Biomaterials (v.32, #23)
A peptide – stainless steel reaction that yields a new bioorganic – metal state of matter
by Elisabeth M. Davis; Dong-yang Li; Randall T. Irvin (pp. 5311-5319).
A synthetic peptide derived from the native protein sequence of a metal binding bacterial pilus was observed to spontaneously react with stainless steel via a previously unreported type of chemical interaction to generate an altered form of stainless steel which we term bioorganic stainless steel. Bioorganic stainless steel has a significantly increased electron work function (4.9 ± 0.05 eV compared to 4.79 ± 0.07 eV), decreased material adhesive force (19.4 ± 8.8 nN compared to 56.7 ± 10.5 nN), and is significantly harder than regular 304 stainless steel (∼40% harder). A formal or semi-formal organo–metallic covalent bond is generated between a pilin receptor binding domain and stainless steel based on XPS analysis which indicates that the electronic state of the surface is altered. Further, we establish that the peptide–steel reaction demonstrates a degree of stereospecificity as the reaction of nativel-peptide,d-peptide and a retro-inverso-d-peptide yields bioorganic steel products that can be differentiated via the resulting EWF (4.867 ± 0.008 eV, 4.651 ± 0.008 eV, and 4.919 ± 0.007 eV, respectively). We conclude that electron sharing between the peptide and steel surface results in the stabilization of surface electrons to generate bioorganic steel that displays altered properties relative to the initial starting material. The bioorganic steel generated from the retro-inverso-d-peptide yields a protease stable product that is harder (41% harder at a 400 μN load), and has a 50% lower corrosion rate compared with regular stainless steel (0.11 ± 0.03 mpy and 0.22 ± 0.04 mpy, respectively). Bioorganic steel is readily fabricated.
Keywords: Peptide; Stainless steel; Pilin receptor binding domain; Electron work function; XPS; Adhesive force; Corrosion
A 3D aligned microfibrous myocardial tissue construct cultured under transient perfusion
by Halime Kenar; Gamze T. Kose; Mehmet Toner; David L. Kaplan; Vasif Hasirci (pp. 5320-5329).
The goal of this study was to design and develop a myocardial patch to use in the repair of myocardial infarctions or to slow down tissue damage and improve long-term heart function. The basic 3D construct design involved two biodegradable macroporous tubes, to allow transport of growth media to the cells within the construct, and cell seeded, aligned fiber mats wrapped around them. The microfibrous mat housed mesenchymal stem cells (MSCs) from human umbilical cord matrix (Wharton’s Jelly) aligned in parallel to each other in a similar way to cell organization in native myocardium. Aligned micron-sized fiber mats were obtained by electrospinning a polyester blend (PHBV (5% HV), P(L-D,L)LA (70:30) and poly(glycerol sebacate) (PGS)). The micron-sized electrospun parallel fibers were effective in Wharton’s Jelly (WJ) MSCs alignment and the cells were able to retract the mat. The 3D construct was cultured in a microbioreactor by perfusing the growth media transiently through the macroporous tubing for two weeks and examined by fluorescence microscopy for cell distribution and preservation of alignment. The fluorescence images of thin sections of 3D constructs from static and perfused cultures confirmed enhanced cell viability, uniform cell distribution and alignment due to nutrient provision from inside the 3D structure.
Keywords: Cardiac tissue engineering; Stem cell; Cell morphology; Polyhydroxybutyric acid; Polylactic acid; DMA (dynamic mechanical analysis)
The effect of anisotropic collagen-GAG scaffolds and growth factor supplementation on tendon cell recruitment, alignment, and metabolic activity
by Steven R. Caliari; Brendan A.C. Harley (pp. 5330-5340).
Current surgical and tissue engineering approaches for treating tendon injuries have shown limited success, suggesting the need for new biomaterial strategies. Here we describe the development of an anisotropic collagen-glycosaminoglycan (CG) scaffold and use of growth factor supplementation strategies to create a 3D platform for tendon tissue engineering. We fabricated cylindrical CG scaffolds with aligned tracks of ellipsoidal pores that mimic the native physiology of tendon by incorporating a directional solidification step into a conventional lyophilization strategy. By modifying the freezing temperature, we created a homologous series of aligned CG scaffolds with constant relative density and degree of anisotropy but a range of pore sizes (55–243 μm). Equine tendon cells showed greater levels of attachment, metabolic activity, and alignment as well as less cell-mediated scaffold contraction, when cultured in anisotropic scaffolds compared to an isotropic CG scaffold control. The anisotropic CG scaffolds also provided critical contact guidance cues for cell alignment. While tendon cells were randomly oriented in the isotropic control scaffold and the transverse (unaligned) plane of the anisotropic scaffolds, significant cell alignment was observed in the direction of the contact guidance cues in the longitudinal plane of the anisotropic scaffolds. Scaffold pore size was found to significantly influence tendon cell viability, proliferation, penetration into the scaffold, and metabolic activity in a manner predicted by cellular solids arguments. Finally, the addition of the growth factors PDGF-BB and IGF-1 to aligned CG scaffolds was found to enhance tendon cell motility, viability, and metabolic activity in dose-dependent manners. This work suggests a composite strategy for developing bioactive, 3D material systems for tendon tissue engineering.
