Theses supervised by Prof. Dr. Havva Yağcı Acar
10 theses · Koç University
Development of carbon dots for security inks
Carbon Dots (CDs) emerged as metal-free promising nanoparticles creating new opportunities in optoelectronics, bio-applications, catalysis, sensing, security applications, etc. However, a profound understanding of the physical and chemical properties still pose a significant challenge. The origin of optical properties and emission tunning is not straightforward. The fundamental problem of this thesis work is towards elucidating the relationship between the structural/chemical features and the optical properties of CDs. This thesis also addresses a daily problem: Authentication of valuable papers such as diplomas, banknotes, passports, etc. to prevent counterfeiting. Therefore, CD`s developed in this thesis was evaluated in inks and binders towards developing photostable optical tags. The documents were authenticated by measuring or reading these tags with special optical sensors or lamps.
Development of luminescent carbon nanoparticles for authentication of petroleum products and investigation of phototherapy potential of nanoparticles
Nanomaterials were exploited in many engineered materials since their properties can be controlled and modified by tunning their size. The relatively new member of the nanomaterials portfolio is the luminescent carbon dots (CD) which are more or less considered biocompatible, non-toxic, and environmentally friendly. However, the development of CDs with different luminescence properties can be challenging. In this thesis, the effects of small organic precursors, N-, O-, P- doping, and solvent (polarity and reducing power) on the emission wavelength of CDs were investigated. In literature, most CDs have blue emission. Examples to CDs with different emission colors exist, yet most of them have major drawbacks such as non-reproducibility, low colloidal stability. A portfolio of colloidally stable, reproducible, easily manufactured CDs to address these disadvantages was developed in this thesis. The second part of the thesis focuses on using CDs in the authentication of petroleum products as photostable optical tags. In the third part of the thesis, one of the most popular photosensitizers, BODIPY, was loaded to highly biocompatible superparamagnetic iron oxide nanoparticles for enhanced delivery of BODIPY to cancer cells and achieve highly selective and enhanced photodynamic therapy. Lastly, the phototherapy potential of aqueous red-emitting CD produced in this thesis was investigated.
Nanoparticle mediated diagnosis and combination therapy of bacterial infections
Treatment of bacterial infections has become one of the challenges over the decades. Adhesion on different surfaces of bacteria and biofilm formation have led to the bacteria species to obtain increased survival rates with high morbidity especially in hospital-related infections. Insufficient treatments result in recurrence of the infection or even death of the patient. Current antibiotic drugs are not capable of eradicating the complex biofilm structure due to the acquired resistance mechanisms of bacteria. Therefore studies have been conducted for alternative therapies against multi-drug resistance bacteria and their biofilm-related infections. Photodynamic therapy (PDT) is noninvasive, localized, and promising alternative method. There have been extensive studies of PDT for the treatment of cancer and bacterial infections. PDT is composed of a photosensitizer (PS), molecular oxygen, and the appropriate light source. PS is excited from ground state to the triplet state by the light source to produce reactive oxygen species (ROS) with the interaction of molecular oxygen in the environment. Following ROS production, cellular components are exposed to produced ROS and consequently cell death. PDT can be utilized alone or in combination with chemotherapy or photothermal therapy (PTT) for the treatment of bacterial infections. Photothermal therapy is another alternative, wherein the PS converts light-to-heat and induce local temperature increase, resulting in cell