Browsing by Subject "Peptides."

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Open Access
Biomimetic self-assembled peptide nanofibers for bone regeneration
(2012) Kocabey, Samet
Self-assembled peptide nanofibers are exploited in regenerative medicine applications due to their versatile, biofunctional and extracellular-matrixresembling structures. These properties provide peptide nanofibers with osteoinductive and osteoconductive behaviors for bone regeneration applications through several approaches. In this thesis, two different approaches were discussed, which were developed to induce bone regeneration and mineralization including extracellular matrix mimicking peptide nanofibers based 2-D gel formation and surface functionalization of titanium implants. For this purpose, we designed glycosaminoglycan-mimetic peptide nanofibers inspired by chemical structure of glycosaminoglycans present in the bone extracellular matrix. We demonstrated that glycosaminoglycan-mimetic peptide nanofibers interact with BMP-2, a critical growth factor for osteogenic activity. Glycosaminoglycan-mimicking ability of the peptide nanofibers and their interaction with BMP-2 promoted osteogenic activity of and mineralization by osteoblastic cells. ALP activity, Alizarin Red Staining and EDAX spectroscopy indicated efficacy of the peptide nanofibers for inducing mineralization. We also developed a hybrid osteoconductive system for titanium biomedical implants inspired by mussel adhesion mechanism in order to overcome bone tissue integration problems. For this purpose, Dopa conjugated peptide nanofiber coating was used along with bioactive peptide sequences for osteogenic activity to enhance osseointegration of titanium surface. Dopamediated immobilization of osteogenic peptide nanofibers on titanium surfaces created an osteoconductive interface between osteoblast-like cells and inhibited adhesion and viability of soft tissue forming fibroblasts compared to the uncoated titanium substrate. In summary, osteoinductive and osteoconductive self-assembled peptide nanofibers were developed to promote osteogenic activity and mineralization of osteogenic cells. These bioactive nanofibers provide a potent platform in clinical applications of bone tissue engineering.
Open Access
Cell penetrating peptide amphiphile integrated liposomal systems for enhanced delivery of cargo to tumor cells
(2013) Kılınç, Murat
Liposomes have been extensively utilized as effective nanocarriers due to their enhanced solubility, higher stability and greater ability to facilitate the slow release of encapsulated drugs compared to free drug administrations. Liposomes are also preferred as drug vectors due to their non-toxic nature, biodegradability and structural resemblance to the cell membrane. However, their low internalization efficiencies pose an important challenge for their use in drug delivery applications. Internalization issues inherent in many liposomal systems can be circumvented by the use of cell penetrating peptides, which non-covalently attach on the liposome surface and greatly enhance liposomal uptake in a receptor- and charge-dependent manner. In this study, we examined the liposomal dynamics effected through the integration of an amphiphilic cell penetrating peptide into a simple liposome system. Peptide amphiphiles with a cell penetrating arginine-rich domain were incorporated into liposomal membranes formed by negatively charged dioleoylphosphoglycerol (DOPG) phospholipids in the presence of cholesterol. Throughout the present study, we sought to analyze the effect of peptide incorporation on (a) the physical characteristics, such as size, surface potential and membrane polarity, of the liposomal membrane, (b) the alterations in the encapsulation and delivery mechanisms of hydrophilic (Rhodamine B) and hydrophobic (Nile Red) drug models and (c) the enhancement of therapeutic capability in liposomes loaded with the drugs Doxorubicin-HCl and Paclitaxel. Our results revealed that the modification of liposomes by cell penetrating peptide amphiphiles results in the improvement of cargo delivery and the enhancement of the therapeutic effects of the anticancer drugs Doxurubicin and Paclitaxel.
