Theses supervised by Prof. Dr. Mustafa Çulha
22 theses · Yeditepe University
Investigation of influence of drugs on cellular metabolism in 2Dc cell culture using surface-enhanced raman scattering
Raman spektroskopisi (RS), etiketsiz bir numuneden moleküler düzeyde bilgi sağlayan güçlü bir titreşim spektroskopik tekniktir. yüzeyde zenginliştirilmiş Raman saçılımı (SERS), Raman Spektroskopisinin oldukça hassas bir modudur ve yüzeydeki moleküler bileşimi veya çok yakın çevresindeki soy metal partiküllerin boyutu, saniyede iki büyüklükten daha az büyüklükte tanımlanmasına yardımcı olabilir. Metformin (MET), mitokondriyal solunum zinciri kompleksi I, hücre proliferasyonu, protein sentezi ve glukoneogenezi etkileyen bir ilaç molekülüdür. 6-Merkaptopürin (6-MP), antijen uyarımını takiben serideki nükleik asit metabolizması ve protein senteziyle ilgisi ve hücreler üzerindeki sitotoksik aktivitesi ile düzenlenebilen immünosüpresif özelliklere sahiptir. Bu çalışmada, SERS'in ilaçların hücre metabolizması üzerindeki etkisini izleyebilme potansiyeli, model ilaçlar olarak metformin ve 6-Merkaptopurin kullanılarak araştırılmıştır. Çalışma için iki hücre hattı, A549 ve Beas-2b seçildi. Ortalama boyutu 50 nm olan altın nanopartiküller, SERS substratları olarak seçildi. Metformin tedavisinde nükleik asitler, amino asitler ve fosfolipid zirveleri değişti. Bu değişikliğin nedeni, proliferasyonun inhibisyonunu ve protein sentezini ve lipid ve kolesterol biyosentezinin baskılanmasını sağlayan MET mekanizmalarıdır. Nükleik asitlere ve proteinlere karşılık gelen SERS pikleri, 6-Merkaptopürin tedavisi ile değiştirildi. Gözlemlenen spektral değişiklikler, de novo purin sentezinin ve protein sentezinin engellenmesine atfedilir. Hücrelerin artan konsantrasyonlarda ilaç moleküllerine maruz kalması üzerine gözlemlenen spektral modeldeki değişiklikler, bilinen ilaç metabolizması mekanizmalarıyla ilişkilendirilmeye çalışılır. Sonuçlar, MET ve 6-MP durumunda gösterildiği gibi, SERS'in ilaç moleküllerine maruz kalması üzerine hücresel düzeyde metabolik değişiklikleri izlemek için kullanılabileceğini göstermektedir.
Surface enhanced raman scattering based endosomal tracking of gold nanoparticles
Understanding the interaction of nanomaterials with living systems is critical to use these novel materials in various fields including nanomedicine. Due to being the first step at the interaction interface, endocytosis has a major role governing the intracellular destinations and behaviors of molecular and non-molecular species including nanoparticles. In this thesis, origin of the intracellular surface-enhanced Raman spectroscopy (SERS) spectra was investigated based on the protein corona structure on the AuNPs surface, endocytosis pathway development and pathway choice of AuNPs. For this purpose, firstly protein corona formation on AuNPs were examined with different cell culture conditions by SERS and effect of the protein corona on the obtained intracellular SERS spectra was investigated. Then, endocytosis pathway choice of AuNPs was investigated with the help of specific inhibitors for each endocytosis pathway. It is aimed to understand whether SERS spectra can tell us endocytosis route into a living cell. For this, endocytosis pathways are inhibited and the obtained SERS spectra are evaluated. Further, the source of the obtained SERS spectra is examined by isolating endosomes and comparing obtained SERS spectra with cellular spectra as time dependent. Finally, the effect of the endocytosis on the macrophage activation and AuNPs phagocytosis is investigated. From the results, it is concluded that protein corona alterations based on different cell culture conditions can be tracked by SERS, and it can affect the intracellular SERS spectra directly. It is also found that SERS can significantly contribute to the investigation of different endosomal pathways, and macrophage activation from single living cells. Furthermore, it is also seen that source of the SERS cannot be thought as only from AuNPs in the endosomes but also from escaped AuNPs, which are evidenced to be found in both cytosol and nucleus accumulated in a time dependent manner. Thus, the origin of the intracellular SERS spectra cannot be considered only from endosomes. Consequently, it is revealed that SERS can distinguish significantly not only protein corona alterations and interference with the intracellular spectra, but also pathways used for nanoparticle uptake and different metabolisms including macrophage activation and phagocytosis.
