RGD Moieties Research Articles

Tumor phagocytosis-driven STING activation invigorates antitumor immunity and reprograms the tumor micro-environment

Immunogenic cell death (ICD) holds the potential for in situ tumor vaccination while concurrently eradicating tumors and stimulating adaptive immunity. Most ICD inducers, however, elicit insufficient immune responses due to negative feedback against ICD biomarkers, limited infiltration of antitumoral immune cells, and the immunosuppressive tumor micro-environment (TME). Recent findings highlight the pivotal roles of stimulators of interferon gene (STING) activation, particularly in stimulating antigen-presenting cells (APCs) and TME reprogramming, addressing ICD limitations. Herein, we introduced ‘tumor phagocytosis-driven STING activation’, which involves the activation of STING in APCs during the recognition of ICD-induced cancer cells. We developed a polypeptide-based nanocarrier encapsulating both doxorubicin (DOX) and diABZI STING agonist 3 (dSA3) to facilitate this hypothesis in vitro and in vivo. After systemic administration, nanoparticles predominantly accumulated in tumor tissue and significantly enhanced anticancer efficacy by activating tumor phagocytosis-driven STING activation in MC38 and TC1 tumor models. Immunological activation of APCs occurred within 12 h, subsequently leading to the activation of T cells within 7 days, observed in both the TME and spleen. Furthermore, surface modification of nanoparticles with cyclic RGD (cRGD) moieties, which actively target integrin αvβ3, enhances tumor accumulation and eradication, thereby verifying the establishment of systemic immune memory. Collectively, this study proposes the concept of tumor phagocytosis-driven STING activation and its effectiveness in generating short-term and long-term immune responses.

3D printing of gellan-dextran methacrylate IPNs in glycerol and their bioadhesion by RGD derivatives.

The ever-growing need for new tissue and organ replacement approaches paved the way for tissue engineering. Successful tissue regeneration requires an appropriate scaffold, which allows cell adhesion and provides mechanical support during tissue repair. In this light, an interpenetrating polymer network (IPN) system based on biocompatible polysaccharides, dextran (Dex) and gellan (Ge), was designed and proposed as a surface that facilitates cell adhesion in tissue engineering applications. The new matrix was developed in glycerol, an unconventional solvent, before the chemical functionalization of the polymer backbone, which provides the system with enhanced properties, such as increased stiffness and bioadhesiveness. Dex was modified introducing methacrylic groups, which are known to be sensitive to UV light. At the same time, Ge was functionalized with RGD moieties, known as promoters for cell adhesion. The printability of the systems was evaluated by exploiting the ability of glycerol to act as a co-initiator in the process, speeding up the kinetics of crosslinking. Following semi-IPNs formation, the solvent was removed by extensive solvent exchange with HEPES and CaCl2, leading to conversion into IPNs due to the ionic gelation of Ge chains. Mechanical properties were investigated and IPNs ability to promote osteoblasts adhesion was evaluated on thin-layer, 3D-printed disk films. Our results show a significant increase in adhesion on hydrogels decorated with RGD moieties, where osteoblasts adopted the spindle-shaped morphology typical of adherent mesenchymal cells. Our findings support the use of RGD-decorated Ge/Dex IPNs as new matrices able to support and facilitate cell adhesion in the perspective of bone tissue regeneration.

Nutritionally enhanced fibrous scaffolds by rolling droplet-interfacial polyelectrolyte complexation (RD-IPC)

The challenge of producing protein in a more sustainable manner than what is currently afforded by meat from livestock has led to the advent of cell-based or cultured meat. One method of obtaining whole-cuts of meat would be to culture the relevant cells on fibrous scaffolds to obtain meat-like textures. In the present work, a novel process of rolling droplet interfacial polyelectrolyte complexation (RD-IPC) is used to make aligned fibrous constructs that can be incorporated with nutritional components. Fiber is drawn by a falling droplet consisting of two oppositely charged polyelectrolytes that are continuously delivered by means of a syringe pump. The fibers are collected individually as they form and accumulate on a rotating collecting device to form planar constructs. Scaffolds incorporating gelatin, soybean flour, potato starch and garlic could be made by dispersing the respective powders into the polyelectrolyte solutions. Following the formation of the constructs, a heating step was found to improve their mechanical properties, in particular the potato starch-incorporated constructs, which exhibited higher tensile moduli due to a gelatinization process. Scaffolds imbued with gelatin and RGD moieties supported the adhesion, alignment, and differentiation of mouse myoblasts, while scaffolds incorporating gelatin alone sufficed for the differentiation of preadipocytes. RD-IPC provides for a scalable, continuous process that allows the incorporation of nutritional components into scaffolds possessing good mechanical and cell-adhesive properties. These fibrous scaffolds are constructed using edible polyelectrolyte components and aqueous solutions at relatively mild process conditions, underlining their promise for the nutritional enhancement of cultured meat.

