Biomaterials (v.31, #26)

Examination of cell–host–biomaterial interactions via high-throughput technologies: A re-appraisal by Karen A. Power; Kathleen T. Fitzgerald; William M. Gallagher (6667-6674).
Biomaterials are required to act harmoniously when exposed to the body or bodily fluids. Investigating cellular or in vivo phenotypic responses and protein adsorption to the material surface helps to determine the associated biocompatibility. Past limitations on progress in this field include time-consuming cell-based screening tools and a limited understanding of the complex nature of cell–biomaterial interactions. While high-throughput technologies by their nature are a rapid tool to derive meaning from multifaceted systems and, in recent years, the biomaterial community is beginning to take advantage of these technologies, the key observation in this Leading Opinion Paper is that the biomaterials community has been slow to accept these methods as an addition to their traditional experimentation workflow. The purpose of this paper is to review the definition and recent usage of high-throughput experiments in order to examine biomaterial interactions at the cellular and wider host level, especially as they become more relevant within the biomaterials arena encapsulating tissue engineering, gene, drug and stem cell delivery systems. The technologies under focus include rapid cell-based screening, transcriptomics and proteomics.
Keywords: Biocompatibility; Transcriptomics; DNA microarrays; Proteomics; Mass spectrometry; Cell interaction;

Biodegradable poly(ethylene glycol) hydrogels based on a self-elimination degradation mechanism by Manjeet Deshmukh; Yashveer Singh; Simi Gunaseelan; Dayuan Gao; Stanley Stein; Patrick J. Sinko (6675-6684).
Two vinyl sulfone functionalized crosslinkers were developed for the purpose of preparing degradable poly(ethylene glycol) (PEG) hydrogels (EMXL and GABA-EMXL hydrogels). A self-elimination degradation mechanism in which an N-terminal residue of a glutamine is converted to pyroglutamic acid with subsequent release of diamino PEG (DAP) is proposed. The hydrogels were formed via Michael addition by mixing degradable or nondegradable crosslinkers and copolymer {4% w/v; poly[PEG-alt-poly(mercapto-succinic acid)]} at room temperature in phosphate buffer (PB, pH = 7.4). Hydrogel degradation was characterized by assessing diamino PEG release and examining morphological changes as well as the swelling and weight loss ratio under physiological conditions (37 °C). Degradation of EMXL and GABA-EMXL hydrogels occurred by surface erosion (confirmed by SEM). GABA-EMXL degradation was significantly faster (∼3-fold) than EMXL; however, the degradation of both hydrogels in mouse plasma was 12-times slower than in PBS. The slower degradation rate in plasma as compared to buffer is consistent with the presence of γ-glutamyltransferase, γ-glutamylcyclotransferase and/or glutaminyl cyclase (QC), which have been shown to suppress pyroglutamic acid formation. The current studies suggest that EMXL and GABA-EMXL hydrogels may have biomedical applications where 1–2 week degradation timeframes are optimal.
Keywords: Degradable crosslinkers; Hydrogel; Michael addition; Self-elimination mechanism;

Hindered amine light stabilizers: An alternative for radiation cross-linked UHMwPE implants by Pieter Gijsman; Harold J. Smelt; Detlef Schumann (6685-6691).
To reduce wear UHMwPE implants are commonly cross-linked by use of gamma or e-beam irradiation. After irradiation however, radicals are still present that may cause oxidative degradation of the polymer. A way to reduce oxidative degradation could be to add a stabilizer to the polymer. Currently Vitamin E is the state of the art in stabilizing irradiation cross-linked UHMwPE implants. However, this stabilizer has some drawbacks. It interferes with the cross-link chemistry, leading to a lower cross-link density, it will be consumed and it results in the discolouration of the UHMwPE compound due to Vitamin E conversion products. It has been shown that all these drawbacks can be overcome by using HALS stabilizers. Due to their different mechanism of action, HALS stabilizers do not interfere with the cross-link chemistry. As part of the stabilization mechanism the radical scavenging molecules are actually regenerated, which allows for using a lower total concentration. The HALS conversion products do not result in discoloration of the polymer compound. Although their biocompatibilities still have to be evaluated, HALS stabilizers are potential alternative stabilizers for UHMwPE implants.
Keywords: Ageing; Degradation; Cross-link density; Hindered amine light stabilizers;

