Theses supervised by Prof. Dr. Amitav Sanyal
12 theses · Boğaziçi University
Fabrication of cleavable covalently crosslinked colloidosomes
In this study, cleavable microcapsules were obtained using self-assembly and crosslinking of magnetic nanoparticles at the oil-water interface. To achieve this, iron oxide nanoparticles coated with oleic acid were synthesized and they were coated with three different linkers including azide and alkyne parts, one of which contained a disulfide bond linkage. Since disulfide bonds can be degraded with thiol compounds, they render these microcapsules cleavable. A water in toluene emulsion system including both nanoparticles and Cu(I) catalyst was prepared. By shaking the emulsion, microcapsules were fabricated via Cu(I) catalyzed Huisgen reaction involving cycloaddition of azides and alkynes. Thus, stable microcapsules made through covalent crosslinking of coated iron oxide nanoparticles were obtained. These microcapsules were characterized using microscope and SEM images. Also, FITC labeled dextran was encapsulated within these microcapsules and their release in the presence of dithiothreitol was demonstrated using fluorescence microscopy.
Fabrication of polymeric microgels using microfluidics:Applications in biological sensing and drug delivery
This thesis focuses on developing polymeric microgels designed to serve various biomedical applications. The fabrication involves microfluidics technology, where unique microchannels are designed and manufactured to produce these microgels. One notable aspect of polymeric microgels is their chemistries, allowing further functionalization to suit specific applications. Using microfluidics technology in the fabrication process ensures precise control and scalability, enhancing these polymeric microgels' potential applications and significant influence in the biomedical field. In the first chapter, we demonstrated monodispersed non-degradable microgels produced by flow-focusing microfluidics channels for protein catch-and-release studies. Following, we synthesized microgels of varying porosities using a Y-shaped microfluidic channel and identified the most porous material for comprehensive in vitro drug delivery studies. The succeeding chapter demonstrates degradable microgels that undergo cross-linking as they traverse the serpentine channels within the microfluidics device. These degradable polymeric materials were accompanied by another reducing agent carrying microgels system. The final chapter is about another kind of polymeric material, imprinted polymers, which were fabricated using different polymerization methods. Obtained materials with different sizes were evaluated by their biomarker-catching performances. In summary, this thesis explores various polymeric microgel systems fabricated by distinct microfluidics channel designs. Each has unique features to be functionalized for multiple biomedical applications.
Design and synthesis of therapeutic agents: From small molecules to polymeric nanomaterials
Novel approaches are needed to address the limitations of traditional therapy methods that are in practice today, for improved treatment of diseases. This thesis evaluates such approaches through the development of new molecular and materials-based systems. The first part of this thesis focuses on the synthesis of a prodrug molecule for pancreatic cancer treatment while the second part includes polymeric nanomaterial-containing delivery systems for biological applications. In the first part, two synthetic pathways were investigated to synthesize a prodrug system comprised of gemcitabine, a chemotherapy agent, and hydroxychloroquine which is known to eradicate pancreatic cancer stem cells. Products linked with acid-sensitive linkages with and without disulfide bonds between them were synthesized, and evaluated for their in vitro cytotoxicity against the Bx-PC3 cell line. Another approach to specifically deliver drugs involves the utilization of localized delivery systems. In this regard, in the second part of this thesis, polymeric nanomaterials were evaluated as drug delivery platforms. As nanomaterials, carbon nanotubes (CNTs), magnetic iron-oxide nanoparticles (IONPs) and reduced graphene oxides (rGOs) were utilized in four distinct projects. CNTs wrapped with a reactive polymer using physical interactions were fabricated, and evaluated for attachment of bioactive molecules using the thiol-maleimide reaction. In another project, rGO-embedded photothermal hydrogels were synthesized using a thiol-ene reaction and were evaluated for on-demand active release using near-infrared (NIR) light to release antibacterial agents inside the hydrogel. In the last two projects, IONPs were coated with two different polymers using reversible addition-fragmentation chain transfer (RAFT) polymerization and these constructs were used to design different drug delivery and release systems. While one polymer contains furan groups on the side chains, the second polymer includes maleimide units for the post-polymerization functionalization of the surface. Overall, this thesis puts forth new drug delivery platforms that may find therapeutic applications.