Functionalization of 3D-Printed Titanium Scaffolds with Elastin-like Recombinamers to Improve Cell Colonization and Osteoinduction.

The 3D printing of titanium (Ti) offers countless possibilities for the development of personalized implants with suitable mechanical properties for different medical applications. However, the poor bioactivity of Ti is still a challenge that needs to be addressed to promote scaffold osseointegration. The aim of the present study was to functionalize Ti scaffolds with genetically modified elastin-like recombinamers (ELRs), synthetic polymeric proteins containing the elastin epitopes responsible for their mechanical properties and for promoting mesenchymal stem cell (MSC) recruitment, proliferation, and differentiation to ultimately increase scaffold osseointegration. To this end, ELRs containing specific cell-adhesive (RGD) and/or osteoinductive (SNA15) moieties were covalently attached to Ti scaffolds. Cell adhesion, proliferation, and colonization were enhanced on those scaffolds functionalized with RGD-ELR, while differentiation was promoted on those with SNA15-ELR. The combination of both RGD and SNA15 into the same ELR stimulated cell adhesion, proliferation, and differentiation, although at lower levels than those for every single moiety. These results suggest that biofunctionalization with SNA15-ELRs could modulate the cellular response to improve the osseointegration of Ti implants. Further investigation on the amount and distribution of RGD and SNA15 moieties in ELRs could improve cell adhesion, proliferation, and differentiation compared to the present study.

Detection of U-87 Tumor Cells by RGD-Functionalized/Gd-Containing Giant Unilamellar Vesicles in Magnetization Transfer Contrast Magnetic Resonance Images.

The targeting of tumor cells and their visualization with magnetic resonance imaging (MRI) is an important task in biomedicine. The low sensitivity of this technique is a significant drawback and one that may hamper the detection of the imaging reporters used.To overcome this sensitivity issue, this work explores the synergy between 2 strategies: (1) arginine, glycine, aspartic acid peptide (RGD)-functionalized giant unilamellar vesicles (GUVs) loaded with Gd complexes to accumulate large amounts of MRI contrast agent at the targeting site; and (2) the use of magnetization transfer contrast (MTC), which is a sensitive MRI technique for the detection of Gd complexes in the tumor region. Giant unilamellar vesicles were prepared using the gentle swelling method, and the cyclic RGD targeting moiety was introduced onto the external membrane. Paramagnetic Gd-containing complexes and the fluorescent probe rhodamine were both part of the vesicle membranes and Gd-complexes were also the payload within the inner aqueous cavity. Giant unilamellar vesicles that were loaded with the imaging reporters, but devoid of the RGD targeting moiety, were used as controls. U-87 MG human glioblastoma cells, which are known to overexpress the targets for RGD moieties, were used. In the in vivo experiments, U-87 MG cells were subcutaneously injected into nu/nu mice, and the generated tumors were imaged using MRI, 15 days after cell administration. Magnetic resonance imaging was carried out at 7 T, and T2W, T1W, and MTC/Z-spectra were acquired. Confocal microscopy images and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) were used for result validation. In vitro results show that RGD GUVs specifically bind to U-87 MG cells. Microscopy demonstrates that (1) RGD GUVs were anchored onto the external surface of the tumor cells without any internalization; (2) a low number of GUVs per cell were clustered at specific regions; and (3) there is no evidence for macrophage uptake or cell toxicity. The MRI of cell pellets after incubation with RGD GUVs and untargeted ctrl-GUVs was performed. No difference in T1 signal was detected, whereas a 15% difference in MT contrast is present between the RGD GUV-treated cells and the ctrl-GUV-treated cells.Magnetic resonance imaging scans of tumor-bearing mice were acquired before and after (t = 0, 4 hours and 24 hours) the administration of RGD GUVs and ctrl-GUVs. A roughly 16% MTC difference between the 2 groups was observed after 4 hours. Immunofluorescence analyses and ICP-MS analyses (for Gd-detection) of the explanted tumors confirmed the specific accumulation of RGD GUVs in the tumor region. RGD GUVs seem to be interesting carriers that can facilitate the specific accumulation of MRI contrast agents at the tumor region. However, the concentration achieved is still below the threshold needed for T1w-MRI visualization. Conversely, MTC proved to be sufficiently sensitive for the visualization of detectable contrast between pretargeting and posttargeting images.

