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Regulatory macrophages (Mregs) are unique in that they have anti-inflammatory and immunosuppressive properties. Thus, treating inflammatory diseases using Mregs is an area of active research. Human Mregs are usually generated by culturing peripheral blood monocytes stimulated using a macrophage colony-stimulating factor with interferon (IFN)-γ. Herein, we generated Mregs with an elongated cell morphology from THP-1 cells that were stimulated with phorbol 12-myristate 13-acetate and cultured with both arginylglycylaspartic acid and vitamin D3. These Mregs regulated macrophage function, and respectively downregulated and upregulated the expression of pro-inflammatory and immunosuppressive mediators. They also expressed Mregs-specific markers, such as dehydrogenase/reductase 9, even when exposed to such inflammatory stimulants as IFN-γ, lipopolysaccharide, purified xenogeneic antigen, and xenogeneic cells. The Mregs also exerted anti-inflammatory and anticoagulatory actions in response to xenogeneic cells, as well as exerting immunosuppressive effects on mitogen-induced Jurkat T-cell proliferation. Our method of generating functional Mregs in vitro without cytokines is simple and cost-effective.

The therapeutic precision and clinical applicability of drug-eluting coatings can be substantially improved by facilitating tunable drug delivery. However, the design of coatings which allows for precise control over drug release kinetics is still a major challenge. Here, a double-layered silk fibroin (SF) coating system was constructed by sequential electrophoretic deposition. A mixture of dissolved Bombyx mori SF (bmSF) molecules and pre-made bmSF nanospheres at different ratios was deposited as under-layer. Subsequently, this underlayer was covered by a top-layer comprising Antheraea pernyi SF (apSF) molecules (rich in arginylglycylaspartic acid, RGD) to improve the cellular response of the resulting double-layered coatings. Additionally, model drug doxycycline was either pre-mixed with dissolved bmSF molecules or pre-loaded into pre-made bmSF nanospheres at the same amount before their mixing and deposition. The thickness and nanosphere content of the under-layer architecture were proportional to the deposition time and nanosphere concentration in precursor mixtures, respectively. The surface topography, wettability, degradation rate and adhesion strength were comparable within the double-layered coating system. As expected, RGD-rich apSF top-layer improved cell adhesion, spreading and proliferation compared with bmSF top-layer. Furthermore, the amount and duration of drug release increased linearly with increasing nanosphere concentration at fixed deposition time, whereas drug release amount increased linearly with increasing deposition time. These results indicate that the dosage and kinetics of loaded drugs can be quantitatively tailored by altering nanosphere concentration and deposition time as main processing parameters. Overall, this study illustrates the strong potential of pre-defining coating architecture to facilitate control over drug delivery., Graphical abstract Image 1, Highlights • Multi-layered silk fibroin coatings can be achieved by electrophoretic deposition. • Drugs can be loaded in silk fibroin solution or nanoparticles before deposition. • Drug release can be easily regulated by the amount of nanoparticles in coatings. • RGD-rich silk fibroin as a top layer improves cellular attachment and proliferation.

Polymeric vectors are safer alternatives for gene delivery owing to their advantages as compared to viral vectors. To improve the stability and transfection efficiency of poly(lactic-co-glycolic acid) (PLGA)- and poly(ethylenimine) (PEI)-based vectors, poly(ethylene glycol) (PEG), folic acid (FA), arginylglycylaspartic acid (RGD) peptides and isoleucine-lysine-valine-alanine-valine (IKVAV) peptides were employed and PLGA-PEI-PEG-FA and PLGA-PEI-PEG-RGD copolymers were synthesized. PLGA-PEI-PEG-FA/DNA, PLGA-PEI-PEG-RGD/DNA and PLGA-PEI-PEG-RGD/IKVAV/DNA nanocomplexes (NCs) were formed through bulk mixing. The structure and properties, including morphology, particle size, surface charge and DNA encapsulation, of NCs were studied. Robust NCs with spherical shape, uniform size distribution and slightly positive charge were able to completely bind DNA above their respective N/P ratios. The critical N/P ratio for PLGA-PEI-PEG-FA/DNA, PLGA-PEI-PEG-RGD/DNA and PLGA-PEI-PEG-RGD/IKVAV/DNA NCs was identified to be 12:1, 8:1 and 10:1, respectively. The covalent modification of PEI through a combination of biodegradable PLGA, hydrophilic PEG and targeting motifs significantly decreased the cytotoxicity of PEI. The developed NCs showed both N/P ratio and cell type-dependent transfection efficiency. An increase in N/P ratio resulted in increased transfection efficiency, and much improved transfection efficiency of NCs was observed above their respective critical N/P ratios. This study provides a promising means to produce polymeric vectors for gene delivery.

Gadolinium neutron capture therapy (GdNCT) is considered as a new promising cancer therapeutic technique. Nevertheless, limited GdNCT applications have been reported so far. In this study, surface-modified ultrasmall gadolinium oxide nanoparticles (UGNPs) with cancer-targeting ability (davg = 1.8 nm) were for the first time applied to the in vivo GdNCT of cancer using nude model mice with cancer, primarily because each nanoparticle can deliver hundreds of Gd to the cancer site. For applications, the UGNPs were grafted with polyacrylic acid (PAA) for biocompatibility and colloidal stability, which was then conjugated with cancer-targeting arginylglycylaspartic acid (RGD) (shortly, RGD-PAA-UGNPs). The solution sample was intravenously administered into the tails of nude model mice with cancer. At the time of the maximum accumulation of the RGD-PAA-UGNPs at the cancer site, which was monitored using magnetic resonance imaging, the thermal neutron beam was locally irradiated onto the cancer site and the cancer growth was monitored for 25 days. The cancer growth suppression was observed due to the GdNCT effects of the RGD-PAA-UGNPs, indicating that the surface-modified UGNPs with cancer-targeting ability are potential materials applicable to the in vivo GdNCT of cancer.

