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Biomaterials (v.32, #35)

Editorial board (pp. ifc).

Oh the irony: Iron as a cancer cause or cure? by Susan P. Foy; Vinod Labhasetwar (pp. 9155-9158).
Iron-oxide nanoparticles facilitate cancer diagnosis through enhanced contrast, selectively enhance tumor cell death with magnetic hyperthermia, and improve drug delivery with magnetic drug targeting. One application that remains largely unexplored is using the iron-oxide nanoparticles themselves to selectively inhibit tumor growth. In this leading opinion paper, we propose that high doses of iron-oxide nanoparticles can be used as a treatment for cancer by generating an oxidative assault against cancer. This proposal may be met with resistance considering the controversy surrounding iron in the field of cancer. Iron generates reactive oxygen species through the Fenton reaction, which may both cause – or cure cancer. Additionally, high demand for iron by cancer cells leads to contradictory therapeutic approaches: iron deprivation or overdose are both potential cancer therapies.

Keywords: Cancer therapy; Iron; Magnetic nanoparticles; Oxidative stress; Reactive oxygen species

Pathways of macrophage apoptosis within the interface membrane in aseptic loosening of prostheses by Fei Yang; Wen Wu; Lei Cao; Yan Huang; Zhenan Zhu; Tingting Tang; Kerong Dai (pp. 9159-9167).
Aseptic loosening is a major cause of failure of total hip arthroplasty (THA). Macrophage apoptosis in interface membrane has been proved to play an important role in the pathogenesis of aseptic loosening. The purpose of current study was to identify the apoptotic mechanism of macrophages in the interface membrane of aseptic loosening. We collected periprosthetic interface membrane from 23 patients undergoing the revision operations for aseptic loosening of hip joint prostheses. To serve as the control group, samples of capsule were collected from 18 patients undergoing the primary hip arthroplasties for osteoarthritis (OA). The ultrastructure of interface membrane was examined by transmission electron microscopy (TEM), and in situ apoptotic macrophage identification was performed by TUNEL staining. Furthermore, using immunohistochemical methods we investigated the expression of some apoptosis-related markers such as inducible nitric oxide synthase (iNOS), peroxynitrite (ONOO), cleaved caspase-3/4/8/9, cytochrome c, glucose regulated protein 78 (GRP78), and growth arrest and DNA damage-inducible gene 153 (GADD153) in macrophages. These markers were regarded as apoptotic inducers or specific indicators of different apoptotic pathways such as death receptor pathway, mitochondrial pathway and endoplasmic reticulum (ER) stress pathway. TEM showed that a great deal of wear debris was phagocytosed by macrophages, which displayed morphological changes characteristic of apoptosis. The results of TUNEL staining demonstrated that there were more apoptotic macrophages in interface membrane. The expression levels of iNOS, ONOO, cleaved caspase-3/4/8/9, cytochrome c, GRP78 and GADD153 in macrophages in interface membrane were significantly higher than those in the control samples ( p < 0.05). Our results suggest that death receptor pathway, mitochondria/cytochrosome c caspase-dependent pathway and ER stress pathway are involved in the process of macrophage apoptosis. A therapeutic target to modulate the apoptotic pathways in macrophages may be a strategy to prevent and treat aseptic loosening.

Keywords: Wear debris; Death receptor; Endoplasmic reticulum stress apoptosis; Macrophage

The impact of dendrimer-grafted modifications to model silicon surfaces on protein adsorption and bacterial adhesion by Mirjam Eichler; Verena Katzur; Lutz Scheideler; Michael Haupt; Juergen Geis-Gerstorfer; Gottfried Schmalz; Stefan Ruhl; Rainer Müller; Frank Rupp (pp. 9168-9179).
In the oral cavity, omnipresent salivary protein films (pellicle) mediate bacterial adhesion and biofilm formation on natural tissues as well as on artificial implant surfaces, which may cause serious infectious diseases like periimplantitis. The purpose of this in vitro study was to investigate the adsorption/desorption behaviour of human saliva on model surfaces grafted with polyamidoamine (PAMAM) dendrimer molecules compared to self-assembled monolayers (SAMs) exhibiting the same terminal functions (–NH2, –COOH) by two complementary analytical methods. Furthermore, the role of saliva conditioning of PAMAM and analogous SAM modifications on the adhesion of Streptococcus gordonii DL1, an early oral colonizer, was investigated. In contrast to SAMs, PAMAM-grafted surfaces showed reduced streptococcal adherence in the absence of pre-adsorbed saliva similar to the level obtained for poly(ethylene glycol) (PEG) coatings. Moreover, coatings of PAMAM-NH2 maintained their bacteria-repellent behaviour even after saliva-conditioning. As a general outcome, it was found that lower amounts of protein adsorbed on PAMAM coatings than on analogous SAMs. Since this study demonstrates that covalently bound PAMAM dendrimers can modulate the oral bacterial response, this approach has significant potential for the development of anti-adhesive biomaterial surfaces that are conditioned with proteinaceous films.

Keywords: Polyamidoamine (PAMAM) dendrimer; Saliva conditioning film; Quartz crystal microbalance (QCM-D); Bacterial adhesion; Streptococcus gordonii; DL1; Dental implant

Pluripotent stem cell-derived cardiac tissue patch with advanced structure and function by Brian Liau; Nicolas Christoforou; Kam W. Leong; Nenad Bursac (pp. 9180-9187).
Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells. However, tissue engineering methodologies to assemble cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. In this study, we introduced 3D cell alignment cues in a fibrin-based hydrogel matrix to engineer highly functional cardiac tissues from genetically purified mouse embryonic stem cell-derived cardiomyocytes (CMs) and cardiovascular progenitors (CVPs). Procedures for CM and CVP derivation, purification, and functional differentiation in monolayer cultures were first optimized to yield robust intercellular coupling and maximize velocity of action potential propagation. A versatile soft-lithography technique was then applied to reproducibly fabricate engineered cardiac tissues with controllable size and 3D architecture. While purified CMs assembled into a functional 3D syncytium only when supplemented with supporting non-myocytes, purified CVPs differentiated into cardiomyocytes, smooth muscle, and endothelial cells, and autonomously supported the formation of functional cardiac tissues. After a total culture time similar to period of mouse embryonic development (21 days), the engineered cardiac tissues exhibited unprecedented levels of 3D organization and functional differentiation characteristic of native neonatal myocardium, including: 1) dense, uniformly aligned, highly differentiated and electromechanically coupled cardiomyocytes, 2) rapid action potential conduction with velocities between 22 and 25cm/s, and 3) significant contractile forces of up to 2mN. These results represent an important advancement in stem cell-based cardiac tissue engineering and provide the foundation for exploiting the exciting progress in pluripotent stem cell research in the future tissue engineering therapies for heart disease.

Keywords: Stem cell; Cardiac tissue engineering; Fibrin; Hydrogel; Contractile function; Optical mapping

The determination of stem cell fate by 3D scaffold structures through the control of cell shape by Girish Kumar; Christopher K. Tison; Kaushik Chatterjee; P. Scott Pine; Jennifer H. McDaniel; Marc L. Salit; Marian F. Young; Carl G. Simon Jr. (pp. 9188-9196).
Stem cell response to a library of scaffolds with varied 3D structures was investigated. Microarray screening revealed that each type of scaffold structure induced a unique gene expression signature in primary human bone marrow stromal cells (hBMSCs). Hierarchical cluster analysis showed that treatments sorted by scaffold structure and not by polymer chemistry suggesting that scaffold structure was more influential than scaffold composition. Further, the effects of scaffold structure on hBMSC function were mediated by cell shape. Of all the scaffolds tested, only scaffolds with a nanofibrous morphology were able to drive the hBMSCs down an osteogenic lineage in the absence of osteogenic supplements. Nanofiber scaffolds forced the hBMSCs to assume an elongated, highly branched morphology. This same morphology was seen in osteogenic controls where hBMSCs were cultured on flat polymer films in the presence of osteogenic supplements (OS). In contrast, hBMSCs cultured on flat polymer films in the absence of OS assumed a more rounded and less-branched morphology. These results indicate that cells are more sensitive to scaffold structure than previously appreciated and suggest that scaffold efficacy can be optimized by tailoring the scaffold structure to force cells into morphologies that direct them to differentiate down the desired lineage.