A Targeted and pH-Responsive Bortezomib Nanomedicine in the Treatment of Metastatic Bone Tumors.

Bortezomib is a boronate proteasome inhibitor widely used as an efficient anticancer drug; however, the clinical use of bortezomib is hampered by its adverse effects such as hematotoxicity and peripheral neuropathy, and low efficacy on solid tumors due to unfavorable pharmacokinetics and poor penetration in the solid tumors. In this study, we developed a tripeptide Arg-Gly-Asp (RGD)-targeted dendrimer conjugated with catechol and poly(ethylene glycol) groups for the targeted delivery of bortezomib to metastatic bone tumors. Bortezomib was loaded on the dendrimer via a boronate-catechol linkage with pH-responsive property, which plays an essential role in the control of bortezomib loading and release. The nontargeted bortezomib nanomedicine showed minimal cytotoxicity at pH 7.4, but significantly increased anticancer activity when cyclic RGD (cRGD) moieties were anchored on the dendrimer surface. The ligand cRGD enabled efficient internalization of the bortezomib complex by breast cancer cells such as MDA-MB-231 cells. The targeted nanomedicine efficiently depressed the progression of metastatic bone tumors and significantly inhibited the tumor-associated osteolysis in a model of bone tumors. This study provided an insight into the development of nanomedicine for metastatic bone tumors.

Near infrared fluorescent peptide nanoparticles for enhancing esophageal cancer therapeutic efficacy

Various types of nanoparticles have been proposed for targeted drug delivering, imaging, and tracking of therapeutic agents. However, highly biocompatible nanoparticles with structure-induced fluorescence and capability to conjugate with biomarkers and drugs remain lacking. This research proposes and synthesizes fluorescent nanoparticles (f-PNPs) assembled by cyclic peptides to combine imaging and drug delivering for esophageal cancer (EC). To achieve tumor targeting, f-PNPs are first conjugated with RGD moieties to selectively target EC cells via αvβ3 integrin; the nanoparticles are then embedded with epirubicin (EPI). Cell viability assays and analysis of tissue histology reveal that EPI-loaded RGD-f-PNPs (RGD-f-PNPs/EPI) led to significantly reduced cardiotoxicity and improved anti-tumor activity compared to EPI alone. Moreover, the drug delivery to tumor sites and therapeutic responses could be monitored with near-infrared fluorescence using RGD-f-PNPs/EPI. This unique nanoparticle system may lead to potential approaches for bioorganic fluorescence-based delivering, imaging, and drug release tracking.

Electrospun Polythiophene Phenylenes for Tissue Engineering.

This research focuses on the design of biocompatible materials/scaffold suitable for use for tissue engineering. Porous fiber mats were produced through electrospinning of polythiophene phenylene (PThP) conducting polymers blended with poly(lactide- co-glycolic acid) (PLGA). A peptide containing an arginylglycylaspartic acid (RGD) fragment was synthesized using solid phase peptide synthesis and subsequently grafted onto a PThP polymer using azide-alkyne "click" chemistry. The obtained RGD functionalized PThP was also electrospun into a fiber mat. The electrospun mats' morphology, roughness and stiffness were studied by means of scanning electron microscopy (SEM) and atomic force microscopy (AFM) and their electroactivity by cyclic voltammetry. The fibers show excellent cytocompatibility in culture assays with human dermal fibroblasts-adult (HDFa) and human epidermal melanocytes-adult (HEMa) cells. The electrospun fibers' roughness and stiffness changed after exposing the fiber mats to the cell culture medium (measured in dry state), but these changes did not affect the cell proliferation. The cytocompatibility of our porous scaffolds was established for their applicability as cell culture scaffolds by means of investigating mitochondrial activity of HDFa and HEMa cells on the scaffolds. The results revealed that the RGD moieties containing PThP scaffolds hold a promise in biomedical applications, including skin tissue engineering.

