Biomaterials (v.33, #11)

Multifunctional unimolecular micelles for cancer-targeted drug delivery and positron emission tomography imaging by Yuling Xiao; Hao Hong; Alireza Javadi; Jonathan W. Engle; Wenjin Xu; Yunan Yang; Yin Zhang; Todd E. Barnhart; Weibo Cai; Shaoqin Gong (3071-3082).
A multifunctional unimolecular micelle made of a hyperbranched amphiphilic block copolymer was designed, synthesized, and characterized for cancer-targeted drug delivery and non-invasive positron emission tomography (PET) imaging in tumor-bearing mice. The hyperbranched amphiphilic block copolymer, Boltorn® H40-poly(L-glutamate-hydrazone-doxorubicin)-b-poly(ethylene glycol) (i.e., H40-P(LG-Hyd-DOX)-b-PEG), was conjugated with cyclo(Arg-Gly-Asp-D-Phe-Cys) peptides (cRGD, for integrin αvβ3 targeting) and macrocyclic chelators (1,4,7-triazacyclononane-N, N’, N’’-triacetic acid [NOTA], for 64Cu-labeling and PET imaging) (i.e., H40-P(LG-Hyd-DOX)-b-PEG-OCH3/cRGD/NOTA, also referred to as H40-DOX-cRGD). The anti-cancer drug, doxorubicin (DOX) was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms (i.e., PLG) via a pH-labile hydrazone linkage to enable pH-controlled drug release. The unimolecular micelles exhibited a uniform size distribution and pH-sensitive drug release behavior. cRGD-conjugated unimolecular micelles (i.e., H40-DOX-cRGD) exhibited a much higher cellular uptake in U87MG human glioblastoma cells due to integrin αvβ3-mediated endocytosis than non-targeted unimolecular micelles (i.e., H40-DOX), thereby leading to a significantly higher cytotoxicity. In U87MG tumor-bearing mice, H40-DOX-cRGD-64Cu also exhibited a much higher level of tumor accumulation than H40-DOX-64Cu, measured by non-invasive PET imaging and confirmed by biodistribution studies and ex vivo fluorescence imaging. We believe that unimolecular micelles formed by hyperbranched amphiphilic block copolymers that synergistically integrate passive and active tumor-targeting abilities with pH-controlled drug release and PET imaging capabilities provide the basis for future cancer theranostics.
Keywords: Unimolecular micelles; Drug delivery; Theranostic nanocarriers; Hyperbranched amphiphilic block copolymer; Positron emission tomography (PET); Cyclic arginine-glycine-aspartic acid (cRGD) peptide;

The antithrombotic and antimicrobial properties of PEG-protected silver nanoparticle coated surfaces by V.M. Ragaseema; S. Unnikrishnan; V. Kalliyana Krishnan; Lissy K. Krishnan (3083-3092).
Cardiovascular implant-associated complications such as infection and thrombosis may be reduced by modification of device surfaces using antimicrobial and antithrombotic agents. Silver nanoparticles (SNPs) are well accepted for its broad-spectrum antimicrobial effect. A recent report suggested its antiplatelet effect also. So the hypothesis of this study is that polyethylene glycol (PEG) protected SNPs can be incorporated with biomaterials to attain dual properties; and by adjusting an optimum concentration, its cytotoxicity to tissues and cells can be prevented. To prove this, detailed study of PEG–SNP was done at three levels: (i) direct inhibitory effect on platelet activation, aggregation and biochemical pathways when PEG–SNP is added into platelet suspension; (ii) inhibition of platelet adhesion to PEG–SNP incorporated biological matrix and polymer scaffold and (iii) non-cytotoxic behavior of immobilized PEG–SNP in fibrin matrix. Inhibitory effects demonstrated are on: platelet function by aggregometry, exposure of activation and apoptosis markers by flow cytometry, biochemical pathway by malondealdehyde (MDA) estimation and protein phosphorylation by Western blot. Reduced platelet adhesion onto PEG–SNP incorporated scaffold is shown using scanning electron microscopy (SEM). Non-toxic behavior of endothelial cells (EC) and smooth muscle cells (SMC) grown on PEG–SNP-fibrin disc is shown by fluorescence microscopy and cell phenotype stability by real-time polymerase chain reaction (PCR).
Keywords: Silver nanoparticles; Antimicrobial activity; Antithrombotic activity; Vascular tissue engineering; Biomaterial surface modification;

The influence of chitosan hydrogel on stem cell engraftment, survival and homing in the ischemic myocardial microenvironment by Zhiqiang Liu; Haibin Wang; Yan Wang; Qiuxia Lin; Anning Yao; Feng Cao; Dexue Li; Jin Zhou; Cuimi Duan; Zhiyan Du; Yanmeng Wang; Changyong Wang (3093-3106).
One challenge of cellular cardiomyoplasty for myocardial infarction (MI) is how to improve MI microenvironment to facilitate stem cell engraftment, survival and homing for myocardial repair. The application of injectable hydrogels is an effective strategy. However, it has not been thoroughly investigated on the role of the injectable scaffolds, in improving MI microenvironment, providing space and guidance for cell survival, engraftment and homing. We explored an injectable chitosan hydrogel for stem cell delivery into ischemic heart and investigated the beneficial effects and mechanisms of the hydrogel. In vitro, H2O2-treatment was used to mimic reactive oxygen species (ROS) microenvironment. The influence of ROS and protection of chitosan components on adipose-derived mesenchymal stem cells (ADSCs) was analyzed too. In vivo, MI was induced by the left anterior descending artery ligation in SD rats. PBS, chitosan hydrogel, ADSC/PBS and ADSC/chitosan hydrogel were injected into the border of infracted hearts respectively. Multi-techniques were used to assess the beneficial effects of chitosan hydrogel after transplantation. We observed that ROS generated by ischemia would impair ADSC adhesion molecules, including integrin-related adhesion molecules integrin αV and β1, focal adhesion-related molecules p-FAK and p-Src, and corresponding ligands of host myocardium ICAM1 and VCAM1. Chitosan hydrogel could rescue these molecules through ROS scavenging and recruit key chemokine for stem cell homing, such as SDF-1. The results suggest that chitosan hydrogel could improve MI microenvironment, enhance stem cell engraftment, survival and homing in ischemic heart through ROS scavenging and chemokine recruitment, contributing to myocardial repair.
Keywords: Temperature-responsive chitosan hydrogel; Stem cell homing; Reactive oxygen species; Myocardial infarction; Cell adhesion;

