Biomaterials (v.31, #5)

The role of protein assembly in dynamically tunable bio-optical tissues by Andrea R. Tao; Daniel G. DeMartini; Michi Izumi; Alison M. Sweeney; Amanda L. Holt; Daniel E. Morse (793-801).
Cephalopods are nicknamed the “masters of disguise” for their highly evolved camouflage mechanisms, including the hallmark ability to rapidly change the color and reflectance of their skin. Previously, reflectin proteins were identified as the major biomaterial component of iridosomes , specialized light-reflecting architectures that contribute intense structural color to squid skin, eyes, and organs . Supramolecular assembly of reflectin has been recognized as a key property in the protein's function . Here, we report the first cloning and expression of a specific reflectin protein found in the responsive iridophore cells of the squid Loligo pealeii, which are unique in their ability to switch on/off and change color. We demonstrate that these iridophores can be chemically tuned to reflect the entire visible spectrum. By examining recombinant reflectin, we show that this dynamic optical function is facilitated by the hierarchical assembly of nanoscale protein particles that elicit large volume changes upon condensation. These findings provide insight into the design and synthesis of biomaterials for complex, responsive function in optical applications.
Keywords: Nanoparticle; Self Assembly; Protein; Biomimetic material; Structural Color; Lens;

An artificial extracellular matrix created by hepatocyte growth factor fused to IgG-Fc by Koji Azuma; Masato Nagaoka; Chong-Su Cho; Toshihiro Akaike (802-809).
The design of artificial extracellular matrices (ECM) has attracted much attention in tissue engineering and regenerative medicine as well as in molecular biology research. A recombinant hepatocyte growth factor (HGF), fused to an immunoglobulin G (IgG) Fc region (abbreviated as AeHGF-Fc) was constructed and confirmed by Western blot assay. Almost similar amounts of HepG2 cells adhered to AeHGF-Fc-coated surface compared to collagen-coated one with large morphological changes. Immobilized AeHGF-Fc continuously activated Akt in HepG2 cells whereas Akt activation induced by soluble HGF rapidly decreased with time, indicating that immobilized AeHGF-Fc follows different signal transduction pathways compared to soluble HGF.
Keywords: Hepatocyte growth factor; ECM (extracellular matrix); Cell morphology; HepG2; c-Met; Akt;

Polyketal microparticles for therapeutic delivery to the lung by Vincent F. Fiore; Megan C. Lofton; Susanne Roser-Page; Stephen C. Yang; Jesse Roman; Niren Murthy; Thomas H. Barker (810-817).
Inflammation in the setting of interstitial lung disease (ILD) occurs in the distal alveolar spaces of the lung, which presents significant challenges for therapeutic delivery. The development of aerosolizable microparticles from non-immunogenic polymers is needed to enable the clinical translation of numerous experimental therapeutics that require localization to the deep lung and repeated delivery for optimal efficacy. Polyketals (PK), a family of polymers, have several unique properties that make them ideal for lung delivery, specifically their hydrolysis into non-acidic, membrane-permeable compounds and their capacity to form microparticles with the aerodynamic properties needed for aerosolization. In this study, we tested the lung biocompatibility of microparticles created from a polyketal polymer, termed PK3, following intratracheal instillation in comparison to commonly used PLGA microparticles. We furthermore tested the initial efficacy of PK3 microparticles to encapsulate and effectively deliver active superoxide dismutase (SOD), a free radical scavenging enzyme, in a model of lung fibrosis. Our findings indicate that PK3 microparticles display no detectable level of alveolar or airway inflammation, whereas PLGA induced a small inflammatory response. Furthermore, SOD-loaded into PK3 microparticles maintained its activity upon release and, when delivered via PK3 microparticles, inhibited the extent of lung fibrosis.
Keywords: Biocompatibility; Drug delivery; Fibrosis; Lung; Microsphere; Superoxide;

Metabolism of TEGDMA and HEMA in human cells by Jürgen Durner; Udo I. Walther; Johannes Zaspel; Reinhard Hickel; Franz-Xaver Reichl (818-823).
Previous in vivo studies have shown that the comonomers triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) from dental materials can be metabolised to CO2 by two postulated pathways: an epoxide and a valine pathway. In the epoxide pathway the formation of pyruvate is postulated and in valine pathway the formation of l-malate. The aim of this investigation was to quantify the formation of the intermediates pyruvate and l-malate to show which pathway may be preferred in A549 cells. Therefore A549 cells were incubated with TEGDMA or HEMA (with a tracer dose 14C-TEGDMA or 14C-HEMA) and afterwards 14C-TEGDMA or 14C-HEMA, 14C-methacrylate, 14C-l-malate and 14C-pyruvate were identified and quantified by thin layer chromatography at different time intervals from the extracellular and intracellular fluid. Our results show that in the metabolism of both comonomers more 14C-pyruvate was formed compared to 14C-l-malate for 14C-HEMA metabolisation during 0.5 up to 6 h after 14C-HEMA exposure and for 14C-TEGDMA metabolisation >4 h after 14C-TEGDMA exposure. Therefore the epoxide pathway with formation of the epoxy-intermediate 2,3-epoxymethacrylic acid is the main route of metabolisation of HEMA and TEGDMA.
Keywords: Degradation; Dental restorative material; Hydroxyethylmethacrlate;