The contribution of the capillary endothelium to blood clearance and tissue deposition of anionic quantum dots in vivo by Marc Praetner; Markus Rehberg; Peter Bihari; Max Lerchenberger; Bernd Uhl; Martin Holzer; Martin E. Eichhorn; Robert Fürst; Tamara Perisic; Christoph A. Reichel; Ulrich Welsch; Fritz Krombach (6692-6700).
The increasing interest in biomedical applications of semiconductor quantum dots (QDs) is closely linked to the use of surface modifications to target specific sites of the body. The immense surface area of vascular endothelium is a possible interaction platform with systemically administered QDs. Therefore, the aim of this study was to investigate the microvascular distribution of neutral, cationic, and anionic QDs in vivo. QDs with carboxyl-, amine- and polyethylene glycol surface coatings were injected into the blood circulation of mice. In vivo microscopy of the cremaster muscle, two-photon microscopy of skeletal and heart muscle, as well as quantitative fluorescence measurements of blood, excreta, and tissue samples were performed. Transmission electron microscopy was used to detect QDs at the cellular level. The in vitro association of QDs with cultured endothelial cells was investigated by flow cytometry and confocal microscopy. Anionic QDs exhibited a very low residence time in the blood stream, preferably accumulated in organs with a prominent mononuclear phagocytic component, but were also found in other tissues with low phagocytic properties where they were predominantly associated with capillary endothelium. This deposition behavior was identified as a new, phagocyte-independent principle contributing to the rapid clearance of anionic QDs from the circulation.
Keywords: Quantum dots; Nanoparticles; Surface modifications; Blood kinetics; In vivo microscopy; Microcirculation;

Regulation of ciliary differentiation of human respiratory epithelial cells by the receptor for hyaluronan-mediated motility on hyaluronan-based biomaterials by Tsung-Wei Huang; Po-Wen Cheng; Yen-Hui Chan; Te-Huei Yeh; Yi-Ho Young; Tai-Horng Young (6701-6709).
Selecting a scaffold that facilitates ciliary differentiation of respiratory epithelial cells (RECs) is crucial in developing tissue engineered respiratory epithelium. Hyaluronan derivative membranes, consisting of an esterified form of hyaluronan (HYAFF®), have been proved to promote ciliary differentiation of RECs in the presence of retinoic acid (RA). However, the regulatory mechanism of ciliary differentiation-promoting effect of hyaluronan-based biomaterials remains unknown. In addition to investigating the ciliary differentiation of RECs on HYAFF with/without RA compared with that on collagen with/without RA, this study elucidates the role of the receptor for hyaluronan-mediated motility (RHAMM) in promoting ciliary differentiation of RECs. Analytical results of culturing RECs on collagen and HYAFF indicate that only HYAFF can increase the ciliary differentiation of RECs under RA-free conditions. The expression level of RHAMM mRNA of RECs more significantly decreases on collagen than that on HYAFF without RA. Therefore, by using lentiviral vector-based short hairpin RNA targeting RHAMM, the study further reveals that knockdown of RHAMM obviously inhibits the ciliary differentiation of RECs on collagen with RA and on HYAFF with/without RA. In addition to demonstrating that hyaluronan-based biomaterials partially “replace” RA in the ciliary differentiation of RECs, which is regulated by RHAMM, this study establishes that RHAMM regulates the ciliary differentiation-promoting effect of RA on RECs.
Keywords: Respiratory epithelial cells (RECs); HYAFF; Ciliogenesis; Retinoic acid (RA); Receptor for hyaluronan-mediated motility (RHAMM);

The induction of thrombus generation on nanostructured neutral polymer brush surfaces by Benjamin F.L. Lai; A. Louise Creagh; Johan Janzen; Charles A. Haynes; Donald E. Brooks; Jayachandran N. Kizhakkedathu (6710-6718).
Surface induced thrombus generation is a major clinical concern associated with vascular medical devices and implants. Here, we show that high graft density hydrophilic non-charged poly (N,N-dimethylacrylamide) (PDMA) brushes prevent the initiation of blood coagulation on synthetic surfaces. Using a multi-faceted analysis approach, we have identified that PDMA brushes greater than 0.27 chains/nm2 graft density showed this highly desired property. Non-specific protein adsorption is greatly reduced on high density brushes compared to bare surface as evident from isothermal titration calorimetry, gel electrophoresis, and proteomic analyses. We have identified approximately 129 proteins of various types on bare and PDMA brush coated surfaces at a range of surface concentrations. Thromboelastography, platelet activation, and aggregation analyses show that only high graft density brushes are neutral to blood coagulation. Since the polymer brush synthesis can be adapted to most currently used biomedical materials, these results have significant impact in the design of highly hemocompatible surfaces.
Keywords: Polymer brushes; Blood-biomaterial interaction; Isothermal titration calorimetry; Proteomics; Blood coagulation; Platelet adhesion;

