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Biomaterials (v.32, #28)

Editorial board (pp. ifc).

The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins by Megan S. Lord; Bill Cheng; Simon J. McCarthy; MoonSun Jung; John M. Whitelock (pp. 6655-6662).
Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by αIIbβ3 integrins, while α2β1 integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of αIIbβ3 integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.► We investigated platelet adhesion to chitosan pre-adsorbed with plasma or extracellular matrix proteins. ► Platelet adhesion to chitosan was enhanced in the presence of adsorbed plasma and extracellular matrix proteins. ► Platelets bound to perlecan coated chitosan through integrin α2β1. ► Chitosan activated platelets, while the extent of activation was modulated by the presence of proteins.

Keywords: Chitosan; Platelets; Integrins; Protein adsorption


Spatio-temporal detachment of single cells using microarrayed transparent electrodes by Junji Fukuda; Yoshiaki Kameoka; Hiroaki Suzuki (pp. 6663-6669).
This paper describes a dynamic cell manipulation approach by which cells can be selectively detached from transparent indium tin oxide (ITO) electrodes via electrochemical desorption of a self-assembled monolayer (SAM) of alkanethiol. Changes in the surface properties of ITO following modification and electrical desorption of the SAM were characterized. By using these reactions on ITO, cells were readily attached and then detached from ITO electrodes in a very rapid manner, with greater than 90% of the cells being detached within 5 min. Furthermore, we fabricated micropatterns of ITO electrodes using photolithography. Cells on the micropatterned ITO electrodes could be detached with single-cell resolution. This approach could potentially lead to on-demand harvesting or elimination of one population from others under microscopy, for a wide range of purposes.

Keywords: Indium tin oxide; Electrochemistry; Gold-thiolate bond; Self-assembled monolayer; Cell detachment; Tissue engineering


The Goldilocks Surface by Erwin A. Vogler (pp. 6670-6675).
A minimum in the biological response to materials that is observed to occur within a narrow surface energy range is related to the properties of water at these biology-contacting surfaces. Wetting energetics are calculated using a published theory from which it is further estimated that water molecules bind to these special surfaces through a single hydrogen bond, leaving three other hydrogen bonds to interact with proximal water molecules. It is concluded that, at this Goldilocks Surface, the local chemical environment of surface-bound water is nearly identical to that experienced in bulk water; neither deprived of hydrogen bond opportunities, as it is in contact with a more hydrophobic surface, nor excessively hydrogen bonded to a more hydrophilic surface. A minimum in the biological response occurs because water vicinal (near) to the Goldilocks Surface is not chemically different than bulk water. A more precise definition of the relative terms hydrophobic and hydrophilic for use in biomaterials becomes evident from calculations: >1.3 kJ/mole-of-surface-sites is expended in wetting a hydrophilic surface whereas <1.3 kJ/mole-of-surface-sites is expended in wetting hydrophobic surfaces; hydrophilic surfaces wet with >1 hydrogen bond per water molecule whereas hydrophobic surfaces wet with <1 hydrogen bond per water molecule.

Keywords: Biological response; Biocompatible; Surface energy; Water wetting; Hydrophilic; Hydrophobic


Polyamidoamine dendrimer-conjugated quantum dots for efficient labeling of primary cultured mesenchymal stem cells by Yuriko Higuchi; Can Wu; Kai-Ling Chang; Kei Irie; Shigeru Kawakami; Fumiyoshi Yamashita; Mitsuru Hashida (pp. 6676-6682).
Monitoring of cells in vivo after transplantation could supply important information for determining the efficacy of stem cell therapy. The use of quantum dots (QDs) has several advantages for in vivo imaging, such as remarkable resistance to photo bleaching, high fluorescence efficiency, and size-tunable emission. After they are taken up by cells via endocytosis, QDs lose their fluorescence intensity in endosomes/lysosomes at low pH because the intensity cannot survive under acidic conditions. Moreover, the amount of QD uptake by mesenchymal stem cells (MSCs) is extremely small. Therefore, for effective labeling of MSCs and long observation of MSCs labeled by QDs in vivo, it is essential both to increase cellular uptake of QDs and to promote endosomal escape into the cytosol. The polyamidoamine (PAMAM) dendrimer had plenty of cationic charge, which promoted cellular uptake though electrostatic interactions, and a “buffering capacity,” which enhanced endosomal escape into the cytosol. In this study, QDs were modified with PAMAM dendrimer for the efficient labeling of MSCs by QDs. The uptake efficiency and cytosolic distribution of QDs in primary cultured MSCs were increased by the modification of the PAMAM dendrimer. The fluorescence intensity in MSCs labeled by PAMAM dendrimer-conjugated QDs lasted for a longer time in harvested culture plates or in cell-transplanted mice than that in MSCs labeled by non-conjugated QDs.

