Biomaterials (v.32, #25)

Prevention of capsular opacification after accommodative lens refilling surgery in rabbits by Steven A. Koopmans; Thom Terwee; Theo G. van Kooten (5743-5755).
Silicone gel-like polymers have been proposed to replace the cataractous lens and therewith restore both vision and accommodation. Lens replacement is associated with opacification of the capsular bag due to the lens epithelial cell response. In this study, the in vivo effectiveness of a 5 min treatment with actinomycin D and/or cycloheximid to prevent the development of capsular opacification after filling the capsular bag with a silicone polymer as an accommodating lens was studied. It was found that treating the inside of the capsular bag with a solution containing actinomycin D reduced the development of visible capsular opacification for three months. In some animals, the lens capsules were completely clear, indicating the potential of this method. Side effects of the treatment in the form of visible cornea opacification occurred and ranged from mild to severe in some animals, while in other animals no toxicity occurred. This indicates that a safe application of the cytotoxic substances is feasible. In view of the side effects and the fact that not all lens capsules of the animals treated with actinomycin D were clear, improvements in the methods used are necessary and seem to be possible.
Keywords: Intra-ocular lens; Lens epithelial cell; Silicone gel; Capsular opacification; Accommodating; Injectable;

Three-dimensional epithelial tissue equivalents tend to lose their self-renewing potential progressively during culture as their epithelial cells lose their proliferative capacity with time. Even though the tissue engineered construct can mimic the native tissue well, it rapidly degrades after implantation due to the insufficient number of proliferating cells in the equivalent. In the present study we demonstrate for the first time that the use of an elastin-like recombinant polymer (ELR) engineered to contain the cell adhesion peptide RGD can result in a 3D tissue equivalent with high self-renewing potential, containing as many proliferative cells as the native tissue itself. The 3D tissue equivalent was reconstructed by the coculture of human lamina propria fibroblasts and oral epithelial cells in the nanofibrous ELR-collagen scaffold. Histological, immunohistological and transmission electron microscopic analyses of this oral mucosa equivalent demonstrated the expression of markers characteristic of epithelial proliferation (Ki67) and differentiation (keratin 13), and also the presence of a pluristratified epithelium and an ultrastructurally well-organized basement membrane expressing laminin 332. The synthesis of new extracellular matrix by the fibroblasts was also demonstrated. The scaffold proposed here presents great potential for tissue engineering applications, and also for studies of epithelial proliferation, and epithelial disorders including carcinogenesis.
Keywords: Elastin-like recombinant polymer; Nanofibrous scaffold; Cell proliferation; 3D tissue equivalent;

As the neurons in the central nervous system (CNS) form a neuronal network in a three-dimensional (3D) manner, a 3D proliferation culture system for differentiated neurons has been desired. Although differentiated neurons were previously thought to never proliferate, differentiated horizontal interneurons of Rb–/–; p107+/−; p130−/− (p107-single) retina clonally proliferated without dedifferentiation in vivo. In the present study, we developed a basement membrane-like matrix sponge (BM-sponge) for the 3D proliferation culture of differentiated horizontal interneurons. p107-single horizontal interneurons, but not other types of retinal neurons, proliferated in the BM-sponge in a 3D manner. These interneurons expressed presynaptic marker and developed synaptic vesicles. These data demonstrated that p107-single horizontal interneurons cultured in the BM-sponge proliferate while maintaining their differentiated features. We described here the 3D proliferation culture system for differentiated neurons.
Keywords: Neural cell; ECM (extra-cellular matrix); Cell proliferation; Cell culture;

Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering by Nandana Bhardwaj; Quynhhoa T. Nguyen; Albert C. Chen; David L. Kaplan; Robert L. Sah; Subhas C. Kundu (5773-5781).
The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.
Keywords: Silk fibroin; Chitosan; Scaffolds; Chondrocytes; Cartilage; Tissue engineering;