The stimulation of angiogenesis and collagen deposition by copper by Catherine Gérard; Louis-Jean Bordeleau; Jake Barralet; Charles J. Doillon (824-831).
Copper is known to trigger endothelial cells towards angiogenesis. Different approaches have been investigated to develop vascularisation in biomaterials. The angiogenic and healing potential of copper ions in combination with two major angiogenic factors was examined. A 3D culture system in which, under stimulation by FGF-2 and to a lesser degree with VEGF, endothelial cells assembled into structures resembling to an angiogenic process was used. The combination of CuSO4 with increasing doses of VEGF or FGF-2 enhanced the complexity of angiogenic networks in a significant manner. In vivo studies were also conducted by incorporating FGF-2 with CuSO4 in a cylindrical collagen-based scaffold. CuSO4 enhanced significantly the invasion of microvessel compared to control implants and to 20 ng FGF-2 ± CuSO4. Vascular infiltration was also significantly improved by combination of CuSO4 with FGF-2, compared to FGF-2 alone (0.2 and 1 μg). Nevertheless, in comparison with CuSO4 alone, there was a significant increase only with 1 μg of FGF-2 combined with CuSO4. Significantly, collagen fiber deposition was enhanced following the combinatory loading in comparison to that with FGF-2 alone but not with CuSO4 only. Thus, copper associated with growth factors may have synergistic effects which are highly attractive in the fields of tissue engineering (e.g., bone) and biomaterials.
Keywords: Collagen scaffold; Angiogenesis; Growth factor; Copper; 3D angiogenesis assay; Collagen deposition;

While platelet adhesion to biomaterial surfaces is widely recognized to be related to adsorbed fibrinogen (Fg), it has remained controversial whether platelet adhesion is in response to the adsorbed amount or the adsorbed conformation of this protein. To address this issue, we designed a series of platelet adhesion studies to clearly separate these two factors, thus enabling us to definitively determine whether it is the amount or the conformation of adsorbed Fg that mediates platelet response. Fg was adsorbed to a broad range of surface chemistries from a wide range of solution concentrations, with the amount and conformation of adsorbed Fg determined by absorbance and circular dichroism (CD) spectropolarimetry, respectively. Platelet adhesion response was determined by lactate dehydrogenase (LDH) assay and scanning electron microscopy (SEM). Our results show that platelet adhesion is strongly correlated with the degree of adsorption-induced unfolding of Fg (r 2  = 0.96) with essentially no correlation with the amount of Fg adsorbed (r 2  = 0.04). Platelet receptor inhibitor studies using an RGDS peptide reduced platelet adhesion by only about 50%, and SEM results show that adherent platelets after RGDS blocking were much more rounded with minimal extended filopodia compared with the unblocked platelets. These results provide definitive proof that the conformation of adsorbed Fg is the critical determinant of platelet adhesion, not the amount of Fg adsorbed, with adsorption-induced unfolding potentially exposing two distinctly different types of platelet binding sites in Fg; one that induces platelet adhesion alone and one that induces both platelet adhesion and activation.
Keywords: Adsorption; Fibrinogen; Protein adsorption; Platelet adhesion; Haemocompatibility;

Fatigue and human umbilical cord stem cell seeding characteristics of calcium phosphate–chitosan–biodegradable fiber scaffolds by Liang Zhao; Elena F. Burguera; Hockin H.K. Xu; Nikhil Amin; Heon Ryou; Dwayne D. Arola (840-847).
Calcium phosphate cement (CPC) has in situ-setting ability and bioactivity, but the brittleness and low strength limit CPC to only non-load-bearing bone repairs. Human umbilical cord mesenchymal stem cells (hUCMSCs) can be harvested without an invasive procedure required for the commonly studied bone marrow MSCs. However, little has been reported on hUCMSC delivery via bioactive scaffolds for bone tissue engineering. The objectives of this study were to develop CPC scaffolds with improved resistance to fatigue and fracture, and to investigate hUCMSC delivery for bone tissue engineering. In fast fracture, CPC with 15% chitosan and 20% polyglactin fibers (CPC–chitosan–fiber scaffold) had flexural strength of 26  mPa, higher than 10  mPa for CPC control (p  < 0.05). In cyclic loading, CPC–chitosan–fiber specimens that survived 2 × 106 cycles had the maximum stress of 10 MPa, compared to 5 MPa of CPC control. CPC–chitosan–fiber specimens that failed after multiple cycles had a mean stress-to-failure of 9 MPa, compared to 5.8 MPa for CPC control (p  < 0.05). hUCMSCs showed excellent viability when seeded on CPC and CPC–chitosan–fiber scaffolds. The percentage of live cells reached 96–99%. Cell density was about 300 cells/mm2 at day 1; it proliferated to 700 cells/mm2 at day 4. Wst-1 assay showed that the stronger CPC–chitosan–fiber scaffold had hUCMSC viability that matched the CPC control (p  > 0.1). In summary, this study showed that chitosan and polyglactin fibers substantially increased the fatigue resistance of CPC, and that hUCMSCs had excellent proliferation and viability on the scaffolds.
Keywords: Calcium phosphate cement; Fatigue; Load-bearing; Bioactive scaffolds; Human umbilical cord stem cells; Bone regeneration;