Keywords: Quantum dot; Stem cell; In vivo; imaging; PAMAM dendrimer; Endosomal escape; Buffering effect


The generation of iPS cells using non-viral magnetic nanoparticlebased transfection by Chang Hyun Lee; Jung-Hyun Kim; Hyun Joo Lee; Kilsoo Jeon; HyeJin Lim; Hye yeon Choi; Eung-Ryoung Lee; Seung Hwa Park; Jae-Yong Park; Sunghoi Hong; Soonhag Kim; Ssang-Goo Cho (pp. 6683-6691).
Induced pluripotent stem (iPS) cells have been generated from various somatic cells; however, a major restriction of the technology is the use of potentially harmful genome-integrating viral DNAs. Here, without a viral vector, we generated iPS cells from fibroblasts using a non-viral magnetic nanoparticle-based transfection method that employs biodegradable cationic polymer PEI-coated super paramagnetic nanoparticles (NP). Our findings support the possible use of transient expression of iPS genes in somatic cells by magnet-based nanofection for efficient generation of iPS cells. Results of dynamic light scattering (DLS) analysis and TEM analyses demonstrated efficient conjugation of NP with iPS genes. After transfection, nanofection-mediated iPS cells showed ES cell-like characteristics, including expression of endogenous pluripotency genes, differentiation of three germ layer lineages, and formation of teratomas. Our results demonstrate that magnet-based nanofection may provide a safe method for use in generation of virus-free and exogenous DNA-free iPS cells, which will be crucial for future clinical applications in the field of regenerative medicine.► We conduct a non-viral magnetic nanoparticle-based transfection to induce iPS from MEF. ► The magnetic nanoparticles are conjugated with 4 different iPS genes. ► The magnetic nanoparticle-based transfection of iPS genes provides safe and efficient generation of iPS.

Keywords: Induced pluripotent stem cells; Super paramagnetic nanoparticles; Non-viral transfection; Embryonic stem cells


Immune responses to implants – A review of the implications for the design of immunomodulatory biomaterials by Sandra Franz; Stefan Rammelt; Dieter Scharnweber; Jan C. Simon (pp. 6692-6709).
A key for long-term survival and function of biomaterials is that they do not elicit a detrimental immune response. As biomaterials can have profound impacts on the host immune response the concept emerged to design biomaterials that are able to trigger desired immunological outcomes and thus support the healing process. However, engineering such biomaterials requires an in-depth understanding of the host inflammatory and wound healing response to implanted materials.One focus of this review is to outline the up-to-date knowledge on immune responses to biomaterials. Understanding the complex interactions of host response and material implants reveals the need for and also the potential of “immunomodulating” biomaterials. Based on this knowledge, we discuss strategies of triggering appropriate immune responses by functional biomaterials and highlight recent approaches of biomaterials that mimic the physiological extracellular matrix and modify cellular immune responses.

Keywords: Immune response to biomaterials; Immunomodulation; Extracellular matrix; Biomaterial integrationAbbreviations; aECM; artificial ECM; BMP; bone morphogenetic protein; CCL; CC chemokine ligand; COX-2; cyclooxygenase-2; CS; chondroitin sulfate; CXCL; CXC chemokine ligand; DC; dendritic cells; DC-STAMP; dendritic cell-specific transmembrane protein; ECM; extracellular matrix; EGF; epidermal growth factor; ENA-78; epithelial cell-derived neutrophil attractant-78; ERK; extracellular signal-regulated kinases; FBGC; foreign body giant cells; FBR; foreign body response; FGF; fibroblast growth factor; GAGs; glycosaminoglycans; GM-CSF; granulocyte macrophage colony stimulating factor; HA; hyaluronic acid; HMW-HA; high molecular weight HA; IL; interleukin; IL-1ra; IL-1 receptor antagonist; IFNγ; interferon gamma; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MCP; monocyte chemotactic protein; MDC; macrophage-derived chemokine; MFI; mean fluorescence index; MHC-II; major histocompatibility complex class II molecules; MIP; macrophage inflammatory protein; MMPs; matrix metalloproteinases; NO; nitric oxide; NOS2; nitric oxide synthase 2; PAMPs; pathogen-associated molecular patterns; PDGF; platelet-derived growth factor; PEG; polyethylene glycol; PEO; poly(ethylene oxide); PGs; proteoglycans; PMN; polymorphonuclear leukocytes; PRR; pattern recognition receptors; RGD; arginine–glycine–aspartic acid; ROS; reactive oxygen species; TF; tissue factor; TGF-β; transforming growth factor beta; TH1; T helper 1 cells; TIMP; tissue inhibitor of metalloproteinases; TLR; toll-like receptors; TNFα; tumor-nekrose-faktor alpha; T; Reg; regulatory T lymphocytes; VEGF; vascular endothelial growth factor