Keywords: Collagen; Scaffold; Tendon; Growth factors; Porosity
Hybrid hydrogels self-assembled from graft copolymers containing complementary β-sheets as hydroxyapatite nucleation scaffolds
by Larisa C. Wu; Jiyuan Yang; Jindřich Kopeček (pp. 5341-5353).
A biomimetic material that can assist bone tissue regeneration was proposed. A bone scaffold based on a hybrid hydrogel self-assembled from N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers grafted with complementary β-sheet peptides was designed. Investigation of self-assembly by circular dichroism spectroscopy suggested that hydrogel formation was triggered through association of the complementary β-sheet motifs. Congo Red and thioflavin T binding, as well as transmission electron microscopy confirmed the formation of a fibril network. Besides mimicking the natural bone extracellular matrix and maintaining preosteoblast cells viability, this hydrogel, as shown by scanning electron microscopy and Fourier transform infrared spectroscopy, provided surfaces characterized by epitaxy that favored hydroxyapatite-like crystal nucleation and growth potentially beneficial for biointegration.
Keywords: Graft copolymers; HPMA; β-Sheet peptides; Self-assembly; Scaffold; Biomineralization
Multi-membrane chitosan hydrogels as chondrocytic cell bioreactors
by S.G. Ladet; K. Tahiri; A.S. Montembault; A.J. Domard; M.-T.M. Corvol (pp. 5354-5364).
We investigated the bioactivity of new chitosan-based multi-membrane hydrogel (MMH) architectures towards chondrocyte-like cells. The microstructure of the hydrogels constituting the membranes precludes any living cell penetration, whereas their lower scale architecture allows the protein diffusion. The biological behavior of chondrocytes implanted within the MMH inter-membrane spaces was studied for 45 days in culture. Chondrocytes formed cell aggregates and proliferated without loosing their chondrogenic phenotype as illustrated by collagen II and aggrecan expressions at the mRNA and protein levels. Cells produced neo-formed alcyan blue matrix proteins filling MMH interspaces. The HiF-2α/SOX9 pattern of expression suggested that the elevated chondrocytic phenotype in MMH could be related to a better hypoxic local environment than in classical culture conditions. Pro-inflammatory markers were not expressed during the period of culture. The low level of nitric oxide accumulation within the inter-membrane spaces and in the incubation medium implied that chitosan consumed nitrites produced by entrapped chondrocytes, in relation with the decrease of its molecular weight of 50%. Our data suggest that MMH structures may be considered as complex chondrocytic cell bioreactors; “active decoys of biological media”, potentially promising for various biomedical applications like the inter-vertebral disk replacement.
Keywords: Multi-membrane hydrogel; Chitosan; Chondrocytes; Inter-vertebral disc; Tissue engineering; Onion-like bioreactor
Delineating the roles of the GPIIb/IIIa and GP-Ib-IX-V platelet receptors in mediating platelet adhesion to adsorbed fibrinogen and albumin
by Balakrishnan Sivaraman; Robert A. Latour (pp. 5365-5370).
Platelet adhesion to adsorbed plasma proteins, such as fibrinogen (Fg), has been conventionally thought to be mediated by the GPIIb/IIIa receptor binding to Arg-Gly-Asp (RGD)-like motifs in the adsorbed protein. In previous studies, we showed that platelet adhesion response to adsorbed Fg and Alb was strongly influenced by the degree of adsorption-induced protein unfolding and that platelet adhesion was only partially blocked by soluble RGD, with RGD-blocked platelets adhering without activation. Based on these results, we hypothesized that in addition to the RGD-specific GPIIb/IIIa receptor, which mediates both adhesion and activation, a non-RGD-specific receptor set likely also plays a role in platelet adhesion (but not activation) to both Fg and albumin (Alb). To identify and elucidate the role of these receptors, in addition to GPIIb/IIIa, we also examined the GPIb-IX-V receptor complex, which has been shown to mediate platelet adhesion (but not activation) in studies by other groups. The platelet suspension was pretreated with either a GPIIb/IIIa-antagonist drug Aggrastat® or monoclonal antibodies 6B4 or 24G10 against GPIb-IX-V prior to adhesion on Fg- and Alb-coated OH- and CH3-functionalized alkanethiol self-assembled monolayer surfaces. The results revealed that GPIIb/IIIa is the primary receptor set involved in platelet adhesion to adsorbed Fg and Alb irrespective of their degree of adsorption-induced unfolding, while the GPIb-IX-V receptor complex plays an insignificant role. Overall, these studies provide novel insights into the molecular-level mechanisms mediating platelet interactions with adsorbed plasma proteins, thereby assisting the biomaterials field develop potent strategies for inhibiting platelet-protein interactions in the design of more hemocompatible cardiovascular biomaterials and effective anti-thrombotic therapies.
