system, PEG-CH12K18. Inclusion of poly-l-histidine increased the buffering capacity of PEG-CH12K18 to levels comparable with branched polyethyleneimine. PEG-CH12K18 compacted DNA into rod-shaped DNA nanoparticles with similar morphology and colloidal stability as PEG-CK30 DNA nanoparticles. PEG-CH12K18 DNA nanoparticles entered human bronchial epithelial cells (BEAS-2B) that lack surface nucleolin by a clathrin-dependent endocytic mechanism followed by endo-lysosomal processing. Despite trafficking through the degradative endo-lysosomal pathway, PEG-CH12K18 DNA nanoparticles improved the in vitro gene transfer by ∼20-fold over PEG-CK30 DNA nanoparticles, and in vivo gene transfer to lung airways in BALB/c mice by ∼ 3-fold, while maintaining a favorable toxicity profile. These results represent an important step toward the rational development of an efficient gene delivery platform for the lungs based on highly compacted DNA nanoparticles.
Keywords: Cystic fibrosis; Gene therapy; Non-viral; pH buffering;

In this study, the aminated gelatin was grafted with carboxylic end-capped poly(N-isopropylacrylamide) (PN) via a carbodiimide-mediated coupling reaction to fabricate biodegradable in situ forming delivery systems for intracameral administration of antiglaucoma medications. The chemical structure of the graft copolymers (GN) was confirmed by Fourier transform infrared (FTIR) spectroscopy. When the feed molar ratio of NH2/COOH was 0.36, the grafting ratio, efficiency and degree of grafting, and weight ratio of PN to aminated gelatin was 25.6, 18.6%, 52.6%, and 1.9, respectively. As compared to PN, the GN samples possessed better thermal gelation ability and adherence, indicating remarkable phase transition properties. Under gelatinase degradation, the remaining weight of GN was significantly lower than those of PN at each time point from 8 h to 4 weeks. Cytocompatibility studies showed that the culture of anterior segment cells with both in situ forming gels does not affect proliferation and has little effect on inflammation. Higher encapsulation efficiency (∼62%) and cumulative release (∼95%) were achieved for GN vehicles, which was attributed to initial fast temperature triggered capture of pilocarpine and subsequent progressive degradation of gelatin network. In a rabbit glaucoma model, the performance of delivery carriers was evaluated by biomicroscopy, intraocular pressure (IOP), and pupil size change. Intracameral administration of pilocarpine using GN was found to be more effective than other methods such as instillation of eye drop and injection of free drug or PN containing drug in improving ocular bioavailability and extending the pharmacological responses (i.e., miosis and IOP lowering effect and preservation of corneal endothelial cell density).
Keywords: Gelatin-g-PNIPAAm; Biodegradable in situ forming gel; Intracameral delivery system; Pilocarpine; Glaucoma therapy;

Structure-property relationships in manganese oxide - mesoporous silica nanoparticles used for T1-weighted MRI and simultaneous anti-cancer drug delivery by Yu Chen; Hangrong Chen; Shengjian Zhang; Feng Chen; Shikuan Sun; Qianjun He; Ming Ma; Xia Wang; Huixia Wu; Lingxia Zhang; Linlin Zhang; Jianlin Shi (2388-2398).
The extremely low longitudinal relaxivity (r 1) of manganese oxide has severely impeded their substitution for cytotoxic gadolinium-based contrast agents for safe clinical magnetic resonance imaging (MRI). Here, we report on a synthetic strategy of chemical oxidation/reduction reaction in-situ in mesopores, followed by hydrogen reduction, for the fabrication of non-toxic manganese oxide/MSNs-based MRI-T1 contrast agents with highly comparable imaging performance to commercial Gd-based agents. This strategy involves a “soft-templating” process to prepare mesoporous silica nanoparticles, in-situ reduction of MnO 4 − by the “soft templates” in mesopores and heat treatment under reducing atmosphere, to disperse manganese oxide nanoparticles within mesopores. This special nanostructure combines the merits of nanopores for maximum manganese paramagnetic center accessibility for water molecules for enhanced MRI performance and encapsulation/sustained release/intracellular delivery of drugs. The synthesized manganese oxide/MSNs were successfully assessed as a high performance contrast agent for MRI-T1 both in intro and in vivo, and meanwhile, was also demonstrated as an effective anti-cancer drug delivery (doxorubicin) vehicle, therefore, a family of manganese-based theranostics was successfully demonstrated based on the manganese oxide/MSNs composite.
Keywords: Magnetic resonance imaging; Manganese oxide; Mesoporous silica; Drug delivery; Diagnosis;

Pathological mechanisms of liver injury caused by continuous intraperitoneal injection of silica nanoparticles by Tianlong Liu; Linlin Li; Changhui Fu; Huiyu Liu; Dong Chen; Fangqiong Tang (2399-2407).
Crystalline silica is well known to induce chronic lung inflammation by inhalation that can progress to silicosis. Recently, we reported that silica nanoparticles (SN) cause more damage to liver instead of lung when they enter the body by intravenous injection. However, this mechanism is still unclear. In the present study, liver damages caused by mesoporous hollow silica nanoparticles (MHSNs) were demonstrated after continuous intraperitoneal injection into mice twice a week for 6 weeks. The administration of MHSNs at 50 mg/kg increased liver injury markers in serum, such as alanine aminotransferase (ALT), inflammatory cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). Histological analysis revealed lymphocytic infiltration and silicotic nodular like lesions in liver. Collagen fibers were observed around the silicotic nodular like lesion, and hydroxyproline level in liver was also increased dramatically. We also found that activated kupffer cells (KCs) played a key role in the liver damage caused by SNs similar to alveolar macrophage in the process of silicosis. These suggest that the mechanism of liver damage caused by SNs is in consonance with the occurrence of silicosis. These findings may provide useful information for the further toxicity and bioapplication research of nanoparticles.
Keywords: Mesoporous hollow silica; Nanoparticles; Kupffer cells; Hepatotoxicity; Silicotic nodular;