death. It is again controlled, highly-localized, and noninvasive method which has been combined with different therapeutics such as PDT, chemotherapy, immunotherapy or radiotherapy for infectious diseases. Some nanoparticles act as photothermal agents, namely as photothermal nanotherapeutics (PTN). Superparamagnetic iron oxide nanoparticles (SPIONs), mostly known as Magnetic Resonance Imaging (MRI) agents, have been also recognized as PTT agents recently. They are the most studied nanoparticles in nanotherapeutics because of the FDA-approval and high biocompability. SPIONs may also be used as nano-carriers by tailoring surface coating with targeting agents or ligands for the targeted delivery of drugs, genes, and DNA/RNA. Silver sulfide quantum dots (Ag2S-QDs) are semiconductor nanocrystals with broad absorption in UV-visible region and NIR-emission. Their luminescent properties are superior to commercial fluorescent dyes in particular with properties such as resistance to the photobleaching, higher quantum yield, and stability. They have also been recognized as PTT agents and extensively studied for the treatment of cancer. QDs are another class of nano-carriers which can be used for the targeted delivery of therapeutics with bioimaging properties. 5-Aminolevulinic acid (ALA) is an FDA-approved PS with strong fluorescence in visible region for diagnosis and fast accumulation in the tumor and infectious tissue. ALA, itself is not an active PS molecule but it is internalized and converted into active protoporphyrin IX (PpIX) molecule in cells. ALA is also an endogeneous molecule in heme biosynthetic pathway of cells. However, high solubility and rapid clearance from tissues reduces effect of PDT. Additionally, higher amounts of PpIX accumulation is needed for successful PDT applications. In this thesis, antibiotic free treatment of bacterial infections via combination of PTT and PDT is proposed. The combination is expected to bring about a synergestic effect at lower doses of PS and mild hyperthermia. In case of the biofilms, permeabilization of the biofilm via mild PTT is expected to take place and enhance the treatment efficiency. For this purpose, ALA loaded SPIONs were produced. Planktonic cells and biofilms of gram-positive (Staphylococcus epidermidis) and gram-negative (Pseudomonas aeruginosa) bacteria were treated with ALA loaded SPIONs and subjected to dual laser irradiation (640 + 808 nm). ALA-PDT takes place at 640 nm irradiation. SPION-PTT is usually achieved at 808 nm, but here it was determined that light-to-heat conversion efficiency of SPION at 640 nm is also 89.73\%. Combination therapy resulted in a higher growth inhibition rate with complete eradication of planktonic cells of P.aeruginosa. Significant growth inhibition rates were achieved for biofilms of S.epidermidis and P.aeruginosa (10-log and 13-log, respectively). Fluorescence images supported the enhanced delivery of ALA into P.aeruginosa biofilms. It is important to emphasize that SPIONs are the only FDA-approved nanoparticles, and they show low cytotoxicities. Therefore, they can be good candidates for the treatment of multi-drug resistant bacteria infections in vivo and in clinical studies by utilizing combination therapy of aPDT and aPTT. An alternative composition for combination therapy wherein a single wavelength irradiation would be sufficient is also proposed in this thesis: ALA loaded Ag2S-QDs coupled with 640 nm irradiation. PTT potential of QDs at 640 nm laser irradiation was investigated and reported as 65.7%. These QDs were mostly effective against gram-positive bacteria. Significant growth inhibition rates were observed as 8-and 6-log for P.aeruginosa planktonic cells and biofilms, respectively. Confocal microscopy images showed strong NIR emission highlighting the imaging potential of the QDs. It is also shown that these QDs do not have any cytotoxicity against healthy cell line, Vero. Thus, ALA loaded NAC coated Ag2S-QDs are promising candidates for in vivo and clinical phototherapies against gram-positive bacteria related infections with the advantage of imaging in the NIR region.