Keywords: Platelet adhesion; Blood compatibility; Thrombosis; Fibrinogen; Albumin
Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair
by Jeffrey M. Caves; Wanxing Cui; Jing Wen; Vivek A. Kumar; Carolyn A. Haller; Elliot L. Chaikof (pp. 5371-5379).
Artificial composites designed to mimic the structure and properties of native extracellular matrix may lead to acellular materials for soft tissue repair and replacement, which display mechanical strength, stiffness, and resilience resembling native tissue. We describe the fabrication of thin lamellae consisting of continuous collagen microfiber embedded at controlled orientations and densities in a recombinant elastin-like protein polymer matrix. Multilamellar stacking affords flexible, protein-based composite sheets whose properties are dependent upon both the elastomeric matrix and collagen content and organization. Sheets are produced with properties that range over 13-fold in elongation to break (23–314%), six-fold in Young’s modulus (5.3–33.1 MPa), and more than two-fold in tensile strength (1.85–4.08 MPa), exceeding that of a number of native human tissues, including urinary bladder, pulmonary artery, and aorta. A sheet approximating the mechanical response of human abdominal wall fascia is investigated as a fascial substitute for ventral hernia repair. Protein-based composite patches prevent hernia recurrence in Wistar rats over an 8-week period with new tissue formation and sustained structural integrity.
Keywords: Elastin; Collagen; Mechanical properties; Fiber-reinforced composite; Surgical mesh; Recombinant protein
Effects of freezing-induced cell–fluid–matrix interactions on the cells and extracellular matrix of engineered tissues
by Ka Yaw Teo; Tenok O. DeHoyos; J. Craig Dutton; Frederick Grinnell; Bumsoo Han (pp. 5380-5390).
The two most significant challenges for successful cryopreservation of engineered tissues (ETs) are preserving tissue functionality and controlling highly tissue-type dependent preservation outcomes. In order to address these challenges, freezing-induced cell–fluid–matrix interactions should be understood, which determine the post-thaw cell viability and extracellular matrix (ECM) microstructure. However, the current understanding of this tissue-level biophysical interaction is still limited. In this study, freezing-induced cell–fluid–matrix interactions and their impact on the cells and ECM microstructure of ETs were investigated using dermal equivalents as a model ET. The dermal equivalents were constructed by seeding human dermal fibroblasts in type I collagen matrices with varying cell seeding density and collagen concentration. While these dermal equivalents underwent an identical freeze/thaw condition, their spatiotemporal deformation during freezing, post-thaw ECM microstructure, and cellular level cryoresponse were characterized. The results showed that the extent and characteristics of freezing-induced deformation were significantly different among the experimental groups, and the ETs with denser ECM microstructure experienced a larger deformation. The magnitude of the deformation was well correlated to the post-thaw ECM structure, suggesting that the freezing-induced deformation is a good indicator of post-thaw ECM structure. A significant difference in the extent of cellular injury was also noted among the experimental groups, and it depended on the extent of freezing-induced deformation of the ETs and the initial cytoskeleton organization. These results suggest that the cells have been subjected to mechanical insult due to the freezing-induced deformation as well as thermal insult. These findings provide insight on tissue-type dependent cryopreservation outcomes, and can help to design and modify cryopreservation protocols for new types of tissues from a pre-developed cryopreservation protocol.
Keywords: Cryopreservation; Dermal equivalent; Fibroblast; ECM; Cell viability; Cell morphology
Microstructured templates for directed growth and vascularization of soft tissue in vivo
by Ying Zheng; Peter W. Henderson; Nak Won Choi; Lawrence J. Bonassar; Jason A. Spector; Abraham D. Stroock (pp. 5391-5401).
Tissue templates for reconstruction and regeneration in vivo have achieved clinical successes for homogeneous tissues in well vascularized regions. Outstanding challenges exist for applications in poorly vascularized regions and for spatially differentiated tissues. Here, we present a strategy to control the spatial patterns of cell and vascular ingrowth throughout the volume of a bioremodelable and resorbable matrix via well-defined micropores and networks of microchannels. Our presentation of this approach includes: a description of a lithographic technique to form deterministic microstructures within a matrix of native collagen; elucidation of multistep process by which microstructures drive rapid invasion and vascularization; and demonstration of long range guidance of invasion through the full thickness of patterned templates. Experiments were performed in a murine wound model. These microstructured tissue templates (MTTs) could improve outcomes in reconstructive surgery and open paths to directed regeneration of spatially heterogeneous tissues or organs.