Bor nitrür tabanlı nanomalzemelerin doku mühendisliği yapı iskelelerinde kullanımlarının araştırılması
Yenileyici tıbbın gelecek vaat eden ve disiplinler arası bir alanı olan doku mühendisliği, kaybolan veya hasar gören doku veya organların geri kazanılması/yenilenmesi için gereken işlevsellik dahilinde doğal, sentetik veya yarı sentetik malzemeler kullanarak doku/organı taklit ederek çözümler geliştirmeyi amaçlamaktadır. Doku mühendisliğinde halihazırda kullanılan iskeleler, hücre tutunması, penetrasyonu, büyümesi ve yenilenmesini sağlamak için daha fazla fiziksel, kimyasal ve biyolojik geliştirmeye ihtiyaç duyulduğundan, bazı yetersizliklere sahiptir. Bu noktada nanoteknoloji fikri, nanomalzemelerin takviye malzemesi olarak doku mühendisliği uygulamalarında kullanılmasıyla devreye girmektedir. Nanomalzemeler, üstün mekanik, kimyasal, fiziksel ve biyolojik özelliklerinin yanı sıra daha yüksek biyouyumlulukları nedeniyle özel fiziksel, kimyasal ve biyolojik özelliklere sahip yüksek performanslı biyomalzemeler üretmek için tercih edilebilir. Bu çalışmada ilk olarak, biyomedikal uygulamalarda kullanılmak üzere hem baryum titanatlar (BaTiO3) hem de bor nitrür nanotüpler (BNNT) içeren yeni ve toksik olmayan bir piezoelektrik kompozitin geliştirilmesi amaçlanmıştır. Daha sonra, BaTiO3, BNNT'ler ve yeni kompozit olmak üzere üç farklı nanomateryal, yüksek piezoelektrik özelliklerinden dolayı poli (Ɛ-kaprolakton) (PCL) ve kitosan polimerlerinin içine onlara piezoelektrik özelliği kazandırmak için gömülerek kullanılmıştır. Bu piezo-kompozit yapı iskelelerinin etkileri, elektriğe duyarlı İnsan Osteoblast hücreleri (HOb) üzerinde araştırılmıştır. Yapı iskeleleri üzerindeki HOb hücreleri, hücre büyümesi sırasında düşük frekanslı ultrasonik ses dalgaları ile uyarılmıştır. Piezo-kompozit yapı iskelelerinin biyouyumluluk, hücre tutunması, alkalin fosfataz aktiviteleri ve mineralizasyonu HOb hücreler üzerinde incelenmiştir. Bu piezo-kompozit yapı iskeletlerinin kullanılması, in vivo elektrik stimülasyonunun geleneksel invazivliğini azaltarak elektriğe duyarlı hücrelerde veya dokularda piezoelektrik fenomenini kullanan kablosuz doku stimülasyon çalışmalarına oldukça katkı sağlayacaktır.
Influence of oligonuclotide and peptide interactions on electronic properties of single walled carbon nanotubes
Carbon nanotubes have been a point of attention since their discovery by Iijima in 1991. However, the biggest problem ahead for the applications of carbon nanotubes is their low solubility properties. Detergents are considered as surfactants which can be used to gain solubility for carbon nanotubes. On the other hand, DNA is a more significant solubilizing ligand which can be enrolled in cooperative binding interactions with SWCNTs. Raman spectroscopy is a powerful tool that can be used to study molecular systems, such as oligonucleotide/SWCNT structures. The changes in the electronic properties of SWCNTs dur to the interactions between oligonucleotides can be detected by Raman spectroscopy.In this study, the solubilization difficulties of SWCNTs are reduced by DNA interaction and covalent attachment to SWCNTs. The changes in the electronic properties of SWCNTs after the non-covalent interactions between oligonucleotides and SWCNTs and the covalent attachment of oligonucleotides to SWCNT are investigated by Raman spectroscopy. SWCNTs are interacted with short (10 bases) and long (25 bases) oligonucleotides. Also, to investigate the selectivity of SWCNT and oligonucleotide interactions, the non-covalent interactions between selected peptides and SWCNTs were studied by Raman spectroscopy. The pristine, carboxyl functionalized and covalently oligonucleotide attached SWCNTs are characterized by IR and X-ray photoelectron spectroscopy.