The significance of integrin ligand nanopatterning on lipid raft clustering in hematopoietic stem cells by Eva Altrock; Christine A. Muth; Gerd Klein; Joachim P. Spatz; Cornelia Lee-Thedieck (3107-3118).
Hematopoietic stem cells (HSCs) are the vital, life-long source of all blood cell types. They are found in stem cell niches, specific anatomic locations that offer all the factors and signals necessary for the maintenance of the stem cell potential of HSCs. Much attention has been paid to the biochemical composition of the niches, but only little is known about the influence of physical parameters, such as ligand nanopatterns, on HSCs. To investigate the impact of nanometer-scale spacing between cell ligands on HSC adhesion, integrin distribution and signal transduction, we employed geometrically defined, nanostructured, bio-functionalized surfaces. HSCs proved to be sensitive to the lateral distance between the presented ligands with regard to adhesion and lipid raft clustering, the latter being a prerequisite for the formation of signaling complexes. Furthermore, an extensive redistribution of stem cell markers, integrins and phosphorylated proteins in HSCs was observed. In conclusion, integrin-mediated adhesion and signaling of HSCs proved to depend on the nanostructured presentation of ligands in their environment. In this work, we show that the nanostructure of the matrix is an important parameter influencing HSC behavior that should be integrated into biomaterial-based approaches aiming at HSC multiplication or differentiation.
Keywords: Substrate nanostructure; Cell adhesion; Integrin; Hematopoietic stem cell; Lipid raft; Cell signaling;

BDNF blended chitosan scaffolds for human umbilical cord MSC transplants in traumatic brain injury therapy by Wei Shi; Dekang Nie; Guohua Jin; Weiwei Chen; Liang Xia; Xiujie Wu; Xing Su; Xide Xu; Lanchun Ni; Xianan Zhang; Xinhua Zhang; Jian Chen (3119-3126).
This study tested the cytotoxicity of a BDNF blended chitosan scaffold with human umbilical cord mesenchymal stem cells (hUC-MSCs), and the in vitro effect of BDNF blended chitosan scaffolds on neural stem cell differentiation with the aim of contributing alternative methods in tissue engineering for the treatment of traumatic brain injury (TBI). The chitosan scaffold based on immobilization of BDNF by genipin (GP) as a crosslinking agent referred to hereafter as a CGB scaffold was prepared by freezing-drying technique. hUC-MSCs were co-cultured with the CGB scaffold. Fluorescent nuclear staining (Hoechst 33342) was employed to determine the attachment of the hUC-MSCs to CGB scaffolds on the 1st, 3rd, 7th and 10th day of co-culture. The viability of hUC-MSCs adhered to the CGB scaffold was determined by digesting with 0.25% trypsin and evaluating with the cell counting kit-8 (CCK-8). Prior to this, the diameter and porosity of CGB scaffolds were measured. The amount of BDNF released from CGB over a 30 day period was determined by ELISA. Finally, we investigated whether the released BDNF can induce NSC to differentiate into neurons. There were no significant differences in diameter and porosity of individual CGB scaffolds (P > 0.05). There were on average more cells on the CGB scaffold on the first day than on any other day sampled (P < 0.05). The CGB scaffolds released BDNF in a uniform profile, whereas the CB scaffolds only released BDNF during the first 3 days. BDNF released from CGB scaffold promoted neuronal differentiation of NSCs and led to significant differences in differentiation rate and average neuron perimeter compared with the control group. The results of this study demonstrate that CGB scaffolds are biocompatible with hUC-MSCs and that granular CGB scaffolds covered with hUC-MSCs are expected to generate new advances for future treatment of traumatic brain injury.
Keywords: Human umbilical cord mesenchymal stem cells; Chitosan scaffold; Co-culture; Brain-derived neurotrophic factor; Genipin; Neural stem cell;

Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers by Yoke Chin Chai; Scott J. Roberts; Eline Desmet; Greet Kerckhofs; Nick van Gastel; Liesbet Geris; Geert Carmeliet; Jan Schrooten; Frank P. Luyten (3127-3142).
Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell–host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca2+) and phosphate (Pi) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca2+ and Pi supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (μCT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell–host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials.
Keywords: Ectopic bone formation; Calcium phosphate; Stem cell–host-material interaction; Mesenchymal stem cells; Osteogenic differentiation;