Zwitterionic stealth peptide-capped 5-aminolevulinic acid prodrug nanoparticles for targeted photodynamic therapy

5-Aminolevulinic acid (ALA) is a FDA-approved photodynamic therapy (PDT) precursor of protoporphyrin IX (PpIX) used for treating various cancers. However, the internalization of ALA is a big challenge due to its hydrophilic nature and low specificity to cancer cells. In this work, ALA conjugated prodrug nanoparticles were prepared by conjugation of thiolated stealth peptide sequence CPPPPEKEKEKEKEKEDGR and hydrazone-containing ALA to gold nanoparticles (AuNPs). Remarkable anti-fouling ability of ALA prodrug nanoparticles in complex environment was achieved owing to the zwitterionic stealth peptide sequence EKEKEKEKEK. The release of ALA could be greatly accelerated upon incubation of ALA prodrug nanoparticles in lysosomal/endosomal pH (pH 5.5). Meanwhile, the cellular internalization could be greatly enhanced by RGD moieties. MTT results demonstrated that ALA prodrug nanoparticles exhibited better photodynamic cytotoxicity than free ALA after light irradiation, suggesting enhanced photodynamic therapeutic efficacy.

RGD and polyhistidine tumor homing peptides potentiates the action of human Maspin as an antineoplastic candidate.

Maspin, a non-inhibitory member of serine protease family, acts as an effective tumor suppressor by inhibiting cell inhesion and mobility. We found that exogenous wild-type rMaspin had a low effect on tumor growth in vivo. However, when the peptide Arg-Gly-Asp-hexahistidine (RGD-6His) was introduced into rMaspin, the modified rMaspin showed significant inhibitory activity in angiogenic assays and tumor-bearing animal models. Overall, our data suggested that both the RGD and hexahistidine fragments contributed to improve the fusion protein activity and polyhistidine peptide could be considered as flexible linker to separate RGD and Maspin moieties to avoid function interference. Besides, it is an efficient tag to achieve purified recombinant proteins. Furthermore, rMaspin fusing with RGD and hexahistidine could be a viable anticancer candidate.

Extracellular-Matrix-Based and Arg-Gly-Asp–Modified Photopolymerizing Hydrogels for Cartilage Tissue Engineering

Articular cartilage damage is a persistent and increasing problem with the aging population. Strategies to achieve complete repair or functional restoration remain a challenge. Photopolymerizing-based hydrogels have long received an attention in the cartilage tissue engineering, due to their unique bioactivities, flexible method of synthesis, range of constituents, and desirable physical characteristics. In the present study, we have introduced unique bioactivity within the photopolymerizing-based hydrogels by copolymerizing polyethylene glycol (PEG) macromers with methacrylated extracellular matrix (ECM) molecules (hyaluronic acid and chondroitin sulfate [CS]) and integrin binding peptides (RGD peptide). Results indicate that cellular morphology, as observed by the actin cytoskeleton structures, was strongly dependent on the type of ECM component as well as the presence of integrin binding moieties. Further, CS-based hydrogel with integrin binding RGD moieties increased the lubricin (or known as superficial zone protein [SZP]) gene expression of the encapsulated chondrocytes. Additionally, CS-based hydrogel displayed cell-responsive degradation and resulted in increased DNA, GAG, and collagen accumulation compared with other hydrogels. This study demonstrates that integrin-mediated interactions within CS microenvironment provide an optimal hydrogel scaffold for cartilage tissue engineering application.