Biodegradable and functionalizable 3D-printed stimuli-responsive hydrogels
In this study, we developed innovative biodegradable and functionalizable 3D-printed stimuli-responsive hydrogels for advanced biomedical applications such as drug delivery and tissue engineering. Utilizing polyethylene glycol (PEG) polymers, the hydrogels incorporated redox-sensitive disulfide linkages for stimuli-responsive behavior. The custom synthesized PEG-disulfide crosslinker (PEGSSDMa) provided stability and redox responsiveness. PEGMEMa enhanced porosity and swelling ratios, while PEGDMa added network stability. The fabrication used extruder-based 3D printing for precision, with gellan gum increasing the viscosity of the hydrogel precursor solution to maintain structural fidelity. UV crosslinking post-printing enhanced mechanical integrity and resistance to dissolution. These hydrogels demonstrated a balanced combination of robustness and biodegradability, tailored to degrade at controlled rates. The redox-responsive nature enabled targeted release of therapeutic agents in specific physiological environments, enhancing treatment efficacy and safety. Functionalizing hydrogels with bioactive molecules promoted cell adhesion, proliferation, and differentiation, essential for tissue regeneration. This study advances biomedical engineering by developing multifunctional hydrogels that integrate biodegradability, functionalizability, and stimuli-responsiveness, paving the way for new therapeutic strategies.
Degradable hydrogels for local drug delivery
Hydrogels are widely utilized in biomedical applications owing to their controllable physical and chemical properties. Their physical strength, elasticity, porosity, water uptake capacity, degradability and biocompatibility can be tuned according to the application of interest. Tunability of the final properties makes them useful constructs for cancer related applications such as tumor models, diagnostic systems and smart drug delivery platforms. The aim of this thesis was to obtain stimuli-responsive degradable hydrogels for local drug delivery in cancer therapy. For this purpose, a functionalizable copolymer, poly(PEGMEMA-co-SCEDEMA), was synthesized via RAFT polymerization. Hydrogel formation was achieved by crosslinking this copolymer with PEG-diamine through a disulfide-based redox and carbamate-based acid sensitive linker. In order to show the tunability of physical properties, PEG-based crosslinker length was chosen as variable. Hydrogels were shown to have lower physical strength but greater porosity and water uptake with increasing crosslinker length. This also had a considerable impact on degradation rate which was expected to influence drug release behavior. To examine this, DOX was chemically bound to hydrogels via the same dual stimuli responsive, disulfide and carbamate bearing side chain which was also utilized for crosslinking as well. Dual- responsive hydrogels were desired in order to introduce a tumor environment sensitive character into hydrogels. This responsivity was designed considering the slightly acidic and reducing conditions of tumor environment. Drug release was investigated under physiological (pH = 7.4), acidic (pH =5.5) and GSH containing acidic environment. As expected, sustainable release of DOX was achieved reaching around 80% cumulative release under GSH containing acidic conditions accompanied by the total degradation of hydrogel construct.
Polymer brush coated iron oxide nanoparticles for biomedical applications
In recent years, the use of iron oxide nanoparticles has gained importance for biological applications such as hyperthermia, targeted drug delivery, drug release, bio-separation, bio-imaging etc. since they are mono-disperse, biocompatible and more importantly they have magnetic properties. In this thesis, water dispersible, monodisperse, disulfide-containing thiol reactive iron oxide nanoparticles have been synthesized. The disulfide groups were introduced either close to nanoparticle surface or on the exterior of the polymeric coating. Additionally, for surface functionalized polymer brush coated nanoparticles, NHS activated carboxylic acid functionalized were also introduced to enable non-cleavable functionalization using molecules containing amine groups. For the synthesis of such functional molecules, chain transfer agent with/without disulfide bond that is used for RAFT polymerization was modified with catechol anchoring group. These compounds were immobilized onto oleic acid stabilized iron oxide nanoparticles via ligand-exchange reaction. PEG based polymers were grafted from the surface of iron oxide nanoparticles by RAFT polymerization in order to make them water dispersible. The end groups of these polymers are trithiocarbanate groups, which can be modified by azo initiators by radical exchange reactions. For this reason, NHS activated carboxylic acid and disulfide functionalized azo initiators were synthesized and used for the functionalization of grafted polymers. After functionalization of polymer-coated iron oxide nanoparticles, they were able to react with thiol and amine bearing molecules. By using the same chemistry methods, disulfide bearing chain transfer agents were immobilized onto the nanoparticles and PEG based polymers were grown from the surface of nanoparticles by RAFT polymerization and grafting-from approach. Disulfide bearing polymers were cleaved from oxide nanoparticles by using reducing agents. Additionally, this method provides an excellent way to measure the molecular weights of the polymers that are grown by grafting-from approach.