Manipulation of dynamics in a droplet for label-free detection of proteins using surface-enhanced raman scattering and protein melting profiles
Detection and identification of biomacromolecules have critical importance in many fields ranging from biotechnology to medicine. Surface-enhanced Raman scattering (SERS) is an emerging technique for the label-free detection and identification of biological molecules and structures with its fingerprinting properties and high sensitivity. However, there are a number of obstacles for its applications for biological macromolecules due to the complexity of biological samples. In this report, manipulation of microscopic processes in play during the drying of a sessile droplet to influence the nanoparticle-macromolecule packing, which has dramatic effect on SERS performance, before the SERS acquisition is demonstrated. A process known as the ?coffee ring phenomenon? jams all particles and molecular species to the edges of the droplet during drying. This uncontrolled process has dramatic effects on a SERS experiment, using colloidal metal nanoparticles as substrates, by sweeping everything to the edges and influencing the packing of nanoparticles in the droplet area.In this study, the dynamics of silver nanoparticles-protein structures in a drying droplet was explored under two different experimental conditions and the effects of these conditions on SERS performance were investigated. Denaturation profiles of proteins with SERS were also investigated and obtained data was used to detect proteins in multiple protein mixtures. Negatively and positively charged proteins were used as model biomacromolecules in this study. A detection limit of 0.05 ?g/mL is obtained for the model proteins with using one of the presented methods.
Identification of bacteria in a bacterial mixture using Raman spectroscopy
The need for a quick, accurate and reliable method for detection of pathogenic bacteria, especially in mixture, is increasing day by day. A lot of detection methods have been developed since the discovery of the first microorganism, most of which are based on biochemical and immunological properties. However, their long sample preparation and procedure times, expensive and heavy instrumentation and need for both well- educated and trained staff are some of the disadvantages of these methods.Raman spectroscopy is a promising technique for fast detection and identification of bacterial cells. It can be applied directly on the sample and there is no need for pre- test procedures.Considering the facts about Raman spectrometry which is a fast, reliable and feasible method for identification of bacterial cells in a mixture was the goal of this study. To achieve this goal five different bacteria (BFK13, BHK7 and DH5alpha varieties of Escherichia coli, Proteus vulgaris and Shigella sonnei) were used together as a model. First, the binary and ternary mixtures of bacteria were prepared. Then, Raman spectra were obtained from particular microorganism and their binary and ternary mixtures. When collected data were processed and statistically analyzed using SPSS software they were applied to plot 2D charts of Euclidean distance. As a result, it was shown that Raman spectra of each bacterium and their mixtures are very similar. However, each spot representing spectra of species fall at different coordinate on 2D charts of Euclidean distance.
Development of a BioChip System with Multiplexing Capability for Detection of Single Nucleotide Polymorphisms (SNPs) and Mutations Based on Surface-Enhanced Raman Scattering
Human genome is exposed to several types of genetic abnormalities including single nucleotide polymorphisms (SNPs), which are the most common polymorphisms throughout the human genome. Because SNPs are abundant and stable, they are widely used in diagnostics of several diseases like familial Mediterranean fever (FMF), Parkinson, and Alzheimer. Identification of SNPs provides opportunities in the diagnosis, prevention and treatment of related diseases. Current methods that are used to detect SNPs are time-consuming and expensive. Therefore, there is a need to improve the speed and reduce the cost. This can only be achieved with a technique capable of multiplexing. Surface-enhanced Raman scattering (SERS) is a vibrational spectroscopic technique, which provides specific information about a molecule's structure due to its "fingerprinting property". The multiplexing property and high sensitivity of SERS is used to reduce the number of spots on the array chip. Considering great number of SNPs present on the human genome, achievement of the multiplex SNP detection will cut the cost and time spent for the screening SNPs. The development of an assay for the detection of SNP mutations has been attempted based on SERS in this study. To test the feasibility of approach, M694V SNP on 10 th exon of MEFV gene is investigated as a model. The promising results indicate that it is possible to detect SNPs using this assay.
Differentiation of healthy and cancerous renal cells using surface-enhanced Raman scattering
Surface-enhanced Raman scattering is used for the differentiation of human kidney adenocarcinoma (ACHN), human kidney carcinoma (A-498) and non-cancerous human kidney embryonic cells (HEK 293). Silver nanoparticles (AgNPs) are used as substrates in the experiments. A volume of colloidal suspension containing AgNPs is added onto the cultured cells on CaF2 slide and the slide is dried at the overturned position. A number of SERS spectra acquired from the three different cell lines are statistically analyzed to differentiate the cells. Principal component analysis (PCA) combined with linear discriminate analysis (LDA) was performed to differentiate the three kidney cell types with a sensitivity and specificity of 88 % and 84 %, respectively. This study demonstrated that SERS could be used to identify renal cancers by combining this new sampling method and LDA algorithms.