Keywords: Vascularization; Tissue template; Microfabrication; Directed invasion
Non-viral gene delivery nanoparticles based on Poly(β-amino esters) for treatment of glioblastoma
by Stephany Y. Tzeng; Hugo Guerrero-Cázares; Elliott E. Martinez; Joel C. Sunshine; Alfredo Quiñones-Hinojosa; Jordan J. Green (pp. 5402-5410).
Glioblastoma (GB) is currently characterized by low survival rates and therapies with insufficient efficacy. Here, we describe biodegradable polymers that can deliver genes to primary GB cells as well as GB tumor stem cells in vitro with low non-specific toxicity and transfection efficiencies of up to 60.6 ± 5% in normal (10%) serum conditions. We developed polymer-DNA nanoparticles that remained more stable in normal serum and could also be stored for at least 3 months in ready-to-use form with no measurable decrease in efficacy, expanding their potential in a practical or clinical setting. A subset of polymers was identified that shows a high degree of specificity to tumor cells compared with healthy astrocytes and human neural stem cells when cultured (separately or in co-culture), yielding higher transfection in GB cells while having little to no apparent effect on healthy cells.
Keywords: Drug delivery; Gene therapy; Nanoparticle; Stem cell; DNA; Cancer
An injectable calcium phosphate cement for the local delivery of paclitaxel to bone
by Marco A. Lopez-Heredia; G.J. Bernard Kamphuis; Peter C. Thüne; F. Cumhur Öner; John A. Jansen; X. Frank Walboomers (pp. 5411-5416).
Bone metastases are usually treated by surgical removal, fixation and chemotherapeutic treatment. Bone cement is used to fill the resection voids. The aim of this study was to develop a local drug delivery system using a calcium phosphate cement (CPC) as carrier for chemotherapeutic agents. CPC consisted of alpha-tricalcium phosphate, calcium phosphate dibasic and precipitated hydroxyapatite powders and a 2% Na2HPO4 hardening solution. Scanning electron microscopy (SEM) was used to observe CPC morphology. X-ray diffraction (XRD) was used to follow CPC transformation. The loading/release capacity of the CPC was studied by a bovine serum albumin-loading model. Release/retention was measured by high performance liquid chromatography and X-ray photoelectron spectrometry. For chemotherapeutic loading, paclitaxel (PX) was loaded onto the CPC discs by absorption. Viability of osteosarcoma U2OS and metastatic breast cancer MDA-MB-231 cells was measured by an AlamarBlue assay. Results of SEM and XRD showed changes in CPC due to its transformation. The loading model indicated a high retention behavior by the CPC composition. Cell viability tests indicated a PX minimal lethal dose of 90 μg/ml. PX released from CPC remained active to influence cell viability. In conclusion, this study demonstrated that CPC is a feasible delivery vector for chemotherapeutic agents.
Keywords: Calcium phosphate cement; Bone repair; Chemotherapy; Drug delivery
Folate-decorated nanogels for targeted therapy of ovarian cancer
by Natalia V. Nukolova; Hardeep S. Oberoi; Samuel M. Cohen; Alexander V. Kabanov; Tatiana K. Bronich (pp. 5417-5426).
Nanogels are comprised of swollen polymer networks and nearly 95% water and can entrap diverse chemical and biological agents for cancer therapy with very high loading capacities. Here we use diblock copolymer poly(ethylene oxide)- b-poly(methacrylic acid) (PEO- b-PMA) to form nanogels with the desired degree of cross-linking. The nanogels are further conjugated to folic acid (FA) and loaded with different types of drugs (cisplatin, doxorubicin). For the first time we demonstrate a tumor-specific delivery and superior anti-tumor effect in vivo of an anti-cancer drug using these polyelectrolyte nanogels decorated with folate-targeting groups. This reinforces the use of nanogels for the therapy of ovarian and other cancers, where folate receptor (FR) is overexpressed.
Keywords: Nanogel; Folate-targeting; Cisplatin; Ovarian cancer; Drug delivery
Designing a binding interface for control of cancer cell adhesion via 3D topography and metabolic oligosaccharide engineering
by Jian Du; Pao-Lin Che; Zhi-Yun Wang; Udayanath Aich; Kevin J. Yarema (pp. 5427-5437).