Regeneration of long-tract axons through sites of spinal cord injury using templated agarose scaffolds by Thomas Gros; Jeff S. Sakamoto; Armin Blesch; Leif A. Havton; Mark H. Tuszynski (6719-6729).
Previously we reported that templated agarose scaffolds can orient and guide local spinal cord axons after injury. In the present study we examined whether growth of long-projecting spinal cord axons could also be promoted into, and then beyond, templated agarose scaffolds placed into a spinal cord lesion site. Ascending spinal cord dorsal column sensory axons were transected at the C4 level. Animals were then subjected to combinatorial therapies consisting of: 1) templated agarose scaffolds implanted into the lesion site, seeded with autologous bone marrow stromal cells expressing a growth factor, neurotrophin-3 (NT-3), 2) lentiviral vectors expressing NT-3 beyond the lesion site (to promote axonal emergence from the scaffold along chemotropic gradients of growth factors), and 3) priming lesions (“conditioning lesions”) of the sensory neuronal cell body to stimulate the endogenous growth state of the injured neuron. Control groups received either non-organized, NT-3-expressing cell suspension grafts in the lesion site, or templated scaffolds plus one of the two components of the combination therapy. Among groups that received templated agarose scaffolds, long-tract sensory axonal regeneration occurred into the spinal cord lesion site, and the growth of these axons was remarkably organized and linear compared to non-organized cell suspension grafts. Axonal penetration was maximal in subjects that received combination therapies; further, 83 + 13% of axons entering the scaffolds in combination-treated subjects continued to grow the full length of the lesion cavity to reach the distal aspect of the scaffold, over a 2 mm distance. In contrast, axons regenerating into cell suspension grafts lacking guidance scaffolds exhibited a parabolic decay of growth as a function of distance, and only 22 + 6% of axons extended the length of the lesion cavity. Moreover, axonal regeneration beyond the lesion site occurred only among subjects that received full combinatorial treatments (p < 0.05). However, axon growth beyond the scaffold was constrained to a reactive cell layer that formed between the distal aspect of the scaffold and host tissue, and did not continue further to re-penetrate the host spinal cord. Thus, templated agarose scaffolds substantially enhance the organization and distance over which long-tract axons extend through a spinal cord lesion site in the presence of combinatorial therapies, but host-scaffold reactive interfaces limit axon re-penetration of the host. Further development must reduce reactive cellular interfaces to support effective axonal penetration of host parenchyma.
Keywords: Guidance channel; Spinal cord injury; Neural interface; Growth factors; Templating; Axon guidance;

The retention of extracellular matrix proteins and angiogenic and mitogenic cytokines in a decellularized porcine dermis by David M. Hoganson; Elisabeth M. O’Doherty; Gwen E. Owens; Dina O. Harilal; Scott M. Goldman; Chris M. Bowley; Craig M. Neville; Russell T. Kronengold; Joseph P. Vacanti (6730-6737).
Decellularized dermis materials demonstrate considerable utility in surgical procedures including hernia repair and breast reconstruction. A new decellularized porcine dermis material has been developed that retains many native extracellular matrix (ECM) proteins and cytokines. This material has substantial mechanical strength with maximum tensile strength of 141.7 ± 85.4 (N/cm) and suture pull through strength of 47.0 ± 14.0 (N). After processing, many ECM proteins remained in the material including collagen III, collagen IV, collagen VII, laminin and fibronectin. Glycosaminoglycans, including hyaluronic acid, were also preserved. Among several cytokines whose levels were quantified, more vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) were retained within this material than in comparable decellularized dermis materials. The retention of bioactivity was demonstrated in a cell culture assay. Because this decellularized porcine dermis material both retains significant strength and has substantial biological activity, it may promote rapid integration and repair in clinical applications.
Keywords: ECM; Cytokine; Surgical mesh; Cell culture;

Bioengineering endothelialized neo-corneas using donor-derived corneal endothelial cells and decellularized corneal stroma by Jin San Choi; James K. Williams; Margaret Greven; Keith A. Walter; Patrick W. Laber; Gilson Khang; Shay Soker (6738-6745).
Corneal transplantation is a common transplant procedure performed to improve visual acuity by replacing the opaque or distorted host tissue by clear healthy donor tissue. However, its clinical utility is limited due to a lack of high quality donor corneas. Bioengineered neo-corneas, created using an expandable population of human donor-derived corneal endothelial cells (HCEC), could address this current shortage. The objectives of this study were to establish HCEC isolation and culture protocols and to investigate the feasibility of bioengineering corneal tissue constructs by seeding the cells on decellularized human corneal stroma. HCECs were removed from the discarded corneas of eye donors by enzymatic digestion. Cells were expanded and evaluated for their expression of Na+/K+-ATPase and zona occludens-1 (ZO-1). Donor corneal stromas were cut to 120–200 μm thickness slices using a microtome and then decellularized. Extracellular matrix components and mechanical properties of the scaffolds were measured after decellularization. To engineer neo-corneas, 130 HCEC/mm2 were seeded on decellularized human corneal stromas. The resulting constructs were placed in growth medium for 14 days and then analyzed using scanning electron microscopy (SEM), histology, and immunocytochemistry. Seeded cells retain expression of the functional markers Na+/K+-ATPase and ZO-1 and constructs have biomechanical properties similar to those of normal corneas. These results indicate that construction of neo-corneas, using HCECs derived from discarded donor corneas and decellularized thin-layer corneal stromas, may create a new source of high quality corneal tissue for transplantation.
Keywords: Cornea; Corneal endothelial cells; Corneal stroma; Decellularization; Tissue engineering;

