1.
Trackable and Targeted Phage as Positron Emission Tomography (PET) Agent for Cancer Imaging.
Source
1. Molecular Imaging Center, Department of Radiology, University of Southern California, Los Angeles 90033, USA.
Abstract
The recent advancement of nanotechnology has provided unprecedented opportunities for the development of nanoparticle enabled technologies for detecting and treating cancer. Here, we reported the construction of a PET trackable organic nanoplatform based on phage particle for targeted tumor imaging. Method: The integrin α(v)β(3) targeted phage nanoparticle was constructed by expressing RGD peptides on its surface. The target binding affinity of this engineered phage particle was evaluated in vitro. A bifunctional chelator (BFC) 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) or 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) was then conjugated to the phage surface for (64)Cu(2+) chelation. After (64)Cu radiolabeling, microPET imaging was performed in U87MG tumor model and the receptor specificity was confirmed by blocking experiments. Results: The phage-RGD demonstrated target specificity based on ELISA experiment. According to the TEM images, the morphology of the phage was unchanged after the modification with BFCs. The labeling yield was 25 ± 4% for (64)Cu-DOTA-phage-RGD and 46 ± 5% for (64)Cu-AmBaSar-phage-RGD, respectively. At 1 h time point, (64)Cu-DOTA-phage-RGD and (64)Cu-AmBaSar-phage-RGD have comparable tumor uptake (~ 8%ID/g). However, (64)Cu-AmBaSar-phage-RGD showed significantly higher tumor uptake (13.2 ± 1.5 %ID/g, P<0.05) at late time points compared with (64)Cu-DOTA-phage-RGD (10 ± 1.2 %ID/g). (64)Cu-AmBaSar-phage-RGD also demonstrated significantly lower liver uptake, which could be attributed to the stability difference between these chelators. There is no significant difference between two tracers regarding the uptake in kidney and muscle at all time points tested. In order to confirm the receptor specificity, blocking experiment was performed. In the RGD blocking experiment, the cold RGD peptide was injected 2 min before the administration of (64)Cu-AmBaSar-phage-RGD. Tumor uptake was partially blocked at 1 h time point. Phage-RGD particle was also used as the competitive ligand. In this case, the tumor uptake was significantly reduced and the value was kept at low level consistently. Conclusion: In this report, we constructed a PET trackable nanoplatform based on phage particle and demonstrated the imaging capability of these targeted agents. We also demonstrated that the choice of chelator could have significant impact on imaging results of nano-agents. The method established in this research may be applicable to other receptor/ligand systems for theranostic agent construction, which could have an immediate and profound impact on the field of imaging/therapy and lay the foundation for the construction of next generation cancer specific theranostic agents.
N-Succinimidyl 4-[(18)F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression.
Source
Department of Medical Imaging and Nuclear Medicine, Fourth Affiliated Hospital, Harbin Medical University, Harbin, 150001, China.
Abstract
RGD peptides, radiolabeled with (18)F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[(18)F]-fluorobenzoate ([(18)F]SFB) or 4-nitrophenyl 2-[(18)F]-fluoropropionate ([(18)F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG(3)-E[c(RGDyK)](2) (PRGD2), using [(18)F]SFMB and evaluated for imaging tumor αvβ3 integrin expression with positron emission tomography (PET). [(18)F]SFMB was prepared in one step using [(18)F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The (18)F-labeled peptide, [(18)F]FMBPRGD2 was prepared by coupling PRGD2 with [(18)F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [(18)F]FMBPRGD2 into a U87MG xenograft mouse model. [(18)F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90 min, which was considerably shorter than the preparation of [(18)F]SFB- and [(18)F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [(18)F]FMBPRGD2 clearly visualized tumors by 15 min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvβ3 integrin expression and for labeling other peptides.
Assessment of molecular imaging of angiogenesis with three-dimensional ultrasonography.
