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Biomaterials (v.33, #15)

Editorial board (pp. ifc).

Mussel-inspired silver-releasing antibacterial hydrogels by Dominic E. Fullenkamp; José G. Rivera; Yong-kuan Gong; K.H. Aaron Lau; Lihong He; Rahul Varshney; Phillip B. Messersmith (pp. 3783-3791).
A silver-releasing antibacterial hydrogel was developed that simultaneously allowed for silver nanoparticle formation and gel curing. Water-soluble polyethylene glycol (PEG) polymers were synthesized that contain reactive catechol moieties, inspired by mussel adhesive proteins, where the catechol containing amino acid 3,4-dihydroxyphenylalanine (DOPA) plays an important role in the ability of the mussel to adhere to almost any surface in an aqueous environment. We utilized silver nitrate to oxidize polymer catechols, leading to covalent cross-linking and hydrogel formation with simultaneous reduction of Ag(I). Silver release was sustained for periods of at least two weeks in PBS solution. Hydrogels were found to inhibit bacterial growth, consistent with the well-known antibacterial properties of silver, while not significantly affecting mammalian cell viability. In addition, thin hydrogel films were found to resist bacterial and mammalian cell attachment, consistent with the antifouling properties of PEG. We believe these materials have a strong potential for antibacterial biomaterial coatings and tissue adhesives, due to the material-independent adhesive properties of catechols.

Keywords: Polyethylene oxide; Silver; Hydrogel; Antibacterial; Biomimetic material; Nanoparticle

Macrophage polarization: An opportunity for improved outcomes in biomaterials and regenerative medicine by Bryan N. Brown; Buddy D. Ratner; Stuart B. Goodman; Salomon Amar; Stephen F. Badylak (pp. 3792-3802).
The host response to biomaterials has been studied for decades. Largely, the interaction of host immune cells, macrophages in particular, with implanted materials has been considered to be a precursor to granulation tissue formation, the classic foreign body reaction, and eventual encapsulation with associated negative impacts upon device functionality. However, more recently, it has been shown that macrophages, depending upon context dependent polarization profiles, are capable of affecting both detrimental and beneficial outcomes in a number of disease processes and in tissue remodeling following injury. Herein, the diverse roles played by macrophages in these processes are discussed in addition to the potential manipulation of macrophage effector mechanisms as a strategy for promoting site-appropriate and constructive tissue remodeling as opposed to deleterious persistent inflammation and scar tissue formation.

Keywords: Foreign body response; Leukocyte; Macrophage; Biocompatibility

Temperature-responsive poly( N-isopropylacrylamide)-grafted microcarriers for large-scale non-invasive harvest of anchorage-dependent cells by Atsushi Tamura; Jun Kobayashi; Masayuki Yamato; Teruo Okano (pp. 3803-3812).
Cell cultivation on the surface of microcarriers in stirred suspension is an essential method for the large-scale culture of anchorage-dependent cells. For applying this method to the field of cell therapy and for obtaining a large number of intact cells, non-invasive cell harvest without proteolytic enzyme treatment is an advantageous method. In this regard, temperature-responsive microcarriers that bearing poly( N-isopropylacrylamide) (PIPAAm)-grafted chains on the outermost surface were developed for harvesting cultured cells by temperature alteration. PIPAAm-grafted beads with the various grafted amount of PIPAAm and various bead diameters were synthesized for optimizing cell proliferation and thermally-induced detachment on the surface. The chinese hamster ovary (CHO-K1) cells adhered on the surface of all PIPAAm-grafted beads at 37 °C, while the adhering cells were found to detach themselves from the surfaces at 20 °C. The efficiency of thermally-induced cell detachment increased with increasing the grafted amount of PIPAAm and the diameter of bead. An efficient cell proliferation on bead surfaces in stirred suspension culture and subsequent thermally-induced cell detachment were achieved by the precise regulation of both the grafted amount of PIPAAm and diameter of bead. The temperature-responsive microcarriers exhibiting temperature-dependent cell adhesion and detachment will be an attractive candidate for the large-scale cell culture of therapeutic cells.

Keywords: Suspension culture; Large-scale culture; Microcarrier; Poly(; N; -isopropylacrylamide); Temperature-sensitivity; Atom transfer radical polymerization