Surface modification and characterization of agglomerated metal oxide particles
Metal Oxide nanomaterials (MONMs) attracted enormous attention due to their unique physicochemical properties and are used in many applications including drugs, paints, biomedical devices and development of novel materials. The surface modification of namomaterials (NMs) is an important step to alter their behavior to further benefit from their unique properties in variety of applications. However, the main problem with them in order to work efficiently is their inefficient dispersion in aqueous environments. In this study, we aimed to understand the surface properties of these materials as we attempt to alter the surface chemistry. Surface coating of the NMs with oligonucleotides, carbohydrates and treatment with hydrogen peroxide (H2O2) help them to stabilize as mono-disperse particles in their colloidal suspensions. The characterization of modified MONMs was performed with Raman Spectroscopy, FTIR Spectroscopy and Dynamic Light Scattering (DLS).
The synthesis and applications of boron nitride nanotubes and hexagonal boron nitrides as nanocarriers and therapeutic agents
In recent years, boron-based nanomaterials, boron nitride nanotubes and hexagonal boron nitrides (BNNTs and hBNs) have gained significant interest for their use in medical and biomedical fields owing to their nanometer size and unique physicochemical properties including biocompatibility, high mechanical strength and chemical stability. These properties make them particularly suitable nanomaterials for their interaction with biomacromolecules and variety of successful biomedical applications. This thesis aims at addressing drug carrying and therapeutic effects of BNNTs and hBNs. Considering their potential applications as drug carriers and therapeutic agents, the route of their interaction with biological environment should be understood at cellular level. Their biocompatibility evaluation revealed that the BNNTs and hBNs are good candidates for medical applications with their non-toxic nature on healthy cells even at relatively high concentrations. In an attempt to use the BNNTs and hBNs as drug carriers, they were modified with doxorubicin (Dox) and folate through nonspecific interactions. Folate was used to increase cellular uptake of BNNT by targeting folate receptors on the cell surface. It was found that the Dox conjugated BNNTs behave as the free Dox molecules while folate conjugation significantly enhanced (2 fold) cancerous cellular uptake. A purer and catalyst free hBNs batch was synthesized to use in the studies. Then, the hBNs were evaluated for their carrier and therapeutic effects. The data obtained from in vitro studies revealed that the level of reactive oxygen species (ROS) in the hBN exposed prostate cancer cells increased 62 per cent, cell death toward apoptosis was 4-fold enhanced and metastasis capacity of cells reduced significantly. These results envision the exploitation of hBNs as therapeutic agents against prostate cancer and pave the way to carry out in vivo studies to disclose their potential as drug carrier and therapeutic agents in cancer treatment.
Multi-gene regulation using DNA-origami-AuNPs nanostructures in breast cancer cells
Gene regulation is a novel approach to cure a gene related disease. Thus, there is an ongoing effort to increase the efficiency of gene delivery. In this thesis, we developed a DNA based nanostructure to deliver gene regulation elements for the up-regulated genes in breast cancer as alternative to the currently used non-viral delivery systems. A tile shaped DNA origami nanostructure is constructed by including morpholino antisense oligonucleotides targeting the HER2, ERα, EGFR, and Ki-67 genes. Then, the sticky ends of the DNA origami nanostructures were hybridized to the complementary oligonucleotide sequence attached to gold nanoparticles (AuNPs) to enhance their cellular uptake. The constructed nanostructure was characterized using agarose gel electrophoresis, AFM, DLS and UV/Vis spectroscopy. Following the stability and toxicity analysis of the structure, the nanocarrier system was used for silencing of the mentioned genes in their overexpressing breast cancer cell lines. It was found that the antisense oligonucleotide embedded DNA origami-AuNPs structure was highly effective for inducing single and multiple-gene silencing in breast cancer cells. The prepared nanocarrier inhibited the expression of target genes approximately 50 per cent at 30 nM morpholino concentration. For multi-gene silencing, more than one morpholino oligos targeting different genes were embedded into DNA nanocarrier and the silencing efficiency increased to about 80 per cent by synergistic effect. After silencing of the target genes alone and in combination, the effects of the gene regulation on breast cancer cell proliferation and cell cycle phase distributions were investigated. It was showed that gene silencing using DNA origami-AuNPs nanostructures inhibited proliferation of the breast cancer cells and altered their cell cycle phase distributions. Silencing efficiency of DNA origami-AuNPs nanostructures was compared with commercial transfection agents and found to be more effective. Furthermore, DNA origami-AuNPs nanostructures did not affect viability of cancer and healthy cells, while the tested commercial systems were highly toxic. The proposed novel nanostructure provides an effective and biocompatible carrier for gene silencing studies.