Abstract
Molecular imaging (MI) with ultrasonography relies on microbubble contrast agents (MCAs) adhering to a ligand-specific target for applications such as characterizing tumor angiogenesis. It is projected that ultrasonic (US) MI can provide information about tumor therapeutic response before the detection of phenotypic changes. One of the limitations of preclinical US MI is that it lacks a comprehensive field of view. We attempted to improve targeted MCA visualization and quantification by performing three-dimensional (3D) MI of tumors expressing αvβ3 integrin. Volumetric acquisitions were obtained with a Siemens Sequoia system in cadence pulse sequencing mode by mechanically stepping the transducer elevationally across the tumor in 800-micron increments. MI was performed on rat fibrosarcoma tumors (n = 8) of similar sizes using MCAs conjugated with a cyclic RGD peptide targeted to αvβ3 integrin. US MI and immunohistochemical analyses show high microbubble targeting variability, suggesting that individual two-dimensional (2D) acquisitions risk misrepresenting more complex heterogeneous tissues. In 2D serial studies, where it may be challenging to image the same plane repeatedly, misalignments as small as 800 microns can introduce substantial error. 3D MI, including volumetric analysis of inter- and intra-animal targeting, provides a thorough way of characterizing angiogenesis and will be a more robust assessment technique for the future of MI.
64Cu Labeled AmBaSar-RGD2 for micro-PET Imaging of Integrin αvβ3 Expression.
Source
Molecular Imaging Center, Department of Radiology, Keck School of Medicine, University of SouthernCalifornia, 1510 San Pablo Street, Room #350, Los Angeles, California 90033, USA; pconti@usc.edu.
Abstract
Integrin αvβ3 plays a critical role in tumor-induced angiogenesis and metastasis. Previously, a 64Cu-AmBaSar- RGD monomer with high in vivo stability compared with 64Cu-DOTA-RGD was developed for integrin αvβ3 PET imaging. It has been established that dimeric RGD peptides have higher receptor-binding affinity and superior in vivo kinetics compared with monomeric RGD peptides due to the polyvalency effect. In this context, we synthesized and evaluated 64Cu-labeled AmBaSar dimeric RGD conjugates (64Cu-AmBaSar-RGD2) for PET imaging of integrin αvβ3 expression. The dimeric RGD peptide was conjugated with a cage-like chelator AmBaSar and labeled with 64Cu. Cell binding, microPET imaging, receptor blocking, and biodistribution studies of 64Cu-AmBaSar-RGD2 were conducted in the U87MG human glioblastoma xenograft model. AmBaSar-RGD2 conjugate was obtained in reasonable yield (45.0 ± 2.5%, n= 4) and the identity was confirmed by HPLC and MS (found 1779.8, calculated m/z for [M+H]+ M: C81H125N27O19 1779.9). 64Cu-AmBaSar-RGD2 was obtained with high radiochemical yield (92.0 ± 1.3%) and purity (≥ 98.0%) under mild conditions (pH 5.0~5.5, 23~37 °C) in 30 min. The specific activity of 64Cu-AmBaSar-RGD2 was estimated to be 15-22 GBq/µmol at the end of synthesis. Based on microPET imaging and biodistribution studies, 64Cu-AmBaSar-RGD2 has demonstrated higher tumor uptake at selected time points than 64Cu-AmBaSar-RGD. At 20 h p.i., the tumor uptake reached 0.65 ± 0.05 %ID/g for 64Cu-AmBaSar-RGD and 1.76 ± 0.38 %ID/g for 64Cu-AmBaSar-RGD2, respectively. The integrin αvβ3 targeting specificity was confirmed by blocking experiments. Therefore, the new tracer 64Cu-AmBaSar- RGD2 exhibited better tumor-targeting efficacy and more favorable in vivo pharmacokinetics than the 64Cu labeled RGD monomer due to the polyvalency effect.
Biomimetic modification of synthetic hydrogels by incorporation of adhesive peptides and calcium phosphate nanoparticles: in vitro evaluation of cell behavior.
Source
Radboud University Nijmegen Medical Center, Department of Biomaterials (309), PO Box 9101, 6500 HB Nijmegen, The Netherlands.j.jansen@dent.umcn.nl.