Open Access
Characterization and corneal tissue engineering application of peptide amphiphiles
(2012) Dağdaş, Yavuz Selim
Molecular self-assembly is a powerful technique for developing novel nanostructures by using non-covalent interactions such as hydrogen bonding, hydrophobic, electrostatic, metal-ligand, π-π and van der Waals interactions. Hydrogen bonding, hydrophobic and electrostatic interactions promote self-assembly of peptide amphiphile molecules into nanofibers. Bundles of nanofibers form a three-dimensional network resulting in gel formation. Concentration and temperature dependent measurements of gel stiffness suggest that the mechanical properties of the gels are determined by a number of factors including the interfiber interactions and mechanical properties of individual nanofibers. Peptide amphiphile molecules provide a convenient model as extracellular matrix mimetic systems for regenerative medicine studies. Since the substrate stiffness is crucial for cellular behaviours such as proliferation, adhesion and differentiation, understanding the mechanisms behind the viscoelastic properties of the gels formed by self-assembling molecules can lead to development of new materials with controlled stiffness. In this study, regeneration of the corneal stroma was used as a model system for utilization of peptide amphiphile molecules in regenerative medicine studies. Corneal stroma is constituted by collagen fiber arrays that are closely packed forming a stiff environment for corneal fibroblasts. The tunability of mechanical properties of self-assembled peptide amphiphile nanostructures was aimed to be utilized in corneal stroma regeneration. Thinning of the corneal stroma is a debilitating problem that can be caused by diseases like keratoconus, infections or accidents. Since corneal stroma has a restricted regenerative capacity, thinning of stroma is usually treated with cornea transplantation, which is limited by the number of donors. In this thesis, I studied mechanical properties of self-assembled peptide amphiphile nanostructures in nanometer and micrometer scale. I found that the divergence in gel stiffness may arise from the difference of strength of interfiber bonds. An injectable, biocompatible, biodegradable and bioactive system that can be used for thickening the corneal stroma was developed. This system that is composed of nanofibers was observed to enhance viability and proliferation of keratocytes in vitro.
Open Access
Characterization of peptide amphiphile nanofibers their interactions with chondroprogenitor cells and morphological analysis of tissues from transgenic animals
(2012) Tombuloğlu, Ayşegül
Peptide amphiphiles, molecules able to self assemble into three dimensional networks resembling to extracellular matrix which is excessive in cartilage tissue, are suitable candidates for overcoming cartilage tissue defects and diseases which constitute central health problems throughout ages. Understanding developmental processes that underlie cartilage formation is also key for regenerating cartilage. In this study, peptide amphiphiles were synthesized, their potential for cartilage regeneration was investigated and a model for cellular aggregation, which is a central process in embryonic cartilage development, was established with chondroprogenitor cells and peptide amphiphile scaffolds. On scaffolds, chondroprogenitor cells aggregated without requiring any additional bioactive factors as opposed to cells grown without scaffolds. Addition of insulin to the medium enhanced the size of the aggregates suggesting scaffolds may be interacting with insulin. Similar to native cartilage tissue, collagen II was massively produced in aggregates. GAG-PA which is designed to mimic glycosaminoglycans and Glu-PA which only presents glutamic acid were used to construct scaffolds with oppositely charged Lys-PA presenting lysine. Formation of aggregates was observed regardless of the PAs used. Use of both GAGPA and Glu-PA induced larger number of aggregates than only Glu-PA. Differential effect of GAG-PA couldn’t be inferred completely and might be investigated in more detail. In a second part of the study, tissue morphologies of lynx3 null mutant mice were studied. Lynx3 is a recently discovered protein belonging to Ly6-superfamily. It is expressed mainly within epithelial lining of respiratory, digestive and genital tracts and is involved in nicotinic acetylcholine receptor desensitization. In this study, morphologies of lynx3 null mice with that of wild type mice were compared to see whether lynx3 has a gross effect on the tissues in which it is expressed. Any significant difference in the morphologies of lung, trachea and thymus cannot be observed. Little variations in esophagus, stomach and female reproductive organ were seen, however, it was not clear whether these variations are related to individual differences or not and the relevance of the variations with lynx3 expression could not be seen clearly. More detailed analysis of tissues may provide additional insight to understand function of lynx3 and the cholinergic mechanisms within various tissues. Short peptides able to pass cell membrane and deliver genes into cells are outstanding alternatives to virus based transfection systems. In the third part of the study, peptide amphiphiles designed to mimic the natural polycationic proteins through forming nanofibers which exhibit positively charged residues at high density, were synthesized. Peptide amphiphiles could form stable complexes with DNA, through neutralization of charges and formation of hydrogen bonds. However, efficient transfection of the gene couldn’t be provided by any complexes in vitro. The study presents primary results upon which more detailed investigation can be built.