Human bone marrow-derived mesenchymal stem cells (hMSCs) are promising cell candidates for cartilage regeneration. Building the appropriate microenvironment for cell differentiation in response to exogenous stimuli is a critical step towards the clinical utilization of hMSCs. In this study, the effects of RGD peptide immobilization onto macro-porous alginate scaffolds on TGF-β1-induced hMSC chondrogenesis were evaluated. The results revealed different cell morphology, viability and proliferation extent in the RGD-immobilized vs. un-modified scaffolds. The TGF-β1-induced activation of both Smad-dependent (SMAD2) and Smad-independent (ERK1/2) signaling pathways was stronger and persisted for over 3 weeks in the RGD-immobilized scaffolds, indicating greater accessibility of the cells to the inducer. By contrast, in the un-modified alginate scaffolds, the cells aggregated into compacted clusters resulting in lesser effects of TGF-β1. The efficient and prolonged exposure to the chondrogenic inducer in the RGD-modified scaffolds ensured the appropriate progression of MSC differentiation from the initial phase of cell condensation until the appearance of committed chondrocytes, at 3 weeks of cultivation. Taken together, our results highlight the fundamental importance of the microenvironment design of the scaffold as well as the presentation of the inductive cue for inducing efficient stem-cell controlled differentiation.
Keywords: Alginate scaffold; Chondrogenesis; Human mesenchymal stem cells; RGD peptide;

Effect of media mixing on ECM assembly and mechanical properties of anatomically-shaped tissue engineered meniscus by Jeffrey J. Ballyns; Timothy M. Wright; Lawrence J. Bonassar (6756-6763).
This study investigated the hypothesis that controlled media mixing will enhance tissue formation and increase mechanical properties of anatomically-shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were seeded in 2% w/v alginate, cross-linked with 0.02 g/mL CaSO4, and injected into molds of menisci. Engineered menisci were incubated for up to 6 weeks. A mixing media bioreactor was designed to ensure proper mixing of culture medium while protecting constructs from the spinning impeller. Impeller speeds were calibrated to produce Reynolds number (Re) of 0.5, 2.9, 5.8, 10.2, and 21.8. Constructs were divided a tested in confined compression and in tension to determine the equilibrium and tensile moduli, respectively. Media stimulation resulted in a 2–5 fold increase in mechanical properties and a 2–3 fold increase in matrix accumulation in constructs over 6 weeks in culture. Benefits from mixing stimulation for collagen accumulation and compressive modulus appeared to peak near Re 2.9, and decreased with increased mixing intensity. This study suggests that fluid mixing can be optimized to enhance mechanical properties of anatomically-shaped engineered constructs.
Keywords: Tissue engineering; Meniscus; Alginate; Media mixing stimulation;

Preparation of Caco-2 cell sheets using plasma polymerised acrylic acid as a weak boundary layer by Ruby Majani; Mischa Zelzer; Nikolaj Gadegaard; Felicity R. Rose; Morgan R. Alexander (6764-6771).
The use of cell sheets for tissue engineering applications has considerable advantages over single cell seeding techniques. So far, only thermoresponsive surfaces have been used to manufacture cell sheets without chemically disrupting the cell-surface interactions. Here, we present a new and facile technique to prepare sheets of epithelial cells using plasma polymerised acrylic acid films. The cell sheets are harvested by gentle agitation of the media without the need of any additional external stimulus. We demonstrate that the plasma polymer deposition conditions affect the viability and metabolic activity of the cells in the sheet and relate these effects to the different surface properties of the plasma polymerised acrylic acid films. Based on surface analysis data, a first attempt is made to explain the mechanism behind the cell sheet formation. The advantage of the epithelial cell sheets generated here over single cell suspensions to seed a PLGA scaffold is presented. The scaffold itself, prepared using a mould fabricated via photolithography, exhibits a unique architecture that mimics closely the dimensions of the native tissue (mouse intestine).
Keywords: Plasma polymerisation; Cell viability; Surface analysis; Electron beam lithography; Intestine; Poly(lactic-co-glycolic acid) (PLGA);