Directed cell attachment by tropoelastin on masked plasma immersion ion implantation treated PTFE by Daniel V. Bax; David R. McKenzie; Marcela M.M. Bilek; Anthony S. Weiss (pp. 6710-6718).
The ability to generate cell patterns on polymer surfaces is critical for the detailed study of cellular biology, the fabrication of cell-based biosensors, cell separation techniques and for tissue engineering. In this study contact tape masking and steel shadow masks were used to exclude plasma immersion ion implantation (PIII) treatment from defined areas of polytetrafluoroethylene (PTFE) surfaces. This process enabled patterned covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. Tropoelastin coating rendered the untreated regions cell adhesive and the PIII-treated area non-adhesive, allowing very fine patterning of cell adhesion to PTFE surfaces. A blocking step, such as with BSA or PEG, was not required to prevent cell binding to the underlying PIII-treated regions as tropoelastin coating alone performed this blocking function. Although tropoelastin coated the entire PTFE surface, the cell binding C-terminus of tropoelastin was markedly less solvent exposed on the PIII-treated, hydrophilic regions. The differential exposure of the C-terminus correlated with the patterned distribution of tropoelastin-mediated cell adhesion. This new methodology specifically enables directed cell behavior on a polymer surface using a simple one-step treatment process, by modulating the adhesive activity of a single extracellular matrix protein.

Keywords: Cell patterning; Tropoelastin; PTFE; Plasma treatment; ECM protein


Selective immobilization of Sonic hedgehog on benzylguanine terminated patterned self-assembled monolayers by Chiu-Wai Kwok; Uwe Strähle; Yujie Zhao; Tim Scharnweber; Simone Weigel; Alexander Welle (pp. 6719-6728).
Patterned two-component, self-assembled monolayers on gold were produced by UV lithography. An oligo(ethylene glycol) terminated disulfide served as inert matrix reducing unspecific protein adsorption and cell adhesion. The second component of the self-assembled monolayer (SAM) presented a benzylguanine moiety for the immobilization of Sonic hedgehog (Shh) fused to a mutant O6-alkylguanine-DNA alkyltransferase (SNAP-tag™). The enzymatic activity of the SNAP-tag allows selective and covalent immobilization of the linked Shh. Time-of-flight secondary ion mass spectrometry verified the correct lateral distribution of the benzylguanine head groups in the patterned SAM. The quantification of unspecific and specific protein binding to mixed SAMs showed increased adsorption of albumin with increasing benzylguanine/(ethylene glycol) ratios. However, the immobilization of SNAP-tagged Shh was not blocked by pre-adsorbed albumin. Furthermore, the obtained micro-patterned substrates permitted direct immobilization of SNAP-tagged Shh even in the presence of many competing proteins from conditioned media of transfected HEK293 cells. Therefore, the presented system is suited for the controlled immobilization of fusion proteins from complex mixtures avoiding purification steps.

Keywords: Site-directed immobilization; Benzylguanine transferase tag; Sonic hedgehog; Micro-patterning; Self-assembled monolayers; Time-of-flight secondary ion mass spectrometry


Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering by Jelena Rnjak-Kovacina; Steven G. Wise; Zhe Li; Peter K.M. Maitz; Cara J. Young; Yiwei Wang; Anthony S. Weiss (pp. 6729-6736).
We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Young’s moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis.

Keywords: Synthetic human elastin; Tropoelastin; Dermal substitute; Electrospinning; Pore size; Porosity