Osteoinductive hydroxyapatite-coated titanium implants by Ugo Ripamonti; Laura C. Roden; Louise F. Renton (pp. 3813-3823).
Previous studies have shown that heterotopic induction of bone formation by calcium phosphate-based macroporous constructs is set into motion by the geometry of the implanted substrata, i.e. a sequence of repetitive concavities assembled within the macroporous spaces. The aim of this study was to construct osteoinductive titanium implants that per se, and without the exogenous application of the osteogenic soluble molecular signals of the transforming growth factor-β supergene family, would initiate the induction of bone formation. To generate intrinsically osteoinductive titanium implants for translation in clinical contexts, titanium grade Ti-6A1-4V cylinders of 15 mm in length and 3.85 mm in diameter, with or without concavities, were plasma sprayed with crystalline hydroxyapatite resulting in a uniform layer of 30 μm in thickness. Before coating, experimental titanium implants were prepared with a sequence of 36 repetitive concavities 1600 μm in diameter and 800 μm in depth, spaced a distance of 1000 μm apart. Mandibular molars and premolars were extracted to prepare edentulous mandibular ridges for later implantation. Planar and geometric hydroxyapatite-coated titanium constructs were implanted in the left and right edentulized hemi-mandibles, respectively, after a healing period of 7–8 months, 3 per hemi-mandible. Three planar and three geometric implants were implanted in the left and right tibiae, respectively; additionally, planar and geometric constructs were also inserted in the rectus abdominis muscle. Six animals were euthanized at 30 and 90 days after implantation; one animal had to be euthanized 5 days after surgery and the remaining animal was euthanized 31 months after implantation. Undecalcified longitudinal sections were precision-sawed, ground and polished to 40–60 μm; all sections were stained with a modified Goldner’s trichrome. Undecalcified specimen block preparation was performed using the EXAKT precision cutting and grinding system. Histomorphometric analyses of bone in contact (BIC) showed that on day 30 there was no difference between the geometric vs. planar control implants; on day 90, the ratio of BIC to surface within the geometric implants was greater than on the standard planar implants in both mandibular and tibial sites; 31 months after implantation, selected concavities cut into the geometric implants harvested from the rectus abdominis muscle showed the spontaneous induction of bone formation with mineralized bone surfaced by osteoid seams. These data in non-human primates indicate that geometrically-constructed plasma-sprayed titanium implants are per se osteogenic, the concavities providing a unique microenvironment to initiate bone differentiation by induction.

Keywords: Primates; Biomimetic matrices; Inductive nanotopography; Plasma sprayed hydroxyapatites; Modified titanium surfaces; Myoblastic stem cells

Microfabrication of complex porous tissue engineering scaffolds using 3D projection stereolithography by Robert Gauvin; Ying-Chieh Chen; Jin Woo Lee; Pranav Soman; Pinar Zorlutuna; Jason W. Nichol; Hojae Bae; Shaochen Chen; Ali Khademhosseini (pp. 3824-3834).
The success of tissue engineering will rely on the ability to generate complex, cell seeded three-dimensional (3D) structures. Therefore, methods that can be used to precisely engineer the architecture and topography of scaffolding materials will represent a critical aspect of functional tissue engineering. Previous approaches for 3D scaffold fabrication based on top-down and process driven methods are often not adequate to produce complex structures due to the lack of control on scaffold architecture, porosity, and cellular interactions. The proposed projection stereolithography (PSL) platform can be used to design intricate 3D tissue scaffolds that can be engineered to mimic the microarchitecture of tissues, based on computer aided design (CAD). The PSL system was developed, programmed and optimized to fabricate 3D scaffolds using gelatin methacrylate (GelMA). Variation of the structure and prepolymer concentration enabled tailoring the mechanical properties of the scaffolds. A dynamic cell seeding method was utilized to improve the coverage of the scaffold throughout its thickness. The results demonstrated that the interconnectivity of pores allowed for uniform human umbilical vein endothelial cells (HUVECs) distribution and proliferation in the scaffolds, leading to high cell density and confluency at the end of the culture period. Moreover, immunohistochemistry results showed that cells seeded on the scaffold maintained their endothelial phenotype, demonstrating the biological functionality of the microfabricated GelMA scaffolds.

Keywords: Microfabrication; Three dimensional printing; Scaffold; Mechanical properties; Confocal microscopy; Cell proliferation

Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment by Wei Seong Toh; Teck Chuan Lim; Motoichi Kurisawa; Myron Spector (pp. 3835-3845).
An injectable and biodegradable hydrogel system comprising hyaluronic acid-tyramine (HA-Tyr) conjugates can safely undergo covalent cross-linking in vivo by the addition of small amounts of peroxidase and hydrogen peroxide (H2O2), with the independent tuning of the gelation rate and degree of cross-linking. Such hydrogel networks with tunable mechanical and degradation properties may provide the additional level of control needed to enhance chondrogenesis and overall cartilage tissue formation in vitro and in vivo. In this study, HA-Tyr hydrogels were explored as biomimetic matrices for caprine mesenchymal stem cells (MSCs) in cartilage tissue engineering. The compressive modulus, equilibrium swelling and degradation rate could be controlled by varying the concentration of H2O2 as the oxidant in the oxidative coupling reaction. Cellular condensation reflected by the increase in effective number density of rounded cells in lacunae was greater in softer hydrogel matrices with lower cross-linking that displayed enhanced scaffold contracture. Conversely, within higher cross-linked matrices, cells adopted a more elongated morphology, with a reduced degree of cellular condensation. Furthermore, the degree of hydrogel cross-linking also modulated matrix biosynthesis and cartilage tissue histogenesis. Lower cross-linked matrix enhanced chondrogenesis with increases in the percentage of cells with chondrocytic morphology; biosynthetic rates of glycosaminoglycan and type II collagen; and hyaline cartilage tissue formation. With increasing cross-linking degree and matrix stiffness, a shift in MSC differentiation toward fibrous phenotypes with the formation of fibrocartilage and fibrous tissues was observed. These findings suggest that the tunable three-dimensional microenvironment of the HA-Tyr hydrogels modulates cellular condensation during chondrogenesis and has a dramatic impact on spatial organization of cells, matrix biosynthesis, and overall cartilage tissue histogenesis.