Hyaluronic acid hydrogels with controlled degradation properties for oriented bone regeneration by Jennifer Patterson; Ruth Siew; Susan W. Herring; Angela S.P. Lin; Robert Guldberg; Patrick S. Stayton (6772-6781).
Non-healing fractures can result from trauma, disease, or age-related bone loss. While many treatments focus on restoring bone volume, few try to recapitulate bone organization. However, the native architecture of bone is optimized to provide its necessary mechanical properties. Hyaluronic acid (HA) hydrogel scaffold systems with tunable degradation properties were developed for the controlled delivery of osteoinductive and angiogenic growth factors, thus affecting the quantity and quality of regenerated tissue. HA hydrogels were designed to degrade at fast, intermediate, and slow rates due to hydrolysis and further provided controlled release of cationic proteins due to electrostatic interactions. Scaffolds delivering bone morphogenetic protein-2 (BMP-2) were evaluated in a rat calvarial bone critical size defect model. BMP-2 delivery from the HA hydrogels had a clear osteoinductive effect in vivo and, for all hydrogel types, BMP-2 delivery resulted in significant mineralization compared to control hydrogels. The temporal progression of this effect could be modulated by altering the degradation rate of the scaffold. All three degradation rates tested resulted in similar amounts of mineral formation at the latest (six week) time point examined. Interestingly, however, the fastest and slowest degrading scaffolds seemed to result in more organized bone than the intermediate degrading scaffold, which was designed to degrade in 6–8 weeks to match the healing time. Additionally, healing could be enhanced by co-delivery of vascular endothelial growth factor along with BMP-2.
Keywords: Angiogenesis; Animal model; Collagen structure; Controlled drug release; Growth factors; Photopolymerisation;

The performance of a hydrogel nucleus pulposus prosthesis in an ex vivo canine model by Niklas Bergknut; Lucas A. Smolders; Leo H. Koole; George Voorhout; Ragnvi E. Hagman; Anne-Sofie Lagerstedt; Ketie Saralidze; Herman A.W. Hazewinkel; Albert J. van der Veen; Björn P. Meij (6782-6788).
A nucleus pulposus prosthesis (NPP) made of the hydrogel N-vinyl-2-pyrrolidinone copolymerized with 2-(4′-iodobenzoyl)-oxo-ethyl methacrylate has recently been developed. The special features of this NPP, i.e. intrinsic radiopacity and its ability to swell in situ to fill the nucleus cavity and restore disc height, were investigated ex vivo in canine spinal specimens. L7-S1 intervertebral discs were isolated from three canine spinal specimens, and the dimensions of the nuclei pulposi were measured. Based on these averaged measurements, the NPP prototype was made and inserted in its dry form (xerogel) into a canine cadaveric spinal segment and allowed to swell overnight at 38 °C. The integrity of the NPP and the filling of the nucleus cavity were assessed before and after swelling, using radiography, computed tomography, and magnetic resonance imaging. The ability of the NPP to restore disc height was assessed on radiographs of 10 spinal specimens. Thereafter the NPP was macroscopically assessed in situ by dissection of the spinal specimen.Both on imaging and macroscopically, 9/10 NPPs appeared to have a near perfect fit and disc height was restored in 8/10 spinal segments. The NPP may thus be an acceptable treatment option for low back patients meeting the requirements for NPP treatment.
Keywords: Intervertebral disc; Spinal surgery; Radiopacity; Hydrogel;

The abnormal aggregation of β-amyloid peptide (Aβ) in the brain is a major histopathological feature of Alzheimer’s disease. Herein, we first report on microfluidic dissociation and clearance analysis of pre-formed Aβ aggregates for parallel screening of aggregate destabilizers. As a proof of the concept for the microfluidic platform, we investigated (1) microfluidics-based clearance of metal ion-induced Aβ aggregates using different types of metal chelators, (2) the clearance effect of deferoxamine on Aβ aggregates within microchannels, (3) comparison between destabilized Aβ dissociated from pre-formed Aβ aggregates and remaining deposits within the microchannels both before and after the clearance, and (4) secondary structure change in Aβ deposits by the clearance treatment. The microfluidics-based clearance system should be suitable for efficient screening of chemical candidates to enhance the clearance of Aβ deposits prior to their in vivo evaluation.
Keywords: Amyloid; Self-assembly; Dissociation; Microfluidics; Metal chelators;