Abstract
The ultimate goal of this work was to develop a biocompatible and biomimetic in situ crosslinkable hydrogel scaffold with an instructive capacity for bone regenerative treatment. To this end, synthetic hydrogels were functionalized with two key components of the extracellular matrix of native bone tissue, i.e. the three-amino acid peptide sequence RGD (which is the principal integrin-binding domain responsible for cell adhesion and survival of anchorage-dependent cells) and calcium phosphate (CaP) nanoparticles in the form of hydroxyapatite (which are similar to the inorganic phase of bone tissue). Rat bone marrow osteoblast-like cells (OBLCs) were encapsulated in four different biomaterials (plain oligo(poly(ethylene glycol) fumarate) (OPF), RGD-modified OPF, OPF enriched with CaP nanoparticles and RGD-modified OPF enriched with CaP nanoparticles) and cell survival, cell spreading, proliferation and mineralized matrix formation were determined via cell viability assay, histology and biochemical analysis for alkaline phosphatase activity and calcium. This study showed that RGD peptide sequences promoted cell spreading in OPF hydrogels and hence play a crucial role in cell survival during the early stage of culture, whereas CaP nanoparticles significantly enhanced cell-mediated hydrogel mineralization. Although cell spreading and proliferation activity were inhibited, the combined effect of RGD peptide sequences and CaP nanoparticles within OPF hydrogel systems elicited a better biological response than that of the individual components. Specifically, both a sustained cell viability and mineralized matrix production mediated by encapsulated OBLCs were observed within these novel biomimetic composite systems.
Biologic Enhancement of a Common Arthroscopic Suture.
Source
Department of Orthopaedic Surgery, University of Connecticut, Farmington, Connecticut, U.S.A.
Abstract
PURPOSE:
The purpose of this study was to investigate the in vitro effects of an arginine-glycine-aspartic acid (RGD) coating on a high-strength nonabsorbable polyester/polyethylene (PE/PEE) suture material commonly used in orthopaedic procedures.
METHODS:
Human bone and tendon specimens were isolated and cultured. The cells were then plated in known densities in the presence of RGD-coated and uncoated PE/PEE suture and allowed to adhere for predetermined time periods. The RGD-coated and uncoated control sutures were then removed and assayed for cell osteoblast and tenocyte adhesion and proliferation.
RESULTS:
The RGD-modified suture showed a statistically significant increase in both adhesion and proliferation of human tenocytes when compared with uncoated controls (P < .05).
CONCLUSIONS:
The RGD peptide sequence can be effectively coupled with commercially available PE/PEE suture. RGD-coated suture is able to stimulate the adhesion and proliferation of human tenocyte cells in vitro, as well as withstand standard sterilization and storage conditions. Furthermore, the acid hydrolysis process did not affect the strength of the suture material.
CLINICAL RELEVANCE:
RGD-modified suture materials have the potential to create favorable biologic responses when used in common orthopaedic procedures.
Copyright © 2011 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.
RGD peptide immobilized on TiO(2) nanotubes for increased bone marrow stromal cells adhesion and osteogenic gene expression.
Source
Department of Prosthodontics, School of Stomatology and Affiliated Ninth People's Hospital, School of Medicine, Shanghai Research Institute of Stomatology, Shanghai Jiao Tong University, Shanghai, 200011, China.
Abstract
Recently, TiO(2) nanotube layers are widely used in orthopedics and dental applications because of their good promotion effect on bone cells. Furthermore, peptide sequences such as arginine-glycine-aspartic acid are used to modify Ti implant for binding to cell surface integrins through motif. In this study, a cellular adhesive peptide of arginine-glycine-aspartic acid-cysteine (RGDC) was immobilized onto anodized TiO(2) nanotubes on Ti to examine its in vitro responses on rat bone marrow stromal cells (BMSCs). Materials were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy techniques. High-resolution C1s scans suggested the presence of RGDC on the surface and SEM images confirmed the nanotubes were not destroyed after modification. BMSCs adhesion and osteogenic gene expression were detected in TiO(2) nanotube layers with and without RGDC modification by fluorescence microscopy, confocal laser scanning microscopy, SEM, and realtime polymerase chain reaction (Real-time PCR). Results showed that the TiO(2) nanotube layers immobilized with RGDC increased BMSCs adhesion compared to nonfunctionalized nanotubes after 4 h of cultivation. Furthermore, the osteogenic gene expression of BMSCs was dramatically enhanced on the TiO(2) nanotube layers immobilized with RGDC (10 mM) compared to the TiO(2) nanotube layers immobilized with RGDC (1 mM) and non-functionalized anodized Ti. Our results from in vitro study provided evidence that Ti anodized to possess nanotubes and then further functionalized with RGDC should be further studied for the design of better biomedical implant surfaces.
Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation.
Source
Mechanobiology Institute, National University of Singapore, 5A Engineering Drive 1, Singapore 117411.
Abstract
Integrin-mediated adhesions are critical for stem cell differentiation, cancer metastasis, and the immune response [Hynes RO (2009) Science 326:1216-1219]. However, the mechanisms of early adhesion formation remain unclear, especially the effects of lateral clustering of integrins and the role of the Src family kinases. Using mobile Arg-Gly-Asp (RGD) peptide ligands on lipid bilayers with nano-fabricated physical barriers [Salaita K, et al. (2010) Science 327:1380-1385], we observe surprising long-range lateral movements of ligated integrins during the process of cell spreading. Initially, RGD-activated integrin clusters stimulate actin polymerization that radiates from the clusters. Myosin II contraction of actin from adjacent clusters produces contractile pairs that move toward each other against barriers. Force generated by myosin II stimulates a Src kinase-dependent lamellipodial extension and outward movement of clusters. Subsequent retraction by myosin II causes inward movement of clusters. The final cell spread area increases with the density of periodic barriers. Early integrin clustering recruits adhesion proteins, talin, paxillin, and FAK, irrespective of force generation. However, recruitment of vinculin is only observed upon contraction. Thus, we suggest that integrin activation and early clustering are independent of lateral forces. Clustering activates Src-dependent actin polymerization from clusters. Myosin contraction of clusters to lines stimulates active spreading with outward forces from actin polymerization followed by a second wave of contraction. Many of these early mechanical steps are not evident in cells spreading on immobilized matrices perhaps because of the low forces involved. These observations can provide new targets to control integrin-dependent adhesion and motility.
Crosstalk between Integrin αvβ3 and Estrogen Receptor-α Is Involved in Thyroid Hormone-Induced Proliferation in Human Lung Carcinoma Cells.
Source
Ordway Signal Transduction, Albany, New York, United States of America.
Abstract
A cell surface receptor for thyroid hormone that activates extracellular regulated kinase (ERK) 1/2 has been identified on integrin αvβ3. We have examined the actions of thyroid hormone initiated at the integrin on human NCI-H522 non-small cell lung carcinoma and NCI-H510A small cell lung cancer cells. At a physiologic total hormone concentration (10(-7) M), T(4) significantly increased proliferating cell nuclear antigen (PCNA) abundance in these cell lines, as did 3, 5, 3'-triiodo-L-thyronine (T(3)) at a supraphysiologic concentration. Neutralizing antibody to integrin αvβ3 and an integrin-binding Arg-Gly-Asp (RGD) peptide blocked thyroid hormone-induced PCNA expression. Tetraiodothyroacetic acid (tetrac) lacks thyroid hormone function but inhibits binding of T(4) and T(3) to the integrin receptor; tetrac eliminated thyroid hormone-induced lung cancer cell proliferation and ERK1/2 activation. In these estrogen receptor-α (ERα)-positive lung cancer cells, thyroid hormone (T(4)>T(3)) caused phosphorylation of ERα; the specific ERα antagonist ICI 182,780 blocked T(4)-induced, but not T(3)-induced ERK1/2 activation, as well as ERα phosphorylation, proliferating-cell nuclear antigen (PCNA) expression and hormone-dependent thymidine uptake by tumor cells. Thus, in ERα-positive human lung cancer cells, the proliferative action of thyroid hormone initiated at the plasma membrane is at least in part mediated by ERα. In summary, thyroid hormone may be one of several endogenous factors capable of supporting proliferation of lung cancer cells. Activity as an inhibitor of lung cancer cell proliferation induced at the integrin receptor makes tetrac a novel anti-proliferative agent.