Integrin αvβ3 is widely expressed in various types of human cancer lines and plays a key role in angiogenesis for tumor growth and metastasis. Delivery of therapeutics to αvβ3-expressing tumors can thus be a promising approach for treating cancer. For targeted delivery of anticancer therapeutics to αvβ3-expressing tumor cells, cyclic arginylglycylaspartic acid (RGD) peptide was covalently conjugated to the surface of carboxylic acid-functionalized carbon nanotubes (fCNTs), and the topoisomerase I inhibitor camptothecin (CPT) was encapsulated in the fCNTs (CPT@fCNT-RGD). CPT@fCNT-RGD was successfully delivered to αvβ3-expressing A375 cells, and compared with nontargeted CPT@fCNT, it provided 3.78- and 3.02-fold increases in the anticancer effect in 2D and 3D culture. Analysis of apoptosis-related gene expression shows that the expression levels of Bax, cleaved caspase-3, and nuclear factor kappa-light-chain-enhancer of activated B cells were significantly increased in A375 cells incubated with CPT@fCNT-RGD compared with those incubated with CPT@fCNT. These results suggest that cyclic RGD-conjugated CNTs encapsulating an anticancer therapeutic can be a promising platform for treating cancer.

Microenvironmental factors play critical roles in regulating stem cell fate, providing a rationale to engineer biomimetic microenvironments that facilitate rapid and effective stem cell differentiation. Three-dimensional (3D) hierarchical microarchitectures have been developed to enable rapid neural differentiation of multipotent human mesenchymal stromal cells (HMSCs) via mechanotransduction. However, low cell viability during long-term culture and poor cell recovery efficiency from the architectures were also observed. Such problems hinder further applications of the architectures in stem cell differentiation. Here, we present improved 3D nanostructured microarchitectures functionalized with cell-adhesion-promoting arginylglycylaspartic acid (RGD) peptides. These RGD-functionalized architectures significantly upregulated long-term cell viability and facilitated effective recovery of differentiated cells from the architectures while maintaining high differentiation efficiency. Efficient recovery of highly viable differentiated cells enabled the downstream analysis of morphology and protein expression to be performed. Remarkably, even after the removal of the mechanical stimulus provided by the 3D microarchitectures, the recovered HMSCs showed a neuron-like elongated morphology for 10 days and consistently expressed microtubule-associated protein 2, a mature neural marker. RGD-functionalized nanostructured microarchitectures hold great potential to guide effective differentiation of highly viable stem cells.

Cellulose nanocrystals (CNCs) are promising bio-derived nanomaterials for the bottom-up fabrication of biomedical constructs. In this report, dicarboxylic acid-functionalized CNC (DCC) was functionalized with arginylglycylaspartic acid (RGD) tripeptide as a motif for improved cell adhesion and targeting. The product (DCC-RGD) self-assembled into a more elongated nanofibrillar structure through lateral and end-to-end association. When added into poly(ethylene imine) (PEI)/pDNA polyplex solution, nanocelluloses interacted electrostatically with positively charged polyplexes without affecting their integrity. The constructs were tested for their potentials as non-viral transfection reagents. Cell viability and transfection efficiency of fibroblast NIH3T3 cells were monitored as a function of CNC concentration where, in general, viability increased as the CNC concentration increased, and transfection efficiency could be optimized. Using wild-type MDCK and αV-knockout MDCK cells, the construct was able to provide targeted uptake of polyplexes. The findings have potential applications, for example, cell-selective in vitro or ex vivo transfection of autologous mesenchymal stem cells for cell therapy, or bottom-up design of future innovative biomaterials.

Aim: Matrix metalloproteinase-2 (MMP2) plays an important role in extracellular matrix remodeling, that is, it increases significantly during cancer progression. In this regard, MMP2 monitoring is important. Experiment: A well-designed MMP2-sensitive polypeptide chain was used to link indium phosphide quantum dots (InP QDs) with upconversion nanoparticles (UCNPs) to form a nanocomposite that was utilized as biosensor. Results: We produced a biosensor that can be recognized by MMP2 and determined the presence or absence of MMP2 in cells by identifying difference in fluorescence wavelength. The InP QDs modified the arginylglycylaspartic acid molecules as targeting ligand based on chitosan. Conclusion: The MMP2-based biosensor, named UCNP-p@InP-cRGD, is sensitive and can be applied for biosensing probes.

Functional peptide molecules or their component functional moieties can be appended to self assembling polymers to mimic the extracellular matrix (ECM) and bring about predictable cell responses. Terminal guanidine functional group renders positive charge to the amino acid arginine, a component of the cell adhesive peptide Arginylglycylaspartic acid (RGD). We present the design and synthesis of guanidine appended polydiacetylene (G-PDA) as a cell adhesive matrix. Human periodontal ligament cell adhesion and differentiation on G-PDA matrix was evaluated to assess the feasibility of using the matrix for endogenous cell homing in periodontal regeneration. Guanidine appended polydiacetylene matrix proves to be a good substrate for cell homing, proliferation and differentiation. A well-designed scaffold using this material can lead to the regeneration of the periodontal bone.

The use of targeted liposomes encapsulating chemotherapy drugs enhances the specific targeting of cancer cells, thus reducing the side effects of these drugs and providing patient-friendly chemotherapy treatment. Targeted pegylated (stealth) liposomes have the ability to safely deliver their loaded drugs to the cancer cells by targeting specific receptors overly expressed on the surface of these cells. Applying ultrasound as an external stimulus will safely trigger drug release from these liposomes in a controlled manner. In this study, we investigated the release kinetics of the model drug "calcein" from targeted liposomes sonicated with low-frequency ultrasound (20 kHz). Our results showed that pegylated liposomes were more sonosensitive compared to nonpegylated liposomes. A comparison of the effect of three targeting moieties conjugated to the surface of pegylated liposomes, namely human serum albumin (HSA), transferrin (Tf) and arginylglycylaspartic acid (RGD), on calcein release kinetics was conducted. The fluorescent results showed that HSA-PEG and Tf-PEG liposomes were more sonosensitive (showing higher calcein release following the exposure to pulsed LFUS) compared to the control pegylated liposomes, thus adding more acoustic benefits to their targeting efficacy.