We describe a method of using a 3D collagen gel scaffold applied at the air–liquid interface to culture dissociated primary tracheal-bronchial ciliated cells into a ciliated epithelial tissue structure (CETS). This 3D collagen gel culture system enables the induction of ciliogenesis and continuously provides support, maintenance, development, differentiation and propagation for the growth of cilia into the CETS. The CETS developed by this system resembles the ciliary metachronal motility and morphological, histological and physiopharmacological characteristics of cells found in native and in vivo ciliated epithelia. The CETS can be sustained for months with a straightforward and simple maintenance protocol. The integrity of the functional ciliary activity of this CETS enables the evaluation of long-term effects of many pulmonary drug candidates without using animals.
Keywords: Ciliated epithelial tissue structure; Ciliogenesis; 3D gel scaffold; Collagen;

The influence of rat mesenchymal stem cell CD44 surface markers on cell growth, fibronectin expression, and cardiomyogenic differentiation on silk fibroin – Hyaluronic acid cardiac patches by Ming-Chia Yang; Nai-Hsin Chi; Nai-Kuan Chou; Yi-You Huang; Tze-Wen Chung; Yu-Lin Chang; Hwa-Chang Liu; Ming-Jium Shieh; Shoei-Shen Wang (854-862).
Since MSCs contain an abundant of CD44 surface markers, it is of interesting to investigate whether CD44 on rat MSC (rMSCs) influenced cell growth, fibronectin expression and cardiomyogenic differentiation on new SF/HA cardiac patches. For this investigation, we examined the influences of rMSCs with or without a CD44-blockage treatment on the aforementioned issues after they were cultivated, and further induced by 5-aza on SF and SF/HA patches. The results showed that the relative growth rates of rMSCs cultured on cultural wells, SF/HA patches without or with a CD44-blockage treatment were 100%, 208.9 ± 7.1 (%) or 48.4 ± 6.0 (%) (n  = 3, for all), respectively, after five days of cultivations. Moreover, rMSCs cultivated on SF/HA patches highly promoted fibronectin expressions (e.g., 1.8 × 105/cell, in fluorescent intensity) while cells with a CD44-blockage treatment markedly diminished the expressions (e.g., 1.1 × 104/cell, in fluorescent intensity) on same patches. For investigating possible influences of CD44 surface markers of rMSCs on their cardiomyogenic differentiation, the expressions of specific cardiac genes of cells were examined by using real-time PCR analysis. The results indicated that 5-aza inducing rMSCs significantly promoted the expressions of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P  < 0.01 or better, n  = 3) on SF/HA patches compared with those expressions on SF patches and for cells with a CD44-blockage treatment on SF/HA patches. Furthermore, the intensity of the expressions of cardiotin and connexin 43 of 5-aza inducing rMSCs were markedly higher than those of cells with a CD44-blockage treatment after they were cultured on SF/HA patches. Through this study, we reported that CD44 surface markers of rMSCs highly influenced the proliferations, fibronectin expressions and cardiomyogenic differentiation of rMSCs cultivated on cardiac SF/HA patches.
Keywords: Silk fibroin/hyaluronic acid patches; CD44 of mesenchymal stem cells; Fibronectin expression; Cardiomyogenic differentiation;

Hydrodynamic spinning of hydrogel fibers by Min Hu; Rensheng Deng; Karl M. Schumacher; Motoichi Kurisawa; Hongye Ye; Kristy Purnamawati; Jackie Y. Ying (863-869).
Hydrogel scaffolds are highly hydrated polymer networks that allow cells to adhere, proliferate and differentiate in the treatment of diseased or injured tissues and organs. Using hydrodynamic shaping and in situ cross-linking of hydrogel precursors, we have developed a highly efficient “hydrodynamic spinning” approach for synthesizing hydrogel fibers of different diameters in a multiphase coaxial flow. A triple-orifice spinneret has been created, and three different types of hydrogel precursors have been examined. Without changing the spinning head, hollow and solid hydrogel fibers with different diameters have been spun by simply manipulating the ratio of input flow rates. Together with the ability of simultaneous cell-seeding in the hydrogel matrix, hydrodynamic spinning can be broadly applied to many hydrogel materials, providing a powerful technique in the preparation of fiber-like and tubule-like hydrogel constructs for tissue engineering.
Keywords: Copolymerization; Hydrogel fibers; Hydrodynamics; In situ cross-linking; Microfluidics;