Keywords: Bone tissue engineering; Cell morphology; Nanotopography; Osteogenesis; Scaffolds; Stem cell

Actin and ERK1/2-CEBPβ signaling mediates phagocytosis-induced innate immune response of osteoprogenitor cells by Heon Goo Lee; Hiroshi Minematsu; Kyung Ok Kim; Ayse B. Celil Aydemir; Mike J. Shin; Saqib A. Nizami; Kook Jin Chung; Anny C. Hsu; Christopher R. Jacobs; Francis Youngin Lee (pp. 9197-9206).
Wear particles at the host bone-implant interface are a major challenge for successful bone implant arthoplasties. Current understanding of aseptic loosening consists of macrophage-mediated inflammatory responses and increasing osteoclastogenesis, which lead to an imbalance between bone formation and resorption.Despite its significant role in bone regeneration and implant osteointegration, the osteoprogenitor response to wear particles has been examined recent years. More specifically, the intracellular mechanism of osteoprogenitor mediated inflammation has not been fully elucidated. In this study, we examined the role of osteoprogenitors and the cellular mechanism by which metal wear particles elicit an inflammatory cascade. Through both in vivo and in vitro experiments, we have demonstrated that osteoprogenitor cells are capable of initiating inflammatory responses by phagocytosing wear particles, which lead to subsequent accumulation of macrophages and osteoclastogenesis, and the ERK_CEBP/β intracellular signaling is a key inflammatory pathway that links phagocytosis of wear particles to inflammatory gene expression in osteoprogenitors. AZD6244 treatment, a potent inhibitor of the ERK pathway, attenuated particle mediated inflammatory osteolysis both in vivo and in vitro. This study advances our understanding of the mechanisms of osteoprogenitor-mediated inflammation, and provides further evidence that the ERK_CEBP/β pathway may be a suitable therapeutic target in the treatment of inflammatory osteolysis.

Keywords: ERK; Actin; IL6; Cox2; Particle

MicroRNAs in the regulation of interfacial behaviors of MSCs cultured on microgrooved surface pattern by Yang Wang; Xian-Li Jiang; Shih-Chi Yang; Xiao Lin; Yu He; Chao Yan; Li Wu; Guo-Qiang Chen; Zhe-Yao Wang; Qiong Wu (pp. 9207-9217).
Cell-substrate interaction is one of the most important aspects of tissue engineering. Changes of MSCs interfacial behaviors were found to be triggered by 10 μm wide grooved pattern on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Global marker genes expression and miRNAs profiling analysis provided insights into the regulation network of the topography induced MSCs’ cell responses including adhesion, proliferation, differentiation and apoptosis. Compared to MSCs cultured on the smooth substrates, MSCs incubated on microgrooved PHBHHx substrates showed increased expression of osteogenesis-related marker genes including cbfa1, col1a1 and bmp2, and decreased expression of vcl, vinculin encoding gene, adipogenesis-related genes including lpl, des and acta2, as well as myogenesis-related genes of myh11 and nse. The miRNA microarrays revealed that 18 differentially-expressed miRNAs on microgrooved pattern had multiple target genes, contributing comprehensively to the cellular regulation process. Similar to the topography-triggered ostegenenesis, co-transfection of the osteogenic miRNAs combination (miR-140, miR-214, miR-320, miR-351 and miR-674-5p) was able to stimulate the expression of osteogenic marker genes. This study elucidated the important roles of miRNAs in the regulation processes of the microenvironment triggered cell behaviors, and provided clues for the PHA biomedical materials development.

Keywords: MicroRNA; Mesenchymal stem cells; Polyhydroxyalkanoates; Microgroove surface pattern; Cell-substrate interactions; Osteogenesis

Patterning human stem cells and endothelial cells with laser printing for cardiac regeneration by Ralf Gaebel; Nan Ma; Jun Liu; Jianjun Guan; Lothar Koch; Christian Klopsch; Martin Gruene; Anita Toelk; Weiwei Wang; Peter Mark; Feng Wang; Boris Chichkov; Wenzhong Li; Gustav Steinhoff (pp. 9218-9230).
Recent study showed that mesenchymal stem cells (MSC) could inhibit apoptosis of endothelial cells in hypoxic condition, increase their survival, and stimulate the angiogenesis process. In this project we applied Laser-Induced-Forward-Transfer (LIFT) cell printing technique and prepared a cardiac patch seeded with human umbilical vein endothelial cells (HUVEC) and human MSC (hMSC) in a defined pattern for cardiac regeneration. We seeded HUVEC and hMSC in a defined pattern on a Polyester urethane urea (PEUU) cardiac patch. On control patches an equal amount of cells was randomly seeded without LIFT. Patches were cultivated in vitro or transplanted in vivo to the infarcted zone of rat hearts after LAD-ligation. Cardiac performance was measured by left ventricular catheterization 8 weeks post infarction. Thereafter hearts were perfused with fluorescein tomato lectin for the assessment of functional blood vessels and stored for histology analyses. We demonstrated that LIFT-derived cell seeding pattern definitely modified growth characteristics of co-cultured HUVEC and hMSC leading to increased vessel formation and found significant functional improvement of infarcted hearts following transplantation of a LIFT-tissue engineered cardiac patch. Further, we could show enhanced capillary density and integration of human cells into the functionally connected vessels of murine vascular system. LIFT-based Tissue Engineering of cardiac patches for the treatment of myocardial infarction might improve wound healing and functional preservation.

Keywords: Human mesenchymal stem cells; Human umbilical vein endothelial cells; Cardiac regeneration; Cardiac patch; Laser-induced forward transfer cell printing

Recombinant exon-encoded resilins for elastomeric biomaterials by Guokui Qin; Amit Rivkin; Shaul Lapidot; Xiao Hu; Itan Preis; Shira B. Arinus; Or Dgany; Oded Shoseyov; David L. Kaplan (pp. 9231-9243).
Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediated di-tyrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative di-tyrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in vivo applications of resilin biomaterials.

Keywords: Resilin; Elastomer; Cross-linking; Biomaterials; Photo-Fenton

Periodontal regeneration using a bilayered PLGA/calcium phosphate construct by Emily C. Carlo Reis; Andréa P.B. Borges; Michel V.F. Araújo; Vanessa C. Mendes; Limin Guan; John E. Davies (pp. 9244-9253).
The regeneration of tissues affected by periodontal disease is a complex process; it encompasses the formation of bone, cementum and periodontal ligament. We developed a semi-rigid PLGA (polylactide- co-glycolide acid)/CaP (calcium phosphate) bilayered biomaterial construct to promote periodontal regeneration, which has a continuous outer barrier membrane and an inner topographically complex component. Our experimental model compared periodontal prophylaxis alone with prophylaxis and biomaterial implantation in the treatment of class II furcation defects in dogs. Clinical evaluation, micro-computed tomography, histology and backscattered electron imaging were used for data analysis. Healing occurred uneventfully and bone volumetric values, trabecular number and trabecular thickness were all significantly greater in the treated group; while trabecular separation was significantly greater in the control group. New cementum, bone, and periodontal ligament with Sharpey fibre insertions were only seen in the treated group. Although periodontal regeneration has been reported elsewhere, the advantages of employing our bilayered PLGA + CaP construct are twofold: 1)it did not collapse into the defect; and, 2) its inner side was able to retain the blood clot throughout the buccal defect. The result was greater periodontal regeneration than has previously been reported with traditional flexible membranes.

Keywords: Periodontal tissue regeneration; PLGA/CaP construct; Macroporous topography; Clot retention; Class II furcation; Canine

The functional performance of microencapsulated human pancreatic islet-derived precursor cells by Pia Montanucci; Ilaria Pennoni; Teresa Pescara; Paolo Blasi; Giovanni Bistoni; Giuseppe Basta; Riccardo Calafiore (pp. 9254-9262).
We have examined long-term cultured, human islet-derived stem/precursor cells (hIPC). Whole human islets (HI) were obtained by multi-enzymatic digestion of cadaveric donor pancreases, plated on tissue flasks, and allowed to adhere and expand for several in vitro passages, in order to obtain hIPC. We detected specific stem cell markers (Oct-4, Sox-2, Nanog, ABCG2, Klf-4, CD117) in both intact HI and hIPC. Moreover, hIPC while retaining the expression of Glut-2, Pdx-1, CK-19, and ICA-512, started re-expressing Ngn3, thereby indicating acquisition of a specific pancreatic islet beta cell-oriented phenotype identity. The intrinsic plasticity of hIPC was documented by their ability to differentiate into various germ layer-derived cell phenotypes (ie, osteocytic, adipocytic and neural), including endocrine cells associated with insulin secretory capacity. To render hIPC suitable for transplantation we have enveloped them within our highly purified, alginate-based microcapsules. Upon intraperitoneal graft in NOD/SCID mice we have observed that the microcapsules acted as three-dimensional niches favouring post-transplant hIPC differentiation and acquisition of beta cell-like functional competence.