This study combines metabolic oligosaccharide engineering (MOE), a technology where the glycocalyx of living cells is endowed with chemical features not normally found in sugars, with custom-designed three-dimensional biomaterial substrates to enhance the adhesion of cancer cells and control their morphology and gene expression. Specifically, Ac5ManNTGc, a thiol-bearing analog of N-acetyl-d-mannosamine (ManNAc) was used to introduce thiolated sialic acids into the glycocalyx of human Jurkat T-lymphoma derived cells. In parallel 2D films and 3D electrospun nanofibrous scaffolds were prepared from polyethersulfone (PES) and (as controls) left unmodified or aminated. Alternately, the materials were malemided or gold-coated to provide bio-orthogonal binding partners for the thiol groups newly expressed on the cell surface. Cell attachment was modulated by both the topography of the substrate surface and by the chemical compatibility of the binding interface between the cell and the substrate; a substantial increase in binding for normally non-adhesive Jurkat line for 3D scaffold compared to 2D surfaces with an added degree of adhesion resulting from chemoselective binding to malemidede-derivatived or gold-coated surfaces. In addition, the morphology of the cells attached to the 3D scaffolds via MOE-mediated adhesion was dramatically altered and the expression of genes involved in cell adhesion changed in a time-dependent manner. This study showed that cell adhesion could be enhanced, gene expression modulated, and cell fate controlled by introducing the 3D topograhical cues into the growth substrate and by creating a glycoengineered binding interface where the chemistry of both the cell surface and biomaterials scaffold was controlled to facilitate a new mode of carbohydrate-mediated adhesion.
Keywords: Cell morphology; Cell spreading; Gold; Leukocyte; Surface modification; Surface nanotopography
Alginate oligosaccharide protects against endoplasmic reticulum- and mitochondrial-mediated apoptotic cell death and oxidative stress
by Solaleh Khoramian Tusi; Leila Khalaj; Ghorbangol Ashabi; Mahmoud Kiaei; Fariba Khodagholi (pp. 5438-5458).
Oxidative stress is a major component of harmful cascades activated in neurodegenerative disorders. We sought to elucidate possible effects of alginate oligosaccharide (AOS) on H2O2-induced cell death and to determine the underlying molecular mechanisms in neuron-like PC12 cells. We found that AOS treatment protected PC12 cells against H2O2-induced endoplasmic reticulum (ER) and mitochondrial-dependent apoptotic cell death. AOS promoted Bcl-2 expression, while blocked Bax expression and inhibited H2O2-induced caspase-3 activation. It also blocked PARP cleavage. AOS acted on key molecules in apoptotic cell death pathway and reduced p53, p38, c-June NH2-terminal kinase phosphorylations, inhibited NFkB, and enhanced Nrf2 activation. These results suggest that treatment of PC12 cells with AOS can block H2O2-induced oxidative stress and caspase-dependent apoptotic cascades originating from both ER and mitochondria. Our in vivo experiments further confirm the neuroprotective potential of AOS against Aβ-induced neural damage. According to our data, the involvement of caspase-independent pathway in AOS-induced protection appears to be unlikely.
Keywords: Alginate oligosaccharide; Caspase-dependent apoptosis; Endoplasmic reticulum; Mitochondria; Oxidative stress; PC12 cellsAbbreviations; AD; Alzheimer’s disease; Aβ; Amyloid beta; AGEs; Advanced glycation end-products; AO/EB; Acridine orange/ethidium bromide; AOS; Alginate oligosaccharide; AREs; Antioxidant responsive elements; CAT; Catalase; ECL; Electrochemiluminescence; ER; Endoplasmic reticulum; ERK; Extracellular signal-regulated kinase; GSH; glutathione; H; 2; O; 2; Hydrogen peroxide; HSP; Heat shock protein; JNK; c-Jun NH; 2; -terminal kinase; Keap1; Kelch-like ECH-associated protein 1; MAPK; Mitogen-activated protein kinases; MDA; Malondialdehyde; MTT; 3-[4, 5-dimethylthiazol-2-yl]-2, 5-dephenyl tetrazolium bromide; NF-κB; Nuclear factor- κB; NGF; nerve growth factor; Nrf2; Nuclear factor-erythroid 2 p45-related factor 2; ROS; reactive oxygen species; SOD; superoxide dismutase
Surface-engineered quantum dots for the labeling of hydrophobic microdomains in bacterial biofilms
by Fadi Aldeek; Christian Mustin; Lavinia Balan; Thibault Roques-Carmes; Marie-Pierre Fontaine-Aupart; Raphaël Schneider (pp. 5459-5470).
Quantum dots (QDs) nanoprobes are emerging as alternatives to small-molecule fluorescent probes in biomedical technology. This paper reports an efficient and rapid method of producing highly dispersed and stable CdSe-core QDs with a hydrophobic gradient. Amphiphilic core/shell CdSe/ZnS QDs were prepared by ligand exchange at the surface of lipophilic CdSe/ZnS QDs using the dihydrolipoic acid (DHLA) dithiol ligand linked to Leucine or Phenylalanine aminoacids. Contact angle relaxations on a hydrophobic surface and surface tension measurements indicated that aqueous dispersions of CdSe/ZnS@DHLA-Leu or CdSe/ZnS@DHLA-Phe QDs exhibit increased hydrophobicity compared to CdSe-core QDs capped by the hydrophilic 3-mercaptopropionic acid (MPA) ligand. We found that the surface functional groups and the ligand density at the periphery of these QDs significantly dictated their interactions with a complex biological matrix called biofilm. Using fluorescence confocal microscopy and an autocorrelation function (semi-variogram), we demonstrated that MPA-capped QDs were homogeneously associated to the biopolymers, while amphiphilic CdSe/ZnS@DHLA-Leu or CdSe/ZnS@DHLA-Phe QDs were specifically confined allowing identification of hydrophobic microdomains of the biofilms. Results obtained clearly point out that the final destination of QDs in biofilms can properly be controlled by an appropriate design of surface ligands.