The promotion of microvasculature formation in poly(ethylene glycol) diacrylate hydrogels by an immobilized VEGF-mimetic peptide by Julia E. Leslie-Barbick; Jennifer E. Saik; Daniel J. Gould; Mary E. Dickinson; Jennifer L. West (5782-5789).
Microvascularization of tissue engineered constructs was achieved by utilizing a VEGF-mimicking peptide, QK, covalently bound to a poly(ethylene glycol) hydrogel matrix. The 15-amino acid peptide, developed by D’Andrea et al., was modified with a PEG-succinimidyl ester linker on the N-terminus of the peptide, then photocrosslinked onto the surface or throughout PEG hydrogels. PEGylation of the peptide increased its solubility and bioactivity, as evidenced by endothelial cell proliferation. PEG-QK showed equal or superior ability to promote angiogenesis in vitro, on the surface of hydrogels and within three-dimensional collagenase-degradable hydrogels, compared to RGDS only or PEG-VEGF hydrogels. Endothelial cells were shown to form tubule structures, migrate, and make cell–cell contacts in response to covalently-bound PEG-QK. In vivo in a mouse cornea micropocket angiogenesis assay, PEG-QK hydrogels promoted more complete coverage of host microvasculature within the hydrogel. PEG-QK was shown to enhance vessel branch points and vessel density as well as space filling properties of fractal dimension and lacunarity. This report shows the ability to promote angiogenesis in tissue engineered constructs using a covalently-bound small peptide rather than a large protein and may point to an advance in designing biomimetic cellular environments.
Keywords: Angiogenesis; Peptide; Poly(ethylene glycol); Vascularization;

Platelet inhibition and endothelial cell adhesion on elastin-like polypeptide surface modified materials by Patrick H. Blit; W. Glenn McClung; John L. Brash; Kimberly A. Woodhouse; J. Paul Santerre (5790-5800).
Platelet adhesion and activation are important early markers of biomaterial blood compatibility, while surfaces that promote enhanced endothelial cell adhesion and eNOS expression are strategic targets for long term vascular graft applications. Materials surface modified with fluorinated surface modifiers, containing peptides inspired from elastin cross-linking domains, have been used for the cross-linking of elastin-like polypeptide 4 (ELP4) macromolecules onto polyurethane surfaces. In the present study, ELP4 modified polyurethanes were evaluated in vitro to assess platelet adhesion, microparticle formation and bulk platelet activation following blood-material interactions. Reduced platelet adhesion and bulk platelet activation were observed following contact between reconstituted human blood and the ELP4 materials, relative to the uncoated base polyurethane controls. ELP4 modified materials also promoted endothelial cell adhesion and retention over a period of one week and showed that the endothelial cells exhibited an organized actin cytoskeleton and enhanced endothelial nitric oxide synthase (eNOS) expression relative to the control surfaces. These results indicate that polyurethane elastomers modified with ELP4 covalently bound to fluorinated surface modifiers provide a promising approach for endowing synthetic elastomers with both reduced blood platelet activation properties and enhanced endothelial cell adhesion for potential use in vascular graft applications.
Keywords: Elastin; Platelet activation; Endothelial cell; Polyurethane; Surface modification;

Engineering of large osteogenic grafts with rapid engraftment capacity using mesenchymal and endothelial progenitors from human adipose tissue by Sinan Güven; Arne Mehrkens; Franziska Saxer; Dirk J. Schaefer; Roberta Martinetti; Ivan Martin; Arnaud Scherberich (5801-5809).
We investigated whether the maintenance in culture of endothelial and mesenchymal progenitors from the stromal vascular fraction (SVF) of human adipose tissue supports the formation of vascular structures in vitro and thereby improves the efficiency and uniformity of bone tissue formation in vivo within critically sized scaffolds. Freshly-isolated human SVF cells were seeded and cultured into hydroxyapatite scaffolds (1 cm-diameter, 1 cm-thickness) using a perfusion-based bioreactor system, which resulted in maintenance of CD34+/CD31+ endothelial lineage cells. Monolayer-expanded isogenic adipose stromal cells (ASC) and age-matched bone marrow stromal cells (BMSC), both lacking vasculogenic cells, were used as controls. After 5 days in vitro, SVF-derived endothelial and mesenchymal progenitors formed capillary networks, which anastomosed with the host vasculature already 1 week after ectopic nude rat implantation. As compared to BMSC and ASC, SVF-derived cells promoted faster tissue ingrowth, more abundant and uniform bone tissue formation, with ossicles reaching a 3.5 mm depth from the scaffold periphery after 8 weeks. Our findings demonstrate that maintenance of endothelial/mesenchymal SVF cell fractions is crucial to generate osteogenic constructs with enhanced engraftment capacity. The single, easily accessible cell source and streamlined, bioreactor-based process makes the approach attractive towards manufacturing of clinically relevant sized bone substitute grafts.
Keywords: Mesenchymal stem cell; Bone tissue engineering; Bioreactor; Bone graft; Adipose tissue; Endothelial cell;