Nanocomposites from a polyester-type waterborne polyurethane (PU) containing various low concentrations (15–75 ppm) of silver nanoparticles (nano Ag, size ∼5 nm) were prepared. The PU–Ag nanocomposites exhibited good nanoparticle dispersion up to 30 ppm of nano Ag, confirmed by the transmission electron microscopy. Distinct surface morphology at different concentrations of nano Ag was shown by the atomic force microscopy. The oxidative degradation of PU–Ag was inhibited in all concentrations of nano Ag tested, especially at 30 ppm (“PU–Ag 30 ppm”). This may be related to the free radical scavenging ability observed for the nanocomposites. PU–Ag 30 ppm showed enhanced fibroblast attachment and endothelial cell response, as well as reduced monocyte and platelet activation, relative to PU alone or nanocomposites at the other silver contents. The rat subcutaneous implantation confirmed the better biocompatibility of the nanocomposites. The adhesion of Bacillus subtilis, Escherichia (E.) coli or Ag+-resistant E. coli on PU–Ag nanocomposites was significantly lower at all concentrations of nano Ag tested. Besides, the nanocomposites demonstrated microbiostatic effect while pure PU did not. The commercial catheters coated with PU–Ag 30 ppm were inserted into rat jugular veins for evaluation. The results showed milder inflammation for PU–Ag after 3 months compared to the non-coated catheters or pure PU-coated catheters. The enhanced performance of PU–Ag over that of pure PU was a result of extensively modified surface morphology in the presence of a very small amount of nano Ag. The dispersion of nano Ag was highly associated with the overall performance.
Keywords: Bacteriostasis; Biocompatibility; Nanocomposites; Silver nanoparticles (nano Ag); Polyurethane (PU);

The biphenyl ethers (BPEs) are the potent inhibitors of TTR fibril formation and are efficient fibril disrupter. However, the mechanism by which the fibril disruption occurs is yet to be fully elucidated. To gain insight into the mechanism, we synthesized and used a new QD labeled BPE to track the process of fibril disruption. Our studies showed that the new BPE-QDs bind to the fiber uniformly and has affinity and specificity for TTR fiber and disrupted the pre-formed fiber at a relatively slow rate. Based on these studies we put forth the probable mechanism of fiber disruption by BPEs. Also, we show here that the BPE-QDs interact with high affinity to the amyloids of Aβ42, lysozyme and insulin. The potential of BPE-QDs in the detection of senile plaque in the brain of transgenic Alzheimer’s mice has also been explored.
Keywords: Transthyretin; Quantum Dots; Amyloidosis; Biphenyl ether; Amyloid;

Dual-emissive quantum dots for multispectral intraoperative fluorescence imaging by Patrick T.K. Chin; Tessa Buckle; Arantxa Aguirre de Miguel; Stefan C.J. Meskers; René A.J. Janssen; Fijs W.B. van Leeuwen (6823-6832).
Fluorescence molecular imaging is rapidly increasing its popularity in image guided surgery applications. To help develop its full surgical potential it remains a challenge to generate dual-emissive imaging agents that allow for combined visible assessment and sensitive camera based imaging. To this end, we now describe multispectral InP/ZnS quantum dots (QDs) that exhibit a bright visible green/yellow exciton emission combined with a long-lived far red defect emission. The intensity of the latter emission was enhanced by X-ray irradiation and allows for: 1) inverted QD density dependent defect emission intensity, showing improved efficacies at lower QD densities, and 2) detection without direct illumination and interference from autofluorescence.
Keywords: Quantum dots; Dual-emissive particles; Multispectral fluorescence imaging; Sentinel lymph node; Prostate cancer;

The effect of surface modification of amorphous silica particles on NLRP3 inflammasome mediated IL-1β production, ROS production and endosomal rupture by Tomohiro Morishige; Yasuo Yoshioka; Hiroshi Inakura; Aya Tanabe; Xinglei Yao; Shogo Narimatsu; Youko Monobe; Takayoshi Imazawa; Shin-ichi Tsunoda; Yasuo Tsutsumi; Yohei Mukai; Naoki Okada; Shinsaku Nakagawa (6833-6842).
Although amorphous silica particles (SPs) are widely used in cosmetics, foods and medicinal products, it has gradually become evident that SPs can induce substantial inflammation accompanied by interleukin-1β (IL-1β) production. Here, to develop safe forms of SPs, we examined the mechanisms of SP-induced inflammation and the relationship between particle characteristics and biological responses. We compared IL-1β production levels in THP-1 human macrophage like cells in response to unmodified SP of various diameters (30- to 1000-nm) and demonstrated that unmodified microsized 1000-nm SP (mSP1000) induced higher levels of IL-1β production than did smaller unmodified SPs. Furthermore, we found that unmodified mSP1000–induced IL-1β production was depended on the sequence of reactive oxygen species (ROS) production, endosomal rupture, and subsequent activation of pro-inflammatory complex NLRP3 inflammasome. In addition, we compared IL-1β production levels in THP-1 cells treated with mSP1000s modified with a functional group (–COOH, –NH2, –SO3H, –CHO). Although unmodified and surface-modified mSP1000s were taken up with similar frequencies equally into the THP-1 cells, surface modification of mSP1000 dramatically suppressed IL-1β production by reducing ROS production. Our results reveal a part of NLRP3 activation pathway and provide basic information that should help to create safe and effective forms of SPs.
Keywords: Cytokine; Inflammation; Interleukin; Macrophage; Nanoparticle; Silica;