Keywords: Microcapsule; Alginate; Cell encapsulation; Diabetes; Stem cell; Transplantation

Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds by Teresa Mortera-Blanco; Athanasios Mantalaris; Alexander Bismarck; Nayef Aqel; Nicki Panoskaltsis (pp. 9263-9270).
Cord blood expansion ex vivo can be achieved in liquid suspension through the addition of cytokines at the expense of often undesirable cell differentiation. In order to derive a cytokine-free dynamic culture system, we hypothesised that a three-dimensional (3D) environment in the form of highly porous scaffolds made of poly (d,l-lactide-co-glycolide) (PLGA) or polyurethane (PU) for the biomimetic growth of cord blood mononuclear cells (CBMNCs), would facilitate expansion of hematopoietic cells without exogenous cytokines. Both scaffolds supported cellular expansion ex vivo. Cytokine-free, long-term culture was best in PU coated with collagen type I (54-fold expansion). In contrast, traditional 2D well-plate cultures collapsed within 4 days in the absence of cytokines. CBMNCs cultured in the scaffolds were visualised by scanning electron microscopy and immunophenotypic/immunostaining analysis and the studies validated the presence of a dynamic culture containing erythroid precursors (CD45/CD71+/CD235a+), hematopoietic stem/progenitor cells (CD38CD34+, CD117+), maturing myeloid cells (CD38+, MPO+), CD4+ and CD8+ T-lymphocytes and megakaryocytes (FVIII+). Colony forming unit (CFU) assays indicated that BFU-E and CFU-GM increased ( p < 0.05) whereas CFU-GEMM were maintained at week 4. In conclusion, this 3D culture system is capable of long-term, cytokine-free expansion of CBMNCs, enabling the study of hematopoiesis and providing a potential platform for drug discovery and therapeutic applications ex vivo.

Keywords: Biomimetic biomaterials; Bone marrow tissue engineering; Cytokine-free; Cord blood stem cells

Human cardiosphere-seeded gelatin and collagen scaffolds as cardiogenic engineered bioconstructs by Isotta Chimenti; Giuseppe Rizzitelli; Roberto Gaetani; Francesco Angelini; Vittoria Ionta; Elvira Forte; Giacomo Frati; Olivier Schussler; Andrea Barbetta; Elisa Messina; Mariella Dentini; Alessandro Giacomello (pp. 9271-9281).
Cardiac tissue engineering (CTE) aims at regenerating damaged myocardium by combining cells to a biocompatible and/or bioactive matrix. Collagen and gelatin are among the most suitable materials used today for CTE approaches. In this study we compared the structural and biological features of collagen (C-RGD) or gelatin (G-FOAM)-based bioconstructs, seeded with human adult cardiac progenitor cells in the form of cardiospheres (CSps). The different morphology between C-RGD (fibrous ball-of-thread-like) and G-FOAM (trabecular sponge-like) was evidenced by SEM analysis and X-ray micro-tomography, and was reflected by their different mechanical characteristics. Seeded cells were viable and proliferating after 1 week in culture, and a reduced expression of cell-stress markers versus standard CSp culture was detected by realtime PCR. Cell engraftment inside the scaffolds was assessed by SEM microscopy and histology, evidencing more relevant cell migration and production of extracellular matrix in C-RGD versus G-FOAM. Immunofluorescence and realtime PCR analysis showed down-regulation of vascular and stemness markers, while early-to-late cardiac markers were consistently and significantly upregulated in G-FOAM and C-RGD compared to standard CSps culture, suggesting selective commitment towards cardiomyocytes. Overall our results suggest that CSp-bioconstructs have suitable mechanical properties and improved survival and cardiogenic properties, representing promising tools for CTE.

Keywords: Cardiac tissue engineering; Collagen; Gelatin; Cardiospheres

Perfluorodecalin-enriched fibrin matrix for human islet culture by Elisa Maillard; Maciej T. Juszczak; Anne Clark; Stephen J. Hughes; Derek R.W. Gray; Paul R.V. Johnson (pp. 9282-9289).
Disruption of microenvironment and decrease in oxygen supply during isolation and culture lead to pancreatic islet injury and their poor survival after transplantation. This study aimed to create a matrix for culturing islets, using fibrin as scaffold and perfluorodecalin as oxygen diffusion enhancing medium. Human pancreatic islets were divided in four groups: control, islets cultured in fibrin, islets in fibrin containing non-emulsified perfluorodecalin, and finally islets in fibrin supplemented with emulsified perfluorodecalin. After an overnight culture, cell damage (viability, proinsulin and insulin unregulated release, apoptosis (caspase-3 activation), secretory function, and presence of hypoxia markers (HIF-1a and VEGF expression) were assessed. Islets cultured in a matrix, had similar islet viability to controls (no matrix) but decreased levels of active caspase-3 and unregulated hormone release, but high level of hypoxia markers expression. Although the supplementation of fibrin with non-emulsified perfluorodecalin improves secretory response, there was no decrease in hypoxia markers expression. In contrast, emulsified perfluorodecalin added to the matrix improved islet function, islet viability and maintained level of hypoxia markers similar to control. Fibrin matrix supplemented with emulsified perfluorodecalin can provide a beneficial physical and chemical environment for improved pancreatic human islet function and viability in vitro.

Keywords: Cell culture; Diabetes; Fibrin; Fluorocarbon; Islet; Scaffold; Transplantation

The amelioration of cardiac dysfunction after myocardial infarction by the injection of keratin biomaterials derived from human hair by Deliang Shen; Xiaofang Wang; Li Zhang; Xiaoyan Zhao; Jingyi Li; Ke Cheng; Jinying Zhang (pp. 9290-9299).
Cardiac dysfunction following acute myocardial infarction is a major cause of advanced cardiomyopathy. Conventional pharmacological therapies rely on prompt reperfusion and prevention of repetitive maladaptive pathways. Keratin biomaterials can be manufactured in an autologous fashion and are effective in various models of tissue regeneration. However, its potential application in cardiac regeneration has not been tested. Keratin biomaterials were derived from human hair and its structure morphology, carryover of beneficial factors, biocompatibility with cardiomyocytes, and in vivo degradation profile were characterized. After delivery into infarcted rat hearts, the keratin scaffolds were efficiently infiltrated by cardiomyocytes and endothelial cells. Injection of keratin biomaterials promotes angiogenesis but does not exacerbate inflammation in the post-MI hearts. Compared to control-injected animals, keratin biomaterials-injected animals exhibited preservation of cardiac function and attenuation of adverse ventricular remodeling over the 8 week following time course. Tissue western blot analysis revealed up-regulation of beneficial factors (BMP4, NGF, TGF-beta) in the keratin-injected hearts. The salient functional benefits, the simplicity of manufacturing and the potentially autologous nature of this biomaterial provide impetus for further translation to the clinic.

Keywords: Heart; Keratin; Scaffold; Angiogenesis; Cardiac tissue engineering

Selective antileukemia effect of stabilized nanohybrid vesicles based on cholesteryl succinyl silane by Yan Ma; Zhifei Dai /; Zhengbao Zha; Yanguang Gao; Xiuli Yue (pp. 9300-9307).
A nanohybrid vesicle was developed from cholesteryl succinyl silane(CSS) in combination of sol–gel process and self-assembly technique. The silicalike surface adds CSS vesicles remarkably high stability against destabilization in blood or leakage of drug cargos. It was found that CSS vesicles alone exhibited selective antiproliferative effects on leukemia cells without destroying normal blood cells. In addition, they are able to encapsulate not only hydrophilic guest species inside the inner water compartment but also hydrophobic molecules in the cholesteryl succinyl bilayer membrane. More importantly, CSS vesicles loaded with doxorubicin enhanced the anticancer efficiency of cancer therapeutics greatly while minimizing the use of inactive materials and lowering the exposure of normal cells to toxic side effects. This makes CSS vesicle a promising carrier for the treatment of cancer, especially for leukemia.