Keywords: Hydrogel; Hyaluronic acid; Chondrogenesis; Mesenchymal stem cells; Cartilage; Tissue engineering

Cartilage repair in transplanted scaffold-free chondrocyte sheets using a minipig model by Goro Ebihara; Masato Sato; Masayuki Yamato; Genya Mitani; Toshiharu Kutsuna; Toshihiro Nagai; Satoshi Ito; Taku Ukai; Miyuki Kobayashi; Mami Kokubo; Teruo Okano; Joji Mochida (pp. 3846-3851).
Lacking a blood supply and having a low cellular density, articular cartilage has a minimal ability for self-repair. Therefore, wide-ranging cartilage damage rarely resolves spontaneously. Cartilage damage is typically treated by chondrocyte transplantation, mosaicplasty or microfracture. Recent advances in tissue engineering have prompted research on techniques to repair articular cartilage damage using a variety of transplanted cells. We studied the repair and regeneration of cartilage damage using layered chondrocyte sheets prepared on a temperature-responsive culture dish. We previously reported achieving robust tissue repair when covering only the surface layer with layered chondrocyte sheets when researching partial-thickness defects in the articular cartilage of domestic rabbits. The present study was an experiment on the repair and regeneration of articular cartilage in a minipig model of full-thickness defects. Good safranin-O staining and integration with surrounding tissues was achieved in animals transplanted with layered chondrocyte sheets. However, tissue having poor safranin-O staining—not noted in the domestic rabbit experiments—was identified in some of the animals, and the subchondral bone was poorly repaired in these. Thus, although layered chondrocyte sheets facilitate articular cartilage repair, further investigations into appropriate animal models and culture and transplant conditions are required.

Keywords: Cartilage tissue engineering; Chondrocyte; Cell culture; Animal model; Transplantation

Molecular mechanism of force induced stabilization of collagen against enzymatic breakdown by Shu-Wei Chang; Brendan P. Flynn; Jeffrey W. Ruberti; Markus J. Buehler (pp. 3852-3859).
Collagen cleavage, facilitated by collagenases of the matrix metalloproteinase (MMP) family, is crucial for many physiological and pathological processes such as wound healing, tissue remodeling, cancer invasion and organ morphogenesis. Earlier work has shown that mechanical force alters the cleavage rate of collagen. However, experimental results yielded conflicting data on whether applying force accelerates or slows down the degradation rate. Here we explain these discrepancies and propose a molecular mechanism by which mechanical force might change the rate of collagen cleavage. We find that a type I collagen heterotrimer is unfolded in its equilibrium state and loses its triple helical structure at the cleavage site without applied force, possibly enhancing enzymatic breakdown as each chain is exposed and can directly undergo hydrolysis. Under application of force, the naturally unfolded region refolds into a triple helical structure, potentially protecting the molecule against enzymatic breakdown. In contrast, a type I collagen homotrimer retains a triple helical structure even without applied force, making it more resistant to enzyme cleavage. In the case of the homotrimer, the application of force may directly lead to molecular unwinding, resulting in a destabilization of the molecule under increased mechanical loading. Our study explains the molecular mechanism by which force may regulate the formation and breakdown of collagenous tissue.

Keywords: Collagen; Collagenolysis; Collagen degradation; Molecular mechanics; Protein structure; Local unfolding

Bridging peripheral nerve defects with a tissue engineered nerve graft composed of an in vitro cultured nerve equivalent and a silk fibroin-based scaffold by Xin Tang; Chengbin Xue; Yaxian Wang; Fei Ding; Yumin Yang; Xiaosong Gu (pp. 3860-3867).
Tissue engineered nerve grafts are considered as a promising alternative to autologous nerve grafts used for peripheral nerve repair. The differences between these two types of nerve grafts are mainly in the regenerative microenvironment established by them. To construct ideal tissue engineered nerve grafts, it is therefore required to develop a better way to introduce biochemical cues into a neural scaffold, as compared to single or combined use of support cells and growth factors. Here, we used a co-culture system of dorsal root ganglia and Schwann cells to create an in vitro formed nerve equivalent, which was introduced into a silk fibroin-based scaffold to furnish a tissue engineered nerve graft (TENG). At 4- and 12- weeks after the TENG was implanted to bridge a 10-mm-long sciatic nerve defect in rats, histological and functional assessments as well as Western blot analysis were performed to evaluate the influences of the TENG on peripheral nerve regeneration. We found that at an early stage of nerve regeneration, the TENG significantly accelerated axonal growth, and up-regulated expressions of N-cadherin and PMP22. Twelve weeks after nerve grafting, the TENG produced a further improved outcome of nerve regeneration and functional recovery, which was more close to that of the autologous nerve graft than that of the silk fibroin-based scaffold. The introduction of an in vitro cultured nerve equivalent into a scaffold might contribute to establishing a native-like microenvironment for nerve regeneration.