Effect of mannan-reduced gold nanoparticles on prostate cancer cells
Prostate cancer is a severe cancer type which kills a large number of males every year. The new nanotechnological therapeutic tools based on the targeting of the over-expressed receptors have showed advances to decrease the side effects on healthy cells. In this thesis, gold nanoparticles were synthesized using mannan (M-AuNPs), which is a polymer of mannose, attaches the mannose 6-phosphate receptor (M6PR) highly expressed in prostate cancer. AuNPs were used as anticancer drug delivery vehicle and their efficiency was compared with other gold nanoparticles reduced with starch and citrate on healthy PNT1A and cancerous DU145 cells. The results showed that the synthesized AuNPs did not influence the viability of the both cell lines. Dox-loaded AuNPs displayed a high rate of toxicity on DU145 cancer cells by inducing cell cycle arrest in G2/M phase. As a conclusion, M-AuNPs can be a promising tool for prostate cancer by combining with photothermal therapy.
In situ biofilm formation and bacterial death monitoring using surface-enhanced raman scattering
Surface-enhanced Raman scattering (SERS) is a powerful technique for characterization of biological molecules and molecular structures due to the fingerprinting property, very narrow spectral bandwidth, and easy sample preparation. In SERS, Raman scattering can be enhanced up to 10 14 times by bringing the molecule or molecular structure of interest close to or in contact with nanostructured noble metals such as gold and silver. SERS can be used for the characterization and identification of components of complex biochemical systems such as bacteria and biofilm. In this thesis, characterization and identification of biofilm components were investigated using SERS. The core AgNP- shell chitosan layer type structure was prepared by coating the AgNPs with a layer of chitosan (c-AgNPs) and they were used as a SERS substrate for biofilm formation and bacterial death monitoring. Due to absorptive property of chitosan, biomaterials that were produced by bacteria can penetrate into shell without saturating the metal surface due to the selective interaction of biofilm components with the AgNPs. The feasibility of in situ monitoring of molecular changes during the bacterial death was also demonstrated. It was found that SERS spectra could provide significant information about the morphologic changes on the bacterial cell wall and released molecular structures such as DNA/RNA bases during the decompositionof bacteria. It was also found that the band at 678 cm -1 could be used for monitoring bacterial death.
Systematic investigation of cellular response to nanoparticle surface chemistry
The interface between nanoparticles (NPs) and living systems is one of the most important determinants of cellular responses. The well-understanding of the interaction between the NP surface chemistry and living systems makes possible to design safer NPs to be used in medical and technological areas. In this study, it was aimed to systematically investigate the cellular response of living cells to subtle surface chemistry changes on AuNPs conjugated with specially designed carbohydrates and peptides. In order to create small differences on AuNP surfaces, spherical AuNPs of 13 nm diameter size were modified with four carbohydrates, D-Glucose, D-Mannose, Lactose and Mannose, and fourteen peptides different in charge, length, isoelectric point, sequence with or without RGD and free end terminus -NH2 or -COOH. The cellular responses of A549, BEAS-2b and MDA-MB-231 cells were investigated by considering cellular uptake, cytotoxicity and cell cycle arrest. Before modification of AuNP surfaces, carbohydrates were thiolated using Lawesson reagent. Then, AuNPs with an average size of 13 nm were conjugated with carbohydrates and peptides in appropriate conditions. The naked AuNPs and AuNP conjugates were characterized with UV/Vis spectroscopy, DLS, agarose gel electrophoresis, FTIR and SERS to understand the nature of the interactions of the used biomacromolecules on the AuNPs surfaces. The peptides designed by placing cysteine to -NH2 or -COOH end showed significantly different binding affinity to AuNPs surfaces. Then, the cellular response of the conjugates were investigated using several molecular techniques including WST-1 cell proliferation assay, Apoptosis/Necrosis assay, Clonogenic assay and cell cycle evaluation assay. It was found that small changes on the surfaces of AuNPs caused varying significant cellular responses depending on surface chemistry, NP concentration and cell line type.