A major goal in the application of hydrogels for tissue engineering scaffolds, especially for load-bearing tissues such as cartilage, is to develop hydrogels with high mechanical strength. In this study, a double-network (DN) strategy was used to engineer strong hydrogels that can encapsulate cells. We improved upon previously studied double-network (DN) hydrogels by using a processing condition compatible with cell survival. The DN hydrogels were created by a two-step photocrosslinking using gellan gum methacrylate (GGMA) for the rigid and brittle first network, and gelatin methacrylamide (GelMA) for the soft and ductile second network. We controlled the degree of methacrylation of each polymer so that they obtain relevant mechanical properties as each network. The DN was formed by photocrosslinking the GGMA, diffusing GelMA into the first network, and photocrosslinking the GelMA to form the second network. The formation of the DN was examined by diffusion tests of the large GelMA molecules into the GGMA network, the resulting enhancement in the mechanical properties, and the difference in mechanical properties between GGMA/GelMA single networks (SN) and DNs. The resulting DN hydrogels exhibited the compressive failure stress of up to 6.9 MPa, which approaches the strength of cartilage. It was found that there is an optimal range of the crosslink density of the second network for high strength of DN hydrogels. DN hydrogels with a higher mass ratio of GelMA to GGMA exhibited higher strength, which shows promise in developing even stronger DN hydrogels in the future. Three dimensional (3D) encapsulation of NIH-3T3 fibroblasts and the following viability test showed the cell-compatibility of the DN formation process. Given the high strength and the ability to encapsulate cells, the DN hydrogels made from photocrosslinkable macromolecules could be useful for the regeneration of load-bearing tissues.
Keywords: Hydrogel; IPN; Photopolymerization; Cell encapsulation; Mechanical properties;

The combined effects of continuous passive motion treatment and acellular PLGA implants on osteochondral regeneration in the rabbit by Nai-Jen Chang; Chih-Chan Lin; Chien-Feng Li; Dong-An Wang; Nontapot Issariyaku; Ming-Long Yeh (3153-3163).
We investigated the active role of clinical rehabilitation in osteochondral regeneration using continuous passive motion (CPM) treatment together with acellular PLGA implants. CPM treatment was performed and compared with immobilization (Imm) treatment and intermittent active motion (IAM) treatment upon full-thickness osteochondral defects either with or without an PLGA implant in the PI (PLGA-implanted) and ED (empty defect) models. The PI and ED tests were performed in 38 rabbits for 4 and 12 weeks. At the end of testing, the PI-CPM group had the best regeneration with nearly normal articular surfaces and no joint contracture or inflammatory reaction. In contrast, degenerated joints, abrasion cartilage surfaces and synovitis were observed in the Imm and IAM groups. The achieved bone volume/tissue volume (BV/TV) ratio, which was measured using micro-CT, was significantly higher in the CPM group compared with the Imm and IAM groups; in particular, the performance of the PI-CPM group exceeds that of the ED-CPM group. The thickness of the trabecular (subchondral) bone was visibly increased in all of the groups from 4 through 12 weeks of testing. However, a histological analysis revealed differences in cartilage regeneration. At week 4, compared with the ED samples, all of the PI groups exhibited better collagen alignment and higher GAG content in the core of their repaired tissues, particularly in the PI-CPM group. At week 12, sound osteochondral repair and hyaline cartilaginous regeneration was observed in the PI-CPM group, and this was marked by type II collagen expression, osteocyte maturation, and trabecular boney deposition. In contrast, the PI-Imm and PI-IAM groups exhibited fibrocartilaginous tissues that had modest GAG content. In summary, this study demonstrates that early CPM treatment together with acellular PLGA implantation has significant positive effects on osteochondral regeneration in rabbit knee joint models.
Keywords: Cartilage; ECM (extracellular matrix); In vivo test; Scaffold;

Self-attaching and cell-attracting in-situ forming dextran-tyramine conjugates hydrogels for arthroscopic cartilage repair by Liliana S. Moreira Teixeira; Suzanne Bijl; Vishnu V. Pully; Cees Otto; Rong Jin; Jan Feijen; Clemens A. van Blitterswijk; Pieter J. Dijkstra; Marcel Karperien (3164-3174).
Small cartilage defects are frequently treated with debridement or left untreated, predisposing to early onset osteoarthritis. We propose to fill these defects with a cell-free injectable hydrogel comprising dextran-tyramine conjugates (Dex-TA) that can be applied during arthroscopic procedures. In this study, we report on the adhesion mechanism between cartilage and Dex-TA hydrogels and enhancement of cell ingrowth by incorporation of Heparin-tyramine (Hep-TA) conjugates. The enzyme-catalyzed crosslinking reaction of Dex-TA and Hep-TA hydrogels is based on covalent bonding of hydroxyphenyl residues. We hypothesized that this reaction results in covalent bonding of the hydroxyphenyl residues in Dex-TA and Hep-TA to tyrosine residues in cartilage matrix proteins. The involvement of TA residues was confirmed by modelling the enzymatic reaction occurring during gelation. The mechanical analysis indicated that higher tyramine content led to stronger binding. Interfacial cartilage-hydrogel morphology and Raman spectroscopy demonstrated collagens’ reorganization and evidenced the coupling of TA to tyrosine residues in collagen. Moreover, the addition of Hep-TA induced cell recruitment. Collectively, in vitro and ex vivo functional studies evidenced the covalent bonding of TA-containing hydrogels to tyrosine residues in cartilaginous matrix proteins. Moreover, the cell-attracting ability of these hydrogels could be explored to guide tissue repair in focal cartilage defects, preventing or delaying the onset osteoarthritis.
Keywords: Adhesion mechanism; Cartilage tissue engineering; Collagen; Cross-linking; Hydrogel; Interface;