The dependence of autologous chondrocyte transplantation on varying cellular passage, yield and culture duration by Gian M. Salzmann; Martin Sauerschnig; Markus T. Berninger; Theresa Kaltenhauser; Martin Schönfelder; Stephan Vogt; Gabriele Wexel; Thomas Tischer; Norbert Sudkamp; Philipp Niemeyer; Andreas B. Imhoff; Philip B. Schöttle (5810-5818).
Matrix-assisted chondrocyte transplantation (m-ACI) still lacks any standardization in its execution in terms of cell passage (P), cell yield (C) and in vitro membrane-holding time (T). It was the goal of this study to analyze the effect of shifting cell culture parameters (P, C, T) on the in vitro as well as in vivo effort of a regulated animal m-ACI. Autologous rabbit knee articular chondrocytes were seeded within bilayer collagen I/III 3-D matrices in variation of P, C and T. Each time, 2 PCT-identical by 2 PCT-identical cell-matrix-constructs (CMC)/animal were created. Simultaneously 2 (PCT-distinct) were re-implanted (CMC-e) autologous into artificial trochlear pristine chondral defects in vivo to remain for 12 weeks while the remaining 2 were harvested (CMC-i) for immediate in vitro analysis at the time of transplantation of their identical twins. mRNA of both, CMC-e regenerates and CMC-i membranes, was analyzed for Collagen-1,-2,-10, COMP, Aggrecan, Sox9 expression by use of a mixed linear model, multiple regression analysis. Generally, CMC-i values were higher than CMC-e values for differentiation targets; the opposite was true for dedifferentiation targets. Regarding individual gene expression, in vivo regenerate cell-matrix properties were significantly dependent on initial cell-matrix in vitro values as a sign of linearity. The parameter membrane-holding time (T) had strongest effects on the resulting mRNA expression with slightly less impact of the parameter passage (P), whereas cell yield (C) had clearly less effects. Noting differences between in vitro and in vivo data, in general, optimal expression patterns concerning chondrogenic differentiation were achieved by few passages, medium cellular yield, short membrane-holding time. Clinical m-ACI may benefit from optimal orchestration of the cell culture parameters passage, yield and time.
Keywords: Chondrocyte; Chondrocyte transplantation; Matrix-assisted; Tissue engineering; Cartilage;

Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model by Yuka Tsumanuma; Takanori Iwata; Kaoru Washio; Toshiyuki Yoshida; Azusa Yamada; Ryo Takagi; Takahiro Ohno; Konghua Lin; Masayuki Yamato; Isao Ishikawa; Teruo Okano; Yuichi Izumi (5819-5825).
Cytotherapeutic approaches have been investigated to overcome the limitations of existing procedures for periodontal regeneration. In this study, cell sheet transplantation was performed using three kinds of mesenchymal tissue (periodontal ligament, alveolar periosteum, and bone marrow)-derived cells to compare the differences between cell sources in a canine severe defect model (one-wall intrabony defect). Periodontal ligament cells (PDLCs), iliac bone marrow mesenchymal stromal cells (BMMSCs), and alveolar periosteal cells (APCs) were obtained from each dog; a total of four dogs were used. Three-layered cell sheets of each cell source supported with woven polyglycolic acid were autologously transplanted to the denuded root surface. One-wall intrabony defects were filled with a mixture of β-tricalcium phosphate (β-TCP) and collagen. Eight weeks after the transplantation, periodontal regeneration was significantly observed with both newly formed cementum and well-oriented PDL fibers more in the PDLC group than in the other groups. In addition, nerve filament was observed in the regenerated PDL tissue only in the PDLC group. The amount of alveolar bone regeneration was highest in the PDLC group, although it did not reach statistical significance among the groups. These results indicate that PDLC sheets combined with β-TCP/collagen scaffold serve as a promising tool for periodontal regeneration.
Keywords: Cell sheet; Periodontal ligament cells; Alveolar periosteal cells; Bone marrow mesenchymal stromal cells; Regenerative medicine; Comparative study;