In the field of molecular imaging, selectivity for target cells is a key determinant of the clarity and accuracy of imaging contrast. Recently, a novel pre-targeted imaging method has been developed whereby target cells can be selectively imaged using a labeled N-glycan that has been ligated with an integrin-targeted cyclic arginylglycylaspartic acid (RGD) peptide on the cell surface in situ. The conceptual basis of this method, its development, and its application to cancerous and noncancerous cells is described in this chapter. This method has the powerful advantage of having the ability to discriminate between various cancerous and non-cancerous cells that cannot be distinguished using conventional RGD ligands. Using this method, various N-glycan molecules, even those with millimolar affinities for their cognate lectins, could be used for selective cancer cell differentiation.

Background: The frequency of skin ulceration makes an important contributor to the morbidity burden in people with sickle cell disease. Many treatment options are available to the healthcare professional, although it is uncertain which treatments have been assessed for effectiveness in people with sickle cell disease. This is an update of a previously published Cochrane Review. Objectives: To assess the clinical effectiveness and harms of interventions for treating leg ulcers in people with sickle cell disease. Search methods: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register. We searched LILACS (1982 to January 2020), ISI Web of Knowledge (1985 to January 2020), and the Clinical Trials Search Portal of the World Health Organization (January 2020). We checked the reference lists of all the trials identified. We also contacted those groups or individuals who may have completed relevant randomised trials in this area. Date of the last search of the Cochrane Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register: 13 January 2020; date of the last search of the Cochrane Wounds Group Trials Register: 17 February 2017. Selection criteria: Randomised controlled trials of interventions for treating leg ulcers in people with sickle cell disease compared to placebo or an alternative treatment. Data collection and analysis: Two authors independently selected studies for inclusion. All three authors independently assessed the risk of bias of the included studies and extracted data. We used GRADE to assess the quality of the evidence. Main results: Six studies met the inclusion criteria (198 participants with 250 ulcers). Each trial investigated a different intervention and within this review we have grouped these as systemic pharmaceutical interventions (L-cartinine, arginine butyrate, isoxsuprine) and topical pharmaceutical interventions (Solcoseryl® cream, arginine-glycine-aspartic acid (RGD) peptide dressing and topical antibiotics). No trials on non-pharmaceutical interventions were included in the review. All trials had an overall unclear or high risk of bias, and drug companies sponsored four of them. We were unable to pool findings due to the heterogeneity in outcome definitions, and inconsistency between the units of randomisation and analysis. Three interventions reported on the change in ulcer size (arginine butyrate, RGD peptide, L-cartinine). Of these, only arginine butyrate showed a reduction of ulcer size compared with a control group, mean reduction -5.10 cm² (95% CI -9.65 to -0.55), but we are uncertain whether this reduces ulcer size compared to standard care alone as the certainty of the evidence has been assessed as very low. Three trials reported on complete leg ulcer closure (isoxsuprine, arginine butyrate, RGD peptide matrix; very low quality of evidence). None reported a clinical benefit. No trial reported on: the time to complete ulcer healing; ulcer-free survival following treatment for sickle cell leg ulcers; quality of life measures; incidence of amputation or harms. Authors' conclusions: Given the very low quality of the evidence identified in this updated Cochrane Review we are uncertain whether any of the assessed pharmaceutical interventions reduce ulcer size or result in leg ulcer closure in treated participants compared to controls. However, this intervention was assessed as having a high risk of bias due to inadequacies in the single trial report. Other included studies were also assessed as having an unclear or high risk of bias. The harm profile of the all interventions remains inconclusive. Copyright © 2021 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.

SummaryData on human skin fibroblast transcriptional responses to external cues were used to reconstruct dynamic gene regulatory networks. The goal of the reconstruction was to determine dynamic network interactions (quantitative predictive relationships of mutual regulatory influences of and on transcription factor expression) from time course data on 56 transcript expression levels obtained following different external cues. The inherently under-determined nature of this problem was addressed in part by excluding putative regulatory motifs that did not appear to be functional in multiple independent experiments from different independent external perturbations. Data were obtained from a previously published experiment in which the 56 transcripts were assayed by bioluminescence in live cells cultured on substrates of varying levels of stiffness and exposed to different levels of arginylglycylaspartic acid (RGD) peptide. The inferred dynamical networks were validated via comparison of predictions to a priori known interactions from gene databases. We discovered that exposures to different substrate stiffnesses and to RGD stimulate responses that are mediated through GATA4, SMAD3/4, ETS-1, and STAT5 and other genes, which can initiate hypertrophic, fibrotic, and inflammatory responses. The developed dynamical system identification method for discovering new mechanotransduction pathways is applicable to the identification of gene regulatory networks in numerous emerging applications where time-series data on multiple state variables and from multiple external perturbations are available.

Bombyx mori silk fibroin (SF) was successfully used for vascular grafts implanted in rats or dogs. Current transgenic technology can be developed to produce SF with improved properties. In this study, the vascular endothelial growth factor (VEGF) or the repeated fibronectin-derived sequence, TGRGDSPAS, and arginylglycylaspartic acid (RGD) were introduced into the SF heavy chain to improve its properties. A blood compatibility assay was performed to study lactose dehydrogenase (LDH) activity for both transgenic and wild type SF. Growth of human umbilical endothelial cells (HUVECs) showed greater enhancement of cellularization behaviour for the transgenic SF samples (VEGF and RGD) than for the wild type (WT) SF. VEGF SF also showed lower platelet adhesion than the RGD SF and WT SF. An in vivo implantation study supported these in vitro results. In particular, early endothelialisation was observed for VEGF transgenic SF, including the occurrence of native tissue organization at three months after implantation in rat abdominal aorta.