Comparison of biological properties of 111In-labeled dimeric cyclic RGD peptides

IntroductionIn this study two 111In-labeled dimeric cyclic RGD peptides, 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2), were evaluated as radiotracers for breast tumor imaging. The objective was to evaluate the impact of SAA, PEG2 and 1,2,3-triazole linkers as compare to PEG4 on the tumor uptake and excretion kinetics of 111In radiotracers. MethodsDOTA-Galacto-RGD2 was prepared by conjugation of Galacto-RGD2 with DOTA-OSu in the presence of diisopropylethylamine. Its integrin αvβ3 binding affinity was determined using a whole-cell displacement assay against 125I-echistatin bound to U87MG glioma cells, and was compared with those of c(RGDfK), DOTA-3P-RGD2 and DOTA-3P-RGK2 (a nonsense peptide conjugate with “scrambled” RGK sequences). 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2) were prepared and evaluated for their tumor-targeting capability and biodistribution properties in athymic nude mice bearing MDA-MB-435 breast tumor xenografts. Planar imaging studies were performed to demonstrate the utility of 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2) for breast tumor imaging. ResultsIC50 values of DOTA-Galacto-RGD2, DOTA-3P-RGD2, and DOTA-3P-RGK2 were calculated to be 27±2, 29±4, 596±48nM, respectively. The tumor uptake values of 111In(DOTA-Galacto-RGD2) (6.79±0.98, 6.56±0.56, 4.17±0.61 and 1.09±0.13 %ID/g at 1, 4, 24 and 72hours p.i., respectively) were almost identical to those of 111In(DOTA-3P-RGD2) (6.17±1.65, 5.94±0.84, 3.40±0.50 and 0.99±0.20 %ID/g, respectively). 111In(DOTA-Galacto-RGD2) had a faster clearance from blood and muscle than 111In(DOTA-3P-RGD2), leading to higher tumor/blood and tumor/muscle ratios. 111In(DOTA-3P-RGD2) had lower liver uptake and better tumor/liver ratios than 111In(DOTA-Galacto-RGD2). The tumor uptake of 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2) was both integrin αvβ3 and RGD-specific. Imaging data suggest that 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2) are useful as radiotracers for imaging integrin αvβ3-positive breast tumors. ConclusionThe results from this study suggest that replacing PEG4 linkers between two RGD moieties with a pair of SAA, PEG2 and 1,2,3-triazole groups has little impact on integrin αvβ3 binding affinity and tumor uptake of 111In-labeled dimeric cyclic RGD peptides. Despite the subtle differences in their excretion kinetics from noncancerous tissues, 111In(DOTA-Galacto-RGD2) and 111In(DOTA-3P-RGD2) are useful radiotracers for imaging integrin αvβ3-positive breast tumors.

Cell-adhesive RGD peptide-displaying M13 bacteriophage/PLGA nanofiber matrices for growth of fibroblasts

BackgroundM13 bacteriophages can be readily fabricated as nanofibers due to non-toxic bacterial virus with a nanofiber-like shape. In the present study, we prepared hybrid nanofiber matrices composed of poly(lactic-co-glycolic acid, PLGA) and M13 bacteriophages which were genetically modified to display the RGD peptide on their surface (RGD-M13 phage).ResultsThe surface morphology and chemical composition of hybrid nanofiber matrices were characterized by scanning electron microscopy (SEM) and Raman spectroscopy, respectively. Immunofluorescence staining was conducted to investigate the existence of M13 bacteriophages in RGD-M13 phage/PLGA hybrid nanofibers. In addition, the attachment and proliferation of three different types of fibroblasts on RGD-M13 phage/PLGA nanofiber matrices were evaluated to explore how fibroblasts interact with these matrices. SEM images showed that RGD-M13 phage/PLGA hybrid matrices had the non-woven porous structure, quite similar to that of natural extracellular matrices, having an average fiber diameter of about 190 nm. Immunofluorescence images and Raman spectra revealed that RGD-M13 phages were homogeneously distributed in entire matrices. Moreover, the attachment and proliferation of fibroblasts cultured on RGD-M13 phage/PLGA matrices were significantly enhanced due to enriched RGD moieties on hybrid matrices.ConclusionsThese results suggest that RGD-M13 phage/PLGA matrices can be efficiently used as biomimetic scaffolds for tissue engineering applications.

Differentiated adipose-derived stem cells act synergistically with RGD-modified surfaces to improve neurite outgrowth in a co-culture model

Peripheral nerve damage is a problem encountered after trauma and during surgery and the development of synthetic polymer conduits may offer a promising alternative to autografts. In order to improve the performance of the polymer to be used for nerve conduits, poly-ε-caprolactone (PCL) films were chemically functionalized with RGD moieties, using a chemical reaction previously developed. In vitro cultures of dissociated dorsal root ganglion (DRG) neurons provide a valid model to study different factors affecting axonal growth. In this work, DRG neurons were cultured on RGD-functionalized PCL films. Adult adipose-derived stem cells differentiated to Schwann cells (dASCs) were initially cultured on the functionalized PCL films, resulting in improved attachment and proliferation. dASCs were also co-cultured with DRG neurons on treated and untreated PCL to assess stimulation by dASCs on neurite outgrowth. Neuron response was generally poor on untreated PCL films, but long neurites were observed in the presence of dASCs or RGD moieties. A combination of the two factors enhanced even further neurite outgrowth, acting synergistically. Finally, in order to better understand the extracellular matrix (ECM)-cell interaction, a β1 integrin blocking experiment was carried out. Neurite outgrowth was not affected by the specific antibody blocking, showing that β1 integrin function can be compensated by other molecules present on the cell membrane. Copyright © 2013 John Wiley & Sons, Ltd.