Myogenic differentiation of human bone marrow mesenchymal stem cells on a 3D nano fibrous scaffold for bladder tissue engineering by Hong Tian; Shantaram Bharadwaj; Yan Liu; Haiyun Ma; Peter X. Ma; Anthony Atala; Yuanyuan Zhang (870-877).
Current strategies for engineering bladder tissues include a bladder biopsy for in vitro cell expansion for use in reconstructive procedures. However, this approach cannot be used in patients with bladder cancer who need a complete bladder replacement. Bone marrow mesenchymal stem cells (BMSC) might be an alternative cell source to better meet this need. We investigated the effects of soluble growth factors, bladder extracellular matrix (ECM), and 3D dynamic culture on cell proliferation and differentiation of human BMSC into smooth muscle cells (SMC). Myogenic growth factors (PDGF-BB and TGF-β1) alone, or combined either with bladder ECM or dynamic cultures, induced BMSC to express smooth muscle-specific genes and proteins. Either ECM or the dynamic culture alone promoted cell proliferation but did not induce myogenic differentiation of BMSC. A highly porous poly-l-lactic acid (PLLA) scaffold provided a 3D structure for maximizing the cell-matrix penetration, maintained myogenic differentiation of the induced BMSC, and promoted tissue remolding with rich capillary formation in vivo. Our results demonstrate that myogenic-differentiated BMSC seeded on a nano fibrous PLLA scaffold can be potentially used for cell-based tissue engineering for bladder cancer patients requiring cystoplasty.
Keywords: Bone marrow; Stem cells; Smooth muscle cell; Bladder; Scaffold;

Local delivery of a collagen-binding FGF-1 chimera to smooth muscle cells in collagen scaffolds for vascular tissue engineering by Yonggang Pang; Xiaoli Wang; Areck A. Ucuzian; Eric M. Brey; Wilson H. Burgess; Kathryn J. Jones; Thomas D. Alexander; Howard P. Greisler (878-885).
We investigated the delivery of R136K–CBD (a collagen-binding mutant chimera of fibroblast growth factor-1) with a type I collagen scaffold as the delivery vehicle to smooth muscle cells (SMCs) for vascular tissue engineering. The binding affinity of R136K–CBD to 3-D collagen scaffolds was investigated both in the presence and absence of cells and/or salts. 2-D and 3-D visualization of delivery of R136K–CBD into SMCs were accomplished by combined fluorescent and reflection confocal microscopy. The mitogenic effect of collagen-immobilized R136K–CBD on SMCs in 3-D collagen was studied by Cyquant assay at different time intervals. In the group devoid of salt and cells, no detectable release of R136K–CBD into overlying culture media was found, compared with burst-and-continuous release of R136K and FGF-1 over a 14-day period in all other groups. The release rate of R136K–CBD was 1.7 and 1.6-fold less than R-136K and FGF-1 when media was supplemented with 2  m salt (P  < 0.0001), and 2.6 and 2.5-fold less in cell-populated collagen hydrogels (P  < 0.0001), respectively. R136K–CBD showed essentially uniform binding to collagen and its distribution was dependent on that of the collagen scaffold. Internalization of R136K–CBD into SMCs was documented by confocal microscopy. 3-D local delivery of collagen-immobilized R136K–CBD increased the proliferation of SMCs in the collagen matrix to significantly greater levels and for a significantly greater duration than R136K or FGF-1, with 2.0 and 2.1-fold more mitogenicity than R136K and FGF-1 respectively (P  < 0.0001) at day 7. The results suggest that our collagen-binding fusion protein is an effective strategy for growth factor delivery for vascular tissue engineering.
Keywords: Controlled drug release; Growth factors; Cell proliferation; Collagen; Smooth muscle cell;

Cytotoxicity of implantable microelectrode arrays produced by laser micromachining by Rylie A. Green; Juan S. Ordonez; Martin Schuettler; Laura A. Poole-Warren; Nigel H. Lovell; Gregg J. Suaning (886-893).
Implantable high-density microelectrode arrays have been successfully fabricated using laser micromachining of conventional implant materials, polydimethylsiloxane (PDMS) and platinum (Pt) foil. This study investigates the impact of modifying PDMS and Pt with high power laser beams and the possible toxicity of by-products that may remain on the implantable device. Materials were characterised both chemically and biologically through x-ray photoelectron spectroscopy (XPS), cell growth inhibition assays and a direct contact cell proliferation assay. It was found that laser micromachining produces oxides of silicon and platinum on the PDMS and Pt respectively. While the chemical properties of materials were altered, there was negligible change in the biological response to either extracts or cell growth directly on the composite electrode array.
Keywords: Laser micromachining; Microelectrodes; Cytotoxicity; High-density array; Neuroprostheses;