Keywords: Nanohybrid vesicles; Active carriers; Selective anticancer effects; Cholesteryl derivative; Leukemia cells

A cell-instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity by Youyun Liang; Jaehyun Jeong; Ross J. DeVolder; Chaenyung Cha; Fei Wang; Yen Wah Tong; Hyunjoon Kong (pp. 9308-9315).
Three dimensional (3D) tumor spheroid models are becoming important biomedical tools for both fundamental and applied cancer studies, but current models do not account for different levels of cancer malignancy. Several studies have reported that the mechanical rigidity of a hydrogel plays a significant role in regulating the phenotypes of cancer cells adhered to the gel surface. This finding suggests that matrix rigidity should also modulate the malignancy of 3D tumor spheroids. However, the role of matrix stiffness is often confounded by concurrent changes in 3D matrix permeability. This study reports an advanced strategy to assemble 3D liver tumor spheroids with controlled intercellular organization, phenotypes, and angiogenic activities using hydrogels with controlled stiffness and minimal differences in molecular diffusivity. The elastic moduli of cell-encapsulated collagen gels were increased by stiffening interconnected collagen fibers with varied amounts of poly(ethylene glycol) di-(succinic acid N-hydroxysuccinimidyl ester). Interestingly, hepatocellular carcinoma cells encapsulated in a fat-like, softer hydrogel formed malignant cancer spheroids, while cells cultured in a liver-like, stiffer gel formed compact hepatoids with suppressed malignancy. Overall, both the hydrogel and the 3D tumor spheroids developed in this study will be greatly useful to better understand and regulate the emergent behaviors of various cancer cells.

Keywords: Three-dimensional hydrogel; Matrix stiffness; Cancer spheroid; Cancer model; Collagen gel

Enriching and characterizing cancer stem cell sub-populations in the WM115 melanoma cell line by Siddarth Chandrasekaran; Lisa A. DeLouise (pp. 9316-9327).
Cutaneous melanoma is an increasingly common and potentially lethal malignancy of melanocytes, the melanin producing cells normally located in the basal layer of the skin epidermis. Despite major advances in cancer chemotherapeutics and immunotherapy, the success in treating metastatic melanoma remains poor. The notion that cancer stem cells (CSCs) play a key role in melanoma progression is well received. Therefore, isolating and characterizing CSCs is of critical importance for designing new therapeutic strategies that target this unique tumor initiating cell sub-population. In this work, we present a simple in vitro method, employing cell culture on polydimethylsiloxane (PDMS) and transfer back onto standard tissue culture plate, to enrich a non-adherent spheroid (NA/S) forming and an adherent monolayer (AM) cell sub-populations from the tumorigenic WM115 melanoma cell line. The phenotypes of the morphologically distinct NA/S and AM sub-populations were further characterized by quantifying the expression of stem cell markers, CD20 and CD271. Flow cytometric analysis found 2.32% of the cells in the NA/S sub-population were CD20+ CD271+ whereas only 0.27% of the cells in the AM sub-population were CD20+ CD271+. When the NA/S sub-population was cultured back onto PDMS it resulted in the further enrichment of CD20+ CD271+ cells to 14.7%. We used microbubble arrays to quantify the in vitro clonogenic potential of the NA/S and AM cell sub-populations. Microbubbles are spherical cavities, ∼160 μm in diameter with 60 μm circular openings, formed in PDMS using the gas expansion molding (GEM) process. Cells from each sub-population were seeded, under limiting dilution conditions, onto separate arrays containing 1215 microbubble wells. After five days in culture, wells seeded with 1, 2, 3 and >3 cells per microbubble well were inspected for cell proliferation. The Extreme Limiting Dilutions Analysis (ELDA) determined a ∼58% clonal survival (1 in every 1.72 cells) for the NA/S sub-population and ∼25% clonal survival (1 in every 3.93 cells) for the AM sub-population (= 176, p = 4.41e-40). These findings taken together add to the existing evidence that melanoma cells propagating as non-adherent/spheroids represent a more aggressive phenotype due to the greater presence of tumor initiating cells.

Keywords: Polydimethylsiloxane; Cancer stem cell; Cell morphology; Flow cytometry; Microbubble arrays

Bioreducible polymer-conjugated oncolytic adenovirus for hepatoma-specific therapy via systemic administration by Pyung-Hwan Kim; Jaesung Kim; Tae-il Kim; Hye Yeong Nam; James W. Yockman; Minjung Kim; Sung Wan Kim; Chae-Ok Yun (pp. 9328-9342).
Systemic administration of adenovirus (Ad) vectors is complicated by host immune responses and viral accumulation in the liver, resulting in a short circulatory virus half-life, low efficacy, and host side effects. Ad surface modification is thus required to enhance safety and therapeutic efficacy. An arginine-grafted bioreducible polymer (ABP) was chemically conjugated to the Ad surface, generating Ad-ΔE1/GFP-ABP. A hepatocellular carcinoma [HCC]-selective oncolytic Ad complex, YKL-1001-ABP, was also generated. Transduction efficiency of Ad-ΔE1/GFP-ABP was enhanced compared to naked Ad-ΔE1/GFP. YKL-1001-ABP elicited an enhanced and specific killing effect in liver cancer cells (Huh7 and HepG2) expressing α-fetoprotein (AFP). Compared with naked Ad, systemic administration of ABP-conjugated Ad resulted in reduced liver toxicity and interleukin (IL)-6 production in vitro and in vivo. Ad-ΔE1/GFP-ABP was more resistant to the neutralizing effects of human serum compared to naked Ad-ΔE1/GFP. ABP conjugation extended blood circulation time 45-fold and reduced anti-Ad Ab neutralization. Moreover, systemic administration of YKL-1001-ABP markedly suppressed growth of Huh7 hepatocellular carcinoma. These results demonstrate that chemical conjugation of ABP to the Ad surface improves safety and efficacy, indicating that ABP-conjugated Ad is a potentially useful cancer therapeutic agent to target cancer via systemic administration.

Keywords: Oncolytic adenovirus; Arginine-grafted bioreducible polymer; Chemical conjugation; Cationic polymer; Systemic delivery of Ad

Preferential accumulation within tumors and in vivo imaging by functionalized luminescent dendrimer lanthanide complexes by Marco A. Alcala; Chad M. Shade; Hyounsoo Uh; Shu Ying Kwan; Matthias Bischof; Zachary P. Thompson; Kristy A. Gogick; Adam R. Meier; Timothy G. Strein; David L. Bartlett; Ruth A. Modzelewski; Yong J. Lee; Stéphane Petoud; Charles K. Brown (pp. 9343-9352).
We have created a dendrimer complex suitable for preferential accumulation within liver tumors and luminescence imaging by substituting thirty-two naphthalimide fluorophores on the surface of the dendrimer and incorporating eight europium cations within the branches. We demonstrate the utility and performance of this luminescent dendrimer complex to detect hepatic tumors generated via direct subcapsular implantation or via splenic injections of colorectal cancer cells (CC531) into WAG/RijHsd rats. Luminescence imaging of the tumors after injection of the dendrimer complex via hepatic arterial infusion revealed that the dendrimer complex can preferentially accumulate within liver tumors. Further investigation indicated that dendrimer luminescence in hepatic tumors persisted in vivo. Due to the incorporation of lanthanide cations, this luminescence agent presents a strong resistance against photobleaching. These studies show the dendrimer complex has great potential to serve as an innovative accumulation and imaging agent for the detection of metastatic tumors in our rat hepatic model.