The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds by Sharon J. Sequeira; David A. Soscia; Basak Oztan; Aaron P. Mosier; Riffard Jean-Gilles; Anand Gadre; Nathaniel C. Cady; Bülent Yener; James Castracane; Melinda Larsen (3175-3186).
Nanofiber scaffolds have been useful for engineering tissues derived from mesenchymal cells, but few studies have investigated their applicability for epithelial cell-derived tissues. In this study, we generated nanofiber (250 nm) or microfiber (1200 nm) scaffolds via electrospinning from the polymer, poly-l-lactic-co-glycolic acid (PLGA). Cell-scaffold contacts were visualized using fluorescent immunocytochemistry and laser scanning confocal microscopy. Focal adhesion (FA) proteins, such as phosphorylated FAK (Tyr397), paxillin (Tyr118), talin and vinculin were localized to FA complexes in adult cells grown on planar surfaces but were reduced and diffusely localized in cells grown on nanofiber surfaces, similar to the pattern observed in adult mouse salivary gland tissues. Significant differences in epithelial cell morphology and cell clustering were also observed and quantified, using image segmentation and computational cell-graph analyses. No statistically significant differences in scaffold stiffness between planar PLGA film controls compared to nanofibers scaffolds were detected using nanoindentation with atomic force microscopy, indicating that scaffold topography rather than mechanical properties accounts for changes in cell attachments and cell structure. Finally, PLGA nanofiber scaffolds could support the spontaneous self-organization and branching of dissociated embryonic salivary gland cells. Nanofiber scaffolds may therefore have applicability in the future for engineering an artificial salivary gland.
Keywords: Cell morphology; ECM; Nanofibers; Organ culture; PLGA; Salivary gland;

Mechanisms underlying the synergistic enhancement of self-assembled neocartilage treated with chondroitinase-ABC and TGF-β1 by Donald J. Responte; Boaz Arzi; Roman M. Natoli; Jerry C. Hu; Kyriacos A. Athanasiou (3187-3194).
Developing a platform for in vitro cartilage formation would enhance the study of cartilage development, pathogenesis, and regeneration. To improve neocartilage formation, our group developed a novel self-assembly process for articular chondrocytes, which has been improved in this study using a novel combination of catabolic and anabolic agents. TGF-β1 was applied in conjunction with the enzyme chondroitinase-ABC (C-ABC) to additively increase tensile properties and synergistically enhance collagen content. Additionally, microarray analysis indicated that TGF-β1 up-regulated MAPK signaling in contrast to C-ABC, which did not enrich genetic pathways. The lack of genetic signaling spurred investigation of the biophysical role of C-ABC, which showed that C-ABC treatment increased collagen fibril diameter and density. After four weeks of culture in nude mice, neocartilage exhibited stability and maturation. This study illustrated an innovative strategy for improving in vitro and in vivo articular cartilage formation and elucidated mechanisms underlying TGF-β1 and C-ABC treatment.
Keywords: Cartilage tissue engineering; Collagen; Glycosaminoglycan; Soft tissue biomechanics;

Protecting against wayward human induced pluripotent stem cells with a suicide gene by Fuyi Cheng; Qiong Ke; Fei Chen; Bing Cai; Yong Gao; Chenghui Ye; Ding Wang; Li Zhang; Bruce T. Lahn; Weiqiang Li; Andy Peng Xiang (3195-3204).
The generation of human induced pluripotent stem cells (hiPSCs) opens a prospect for regenerative medicine. However, transplantation of somatic cells derived from hiPSCs still harbor many risks such as cells’ incorrect differentiation or over-proliferation, and the worst, tumor formation. Therefore, it’s essential to ravel out these obstacles before their clinical application. Herein, we genetically modified hiPSCs and human embryonic stem cells (hESCs) with a truncated herpes simplex virus delta thymidine kinase (deltaTK) gene driven by EF1α or Nanog promoter to selectively ablate wayward pluripotent stem cells. The results showed that insertion of deltaTK gene did not alter their pluripotency and self-renewal capacity but rendered them sensitive to ganciclovir, which induced elimination of deltaTK+ cells in vitro in a dose and time-dependent manner, most importantly, facilitated both prevention and ablation of tumors in vivo. Furthermore, comparative analysis between transduced hiPSCs and hESCs showed that there was no difference in ganciclovir sensitivity between them. This approach may help to develop safety strategies for clinical application of hiPSCs in regenerative medicine in the future.
Keywords: Human induced pluripotent stem cells; Suicide gene; Thymidine kinase; Nanog;

A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells by Ana M.C. Barradas; Hugo A.M. Fernandes; Nathalie Groen; Yoke Chin Chai; Jan Schrooten; Jeroen van de Peppel; Johannes P.T.M. van Leeuwen; Clemens A. van Blitterswijk; Jan de Boer (3205-3215).
The response of osteoprogenitors to calcium (Ca2+) is of primary interest for both normal bone homeostasis and the clinical field of bone regeneration. The latter makes use of calcium phosphate-based bone void fillers to heal bone defects, but it is currently not known how Ca2+ released from these ceramic materials influences cells in situ. Here, we have created an in vitro environment with high extracellular Ca2+ concentration and investigated the response of human bone marrow-derived mesenchymal stromal cells (hMSCs) to it. Ca2+ enhanced proliferation and morphological changes in hMSCs. Moreover, the expression of osteogenic genes is highly increased. A 3-fold up-regulation of BMP-2 is observed after only 6 h and pharmaceutical interference with a number of proteins involved in Ca2+ sensing showed that not the calcium sensing receptor, but rather type L voltage-gated calcium channels are involved in mediating the signaling pathway between extracellular Ca2+ and BMP-2 expression. MEK1/2 activity is essential for the effect of Ca2+ and using microarray analysis, we have identified c-Fos as an early Ca2+ response gene. We have demonstrated that hMSC osteogenesis can be induced via extracellular Ca2+, a simple and economic way of priming hMSCs for bone tissue engineering applications.
Keywords: Calcium phosphate; Stem cell; Bone morphogenetic protein; Osteogenesis;