Keywords: Biofilm; ECM (extracellular matrix); Fluorescence; Hydrophilicity; Nanoparticle; Surface modification
One-to-one quantum dot-labeled single long DNA probes
by Shibin He; Bi-Hai Huang; Junjun Tan; Qing-Ying Luo; Yi Lin; Jun Li; Yong Hu; Lu Zhang; Shihan Yan; Qi Zhang; Dai-Wen Pang; Lijia Li (pp. 5471-5477).
Quantum dots (QDs) have been received most attention due to their unique properties. Constructing QDs conjugated with certain number of biomolecules is considered as one of the most important research goals in nanobiotechnology. In this study, we report polymerase chain reaction (PCR) amplification of primer oligonucleotides bound to QDs, termed as QD-based PCR. Characterization of QD-based PCR products by gel electrophoresis and atomic force microscopy showed that QD-labeled long DNA strands were synthesized and only a single long DNA strand was conjugated with a QD. The QD-based PCR products still kept fluorescence properties. Moreover, the one-to-one QD-labeled long DNA conjugates as probes could detect a single-copy gene on maize chromosomes by fluorescence in situ hybridization. Labeling a single QD to a single long DNA will make detection of small single-copy DNA fragments, quantitative detection and single molecule imaging come true by nanotechnology, and it will promote medical diagnosis and basic biological research as well as nano-material fabrication.
Keywords: Nanoparticle; DNA; PCR (polymerase chain reaction); In situ hybridization
The use of glass substrates with bi-functional silanes for designing micropatterned cell-secreted cytokine immunoassays
by Jeong Hyun Seo; Li-Jung Chen; Stanislav V. Verkhoturov; Emile A. Schweikert; Alexander Revzin (pp. 5478-5488).
It is often desirable to sequester cells in specific locations on the surface and to integrate sensing elements next to the cells. In the present study, surfaces were fabricated so as to position cytokine sensing domains inside non-fouling poly(ethylene glycol) (PEG) hydrogel microwells. Our aim was to increase sensitivity of micropatterned cytokine immunoassays through covalent attachment of biorecognition molecules. To achieve this, glass substrates were functionalized with a binary mixture of acrylate- and thiol-terminated methoxysilanes. During subsequent hydrogel photopatterning steps, acrylate moieties served to anchor hydrogel microwells to glass substrates. Importantly, glass attachment sites within the microwells contained thiol groups that could be activated with a hetero-bifunctional cross-linker for covalent immobilization of proteins. After incubation with fluorescently-labeled avidin, microwells fabricated on a mixed acryl/thiol silane layer emitted ∼ 6 times more fluorescence compared to microwells fabricated on an acryl silane alone. This result highlighted the advantages of covalent attachment of avidin inside the microwells. To create cytokine immunoassays, micropatterned surfaces were incubated with biotinylated IFN-γ or TNF-α antibodies (Abs). Micropatterned immunoassays prepared in this manner were sensitive down to 1 ng/ml or 60 pM IFN-γ. To further prove utility of this biointerface design, macrophages were seeded into 30 μm diameter microwells fabricated on either bi-functional (acryl/thiol) or mono-functional silane layers. Both types of microwells were coated with avidin and biotin-anti-TNF-α prior to cell seeding. Short mitogenic activation followed by immunostaining for TNF-α revealed that microwells created on bi-functional silane layer had 3 times higher signal due to macrophage-secreted TNF-α compared to microwells fabricated on mono-functional silane. The rational design of cytokine-sensing surfaces described here, will be leveraged in the future for rapid detection of multiple cytokines secreted by individual immune cells.
Keywords: Cell function; Cytokine release and immune cells; Cytokine detection; Immunoassay; Cell micropatterning; Hydrogel microwells
The binding affinity of anti-Aβ1-42 MAb-decorated nanoliposomes to Aβ1-42 peptides in vitro and to amyloid deposits in post-mortem tissue
by Mara Canovi; Eleni Markoutsa; Adina N. Lazar; Georgios Pampalakis; Carla Clemente; Francesca Re; Silvia Sesana; Massimo Masserini; Mario Salmona; Charles Duyckaerts; Orfeu Flores; Marco Gobbi; Sophia G. Antimisiaris (pp. 5489-5497).