Keywords: Tissue engineered nerve grafts; Peripheral nerve regeneration; In vitro; cultured nerve equivalent; Schwann cells; Dorsal root ganglia

The in vitro preconditioning of myoblasts to enhance subsequent survival in an in vivo tissue engineering chamber model by Daniel J. Tilkorn; E. Michele Davies; Effie Keramidaris; Aaron M. Dingle; Yi-Wen Gerrand; Caroline J. Taylor; Xiao Lian Han; Jason A. Palmer; Anthony J. Penington; Christina A. Mitchell; Wayne A. Morrison; Gregory J. Dusting; Geraldine M. Mitchell (pp. 3868-3879).
The effects of in vitro preconditioning protocols on the ultimate survival of myoblasts implanted in an in vivo tissue engineering chamber were examined. In vitro testing: L6 myoblasts were preconditioned by heat (42 °C; 1.5 h); hypoxia (<8% O2; 1.5 h); or nitric oxide donors: S-nitroso-N-acetylpenicillamine (SNAP, 200 μM, 1.5 h) or 1-[N-(2-aminoethyl)-N-(2-aminoethyl)amino]-diazen-1-ium-1,2-diolate (DETA-NONOate, 500 μM, 7 h). Following a rest phase preconditioned cells were exposed to 24 h hypoxia, and demonstrated minimal overall cell loss, whilst controls (not preconditioned, but exposed to 24 h hypoxia) demonstrated a 44% cell loss. Phosphoimmunoblot analysis of pro-survival signaling pathways revealed significant activation of serine threonine kinase Akt with DETA-NONOate ( p < 0.01) and heat preconditioning ( p < 0.05). DETA-NONOate also activated ERK 1/2 signaling ( p < 0.05). In vivo implantation: 100,000 preconditioned (heat, hypoxia, or DETA-NONOate) myoblasts were implanted in SCID mouse tissue engineering chambers. 100,000 (not preconditioned) myoblasts were implanted in control chambers. At 3 weeks, morphometric assessment of surviving myoblasts indicated myoblast percent volume ( p = 0.012) and myoblasts/mm2 ( p = 0.0005) overall significantly increased in preconditioned myoblast chambers compared to control, with DETA-NONOate-preconditioned myoblasts demonstrating the greatest increase in survival ( p = 0.007 and p = 0.001 respectively). DETA-NONOate therefore has potential therapeutic benefits to significantly improve survival of transplanted cells.

Keywords: Cell preconditioning; In vitro; test of cell survival; Implantation in an; in vivo; tissue engineering chamber; Morphometric assessment of cell survival

Dual functionalized PVA hydrogels that adhere endothelial cells synergistically by Marjan Rafat; Lisa S. Rotenstein; Jin-Oh You; Debra T. Auguste (pp. 3880-3886).
Cell adhesion molecules govern leukocyte-endothelial cell (EC) interactions that are essential in regulating leukocyte recruitment, adhesion, and transmigration in areas of inflammation. In this paper, we synthesized hydrogel matrices modified with antibodies against vascular cell adhesion molecule-1 (VCAM1) and endothelial leukocyte adhesion molecule-1 (E-Selectin) to mimic leukocyte-EC interactions. Adhesion of human umbilical vein ECs to polyvinyl alcohol (PVA) hydrogels was examined as a function of the relative antibody ratio (anti-VCAM1:anti-E-Selectin) and substrate elasticity. Variation of PVA backbone methacrylation was used to affect hydrogel matrix stiffness, ranging from 130 to 720 kPa. Greater EC adhesion was observed on hydrogels presenting 1:1 anti-VCAM1:anti-E-Selectin than on gels presenting either arginine-glycine-asparagine (RGD) peptide, anti-VCAM1, or anti-E-Selectin alone. Engineered cell adhesion - based on complementing the EC surface presentation - may be used to increase the strength of EC-matrix interactions. Hydrogels with tunable and synergistic adhesion may be useful in vascular remodeling.