The promotion of neural progenitor cells proliferation by aligned and randomly oriented collagen nanofibers through β1 integrin/MAPK signaling pathway by Yansong Wang; Meng Yao; Jihui Zhou; Wei Zheng; Changwei Zhou; Daming Dong; Yugang Liu; Zhaowei Teng; Yongqing Jiang; Guojun Wei; Xiaoying Cui (pp. 6737-6744).
In regenerative medicine, accumulating evidence demonstrates that the property of substrates monitors neural stem cells behavior. However, how stem cells sense and interpret biochemical and topographical cues remains elusive. This study aimed to explore the mechanism how nanofibrous scaffold modulated stem cells behavior. Spinal cord derived neural progenitor cells (NPCs) were cultured on electrospun aligned and randomly oriented collagen nanofibrous scaffolds. A 30% increase in proliferation and an elevation of BrdU incorporation were observed in NPCs on collagen nanofibers, compared to that on collagen-coated surface. In particular, NPCs expanded faster on aligned nanofibers in comparison with that on randomly oriented nanofibers. Moreover, an alteration in cell cycle progression with a reduced percentage of cells in G0/G1 phase and increased cell proliferation index (S phase plus G2/M phase) was also detected in NPCs cultured on collagen nanofibers. Incubating NPCs with anti-β1 integrin antibody or U1026 (an inhibitor of mitogen-activated protein kinase kinase, MEK) eliminated the altered cell cycle dynamics and BrdU incorporation induced by collagen nanofibers. In addition, cyclin D1 and cyclin dependent kinase 2 (CDK2), downstream genes of β1 integrin/mitogen-activated protein kinase (MAPK) pathway that control G1/S phase transition, were correspondingly regulated by nanofibers. Collectively, these data suggested that the property of substrate modulated NPCs proliferation by promoting cell cycle through β1 integrin/MAPK pathway. Our findings provide a better understanding of the interaction between NPCs and the substrate and therefore will pave way for regenerative medicine.

Keywords: Collagen; Nanofibers; Neural progenitor; Proliferation; Cell cycle; β1 integrin/MAPK


A phosphate-dependent shift in redox state of cerium oxide nanoparticles and its effects on catalytic properties by Sanjay Singh; Talib Dosani; Ajay S. Karakoti; Amit Kumar; Sudipta Seal; William T. Self (pp. 6745-6753).
Cerium oxide nanoparticles (CeNPs) have shown promise as catalytic antioxidants in cell culture and animal models as both superoxide dismutase and catalase mimetics. The reactivity of the cerium (Ce) atoms at the surface of its oxide particle is critical to such therapeutic properties, yet little is known about the potential for a protein or small molecule corona to form on these materials in vivo. Moreover Ce atoms in these active sites have the potential to interact with small molecule anions, peptides, or sugars when administered in culture or animal models. Several nanomaterials have been shown to alter or aggregate under these conditions, rendering them less useful for biomedical applications. In this work we have studied the change in catalytic properties of CeNPs when exposed to various biologically relevant conditions in vitro. We have found that CeNPs are resistant to broad changes in pH and also not altered by incubation in cell culture medium. However to our surprise phosphate anions significantly altered the characteristics of these nanomaterials and shifted the catalytic behavior due to the binding of phosphate anions to cerium. Given the abundance of phosphate in biological systems in an inorganic form, it is likely that the action of CeNPs as a catalyst may be strongly influenced by the local concentration of phosphate in the cells and/or tissues in which it has been introduced.

Keywords: Cerium oxide nanoparticles; Oxidative stress; Catalysis; Redox active nanomaterials; Reactive oxygen species


Hydrogel-based biomimetic environment for in vitro modulation of branching morphogenesis by Hiroyuki Miyajima; Takuya Matsumoto; Takayoshi Sakai; Satoshi Yamaguchi; Sang Hyun An; Makoto Abe; Satoshi Wakisaka; Kuen Yong Lee; Hiroshi Egusa; Satoshi Imazato (pp. 6754-6763).
The mechanical properties of the cellular microenvironment dramatically alter during tissue development and growth. Growing evidence suggests that physical microenvironments and mechanical stresses direct cell fate in developing tissue. However, how these physical cues affect the tissue morphogenesis remains a major unknown. We explain here that the physical properties of the cell and tissue microenvironment, biomimetically reproduced by using hydrogel, guide the tissue morphogenesis in the developmental submandibular gland (SMG). In particular, the softer gel enhances the bud expansion and cleft formation of SMG, whereas the stiffer gel attenuates them. These morphological changes in SMG tissue are led by soluble factors (FGF7/10) induction regulated by cell traction force derived from the tissue deformation. Our findings suggest that cells sense the mechanics of their surrounding environment and alter their properties for self-organization and the following tissue morphogenesis. Also, physically designed hydrogel material is a valuable tool for producing the biomimetic microenvironment to explore how physical cues affect tissue morphogenesis and to modulate tissue morphogenesis for in vitro tissue synthesis.