We report a pH dependence of degradable silica nanotubes, which dissolved to the biodegradation product monosilicic acid, Si(OH)4. The silica nanotubes, potentially acting as oral-based administration carriers, were resistant to dissolution in the extreme acidic condition of pH 1, but degraded quickly at pH 8, and the degradation rate can be tuned by tailoring the thickness of silica nanotubes with thicker nanotubes dissolving more slowly. Because Gd(OH)3 nanorods were used as templates, the silica nanotubes could be further developed as MR imaging contrast agents as well as drugs carriers. The released Gd3+ ions resulting from the etching of Gd(OH)3 nanorods were chelated by the pre-modified DOTA, yielding Gd-DOTA complexes grafted onto silica nanotubes. The Gd-DOTA grafted silica nanotubes loaded with doxorubicin revealed enhanced T1 imaging contrast and anticancer activity.
Keywords: Silica; Nanotube; Biodegradation; MRI; Drug delivery;

Biodistribution, pharmacodynamics and pharmacokinetics of insulin analogues in a rat model: Oral delivery using pH-Responsive nanoparticles vs. subcutaneous injection by Kiran Sonaje; Kun-Ju Lin; Shiaw-Pyng Wey; Che-Kuan Lin; Tzyy-Harn Yeh; Ho-Ngoc Nguyen; Chia-Wei Hsu; Tzu-Chen Yen; Jyuhn-Huarng Juang; Hsing-Wen Sung (6849-6858).
In this study, we report the biodistribution of aspart-insulin, a rapid-acting insulin analogue, following oral or subcutaneous (SC) administration to rats using the single-photon emission computed tomography (SPECT)/computed tomography (CT). Oral delivery of aspart-insulin was achieved using a pH-responsive nanoparticle (NP) system composed of chitosan (CS) and poly(γ-glutamic acid). The results obtained in the SPECT/CT study indicate that the orally administered aspart-insulin was absorbed into the systemic circulation, while the drug carrier (CS) was mainly retained in the gastrointestinal tract.Via the SC route, the peak aspart-insulin concentration in the peripheral tissue/plasma was observed at 20 min after injection. Within 3 h, half of the initial dose (ID) of aspart-insulin was degraded and excreted into the urinary bladder. In contrast, via oral delivery, there was constantly circulating aspart-insulin in the peripheral tissue/plasma during the course of the study, while 20% of the ID of aspart-insulin was metabolized and excreted into the urinary bladder. In the pharmacodynamic (PD) and pharmacokinetic (PK) evaluation in a diabetic rat model, the orally administered aspart-insulin loaded NPs produced a slower hypoglycemic response for a prolonged period of time, whereas the SC injection of aspart-insulin produced a more pronounced hypoglycemic effect for a relatively shorter duration. Finally, comparison of the PD/PK profiles of the orally administered aspart-insulin with those of the SC injection of NPH-insulin, an intermediate-acting insulin preparation, suggests the suitability of our NP system to be used as a non-invasive alternative for the basal insulin therapy.
Keywords: Diabetes; Oral insulin; Biodistribution; SPECT/CT; Pharmacodynamics; Pharmacokinetics;

We synthesized a series of pH-sensitive vehicles, composed of dimethylaminoethyl methacrylate (DMAEMA) and 2-hydroxyethyl methacrylate (HEMA), to optimize the triggered release of DNA for gene transfection. The purpose of this study was to assess the role of swelling and cationic character independently on transfection; both of which may affect DNA release. Gene transfection was performed by delivering plasmid DNA (pDNA) encoding for luciferase. DNA release was controlled via volumetric swelling by regulating the endosomal pH as a result of inhibiting V ATPases using bafilomycin A1. Increasing the cationic character from 10 to 30 mol% DMAEMA did not increase transfection when swelling was inhibited. Transfection was significantly affected by the rate of pDNA release. pH-sensitive nanocarriers were also compared to vehicles comprised of polyethyleneimine (PEI), dioleoyl triammonium propane (DOTAP), and poly(lactic-co-glycolic acid) (PLGA, 50:50). pDNA encapsulating DMAEMA/HEMA nanoparticles and PEI/pDNA complexes had reduced transfection when V ATPases were inhibited, whereas pDNA encapsulating PLGA nanoparticles showed no endosomal pH dependence. DMAEMA/HEMA nanoparticles cross-linked with 3 mol% tetraethylene glycol dimethacrylate (TEGDMA) reported equivalent or greater gene transfection relative to the nanocarriers tested at 24 and 48 h.
Keywords: Nonviral gene delivery; pH-sensitive nanoparticles; DMAEMA; V ATPase; Bafilomycin A1;