Amyloid β (Aβ) aggregates are considered as possible targets for therapy and/or diagnosis of Alzheimer disease (AD), and nanoparticles functionalized with Aβ-specific ligands are considered promising vehicles for imaging probes and therapeutic agents. Herein, we characterized the binding properties of nanoliposomes decorated with an anti-Aβ monoclonal antibody (Aβ-MAb). The Aβ-MAb was obtained in mice by immunization with Aβ antigen followed by hybridoma fusion. Surface Plasmon Resonance (SPR) studies confirmed the very high affinity of purified Aβ-MAb for both Aβ monomers and fibrils ( KD = 0.08 and 0.13 nm, respectively). The affinity of the biotinylated Aβ-MAb, used thereafter for liposome decoration, was lower although still in the low nanomolar range ( KD = 2.1 and 1.6 nm, respectively). Biotin-streptavidin ligation method was used to decorate nanoliposomes with Aβ-MAb, at different densities. IgG-decorated liposomes were generated by the same methodology, as control. Vesicles were monodisperse with mean diameters 124–134 nm and demonstrated good colloidal stability and integrity when incubated with serum proteins. When studied by SPR, Aβ-MAb-liposomes, but not IgG-liposomes, markedly bound to Aβ monomers and fibrils, immobilized on the chip. KD values (calculated on Aβ-MAb content) were about 0.5 and 2 nm with liposomes at high and low Aβ-MAb density, respectively. Aβ-MAb-liposome binding to Aβ fibrils was additionally confirmed by ultracentrifugation technique, in which interactions occur in solution under physiological conditions. Moreover, Aβ-MAb-liposomes bound amyloid deposits in post-mortem AD brain samples, confirming the potential of these nanoparticles for the diagnosis and therapy of AD.
Keywords: Alzheimer disease (AD); amyloid beta (Aβ); Antibody; Affinity; Liposomes; Surface plasmon resonance (SPR)
Fine tuning of receptor-selectivity for tumor necrosis factor-α using a phage display system with one-step competitive panning
by Yasuhiro Abe; Tomoaki Yoshikawa; Masaki Inoue; Tetsuya Nomura; Takeshi Furuya; Takuya Yamashita; Kazuya Nagano; Hiromi Nabeshi; Yasuo Yoshioka; Yohei Mukai; Shinsaku Nakagawa; Haruhiko Kamada; Yasuo Tsutsumi; Shin-ichi Tsunoda (pp. 5498-5504).
Tumor necrosis factor-α (TNF) is one of the attractive targets for the development of anti-inflammatory and anti-tumor drugs, because it is an important mediator in the pathogenesis of several inflammatory diseases and tumor progression. Thus, there is an increasing need to understand the TNF receptor (TNFR1 and TNFR2) biology for the development of TNFR-selective drugs. Nonetheless, the role of TNFRs, especially that of TNFR2, remains poorly understood. Here, using a unique competitive panning, we optimized our phage display-based screening technique for isolating receptor-selective TNF mutants, and identified several TNFR2-specific TNF mutants with high TNFR2 affinity and full bioactivity via TNFR2. Among these mutants, the R2-7 clone revealed very high TNFR2-selectivity (1.8 × 105 fold higher than that for the wild-type TNF), which is so far highest among the reported TNFR2-selective TNF mutants. Because of its high TNFR2-selectivity and full bioactivity, the TNF mutant R2-7 would not only help in elucidating the functional role of TNFR2 but would also help in understanding the structure-function relationship of TNF/TNFR2. In summary, our one-step competitive panning system is a simple, useful and effective technology for isolating receptor-selective mutant proteins.
Keywords: Cytokine; Molecular biology; Protein; Affinity; Bioactivity
Thermoresponsive nanostructured polycarbonate block copolymers as biodegradable therapeutic delivery carriers
by Sung Ho Kim; Jeremy P.K. Tan; Kazuki Fukushima; Fredrik Nederberg; Yi Yan Yang; Robert M. Waymouth; James L. Hedrick (pp. 5505-5514).