Keywords: Biomimetic material; Mechanical properties; Alginate; Salivary gland; Morphogenesis


Irreversible optical clearing of rabbit dermis for autogenic corneal stroma transplantation by Yuji Tanaka; Dong Shi; Akira Kubota; Yoshimasa Takano; Nobuo Fuse; Masayuki Yamato; Teruo Okano; Kohji Nishida (pp. 6764-6772).
Tissue engineering and transplantation of autogenic grafts have been widely investigated for solving problems on current allograft treatments (i.g., donor shortage and rejection). However, it is difficult to obtain an autogenic corneal stromal replacement that is composed of transparent, tough, and thick collagen constructs by current cell culture-based tissue engineering. Aim of this study is to develop transparent dermis for an autogenic corneal stroma transplantation. This study examined dehydration at 4–8°C and carbodiimide cross-linking on cloudy rabbit dermis (approx. 1.8%–3.8% light transmittance at 550nm) for dermis optical clearing. Transparency of dehydrated rabbit dermis was founded to be approx. 37.9%–41.4% at 550nm. Additional cross-linking treatment on dehydrated dermis prevented from swelling and clouding in saline, and improved its transparency to be 56.9% at 550nm. Rabbit corneal epithelium was found to regenerate on optically cleared dermis in vitro. Furthermore, no abnormal biological response (i.e., inflammation, vascularization, and the barrier defect of epithelia) or no optical functional change on optically cleared dermis was observed during its 4-week autogenic transplantation into rabbit corneal stromal pocket.

Keywords: Autogenic transplantation; Corneal stroma; Dermis; Optical clearing; Chemical cross-linking


The balance between adipogenesis and osteogenesis in bone regeneration by platelet-rich plasma for age-related osteoporosis by Hen-Yu Liu; Alexander T.H. Wu; Ching-Yu Tsai; Kuei-Ru Chou; Rong Zeng; Ming-Fu Wang; Wen-Chang Chang; Shiaw-Min Hwang; Ching-Hua Su; Win-Ping Deng (pp. 6773-6780).
The aim of this study was to develop a new diagnostic and therapeutic approach for the treatment of osteoporosis. Previously, we demonstrated that intraosseous transplantation of platelet-rich plasma (PRP) treated-osteoblast-like cells into ovariectomized senescence-accelerated mice (OVX-SAMP8) prevented the development of osteoporosis. In continuation, we aimed to explore the complex etiology of osteoporosis using this platform. An inverse relationship between bone marrow adipogenesis and osteogenesis has been suggested in the development of osteoporosis but the underlying mechanisms remain poorly described. To address these issues, we used PRP to inhibit adipocyte differentiation by promoting osteoblastic differentiation in adipocytes. In addition, a positive correlation between an increase in bone marrow adipocytes and bone loss was established. We assessed this relationship using an osteoporotic animal disease model which consisted of young (for prevention) and old (for treatment) OVX-SAMP8 mice. This animal model demonstrated that PRP treatment mainly exerted its action via promoting bone regeneration but also appeared to suppress adipogenesis within the marrow. The findings and methodology of this study could potentially be applied in the prevention and treatment of osteoporosis.

Keywords: Osteoporosis; Platelet-rich plasma (PRP); Ovariectomized senescence-accelerated mice (OVX-SAMP8); Adipocyte; Prevention and treatment


Tumor eradication in rat glioma and bypass of immunosuppressive barriers using internal radiation with188Re-lipid nanocapsules by Claire Vanpouille-Box; Franck Lacoeuille; Camille Belloche; Nicolas Lepareur; Laurent Lemaire; Jean-Jacques LeJeune; Jean-Pierre Benoît; Philippe Menei; Olivier F. Couturier; Emmanuel Garcion; François Hindré (pp. 6781-6790).
To date, glioblastoma treatments have only been palliative. In this context, locoregional drug delivery strategies, which allow for blood–brain barrier bypass and reduced systemic toxicity, are of major significance. Recent progress in nanotechnology has led to the development of colloidal carriers of radiopharmaceutics, such as lipid nanocapsules loaded with rhenium-188 (LNC188Re-SSS) that are implanted in the brain. In our study, we demonstrated that fractionated internal radiation using LNC188Re-SSS triggered remarkable survival responses in a rat orthotopic glioma model (cure rates of 83%). We also highlighted the importance of the radioactivity activity gradient obtained by combining a simple stereotactic injection (SI) with convection-enhanced delivery (CED).We assumed that the immune system played a role in the treatment’s efficacy on account of the overproduction of peripheral cytokines, recruitment of immune cells to the tumor site, and memory response in long-term survivor animals. Hence, nanovectorized internal radiation therapy with activity gradients stimulating immune responses may represent a new and interesting alternative for the treatment of solid tumors such as glioblastomas.