We demonstrate that etched microgrooves, with truncated V-shape in cross-section and subsequent acid etching, on titanium substrata alter the expression of various genes and proteins in human primary cells. Etched microgrooves with 30 or 60 μm width and 10 μm depth promoted human gingival fibroblast proliferation and significantly enhanced the osteoblast differentiation of human bone marrow-derived mesenchymal stem cells and human periodontal ligament cells by inducing differential expression of various genes involved in cell adhesion, migration, proliferation, mitosis, cytoskeletal reorganization, translation initiation, vesicular trafficking, proton transportation, transforming growth factor-β signaling, mitogen-activated protein kinase signaling, simvastatin’s anabolic effect on bone, inhibitory guanine nucleotide binding protein (G protein)’s action, sumoylation pathway, survival/apoptosis, mitochondrial distribution, type I collagen production, osteoblast differentiation, and bone remodeling that were verified by the differential display PCR and quantitative real-time PCR. The most influential genes on the enhancement of fibroblast proliferation or osteoblast differentiation were determined by multiple regression analysis, and the expression of relevant proteins was confirmed by western blotting and protein quantitation.
Keywords: Microgrooves; Acid etching; Cell proliferation; Osteoblast differentiation; Differential gene expression; Protein expression;

Antitumor immunologically modified carbon nanotubes for photothermal therapy by Feifan Zhou; Shengnan Wu; Sheng Song; Wei R. Chen; Daniel E. Resasco; Da Xing (3235-3242).
An immunologically modified nanotube system was developed using an immunoadjuvant, glycated chitosan (GC), as surfactant of single-walled carbon nanotube (SWNTs). This SWNT-GC system not only retained both optical properties of SWNTs and immunological functions of GC, but also could enter cells due to the carrier properties of SWNTs. Cellular SWNTs induced thermal destruction of tumor cells when irradiated by a near-infrared laser and, at the same time, cellular GC could serve both as damage associated molecular pattern molecules (DAMPs) and pathogen associated molecular pattern molecules (PAMPs) to enhance the tumor immunogenicity and enhance the uptake and presentation of tumor antigens, leading to special antitumor response. Using this system and a 980 nm laser, we treated tumors, both in vitro and in vivo, and investigated the induced thermal and immunological effects. Laser + SWNT-GC afford a remarkable efficacy in suppressing tumor growth in animal cancer models, in many cases resulting in complete tumor regression and long-term survival. Mice successfully treated by Laser + SWNT-GC could establish resistance to tumor rechallenge. This system forms a multifunctional temporal-spatial continuum, which can synergize photothermal and immunological effects. The Laser + SWNT-GC could represent a promising treatment modality to induce systemic antitumor response through a local intervention, while minimizing the adverse side effects.
Keywords: Carbon nanotubes; Antitumor response; Photothermal therapy; Immunological effects;

Nitric oxide is a cell signaling molecule that can be a potent inducer of cell death in cancers at elevated concentrations. However, NO is also toxic to normal tissues and chronic exposure at low levels can induce tumor growth. We have designed a polymeric carrier system to deliver nitric oxide locoregionally to tumorigenic tissues at micromolar concentrations. A highly water solubility and biodegradable multi-arm polymer nanocarrier, sugar poly-(6-O-methacryloyl-d-galactose), was synthesized using MADIX/RAFT polymerization, and utilized to deliver high concentrations of nitric oxide to xenografts of human head and neck squamous cell carcinoma (HNSCC). The in vitro release of the newly synthesized nitric oxide donor, O 2-(2,4-dinitrophenyl) 1-[4-(2-hydroxy)ethyl]-3-methylpiperazin-1-yl]diazen-1-ium-1,2-diolate and its corresponding multi-arm polymer-based nanoconjugate demonstrated a 1- and 2.3-fold increase in half-life, respectively, compared to the release half-life of the nitric oxide-donor prodrug JS-K. When administered to tumor-bearing nude mice, the subcutaneously injected multi-arm polymer nitric oxide nanoparticles resulted in 50% tumor inhibition and a 7-week extension of the average survival time, compared to intravenous JS-K therapy. In summary, we have developed an effective nitric oxide anti-cancer chemotherapy that could be administered regionally to provide the local disease control, improving prognosis for head and neck cancers.
Keywords: Biocompatibility; Drug delivery; Drug release; Nanoparticle; Nitric oxide; Polymerization;

Paramagnetic dysprosium oxide nanoparticles and dysprosium hydroxide nanorods as T 2 MRI contrast agents by Krishna Kattel; Ja Young Park; Wenlong Xu; Han Gyeol Kim; Eun Jung Lee; Badrul Alam Bony; Woo Choul Heo; Seonguk Jin; Jong Su Baeck; Yongmin Chang; Tae Jeong Kim; Ji Eun Bae; Kwon Seok Chae; Gang Ho Lee (3254-3261).
We report here paramagnetic dysprosium nanomaterial-based T 2 MRI contrast agents. A large r 2 and a negligible r 1 is an ideal condition for T 2 MR imaging. At this condition, protons are strongly and nearly exclusively induced for T 2 MR imaging. The dysprosium nanomaterials fairly satisfy this because they are found to possess a decent r 2 but a negligible r 1 arising from L + S state 4f-electrons in Dy(III) ion (6 H 15/2). Their r 2 will also further increase with increasing applied field because of unsaturated magnetization at room temperature. Therefore, MR imaging and various physical properties of the synthesized d-glucuronic acid coated ultrasmall dysprosium oxide nanoparticles (d avg = 3.2 nm) and dysprosium hydroxide nanorods (20 × 300 nm) are investigated. These include hydrodynamic diameters, magnetic properties, MR relaxivities, cytotoxicities, and 3 tesla in vivo T 2 MR images. Here, MR imaging properties of dysprosium hydroxide nanorods have not been reported so far. These two samples show r 2s of 65.04 and 181.57 s−1mM−1, respectively, with negligible r 1s at 1.5 tesla and at room temperature, no in vitro cytotoxicity up to 100 μM Dy, and clear negative contrast enhancements in 3 tesla in vivo T 2 MR images of a mouse liver, which will be even more improved at higher MR fields. Therefore, d-glucuronic acid coated ultrasmall dysprosium oxide nanoparticles with renal excretion can be a potential candidate as a sensitive T 2 MRI contrast agent at MR field greater than 3 tesla.
Keywords: Dysprosium; Nanomaterials; Paramagnetic; MRI; Imaging agent;