Development of Gold/gold Sulfide Nanoparticles for Photoacoustic Imaging and Radiotherapy
Cancer is a complex and challenging health concern that requires a tailored approach to treatment. The type of cancer, its stage at diagnosis, and the patient's overall health all play a role in determining the most appropriate therapy. Surgical resection followed by chemotherapy and/or radiation therapy and an imaging technique may be used to improve the chances of a successful outcome. Theranostics is an innovative approach to healthcare that combines diagnostic and therapeutic functions in a single formulation. This allows for the simultaneous diagnosis and treatment of a disease, as well as the real-time monitoring of the treatment's progress and effectiveness. The use of theranostic agents can streamline the process of disease management and improve patient outcomes. Photoacoustic imaging (PAI) is a novel medical imaging method that produces images by analyzing local optical absorption. It utilizes either endogenous or exogenous chromophores with distinguishable optical absorption characteristics, and provides high-contrast images. If an exogenous contrast agent is chosen that has an optical absorption unlike endogenous tissue chromophores, then there will be minimal interference from background tissue signals. Gold nanoparticles (GNP) are a prime example of an exogenous contrast agent, due to their unique optical properties suitable for photoacoustic imaging. The absorption of light in GNP is greatest when the frequency of incident light is in resonance with their surface plasmon resonance (SPR). This resonance is seen in the UV-vis-NIR spectrum, often resulting in a high absorption peak of GNP. GNP are known to be excellent light-to-heat converters, contrast agents, and radiosensitizers. Gold-gold sulfide nanoparticles (GGS), which are relatively new in the literature, absorb NIR wavelengths and potential candidates for increasing effectiveness in tumor therapy and optical imaging. However, little knowledge of the structure and difficulty of maintaining stability complicates its use in the biomedical field. In the first chapter of this thesis, a simple, one-step, aqueous synthesis of GGS nanoparticles was developed. 3-mercaptopropionic acid/ methoxy poly (ethylene glycol) thiol (GGS-3MPA), branched poly (ethyleneimine)/ methoxy poly (ethylene glycol) thiol (GGS-bPEI), and bovine serum albumin (GGS-BSA) coatings were optimized to keep GGS stable. Absorbance, size and surface potential was followed for a year and no significant change observed. The NIR absorbance of the GGS is controversial in the literature. To understand whether the shape anisotropy or core-shell structure of the particles causes NIR property, shape, and size distribution studied by TEM analysis and the compositional changes along the particles measured by XPS. TEM images showed that the solution of GGS has spherical, triangular, hexagonal, octagonal, and rod-like particles with varying sizes. There was no change in the chemical states of the gold and sulfur along the etched layers of GGS. Further characterization was done by DLS-zeta, FTIR, and XRD measurements. In the second chapter, we evaluated the potential of GGS as an exogenous contrast agent for photo-acoustic microscopy (PAM) in visible and near-infrared regions. First, the photoacoustic signals were obtained from the solution of the nanoparticles. For the photoacoustic evaluation in the NIR range, all three GGS were compared with gold nanorods (GNR), which are known for their high NIR absorbing ability. Stronger signals were obtained with GGS compared to GNR. Depending on the cytotoxicity results 100 µg GGS/mL concentration was used for all in vitro imaging and therapy experiments. For in vitro imaging with PAM, HeLa, MDA-MB-231, and L929 cell lines were used. PAI revealed the uptake performance of the synthesized GGS with different coatings. Strong signals were obtained with a 532 nm laser; however, 800 nm results were weak. Later, the potential of GGS as a radiosensitizer was evaluated. Radiation dose-dependent radiotherapy showed that especially GGS-3MPA and GGS-bPEI can lower the cell viability to 25% with 0.05 Gy, which is a very low dose of radiation beyond expected. These results imply the conclusion that healthy tissues may not be harmed by eliminating all the side effects expected in radiotherapy applications in the future.
Nanoparticle enabled diagnosis and combination therapy of cancer