Keywords: Rat glioma model; Internal radiotherapy; Activity gradient; Lipid nanocapsules loaded with rhenium-188; Adaptative immune response


The influence of PEG chain length and targeting moiety on antibody-mediated delivery of nanoparticle vaccines to human dendritic cells by Luis J. Cruz; Paul J. Tacken; Remco Fokkink; Carl G. Figdor (pp. 6791-6803).
Targeted delivery of nanoparticles (NPs) carrying vaccine components to dendritic cells (DCs) is a promising strategy to initiate antigen-specific immune responses. Improving the interactions between nanoparticle-carried ligands and receptors on DCs is a major challenge. These NPs are generally coated with poly(ethylene glycol) (PEG), to shield non-specific interactions, and antibodies, to facilitate specific delivery to DC surface receptors. We have devised a strategy to covalently link PEG molecules of various chain length (Mw 2000–20000 g/moL) to poly(lactic-co-)glycolic acid (PLGA) NP vaccines. We coated these NPs with various antibodies recognizing the DC-specific receptor DC-SIGN to study the effects of shielding and antibody type on antibody–receptor interactions. Chemical attachment of PEG to the particle surface was followed by detailed zeta potential, DLS and NMR studies, and analyzed by analytical chemistry. Increasing the PEG chain length increased particle size and polydispersity index and reduced the intracellular degradation rate of encapsulated antigens. Binding and uptake of NPs by human DCs was affected by both PEG chain length and antibody type. NPs coated with PEG-3000 had the optimal chain length for antibody–receptor interactions and induction of antigen-specific T-cell responses. Interestingly, clear differences were observed upon targeting distinct epitopes of the same receptor. Binding and uptake of NPs carrying antibodies recognizing the carbohydrate recognition domain of DC-SIGN was enhanced when compared to those carrying antibodies recognizing the receptor’s neck region. In conclusion, our data show that PEG chains cannot be extended beyond a certain length for shielding purposes without compromising the efficacy of targeted delivery. Thereby, the implications of our findings are not limited to the future design of nanovaccines specifically targeted to DC-SIGN, but apply to the general design of targeted nanocarriers.

Keywords: Delivery vehicle; Nanoparticle; PEG lengths; Targeting; Specific ligands; PLGAAbbreviations; CRD; carbohydrate-recognition domain; DC; dendritic cell; DC-SIGN; Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; DIEA; N,N-diisopropylethylamine; DLS; dynamic light scattering; DMF; N,N-dimethylformamide; DCM; dichloromethane; FITC; fluorescein isothiocyanate; FDA; Food and Drug Administration; HOBt; 1-hydroxybenzotriazole; HPLC; high pressure liquid chromatography; NP; nanoparticle; PBL; peripheral blood lymphocyte; PBMC; peripheral blood mononuclear cell; PEG; polyethylene glycol; PVA; polyvinyl alcohol; TT; tetanus toxoid; EDAC; N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; NHS; (N-hydroxisuccinimide); SMCC; sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate; SATP; (N-hydroxysuccinimide (NHS) esters of S-acetylthioacetic and propionic acid)


Modulation of the immune-related gene responses to protect mice against Japanese encephalitis virus using the antimicrobial peptide, tilapia hepcidin 1-5 by Han-Ning Huang; Venugopal Rajanbabu; Chieh-Yu Pan; Yi-Lin Chan; Cho-Fat Hui; Jyh-Yih Chen; Chang-Jer Wu (pp. 6804-6814).
Japanese encephalitis virus (JEV), a neurotropic flavivirus, is one of the major causes of acute encephalitis in humans. After infection, it is commonly associated with inflammatory reactions and neurological disease. There is still no effective antiviral drug available against Japanese encephalitis virus infection. Recently, a number of investigators found that antimicrobial peptide (AMPs) present a broad range of biological activities including antimicrobial and immunomodulatory activities. In this study, we found that an AMP, tilapia hepcidin (TH)1-5, caused no harm to either cells or test animals during the test course and could control JEV viral infection in BHK-21 cells. Mice co-injected with TH1-5/JEV and subsequently subjected to JEV re-challenge survived and behaved normally. The neuroprotective effects were associated with marked decreases in: (i) the viral load and viral replication within the brain, (ii) neuronal death, and (iii) secondary inflammation resulting from microglial activation. TH1-5 was also determined to enhance adaptive immunity by elevating levels of anti-JEV-neutralizing antibodies in the serum. The microarray data also showed that TH1-5 modulated Socs-6, interleukin (IL)-6, Toll-like receptor (TLR)-1, TLR-7, caspase-4, interferon (IFN)-β1, ATF-3, and several immune-responsive genes to protect mice against JEV infection. In addition, TH1-5 was confirmed to modulate the expressions of several proinflammatory and immune-responsive genes, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, tumor necrosis factor (TNF)-α, IFN-γ and monocyte chemoattractant protein (MCP)-1 at both the transcriptional and translational levels in JEV-infected mice. In conclusion, our findings provide mechanistic insights into the actions of TH1-5 against JEV. Results from our in vivo and in vitro experiments clearly indicate that TH1-5 has antiviral, neuroprotective, anti-inflammatory, and immunomodulatory activities. Furthermore, TH1-5 successfully reduced the severity of disease induced by JEV. Our results point out that TH1-5 is a promising candidate for further development as an antiviral agent against JEV infection.