Toxicological characteristics of nanoparticulate anatase titanium dioxide in mice by Yanmei Duan; Jie Liu; Linglan Ma; Na Li; Huiting Liu; Jue Wang; Lei Zheng; Chao Liu; Xuefeng Wang; Xiaoyang Zhao; Jingying Yan; Sisi Wang; Han Wang; Xueguang Zhang; Fashui Hong (894-899).
In an effort to examine liver injury, immune response, and other physiological effects in mice caused by intragastric administration of nanoparticulate anatase titanium dioxide (5 nm), we assessed T lymphocytes, B lymphocyte and NK lymphocyte counts, hematological indices, biochemical parameters of liver functions, and histopathological changes in nanoparticulate titanium dioxide -treated mice. Indeed, mice treated with higher dose nanoparticulate titanium dioxide displayed a reduction in body weight, an increase in coefficients of the liver and histopathological changes in the liver. Specifically, in these nanoparticulate titanium dioxide -treated mice, interleukin-2 activity, white blood cells, red blood cells, haemoglobin, mean corpuscular haemoglobin concentration, thrombocytes, reticulocytes, T lymphocytes (CD3+, CD4+, CD8+), NK lymphocytes, B lymphocytes, and the ratio of CD4 to CD8 of mice were decreased, whereas NO level, mean corpuscular volume, mean corpuscular haemoglobin, red (cell) distribution width, platelets, hematocrit, mean platelet volume of mice were increased. Furthermore, liver functions were also disrupted, as evidenced by the enhanced activities of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase and cholinesterase, an increase of the total protein, and the reduction of ratio of albumin to globulin, the total bilirubin, triglycerides, and the total cholesterol levels. These results suggested that the liver function damage observed in mice treated with higher dose nanoparticulate titanium dioxide is likely associated with the damage of haemostasis blood system and immune response. However, low dose nanoparticulate anatase TiO2 has little influences on haemostasis blood system and immune response in mice.
Keywords: Mice; Nanoparticulate titanium dioxide; Immune response; Hematological indices; Organs indices; Liver function;

Targeted drug delivery to inflamed or injured vascular endothelial cells (ECs) and smooth muscle cells (SMCs) may provide a precise and effective therapeutic treatment for cardiovascular diseases. Upregulation of cytokine-regulated cell surface receptors, intercellular cell adhesion molecule-1 (ICAM) and endothelial-leukocyte adhesion molecule-1 (ELAM), on ECs and SMCs are used to target drug delivery vehicles. Recent studies demonstrate clustering of these molecules in lipid rafts may affect binding due to a nonhomogenous presentation of antibodies. We hypothesized that altering the antibody ratio for ICAM and ELAM (aICAM:aELAM) and mobility would influence cellular targeting. To alter antibody mobility, liposomes were prepared from either 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, C18:1, T m  = −20 °C) or 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, C16:0, T m  = 42 °C) which are in the liquid crystalline (Lα) and gel phase (Lβ) at 37 °C, respectively. We report that cellular binding of DOPC immunoliposomes by ECs is maximal at an equimolar ratio of aICAM:aELAM whereas DPPC immunoliposomes showed no ratio dependence and binding was reduced by more than 2-fold. SMCs, which do not express ELAM, show a dependence on aICAM surface density. These results suggest that antibody mobility and molar ratio play a key role in increasing receptor-mediated cell targeting.
Keywords: Immunoliposome; Membrane elasticity; Cell adhesion molecule; Endocytosis; Synergy; Mobility;

Trimethylated chitosan-conjugated PLGA nanoparticles for the delivery of drugs to the brain by Zhao H. Wang; Zhan Y. Wang; Chang S. Sun; Chun Y. Wang; Tong Y. Jiang; Si L. Wang (908-915).
Trimethylated chitosan (TMC) surface-modified poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (TMC/PLGA–NP) were synthesized as a drug carrier for brain delivery. TMC was covalently coupled to the surface of PLGA nanoparticles (PLGA–NP) via a carbodiimide-mediated link. The zeta potential of TMC/PLGA–NP was about 20 mV with a mean diameter around 150 nm. 6-coumarin loaded PLGA–NP and TMC/PLGA–NP were injected into the caudal vein of mice, and fluorescent microscopy of brain sections showed a higher accumulation of TMC/PLGA–NP in the cortex, paracoele, the third ventricle and choroid plexus epithelium, while no brain uptake of PLGA–NP was observed. There was no pronounced difference in cell viability between TMC/PLGA–NP and PLGA–NP as shown by MTT assay. Behavioral testing showed that the injection of coenzyme Q10 loaded TMC/PLGA–NP greatly improved memory impairment, restoring it to a normal level, but the efficacy was slight for loaded PLGA–NP, without TMC conjugation. The senile plaque and biochemical parameter tests confirmed the brain-targeted effects of TMC/PLGA–NP. These experiments show that TMC surface-modified nanoparticles are able to cross the blood–brain barrier and appear to be a promising brain drug delivery carrier with low toxicity.
Keywords: Nanoparticle; Brain targeting; Trimethylated chitosan; Neuroprotective effects; Biochemical parameters; Co-Q10;