α(V)β(3) integrin-targeted PLGA-PEG nanoparticles for enhanced anti-tumor efficacy of a Pt(IV) prodrug.

Targeted delivery of therapeutics to tumor neovasculature is potentially a powerful approach for selective cancer treatment. Integrins are heterodimeric transmembrane proteins involved in cell adhesion and cell signaling, and their expression is commonly upregulated in cancers and inflammatory diseases. The α(v)β(3) integrin is differentially upregulated on angiogenic endothelial cells as well as on many cancer cells. Here we demonstrate the differential targeting of cisplatin prodrug-encapsulated poly(d,l-lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) to the α(v)β(3) integrin on cancer cells using the cyclic pentapeptide c(RGDfK). Cisplatin is one of the most widely used anticancer drugs, and approaches that can improve its therapeutic index are of broad importance. The RGD-targeted Pt(IV)-encapsulated NPs displayed enhanced cytotoxicity as compared to cisplatin administered in its conventional dosage form in model prostate and breast cancer epithelial cells in vitro. Cytotoxicities were also elevated in comparison to those of previously reported systems, a small molecule Pt(IV)-RGD conjugate and a Pt(IV) nanoscale coordination polymer carrying RGD moieties. This result encouraged us also to evaluate the anticancer effect of the new construct in an animal model. The RGD-targeted PLGA-PEG NPs were more efficacious and better tolerated by comparison to cisplatin in an orthotopic human breast cancer xenograft model in vivo.

Are there Technical/Clinical Tools to Improve the Present Vascular Access Outcome in Haemodialysis Patients?

When native vein and artery are not available due to previous harvest, anatomical limitations, or disease progression, synthetic materials such as Dacron or ePTFE have been used with varying degrees of success. Synthetic graft materials are used with great success in larger diameter applications such as aortic or iliac reconstruction, but they have demonstrated unacceptably poor performance in most small diameter applications (below 6 mm inside diameter). The poor efficacy of small diameter synthetics is linked to short-term thrombosis, increased rate of infection, chronic inflammatory responses to the foreign materials, and compliance mismatch between the native tissue and the prosthetic material. These problems are well illustrated in A-V access grafts, where the intervention rates for synthetic grafts are three-fold higher than for native vein fistulas [1]. Attempts to improve the durability of prosthetic grafts began in the 1970s with the concept of seeding the luminal surface of the graft, considered to be thrombogenic, with endothelial cells [2]. The major technical feat overcome by extensive work in the 1980s and 1990s centered on preventing the cells from being dislodged by luminal blood flow on implantation of the graft. Strategies to overcome this problem include precoating the graft with various adhesives, pressure sodding, modification of the graft surface with RGD moieties, prolonged culture of the graft, and flow conditioning. The field of Cardiovascular Tissue Engineering has attempted to produce a clinically viable synthetic conduit by using a variety of in vitro approaches that typically combine living cells seeded into reconstituted scaffolds to create living tissue engineered blood vessels (TEBVs.

Bioactivation of porous polyurethane scaffolds using fluorinated RGD surface modifiers

Biomaterial scaffolds for tissue engineering require appropriate cell adhesion, proliferation, and infiltration into their three-dimensional (3D) porous structures. Surface modification techniques have the potential to enhance cell infiltration into synthetic scaffolds while retaining bulk material properties intact. The objective of this work was to assess the potential of achieving a uniform surface modification in 3D porous constructs through the blending of surface-modifying additives known as bioactive fluorinated surface modifiers (BFSMs) with a base polyurethane material. By coupling RGD peptides to the fluorinated surface modifiers to form RGD-BFSMs, the BFSMs can act as a vehicle for the delivery of RGD moieties to the surface without direct covalent attachment to the polymer substrate. Fluorescent RGD-BFSMs were shown to migrate to the polymer-air interfaces within the porous scaffolds by two-photon confocal microscopy. A-10 rat aortic smooth muscle cells were cultured for 4 weeks on nonmodified and RGD-BFSM-modified porous scaffolds, and cell adhesion, proliferation, and viability were quantified at different depths. RGD-BFSM-modified scaffolds showed significantly greater cell numbers within deeper regions of the scaffolds, and this difference became more pronounced over time. This study demonstrates an effective approach to promote cell adhesion and infiltration within thick (approximately 0.5 cm) porous synthetic scaffolds by providing a uniform distribution of adhesive peptide throughout the scaffolds without the use of covalent surface reaction chemistry.