Keywords: Tilapia hepcidin 1-5; Antimicrobial peptides; Japanese encephalitis virus; Antiviral agent; Proinflammatory genes


Activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants by Latrisha K. Petersen; Amanda E. Ramer-Tait; Scott R. Broderick; Chang-Sun Kong; Bret D. Ulery; Krishna Rajan; Michael J. Wannemuehler; Balaji Narasimhan (pp. 6815-6822).
Techniques in materials design, immunophenotyping, and informatics can be valuable tools for using a molecular based approach to design vaccine adjuvants capable of inducing protective immunity that mimics a natural infection but without the toxic side effects. This work describes the molecular design of amphiphilic polyanhydride nanoparticles that activate antigen presenting cells in a pathogen-mimicking manner. Biodegradable polyanhydrides are well suited as vaccine delivery vehicles due to their adjuvant-like ability to: 1) enhance the immune response, 2) preserve protein structure, and 3) control protein release. The results of these studies indicate that amphiphilic nanoparticles possess pathogen-mimicking properties as evidenced by their ability to activate dendritic cells similarly to LPS. Specific molecular descriptors responsible for this behavior were identified using informatics analyses, including the number of backbone oxygen moieties, percent of hydroxyl end groups, polymer hydrophobicity, and number of alkyl ethers. Additional findings from this work suggest that the molecular characteristics mediating APC activation are not limited to hydrophobicity but vary in complexity (e.g., presentation of oxygen-rich molecular patterns to cells) and elicit unique patterns of cellular activation. The approach outlined herein demonstrates the ability to rationally design pathogen-mimicking nanoparticle adjuvants for use in next-generation vaccines against emerging and re-emerging diseases.

Keywords: Combinatorial; High throughput; Polyanhydrides; Vaccine adjuvants; Nanoparticles


Infusion pressure and pain during microneedle injection into skin of human subjects by Jyoti Gupta; Sohyun S. Park; Brian Bondy; Eric I. Felner; Mark R. Prausnitz (pp. 6823-6831).
Infusion into skin using hollow microneedles offers an attractive alternative to hypodermic needle injections. However, the fluid mechanics and pain associated with injection into skin using a microneedle have not been studied in detail before. Here, we report on the effect of microneedle insertion depth into skin, partial needle retraction, fluid infusion flow rate and the co-administration of hyaluronidase on infusion pressure during microneedle-based saline infusion, as well as on associated pain in human subjects. Infusion of up to a few hundred microliters of fluid required pressures of a few hundred mmHg, caused little to no pain, and showed weak dependence on infusion parameters. Infusion of larger volumes up to 1 mL required pressures up to a few thousand mmHg, but still usually caused little pain. In general, injection of larger volumes of fluid required larger pressures and application of larger pressures caused more pain, although other experimental parameters also played a significant role. Among the intradermal microneedle groups, microneedle length had little effect; microneedle retraction lowered infusion pressure but increased pain; lower flow rate reduced infusion pressure and kept pain low; and use of hyaluronidase also lowered infusion pressure and kept pain low. We conclude that microneedles offer a simple method to infuse fluid into the skin that can be carried out with little to no pain.

Keywords: Flow conductivity; Hollow microneedle; Infusion pressure; Intradermal injection; Pain; Saline


Dual targeting effect of Angiopep-2-modified, DNA-loaded nanoparticles for glioma by Shixian Huang; Jianfeng Li; Liang Han; Shuhuan Liu; Haojun Ma; Rongqin Huang; Chen Jiang (pp. 6832-6838).
Gene therapy offers a promising cure of brain glioma and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce cell apoptosis of glioma selectively without affecting the normal cells. In this study, the nanoscopic high-branching dendrimer, polyamidoamine (PAMAM), was selected as the principal vector. Angiopep-2, which can target to the low-density lipoprotein receptor-related protein-1 (LRP1) expressed on BCECs and glial cells, was exploited as the targeting ligand to conjugate PAMAM via bifunctional polyethyleneglycol (PEG) and then complexed with the DNA, designated as PAMAM-PEG-Angiopep/DNA nanoparticles (NPs). The cellular uptake mechanism explored in glial cells showed that the DNA of PAMAM-PEG-Angiopep/DNA NPs entered into the nuclei through the endosome/lysosome pathway. The in vivo biodistribution of PAMAM-PEG-Angiopep/DNA NPs in the brain especially the tumor site was higher than that of PAMAM-PEG/DNA NPs and PAMAM/DNA NPs. Furthermore, the TUNEL analysis showed a more wide-extended apoptosis in the PAMAM-PEG-Angiopep/pORF-TRAIL NPs treated group, compared to other groups including commercial Temozolomide-treated one. The median survival time of PAMAM-PEG-Angiopep/pORF-TRAIL NPs and Temozolomide treated on brain tumor-bearing mice was 61 and 49 days respectively, significantly longer than that of other groups. Besides, the NPs suggested low cytotoxicity after in vitro transfection. Thus, the results showed that Angiopep-2 could be exploited as a specific ligand to cross the BBB and targeted to glial cells, and PAMAM-PEG-Angiopep/DNA NPs can be a potential non-viral delivery system for gene therapy of glial tumor.