Keywords: Animal model; Functionalized dendrimer; Luminescence imaging; Hepatic tumor; Hepatic arterial infusionAbbreviations; CCD; charged coupled device; CE; capillary electrophoresis; CRC; colorectal cancer; CZE; capillary zone electrophoresis; DAPI; 4′,6-diamidino-2-phenylindole; DIPEA; diisopropylethylamine; DMEM; Dulbecco’s modified eagle medium; DMF; N,N; -dimethylformamide; DMSO; dimethyl sulfoxide; Eu-G3P4A18N; generation-3 PAMAM 4-amino-1,8-naphthalimide dendrimer contains europium ions; GDA; gastroduodenal artery; GFP; green fluorescence protein; HATU; 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; H & E; hematoxylin and eosin; 1; H-NMR; proton nuclear magnetic resonance; LED; light-emitting diode; LLL; left lateral lobe; MWCO; molecular-weight cutoff; PAMAM; poly(aminoamine); PBS; phosphate buffered saline; RML; right medial lobe; TACE; transarterial chemoembolization; TLC; thin-layer chromatography; UV; ultraviolet; Vis; visible

The effects of aggregation and protein corona on the cellular internalization of iron oxide nanoparticles by M. Safi; J. Courtois; M. Seigneuret; H. Conjeaud; J.-F. Berret (pp. 9353-9363).
Engineered inorganic nanoparticles are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient: the particles become either coated by a corona of serum proteins or precipitate out of the solvent. In the present paper, we show that by changing the coating of iron oxide nanoparticles from a low-molecular weight ligand (citrate ions) to small carboxylated polymers (poly(acrylic acid)), the colloidal stability of the dispersion is improved and the adsorption/internalization of iron toward living mammalian cells is profoundly affected. Citrate-coated particles are shown to destabilize in all fetal-calf-serum based physiological conditions tested, whereas the polymer coated particles exhibit an outstanding dispersibility as well as a structure devoid of protein corona. The interactions between nanoparticles and human lymphoblastoid cells are investigated by transmission electron microscopy and flow cytometry. Two types of nanoparticle/cell interactions are underlined. Iron oxides are found either adsorbed on the cellular membranes, or internalized into membrane-bound endocytosis compartments. For the precipitating citrate-coated particles, the kinetics of interactions reveal a massive and rapid adsorption of iron oxide on the cell surfaces. The quantification of the partition between adsorbed and internalized iron was performed from the cytometry data. The results highlight the importance of resilient adsorbed nanomaterials at the cytoplasmic membrane.

Keywords: Magnetic nanoparticles; Polymer coating; Protein corona; Flow cytometry; Living cells; Endocytic compartment

Polymer encapsulated upconversion nanoparticle/iron oxide nanocomposites for multimodal imaging and magnetic targeted drug delivery by Huan Xu; Liang Cheng; Chao Wang; Xinxing Ma; Yonggang Li; Zhuang Liu (pp. 9364-9373).
Multimodal imaging and imaging-guided therapies have become a new trend in the current development of cancer theranostics. In this work, we encapsulate hydrophobic upconversion nanoparticles (UCNPs) together with iron oxide nanoparticles (IONPs) by using an amphiphilic block copolymer, poly (styrene-block-allyl alcohol) (PS16- b-PAA10), via a microemulsion method, obtaining an UC-IO@Polymer multi-functional nanocomposite system. Fluorescent dye and anti-cancer drug molecules can be further loaded inside the UC-IO@Polymer nanocomposite for additional functionalities. Utilizing the Squaraine (SQ) dye loaded nanocomposite (UC-IO@Polymer-SQ), triple-modal upconversion luminescence (UCL)/down-conversion fluorescence (FL)/magnetic resonance (MR) imaging is demonstrated in vitro and in vivo, and also applied for in vivo cancer cell tracking in mice. On the other hand, a chemotherapy drug, doxorubicin, is also loaded into the nanocomposite, forming an UC-IO@Polymer-DOX complex, which enables novel imaging-guided and magnetic targeted drug delivery. Our work provides a method to fabricate a nanocomposite system with highly integrated functionalities for multimodal biomedical imaging and cancer therapy.

Keywords: Upconversion nanoparticles; Iron oxide; Composite nanostructures; Multimodal imaging; Drug delivery; Magnetic targeting

The promotion of neurite sprouting and outgrowth of mouse hippocampal cells in culture by graphene substrates by Ning Li; Xuemin Zhang; Qin Song; Ruigong Su; Qi Zhang; Tao Kong; Liwei Liu; Gang Jin; Mingliang Tang; Guosheng Cheng (pp. 9374-9382).
Graphene has been demonstrated in many biomedical applications and its potentials for neural interfacing. Emerging concerns on graphene, as a biomedical material, are its biocompatibility and how biologically targeted tissue/cells respond to it. Relatively few studies attempted to address the interactions of graphene or its derivatives with the tissues/cells, while very few reports on neural system. In this study, we tried to explore how neurites, one of the key structures for neural functions, are affected by graphene during the development until maturation in a mouse hippocampal culture model. The results reveal that graphene substrates exhibited excellent biocompatibility, as cell viability and morphology were not affected. Meanwhile, neurite numbers and average neurite length on graphene were significantly enhanced during 2–7 days after cell seeding compared with tissue culture polystyrene (TCPS) substrates. Especially on Day 2 of the neural development period, graphene substrates efficiently promoted neurite sprouting and outgrowth to the maximal extent. Additionally, expression of growth-associate protein-43 (GAP-43) was examined in both graphene and TCPS groups. Western blot analysis showed that GAP-43 expression was greatly enhanced in graphene group compared to TCPS group, which might result in the boost of neurite sprouting and outgrowth. This study suggests the potential of graphene as a material for neural interfacing and provides insight into the future biomedical applications of graphene.

Keywords: Neuron-favorable CVD graphene; GAP-43; Neurite sprouting & outgrowth; Hippocampus

Real time monitoring of biomaterial-mediated inflammatory responses via macrophage-targeting NIR nanoprobes by Jun Zhou; Yi-Ting Tsai; Hong Weng; David W. Baker; Liping Tang (pp. 9383-9390).
Medical implant-mediated inflammatory responses, often involving high levels of macrophages, are typically determined by histological analyses. These methods however are time consuming and require many animals to monitor the kinetics of inflammatory reactions and to generate reproducible outcomes. Recent studies have shown that activated macrophages in inflamed tissue express high levels of folate receptor (FR). In this study, FR-targeting NIR nanoprobes were fabricated and then tested for their ability to detect and quantify the extent of biomaterial-mediated inflammatory responses in vivo. Indeed, FR-targeting nanoprobes preferentially accumulate on activated macrophage surfaces. When administered intravenously, we found that the FR-targeting nanoprobes distinctively gathered in the inflamed tissues and that a different extent of FR-targeting nanoprobe gathering could be found in tissues implanted with different types of biomaterials. Most importantly, we found that there was a good relationship between the extent of inflammatory reactions and the intensity of nanoprobe-associated NIR signal in tissue. Our results support that FR-targeting NIR nanoprobes can be used to monitor and quantify the extent of macrophage recruitment and the degree of an implants’ biocompatibility in real time.

Keywords: In vivo imaging; Nanoparticle; Macrophage; Inflammation; Biocompatibility

MRI of transplanted surface-labeled pancreatic islets with heparinized superparamagnetic iron oxide nanoparticles by Min Jin Jung; Seung Soo Lee; Yong Hwa Hwang; Hae Song Jung; Jin Wook Hwang; Min Jun Kim; Sangwoo Yoon; Dong Yun Lee (pp. 9391-9400).
Transplantation of insulin-secreting pancreatic islets can provide real-time regulation of blood glucose in patients with type 1 diabetes mellitus. Currently, noninvasive and repetitive monitoring of islet engraftment and function is an emerging and promising modality for successful islet transplantation. Here we report a new technique for highly sensitive in vivo magnetic resonance (MR) imaging of transplanted pancreatic islets. To this end, heparinized superparamagnetic iron oxide (heparin-SPIO) nanoparticle was newly synthesized for chemical conjugation onto islet surface. Compared to typical cellular labeling of Feridex® via random endocytosis, chemical conjugation of heparin-SPIO was stable and improved the hypointensity of transplanted islets due to surface modification of every islet. These heparin-SPIO-conjugated islets showed normal viability and insulin secretion, and were quantified by spin echo T2-weighted MR imaging with linear correlation depending on transplanted islet mass in vitro and in vivo for 30 days. Also, from the immunohistochemistry, we confirmed the existence of heparin-SPIO and insulin biosynthesis in transplanted islets. However, Feridex-uptake islets showed late glucose responsiveness according to changing glucose concentration although they could normally control the blood glucose levels in diabetic mouse. Thus, we anticipate that this surface labeling with heparin-SPIO can be directly applicable for MR imaging of transplanted islets.