Design of ultrasonically-activatable nanoparticles using low boiling point perfluorocarbons by Paul S. Sheeran; Samantha H. Luois; Lee B. Mullin; Terry O. Matsunaga; Paul A. Dayton (3262-3269).
Recently, an interest has developed in designing biomaterials for medical ultrasonics that can provide the acoustic activity of microbubbles, but with improved stability in vivo and a smaller size distribution for extravascular interrogation. One proposed alternative is the phase-change contrast agent. Phase-change contrast agents (PCCAs) consist of perfluorocarbons (PFCs) that are initially in liquid form, but can then be vaporized with acoustic energy. Crucial parameters for PCCAs include their sensitivity to acoustic energy, their size distribution, and their stability, and this manuscript provides insight into the custom design of PCCAs for balancing these parameters. Specifically, the relationship between size, thermal stability and sensitivity to ultrasound as a function of PFC boiling point and ambient temperature is illustrated. Emulsion stability and sensitivity can be ‘tuned’ by mixing PFCs in the gaseous state prior to condensation. Novel observations illustrate that stable droplets can be generated from PFCs with extremely low boiling points, such as octafluoropropane (b.p. −36.7 °C), which can be vaporized with acoustic parameters lower than previously observed. Results demonstrate the potential for low boiling point PFCs as a useful new class of compounds for activatable agents, which can be tailored to the desired application.
Keywords: Ultrasound; Nanoparticle; Fluorocarbon; Phospholipid; Thermally responsive material; Acoustically responsive material;

Gold nanomaterials conjugated with indocyanine green for dual-modality photodynamic and photothermal therapy by Wen-Shuo Kuo; Yi-Ting Chang; Keng-Chi Cho; Kuo-Chih Chiu; Chi-Hsiang Lien; Chen-Sheng Yeh; Shean-Jen Chen (3270-3278).
Light-exposure-mediated higher temperatures that markedly accelerate the degradation of indocyanine green (ICG) in aqueous solutions by thermal decomposition have been a serious medical problem. In this work, we present the example of using gold nanorods (Au NRs) and gold nanoparticles (Au NPs) simultaneously serving as photodynamic and photothermal agents to destroy malignant cells. Au NRs and Au NPs were successfully conjugated with hydrophilic photosensitizer, indocyanine green (ICG), to achieve photodynamic therapy (PDT) and photothermal therapy (PTT). We also demonstrated that Au NRs and Au NPs conjugated with ICG displayed high chemical stability and acted as a promising diagnostic probe. Moreover, the photochemical destruction ability would have a gradually increase depending on different sizes of Au NPs. Due to its stability even via higher temperatures mediated by laser irradiation, the combination of PTT and PDT proved to be efficiently killing cancer cells as compared to PTT or PDT treatment alone and enhanced the effectiveness of photodestruction and was demonstrated to enhance its photostability. As a result, the preparation of Au-based nanomaterials conjugated with ICG as well as their use in biomedical applications is valuable developments in multifunctional nanomaterials.
Keywords: Indocyanine green; Gold nanorod; Gold nanoparticles; Photodestruction; Photostability;

Alginate derivatization: A review of chemistry, properties and applications by Siddhesh N. Pawar; Kevin J. Edgar (3279-3305).
Alginates have become an extremely important family of polysaccharides because of their utility in preparing hydrogels at mild pH and temperature conditions, suitable for sensitive biomolecules like proteins and nucleic acids, and even for living cells such as islets of Langerhans. In addition, the complex monosaccharide sequences of alginates, and our growing ability to create controlled sequences by the action of isolated epimerases upon the alginate precursor poly(mannuronic acid), create remarkable opportunities for understanding the relationship of properties to sequence in natural alginates (control of monosaccharide sequence being perhaps the greatest synthetic challenge in polysaccharide chemistry). There is however a trend in recent years to create “value-added” alginates, by performing derivatization reactions on the polysaccharide backbone. For example, chemical derivatization may enable alginates to achieve enhanced hydroxyapatite (HAP) nucleation and growth, heparin-like anticoagulation properties, improved cell–surface interactions, degradability, or tuning of the hydrophobic-hydrophilic balance for optimum drug release. The creation of synthetic derivatives therefore has the potential to empower the next generation of applications for alginates. Herein we review progress towards controlled synthesis of alginate derivatives, and the properties and applications of these derivatives.
Keywords: Alginate; Bioartificial pancreas; Drug delivery; Drug release; Hydrogel; Polysaccharide;