Cancer causes approximately 10 million deaths yearly, with even more people diagnosed, comprising one-sixth of global mortality. It is both an economical and public health burden. Despite the developments in conventional therapies, which include surgery, chemotherapy and radiotherapy, the treatment outcome still falls behind the disease progression. As such, novel therapeutic approaches are needed to address the insufficiencies associated with conventional treatments, such as tumor specificity, poor prognosis, and side effects. Light-based therapies for different pathologies date back to ancient. Two main approaches, photodynamic therapy and photothermal therapy, have been highly popular in recent years. Photodynamic therapy mainly exploits the interaction between light, photosensitizer and oxygen, which produces reactive oxygen species killing the tumor cells via direct and indirect pathways. Photothermal therapy relies on localized temperature increase upon irradiation of a photosensitizer with light. Photothermal therapy does not require oxygen to be effective, thus, it has the potential to treat hypoxic tumors as well. For both modalities, photosensitizers that can work with near-infrared light is highly desired for safety and deep tissue penetration. Semiconducting nanocrystals, namely quantum dots (QDs), were initially designed as luminescent nanoprobes, and recently they are being investigated for theranostic approaches as drug delivery vehicles and photosensitizers for photothermal therapy. The first generation of QDs based on Cd, Hg or Pb suffered from toxicity. As a result of extensive research efforts, novel, heavy metal free Ag2S (AS) QDs emerged as a new class of theranostic QDs which may be tracked optically in NIR and induce local temperature increase upon irradiation in NIR. This thesis work was dedicated to exploit the theranostic applications of AS QDs in cancer treatment. In the first part, they were utilized as hydrophobic drug carriers, to efficiently deliver a commonly used chemotherapeutic agent, paclitaxel (PTX) and provide PTT at the same time. Since PTX suffers from low aqueous solubility, a novel design idea was proposed to produce small-sized, colloidally stable, NIR emitting AS QDs. Inspired by clinically approved albumin bound PTX structure, Abraxane®, bovine serum albumin (BSA) was covalently conjugated on the surface of the QDs and PTX was electrostatically loaded on the BSA. PTT/chemotherapy potential of this optically trackable nanoparticle (AS-BSA-PTX) was tested on SKBR3 and MDA-MB-231 breast cancer cells in vitro, utilizing a laser irradiation at 808 nm, 700 mW for 10 min, after 24 h incubation. For both cell lines, a significant difference between only PTT and combined treatment was observed at low doses. The IC50 value of PTX, 100 ng/mL, was reduced down 5-fold to 20 ng/mL with combination therapy. This design holds great potential as it can be adapted to deliver other hydrophobic species as well. In the second part of this thesis work, another chemotherapy drug, Cisplatin (CPt), was delivered to HER2 overexpressing breast cancer cells selectively via a targeted approach using Herceptin (Her) as a targeting molecule. Glutathione (GSH) coated AS QDs were utilized as imaging, drug delivery and PTT agents. Her was covalently conjugated on the QD surface and CPt was electrostatically loaded. AS-Her/CPt QDs were small-sized, colloidally stable, had strong NIR emission at 900 nm and provided sufficient temperature increase in the solution when irradiated with 808 nm (215 mW) laser. In vitro experiments showed 2-fold enhanced intracellular uptake of the QDs in HER2(+) SKBR3 cells compared to HER2(-) MDA-MB-231 cells due to receptor mediated internalization. The viability of the SKBR3 cells was reduced by 75% with PTT/chemotherapy combination at 100 μg/mL [Ag] concentration, with only 4 h of incubation and 808 nm (700 mW) laser irradiation for 10 min. Following up on the promising in vitro results, a safe dose of ASHer/CPt QDs was injected in vivo on tumor xenograft nude mice of SKBR3 cells. Selective accumulation on the tumor site was confirmed with IVIS imaging throughout the treatment period. Enhanced treatment efficiency was observed compared to mono therapies, chemotherapy and PTT with AS-Her QDs. Only chemotherapy treatment (only CPt at 0.7 mg/kg without laser irradiation) and only PTT treatment with AS-Her at 30 mg/kg dose (2.0 W/cm2, 10 min laser irradiation) resulted in 40% and 30% tumor volume decrease, respectively. When PTT/chemotherapy combination treatment was applied, complete tumor regression was observed at the same treatment parameters. Thus, AS-Her QDs were proposed as a novel, efficient PTT agents for the treatment of HER2 overexpressing tumors and delivering therapeutic cargo to tumor site. The third part of this thesis was focused on the delivery of a novel PDT agent, brominated hemicyanine (Hemi-Br) for the image-guided, combined treatment of folate receptor (FR) positive HeLa cells. For this purpose, AS-GSH QDs were decorated with folic acid (FA) and electrostatically loaded with Hemi-Br. AS-GSH-FA/Hemi-Br QDs were colloidally stable over one year, small-sized (<100 nm) and had dual emission at 705 and 900 nm from Hemi-Br and AS QDs, respectively. PTT and PDT potential of the final nanoparticle was first investigated in solution. With laser irradiation at 640 nm (215 mW),17.74oC temperature increase was observed with the combined nanoparticle compared to 6.67oC and 13.6oC with free Hemi-Br and AS-GSH-FA, respectively. This study signifies as the first study to investigate light-to-heat conversion efficiency of AS QDs at this wavelength. The singlet oxygen formation with laser irradiation of Hemi-Br was also confirmed in the solution. After promising results in the solution, particles were facilitated as FR targeted, NIR-emitting, PTT/PDT dual therapy agents in FR(+) HeLa and FR(-) A549 