Observing suppression of microbial growth with surface-enhanced raman scattering
Microorganisms can be both harmful and beneficial for human depending on their nature. If the mechanisms that make them harmful are known, their detrimental effects can be minimized, which makes the study of microbial growth crucial. The amount of nutrients plays an important role on the growth as well as presence of different chemicals that are toxic to them. Observation of microbial growth in different environmental conditions is important in terms of understanding the growth mechanisms of microorganisms. Among many analytical and spectroscopic methods, Surface-Enhanced Raman Scattering (SERS) is a promising technique to work with biological samples. It gives valuable information about molecular structures based on their vibrational modes. While working with biological samples, it has many advantages such as simple sample preparation, no interference from water and achieving low limits of detection. In the thesis presented below, microbial growth of bacteria and yeast cells in the presence of a biocidal agent were investigated with SERS. Maltose-reduced silver nanoparticles (mAgNPs) were used as biocidal agent. Escherichia coli (E.coli) and Saccharomyces cerevisiae (S.cerevisiae) were selected as model organisms for bacteria and yeast, respectively. The effect of three different concentrations (5, 10, and 20 ppm) of m-AgNPs were tested on the mentioned microorganisms. Their microbial growth was monitored by both measuring optical density (OD) and collecting their Raman scattering under a laser beam. The spectral changes with respect to time were analyzed and the bands related to certain metabolites of microorganisms such as amino acids, carbohydrates, proteins and genetic material were used to monitor the changes in the chemical composition of cell cultures. Results of both analysis revealed that microbial growth of E.coli is suppressed when the cells are treated with 5 and 10 ppm m-AgNPs, and is inhibited in the presence of 20 ppm m-AgNPs. The results obtained from S.cerevisiae demonstrates suppression of its growth increses with the increasing concentration of m-AgNPs as well.
Development of SERS based methods for early cancer detection
The applicability of SERS in early cancer detection by means of differentiating cancerous tissue from healthy tissues was systematically investigated by employing three different sample preparation strategies. In the first approach, a 5-µm thick cryosectioned tissue specimen was placed on a PDMS layer coated glass slide before adding a volume of 20-µl 16× concentrated AgNPs containing colloidal suspension onto the tissue. Then, the colloidal suspension was dried at the suspended position to achieve optimal distribution of the AgNPs in the droplet area. In the second approach, in situ synthesis of AgNPs in the homogenized tissue sample was aimed. A 10-µl from each of 4.36x10-1 M AgNO3, 3.55x10-2 M HONH2·HCl and 3x10-2 M NaOH was successively added into the 10-µl of homogenized tissue suspension to reduce Ag+ ions into AgNPs in the homogenized tissue. Then, a 2µl of this mixture was placed onto a CaF2 slide and dried before the SERS measurements. The first approach was optimized through parameters including substrate used to place tissue specimen (PDMS, Al-foil or CaF2), signal collection type (random selection versus mapping), spectral range and acquired-mapping size while the latter approach optimized through the acquisition type and concentrations of AgNO3, HONH2·HCl and NaOH solutions. Then, the data acquired with each sampling method was analyzed with PC-LDA classification models and the coefficients by comparing intra- and inter-method reproducibility, accuracy, and SERS performance indicators of spectral richness and SNR. Finally, the data acquired from these two methods were compared with our previously developed approach of Crashed-liquefied consisting of mixing a 5-µl 32× concentrated colloidal AgNP suspension with homogenized tissue sample by utilizing human thyroid biopsies (n=64). The results showed that in situ approach indicated a higher classification accuracy compared to other approaches for malignant vs. healthy tissue and benign vs. healthy tissue diagnostic combinations by using full spectra region of spectra while the best classification performance for benign vs. malignant tumors was obtained by using Cryosectioned-PDMS and Crashed-liquefied approaches. The findings of this study clearly indicate that SERS is a suitable technique to employ in cancer diagnosis.