Keywords: Islet; Transplantation; Superparamagnetic iron oxide (SPIO); MRI; Conjugation

The effect of static magnetic fields on the aggregation and cytotoxicity of magnetic nanoparticles by Ji-Eun Bae; Man-Il Huh; Byung-Kyu Ryu; Ji-Yeon Do; Seong-Uk Jin; Myung-Jin Moon; Jae-Chang Jung; Yongmin Chang; Eungseok Kim; Sung-Gil Chi; Gang-Ho Lee; Kwon-Seok Chae (pp. 9401-9414).
Biomedical applications of magnetic nanoparticles (MNP), including superparamagnetic nanoparticles, have expanded dramatically in recent years. Systematic and standardized cytotoxicity assessment to ensure the biosafety and biocompatibility of those applications is compulsory. We investigated whether exposure to static magnetic field (SMF) from e.g. magnetic resonance imaging (MRI) could affect the cytotoxicity of superparamagnetic iron oxide (SPIO) nanoparticles using mouse hepatocytes and ferucarbotran, a liver-selective MRI contrast agent as a model system. We show that while the SPIO satisfied the conventional cytotoxicity assessment, clinical doses combined with SMF exposure exerts synergistic adverse effects such as reduced cell viability, apoptosis, and cell cycle aberrations on hepatocytes in vitro and in vivo. Concomitant treatments with the SPIO and SMF generated SPIO aggregates, which demonstrated enhanced cellular uptake, was sufficient to induce the cytotoxicity without further SMF, emphasizing that the SPIO aggregates were the predominant source of the cytotoxicity. Interestingly, the apoptotic effect was dependent on levels of reactive oxygen species (ROS) and SPIO uptake while the reduced cell viability was independent of these factors. Moreover, long-term monitoring showed a significant increase in multinuclear giant cells in the cells concomitantly treated with the SPIO and SMF compared with the control. The results demonstrate that the SPIO produces unidentified cytotoxicity on liver in the presence of SMF and the SPIO aggregates predominantly exert the effect. Since aggregation of MNP in biological milieu in the presence of strong SMF is inevitable, a fundamentally different approach to surface fabrication is essential to increase the biocompatibility of MNP.

Keywords: Magnetic nanoparticles; Static magnetic field; Cytotoxicity; MRI; Electromagnetic biology; Aggregates

The use of injectable sonication-induced silk hydrogel for VEGF165 and BMP-2 delivery for elevation of the maxillary sinus floor by Wenjie Zhang; Xiuli Wang; Shaoyi Wang; Jun Zhao; Lianyi Xu; Chao Zhu; Deliang Zeng; Jake Chen; Zhiyuan Zhang; David L. Kaplan; Xinquan Jiang (pp. 9415-9424).
Sonication-induced silk hydrogels were previously prepared as an injectable bone replacement biomaterial, with a need to improve osteogenic features. Vascular endothelial growth factor (VEGF165) and bone morphogenic protein-2 (BMP-2) are key regulators of angiogenesis and osteogenesis, respectively, during bone regeneration. Therefore, the present study aimed at evaluating in situ forming silk hydrogels as a vehicle to encapsulate dual factors for rabbit maxillary sinus floor augmentation. Sonication-induced silk hydrogels were prepared in vitro and the slow release of VEGF165 and BMP-2 from these silk gels was evaluated by ELISA. For in vivo studies for each time point (4 and 12 weeks), 24 sinus floors elevation surgeries were made bilaterally in 12 rabbits for the following four treatment groups: silk gel (group Silk gel), silk gel/VEGF165 (group VEGF), silk gel/BMP-2 (group BMP-2), silk gel/VEGF165/BMP-2 (group V + B) ( n = 6 per group). Sequential florescent labeling and radiographic observations were used to record new bone formation and mineralization, along with histological and histomorphometric analysis. At week 4, VEGF165 promoted more tissue infiltration into the gel and accelerated the degradation of the gel material. At this time point, the bone area in group V + B was significantly larger than those in the other three groups. At week 12, elevated sinus floor heights of groups BMP-2 and V + B were larger than those of the Silk gel and VEGF groups, and the V + B group had the largest new bone area among all groups. In addition, a larger blood vessel area formed in the remaining gel areas in groups VEGF and V + B. In conclusion, VEGF165 and BMP-2 released from injectable and biodegradable silk gels promoted angiogenesis and new bone formation, with the two factors demonstrating an additive effect on bone regeneration. These results indicate that silk hydrogels can be used as an injectable vehicle to deliver multiple growth factors in a minimally invasive approach to regenerate irregular bony cavities.

Keywords: Silk fibron; Hydrogels; Sonication; VEGF; BMP-2; Maxillary sinus augmentation

Cationic amylose-encapsulated bovine hemoglobin as a nanosized oxygen carrier by Wei Gao; Baoyong Sha; Wei Zou; Xuan Liang; Xiangzhong Meng; Hao Xu; Jun Tang; Daocheng Wu; Lixian Xu; Hui Zhang (pp. 9425-9433).
Nanosized hemoglobin-based oxygen carriers are one of the most promising blood substitutes. In the present study, a comprehensive strategy for the preparation of nanosized cationic amylose-encapsulated hemoglobins (NCAHbs) was developed. First, cationic amylase (CA) was synthesized from amylose and quaternary ammonium salt by an etherification reaction. The structure of CA was characterized using Fourier transform infrared spectrophotometry (FTIR) and proton nuclear magnetic resonance spectrophotometry (1H NMR). The degree of substitution and the zeta potential were also measured. Then, the NCAHbs were prepared by electrostatic adhesion, reverse micelles and cross-linking. The UV–visible spectrophotometer was used to measure the entrapment efficiency (EE%) and drug loading efficiency (DL%) of the NCAHbs. Transmission electron microscopy and Malvern Nano-zs 90 analyzer were used to observe the size distribution and morphology of particles. Chemical structure was determined from the FTIR spectrum. A Hemox analyzer was used to measure the P50 and Hill coefficients. A lethal hemorrhagic shock model in rats was used to evaluate the therapeutic effect of the NCAHbs. The results showed that the combined methods improved the size, stability, EE%, DL%, and oxygen-carrying capacity of the NCAHbs. The average diameter of the NCAHbs was 92.53 ± 3.64 nm, with a narrow polydispersity index of 0.027. The EE% was 80.05% ± 1.56% and DL% was 61.55% ± 1.41%. The P50 and Hill coefficient were equal to 28.96 ± 1.33 mmHg and 2.55 ± 0.22, respectively. The size of NCAHbs remained below 200 nm for six days in PBS solution. The NCAHbs could effectively prevent lung injury from progressing to lethal hemorrhagic shock because they acted as both a volume expander and an oxygen carrier.

Keywords: Nanoparticle; Blood; Micelles; Starch

The comparative effects of mesoporous silica nanoparticles and colloidal silica on inflammation and apoptosis by Soyoung Lee; Hui-Suk Yun; Sang-Hyun Kim (pp. 9434-9443).
Mesoporous silica (MPS), synthesized via the supramolecular polymer templating method, is one of the most attractive nanomaterials for biomedical applications, such as drug delivery systems, labeling, and tissue engineering. The significant difference between MPS and general silica (colloidal silica) is the pore architectures, such as specific surface area and pore volume. The pore structures of nanomaterials have been considered to be one of the key conditions, causing nanotoxicity due to their different efficiency of cellular uptake and immune response. We first studied the influence of pore structural conditions of silica nanoparticles on both inflammation and apoptosis, in vitro and in vivo, by comparing MPS and colloidal silica, and defined underlying mechanisms of action. Both the MPS and colloidal silica nanoparticles are produced by almost similar synthetic conditions, except the use of polymer template for MPS. The specific surface area of colloidal silica and MPS was 40 and 1150 m2 g−1, respectively, while other conditions, including particle size (100 nm) and shape (spherical), were kept constant. In both MTT assay and FACS analysis, MPS nanoparticles showed significantly less cytotoxicity and apoptotic cell death than colloidal silica nanoparticles. MPS nanoparticles induced lower expression of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, in macrophages. The reduced inflammatory response and apoptosis elicited by MPS nanoparticles were resulting from the reduction of mitogen-activated protein kinases, nuclear factor-κB, and caspase 3. In addition, using the local lymph node assay, a standalone in vivo method for hazard identification of contact hypersensitivity, we showed that colloidal silica nanoparticles act as an immunogenic sensitizer and induce contact hypersensitivity but not MPS nanoparticles. In conclusion, the pore architecture of silica nanoparticles greatly influences their biocompatibility and should be carefully designed. The MPS nanoparticles exhibit better biocompatibility than colloidal silica and promise excellent potential usage in the field of biomedical and biotechnological applications.