A new strategy for designing contrast agents (CAs) based on geometrical confinement will become a competent way to improve the relaxivity of CAs. Herein, a magnetic resonance imaging (MRI) nanoconstruct is fabricated through loading Gd2O3 nanoparticles into mesoporous carbon nanospheres, followed by conjugation of poly(ethylene glycol) (PEG) and the c(RGDyK) peptide (Gd2O3@OMCN-PEG-RGD), which could prolong the blood circulation half-life as well as improve the tumor-targeting ability. As a result, the Gd2O3@OMCN-PEG-RGD exhibits an outstandingly high relaxivity (r1 = 68.02 mM–1 s–1), which is ∼5.3 times higher than that of Gd2O3 nanoparticles (r1 = 12.74 mM–1 s–1). Afterward, both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test and H&E staining show that the Gd2O3@OMCN-PEG-RGD has wonderful biocompatibility in vitro and in vivo. Moreover, the in vivo MR images indicate that the Gd2O3@OMCN-PEG-RGD could accumulate in the tumor region more rapidly than Gd2O3@OMCN-PEG. This study prese...

As a conductive nanomaterial, graphene has huge potentials in nerve function restoration by promoting electrical signal transduction and metabolic activities with unique topological properties. Polydopamine (PDA) and arginylglycylaspartic acid (RGD) can improve cell adhesion in tissue engineering. Here we report an integrated 3D printing and layer-by-layer casting (LBLC) method in multi-layered porous scaffold fabrication. The scaffold is composed of single-layered graphene (SG) or multi-layered graphene (MG) and polycaprolactone (PCL). The electrically conductive 3D graphene scaffold can significantly improve neural expression both in vitro and in vivo. It promotes successful axonal regrowth and remyelination after peripheral nerve injury. These findings implicate that graphene-based nanotechnology have great potentials in peripheral nerve restoration in preclinical and clinical application., Graphene, as a conductive nanomaterial, has potential applications in the restoration of nerve function following physical injury. Here the authors design a graphene scaffold that can improve nerve regeneration.

Although microfiber (MF) loaded polypeptides have been widely studied in the field of medicine, load control and slow release remain significant challenges. Herein, polybutylene adipate terephthalate (PBAT) hollow porous MFs (HPMFs) were prepared by improving and regulating the groups and structures of the MFs to address these challenges. The capacity of the HPMFs for the loading of polypeptide can be improved. Measurements involving the use of X-ray photoelectron, energy-dispersive, and ultraviolet (UV) spectroscopies in conjunction with scanning electron microscopy measurements showed that the amount of polypeptide (arginylglycylaspartic acid, RGD) loaded on the HPMFs was significantly higher than that loaded on the MFs. Animal cell experiments revealed that PBAT grafted MAH has good bioactivity. By loading RGD onto HPMFs and MFs, RGD@HPMFs and RGD@MFs were obtained. A study on HeLa and A549 cancer cells showed that the inhibition rates of RGD@HPMFs were higher than that of RGD@MFs by 14.1% and 6.9%, respectively. The results obtained herein show that HPMF scaffold preparation by improving the material groups and regulating the structure of MFs can address the challenges associated with control of the load and sustained release of polypeptides and other drugs.

Upconversion NaLuF4 nanophosphors (UCNPs) with excellent upconversion luminescence (UCL) and good biocompatibility were synthesized by the solvothermal method. The surface of the NaLuF4 UCNPs was modified with the tripeptide arginylglycylaspartic acid (RGD) nanocrystals for targeted tumor cells based on the high and specific affinity to the transmembrane receptor αvβ3 integrin. The morphology, size and UCL properties of the UCNPs were investigated in detail. The methyl thiazolyl tetrazolium (MTT) assay showed low cytotoxicity of the UCNPs to tumor cells and normal cells, indicative of suitability for bioapplication. The RGD modified NaLuF4 nanocrystals have been used for UCL targeted cell imaging in U87MG tumor cells. The results demonstrated that RGD modification allowed the NaLuF4 UCNPs for targeted UCL cell imaging under excitation at 980 nm owing to the high and specific affinity between RGD and the αvβ3 integrin.

Chemotherapy of bladder cancer has limited efficacy because of the short retention time of drugs in the bladder during therapy. In this research, nanoparticles (NPs) with a new core/shell/corona nanostructure have been synthesized, consisting of iron oxide (Fe3O4) as the core to providing magnetic properties, drug (doxorubicin) loaded calcium phosphate (CaP) as the shell for pH-responsive release, and arginylglycylaspartic acid (RGD)-containing peptide functionalized alginate as the corona for cell targeting (with the composite denoted as RGD-Fe3O4/CaP/Alg NPs). We have optimized the reaction conditions to obtain RGD-Fe3O4/CaP/Alg NPs with high biocompatibility and suitable particle size, surface functionality, and drug loading/release behavior. The results indicate that the RGD-Fe3O4/CaP/Alg NPs exhibit enhanced chemotherapy efficacy toward T24 bladder cancer cells, owing to successful magnetic guidance, pH-responsive release, and improved cellular uptake, which give these NPs great potential as therapeu...

Preclinical Research Doxorubicin (DOX) is commonly used for the treatment of breast cancer and lymphoma. However, its clinical use has been severely limited due to cardiotoxicity, requiring the development of safer and more efficient pharmaceutical formulations of DOX. Advances in nanotechnology have provided new ways to administer chemotherapeutic drugs like DOX are conveyed into the body and to tumor sites. These Nanotechnology approaches have aided in the selective accumulation of DOX into tumor sites via the enhanced permeability and retention. However, the absence of active targeting ligands still hinders the effective delivery of DOX. Among all active targeting ligands developed to date, RGD peptide (Arginylglycylaspartic acid) occupies a unique position owing to its inherent safety, biocompatibility, and targeting ability. Accordingly, modification of DOX with RGD ligand is anticipated to improve transport of DOX into tumor cells. In this review, we discuss using RGD peptide for improving the therapeutic efficacy of DOX nanomedicine. Drug Dev Res 78 : 283-291, 2017. © 2017 Wiley Periodicals, Inc.