Combination of Integrin-Binding Peptide and Growth Factor Promotes Cell Adhesion on Electron-Beam-Fabricated Patterns.
Source
Department of Chemistry and Biochemistry and the California NanoSystems Institute, University of California , Los Angeles, 607 Charles E. Young Drive South, Los Angeles, California 90095, United States.
Abstract
Understanding and controlling cell adhesion on engineered scaffolds is important in biomaterials and tissue engineering. In this report we used an electron-beam (e-beam) lithography technique to fabricate patterns of a cell adhesive integrin ligand combined with a growth factor. Specifically, micron-sized poly(ethylene glycol) (PEG) hydrogels with aminooxy- and styrene sulfonate-functional groups were fabricated. Cell adhesion moieties were introduced using a ketone-functionalized arginine-glycine-aspartic acid (RGD) peptide to modify the O-hydroxylamines by oxime bond formation. Basic fibroblast growth factor (bFGF) was immobilized by electrostatic interaction with the sulfonate groups. Human umbilical vein endothelial cells (HUVECs) formed focal adhesion complexes on RGD- and RGD and bFGF-immobilized patterns as shown by immunostaining of vinculin and actin. In the presence of both bFGF and RGD, cell areas were larger. The data demonstrate confinement of cellular focal adhesions to chemically and physically well-controlled microenvironments created by a combination of e-beam lithography and "click" chemistry techniques. The results also suggest positive implications for addition of growth factors into adhesive patterns for cell-material interactions.
Anti-tumor activity of paclitaxel through dual-targeting carrier of cyclic RGD and transferrin conjugated hyperbranched copolymer nanoparticles.
Source
CAS Key Lab for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China.
Abstract
Targeted delivery strategies are becoming increasingly important. Herein, a novel hyperbranched amphiphilic poly[(amine-ester)-co-(d,l-lactide)]/1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine copolymer (HPAE-co-PLA/DPPE) with RGD peptide (cRGDfK) and transferrin (Tf) on the periphery was synthesized and used to prepare paclitaxel-loaded nanoparticles (NPs) for dual-targeting chemotherapy. These NPs show satisfactory size distribution, high encapsulated efficiency and a pH-dependent release profile. The intrinsic fluorescence of the hyperbranched copolymer renders the detection and tracking of NPs in vitro and in vivo conveniently. In vitro cytotoxicity studies proved that the presence of cRGDfK enhanced the cytotoxic efficiency by 10 folds in α(ν)β(3) integrin over-expressed human umbilical vein endothelial cells, while Tf improved cytotoxicity by 2 folds in Tf receptor over-expressed human cervical carcinoma cells. The drug-loaded NPs can be efficiently transported into the vascular endothelial cells and the target tumor cells. These results indicate that the cRGDfK and Tf decorated HPAE-co-PLA/DPPE could deliver chemotherapies specifically inside the cell via receptor-mediated endocytosis with greater efficacy. Therefore, such a fluorescent nanocarrier prepared from non-cytotoxic and biodegradable polymers is promising for drug delivery in tumor therapy.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Physiological type I collagen organization induces the formation of a novel class of linear invadosomes.
Source
INSERM, U1053, F-33076 Bordeaux, France Université Bordeaux Segalen, F-33076 Bordeaux, France European Institute of Chemistry and Biology, F-33600 Pessac, France Institut Albert Bonniot, Université Joseph Fourier, CNRS ERL5284, INSERM, U823 Site Santé BP 170, Grenoble 38042, Cedex 9, France CHU de Bordeaux, F-33076 Bordeaux, France.