Open Access
Programming microenvironmental signals with bioactive peptide amphiphiles for skeletal and cardiac myogenesis
(2014) Garip, İmmihan Ceren
The extracellular matrix (ECM) is crucial for the coordination and regulation of various cellular processes, including cell adhesion, recruitment, differentiation and death. ECM components structurally support tissue function and regeneration by acting as a substrate for cell migration and differentiation. In addition, by facilitating the fine localization of signals within their structural framework, these components activate receptors on the cell membrane for the initiation of signal transduction cascades. As such, cell-matrix interactions and matrix-associated signals are important for the normal functioning of cells, as well as for natural or artificially assisted tissue regeneration. In keeping with this ECM-centric approach, we designed and synthesized peptide amphiphiles with bioactive epitopes to resemble the native microenvironment of muscle tissue and to examined their potential in the induction of progenitor cell differentiation into skeletal myotubes and cardiac myocytes. The formation of skeletal myotubes was promoted through the use of basal laminamimetic peptide nanofibers inspired by the chemical structures of laminin and fibronectin, two proteins strongly represented in the skeletal muscle extracellular matrix. We demonstrated that our basal lamina mimetic peptide nanofiber system actively interacts with the cells it contains and enhances their differentiation within 3 days. Morphological analysis and immunocytochemical stainings indicated the formation of differentiated myotubes.We also designed glycosaminoglycan-mimetic peptide amphiphiles to mimic the glycosaminoglycans found in the myocardium. Glycosaminoglycans have been reported to play substantial roles in growth factor binding and the induction of angiogenesis, and their mimicry through peptide amphiphile nanofibers is promising as a combined approach for generating multifunctional cardiovascular tissue engineering scaffolds. We demonstrated that peptide nanofibers enhance the adhesion of cells to the surface and induce cardiac myoblast cells to differentiate into cardiomyocytes through both gene expression analysis and immunostainings. In summary, myogenic platforms were developed by programming signal rich environment from self-assembled peptide nanofibers inspired from the components of the ECM to induce the differentiation of cells. These bioactive nanofiber systems serve as promising platforms for muscle tissue engineering applications.
Open Access
Selective fluorescence sensing of biological thiols using a bodipy based bifunctional probe and the catalytic activity of short peptide amphiphile nanostructures : implications on the oring of life
(2013) Altay, Yiğit
Chemosensor development is an attractive field of modern chemistry and there exist large amount of contribution from all over the world. The biological importance of thiols triggered the development of sensors to differentiate especially cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) which play key roles in biological systems. Concentration of those thiols results in number of diseases and their structural similarity complicates the differentiation. Optical probes especially fluorescent ones are widely employed for that purpose since it offers simplicity, sensitivity and low detection limits as well as real time analysis. BODIPY core is decorated with a Michael acceptor nitro-styrene group to covalent incorporation of thiols and with an aza-crown moiety to recognition of N-terminus of them. The work in this thesis is the first example in which one of them is separated from others or three of them separated from each other’s by chain length difference using fluorescence spectrometry. Formation of short peptides (2-4 aa residues) is considered to be likely under primordial conditions, following a number of scenarios. In this work, it is constructed a short peptide library limiting our choice of amino acids to those believed to be available at larger concentrations such as Gly, Ala, Asp and Cys. It is demonstrated that when acylated at the N-terminus, nanostructures of varying size and shapes were formed. Investigations on the catalytic activity of these nanostructures under different conditions are presented. The findings on the correlation of peptide structure and nanostructure formation and/or catalytic activity are presented.