Here, we report a peptide aspartic acid side-chain benzyl ester as a useful precursor that can be efficiently converted into various functional groups, including acid, amide, carbonyl hydrazide, carbonyl azide, or thio ester groups, without other protection for the peptide. With this strategy, we synthesized a series of novel branched and cyclic arginylglycylaspartic acid peptides through successive peptide C-terminal ligation and side-chain ligation based on a side-chain carbonyl azide or thio ester.

RSC Advances 10(8), 4190 - 4200 (2020). doi:10.1039/C9RA08169K, Fibrin is a well-known tool in tissue engineering, but the structure of its modifications created to improve its properties remains undiscussed despite its importance, e.g. in designing biomaterials that ensure cell migration and lumenogenesis. We sought to uncover the structural aspects of PEGylated fibrin hydrogels shown to contribute to angiogenesis. The analysis of the small-angle X-ray scattering (SAXS) data and ab initio modeling revealed that the PEGylation of fibrinogen led to the formation of oligomeric species, which are larger at a higher PEG : fibrinogen molar ratio. The improvement of optical properties was provided by the decrease in aggregates' sizes and also by retaining the bound water. Compared to the native fibrin, the structure of the 5 : 1 PEGylated fibrin gel consisted of homogenously distributed flexible fibrils with a smaller space between them. Moreover, as arginylglycylaspartic acid (RGD) sites may be partly bound to PEG-NHS or masked because of the oligomerization, the number of adhesion sites may be slightly reduced that may provide the better cell migration and formation of continuous capillary-like structures., Published by RSC Publishing, London

A new biomimetic nanointerface was constructed by facile grafting the bioactive arginylglycylaspartic acid (RGD) peptide on the graphene oxide (GO) surface through carbodiimide and N-hydroxysuccinimide coupling amidation reaction. The formed RGD-GO nanocomposites own unique two-dimensional structure and desirable electrochemical performance. The linked RGD peptides could improve GO’s biocompatibility and support the adhesion and proliferation of human periodontal ligament fibroblasts (HPLFs) on RGD-GO biofilm surface. Furthermore the biologically active RGD-GO nanocomposites were demonstrated as a potential biomimetic nanointerface for monitoring cell biobehaviors by electrochemical impedance spectroscopy (EIS). By analysis of the data obtained from equivalent circuit-fitting impedance spectroscopy, the information related to cell membrane capacitance, cell-cell gap resistance, and cell-electrode interface gap resistance in the process of cell adhesion and proliferation could be obtained. Besides, this proposed impedance-based cell sensor could be used to assess the inhibition effect of the lipopolysaccharide (LPS) on the HPLFs proliferation. Findings from this work suggested that RGD peptide functionalized GO nanomaterials may be not only applied in dental tissue engineering but also used as a sensor interface for electrochemical detection and analysis of cell behaviors in vitro.

In order to stimulate the cellular response to implant materials, extracellular matrix (ECM) proteins, such as collagen and fibronectin (FN), are immobilized on the implant surface. Amongst all ECM proteins used for biomimetic materials for medical applications, FN is one of the most investigated proteins thanks to its ability to promote cell adhesion and its contribution to important physiological processes. However, its conformation and hence its bioactivity strongly depend on the hydrophilic/hydrophobic nature of the surface as well as on immobilization strategies. This work investigates the effect of these two parameters, as well as the effect of the crosslinker length. FN was grafted onto silicon wafers using eights different linking arms presenting different lengths, hydrophilic/hydrophobic characters and binding sites. The protein was linked through either its amino groups (lysine amino acids) or sulfhydryl functionalities (cysteine amino acids). The grafting of each crosslinker and subsequent FN conjugation onto the surfaces was evidenced by X-ray photoelectron spectroscopy, while the surface hydrophilicity was determined by contact angle measurements. Moreover, atomic force microscopy images revealed that the conformation of surface conjugated FN only depends on the hydrophilicity of the linking arm. The FN conformation was also probed by enzyme-linked immunosorbent assays (ELISA). ELISA data demonstrated that all of the three investigated parameters linking arm parameter (length, hydrophobic/hydrophilic character, and terminal end-group) somewhat influence the RGD accessibility.

Objective The purpose of this study was to produce an arginylglycylaspartic acid (RGD) peptide-modified ultra-small superparamagnetic iron oxide (Fe3O4) nanoparticles (NPs) for targeted magnetic resonance (MR) imaging of hepatocellular carcinoma (HCC) cells and verify its utility as a T1 positive MRI imaging contrast agent in vitroand in vivo.Methods The carboxylated Fe3O4 NPs stabilized with sodium citrate were conjugated with polyethylene glycol (PEG)-linked RGD nanoparticles to form a novel target contrast agent Fe3O4-PEG-RGD NPs. The specificity of Fe3O4-PEG-RGD to bind RGD receptor was investigated in vitro by HepG2 cellular uptake and cell MR imaging, and in vivo by MR imaging of subcutaneous HepG2 tumors of nude mice.Results The formed Fe3O4-PEG-RGD NPs displayed good biocompatibility, and the ultrahigh r1 relaxivity was 1.37 mM -1S -1. The synthesized Fe3O4-PEG-RGD NPs were demonstrated spherical-like with an approximate diameter of 2.7 nm in similar size. The targeting effect to HepG2 cells was confirmed by in vitro cellular uptake and cell MR imaging. The in vivo MR imaging of nude mice demonstrated that the MR signal intensity enhancement of HepG2 tumor in Fe3O4-PEG-RGD NPs treated mice was significantly higher than in mice treated with non-targeted Fe3O4-mPEG NPs at the same post-administration time point. Conclusion The results indicate that the Fe3O4-PEG-RGD particles have potential utility as T1 positive contrast agent in targeted MR imaging.