Mechanistic study of transfection of chitosan/DNA complexes coated by anionic poly(γ-glutamic acid) by Zi-Xian Liao; Shu-Fen Peng; Yi-Cheng Ho; Fwu-Long Mi; Barnali Maiti; Hsing-Wen Sung (3306-3315).
Chitosan (CS) has been investigated as a non-viral carrier for gene delivery, but resulting in a relatively low transfection. To address this concern, we developed a ternary system comprised the core of CS/DNA complex and the outer coating of an anionic polymer, poly(γ-glutamic acid) (γ-PGA). In molecular dynamic (MD) simulations, we found that γ-PGA was entangle tightly with the excess CS emanating from the surface of test complexes, thus making them more compact. With γ-PGA coating, the extent of test complexes internalized and their transfection efficiency were evidently enhanced. Trypsin treatment induced a concentration-dependent decrease in internalization of the γ-PGA-coated complexes, suggesting a specific protein-mediated endocytosis. The endocytosis inhibition study indicates that the γ-glutamyl transpeptidase (GGT) present on cell membranes was responsible for the uptake of test complexes. The amine group in the N-terminal γ-glutamyl unit on γ-PGA played an essential role in the interaction with GGT. When entangled with CS, the free N-terminal γ-glutamyl unit of γ-PGA on test complexes was exposed and might thus be accommodated within the γ-glutamyl binding pocket of the membrane GGT. Above results suggest that the γ-PGA coating on CS/DNA complexes can significantly enhance their cellular uptake via a specific GGT-mediated pathway. Knowledge of the uptake mechanism is crucial for the development of an efficient vector for gene transfection.
Keywords: Gene transfection; γ-glutamyl transpeptidase; Receptor-mediated endocytosis; γ-glutamyl unit; Poly(γ-glutamic acid);

Despite viral vectors’ predominant use in clinical trials, due to higher gene delivery efficiency than nonviral counterparts, intrinsic immunogenicity and limited tunability for multi-modal effects are major concerns for their usage in gene therapy. An adeno-associated viral (AAV) particle was shielded with acid-degradable, siRNA-encapsulating polyketal (PK) shell, resulting in core–shell viral/nonviral chimeric nanoparticles (ChNPs). The AAV core of a ChNP is protected from immune responses by the PK shell which also facilitates the intracellular trafficking of the AAV core and efficiently releases the encapsulated siRNA into the cytoplasm. ChNPs led to significantly enhanced gene transduction, compared to unmodified free AAVs, and simultaneous silencing of a target gene, while avoiding inactivation by recognition from the immune system. Furthermore, conjugation of sialic acid (SA) on the surface of ChNPs enabled receptor-mediated targeted gene delivery to CD22-expressing cells. The ChNPs developed in this study combine the advantages of both viral and nonviral vectors and are a promising platform for targeted co-delivery of DNA and siRNA in inducing synergistic therapeutic effects by simultaneous expression and silencing of multiple genes.
Keywords: Adeno-associated virus; Combined viral/nonviral chimeric nanoparticle; Gene transfer; Simultaneous expression and silencing; siRNA; Stimuli-responsive delivery;

The targeted delivery of anticancer drugs to brain glioma by PEGylated oxidized multi-walled carbon nanotubes modified with angiopep-2 by Jinfeng Ren; Shun Shen; Dangge Wang; Zhangjie Xi; Liangran Guo; Zhiqing Pang; Yong Qian; Xiyang Sun; Xinguo Jiang (3324-3333).
In this study, a dual-targeting drug delivery system based on PEGylated oxidized multi-walled carbon nanotubes (O-MWNTs) modified with angiopep-2 (O-MWNTs-PEG-ANG) was successfully developed for treatment of brain glioma. O-MWNTs can not only distribute in brains but also accumulate in tumors, and have ultrahigh surface area with remarkably high loading anticancer drug of doxorubicin (DOX), which was selected as drug carrier. Angiopep-2 can specifically combine to the low-density lipoprotein receptor-related protein (LRP) receptor overexpressed on the blood–brain barrier (BBB) and glioma cells, which was selected as targeting ligand. The cooperative dual-targeting to brain glioma by O-MWNTs-PEG-ANG was evaluated by intracellular tracking in vitro and fluorescence imaging in vivo, which demonstrated that the combination of O-MWNTs-PEG and angiopep-2 constituted an ideal dual-targeting drug delivery system. The anti-glioma effect of DOX-loaded O-MWNTs-PEG-ANG (DOX-O-MWNTs-PEG-ANG) was assessed by C6 cytotoxicity and median survival time of glioma bearing mice, which showed a better anti-glioma effect than DOX. The biological safety of O-MWNTs-PEG-ANG was evaluated by BCEC and C6 cytotoxicity, hematology analysis and CD68 immunohistochemical analysis, which proved O-MWNTs-PEG-ANG was good biocompatibility and low toxicity. The biological safety of DOX-O-MWNTs-PEG-ANG was evaluated by histopathological analysis, which suggested a lower cardiac toxicity than DOX. In conclusion, O-MWNTs-PEG-ANG is a promising dual-targeting carrier to deliver DOX for the treatment of brain tumor.
Keywords: Dual-targeting; Carbon nanotubes; Angiopep; Doxorubicin; Blood–brain barrier; Brain glioma;

Translocation mechanisms of chemically functionalised carbon nanotubes across plasma membranes by Lara Lacerda; Julie Russier; Giorgia Pastorin; M. Antonia Herrero; Enrica Venturelli; Hélène Dumortier; Khuloud T. Al-Jamal; Maurizio Prato; Kostas Kostarelos; Alberto Bianco (3334-3343).
Understanding the mechanisms responsible for carbon nanotube (CNT) internalisation into live cells is considered critical both from a fundamental point of view and for further engineering of CNT-based delivery systems to intracellular targets. While several studies are focused on the development of such CNT-based delivery systems, attempts to systematically elucidate the cellular uptake mechanisms of CNTs are still rather limited. The aim of the present study is to evaluate the cellular internalisation of chemically functionalised multi-walled carbon nanotubes (f-MWCNTs) in the presence of different well-known cellular uptake inhibitors. Our data reveal how f-MWCNTs are able to translocate across cell membranes of both phagocytic and non-phagocytic cell lines. We have evidenced that at least 30–50% of f-MWCNTs are taken up by cells through an energy-independent mechanism. This characteristic makes nanotubes loaded with therapeutic or diagnostic cargos extremely interesting as the release of active molecules directly into the cytoplasm increase their biological activity and therapeutic efficacy.
Keywords: Nanomaterials; Carbon nanotubes; Cell uptake; Inhibitors; Phagocytosis; Endocytosis;