Human hepatocellular carcinoma (HCC) is one of the most causes of cancer-related death and is well known because of resistant to chemotherapeutic drug. Co-delivery of antitumor agent docetaxel and iSur-pDNA, a suppressor of metastatic and resistance-related protein survivin, was postulated to achieve synergistic/combined effect of antitumor drug and gene therapeutics. To valid this hypothesis, a folate-modified multifunctional nanoassembly (FNA) loading both docetaxel and iSur-pDNA was constructed and evaluated as a therapeutic approach for HCC. The FNAs were prepared with folate-modified lipid FA-PEG-DSPE as the target to tumor, protamine sulfate (PS) as the condenser to protect and enhance the nuclear transfer of iSur-pDNA, and DOPE-based lipid envelope as the carrier of doctaxel and PS/DNA complex to achieve their co-delivery and enhance internalization into hepatoma cells. FNAs showed the particle size about 200 nm with encapsulation efficiency >90%. Blank nanoassemblies (BNAs) loading only reporter gene revealed higher transfection efficiency with neglectable cytotoxicity compared with Lipofectamine™ 2000, which could result from enhanced cellular uptake via ligand-receptor recognition and efficient nuclear delivery mediated by PS. Cytotoxicity of FNAs against hepatocellular carcinoma cell line BEL 7402 was much higher than either docetaxel or non-docetaxel FNAs (nFNAs) loading only iSur-pDNA, and was also superior to the combined treatment with free docetaxel and nFNAs. Better antitumor efficacy of FNAs with low systemic toxicity was also observed on mouse hepatocellular carcinoma xenograft model. These results suggested that co-delivery of docetaxel and iSur-pDNA with FNAs could be a safer and more efficient strategy for the treatment of locally advanced and metastatic HCC.
Keywords: Hepatocellular carcinoma; Chemotherapy; Gene therapy; Co-delivery; Docetaxel; Survivin;

Polymeric micelles with cross-linked ionic cores of poly(methacrylic acid) and nonionic shell of poly(ethylene oxide) (cl-micelles) are shown here to readily internalize in epithelial cancer cells but not in normal epithelial cells that form tight junctions (TJ). The internalization of such cl-micelles in the cancer cells proceeded mainly through caveolae-mediated endocytosis. In confluent normal epithelial cells this endocytosis route was absent at the apical side and the cl-micelles sequestered in TJ regions of the cell membrane without entering the cells for at least 24 h. Disruption of the TJ by calcium deprivation resulted in redistribution of cl-micelles inside the cells. In cancer cells following initial cellular entry the cl-micelles bypassed the early endosomes and reached the lysosomes within 30 min. This allowed designing cl-micelles with cytotoxic drug, doxorubicin, linked via pH-sensitive hydrazone bonds, which were cleaved in the acidic environment of lysosomes resulting in accumulation of the drug in the nucleus after 5 h. Such pH-sensitive cl-micelles displayed selective toxicity to cancer cells but were non-toxic to normal epithelial cells. In conclusion, we describe major difference in interactions of cl-micelles with cancer and normal cells that can lead to development of novel drug delivery system with reduced side effects and higher efficacy in cancer chemotherapy.
Keywords: Nanomaterials; Endocytosis; Drug delivery; Tight junctions; Caveolae; Nanoparticulate materials; Nanoparticles;

Ring-opening metathesis polymerization-based synthesis of polymeric nanoparticles for enhanced tumor imaging in vivo: Synergistic effect of folate-receptor targeting and PEGylation by Koji Miki; Kazuaki Oride; Satoru Inoue; Yoshiaki Kuramochi; Rati R. Nayak; Hideki Matsuoka; Hiroshi Harada; Masahiro Hiraoka; Kouichi Ohe (934-942).
We have synthesized amphiphilic copolymers using ring-opening metathesis polymerization (ROMP), a copper-catalyzed dipolar click reaction, and osmium-catalyzed dihydroxylation. The resulting copolymers were easily conjugated with folate and dye (indocyanine green) moieties, using a transamidation method. The copolymers exhibited high water solubility and formed nanometer-sized self-assemblies in aqueous medium. The amphiphilic copolymers modified by dihydroxylation of the polymer backbone exhibited much lower cmc values than the non dihydroxylated copolymer. Copolymers conjugated with folate moieties reduced the fluorescence intensity of aqueous polymer solutions both in vitro and in vivo, but their self-assemblies efficiently accumulated at tumor sites because of folate-receptor recognition at tumor tissue. The PEGylation of copolymers improved the stability of the self-assemblies in aqueous medium as well as the tumor site selectivity in vivo. Furthermore, the fluorescent nanoparticles consisting of PEG- and folate-conjugated ROMP-based copolymers accumulated in tumor tissue selectively and efficiently, whereas accumulation in all other normal organs was reduced. The PEGylation and folate conjugation can synergistically improve the in vivo tumor site selectivity of ROMP-based copolymers.
Keywords: Tumor imaging; Amphiphilic copolymer; Micelle; Folate; Metathesis;