RGD-mediated adhesive interactions are important for peripheral axon outgrowth in vivo

We identify an essential role for the RGD (Arg-Gly-Asp tripeptide) moiety in vivo during adult peripheral neuron regenerative growth. Beyond a peripheral nerve transection there were rises in the fibronectin extracellular matrix, and striking rises in the mRNA and protein expression of integrin subunits sensitive to RGD/fibronectin signalling. Neuron perikarya, axons and Schwann cells all expressed RGD/fibronectin sensitive integrins after injury. To evaluate the significance of RGD/fibronectin-integrin interactions, we infused solutions of a pentapeptide including the RGD motif (sRGD) serially and directly within the milieu of early axon growth across rat sciatic transection injuries. While low dose infusions of sRGD facilitated early axon ingrowth, we encountered inhibition of ingrowth and bridge formation with higher doses of sRGD indicating competitive disruption of RGD/fibronectin-integrin signalling. Fibronectin RGD moieties serve a critical and important role during peripheral axon outgrowth.

Chemical Conjugation of Linear and Cyclic RGD Moieties to a Recombinant Elastin‐Mimetic Polypeptide ‐ A Versatile Approach towards Bioactive Protein Hydrogels

An elastin-mimetic polypeptide, (EMM)(7), with the amino-acid sequence GRDPSS [VPGVG VPGKG VPGVG VPGVG VPGEG VPGIG](7) was used for chemical conjugation of various integrin ligands (RGD peptides) to prepare bioactive hydrogels. The chemical approach involved (1) chemical protection of lysine residues with Fmoc or Boc groups, (2) chemical ligation of a protected linear or cyclic RGD ligand, with or without a hexanoic-acid spacer to the glutamic acid residue, (3) deprotection of the lysine functionalities and the RGD moieties and (4) cross-linking to form a bioactive hydrogel. (1)H NMR spectroscopy was used to quantify the multiple steps in the reaction. The chemical protection was found to be between 65 and 93% for Fmoc and Boc, respectively. The ligands studied included linear RGD cell-binding [H-FGRGDS-OH (1-l-RGD), H-Ahx--FGRGDS-OH (2-Ahx-FGRGDS) and a cyclic -H(2)N-(CH(2))(6)COHN-cyclo(-RGDfK-) (H-Ahx-c(-RGDfK-)) peptide also with a hexanoic-acid spacer. Cell adhesion with mouse osteoblast cells was dependent on the ligand type, ligand density and the use of a spacer.

Surface functionalization of titanium with hyaluronic acid/chitosan polyelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion

Titanium (Ti) and its alloys are used extensively in orthopedic implants due to their excellent biocompatibility and mechanical properties. However, titanium-based implant materials have specific complications associated with their applications, such as the loosening of implant–host interface owing to unsatisfactory cell adhesion and the susceptibility of the implants to bacterial infections. Hence, a surface which displays selective biointeractivity, i.e. enhancing beneficial host cell responses but inhibiting pathogenic microbial adhesion, would be highly desirable. This present study aims to improve biocompatibility and confer long-lasting antibacterial properties on Ti via polyelectrolyte multilayers (PEMs) of hyaluronic acid (HA) and chitosan (CH), coupled with surface-immobilized cell-adhesive arginine-glycine-aspartic acid (RGD) peptide. The HA/CH PEM-functionalized Ti is highly effective as an antibacterial surface but the adhesion of bone cells (osteoblasts) is poorer than on pristine Ti. With additional immobilized RGD moieties, the osteoblast adhesion can be significantly improved. The density of the surface-immobilized RGD peptide has a significant effect on osteoblast proliferation and alkaline phosphatase (ALP) activity, and both functions can be increased by 100–200% over that of pristine Ti substrates while retaining high antibacterial efficacy. Such substrates can be expected to have good potential in orthopedic applications.