Keywords: Mesoporous silica nanoparticles; Colloidal silica nanoparticles; Apoptosis; Inflammation

Synergistic effect of folate-mediated targeting and verapamil-mediated P-gp inhibition with paclitaxel -polymer micelles to overcome multi-drug resistance by Feihu Wang; Dianrui Zhang; Qiang Zhang; Yuxuan Chen; Dandan Zheng; Leilei Hao; Cunxian Duan; Lejiao Jia; Guangpu Liu; Yue Liu (pp. 9444-9456).
Multidrug resistance (MDR) in tumor cells is a significant obstacle for successful cancer chemotherapy. Overexpression of drug efflux transporters such as P-glycoprotein (P-gp) is a key factor contributing to the development of tumor drug resistance. Verapamil (VRP), a P-gp inhibitor, has been reported to be able to reverse completely the resistance caused by P-gp. For optimal synergy, the drug and inhibitor combination may need to be temporally colocalized in the tumor cells. Herein, we investigated the effectiveness of simultaneous and targeted delivery of anticancer drug, paclitaxel (PTX), along with VRP, using DOMC-FA micelles to overcome tumor drug resistance. The floate-functionalized dual agent loaded micelles resulted in the similar cytotoxicity to PTX-loaded micelles/free VRP combination and co-administration of two single-agent loaded micelles, which was higher than that of PTX-loaded micelles. Enhanced therapeutic efficacy of dual agent micelles could be ascribe to increased accumulation of PTX in drug-resistant tumor cells. We suggest that the synergistic effect of folate receptor-mediated internalization and VRP-mediated overcoming MDR could be beneficial in treatment of MDR solid tumors by targeting delivery of micellar PTX into tumor cells. As a result, the difunctional micelle systems is a very promising approach to overcome tumor drug resistance.

Keywords: Paclitaxel; Polymeric micelles; Targeted delivery; Multidrug resistance; Drug efflux; Verapamil

Self-aggregated pegylated poly (trimethylene carbonate) nanoparticles decorated with c(RGDyK) peptide for targeted paclitaxel delivery to integrin-rich tumors by Xinyi Jiang; Xianyi Sha; Hongliang Xin; Liangcen Chen; Xihui Gao; Xiao Wang; Kitki Law; Jijin Gu; Yanzuo Chen; Ye Jiang; Xiaoqing Ren; Qiuyue Ren; Xiaoling Fang (pp. 9457-9469).
Cyclic RGD peptide-decorated polymeric micellar-like nanoparticles (MNP) based on PEGylated poly (trimethylene carbonate) (PEG-PTMC) were prepared for active targeting to integrin-rich cancer cells. An amphiphilic diblock copolymer, α-carboxyl poly (ethylene glycol)-poly (trimethylene carbonate) (HOOC-PEG-PTMC),was synthesized by ring-opening polymerization. The c(RGDyK) ligand, a cyclic RGD peptide that can bind to the integrin proteins predominantly expressed on the surface of tumor cells with high affinity and specificity, was conjugated to the NHS-Activated PEG terminus of the copolymer. The c(RGDyK)-functionalized PEG-PTMC micellar nanoparticles encapsulating PTX (c(RGDyK)-MNP/PTX) was fabricated by the emulsion/solvent evaporation technique and characterized in terms of morphology, size and zeta potential. Cellular uptake of c(RGDyK)-MNP/PTX was found to be higher than that of MNP/PTX due to the integrin protein-mediated endocytosis effect. In vitro cytotoxicity, cell apoptosis and cell cycle arrest studies also revealed that c(RGDyK)-MNP/PTX was more potent than those of MNP/PTX and Taxol. Pharmacokinetic study in rats demonstrated that the polymeric micellar nanoparticles significantly enhanced the bioavailability of PTX than Taxol. In vivo multispectral fluorescent imaging indicated that c(RGDyK)-MNP/PTX had high specificity and efficiency in tumor active targeting. Therefore, the results demonstrated that c(RGDyK)-decorated PEG-PTMC MNP developed in this study could be a potential vehicle for delivering hydrophobic chemotherapeutic agents to integrin-rich tumors.

Keywords: Nanoparticles; PEG-PTMC; Integrin protein; Cyclic RGD; Paclitaxel

The sequestration of hydroxyapatite nanoparticles by human monocyte-macrophages in a compartment that allows free diffusion with the extracellular environment by Michael Motskin; Karin H. Müller; Christel Genoud; Alexander G. Monteith; Jeremy N. Skepper (pp. 9470-9482).
Calcium phosphate and hydroxyapatite nanoparticles are extensively researched for medical applications, including bone implant materials, DNA and SiRNA delivery vectors and slow release vaccines. Elucidating the mechanisms by which cells internalize nanoparticles is fundamental for their long-term exploitation. In this study, we demonstrate that hydrophilic hydroxyapatite nanoparticles are sequestered within a specialized compartment called SCC (surface-connected compartment). This membrane-bound compartment is an elaborate labyrinth-like structure directly connected to the extracellular space. This continuity is demonstrated by in vivo 2-photon microscopy of ionic calcium using both cell-permeable and cell-impermeable dyes and by 3-D reconstructions from serial block-face SEM of fixed cells. Previously, this compartment was thought to be initiated specifically by exposure of macrophages to hydrophobic nanoparticles. However, we show that the SCC can be triggered by a much wider range of nanoparticles. Furthermore, we demonstrate its formation in A549 human lung epithelial cells, which are considerably less phagocytic than macrophages. EDX shows that extensive amounts of hydroxyapatite nanoparticles can be sequestered in this manner. We propose that SCC formation may be a means to remove large amounts of foreign material from the extracellular space, followed by slow degradation, may be to avoid excessive damage to surrounding cells or tissues.

Keywords: Serial block-face SEM (SBFSEM); Surface-connected compartment; Hydroxyapatite; Nanoparticles; Human monocyte-macrophages; 3D reconstruction

Nanoparticle-mediated gene transfer specific to retinal pigment epithelial cells by Adarsha Koirala; Rasha S. Makkia; Mark J. Cooper; Muna I. Naash (pp. 9483-9493).
Previously, we demonstrated that CK30PEG10k-compacted DNA nanoparticles (NPs) efficiently target photoreceptor cells and improve visual function in a retinitis pigmentosa model. Here, we test the ability of these NPs in driving transgene expression in the retinal pigment epithelium (RPE), using an RPE-specific reporter vector (VMD2-eGFP). NPs, uncompacted plasmid, or saline were subretinally delivered to adult BALB/c mice. NP-based expression was specific to RPE cells and caused no deleterious effects on retinal structure and function. eGFP expression levels in NP-injected eyes peaked at post-injection day 2 (PI-2), stabilized at levels ∼3-fold higher than in naked DNA-injected eyes, and remained elevated at the latest time-point examined (PI-30). Unlike naked DNA, which only transfected cells at the site of injection, NPs were able to transfect cells throughout the RPE. Subretinal injections of rhodamine labeled NPs and naked DNA showed comparable initial uptake into RPE cells. However, at PI-7 and -30 days significantly more fluorescence was detected inside the RPE of NP-injected eyes compared to naked DNA, suggesting NPs are stable inside the cell which could possibly lead to higher and sustained expression. Overall, our results demonstrate that NPs can efficiently deliver genes to the RPE and hold great potential for the treatment of RPE-associated diseases.

Keywords: Gene therapy; Retinal pigment epithelium; DNA nanoparticle; Subretinal injection; Polyethylene glycol (PEG)

Induction of immunotolerance via mPEG grafting to allogeneic leukocytes by Duncheng Wang; Wendy M. Toyofuku; Audrey M. Chen; Mark D. Scott (pp. 9494-9503).
The induction of anergy or tolerance to prevent allorecognition is of clinical interest. To this end, the effects of methoxypoly(ethylene glycol) [mPEG] grafting to allogeneic lymphocytes on proliferation and phenotype (Th17 and Treg) was examined in vitro and in vivo. Control studies demonstrated that PEGylation did not affect cells viability or proliferation (mitogen) potential. Conditioned media (1° MLR) collected at 72 h from resting PBMC demonstrated no immunomodulatory effects whereas the control MLR demonstrated significant ( p < 0.001) pro-proliferative potential and significantly increased in IL-2, TNF-α, and INF-γ. However, 1° media from either resting mPEG-PBMC or the PEGylated MLR resulted in a significant inhibitory effect ( p < 0.001) in the 2° MLR and no increase in cytokines. PEGylation of donor murine splenocytes resulted in significant in vivo immunosuppressive effects in H2-disparate mice. While unmodified allogeneic splenocytes resulted in a significant in vivo decrease in Treg and increased Th17 lymphocytes, PEGylated allogeneic splenocytes yielded significantly increased Tregs and baseline levels of Th17 lymphocytes. This effect was persistent to at least 30 days post challenge and was not reversed by unmodified allogeneic cells. These studies demonstrate that PEGylation of allogeneic lymphocytes induced an immunoquiescent state both in vitro and in vivo.