Keywords: Cell adhesion; Polyvinyl alcohol; Photopolymerization; Endothelial cell; Hydrogel; Adhesion molecule

Tissue regeneration in vivo within recombinant spidroin 1 scaffolds by Mikhail M. Moisenovich; Olga Pustovalova; Julia Shackelford; Tamara V. Vasiljeva; Tatiana V. Druzhinina; Yana A. Kamenchuk; Vitaly V. Guzeev; Olga S. Sokolova; Vladimir G. Bogush; Vladimir G. Debabov; Mikhail P. Kirpichnikov; Igor I. Agapov (pp. 3887-3898).
One of the major tasks of tissue engineering is to produce tissue grafts for the replacement or regeneration of damaged tissue, and natural and recombinant silk-based polymer scaffolds are promising candidates for such grafts. Here, we compared two porous scaffolds made from different silk proteins, fibroin of Bombyx mori and a recombinant analog of Nephila clavipes spidroin 1 known as rS1/9, and their biocompatibility and degradation behavior in vitro and in vivo. The vascularization and intergrowth of the connective tissue, which was penetrated with nerve fibers, at 8 weeks after subcutaneous implantation in Balb/c mice was more profound using the rS1/9 scaffolds. Implantation of both scaffolds into bone defects in Wistar rats accelerated repair compared to controls with no implanted scaffold at 4 weeks. Based on the number of macrophages and multinuclear giant cells in the subcutaneous area and the number of osteoclasts in the bone, regeneration was determined to be more effective after the rS1/9 scaffolds were implanted. Microscopic examination of the morphology of the matrices revealed differences in their internal microstructures. In contrast to fibroin-based scaffolds, the walls of the rS1/9 scaffolds were visibly thicker and contained specific micropores. We suggest that the porous inner structure of the rS1/9 scaffolds provided a better micro-environment for the regenerating tissue, which makes the matrices derived from the recombinant rS1/9 protein favorable candidates for future in vivo applications.

Keywords: Silk-based polymer scaffolds; Fibroin; Recombinant spidroin; Tissue engineering; In vivo; regeneration

A dual-targeting nanocarrier based on poly(amidoamine) dendrimers conjugated with transferrin and tamoxifen for treating brain gliomas by Yan Li; Hai He; Xinru Jia; Wan-Liang Lu; Jinning Lou; Yen Wei (pp. 3899-3908).
A pH-sensitive dual-targeting drug carrier (G4-DOX-PEG-Tf-TAM) was synthesized with transferrin (Tf) conjugated on the exterior and Tamoxifen (TAM) in the interior of the fourth generation PAMAM dendrimers for enhancing the blood–brain barrier (BBB) transportation and improving the drug accumulation in the glioma cells. It was found that, on average, 7 doxorubicine (DOX) molecules, over 30 PEG1000 and PEG2000 chains and one Tf group were bonded on the periphery of each G4 PAMAM dendrimer, while 29 TAM molecules were encapsulated into the interior of per dendrimer. The pH-triggered DOX release was 32% at pH 4.5 and 6% at pH 7.4, indicating a comparatively fast drug release at weak acidic condition and stable state of the carrier at physiological environment. The in vitro assay of the drug transport across the BBB model showed that G4-DOX-PEG-Tf-TAM exhibited higher BBB transportation ability with the transporting ratio of 6.06% in 3 h. The carrier was internalized into C6 glioma cells upon crossing the BBB model by the coactions of TfR-mediated endocytosis and the inhibition effect of TAM to the drug efflux transports. Moreover, it also displayed the in vitro accumulation of DOX in the avascular C6 glioma spheroids made the tumor volume effectively reduced.

Keywords: Blood-brain barrier (BBB); PAMAM; Dual-targeting; pH-sensitive; Gliomas

Prevention of colorectal cancer liver metastasis by exploiting liver immunity via chitosan-TPP/nanoparticles formulated with IL-12 by Qiongming Xu; Lingchuan Guo; Xinhua Gu; Biao Zhang; Xin Hu; Jiajia Zhang; Jinghong Chen; Yi Wang; Cheng Chen; Bei Gao; Yuting Kuang; Shouli Wang (pp. 3909-3918).
The development of effective therapies for the prevention of colorectal cancer (CRC) liver metastasis is of great importance. Recently, chitosan (CS) nanoparticles have been utilized as carriers of interluekin-12 (IL-12) administered locally to deliver therapeutic proteins and genes. In this study, we encapsulated IL-12 by incorporation using tripolyphosphate (TPP) as the coacervated crosslinking agent to form CS-TPP/IL-12 nanoparticles. We further characterized the association efficiency, rate of release, liver-targeting, and toxicity, which were predominantly dependent on the factors of particle size, zeta potential, pH of solution, and whether or not modified with TPP. Systemic delivery of CS-TPP/IL-12 nanoparticles significantly reduced the number and volume of CRC liver metastasis foci compared to the CS-TPP treated mouse group. Although delivery of IL-12 alone also inhibited the number of CRC liver metastasis observed, further study of the change in hepatic metastasis volume demonstrated no significant differences between the groups treated with CS-TPP or IL-12 alone. Mechanistically, CS-TPP nanoparticles blocked the toxicity of IL-12 and induced infiltration of NK cells and some T cells, which are most likely the effector cells that mediate tumor metastasis inhibition during CS-TPP/IL-12 immunotherapy. The results obtained from this study demonstrate the potential benefit of using chitosan modification technology as a cytokine delivery system for the successful prevention of CRC liver metastasis by exploiting liver immunity.