cells. No cytotoxic effect was observed in the absence of laser irradiation. On the other hand, complete cell death was observed for the targeted HeLa cell line at 10 μg/mL Hemi-Br (57 μg/mL [Ag]) dose when PDT/PTT combined therapy was applied. Whereas the viability was approximately 70% for untargeted A549 cells at the same concentration. The cell death mechanism was further investigated to show the effects of mono therapies. The fourth and the last chapter was aimed to enhance the PDT efficiency by improving the tumor hypoxia. For this purpose, oxygen and hydroxyl radical producing MnO2 was produced on the BSA coated AS QDs by the reduction of KMnO4, and a hybrid structure of AS-BSA-MnO2 was produced. To provide PDT, 5-aminolevulinic acid (ALA) was electrostatically loaded to these particles. Final nanoparticle had NIR emission and small hydrodynamic size with an ability to perform PDT and PTT in the solution. No significant cytotoxicity was observed on SKBR3 and MDA-MB-231 breast cancer cell lines without the laser irradiation, indicating the biocompatible nature of the nanoparticles. In the presence of both MnO2 and AS QDs, significant enhancement in the ALA-PDT treatment efficiency was observed for both cell lines with co-irradiation at 640 and 808 nm (300 and 700 mW) for 5 min. Additionally, the superiority of the enhanced combination treatment compared to mono therapies was shown. These findings demonstrated that AS-BSA-MnO2-ALA had excellent image-guided PTT/PDT potential against breast cancer cells.
Development of gold/gold sulfide nanoparticles for photothermal and photodynamic therapy
Cancer is a high-mortality disease. There is an urgent need for alternative or adjuvant therapies with high specificity and efficacy. Light-based therapies offer an excellent locality and turn on/off modality to the treatment. In photothermal therapy, the nanoparticle is irradiated with a light source (LED or laser) at a specific wavelength, which is absorbed by the nanoparticle and converted into heat, causing damage to the surrounding tissue. In Photodynamic therapy (PDT), the irradiated photosensitizer, which is usually an organic molecule, generates reactive oxygen species (ROS) which cause apoptosis, DNA damage, and cell death. PDT is used in the clinic for specific cancer types, such as head-and-neck cancer. Nanoparticles are very attractive for various applications due to their tunable physical, chemical, and biological features. Gold nanoparticles are one of the most studied due to their inert and biocompatible nature and are used in different fields such as catalysis, energy, and medicine as a theranostic agent. In this thesis, a new type of colloidally stable gold nanoparticle with near-infrared absorbance, namely gold-gold sulfide (GGS) nanoparticles were developed. Then, these GGS nanoparticles were developed into a multifunctional nanoparticle (NP) to offer combination phototherapy; PTT and PDT for enhanced therapeutic outcome. In vitro toxicity studies of these nanoparticles and the efficacy of PTT/PDT therapy combinations of prostate cancer cell lines have been discussed.
Development of carbon quantum dots for the selective and sensitive detection of nitrite ions
Nitrite ion is used as a preservative in food and beverages and is also a common pollutant emerging from industrial processes. The concentration of nitrite requires strict control due to its toxicity and reactivity. It is also an indicator of infections in the human body. Detection of nitrite concentration via effective, cheap, and sustainable methods will help regulation of industrial uses and early diagnosis of diseases mostly related with the respiratory system. Carbon dots are a relatively new class of luminescent nanoparticles and are highly promising in sensing applications due to their tunable, functional group-rich surface and fluorescence properties. The lack of heavy metals, ease of production and biocompatibility, the lack of surface capping agents make the luminescence of carbon dots highly sensitive to the surrounding media and favorable for sensing applications. The development of carbon dots from various carbon sources including small molecules and polyolefins has been studied to produce stable, reproducible, low-cost carbon dots with high quantum yield and various emission profiles. Red luminescent carbon dots were developed for highly selective and sensitive nitrite sensing with 0.01 ppm limit of detection (LOD). From the emergence of plastics in the early 20th century, plastics replaced many materials due to cost, light weight, durability, etc. Today, humanity faces the problem of plastic pollution as a result of the large volume of production but insufficient recycling. Chemical stability and corrosion resistance of plastics especially polyolefins like polyethylene (PE) and polypropylene (PP), make the recycle processes highly expensive and inefficient. Conversion of these polyolefins to the value-added carbon nanoparticles proposes an efficient way to handle plastic pollution. Upcycling of polyolefins to luminescent carbon dots with high quantum and conversion efficiency is demonstrated in this thesis as an alternative to polymer recycling.