Suppression of Runx2 protein degradation by fibrous engineered matrix by Joung-Hwan Oh; Jihye Seo; Won-Joon Yoon; Je-Yoel Cho; Jeong-Hwa Baek; Hyun-Mo Ryoo; Kyung Mi Woo (5826-5836).
The fibre structure of engineered matrix that mimic the morphology of type I collagen has exhibited good biological performance for bone regeneration. However, the mechanism by which synthetic fibres promote osteoblast differentiation has yet to be determined. In this study, we demonstrate that fibre structure of an engineered matrix suppresses the degradation of Runx2, a master transcription factor that can turn on to osteoblast differentiation. MC3T3-E1 pre-osteoblasts grown on a fibrous collagen matrix sustained a higher level of Runx2 protein than those on tissue culture dishes or on a collagenase-treated, non-fibrous collagen matrix. The ubiquitin-dependent degradation of Runx2 was profoundly decreased in cells grown on the fibrous collagen matrix. The forced expression of Smurf1, an ubiquitin ligase responsible for Runx2 degradation, abrogated the collagen fibre-induced increase of Runx2. We also prepared a polystyrene fibre matrix, and confirmed that the fibre matrix stabilised the Runx2 protein in MC3T3-E1. Furthermore, we genetically modified C2C12 myoblasts with Runx2, cultured the cells on polystyrene fibre matrix, and observed that the fibre matrix stabilised and sustained exogenous Runx2, which led to the promotion of osteoblast differentiation. Our findings in this study provide evidence that the fibre structure of an engineered matrix contributes to osteoblast differentiation by stabilising the Runx2 protein.
Keywords: Bone tissue engineering; Surface topology; Fibrous structure; Collagen structure; Runx2; Protein degradation;

The structural and biological properties of hydroxyapatite-modified titanate nanowire scaffolds by Haixin Zhao; Wenjun Dong; Yingying Zheng; Aiping Liu; Juming Yao; Chaorong Li; Weihua Tang; Benyong Chen; Ge Wang; Zhan Shi (5837-5846).
Hydroxyapatite-modified titanate nanowire scaffolds as alternative materials for tissue engineering have been developed via a titanate nanowire matrix assisted electrochemical deposition method. The macroporous titanate nanowire matrix on Ti metal was fabricated by a hydrothermal method, and then followed by an electrochemical synthesis of hydroxyapatite nanoparticles on titanate nanowire. The incorporation of titanate nanowire matrix with high oriented hydroxyapatite nanoparticles generates hierarchical scaffolds with highly osteogenic, structural integrity and excellent mechanical performance. As-prepared porous three dimensional interconnected hydroxyapatite-modified titanate nanowire scaffolds, mimicking the nature’s extracellular matrix, could provide a suitable microenvironment for tissue cell ingrowth and differentiation. The ceramic titanate nanowire core with HA nanoparticle sheath structure displays superhydrophilicity, which facilitates the cell attachment and proliferation, and induces the in vitro tissue-engineered bone. Human osteoblast-like MG63 cells were cultured on the hydroxyapatite-modified titanate nanowire scaffolds, and the results showed that the scaffolds highly promote the bioactivity, osteoconductivity and osteoblast differentiation.
Keywords: Scaffold; Hydroxyapatite; Titanate nanowire; Biomaterial;

Regenerative potentials of platelet-rich plasma enhanced by collagen in retrieving pro-inflammatory cytokine-inhibited chondrogenesis by Chia-Che Wu; Wei-Hong Chen; Bin Zao; Pei-Lun Lai; Tzu-Chieh Lin; Hung-Yao Lo; Ying-Hua Shieh; Chih-Hsiung Wu; Win-Ping Deng (5847-5854).
This study was undertaken to evaluate the role of collagen matrix to enhance platelet-rich plasma (PRP) effects on pro-inflammatory cytokine-induced arthritic model. We have previously demonstrated the highly regenerative roles of PRP to restore disc degeneration and osteoporosis. In this study, PRP modulated by collagen matrix was used as a regenerative and anti-inflammatory mediator to rescue the chondrocyte degeneration induced by pro-inflammatory cytokines IL-1β (10 ng/ml)+TNF-α (20 ng/ml). First, the MTT result indicated that 1 ng/ml TGF-β1 in PRP showed an optimal dosage for chondrocytes proliferation. The chondrogenic-specific gene expressions were rescued by PRP from the inhibition of IL-1β+TNF-α, especially under the modulation of collagen matrix. The inflammatory molecules activated by IL-1β+TNF-α were also significantly diminished by PRP with collagen matrix. The membrane receptors integrin α1β1 and CD44 were strongly inhibited by IL-1β+TNF-α, while this inhibition was then recovered by PRP in collagen coating condition. In a 3D model encapsulated with collagen, PRP-induced chondrogenesis were highly enhanced, such as strong restoration of type II collagen and proteoglycan from the inhibition of IL-1β+TNF-α. The result indicated that collagen matrix enhances the effect of PRP on chondrogenesis in response to pro-inflammatory cytokines. The combination of PRP and collagen matrix might facilitate a physiological microenvironment beneficial for maintaining chondrocyte homeostasis and represents an advanced osteoarthritis therapy for clinical applications.
Keywords: Osteoarthritis (OA); Platelet-rich plasma (PRP); Collagen matrix; Physiological microenvironment; Pro-inflammatory cytokines;