Keywords: Chitosan; IL-12; Nanoparticles; Colorectal cancer; Metastasis; Systemic delivery

An epirubicin–conjugated nanocarrier with MRI function to overcome lethal multidrug-resistant bladder cancer by Hung-Wei Yang; Mu-Yi Hua; Hao-Li Liu; Rung-Ywan Tsai; See-Tong Pang; Po-Hong Hsu; Hsiang-Jun Tang; Tzu-Chen Yen; Cheng-Keng Chuang (pp. 3919-3930).
Multidrug resistance (MDR) presents a major obstacle to curing cancer. Chemotherapy failure can occur through both cell membrane drug resistance (CMDR) and nuclear drug resistance (NDR), and anticancer effectiveness of chemotherapeutic agents is especially reduced by their nuclear export. Here we report an exciting magnetically-targeted nanomedicine formed by conjugation of epirubicin (EPI) to non-toxic and high-magnetization nanocarrier (HMNC). Strikingly, HMNC-EPI overcomes both CMDR and NDR in human bladder cancer cell models, without using P-glycoprotein (P-gp) and nuclear pore inhibitors. Besides, the half-life of drug is prolonged ∼1.8-fold (from 45 h to 81 h) at 37 °C, with a ∼10-fold increase in concentration at the tumor site through magnetic targeting (MT). Moreover, malignant NDR bladder cancer can be effectively inhibited after 14 days in mice by just two injections and MT. We are the first to demonstrate the nanomedical strategy that can overcome the CMDR and NDR bladder cancers simultaneously, and proceed to the excellent MT therapy, significantly reducing the dosage and cardiotoxicity and holding great promise for incurable human MDR bladder cancer.

Keywords: Bladder cancer; Chemotherapy; Multidrug resistance; Magnetic targeting; MRI

Tumor-targeted drug delivery using MR-contrasted docetaxel – Carboxymethylcellulose nanoparticles by Mark J. Ernsting; Warren D. Foltz; Elijus Undzys; Tatsuaki Tagami; Shyh-Dar Li (pp. 3931-3941).
A carboxymethylcellulose-based polymer conjugate with nanoparticle forming properties (Cellax) has been shown to enhance the pharmacokinetics, specificity of biodistribution, anti-tumor efficacy and safety of docetaxel (DTX) in comparison to the Taxotere™ formulation. We examined Cellax and Taxotere efficacy in four tumor models (EMT-6, B16F10, PC3, and MDA-MB-231), and observed variances in efficacy. To explore whether differences in tumor uptake of Cellax were responsible for these effects, we incorporated superparamagnetic iron oxide nanoparticles (SPIONs) into Cellax particles to enable magnetic resonance (MR) imaging (Cellax-MR). In the EMT-6 tumor model, Cellax-MR nanoparticles exhibited peak tumor accumulation 3–24 h post intravenous injection, and 3 days post-treatment, significant MR contrast was still detected. The amount of Cellax-MR deposited in the EMT-6 tumors was quantifiable as a hypointense volume fraction, a value positively correlated with drug content as determined by LC/MS analysis ( R2 = 0.97). In the four tumor models, Cellax-MR uptake was linearly associated with anti-tumor efficacy ( R2 > 0.9), and was correlated with blood vessel density ( R2 > 0.9). We have affirmed that nanoparticle uptake is variable in tumor physiology, and that this efficacy-predictive parameter can be non-invasively estimated in real-time using a theranostic variant of Cellax.

Keywords: Docetaxel; Carboxymethylcellulose; Conjugated Polymers; Magnetic nanoparticles

Polyethyleneimine-lipid conjugate-based pH-sensitive micellar carrier for gene delivery by Rupa R. Sawant; Shravan Kumar Sriraman; Gemma Navarro; Swati Biswas; Riddhi A. Dalvi; Vladimir P. Torchilin (pp. 3942-3951).
A low molecular weight polyethyleneimine (PEI 1.8 kDa) was modified with dioleoylphosphatidylethanolamine (PE) to form the PEI-PE conjugate investigated as a transfection vector. The optimized PEI-PE/pDNA complexes at an N/P ratio of 16 had a particle size of 225 nm, a surface charge of +31 mV, and protected the pDNA from the action of DNase I. The PEI-PE conjugate had a critical micelle concentration (CMC) of about 34 μg/ml and exhibited no toxicity compared to a high molecular weight PEI (PEI 25 kDa) as tested with B16-F10 melanoma cells. The B16-F10 cells transfected with PEI-PE/pEGFP complexes showed protein expression levels higher than with PEI-1.8 or PEI-25 vectors. Complexes prepared with YOYO 1-labeled pEGFP confirmed the enhanced delivery of the plasmid with PEI-PE compared to PEI-1.8 and PEI-25. The PEI-PE/pDNA complexes were also mixed with various amounts of micelle-forming material, polyethylene glycol (PEG)-PE to improve biocompatibility. The resulting particles exhibited a neutral surface charge, resistance to salt-induced aggregation, and good transfection activity in the presence of serum in complete media. The use of the low-pH-degradable PEG-hydrazone-PE produced particles with transfection activity sensitive to changes in pH consistent with the relatively acidic tumor environment.