Abstract
Invadosomes are F-actin structures able to degrade the matrix through the activation of matrix metalloproteases. As fibrillar type I collagen promotes pro-MMP2 activation by MT1-MMP, we aimed at investigating the functional relationships between collagen I organization and invadosomes induction. We found that fibrillar collagen I induced linear F-actin structures, distributed along the fibrils, on endothelial cells, macrophages, fibroblasts and tumour cells. These structures share features with conventional invadosomes as they express cortactin, N-WASP and accumulate the scaffold protein Tks5, which proved essential for their formation. Based on their ability to degrade extracellular matrix elements and their original architecture, we named these structures linear invadosomes. Interestingly, podosomes or invadopodia were replaced by linear invadosomes upon contact of the cells with fibrillar collagen I. However, linear invadosomes clearly differ from classical invadosomes as they do not contain paxillin, vinculin and β1/β3 integrins. Using knock out MEFs and RGD peptide, we demonstrate that linear invadosome formation and activity are independent of β1 and β3 integrins. Finally, linear invadosomes were also formed in a 3D collagen matrix. This study demonstrates that fibrillar collagen I is the physiological inducer of a novel class of invadosomes.
Cell chip with nano-scale peptide layer to detect dopamine secretion from neuronal cells.
Source
Interdisciplinary Program of Integrated Biotechnology, Sogang University, #1 Shinsu-dong Mapo-gu, Seoul 121-742, Republic of Korea.
Abstract
A cell chip with a nano-scaled thin film of cysteine modified synthetic oligopeptide C(RGD)4 was fabricated to detect dopamine secretion from neuronal cells. Thin C(RGD)4 peptide layer was fabricated on chip surface for increasing the binding affinity of cells to gold electrode surface, which is essential for the electrochemical detection of dopamine released from PC12 cells. The structural formation of the peptide thin film was confirmed by both atomic force microscopy (AFM) and scanning electron microscopy (SEM). Redox characteristics of chemical dopamine were firstly characterized by voltammetric tool to compare the dopamine released from PC12 cells. Cells grown on the chip were then subjected to cyclic voltammetric (CV) analysis after 48 hours of incubation. The intensities of reduction peaks were found to be increased with increasing the concentrations of PC12 cells. In addition, the electrochemical redox signal increased more in the cells treated with glucose and potassium compared to the control group. Hence, the developed cell chip can be used to determine the effects of drugs on living cells electrochemically.
- PMID:
- 22103130
- [PubMed - indexed for MEDLINE]
RGD peptide-modified adenovirus expressing HGF and XIAP improves islet transplantation.
Source
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, USA.
Abstract
BACKGROUND:
Islet transplantation has the potential to treat type I diabetes; however, its widespread clinical application is limited by the massive apoptotic cell death and poor revascularization of transplanted islet grafts.
METHODS:
We constructed a surface-modified adenoviral vector with Arg-Gly-Asp (RGD) sequences encoding human X-linked inhibitor of apoptosis and hepatocyte growth factor (RGD-Adv-hHGF-hXIAP). The in vitro transgene expression in human islets was determined by ELISA. RGD-Adv-hHGF-hXIAP-transduced human islets were transplanted under the kidney capsule of streptozotocin-induced diabetic NOD-SCID mice. The blood glucose levels of mice were measured weekly. The kidneys bearing islets were isolated at the end of the experiment and subjected to immunofluorescence staining.
RESULTS:
The transduction efficiency on human islets was significantly improved using RGD-modified adenovirus. HGF and XIAP gene expressions were dose dependent after viral transduction. When exposed to a cocktail of inflammatory cytokines, RGD-Adv-hHGF-hXIAP-transduced human islets showed decreased caspase 3 activity and reduced apoptotic cell death. Prolonged normoglycemic control could be achieved by transplanting RGD-Adv-hHGF-hXIAP-transduced human islets. Immunofluorescence staining of kidney sections bearing RGD-Adv-hHGF-hXIAP-transduced islets was positive for insulin and von-willebrand factor (vWF) at 200 days after transplantation.
CONCLUSION:
These results indicated that ex vivo transduction of islets with RGD-Adv-hHGF-hXIAP decreased apoptotic islet cell death, improved islet revascularization, and eventually might improve the outcome of human islet transplantation. Copyright © 2011 John Wiley & Sons, Ltd.
Copyright © 2011 John Wiley & Sons, Ltd.
Functionalized STAT1 siRNA nanoparticles regress rheumatoid arthritis in a mouse model.
Source
Department of Pharmaceutical Sciences, University of Colorado Denver, 12850 E Montview Blvd, CO C238-V20, USA.