In vivo distribution, pharmacokinetics, and toxicity of aqueous synthesized cadmium-containing quantum dots by Yuanyuan Su; Fei Peng; Ziyun Jiang; Yiling Zhong; Yimei Lu; Xiangxu Jiang; Qing Huang; Chunhai Fan; Shuit-Tong Lee; Yao He (5855-5862).
Fluorescent Ⅱ–Ⅳ Quantum dots (QDs) have demonstrated to be highly promising biological probes for various biological and biomedical applications due to their many attractive merits, such as robust photostabilty, strong photoluminescence, and size-tunable fluorescence. Along with wide ranging bioapplications, concerns about their biosafety have attracted increasingly intensive attentions. In comparison to full investigation of in vitro toxicity, there has been only scanty information regarding in vivo toxicity of the QDs. Particularly, while in vivo toxicity of organic synthesized QDs (orQDs) have been investigated recently, there exist no comprehensive studies concerning in vivo behavior of aqueous synthesized QDs (aqQDs) up to present. Herein, we investigate short- and long-term in vivo biodistribution, pharmacokinetics, and toxicity of the aqQDs. Particularly, the aqQDs are initially accumulated in liver after short-time (0.5–4 h) post-injection, and then are increasingly absorbed by kidney during long-time (15–80 days) blood circulation. Moreover, obviously size-dependent biodistribution is observed: aqQDs with larger sizes are more quickly accumulated in the spleen. Furthermore, histological and biochemical analysis, and body weight measurement demonstrate that there is no overt toxicity of aqQDs in mice even at long-time exposure time. Our studies provide invaluable information for the design and development of aqQDs for biological and biomedical applications.
Keywords: Quantum dots; Aqueous synthesis; In vivo; Biodistribution; Toxicity;

FMN-coated fluorescent iron oxide nanoparticles for RCP-mediated targeting and labeling of metabolically active cancer and endothelial cells by Jabadurai Jayapaul; Michael Hodenius; Susanne Arns; Wiltrud Lederle; Twan Lammers; Peter Comba; Fabian Kiessling; Jessica Gaetjens (5863-5871).
Riboflavin is an essential vitamin for cellular metabolism and is highly upregulated in metabolically active cells. Consequently, targeting the riboflavin carrier protein (RCP) may be a promising strategy for labeling cancer and activated endothelial cells. Therefore, Ultrasmall SuperParamagnetic Iron Oxide nanoparticles (USPIO) were adsorptively coated with the endogenous RCP ligand flavin mononucleotide (FMN), which renders them target-specific and fluorescent. The core diameter, surface morphology and surface coverage of the resulting FMN-coated USPIO (FLUSPIO) were evaluated using a variety of physico-chemical characterization techniques (TEM, DLS, MRI and fluorescence spectroscopy). The biocompatibility of FLUSPIO was confirmed using three different cell viability assays (Trypan blue staining, 7-AAD staining and TUNEL). In vitro evaluation of FLUSPIO using MRI and fluorescence microscopy demonstrated high labeling efficiency of cancer cells (PC-3, DU-145, LnCap) and activated endothelial cells (HUVEC). Competition experiments (using MRI and ICP-MS) with a 10- and 100-fold excess of free FMN confirmed RCP-specific uptake of the FLUSPIO by PC-3 cells and HUVEC. Hence, RCP-targeting via FMN may be an elegant way to render nanoparticles fluorescent and to increase the labeling efficacy of cancer and activated endothelial cells. This was shown for FLUSPIO, which due to their high T2-relaxivity, are favorably suited for MR cell tracking experiments and cancer detection in vivo.
Keywords: Cell tracking; Flavin mononucleotide; Iron oxide nanoparticles; Magnetic resonance imaging; Molecular imaging; Riboflavin carrier protein;