Keywords: Polyethyleneimine; Transfection; pH-sensitive; Micelles; Cancer

Post-nuclear gene delivery events for transgene expression by biocleavable polyrotaxanes by Yuma Yamada; Taku Nomura; Hideyoshi Harashima; Atsushi Yamashita; Nobuhiko Yui (pp. 3952-3958).
A quantitative comparison between nuclear DNA release from carriers and their transfection activity would be highly useful for improving the effectiveness of non-viral gene vectors. We previously reported that, for condensed DNA particles, a close relationship exists between the efficiency of DNA release and transfection activity, when biocleavable polyrotaxanes (DMAE-SS-PRX), in which the cationic density can be easily controlled. In this study, we first investigated the efficiencies of DNA release from condensed DNA particles with various types of DMAE-SS-PRX. The findings indicate that an optimal cationic density in DMAE-SS-PRX exists for DNA release. We then packaged condensed DNA particles in a multifunctional envelope-type nano device (MEND), and evaluated their transfection activities. The results showed that the transfection activity was increased and this increase was, to some extent, dependent on the efficiency of the DNA release. However, transfection activity decreased, when the value for the efficiency of DNA release was higher than a certain value. An investigation of the fate of intranuclear DNA indicated that a very high efficiency of DNA release has a positive influence on transcription, however, it would inhibit the post-transcription process; nuclear mRNA export, translation and related processes. Such information provides a new viewpoint for the development of cationic polymer-based vectors.

Keywords: Non-viral vector; Multifunctional envelope-type nano device; Biocleavable polyrotaxane; Nuclear DNA release; Transgene expression

The targeted intracellular delivery of cytochrome C protein to tumors using lipid-apolipoprotein nanoparticles by Sang Kyoon Kim; Michael B. Foote; Leaf Huang (pp. 3959-3966).
Intracellular-acting therapeutic proteins offer a promising clinical alternative to extracellular-acting agents, but are limited in efficacy by their low permeability into the cell cytoplasm. We have developed a nanoparticle (NP) composed of lipid (DOTAP/DOPE) and apolipoprotein (APOA-I) to mediate the targeted delivery of intracellular-acting protein drugs to non-small cell lung tumors. NPs were produced with either GFP, a fluorescent model protein, or cytochrome C (cytC), an inducer of apoptosis in cancer cells. GFP and cytC were separately conjugated with a membrane permeable sequence (MPS) peptide and were admixed with DOPE/DOTAP nanoparticle formulations to enable successful protein loading. Protein-loaded NPs were modified with DSPE-PEG-Anisamide to enable specific NP targeting to the tumor site in a xenograft model. The resulting particle was 20–30 nm in size and exhibited a 64–75% loading efficiency. H460 cells treated with the PEGylated MPS-cytC-NPs exhibited massive apoptosis. When MPS-GFP-NPs or MPS-cytC-NPs were intravenously administered in H460 tumor bearing mice, a specific tumor targeting effect with low NP accumulation in the liver was observed. In addition, MPS-cytC-NP treatment provoked a tumor growth retardation effect in H460 xenograft mice. We conclude that our NP enables targeted, efficacious therapeutic protein delivery for the treatment of lung cancer.

Keywords: Nanoparticle; Liposome; Apolipoprotein; PEGylation; Protein delivery; Anisamide

The potential of laminin-2-biomimetic short peptide to promote cell adhesion, spreading and migration by inducing membrane recruitment and phosphorylation of PKCδ by Sung Youn Jung; Jin-Man Kim; Seung-Ki Min; O. Bok Kim; Da Hyun Jang; Hyun Ki Kang; Byung-Moo Min ∗ (pp. 3967-3979).
Laminin α2 chain plays an important role in basement membrane assembly and peripheral myelinogenesis; however, the integrin binding motif within human laminin α2 chain and the signaling pathways downstream of this ligand–receptor interaction are poorly understood. We identified a motif, RNIPPFEGCIWN (Ln2-LG3-P2), within LG3 domain of human laminin α2 chain as a major site for both α3β1 integrin and cellular activities such as cell adhesion, spreading, and migration. Binding of α3β1 integrin with Ln2-LG3-P2 induced the membrane recruitment of protein kinase Cδ (PKCδ) and stimulated its tyrosine phosphorylation. The cellular activities induced by Ln2-LG3-P2 and the phosphorylation of focal adhesion kinase (FAK) were inhibited by rottlerin, a PKCδ inhibitor, but not by Gö6976, a PKCα/β inhibitor. These results indicate that RNIPPFEGCIWN motif within human laminin α2 chain is a major ligand for α3β1 integrin, and that binding of α3β1 integrin mediates cellular activities through membrane recruitment and tyrosine phosphorylation of PKCδ and FAK phosphorylation.