Methods for efficient detachment of cells avoiding damage are required in tissue engineering and regenerative medicine. We introduce a bottom–up approach to build plasmonic substrates using micellar block copolymer nanolithography to generate a 2D array of Au seeds, followed by chemical growth leading to anisotropic nanoparticles. The resulting plasmonic substrates show a broad plasmon band covering a wide part of the visible and near‐infrared (NIR) spectral ranges. Both human and murine cells were successfully grown on the substrates. A simple functionalization step of the plasmonic substrates with the cyclic arginylglycylaspartic acid (c‐RGD) peptide allowed us to tune the morphology of integrin‐rich human umbilical vein endothelial cells (HUVEC). Subsequent irradiation with a NIR laser led to highly efficient detachment of the cells with cell viability confirmed using the MTT assay. We thus propose the use of such plasmonic substrates for cell growth and controlled detachment using remote near‐IR irradiation, as a general method for cell culture in biomedical applications.

We report a convenient approach to prepare ultrasmall Fe3O4 nanoparticles (NPs) functionalized with an arginylglycylaspartic acid (RGD) peptide for in vitro and in vivo magnetic resonance (MR) imaging of gliomas. In our work, stable sodium citrate-stabilized Fe3O4 NPs were prepared by a solvothermal route. Then, the carboxylated Fe3O4 NPs stabilized with sodium citrate were conjugated with polyethylene glycol (PEG)-linked RGD. The formed ultrasmall RGD-functionalized nanoprobe (Fe3O4-PEG-RGD) was fully characterized using different techniques. We show that these Fe3O4-PEG-RGD particles with a size of 2.7 nm are water-dispersible, stable, cytocompatible and hemocompatible in a given concentration range, and display targeting specificity to glioma cells overexpressing αvβ3 integrin in vitro. With the relatively high r1 relaxivity (r1 = 1.4 mM(-1) s(-1)), the Fe3O4-PEG-RGD particles can be used as an efficient nanoprobe for targeted T1-weighted positive MR imaging of glioma cells in vitro and the xenografted tumor model in vivo via an active RGD-mediated targeting pathway. The developed RGD-functionalized Fe3O4 NPs may hold great promise to be used as a nanoprobe for targeted T1-weighted MR imaging of different αvβ3 integrin-overexpressing cancer cells or biological systems.

Doxorubicin (Dox) clinical use is limited by dose-related cardiomyopathy, becoming more prevalent with increasing cumulative doses. Previously, we developed Dox-loaded lipid-core nanocapsules (Dox-LNC) and, in this study, we hypothesized that self-assembling and interfacial reactions could be used to obtain arginylglycylaspartic acid (RGD)-surface-functionalized-Dox-LNC, which could target tumoral cells overexpressing αvβ3 integrin. Human breast adenocarcinoma cell line (MCF-7) and human glioblastoma astrocytoma (U87MG) expressing different levels of αvβ3 integrin were studied. RGD-functionalized Dox-LNC were prepared with Dox at 100 and 500 mg·mL−1 (RGD-MCMN (Dox100) and RGD-MCMN (Dox500)). Blank formulation (RGD-MCMN) had z-average diameter of 162 ± 6 nm, polydispersity index of 0.11 ± 0.04, zeta potential of +13.2 ± 1.9 mV and (6.2 ± 1.1) × 1011 particles mL−1, while RGD-MCMN (Dox100) and RGD-MCMN (Dox500) showed respectively 146 ± 20 and 215 ± 25 nm, 0.10 ± 0.01 and 0.09 ± 0.03, +13.8 ± 2.3 and +16.4 ± 1.5 mV and (6.9 ± 0.6) × 1011 and (6.1 ± 1.0) × 1011 particles mL−1. RGD complexation was 7.73 × 104 molecules per nanocapsule and Dox loading were 1.51 × 104 and 7.64 × 104 molecules per nanocapsule, respectively. RGD-functionalized nanocapsules had an improved uptake capacity by U87MG cells. Pareto chart showed that the cell viability was mainly affected by the Dox concentration and the period of treatment in both MCF-7 and U87MG. The influence of RGD-functionalization on cell viability was a determinant factor exclusively to U87MG.

Aggressive tumors are characterized by angiogenesis that promotes the migration and dissemination of tumor cells. Our aim was to develop a dual-targeted microbubble system for non-invasive evaluation of tumor angiogenesis in ultrasound. Avidinylated microbubbles were conjugated with biotinylated arginylglycylaspartic acid and vascular endothelial growth factor receptor 2 (VEGFR2) antibodies. Subcutaneous MHCC-97H liver carcinoma models were established. Non-targeted, αvβ3-targeted, VEGFR2-targeted and dual-targeted microbubbles was intravenously injected in series while acquiring ultrasound images of the tumor. The microbubbles were destroyed by a high-mechanical-index pulse 4 min after the injection. Peak intensity (PI) before and after the destructive pulse was recorded to compare contrast enhancement by different microbubbles. The targeting rates of the integrin-targeted, VEGFR2-targeted and dual-targeted groups were 95.02%, 96.04% and 94.23%, respectively, with no significant differences. Tumors in all groups were significantly enhanced. The time-intensity curve indicated no significant differences in arrival time, PI, area under the curve, amplitude and mean transit time. The difference in ultrasound signal intensity before and after the destructive pulse (⊿PI) for all targeted microbubble groups was significantly greater than that for the non-targeted microbubble group (all p values 0.05), and the difference for the dual-targeted microbubble group was significantly greater than those of both mono-targeted groups (p 0.05).