Peptide-conjugated polymeric micellar nanoparticles for Dual SPECT and optical imaging of EphB4 receptors in prostate cancer xenografts by Rui Zhang; Chiyi Xiong; Miao Huang; Min Zhou; Qian Huang; Xiaoxia Wen; Dong Liang; Chun Li (5872-5879).
EphB4, a member of the largest family of receptor tyrosine kinases, is overexpressed in numerous tumors. In this study, we developed a new class of multimodal nanoplatform for dual single photon emission computed tomography (SPECT) and near-infrared fluorescence imaging of EphB4. EphB4-binding peptide TNYL-FSPNGPIARAW (TNYL-RAW) was conjugated to polyethylene glycol-coated, core-crosslinked polymeric micelles (CCPM) dually labeled with near-infrared fluorescence fluorophores (Cy7) and a radioisotope (indium 111). In vitro, TNYL-RAW-CCPM selectively bound to EphB4-positive PC-3M prostate cancer cells, but not to EphB4-negative A549 lung cancer cells. In vivo, PC-3M tumors were clearly visualized by both SPECT and near-infrared fluorescence tomography after intravenous administration of 111In-labeled TNYL-RAW-CCPM. In contrast, there was little signal in A549 tumors of mice injected with 111In-labeled TNYL-RAW-CCPM or in PC-3M tumors of mice injected with 111In-labeled CCPM. The high accumulation of 111In-labeled TNYL-RAW-CCPM in PC-3M tumor could be significantly reduced after co-injection with an excess amount of TNYL-RAW peptide. Immunohistochemical analysis showed that fluorescence signal from the nanoparticles correlated with their radioactivity count, and co-localized with the EphB4 expressing region. 111In-labeled TNYL-RAW-CCPM allowed visualization of cancer cells overexpressing EphB4 by both nuclear and optical techniques. The complementary information acquired with multiple imaging techniques should be advantageous in early detection of cancer.
Keywords: Micelle; Nanoparticle; Fluorescence; Molecular imaging;

Aggregation-enhanced fluorescence in PEGylated phospholipid nanomicelles for in vivo imaging by Dan Wang; Jun Qian; Sailing He; Jin Sun Park; Kwang-Sup Lee; Sihai Han; Ying Mu (5880-5888).
We report polymeric nanomicelles doped with organic fluorophores (StCN, (Z)-2,3-bis[4-(N-4-(diphenylamino)styryl)phenyl]-acrylonitrile), which have the property of aggregation-enhanced fluorescence. The fluorescent nanomicelles have two unique features: (1) They give much brighter fluorescence emission than mono-fluorophores. (2) The nanomicelles with amphiphilic copolymers [e.g., phospholipids-PEG (polyethylene glycol)] make the encapsulated fluorophores more stable in various bio-environments and easy for further conjugation with bio-molecules. After chemical and optical characterization, these fluorescent nanomicelles are utilized as efficient optical probes for in vivo sentinel lymph node (SLN) mapping of mice. The StCN-encapsulated nanomicelles, as well as their bioconjugates with arginine-glycine-aspartic acid (RGD) peptides, are used to target subcutaneously xenografted tumors in mice, and in vivo fluorescence images demonstrate the potential to use PEGylated phospholipid nanomicelles with aggregation-enhanced fluorescence as bright nanoprobes for in vivo diagnosis of tumors.
Keywords: Aggregation-enhanced fluorescence; Phospholipid-PEG nanomicelles; Bright nanoprobes; In vivo imaging;

Enhanced gene expression in tumors after intravenous administration of arginine-, lysine- and leucine-bearing polypropylenimine polyplex by Hibah Aldawsari; RuAngelie Edrada-Ebel; David R. Blatchford; Rothwelle J. Tate; Laurence Tetley; Christine Dufès (5889-5899).
The possibility of using non-viral gene delivery systems for the treatment of cancer is currently limited by their lower transfection efficacy compared to viral systems. On the basis that amino acids such as arginine, lysine and leucine were involved in enhancing DNA transportation into cells, we hypothesized that the grafting of these amino acids to the highly promising generation 3 diaminobutyric polypropylenimine (DAB) dendrimer would improve its transfection efficacy in cancer cells. In this work we demonstrated that the conjugation of arginine, lysine and leucine to the dendrimer led to an enhanced anti-proliferative activity of the polyplexes, by up to 47-fold for DAB-Lys in T98G cancer cells compared to the unmodified polyplex in vitro. In vivo, the intravenous administration of amino acid-bearing DAB polyplexes resulted in a significantly improved tumor gene expression, with the highest gene expression level observed after treatment with DAB-Lys polyplex. Arginine, lysine and leucine-bearing generation 3 polypropylenimine polymers are therefore highly promising gene delivery systems for gene transfection in tumors.
Keywords: Amino acid; Gene expression; Nanoparticles; Dendrimer; DNA;