pH-Sensitive fusogenic polymer-modified liposomes as a carrier of antigenic proteins for activation of cellular immunity by Eiji Yuba; Chie Kojima; Atsushi Harada; Tana; Shinobu Watarai; Kenji Kono (943-951).
By modification of liposomes with poly(glycidol) derivatives such as succinylated poly(glycidol) and 3-methylglutarylated poly(glycidol), we have developed functional liposomes that generate fusion ability at mildly acidic pH. We investigated the feasibility of these polymer-modified liposomes as a carrier of antigenic proteins for induction of cellular immunity. These pH-sensitive fusogenic liposomes encapsulating ovalbumin (OVA) were applied to DC2.4 cells, a murine dendritic cell line. Observation with confocal laser scanning microscopy showed that these polymer-modified liposomes were taken up efficiently by the cells, thereafter delivering their contents into the cytosol, probably through fusion with endosomal membranes. Murine bone marrow-derived dendritic cells treated with polymer-modified liposomes encapsulating OVA stimulated CD8-OVA1.3 cells more strongly than OT4H.1D5 cells, indicating that the liposomes induced MHC class I-restricted presentation. Furthermore, administration of the polymer-modified, OVA-loaded liposomes from nasal cavities of mice induced stronger cellular immune responses than the OVA-loaded plain liposomes. Because the ability of the polymer-modified liposomes to activate cellular immunity was comparable to that of Freund's complete adjuvant, which is a widely used adjuvant, they potentially have use in production of efficient vaccines for immunotherapy.
Keywords: Vaccine; Dendritic cell; pH-Sensitive polymer; Cytoplasmic delivery; Fusogenic liposome;

Polyethylenimine–PEG coated albumin nanoparticles for BMP-2 delivery by Sufeng Zhang; Cezary Kucharski; Michael R. Doschak; Walter Sebald; Hasan Uludağ (952-963).
Bone Morphogenetic Protein-2 (BMP-2) plays an important role in stimulating new bone formation, and has been utilized in clinical bone repair by implantation. In this study, we report a nanoparticulate (NP) system for BMP-2 delivery based on bovine serum albumin (BSA) NPs stabilized with a poly(ethylene glycol) modified polyethylenimine (PEI–PEG) coating. PEI–PEG with different PEG substitutions were synthesized, and the cell viability assay showed PEG substitution greatly reduced the cytotoxicity of the native PEI. Furthermore, PEI–PEG coated BSA NPs demonstrated smaller size and decreased zeta potential compared to PEI-coated NPs. The bioactivity of the encapsulated BMP-2 and the toxicity of PEI–PEG coated NPs were examined by the alkaline phosphatase (ALP) induction assay and the MTT assay, respectively, using human C2C12 cells. The results indicated that BMP-2 remained bioactive in NPs and PEI–PEG coating was advantageous in reducing the NP toxicity as compared to PEI. A 7-day pharmacokinetics study showed the BMP-2 retention in PEI–PEG coated NPs was similar to the uncoated NPs, but lower than that of the PEI-coated NPs. The osteoinductivity of BMP-2 delivered in NPs was determined by subcutaneous implantation in rats, and the results revealed that PEI–PEG coated BSA NPs induced significant de novo bone formation after implantation, while PEI-coated NPs demonstrated much less bone formation. We conclude that BMP-2 delivered by PEGylated PEI-coated BSA NPs displays favorable biocompatibility and promotes new bone formation after implantation.
Keywords: Bone morphogenetic protein-2; Polyethylenimine–PEG; Bovine serum albumin; Nanoparticle; In vivo; Ectopic bone formation;

Doxycycline loaded poly(ethylene glycol) hydrogels for healing vesicant-induced ocular wounds by SivaNaga S. Anumolu; Andrea S. DeSantis; Anupa R. Menjoge; Rita A. Hahn; John A. Beloni; Marion K. Gordon; Patrick J. Sinko (964-974).
Half mustard (CEES) and nitrogen mustard (NM) are commonly used surrogates and vesicant analogs of the chemical warfare agent sulfur mustard. In the current study, in situ forming poly(ethylene glycol) (PEG)-based doxycycline hydrogels are developed and evaluated for their wound healing efficacy in CEES and NM-exposed rabbit corneas in organ culture. The hydrogels, characterized by UV–Vis spectrophotometry, rheometry, and swelling kinetics, showed that the hydrogels are optically transparent, have good mechanical strength and a relatively low degree of swelling (<7%). In vitro doxycycline release from the hydrogel disks (0.25% w/v) was found to be biphasic with release half times of ∼12 and 72 h, respectively, with 80–100% released over a 7-day period. Permeation of doxycycline through vesicant wounded corneas was found to be 2.5 to 3.4 fold higher than non-wounded corneas. Histology and immunofluorescence studies showed a significant reduction of matrix metalloproteinase-9 (MMP-9) and improved healing of vesicant-exposed corneas by doxycycline hydrogels compared to a similar dose of doxycycline delivered in phosphate buffered saline (PBS, pH 7.4). In conclusion, the current studies demonstrate that the doxycycline-PEG hydrogels accelerate corneal wound healing after vesicant injury offering a therapeutic option for ocular mustard injuries.
Keywords: Hydrogel; Ocular drug delivery; Doxycycline; Poly(ethylene glycol); Cornea organ culture model; Sustained release;