Zhiwang Song, Yun Lin, Xia Zhang, Chan Feng, Yonglin Lu, Yong Gao, Chunyan Dong Department of Oncology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China Abstract: Apatinib is an oral tyrosine kinase inhibitor, which selectively targets vascular endothelial growth factor receptor 2 and has the potential to treat many tumors therapeutically. Cyclic arginylglycylaspartic acid (cRGD)- and polyethylene glycol (PEG)-modified liposomes (cRGD-Lipo-PEG) were constructed to act as a targeted delivery system for the delivery of apatinib to the human colonic cancer cell line, HCT116. These cRGD-modified liposomes specifically recognized integrin αvβ3 and exhibited greater uptake efficiency with respect to delivering liposomes into HCT116 cells when compared to nontargeted liposomes (Lipo-PEG), as well as greater death of tumor cells and apoptosis. The mechanism by which cRGD-Lipo-PEG targets cells was elucidated further with competition assays. To determine the anticancer efficacy in vivo, nude mice were implanted with HCT116 xenografts and treated with apatinib-loaded liposomes or free apatinib intravenously or via intragastric administration. The active and passive targeting of cRGD-Lipo-PEG led to significant tumor treatment targeting ability, better inhibition of tumor growth, and less toxicity when compared with treatments using uncombined apatinib. The results presented strongly support the case for cRGD-Lipo-PEG representing a targeted delivery system for apatinib in the treatment of colonic cancer. Keywords: integrin αvβ3, cRGD, targeted oral therapy, apatinib, colorectal cancer

A systematic analysis of results available from in vitro, in vivo and clinical trials on the effects of biocompatible calcium phosphate (CaP) coatings is presented. An overview of the most frequently used methods to prepare CaP-based coatings was conducted. Dense, homogeneous, highly adherent and biocompatible CaP or hybrid organic/inorganic CaP coatings with tailored properties can be deposited. It has been demonstrated that CaP coatings have a significant effect on the bone regeneration process. In vitro experiments using different cells (e.g. SaOS-2, human mesenchymal stem cells and osteoblast-like cells) have revealed that CaP coatings enhance cellular adhesion, proliferation and differentiation to promote bone regeneration. However, in vivo, the exact mechanism of osteogenesis in response to CaP coatings is unclear; indeed, there are conflicting reports of the effectiveness of CaP coatings, with results ranging from highly effective to no significant or even negative effects. This review therefore highlights progress in CaP coatings for orthopaedic implants and discusses the future research and use of these devices. Currently, an exciting area of research is in bioactive hybrid composite CaP-based coatings containing both inorganic (CaP coating) and organic (collagen, bone morphogenetic proteins, arginylglycylaspartic acid etc.) components with the aim of promoting tissue ingrowth and vascularization. Further investigations are necessary to reveal the relative influences of implant design, surgical procedure, and coating characteristics (thickness, structure, topography, porosity, wettability etc.) on the long-term clinical effects of hybrid CaP coatings. In addition to commercially available plasma spraying, other effective routes for the fabrication of hybrid CaP coatings for clinical use still need to be determined and current progress is discussed.

The adsorption of peptides on metal oxides is an area of significant interest, both fundamentally and in a number of technologically important areas. These range from the integration of biomaterials in the body, to denaturation of protein therapeutics and the use of biomolecules and bioinspired materials in synthesis and stabilization of novel nanomaterials. Here we present a study of the tripeptide arginylglycylaspartic acid (RGD) on the surfaces of vacuum-prepared single crystalline TiO2(110), pyrocatechol-capped TiO2(110), and model SLA and SLActive dental implant samples. X-ray Photoelectron Spectroscopy and Scanning Tunneling Microscopy show that the RGD adsorption mode on the single crystal is consistent with bonding through the deprotonated carboxylate groups of the peptide to surface Ti atoms of the substrate. Despite the increased hydrophobicity of the pyrocatechol-capped TiO2(110) surface RGD adsorption from solution increases following this surface treatment. RGD adsorption on SLA and SLActive surfaces shows that the SLActive surface has a greater uptake of RGD. The RGD uptake on the pyrocatechol capped single crystal and the model implant surfaces suggest that the ease with which surface contaminant hydrocarbons are removed from the surface has a greater influence on peptide adsorption than hydrophobicity/hydrophilicity of the surface.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy. The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

Implanted polymeric materials, such as medical devices, provoke the body to initiate an inflammatory reaction, known as the foreign body response (FBR), which causes several complications. In this study, polyurethane (Tecoflex�, PU) surface modified with the nonionic surfactant Tween80� (PU/T80) and the cell adhesive PLL-RGD peptide (PU/PLL-RGD) by a previously described entrapment technique were implanted in the peritoneal cavity of Wistar rats for 30 days. Implants were retrieved and examined for tissue reactivity and cellular adherence by various microscopic and analytical techniques. Surface-induced inflammatory response was assessed by real-time PCR based quantification of proinflammatory cytokine transcripts, namely, TNF-? and IL-1?, normalized to housekeeping gene GAPDH. Cellular adherence and their distribution profile were assessed by microscopic examination of H&E stained implant sections. It was observed that PU/PLL-RGD followed by the bare PU surface exhibited severe inflammatory and fibrotic response with an average mean thickness of 19 and 12 I?m, respectively, in 30 days. In contrast, PU/T80 surface showed only a cellular monolayer of 2-3 I?m in thickness, with a mild inflammatory response and no fibrotic encapsulation. The PU/PLL-RGD peptide-modified substrate promoted an enhanced rate of macrophage cell fusion to form foreign body giant cell (FBGCs), whereas FBGCs were rarely observed on Tween80�-modified substrate. The expression levels of proinflammatory cytokines (TNF-? and IL-1?) were upregulated on PU/PLL-RGD surface followed by bare PU, whereas the cytokine expressions were significantly suppressed on PU/T80 surface. Thus, our study highlights modulation of foreign body response on polyurethane surfaces through surface entrapment technique in the form of differential responses observed on PLL-RGD and Tween80� modified surfaces with the former effective in triggering tissue cell adhesion thereby fibrous encapsulation, while the later being mostly resistant to this phenomenon. � 2010 Wiley Periodicals, Inc.