The design of protein-imprinted polymers as antibody substitutes for investigating protein–protein interactions by Junfei Gao; Huijuan Tian; Ying Wang; Qian Yang; Dejing Liu; Ying Wang; Huaifeng Mi (3344-3352).
Co-immunoprecipitation is a very effective method for studying protein–protein interactions. However, the preparation of antibodies in this method involves the injection of antigen into mammals, and requires the use of the expensive protein A-Sepharose 4B. Molecular imprinting polymer can compensate for these deficiencies. In this paper, a new strategy for studying protein interactions is reported; this method is based on the use of protein-imprinted polymers (PIPs). PIP is a proper substitute for antibody. We designed and synthesized assistant recognition polymer chains (ARPCs), which were limited length polymer chains with randomly distributed recognition and immobilizing sites. The template protein was selectively assembled with ARPCs. The assemblies were adsorbed by macroporous microspheres, and were immobilized by cross-linking polymerization. After removing the templates, the two kinds of synthesized PIPs were used to adsorb natural BiP or FKBP23 from ER extract; both showed high selectivity. Furthermore, we investigated the binding specificity of BiP to FKBP23, using synthesized PIPs. The results showed that FKBP23 could bind to BiP in ER in a process regulated by the concentration of Ca2+, which was consistent with the immunoprecipitation results. This strategy may provide a general solution for investigating protein interactions.
Keywords: Protein-imprinted polymer; Assistant recognition polymer chains; Immunoglobulin binding protein; FK506 binding protein 23; Concentration of Ca2+;

The mucoadhesive and gastroretentive properties of hydrophobin-coated porous silicon nanoparticle oral drug delivery systems by Mirkka P. Sarparanta; Luis M. Bimbo; Ermei M. Mäkilä; Jarno J. Salonen; Päivi H. Laaksonen; A.M. Kerttuli Helariutta; Markus B. Linder; Jouni T. Hirvonen; Timo J. Laaksonen; Hélder A. Santos; Anu J. Airaksinen (3353-3362).
Impediments to intestinal absorption, such as poor solubility and instability in the variable conditions of the gastrointestinal (GI) tract plague many of the current drugs restricting their oral bioavailability. Particulate drug delivery systems hold great promise in solving these problems, but their effectiveness might be limited by their often rapid transit through the GI tract. Here we describe a bioadhesive oral drug delivery system based on thermally-hydrocarbonized porous silicon (THCPSi) functionalized with a self-assembled amphiphilic protein coating consisting of a class II hydrophobin (HFBII) from Trichoderma reesei. The HFBII-THCPSi nanoparticles were found to be non-cytotoxic and mucoadhesive in AGS cells, prompting their use in a biodistribution study in rats after oral administration. The passage of HFBII-THCPSi nanoparticles in the rat GI tract was significantly slower than that of uncoated THCPSi, and the nanoparticles were retained in stomach by gastric mucoadhesion up to 3 h after administration. Upon entry to the small intestine, the mucoadhesive properties were lost, resulting in the rapid transit of the nanoparticles through the remainder of the GI tract. The gastroretentive drug delivery system with a dual function presented here is a viable alternative for improving drug bioavailability in the oral route.
Keywords: Silicon; Hydrophobin; Nanoparticle; Drug delivery; Adhesion; Biocompatibility;

Non-viral delivery of genes involved in stimulation of bone formation has been pursued for clinical bone repair, but no effort has been made to assess protein expression levels after in vivo delivery. This is critical to better understand gene delivery efficiencies and to compare different modes of non-viral delivery. This study investigated expression levels of basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) after delivering expression vectors (plasmid DNA) with polymeric carriers in a rat subcutaneous implant model. The polymers used were a 2 kDa molecular weight polyethylenimine modified with linoleic acid (PEI-LA) and the 25 kDa PEI (PEI25) used for non-viral gene delivery in animal models. The PEI-LA mediated delivery of the plasmid DNAs in 293T cells led to ∼3.5 and ∼13 ng/106 cells/day secretion of bFGF and BMP-2 in vitro, respectively. Using the reporter protein, Green Fluorescence Protein (GFP), transfection in implants was readily detected by the presence of GFP-positive cells and a polymeric carrier was needed for this GFP expression. No bFGF and BMP-2 were detected in the scaffolds due to high background in detection assays and/or rapid diffusion of the secreted proteins from the implant site. However, using an ex vivo culture system, significant levels of BMP-2, but not bFGF, secretion were observed from the scaffolds. The BMP-2 secretion from PEI-LA delivered expression vector was equivalent and/or superior to PEI25 depending on the plasmid DNA implant dose. Gelatin scaffolds were able to sustain ∼0.3 ng/sponge/day BMP-2 secretion as compared to collagen scaffolds (∼0.1 ng/sponge/day). These values were equivalent to secretion rates reported with some viral delivery systems from independent studies.
Keywords: In vivo gene transfer; Fibroblast growth factor; Bone morphogenetic protein; Recombinant protein; Gelatin; Gene expression;