Molecular design of chitosan gene delivery systems with an optimized balance between polyplex stability and polyplex unpacking by Sabina P. Strand; Sylvie Lelu; Nina K. Reitan; Catharina de Lange Davies; Per Artursson; Kjell M. Vårum (975-987).
Chitosan is an attractive gene delivery vehicle, but the criteria and strategies for the design of efficient chitosan gene delivery systems remain unclear. The purpose of this work was to investigate how the strength of the charge-based interaction between chitosan and DNA determines the gene expression levels and to design chitosan vectors with an optimized balance between polyplex stability and polyplex unpacking. Using 21 formulations based on low molecular weight chitosans with constant charge density and a number-average degree of polymerization (DPn) in the range of 21–88 (M w 4.7–33 kDa), we studied the relationship between the chain length and the formulation properties, cellular uptake of polyplexes and gene transfer efficacy. We were able to identify a narrow interval of DPn31-42 that mediated the maximum level of transgene expression. An increase in chain length and/or the amino-phosphate (A/P) ratio reduced and delayed transgene expression. Compared to DPn31, transfection with the same amount of DPn72 or DPn88 resulted in 10-fold-lower expression levels. The gene transfer pattern correlated with the ability of heparin to release DNA from the polyplexes. As a tool to facilitate the unpacking of the polyplexes, we substituted the chitosans with uncharged oligosaccharides that reduced the interaction with DNA. The substitution of chitosans that originally yielded too stable polyplexes, such as DPn72 and DPn88 resulted in a 5–10-fold enhancement of the expression levels. However, the substitution of chitosans shorter than DP28 completely abolished transfection. Tailoring of the chain length and the substitution of chitosan were shown to be feasible tools to modulate the electrostatic interactions between the chitosan and DNA and to design chitosans with an optimized balance between polyplex stability and polyplex unpacking.
Keywords: Chitosan; Gene transfer; Nanoparticles; DNA;

Biodegradable branched poly(ethylenimine sulfide) for gene delivery by Heebeom Koo; Geun-woo Jin; Hyunseo Kang; Yan Lee; Kihoon Nam; Cheng Zhe Bai; Jong-Sang Park (988-997).
We synthesized biodegradable b-PEIS (branched poly(ethylenimine sulfide)) by crosslinking linear PEIS. We controlled the degree of crosslinking and molecular weight by adjusting the amount of the crosslinker, bisepoxide. The b-PEIS was readily degradable under reductive conditions (5 mm glutathione solution) and the degradation time was dependent on the degree of crosslinking. We controlled the molecular weights of the b-PEIS by regulating the amount of crosslinker and thus, the degree of crosslinking. Our titration data showed that there was almost no loss in buffering ability before or after bisepoxide crosslinking. We verified the degradation of this polymer by MALLS and gel electrophoresis, and confirmed that there was a high transfection efficiency and low cytotoxicity based on cellular data. Intracellular trafficking was observed by image restoration microscopy, demonstrating that b-PEIS does not accumulate in the cell interior.
Keywords: Gene transfer; Biodegradation; Cytotoxicity; Cell viability; Biocompatibility;

Tissue-specific gene delivery via nanoparticle coating by Todd J. Harris; Jordan J. Green; Peter W. Fung; Robert Langer; Daniel G. Anderson; Sangeeta N. Bhatia (998-1006).
The use of biomaterials for gene delivery can potentially avoid many of the safety concerns with viral gene delivery. However, the efficacy of polymeric gene delivery methods is low, particularly in vivo. One significant concern is that the interior and exterior composition of polymeric gene delivery nanoparticles are often coupled, with a single polymer backbone governing all functions from biophysical properties of the polymer/DNA particle to DNA condensation and release. In this work we develop electrostatically adsorbed poly(glutamic acid)-based peptide coatings to alter the exterior composition of a core gene delivery particle and thereby affect tissue-specificity of gene delivery function in vivo. We find that with all coating formulations tested, the coatings reduce potential toxicity associated with uncoated cationic gene delivery nanoparticles following systemic injection. Particles coated with a low 2.5:1 peptide:DNA weight ratio (w/w) form large 2 μ sized particles in the presence of serum that can facilitate specific gene delivery to the liver. The same particles coated at a higher 20:1 w/w form small 200 nm particles in the presence of serum that can facilitate specific gene delivery to the spleen and bone marrow. Thus, variations in nanoparticle peptide coating density can alter the tissue-specificity of gene delivery in vivo.
Keywords: Nanoparticle; Targeting; Gene delivery; Polymer; Peptide; Self-assembly;

There is increasing attention in the unique biological and medical properties of graphene, and it is expected that biomaterials incorporating graphene will be developed for the graphene-based drug delivery systems and biomedical devices. Despite the importance of biomolecules–graphene interactions, a detailed understanding of the adsorption mechanism and features of biomolecules onto the surfaces of graphene is lacking. To address this, we have performed density functional theory (DFT) and molecular dynamics (MD) methods exploring the adsorption geometries, adsorption energies, electronic band structures, adsorption isotherms, and adsorption dynamics of l-leucine (model biomolecule)/graphene composite system. DFT calculations confirmed the energetic stability of adsorption model and revealed that electronic structure of graphene can be controlled by the adsorption direction of l-leucine. MD simulations further investigate the potential energy and van der Waals energy for the interaction processes of l-leucine/graphene system at different temperatures and pressures. We find that the van der Waals interaction between the l-leucine and the graphene play a dominant role in the adsorption process under a certain range of temperature and pressure, and the l-leucine molecule could be adsorbed onto graphene spontaneously in aqueous solution.
Keywords: Graphene; l-Leucine; Adsorption; Density functional theory; Molecular dynamics;