Keywords: Angiopep; Brain-targeting; Glioma-targeting; Gene therapy; pORF-TRAIL; Polyamidoamine


Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides by Boonchoy Soontornworajit; Jing Zhou; Matthew P. Snipes; Mark R. Battig; Yong Wang (pp. 6839-6849).
Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer–protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer–protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.► We studied the effects of molecular hybridization on protein-aptamer dissociation. ► Both oligonucleotide tail and hybridizing length are important factors. ► Importantly, pegylated complementary oligonucleotides can trigger protein release. ► In addition, the triggered release can be achieved at multiple time points.

Keywords: Hydrogel; Affinity; Growth factor; Drug delivery; Controlled drug release


A strategy to establish a gene-activated matrix on titanium using gene vectors protected in a polylactide coating by Andreas Kolk; Cornelia Haczek; Christian Koch; Stephan Vogt; Martin Kullmer; Christoph Pautke; Herbert Deppe; Christian Plank (pp. 6850-6859).
Bioactive implants are promising tools in regenerative medicine. Here we describe a versatile procedure for preparing a gene-activated matrix on titanium. Lyophilized copolymer-protected gene vectors ( COPROGs) suspended in poly(d,l-lactide) (PDLLA) solutions in ethyl acetate were used to varnish solid surfaces. The gene-activated PDLLA surfaces were first established on polypropylene 96-well plates. Vector release from these surfaces in aqueous buffer, cell viability and gene transfer efficiency to NIH 3T3 fibroblasts was strongly dependent on the vector dose and its ratio to PDLLA film thickness. A detailed analysis of these relationships allowed establishing correlations which can be used to calculate suitable combinations of COPROGs and PDLLA yielding optimal gene transfer efficiency. This was verified with COPROG-activated PDLLA coatings on titanium foils. HEK 293 and mesenchymal stem cells expressed the BMP-2 gene comprised in the gene-activated surface in a manner that was consistent with the predicted dose–response and toxicity profiles found in NIH 3T3 cells. The systematic procedure presented here for identifying optimal coating compositions can be applied to any combination of vector type and coating material.

Keywords: Nonviral gene transfer; Protective copolymer; Gene-activated matrix; Titanium; Polylactic acid; Stem cell


Oral bioavailability, therapeutic efficacy and reactive oxygen species scavenging properties of coenzyme Q10-loaded polymeric nanoparticles by Nitin K. Swarnakar; Amit K. Jain; Raman P. Singh; Chandraiah Godugu; Manasmita Das; Sanyog Jain (pp. 6860-6874).
The present investigation consists in the development and characterization of CoQ10 loaded PLGA nanoparticles (CoQ10-NPs, size < 100 nm) by a scalable emulsion-diffusion-evaporation method. Thermal and crystallinity analysis collectively corroborated that CoQ10 was entrapped into the NPs in amorphous form. The lyophilized CoQ10-NPs were found to be stable for a period of 6 months (at room temperature). In vitro cell culture studies indicated that CoQ10-NPs significantly quenched ROS with nearly 10 fold higher efficacy than free CoQ10. Further, positively charged CoQ10-NPs were localized in two major sources of ROS generation: mitochondria and lysosomes. CoQ10-NPs showed improved oral bioavailability (4.28 times) as compared to free CoQ10. Finally remarkably higher hepatoprotective and anti-inflammatory activity of CoQ10-NPs as compared to free CoQ10 was observed due to mitigation of deleterious effects associated with the generation of free radicals. As elucidated by live noninvasive animal imaging, the higher anti-inflammatory activity of CoQ10-NPs can be attributed to significant accumulation of these NPs in the inflamed tissues.

Keywords: PLGA nanoparticles; CoQ10; Oxidative stress; Hepatoprotective activity; Anti-inflammatory activity; Mitochondria and lysosomes localization

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