The use of cationic nanogels to deliver proteins to myeloma cells and primary T lymphocytes that poorly express heparan sulfate by Kozo Watanabe; Yumiko Tsuchiya; Yoshinori Kawaguchi; Shin-ichi Sawada; Hirohito Ayame; Kazunari Akiyoshi; Takeshi Tsubata (5900-5905).
Fusion proteins containing protein transduction domain (PTD) are widely used for intracellular delivery of exogenous proteins. PTD-mediated delivery requires expression of heparan sulfate on the surface of the target cells. However, some of metastatic tumor cells and primary lymphocytes poorly express heparan sulfate. Here we demonstrate that proteins complexed with nanosize hydrogels formed by cationic cholesteryl group-bearing pullulans (cCHP) are efficiently delivered to myeloma cells and primary CD4+ T lymphocytes probably by induction of macropinocytosis, although these cells are resistant to PTD-mediated protein delivery as a consequence of poor heparan sulfate expression. The anti-apoptotic protein Bcl-xL delivered by cCHP nanogels efficiently blocked apoptosis of these cells, establishing functional regulation of cells by proteins delivered by cCHP nanogels. Thus, cCHP nanogel is a useful tool to deliver proteins for development of new cancer therapy and immune regulation.
Keywords: Cationic cholesteryl group-bearing pullulans; Macropinocytosis; Lymphocyte; Nanoparticle; Hydrogel; Protein delivery;

Encapsulation of curcumin in self-assembling peptide hydrogels as injectable drug delivery vehicles by Aysegul Altunbas; Seung J. Lee; Sigrid A. Rajasekaran; Joel P. Schneider; Darrin J. Pochan (5906-5914).
Curcumin, a hydrophobic polyphenol, is an extract of turmeric root with antioxidant, anti-inflammatory and anti-tumorigenic properties. Its lack of water solubility and relatively low bioavailability set major limitations for its therapeutic use. In this study, a self-assembling peptide hydrogel is demonstrated to be an effective vehicle for the localized delivery of curcumin over sustained periods of time. The curcumin-hydrogel is prepared in-situ where curcumin encapsulation within the hydrogel network is accomplished concurrently with peptide self-assembly. Physical and in vitro biological studies were used to demonstrate the effectiveness of curcumin-loaded β-hairpin hydrogels as injectable agents for localized curcumin delivery. Notably, rheological characterization of the curcumin-loaded hydrogel before and after shear flow have indicated solid-like properties even at high curcumin payloads. In vitro experiments with a medulloblastoma cell line confirm that the encapsulation of the curcumin within the hydrogel does not have an adverse effect on its bioactivity. Most importantly, the rate of curcumin release and its consequent therapeutic efficacy can be conveniently modulated as a function of the concentration of the MAX8 peptide.
Keywords: Peptide; Self-assembly; Curcumin; Hydrogel;