Open Access
Self-assembled peptide template directed synthesis of one-dimensional inorganic nanostructures and their applications
(2012) Acar, Handan
Engineering at the nano scale has been an active area of science and technology over the last decade. Inspired by nature, synthesis of functional inorganic materials using synthetic organic templates constitutes the theme of this thesis. Developing organic template directed synthesis approach for inorganic nanomaterial synthesis was aimed. For this purpose, an amyloid like peptide sequence which is capable of self-assembling into nanofibers in convenient conditions was designed and decorated with functional groups showing relatively high affinity to special inorganic ions, which are presented at the periphery of the one-dimensional peptide nanofiber. These chemical groups facilitated the deposition of targeted inorganic monomers onto the nanofibers yielding one-dimensional organic-inorganic core-shell nanostructures. The physical and chemical properties of the synthesized peptide nanofibers and inorganic nanostructures were characterized using both qualitative and quantitative methods. First, silica nanotubes were obtained by silica mineralization around these peptide nanofiber templates for the construction of sensors for explosives. The fluorescence dye was used to coat the silica nanotubes to detect explosive vapor. The surface of the silica nanotubes were porous enough to adsorb more dye compared to the silica nanoparticles and silica film, and causes faster fluorescence quenching in the presence of explosives like trinitrotoluene and dinitrotoluene. The silica nanotubes which synthesized with this peptide nanofiber templates can be used in catalysis and sensors in which high surface area is advantageous. In the second part of the thesis, titanium dioxide nanotubes were obtained from titania mineralization. They are wellknown with their fascinating properties as a result of the one-dimensional nanostructure, such as more efficient electron transfer and less electron-hole recombination. The sufficient photoactivity of titanium dioxide makes them suitable materials for Dye-Sensitized Solar-Cell construction. It is demonstrated that the peptide nanofiber templated titanium dioxide nanotubes have more than two times more efficiency compared to template-free synthesized titanium dioxide particles. Finally, designed peptide sequence was conducted to a multi-step seeding mediated growth method for gold mineralization around peptide nanofibers. The gold-peptide hybrid nanostructures with different packing characteristics and sizes were synthesized and fully characterized. Further, it was demonstrated that the dry film of these nanostructures showed a resistive switching dominant conductivity, due to the nanogaps in between gold nanoparticles as a result of particle alignment driven by the peptide nanofiber. The results obtained in this thesis encourage use of a new “bottom-up” synthesis approach. Specially designed peptides with desired properties and functional groups were synthesized and peptide nanofibers formed were further used as templates for inorganic mineralization. Not only it is possible to synthesis high amount of nanostructure with this approach, but also formed one-dimensional nanostructures show advance functionalities used in several applications as a part of the thesis scope. This methodology is suitable for many metals and metal oxide based applications.
Open Access
Slow release and delivery of antisense oligonucleotide drug by self-assembled peptide amphiphile nanofibers
(2012) Bulut, Selma
Antisense oligonucleotides are short single stranded DNA sequences and they are suggested to be used for treatment of several disorders including cancer. They could enter the cell and specifically inhibit the target gene, however chemical stability, controlled release and intracellular delivery are areas that has to be focused on to increase their efficacy. Gels composed of nanofibrous peptide network have been previously suggested as carriers for controlled delivery of drugs to improve stability and to provide controlled release, but have not been used for oligonucleotide delivery. In this work, a self-assembled peptide nanofibrous system is formed by mixing a cationic peptide amphiphile (PA) with Bcl-2 antisense oligodeoxynucleotide (ODN), G3139, through electrostatic interactions. The self-assembly of PA-ODN gel was characterized by circular dichroism, rheology, atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM and SEM images revealed establishment of the nanofibrous PA-ODN network. Due to the electrostatic interactions between PA and ODN, ODN release can be controlled by changing PA and ODN concentrations in the PA-ODN gel. Cellular delivery of the ODN by PA-ODN nanofiber complex was observed by fluorescently labeled ODN molecule. Cells incubated with PA-ODN complex had enhanced cellular uptake compared to cells incubated with naked ODN. Furthermore, Bcl-2 mRNA amounts were lower in MCF-7 human breast cancer cells in the presence of PA-ODN complex compared to naked ODN and mismatch ODN evidenced by quantitative RT-PCR studies. These results suggest that PA molecules can control ODN release, enhance cellular uptake and present a novel efficient approach for gene therapy studies and oligonucleotide based drug delivery. In follow-up studies, increase in the internalization efficacy of ODN by incorporation of bioactive sequences, RGDS, to peptide sequence was also shown.