Multifunctional nanoparticles for radiotherapy enhancement and targeted phototherapies
In 2024, the National Institutes of Health reported 2 million new cancer cases and 600,000 cancer-related deaths in the United States. Cancer remains a complex disease and a significant public health challenge. Conventional cancer treatments such as surgery, chemotherapy, and radiotherapy are successful. However, localization of the treatment, tumor specificity, adverse side effects of the treatments, and cancer metastases are still challenging issues. Novel treatment strategies that overcome these limitations are the focus of researchers, which is to increase the survival rate and improve patients' comfort. Radiotherapy (RT), a commonly used cancer treatment, uses ionizing radiation to kill or prevent the growth of malignant cells. It is a relatively local treatment compared to chemotherapy but is usually combined with other treatment methods for more efficient results. Some concerns associated with this treatment are dose heterogeneity, side effects on healthy tissues, and the radio-resistance of specific cancer cells. Radiosensitizers allowing local radiation enhancement at low doses of ionizing radiation and more homogeneous dose distribution within the tumor are highly popular and the subject of continuous research. Light-based therapies, such as photothermal and photodynamic therapies, may be combined with RT to enhance the treatment efficacy. These techniques provide a local, turn-on mechanism for cancer treatment. Photothermal therapy (PTT) is based on local hyperthermia caused by light irradiation of a photosensitizer (PS). Photodynamic therapy (PDT) relies on activating a photosensitizer by light and generating reactive oxygen species. Limitations in these therapies include solubility and bioavailability of the photosensitizer, day-light sensitivity of the patient, and activation of PSs mostly in blue, exerting a significant limitation in penetration depth. Protoporphyrin IX (PpIX), a common PDT photosensitizer typically irradiated with blue or red light, has been recently reported to be activated with X-ray, and this process is called radio dynamic therapy (RDT). This thesis explores the theranostic applications of gold-gold sulfide nanoparticles (GGS) with strong absorbance in the near-infrared, contrast ability in photoacoustic imaging (PAI), and potential for radiosensitization for low-dose RT, and combination of low-dose RT with phototherapies to improve therapeutic outcomes. In this thesis work, a combination of RDT with low-dose RT, bypassing high radiation dose requirements and light penetration limitations, was investigated via 5-aminolevulinic acid (ALA) loaded branched polyethyleneimine-coated GGS (bPEI-GGS) nanoparticles. ALA is a pro-drug converted to PpIX in living cells, more so in cancer cells. In vitro activity assessments were performed on PC3 prostate cancer and MDA-MB-231 breast cancer cell lines at low X-ray doses between 0.5, 1, 2, and 4 Gy. Cell viability dropped below 20% after 0.5 Gy RDT treatment with a low dose of GGS (Au concentration (μg/ml)/ALA concentration (mM): 25/0.22) for both cell lines. ALA-bPEI-GGS nanoparticles represent a promising approach to enhancing RDT effectiveness, especially for deep-seated and radioresistant tumors, with a level of selectivity towards cells overexpressing PEPT1/PEPT2. In the second part of the thesis, tumor-specific delivery of multifunctional nanoparticles providing a combination of photothermal therapy (PTT) and low-dose RT for improved therapeutic outcomes under mild conditions has been demonstrated using theranostic GGS nanoparticles with no further therapeutic agents. Multifunctional 3-mercaptopropionic acid-coated GGS (3MPA-GGS) were tagged with folic acid (FA-3MPA-GGS) to selectively target folate receptor-positive (FR+) cancer cells, enhancing therapeutic specificity and efficacy. FA-3MPA-GGS demonstrated strong NIR absorption, high-temperature increase, photothermal stability, and efficient light-to-heat conversion in the solution when irradiated with 808 nm, 215 mW laser. In vitro experiments performed with FR+ (HeLa and MDA-MB-231) and FR- (A549) cells indicated enhanced uptake, increased ROS generation, and more significant cytotoxicity in