Water-soluble, thermoresponsive block copolymers based on a biodegradable platform were synthesized by the ring opening polymerization of cyclic carbonate monomers functionalized with hydrophilic and hydrophobic groups for application as nanocarriers in medicine. The approach based on cyclic carbonate monomers derived from 2,2-bis(methylol)propionic acid (bis-MPA) allowed a simple and versatile route to functional monomers capable of undergoing ring opening polymerization (ROP). The resulting polymers possessed the predicted molecular weights based on the molar ratio between monomers to initiators and the narrow molecular weight distributions. Transmittance measurement for aqueous polymer solutions provided an evidence for temperature-responsiveness with lower critical solution temperature (LCST) in the range of 36 °C–60 °C, depending on the molecular weight of hydrophilic poly(ethylene glycol) (PEG) chains, compositions of copolymers, molar ratios of hydrophilic to hydrophobic monomers in the corona, and the hydrophobic core. This study showed synthetic advancement toward the design and preparation of biodegradable thermoresponsive polymers with extremely low CMC values for injectable drug delivery systems. TRC350-10,30,60, which possessed an LCST of 36 °C in PBS, was identified as a useful model polymer. Paclitaxel, an anti-cancer drug, was loaded into the micelles efficiently, giving rise to nano-sized particles with a narrow size distribution. Paclitaxel release from the micelles was faster, and cellular uptake of the micelles was higher at the body temperature (i.e. 37 °C) as compared to a temperature below the LCST. While the polymer was not cytotoxic, paclitaxel-loaded micelles killed HepG2 human liver carcinoma cells more efficiently at the body temperature as compared to free paclitaxel and paclitaxel-loaded micelles at the temperature below the LCST. These micelles are ideally suited to deliver anti-cancer drugs to tumor tissues through local injection.
Keywords: Thermally responsive micelles; Ring opening polymerization; Polycarbonate; Biodegradable; Drug delivery
Genomic instability of gold nanoparticle treated human lung fibroblast cells
by Jasmine J. Li; Soo-Ling Lo; Cheng-Teng Ng; Resham Lal Gurung; Deny Hartono; Manoor Prakash Hande; Choon-Nam Ong; Boon-Huat Bay; Lin-Yue Lanry Yung (pp. 5515-5523).
Gold nanoparticles (AuNPs) are one of the most versatile and widely researched materials for novel biomedical applications. However, the current knowledge in their toxicological profile is still incomplete and many on-going investigations aim to understand the potential adverse effects in human body. Here, we employed two dimensional gel electrophoresis to perform a comparative proteomic analysis of AuNP treated MRC-5 lung fibroblast cells. In our findings, we identified 16 proteins that were differentially expressed in MRC-5 lung fibroblasts following exposure to AuNPs. Their expression levels were also verified by western blotting and real time RT-PCR analysis. Of interest was the difference in the oxidative stress related proteins (NADH ubiquinone oxidoreductase (NDUFS1), protein disulfide isomerase associate 3 (PDIA3), heterogeneous nuclear ribonucleus protein C1/C2 (hnRNP C1/C2) and thioredoxin-like protein 1 (TXNL1)) as well as proteins associated with cell cycle regulation, cytoskeleton and DNA repair (heterogeneous nuclear ribonucleus protein C1/C2 (hnRNP C1/C2) and Secernin-1 (SCN1)). This finding is consistent with the genotoxicity observed in the AuNP treated lung fibroblasts. These results suggest that AuNP treatment can induce oxidative stress-mediated genomic instability.
Keywords: Gold nanoparticles; Proteomic analysis; Lung fibroblast; Genotoxicity
Multifunctional nanoassemblies for vincristine sulfate delivery to overcome multidrug resistance by escaping P-glycoprotein mediated efflux
by Peng Zhang; Guixia Ling; Jin Sun; Tianhong Zhang; Yue Yuan; Yongbing Sun; Zhiyuan Wang; Zhonggui He (pp. 5524-5533).
Multifunctional nanoassemblies (MNAs) were successfully developed for controlled delivery of water-soluble cationic vincristine sulfate (VCR) to overcome multidrug resistance (MDR). The incorporation of anionic small molecule of phosphatidylserine (PS) significantly enhanced the encapsulation efficiency of VCR in MNAs up to 94.4% by electrostatic interaction. Obvious sustained-release characteristics were found in VCR-loaded MNAs (VCR-MNAs) as the cumulative release of VCR was 83.2% at 96 h, and burst-release was effectively diminished. In vivo pharmacokinetics in rats following intravenous administration demonstrated that VCR-MNAs had higher AUC and longer t1/2 than VCR solution (VCR-Sol). To investigate the MDR reversal effect and clarify the possible mechanism induced by MNAs, the cytotoxicity, cellular uptake and uptake mechanism experiments were performed in MCF-7 and P-glycoprotein over-expressing MCF-7/Adr cells, respectively. Compared with VCR-Sol, VCR-MNAs efficiently enhanced the cytotoxicity to 36.5-fold by increasing the cellular accumulation of VCR (12.6-fold higher) in MCF-7/Adr cells. The results of endocytosis inhibition experiment proved that VCR-MNAs were uptaken into the resistant cancer cells by clathrin- and caveolae-mediated endocytosis pathways, which escaped the efflux induced by P-gp transporter and thereby overcame the MDR of VCR.
Keywords: Vincristine sulfate; Multifunctional nanoassemblies (MNAs); Pharmacokinetics; Cellular uptake; Multidrug resistance; P-glycoprotein