Development of an in situ sers based method for observation of biofilm formation
A biofilm is an assembly composed of microbial cells and extracellular polymeric substances, which provides and supports microorganisms for attach themselves onto a surface irreversibly and protect them from environmental stress conditions. Monitoring the in situ molecular changes during a biofilm formation can provide valuable insights in the fields including medicine, biology and related industrial processes. The conventional molecular and microscopy techniques are time consuming due to cumbersome sample preparation steps and destructive nature. With the aim of better understanding biofilm formation and possibility of detection, in this study, biofilm formation of clinically important microorganisms, Pseudomonas aeruginosa, Staphylococcus epidermidis and Candida albicans were monitored by utilizing surface-enhanced Raman scattering (SERS). Clinically relevant microorganisms threaten patient health often through biofilm formation on the surface of polymeric medical devices and implants. In this thesis work, the biofilm characteristics of the model microorganisms were identified on agar plates, 2D and 3D poly (methyl methacrylate) substrates and 3D glucose-gelatin scaffolds with the aim of understanding the influence of substrate type on biofilm formation process. The significant concentration changes on carbohydrates, lipids, proteins and genetic materials with increasing incubation time provided information about biofilm formation process. Moreover, the spectral data was attempted to confirm with confocal laser scanning microscopy and scanning electron microscopy analyses. The discrimination of microorganisms was also demonstrated from the SERS spectra using principle component analysis and linear discrimination analysis. A further step was taken in the way to clinical application of the approach by monitoring biofilm formation by using a multi-species sample. In summary, the results present a comprehensive evaluation of the applicability of SERS in clinically relevant biofilm formation with exciting outputs and opportunities.
Development of surface-enhanced Raman scattering-based nanospectroscopic methods for toxicity determination of nanomaterials
The intensive research on nanomaterials (NMs) in the last decades resulted in broad applications in many industries ranging from energy deposition to medicine, cosmetics, food, textile, military or communication. The tremendous utilization of these newly discovered materials, however, also raised concerns about their possible side effects. On the other hand, the efforts to determine NM toxicity faced problems in terms of obtaining accurate results; originating from the physicochemical properties of NMs. Most of the conventional cytotoxicity assays have been reported to give false results due to interaction with assay components or absorbance artefacts. Therefore, in vitro nanotoxicity evaluation field is in need of alternative approaches. In an attempt to propose a new perspective, in the present work, surface-enhanced Raman scattering (SERS) was utilized on four cell lines; lung adenocarcinoma (A549), human and mouse fibroblasts (HSF, L929), and human umbilical vein endothelial cell line (HUVEC), to test in vitro nanotoxicity of a panel of NMs; zinc oxide and titanium dioxide nanoparticles, single- and multi-walled carbon nanotubes, and quantum dots. Before that, SERS substrate localization within the cells as well as the spectral contributors were evaluated. Then, SERS spectra obtained from NM-exposed cells were carefully investigated for possible toxicity markers and seven of them were proposed to be in good correlation with the conventional cytotoxicity assays; apoptosis/necrosis and WST-1 cell proliferation assays, as well as the transmission electron microscopy and enhanced-dark field microscopy images. Altogether the results were in agreement with the nanotoxicity studies in the literature. The seven intensity ratios gave information about the rates of collagen, fibronectin, cholesterol depletion, lipid stability, tyrosine phosphorylation, phenylalanine to tyrosine conversion and protein C-S to S-S bond conversion, all of which can also be utilized for various further cellular SERS studies.
Length-dependent selective killing of brain cancer cells using polyguanine modified gold nanoparticles
Gold nanoparticles (AuNPs) are investigated as a promising therapeutic agent in nanomedicine due to their easy synthesis and surface functionalization. AuNPs also have extraordinary physicochemical properties such as inertness, being plasmonic and biocompatible. AuNPs can be modified using variety of molecules including biological ones, such as deoxyribonucleic acid (DNA), peptides, proteins, carbohydrates for highly exciting applications in various fields of therapeutic nanomedicine such as drug delivery, gene therapy, sensing, detection, and imaging. Among these biological molecules, therapeutic use of oligonucleotides, more specifically guanine-rich (G-rich) oligonucleotides, gain interest due to their anti-proliferative effects in malignant tumor cells. Surface functionalization of AuNPs using G-rich oligonucleotides are being studied in nanomedicine, however, little is known about the cellular response of these hybrid structures as potential therapeutic agents. In this study, we aimed to investigate length-dependent cellular responses of oligonucleotide modified-AuNPs. With this goal in mind, polyadenine-tailed polyguanine sequences (G10 and G20) are used to functionalize the surface of AuNPs. Resulting nanostructures, G10-AuNPs and G20-AuNPs, are used to investigate their effect through cell cycle analysis, apoptosis induction, cellular uptake studies, as well as in vitro cytotoxicity assessments. As a result, increasing number of guanine bases showed enhanced cellular uptake (1.86 fold), increased cell accumulation in S phase (1.67-fold) and G2/M phase (1.34-fold), and elevated induction of apoptosis (two-fold) in glioblastoma (GBM) cells (U87MG and U373) when compared with normal human astrocytes (NHA). In conclusion, the data suggests length-dependent selective killing of GBM cells using polyguanine modified-AuNPs.