FR+ cells. Combining mild PTT with low-dose RT via FA-3MPA-GGS led to synergistic effects, lowering the treatment dose and enabling image-guided precision. Near-complete death of HeLa and MDA-MB-231 were obtained with PTT/RT at 200 μg Au/mL concentration, with 10 min 795 nm (1 W) laser irradiation followed w 0.5 Gy X-ray irradiation. This thesis's third part focuses on phototherapies without the use of ionizing radiation: the combination of nanoparticle-driven PTT and ALA-PDT. A novel dual-mode phototherapy approach for the treatment of prostate cancer using ALA-loaded superparamagnetic iron oxide nanoparticles (ALA-PAA-SPIONs) was demonstrated. Utilizing the upregulation of PEPT1/PEPT2, effective in selective transport of ALA, in prostate cancer cells presents a valuable target for enhancing delivery and specificity in treatment. In vitro experiments on LNCaP, PC3, and DU145 cell lines demonstrated selective cellular uptake of NPs in PEPT1/PEPT2 overexpressed LNCaP and PC3 cells. Formation of reactive oxygen species (ROS) and induction of apoptosis upon laser irradiation (640 nm, 700 mW, 10 minutes) lead to significant loss of cell viability (<10%), specifically in these two cell lines. The results showed the potential of ALA-PAA-SPION as a promising phototherapy agent for prostate cancer treatment, offering improved specificity and minimized systemic effects.
ALA-aracılı fotodinamik terapi ve genlerin özgün polimerler ile taşınması
Cancer is a challenging and complex disease to treat. Conventional therapeutic approaches, namely chemotherapy, radiotherapy and surgery, has limited success. Alternative treatment methods such as local phototherapies and gene therapy are highly promising in the treatment of different cancers as a single treatment modality or combination with conventional methods. In this thesis, in vitro studies for photodynamic therapy and its combination with chemotherapy for the treatment of colorectal cancer and bone targeting polyplexes made by novel copolymers for gene-therapy of osteosarcoma are described. Photodynamic therapy (PDT) is a local, approved and promising approach in the treatment of cancer and bacterial infections. It induces tumor cell death creating reactive oxygen species (ROS), such as singlet oxygen, by photosensitizer excitation at a specific wavelength. It can be applied alone or in combination with other therapeutic techniques such as photothermal therapy (PTT) or chemotherapy for the treatment of colorectal, head, neck and breast cancer. Combination of chemotherapy and photodynamic therapy enhances therapeutic efficiency overcoming multidrug resistance (MDR) at low drug doses. 5-Aminolevulinic acid (ALA), a well-known and FDA approved photosensitizer, is a promising prodrug with the strong fluorescence for diagnosis and fast accumulation in the tumor tissue. ALA is produced in mitochondria as a rate-limited step of the heme biosynthesis pathway and converted to protoporphyrin IX (PpIX), which is a photosensitizer. For efficient PDT, exogenous ALA administration is needed to increase the PpIX concentration to therapeutic levels in the tumor cells. In the first chapter of this thesis, ALA-mediated PDT and its combination with 5FU in the treatment of colorectal cancer is (HCT116 cells) described. Colorectal cancer is the 3rd most diagnosed tumor type and difficult to treat. Dose dependence of PDT, irradiation protocol and suitable cytotoxicity assays were first determined in 2D cell cultures and then applied to 3D early and late tumor models that are more realistic models to predict the efficiency of the treatment. Alamar Blue was determined as the more appropriate method to evaluate in vitro toxicity, 100 μM ALA concentration and 10 min irradiation with blue lamp were found as an effective treatment for effective PDT in 2D and 3D. Besides, PpIX accumulation was significantly increased with an increase in the complexity of cell structures from 2D to late 3D cultures. As a result, ALA-mediated PDT causing apoptotic/necrotic death of HCT116 colorectal cancer cells in 3D tumor models was demonstrated. It was also noticed that ALA to PpIX conversion is hindered with the co-existence of 5FU; therefore, the 1/0.15 mol ratio of ALA/5FU was suggested as a promising recipe for