Delivery of nucleic acids with positively charged lipid nanoparticles ((+)NPs) is widely used as research reagents and potentially for therapeutics due to their ability to deliver nucleic acids into the cell cytoplasm. However, in most reports little attention has been made to their toxic effects. In the present study, we performed comprehensive analyses of the potential toxicity associated with (+)NPs. Mice treated with (+)NPs showed increased liver enzyme release and body weight loss compared to mice treated with neutral or negatively charged NPs ((−)NPs), suggesting hepatotoxicity. Intravenous administration of (+)NPs induced interferon type I response and elevated mRNA levels of interferon responsive genes 15–25-fold higher than neutral and (−)NPs in different subsets of leukocytes. Moreover, treatment with (+)NPs provoked a dramatic pro-inflammatory response by inducing Th1 cytokines expression (IL-2, IFN γ and TNF α) 10–75-fold higher than treatment with control particles. Finally, we showed that activation of TLR4 might serve as the underlying mechanism for induction of an immune response when (+)NPs are used. These results suggest that a careful attention must be made when different types of (+)NPs are being developed as nanotherapeutics.
Keywords: Lipid-based nanoparticles; Immune activation; Interferon response; Cytokine induction; TLR4;

Chondrogenesis of synovium-derived mesenchymal stem cells in gene-transferred co-culture system by Rohan R. Varshney; Ruijie Zhou; Jinghua Hao; Suan Siong Yeo; Wai Hon Chooi; Jiabing Fan; Dong-An Wang (6876-6891).
A co-culture strategy has been developed in this study wherein rabbit synovial mesenchymal stem cells (SMSCs) are co-cultured with growth factor (GF) transfected articular chondrocytes. Toward this end, both SMSCs and early passage rabbit articular chondrocytes that had been adenovirally transduced with transforming growth factor-beta 3 (TGF-β3) gene were separately encapsulated in alginate beads and co-cultured in the same pool of chondrogenic medium. The chondrocytes act as transfected companion cells (TCCs) providing GF supply to induce chondrogenic differentiation of SMSCs that play the role of therapeutic progenitor cells (TPCs). Against the same TCC based TGF-β3 release profile, the co-culture was started at different time points (Day 0, Day 10 and Day 20) but made to last for identical periods of exposure (30 days) so that the exposure conditions could be optimized in terms of initiation and duration. Transfection of TCCs prevents the stem cell based TPCs from undergoing the invasive procedure. It also prevents unpredictable complications in the TPCs caused by long-term constitutive over-expression of a GF. The adenovirally transfected TCCs exhibit a transient GF expression which results in a timely termination of GF supply to the TPCs. The TCC-sourced transgenic TGF-β3 successfully induced chondrogenesis in the TPCs. Real-time PCR results show enhanced expression of cartilage markers and immuno/histochemical staining for Glycosaminoglycans (GAG) and Collagen II also shows abundant extracellular matrix (ECM) production and chondrogenic morphogenesis in the co-cultured TPCs. These results confirm the efficacy of directing stem cell differentiation towards chondrogenesis and cartilage tissue formation by co-culturing them with GF transfected chondrocytes.
Keywords: Co-culture; Chondrogenesis; Mesenchymal stem cells; TGF-beta 3; Adenovirus; Transgene; Chondrocyte;

Liposome–polyethylenimine complexes for enhanced DNA and siRNA delivery by Jens Schäfer; Sabrina Höbel; Udo Bakowsky; Achim Aigner (6892-6900).
The efficient delivery of nucleic acids into cells is critical for successful gene therapy or gene knockdown. Polyethylenimines (PEIs) are positively charged polymers which complex and deliver DNA for gene transfection or small interfering RNAs (siRNAs) for the induction of RNA interference (RNAi), and mediate their endosomal release. Likewise, various liposomes act as transfection reagents, with some lipids showing increased endocytosis and influencing endosomal escape. This study combines the favourable properties of PEI and lipid systems for DNA and siRNA delivery. Various lipids and lipid combinations, which cover a broad range of physicochemical properties and form optimal liposomes, are assessed. By addition of the liposomes to pre-formed polyplexes, based on the low molecular weight PEI F25-LMW, we establish liposome–PEI complexes (lipopolyplexes) and characterise them in comparison to their ‘parent’ polyplexes and liposomes regarding size, shape and zeta-potential. Furthermore, while the lipidation of polyplexes generally decreases their toxicity, our studies on DNA transfection and siRNA-mediated knockdown also establish certain lipopolyplexes based on rigid, negatively charged lipids as particularly efficient vehicles for nucleic acid delivery, further improving DNA transfection. The analysis of their mechanism and kinetics of cellular uptake confirms that the biological properties of lipopolyplexes are mainly determined by the liposome shell. We conclude that certain lipopolyplexes show improved biological properties over PEI complexes, thus representing potentially attractive non-viral vectors for gene therapy and RNAi.
Keywords: Liposomes; Gene delivery; RNAi; siRNAs; Polyethylenimine; Lipid coating;