Nanofiber-mediated controlled release of siRNA complexes for long term gene-silencing applications by Pim-on Rujitanaroj; Yu-Cai Wang; Jun Wang; Sing Yian Chew (5915-5923).
Nanofiber scaffold-mediated delivery of small-interfering RNA (siRNA) holds great potential in regenerative medicine by providing biomimicking topographical signals and enhanced gene silencing effects to seeded cells. While the delivery of naked siRNA was demonstrated previously using poly (ε-caprolactone) (PCL) nanofibers, the resulting siRNA release kinetics and gene knockdown efficiencies were sub-optimal. In this study, we investigated the feasibility of encapsulating siRNA and transfection reagent (TKO) complexes within nanofibers comprising of a copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP, diameter ∼ 400 nm). Sustained release of bioactive naked siRNA and siRNA/TKO complexes were obtained for at least 28 days. By copolymerizing EEP with caprolactone, siRNA release was significantly enhanced (total siRNA that was released by day 49 was ∼ 89.3–97.2% as compared to previously reported 3% by plain PCL nanofiber delivery). Using GAPDH as the model protein, bioactivity analyses by supernatant transfection revealed the partial retention of bioactivity of naked siRNA and siRNA/TKO complexes for at least 30 days. In particular, GAPDH siRNA/TKO supernatant alone induced significant gene silencing (∼40%), indicating the feasibility of co-encapsulating siRNA and transfection reagent within a single scaffold construct for sustained delivery. Direct culture of cells on siRNA incorporated scaffolds for scaffold-mediated gene transfection revealed significant gene knockdown even in the absence of transfection reagent (21.3% knockdown efficiency by scaffolds incorporating naked siRNA only). By encapsulating siRNA/TKO complexes, more significant gene knockdown was obtained (30.9% knockdown efficiency as compared to previously reported 18% by plain PCL scaffold-mediated transfection). Taken together, the results demonstrated the feasibility of co-encapsulating siRNA-transfection reagent complexes within a single nanofiber construct for sustained siRNA delivery and enhanced gene knockdown efficiency. The study also highlights the potential of PCLEEP as a platform for tailoring siRNA release kinetics for long-term gene silencing applications.
Keywords: RNA interference; Scaffold-mediated reverse transfection; Tissue engineering; Electrospinning; Gene knockdown;

C/EBP-α and C/EBP-β–mediated adipogenesis of human mesenchymal stem cells (hMSCs) using PLGA nanoparticles complexed with poly(ethyleneimmine) by Han Na Yang; Ji Sun Park; Dae Gyun Woo; Su Yeon Jeon; Hyun-Jin Do; Hye-Young Lim; Jae-Hwan Kim; Keun-Hong Park (5924-5933).
In this study, to drive efficient adipogenic differentiation, the adipogenic transcription factors C/EBP-α and C/EBP-β fused to green fluorescent protein (GFP) or red fluorescent protein (RFP) were complexed with poly-ethyleneimine (PEI) coupled with biodegradable PLGA nanospheres and delivered to human mesenchymal stem cell (hMSC). FACS analysis revealed that the transfection efficiency of C/EBP-α, C/EBP-β, or both genes complexed with PEI-coated PLGA nanospheres was 12.59%, 21.74%, and 28.96% of hMSCs. Expression and localization of C/EBP-α and C/EBP-β were confirmed by Western blotting and confocal laser microscopy. Overexpression of exogenous C/EBP-α and C/EBP-β significantly elevated adipogenic differentiation processes as indicated by RT-PCR, real-time PCR, Western blotting, histology, and immunofluorescence microscopy. During adipogenesis, PEI-coupled PLGA nanospheres complexed with C/EBP-α and C/EBP-β greatly increased the adipogenic capability of in vitro cultured cells, as well of in vivo transplanted cells. The expression of genes and proteins specific to adipogenic differentiation in hMSCs was significantly elevated compared to the controls.
Keywords: Gene transfection; hMSCs; C/EBP-α; C/EBP-β; PLGA nanospheres;

The increasing global incidence of malignant melanoma combined with the poor prognosis and low survival rates of patients necessitates the development of new chemotherapeutic strategies. Thus, the objective of this present study was to investigate the therapeutic efficacy of Pluronic polymeric micelles encapsulating paclitaxel (PTX) in both B16F10 melanoma subcutaneous mice model and pulmonary metastatic mice model. Herein, we developed a PTX-loaded polymeric micelles (PF-PTX) consisting of Pluronic P 123 and F127 block copolymers with small particle size (∼25 nm), high encapsulation efficiency (>90%), good stability in lyophilized form and pH-dependent in vitro release. Furthermore, influence of PF-PTX on in vitro cytotoxicity was determined by MTT assay using B16F10 melanoma cell line, while cellular distribution of PF-PTX was detected by confocal microscopy. Additionally, C57BL/6 mice bearing subcutaneous or pulmonary B16F10 melanoma tumors were treated with Taxol or PF-PTX, and antitumor effect was compared. It was found that antitumor efficacy of PF-PTX in both tumor models showed significant tumor growth delay and increased survival. In summary, the simple Pluronic-based nanocarrier could be harnessed for the delivery of anticancer drug to melanoma, with increased therapeutic index.
Keywords: Pluronic block copolymers; Paclitaxel; Polymeric micelles; EPR effect; Tumor model;