Keywords: Laminin-2; RNIPPFEGCIWN motif; Cellular activity; Integrin α3β1/PKCδ/FAK signaling; Peripheral nerve regeneration

Tumor-targeting hyaluronic acid nanoparticles for photodynamic imaging and therapy by Hong Yeol Yoon; Heebeom Koo; Ki Young Choi; So Jin Lee; Kwangmeyung Kim; Ick Chan Kwon; James F. Leary; Kinam Park; Soon Hong Yuk; Jae Hyung Park; Kuiwon Choi (pp. 3980-3989).
Tumor-targeted imaging and therapy have been the challenging issue in the clinical field. Herein, we report tumor-targeting hyaluronic acid nanoparticles (HANPs) as the carrier of the hydrophobic photosensitizer, chlorin e6 (Ce6) for simultaneous photodynamic imaging and therapy. First, self-assembled HANPs were synthesized by chemical conjugation of aminated 5β-cholanic acid, polyethylene glycol (PEG), and black hole quencher3 (BHQ3) to the HA polymers. Second, Ce6 was readily loaded into the HANPs by a simple dialysis method resulting in Ce6-loaded hyaluronic acid nanoparticles (Ce6-HANPs), wherein in the loading efficiency of Ce6 was higher than 80%. The resulting Ce6-HANPs showed stable nano-structure in aqueous condition and rapid uptake into tumor cells. In particular Ce6-HANPs were rapidly degraded by hyaluronidases abundant in cytosol of tumor cells, which may enable intracellular release of Ce6 at the tumor tissue. After an intravenous injection into the tumor-bearing mice, Ce6-HANPs could efficiently reach the tumor tissue via the passive targeting mechanism and specifically enter tumor cells through the receptor-mediated endocytosis based on the interactions between HA of nanoparticles and CD44, the HA receptor on the surface of tumor cells. Upon laser irradiation, Ce6 which was released from the nanoparticles could generate fluorescence and singlet oxygen inside tumor cells, resulting in effective suppression of tumor growth. Overall, it was demonstrated that Ce6-HANPs could be successfully applied to in vivo photodynamic tumor imaging and therapy simultaneously.

Keywords: Photodynamic therapy; Imaging; Tumor-targeting; Nanoparticle; Hyaluronic acid; Chlorin e6

The effect of molecular weight, compositions and lectin type on the properties of hyperbranched glycopolymers as non-viral gene delivery systems by Marya Ahmed; Ravin Narain (pp. 3990-4001).
The architectures of gene delivery vectors, in addition to their molecular weights and compositions, can play a critical role in DNA condensation and hence on their gene expression. In general, branched polymers are superior gene delivery vectors as compared to their linear analogs. This study reports the efficacy of cationic hyperbranched glycopolymers for DNA condensation and gene expression. Hyperbranched glycopolymers of varying molecular weights and compositions are synthesized via reversible addition fragmentation chain transfer (RAFT) process and are further explored for their gene expression in vitro. Galactose-based hyperbranched polymers are compared to glucose-derived hyperbranched polymers for their cellular uptake, toxicity and gene expression. It is found that molecular weight of hyperbranched polymers, and carbohydrate content of copolymers are critical factors in determining the gene expression as well as in imparting the specificity to these novel gene delivery vectors. The galactose-based hyperbranched glycopolymer of ∼30 kDa or lower show improved gene expression at varying polymer/plasmid ratios. The incubation of hyperbranched polyplexes in the presence of serum protein show the presence of stable particles and gene expression of these hyperbranched polyplexes is unaffected in the presence of serum proteins. Furthermore, the cellular uptake and gene expression are studied in two different cell lines in the presence of lectins. It is found that polyplexes-lectin conjugates show enhanced cellular uptake in vitro, however their gene expression is cell line and lectin type dependent.

Keywords: Hyperbranched glycopolymers; Reversible addition fragmentation chain transfer (RAFT); Gene delivery; Lectin induced cellular uptake; Specificity

The inhibition of Th17 immune response in vitro and in vivo by the carbosilane dendrimer 2G-NN16 by Rafael Gras; Miguel Relloso; María I. García; F. Javier de la Mata; Rafael Gómez; Luis A. López-Fernández; M. Angeles Muñoz-Fernández (pp. 4002-4009).
We evaluated the 2G-NN16-carbosilane dendrimer activities in Th17 response as a potential therapy for Th17 deregulated pathologies. IL17A, IL17F, IL22, IL23 and other interleukins secreted by Th17 cells CD4+ cells were down regulated when cells were cultured in the presence of this dendrimer. Furthermore, IL17F and IL17A protein levels in splenocytes from mice pretreated with 2G-NN16 dendrimer in a Th17 induction mouse model were lower than those corresponding to PBS treated mice. Treatment of mice with 2G-NN16 inhibited the Th17 response causing much more pathogenicity as indicated by the increase in the number of Candida albicans colonies in the kidneys as compared to PBS-treated mice. All these results suggest a potential pharmacological application for this dendrimer in the therapy of Th17-mediated diseases.

Keywords: Th17; Carbosilane dendrimer; Th17 mouse model; IL17F; IL17AAbbreviation; 2G-NN16; second generation ammonium-terminated carbosilane dendrimer

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