Keywords: Poly(ethylene glycol); Tolerance; Immunocamouflage; Lymphocyte; Regulatory T cells; Th17 lymphocytes

Heteromultivalent liposomal nanoconstructs for enhanced targeting and shear-stable binding to active platelets for site-selective vascular drug delivery by Christa L. Modery; Madhumitha Ravikumar; Timothy L. Wong; Michael J. Dzuricky; Nat Durongkaveroj; Anirban Sen Gupta (pp. 9504-9514).
Activated platelets play multiple roles in vascular diseases. Hence, a delivery vehicle that can specifically target activated platelets and stay retained under a hemodynamic environment can potentially enhance the efficacy of vehicle-encapsulated vascular drug by protecting it from rapid plasma deactivation, reducing its systemic non-specific side-effects, and increasing its therapeutic index at disease sites undergoing platelet hyperactivity. We rationalized that liposomal nanoconstructs surface-modified with two kinds of peptide that simultaneously bind integrin αIIbβ3 and P-selectin on activated platelets can provide synergistic mechanisms for enhanced selectivity to vascular disease sites. We further hypothesized that dual-receptor targeting will enhance binding strength and retention under flow. We tested this using fluorescently-labeled liposomes, surface-modified by an RGD peptide targeted to active αIIbβ3 and an EWVDV peptide targeted to P-selectin. The liposomes were studied for their platelet-specific interactions inside a parallel plate flow chamber at low-to-high shear stresses. The interaction specificity was further confirmed by flow cytometry. Our results indicate that liposomes surface-modified with both RGD and EWVDV simultaneously have higher selectivity as well as retention to activated platelets under flow compared to liposomes bearing any one peptide type. These results establish the potential of our nanoconstructs for enhanced site-selective drug delivery in vascular diseases.

Keywords: Platelets; Vascular disease; Drug delivery; Liposomes; Flow chamber

Cellular uptake of polyurethane nanocarriers mediated by gemini quaternary ammonium by Mingming Ding; Xueling He; Zhigao Wang; Jiehua Li; Hong Tan; Hua Deng; Qiang Fu; Qun Gu (pp. 9515-9524).
The effective passage of drug formulations into tumor cells is a key factor in the development of nanoscale delivery systems. However, rapid cellular uptake with reduced toxicity remains a great challenge for efficient and safe delivery. In this study, we first use gemini quaternary ammonium (GQA) as a cell internalization promoter to enhance the cellular uptake of drug nanocarriers. It is found that a twenty times faster cell internalization could be achieved by introducing GQA into biodegradable multiblock polyurethane nanomicelles, as confirmed by flow cytometry and confocal laser scanning microscopy (CLSM) studies. Meanwhile, an added methoxyl-poly(ethylene glycol) (mPEG) outer corona could protect these cationic micelles from cytotoxicity at high concentrations, as verified by methyl tetrazolium (MTT) assay. Moreover, GQA not only acts as an enhancer for rapid cellular entry, but also plays an important role in controlled self-assembly and high drug loading capacity. Our work offers a new understanding on the role of cationic surfactants; and provides a facile and economical approach for the design of versatile drug nanocarriers to achieve efficient delivery and good biocompatibility.

Keywords: Biodegradable multiblock polyurethane; Nanocarrier; Gemini quaternary ammonium; Cellular uptake; Drug delivery

The effect of hydrophilic chain length and iRGD on drug delivery from poly(ε-caprolactone)-poly(N-vinylpyrrolidone) nanoparticles by Zhenshu Zhu; Chen Xie; Qin Liu; Xu Zhen; Xianchuang Zheng; Wei Wu; Rutian Li; Yin Ding; Xiqun Jiang; Baorui Liu (pp. 9525-9535).
Poly(ε-caprolactone)-b-Poly(N-vinylpyrrolidone) (PCL-b-PVP) copolymers with different PVP block length were synthesized by xanthate-mediated reverse addition fragment transfer polymerization (RAFT) and the xanthate chain transfer agent on chain end was readily translated to hydroxy or aldehyde for conjugating various functional moieties, such as fluorescent dye, biotin hydrazine and tumor homing peptide iRGD. Thus, PCL-PVP nanoparticles were prepared by these functionalized PCL-b-PVP copolymers. Furthermore, paclitaxel-loaded PCL-PVP nanoparticles with satisfactory drug loading content (15%) and encapsulation efficiency (>90%) were obtained and used in vitro and in vivo antitumor examination. It was demonstrated that the length of PVP block had a significant influence on cytotoxicity, anti-BSA adsorption, circulation time, stealth behavior, biodistribution and antitumor activity for the nanoparticles. iRGD on PCL-PVP nanoparticle surface facilitated the nanoparticles to accumulate in tumor site and enhanced their penetration in tumor tissues, both of which improved the efficacy of paclitaxel-loaded nanoparticles in impeding tumor growth and prolonging the life time of H22 tumor-bearing mice.

Keywords: Poly(N-vinyl pyrrolidone); Antitumor; iRGD; Tumor penetration

The transduction of Coxsackie and Adenovirus Receptor-negative cells and protection against neutralizing antibodies by HPMA- co-oligolysine copolymer-coated adenovirus by Chung-Huei K. Wang; Leslie W. Chan; Russell N. Johnson; David S.H. Chu; Julie Shi; Joan G. Schellinger; André Lieber; Suzie H. Pun (pp. 9536-9545).
Adenoviral (AdV) gene vectors offer efficient nucleic acid transfer into both dividing and non-dividing cells. However issues such as vector immunogenicity, toxicity and restricted transduction to receptor-expressing cells have prevented broad clinical translation of these constructs. To address this issue, engineered AdV have been prepared by both genetic and chemical manipulation. In this work, a polymer-coated Ad5 formulation is optimized by evaluating a series of N-(2-hydroxypropyl) methacrylamide (HPMA)- co-oligolysine copolymers synthesized by living polymerization techniques. This synthesis approach was used to generate highly controlled and well-defined polymers with varying peptide length (K5, K10 and K15), polymer molecular weight, and degradability to coat the viral capsid. The optimal formulation was not affected by the presence of serum during transduction and significantly increased Ad5 transduction of several cell types that lack the Coxsackie and Adenovirus Receptor (CAR) by up to 6-fold compared to unmodified AdV. Polymer-coated Ad5 also retained high transduction capability in the presence of Ad5 neutralizing antibodies. The critical role of heparan sulfate proteoglycans (HSPGs) in mediating cell binding and internalization of polymer-coated AdV was also demonstrated by evaluating transduction in HSPG-defective recombinant CHO cells. The formulations developed here are attractive vectors for ex vivo gene transfer in applications such as cell therapy. In addition, this platform for adenoviral modification allows for facile introduction of alternative targeting ligands.

Keywords: Adenovirus; Gene therapy; Copolymer; Polymerization; Immune response

The packaging of siRNA within the mesoporous structure of silica nanoparticles by Xu Li; Qian Reuben Xie; Jixi Zhang; Weiliang Xia; Hongchen Gu (pp. 9546-9556).
Mesoporous silica nanoparticle (MSN) is a promising material for biomedical applications, such as delivering drugs or biological molecules (siRNA or DNA), to the target cells or tissues. With positive-charge functionalization on their surface, MSNs have already been used as vectors for siRNA delivery. Nevertheless, such siRNA packaging strategy avoids utilizing the mesopores and consequently hinders further modifications on the delivery vehicle surface. To solve these problems, we have successfully packaged siRNA into the mesopores of magnetic mesoporous silica nanoparticles (M-MSNs) under a strongly dehydrated solution condition. The siRNA-loaded M-MSNs were mixed with polyethyleneimine (PEI) to form a polymer layer on their external surface. The obtained aggregates were further treated by ultrasonication in acidic solution to prepare well dispersed siRNA delivery vehicles (M-MSN_siRNA@PEI). Such delivery vehicles, with effective siRNA protective effect and negligible cytotoxicity, could be internalized into cancer cells and release siRNA in the cytoplasm. In gene silencing experiments, these delivery vehicles mediated, with high efficiency, knockdown of both exogenous enhanced green fluorescent protein (EGFP) gene and endogenous B-cell lymphoma 2 ( Bcl-2) gene. In summary, our siRNA packaging strategy extends the application potential of M-MSNs and the resulting siRNA delivery vehicles can be further tested for in vivo experiments.

Keywords: siRNA; Delivery; Vehicle; Transfection; Magnetic mesoporous silica nanoparticles (M-MSNs); Polyethyleneimine (PEI)

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