combination therapy. Osteosarcoma is aggressive bone cancer, whose treatment has not changed significantly for the past few decades. Although gene therapy has emerged as a potential treatment route, the need for efficient and non-toxic gene delivery systems targeting osteosarcoma cells remains a challenge. High molecular weight poly (ethyleneimine)s (PEIs), are used as universal transfection agents, however, cause significant cytotoxicity. On the other hand, poly (amidoamine)s (PAAs) are biocompatible, biodegradable Polymers with promising transfection efficiency. In the second chapter, in vitro transfection of osteosarcoma cells with PEI-PAA copolymers with (bis)phosphonic acid groups with the potential to target bone is discussed. A set of novel polymers (PAEI) comprised of low molecular weight branched PEI (Mw = 1800 Da) and PAA macromers which were functionalized in different ratios with 5-amino-1-pentanol (AP) and (bis)phosphonic acid groups were studied. The PAEI polymers synthesized by the group of Prof. Duygu Avcı Semiz were tested on an osteosarcoma cell line (U-2 OS cells), which is one of the hardest cell types to transfect, and on muscle cells (C2C12 cells) as the control. The cytotoxicity and transfection efficiency of PAEIs were adjusted by altering the ratios of phosphonic acid (via APA, aminophosphonic acid) or bisphosphonic acid (via ALE, an amino bisphosphonic acid) content. The highest transfection efficiency was obtained with ALE containing PAEIs. The presence of PAA, PEI and ALE seemed to play a synergistic effect in the transfection performance of PAEIs. The most efficient PAEI polymer, containing a 0.7: 0.3 AP: ALE ratio, displayed a transfection efficiency that was 5-times higher than 25 kDa bPEI. This novel set of polymers can be promising candidates for the gene therapy of osteosarcoma.
İki dağılımlı magnetoreolojik akışkanlar
Superparamagnetic iron oxide nanoparticles (SPION) are exploited in many different fields from automotive to medicine. In every field, there is always a need for better performance that exhibits new challenges to scientists. In this thesis work magnetorheological fluids and magnetic hydrogels are studied to solve the problems and improve the properties of these materials. Magnetorheological Fluids (MRFs) are non-Brownian fluids that consist of micron-sized magnetic particles in carrier fluids, mostly different oils. The biggest challenge in MRFs is the prevention of sedimentation and enhancement of redispersibility to prolong shelf-life as well as life-in-use. This thesis work proposed and demonstrated a method to improve the stability and redisperability of MRFs with high particle loading while having good magnetorheological properties. Bidisperse MRFs composed of micron-sized magnetic particles and functional superparamagnetic nanoparticles which would interact with each other is proposed as the primary strategy. First, the idea was tested on a commercial MRF, 140-CG LORD® (with fatty-acid coated micron-sized particles in hydraulic oil). Poly(acrylic) acid and lauric-acid coated superparamagnetic iron oxide nanoparticles (SPION-PAA and SPION-LA) were synthesized and added in 5-20 weight percentages to 140-CG LORD® to understand the influence of surface coating and SPION content on the sedimentation and magnetorheological properties. Then, new bidisperse MRFs were prepared by adding commercial bare carbonyl iron (CI) and SPION-LA or SPION-PAA to different carrier fluids. Also, CI was coated with LA and bidisperse MRFs were prepared from CI-LA and SPION-LA or SPION-PAA in different carrier oils such as hydraulic oil, silicone oil, mineral oil and glycerol. Magnetorheological properties of prepared bidisperse MRFs were measured with Anton Paar 302 MCR rheometer in both rotational, and frequency modes and mechanical properties, as well as the sedimentation behavior of MRFs, were compared with 140-LORD MRF® as a benchmark. SPION-PAA particles provided the best MRFs in both formulations with enhanced stability for months, better redispersibility and rheological properties at least as good as the commercial one. Selected bidisperse MRFs were used in washing machine MR dampers by the group of Prof. İsmail Lazoglu. This research was funded by TUBITAK and Arçelik (ID: 5150060).