Objective: Crab shell chitosan is a biomaterial used for scaffolding. In Indonesia, Badan Tenaga Nuklir Nasional has made a crab shell chitosan arginylglycylaspartic acid (RGD) scaffold membrane. The purpose of adding RGD was to enhance cell attachment to the scaffold. The objective of this research is to analyze the toxicity of crab shell chitosan RGD scaffold membrane on human dental pulp cells and its effect on their viabilityMethods: Human dental pulp cells were cultured for 5 days in Minimum Essential Medium Alpha (α-MEM) complete containing amphotericin B, penicillin, streptomycin, and fetal bovine serum. Then, the treatment group was exposed to crab shell chitosan RGD scaffold membrane and crab shell chitosan scaffold membrane incubated for 24 h. The toxicity of the crab shell chitosan RGD scaffold membrane was analyzed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Result: The result of this research is that crab shell chitosan RGD scaffold membrane did not decrease the percentage of viability of human dental pulp cells.Conclusion: It is concluded that crab shell chitosan RGD scaffold membrane does not have toxic effects on human dental pulp cells.

The crystal structure of a tetrahydrated form of L-arginyl-glycyl-L-aspartic acid (RGD), the consensus sequence for binding of fibrinogen to cell surface receptors, has been determined from diffractometer data. The tripeptide was crystallized in double zwitterionic form via hanging drop vapor diffusion experiments at a pH near 6.5. The orthorhombic unit cell contains four formula units in space group P2(1)2(1)2(1) with lattice parameters a = 4.852(4), b = 11.376(3), c = 34.083(8)A at RT. The structure was solved by direct methods and refined to a final R = 0.067 based upon 1345 observations with I greater than or equal to 2 sigma(I). Peptide bonds both are trans, omega 2 = 174.2(6) degrees and omega 3 = -169.3(6) degrees. The backbone bends at glycine with phi 2 = -85.5(8) degrees. One of the water molecules sits between the arginyl side chain and the C-terminal carboxylate, forming an intramolecular hydrogen bond to the glycyl carboxyl and linking adjacent molecules through two other H-bond interactions. Comparison of the structure to RGD sequences extracted from 3-D protein structures reveals a diversity of conformations for this tripeptide sequence.

During the past years, we have explored the cellular delivery of kinase inhibitors. Kinase inhibitors have selectivity for specific kinases but they lack cellular selectivity. This is exemplified by recent reports on cardiotoxicity of kinase inhibitors used in cancer treatment. We postulate that targeted cellular delivery of kinase inhibitors can improve their safety/toxicity profiles, as will be exemplified by recent published studies. Cell specific delivery of therapeutics is a quickly growing area of investigation. This innovative strategy employs carrier molecules that bind to receptors exposed on the surface of cell types involved in disease processes. Binding and receptor mediated internalization of the carrier facilitates local accumulation of the product in target cells. Upon systemic administration, this may create local drug depots in specific organs, while other tissues are avoided, thus favoring enhanced localized drug efficacy and reduced side-effects. Synthesis of targeted kinase inhibitor-carrier conjugates was achieved using a new approach, in which kinase inhibitors were bound to a platinum(II) atom, the so-called Universal Linkage System (ULS). We review this novel linkage chemistry and demonstrate the applicability of ULS for drug targeting approaches aiming at angiogenic endothelial cells, hepatic stellate cells, and kidney tubular cells. We will review important issues like drug release mechanism, safety of the linker, and pharmacokinetics of the products in animals. Finally, we review the pharmacological efficacy of the cellular targeted drug conjugates in experimental animal models, especially in renal and liver fibrosis models.

We synthesized a series of RGD peptides and immobilized them to an amine-functional self-assembled monolayer using a modified maleimide-based conjugate technique that minimizes nonspecific interactions. Using a spinning disc apparatus, a trend in the detachment strength (τ50) of RGD peptides of different flanking residues was found: RGDSPK > RGDSVVYGLR ≈ RGDS > RGES. Using blocking monoclonal antibodies, cellular adhesion to the peptides was shown to be primarily αv-integrin-mediated. In contrast, the τ50 value of the cells on fibronectin (Fn)-coated substrates of similar surface density was 6–7 times higher and involved both α5β1 and αvβ3 integrins. Cellular spreading was enhanced on RGD peptides after 1 h when compared to RGE and unmodified substrates. However, no significant differences were observed between the different RGD peptides. Long-term function of MC3T3-E1 cells was also evaluated by measuring alkaline phosphatase (ALP) activity and mineral deposition. Among the four peptides, RGDSPK exhibited the highest level of ALP activity after 11 days and mineralization after 15 days and reached comparable levels as Fn substrates after 15 and 24 days, respectively. These findings collectively illustrate both the advantages and limitations of enhancing cellular adhesion and function by the design of RGD peptides. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2007

Hydrogels possess great potential in biofabrication because they allow cell encapsulation and proliferation in a highly hydrated three-dimensional environment, and they provide biologically relevant chemical and physical signals. However, development of hydrogel systems that mimic the complexity of natural extracellular matrix remains a challenge. In this study, we report the development of a binary hydrogel system containing a synthetic poly(amido amine) (PAMAM) dendrimer and a natural polymer, i.e., hyaluronic acid (HA), to form a fast cross-linking hydrogel. Live cell staining experiment and cell viability assay of bone marrow stem cells demonstrated that cells were viable and proliferating in the in situ formed PAMAM/HA hydrogel system. Furthermore, introduction of a Arginylglycylaspartic acid (RGD) peptide into the hydrogel system significantly improved the cell viability, proliferation, and attachment. Therefore, this PAMAM/HA hydrogel system could be a promising platform for various applications in biofabrication.