Abstract
Aim: To develop and characterize an RGD peptide functionalized poly(lactide-co-glycolytic) acid (PLGA) nanosystem to deliver a STAT1 siRNA to joint tissues in a mouse model of rheumatoid arthritis. Methods: RGD-PLGA polymer was synthesized and used in preparing functionalized nanoparticles loaded with either tracking material or siRNA. The properties of the nanoparticles and stability of siRNA after encapsulation was assessed. Nanoparticle distribution was determined both noninvasively and based on analysis of dissected organs from arthritic and healthy mice. Arthritic mice were treated with weekly doses of STAT1 siRNA-loaded nanoparticles or controls. Clinical disease was assessed. Paws of arthritic mice were sectioned for histology or processed for RNA. STAT1, Mrc-1, and IL-10 mRNA abundance was determined by quantitative PCR. Results: Nanoparticles protected the siRNA from serum degradation. The presence ofRGD peptide on the nanoparticles increased paw tissue uptake in arthritic mice. Furthermore, RGD functionalization increased lung delivery of nanoparticles in arthritic mice but not in control mice. Disease regressed in the STAT1 siRNA-treated animals and progressed in all control groups. STAT1 mRNA levels were decreased in paws of treated animals, while Mrc-1 and IL-10 mRNA levels were increased. Conclusion: RGD functionalized PLGA nanoparticles encapsulating STAT1-targeted siRNAs are efficacious in the treatment of established arthritis, possibly through a selective inhibition of macrophage and dendritic cell activation.
PTTG induces EMT through integrin α(V)β(3)-focal adhesion kinase signaling in lung cancer cells.
Source
James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA.
Abstract
Pituitary tumor transforming gene (PTTG) is a well-studied oncogene for its role in tumorigenesis and serves as a marker of malignancy in several cancer types including lung. In the present study, we defined the role of PTTG in actin cytoskeleton remodeling, cell migration and induction of epithelial mesenchymal transition (EMT) through the regulation of integrin α(V)β(3)-FAK (focal adhesion kinase) signaling pathway. Overexpression of PTTG through an adenovirus vector resulted in a significant increase in the expression of integrins α(V) and β(3), a process that was reversed with the downregulation of PTTG expression through the use of an adenovirus expressing PTTG-specific small interfering RNA (siRNA). Western blot analysis of cells infected with adenovirus PTTG cDNA resulted in increased FAK and enhanced expression of adhesion complex molecules paxillin, metavincullin, and talin. Furthermore, downstream signaling genes Rac1, RhoA, Cdc42 and DOCK180 showed upregulation upon PTTG overexpression. This process was dependent on integrin α(V), as blockage by antagonist echistatin (RGD peptide) or α(V)-specific siRNA resulted in a decrease in FAK and subsequent adhesion molecules. Actin cytoskeleton disruption was detected as a result of integrin-FAK signaling by PTTG as well as enhanced cell motility. Taken together, our results suggest for the first time an important role of PTTG in regulation of integrins α(V) and β(3) and adhesion-complex proteins leading to induction of EMT.Oncogene advance online publication, 14 November 2011; doi:10.1038/onc.2011.488.
Maleimide-thiol coupling of a bioactive peptide to an elastin-like protein polymer.
Source
Department of Biomedical Engineering, Georgia Institute of Technology/Emory University, Atlanta, GA 30332, USA.
Abstract
Recombinant elastin-like protein (ELP) polymers display several favorable characteristics for tissue repair and replacement as well as drug delivery applications. However, these materials are derived from peptide sequences that do not lend themselves to cell adhesion, migration, or proliferation. This report describes the chemoselective ligation of peptide linkers bearing the bioactive RGD sequence to the surface of ELP hydrogels. Initially, cystamine is conjugated to ELP, followed by the temperature-driven formation of elastomeric ELP hydrogels. Cystamine reduction produces reactive thiols that are coupled to the RGD peptide linker via a terminal maleimide group. Investigations into the behavior of endothelial cells and mesenchymal stem cells on the RGD-modified ELP hydrogel surface reveal significantly enhanced attachment, spreading, migration and proliferation. Attached endothelial cells display a quiescent phenotype.
Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Photoactivatable caged cyclic RGD peptide for triggering integrin binding and cell adhesion to surfaces.
Source
Max-Planck-Institut für Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany.
Abstract
We report the synthesis and properties of a photoactivatable caged RGD peptide and its application for phototriggering integrin- and cell-binding to surfaces. We analysed in detail 1) the differences in the integrin-binding affinity of the caged and uncaged forms by quartz crystal microbalance (QCM) studies, 2) the efficiency and yield of the photolytic uncaging reaction, 3) the biocompatibility of the photolysis by-products and irradiation conditions, 4) the possibility of site, temporal and density control of integrin-binding and therefore human cell attachment, and 5) the possibility of in situ generation of cell patterns and cell gradients by controlling the UV exposure. These studies provide a clear picture of the potential and limitations of caged RGD for integrin-mediated cell adhesion and demonstrate the application of this approach to the control and study of cell interactions and responses.
Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Fabrication and in vitro evaluation of the collagen/hyaluronic acid PEM coating crosslinked with functionalized RGD peptide on titanium.
Source
Department of Oral and Maxillofacial Surgery, The Affiliated Stomatology Hospital, College of Medicine, Zhejiang University, Hangzhou 310006, People's Republic of China.
Abstract
Surface modification of titanium (Ti) using biomolecules has attracted much attention recently. In this study, a new strategy has been employed to construct a stable and bioactive coating on Ti. To this end, a derivative of hyaluronic acid (HA), i.e. HA-GRGDSPC-(SH), was synthesized. The disulfide-crosslinked Arg-Gly-Asp (RGD)-containing collagen/hyaluronic acid polyelectrolyte membrane (PEM) coating was then fabricated on Ti through the alternate deposition of collagen and HA-GRGDSPC-(SH) with five assembly cycles and subsequent crosslinking via converting free sulphydryl groups into disulfide linkages (RGD-CHC-Ti group). The assembly processes for PEM coating and the physicochemical properties of the coating were carefully characterized. The stability of PEM coating in phosphate-buffered saline solution could be adjusted by the crosslinking degree, while its degradation behaviors in the presence of glutathione were glutathione concentration dependent. The adhesion and proliferation of MC3T3-E1 cells were significantly enhanced in the RGD-CHC-Ti group. Up-regulated bone specific genes, enhanced alkaline phosphatase activity and osteocalcin production, the increased areas of mineralization were also observed in the RGD-CHC-Ti group. These results indicate that the strategy employed herein may function as an effective way to construct stable, RGD-containing bioactive coatings on Ti.
Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Cell adhesion, spreading, and proliferation on surface functionalized with RGD nanopillar arrays.
Source
Interdisciplinary Program of Integrated Biotechnology, Sogang University, 35 Baekbeom-Ro, Mapo-Gu, Seoul 121-742, Republic of Korea.
Abstract
In this paper, a method was introduced for the fabrication of vertically and spatially-controlled peptide nanostructures that enhance cell adhesion, proliferation, spreading on artificial surfaces. The RGD nanostructures with different heights were fabricated on gold surfaces by self-assembly technique through a nanoporous alumina mask composed of nanoscale-controlled pores. Pore diameter and spatial distribution were controlled by manipulating the pore widening time at a constant voltage during the mask fabrication process. Two-dimensional RGD nanodot, three-dimensional RGD nanorod, and RGD nanopillar arrays were carried out using various concentrations of RGD peptide solution, self-assembly times, and pore sizes, which were 74 nm, 63 nm, and 43 nm in diameter, respectively. The fabricated RGD nanodot, nanorod, and nanopillar arrays were utilized as a cell adhesion layer to evaluate the cell adhesion force, adhesion speed, spreading assay, and phosphorylation of cofilin protein in PC12, HeLa, and HEK293T normal cells. Among the three different nanostructures, RGD nanopillar arrays were found to be suitable for cellular attachment, spreading, and proliferation due to the proper arrangement of the RGD motif, which mimics in vivo conditions. Hence, our newly fabricated RGD nanostructured array can be successfully applied as a bio-platform for improving cellular functions and in in vitro tissue engineering.
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