Investigation of biochemical processes in single cell using surface-enhanced Raman scattering
Single cell analysis is rapidly emerging approach to gain molecular information at the individual cell level. Investigation of specific molecular mechanisms and pathways providing in single cell level is crucial. In this study, we investigated Surface-enhanced Raman scattering (SERS) to study single-cells. Gold nanoparticles (AuNPs) were used as routinely employed SERS substrate. SERS activity of AuNPs depends on size, shape and aggregation status in living cell depending on their uptake profile. We are interested in understanding the biochemical changes on AuNPs or in their aggregates to understand the possibility of monitoring cellular biochemical process in a living cell. Thus, two different sizes of AuNPs with two different surface chemistry were used in the study. The order and combination of AuNPs with different sizes and surface chemistry helped to understand the spectra obtained from a single cell and thus the nature of the biochemical processes taking place on the AuNPs surfaces. This study showed that SERS can be used for single cell analysis and its possible use in molecular biology. In this study, the spectral changes espicially correspond to protein and lipids were observed depending on size, surface chemistry, and aggregation status in time dependent manner.
Preparation of a gold nanoparticle containing electrospun poly (ɛ-caprolactone) Scaffold for stem cell differentiation
Nerve tissue engineering aims to improve the nerve repair and regeneration rate. Electrical stimulation has an influence on the adipose stem cell differentiation into neuronal cells. The developed hypothesis argues that gold nanoparticle (AuNP) incorporated Poly (ɛ- caprolactone) (PCL) scaffolds can be applied in nerve tissue engineering. Although PCL is a non-conductive and highly hydrophobic polymer, it is commonly used for nerve tissue engineering. The hydrophilicity, which is necessary for cell attachment and proliferation is provided by AuNPs. Also, AuNPs give electrical conductivity to non-conductive PCL polymer. In this study, an AuNP containing extracellular matrix for differentiation of human adipose stem cells was developed by preparing a scaffold using electrospinning technique. In order to provide conductivity, the aqueous alkaline sodium borohydride reduced AuNPs were blended into the PCL. The AuNP containing PCL scaffold was composed of fibers in the size range of 3 to 6 μm. The aim was to easily differentiate human adipose stem cells (HASCs) into neuronal cells using electrical stimulation throughout AuNPs. This study is promising a new regeneration method for the neuronal cell differentiation.
The influence of peptide functionalized gold nanoparticles on prostate cancer cells
Nanomaterials have emerged as new tools for theranostic applications. However, their use in biomedical fields is limited by their potential toxic effects on human health. It has been indicated in the literature that functionalizing the surface of NPs is a favorable approach to eliminate the safety issue of the nanomaterials. AuNPs provide a platform for designing therapeutic biomaterials owing to their easy surface chemistry, less toxicity, biocompatibility, high degree of stability and size uniformity. Besides, small differences in the surface chemistry of AuNPs can be observed by UV/Vis spectroscopy due to their unique plasmonic properties. Functionalization of AuNPs with a small biomolecule introduces a good platform to enhance cellular uptake and reduce the toxicity. In this study, a range of custom designed peptides were choosen for modification of the 13 nm AuNPs by taking their sequence, size, and charge into account. To evaluate cellular responses comparatively, both healthy (PNT1A) and cancer (DU145) cell lines were treated with the AuNP-Peptide conjugates. The peptide modified AuNPs indicated more toxic effects on DU145 cells than PNT1A cells, and the position of Cys amino acid in the sequence caused difference in cytotoxicity. Furthermore, the AuNP-Peptides induced the apoptotic cell death in prostate cancer cells in contrast to response of healthy cells. Pep1 (H2N-Glu-Glu-Glu-Cys-COOH) and Pep5 (H2N-Asp-Gly-Arg-Glu-Glu-Glu-Cys-COOH) modified AuNPs significantly induced apoptotic cell death for DU145 cell line compared to Cys-N-terminal peptide modified AuNPs. In addition, the cell cycle of the PNT1A and DU145 cells were also investigated. While the DU145 cells were arrested at G2/M phase in a high ratio, there was no significant arrest for PNT1A cells. The highest arrest in G0/G1 phase of the PNT1A cells obtained with the His containing Cys-C-terminal peptide. In conclusion, it was shown that subtle changes in surface chemistry of AuNPs caused noticeable difference in cellular response, which indicates the importance of tailoring surface chemistry of nanomaterials aimed to be used in nanomedicine.