Functionalized pisa particles for targeted delivery and imaging
Polymer based nanoparticles are widely used in various biomedical applications due to their easily tunable size and morphology. Furthermore, characteristic properties such as solubility or biocompatibility can also be adapted depending on the needs of the application. Desired properties can easily be incorporated with the right choice of material from an extensive pool of available polymers Micellar structures are one of the most utilized examples of polymeric nano-structures with their amphiphilic nature, controllable size, morphology and surface properties on top of their unique capability of encapsulating host molecules. They can be obtained effortlessly with a one pot, straightforward reaction, by using polymerization induced self-assembly (PISA) technique. In this study, our aim is to obtain micellar polymeric nanomaterial with a functionalizable surface and guest molecule encapsulation capability by using the PISA technique. Surface functionalizability will be used to introduce targeting units on the surface of the nanoparticles for specific cancer tissues, whereas the hosting through encapsulation will be used to carry hydrophobic dyes as cargo. For this purpose, p(PEGMEMA)-b-pST copolymers with dibromomaleimide (DBM) end groups were synthesized in a solvent, where they will self-assemble during the polymerization of the second block and form stable nanomaterial. It was observed that the formation of the nanoparticles were dependent on the degree of polymerization of the solvophobic pST block. Nile red, a hydrophobic dye, was also introduced in the media during the polymerization and encapsulated by the nanoparticles in situ. The DBM moieties on the surface were used for further functionalization of the particles as they are well known to give fast and mild reactions with free thiol-containing compounds. Nanoparticles were reacted with two different thiol bearing molecules, one of which was a cysteine containing peptide that has very high affinity for integrin receptors that are overexpressed on cancer tissues. Consequently, a new type of surface functionalized PISA particles with targeting property and guest molecule encapsulation capability are successfully synthesized.
Yara tedavisi için özgün hidrojel sistemler
Hydrogels are cross-linked, three-dimensional hydrophilic polymeric networks. They possess similarity in structure to the soft tissue in the body, thus have found a great number of applications as biomaterials. For wound care applications, hydrogels are considered to be new forms of wound dressings. Hydrogels with designed properties can act as a barrier for wounds while fastening the healing process. They can also cargo macromolecules encapsulated within their structure that are necessary for wound regeneration and protection. The aim of this thesis is to synthesize redox responsive hydrogels that can be applied as wound care materials. Said hydrogels were designed to obtain a tissue regeneration triggering, non-cytotoxic and on-demand degradable hydrogel dressing to avoid disturbance of tissue while the material is physically removed. To do so, hyaluronic acid, a natural polymer constituent of human soft tissue, was modified with furan groups to construct hydrogels through reaction with a maleimide and disulfide functional group containing redox-responsive PEG-based crosslinker by Diels-Alder cycloaddition. The stimuli responsive degradation features are tuned by varying the amount of crosslinker in hydrogels. The change of porosity and disulfide concentration was expected to effect the degradation rates. It was demonstrated that hydrogels with higher crosslinking density exhibited greater strength, along with longer degradation times. All hydrogels exhibited high swelling capacity. To examine the passive and on-demand delivery of tissue regenerative macromolecules from this system, a model protein, namely, FITC-BSA was encapsulated within the hydrogels. The release of these biomolecules were investigated in PBS (pH=7.4), in absence and presence of the reducing agent DTT, to observe the passive and on-demand release profiles. Importantly, complete dissolution of the hydrogel was accomplished under mild conditions upon exposure to a solution containing the reducing agent.
Smart polymeric nanocomposites tailored for targeted delivery, protein separation and biofilm disruption
Fabrication and biomedical applications of polymeric nanomaterials are described. In the first study, transdermal delivery of insulin via rGO confined hydrogels is reported. rGO impregnated PEG-based hydrogels were fabricated by photo-induced FRP of PEGDMA monomers. Hydrogels loaded with insulin were used for on-demand drug release exploiting photothermal heating ability of rGO in the hydrogel matrix upon exposure to NIR irradiation. In the second part, functionalization of rGO with clickable maleimide group is reported. Maleimide was conjugated with a thiol bearing targeting peptide, and a chemotherapy drug was loaded on the surface of rGO via non-covalent interactions. Targeted delivery and cancer cell killing ability of doxorubicin loaded rGO were then evaluated. In the third part, selective cell targeting and imaging with polymer coated MNPs are studied. The surface of MNPs was modified with clickable groups which were further conjugated with a targeting peptide and an imaging agent. Cell targeting and internalization were achieved with these MNPs. In the fourth part, lectin separation using polymer coated MNPs is investigated. The surface of these MNPs was modified with adamantane group, and supramolecular host-guest interactions were exploited for binding of $\beta$-CD-sugar conjugates. A specific lectin from a mixture was separated by sugar containing MNPs. Final work describes phytochemical containing polymer NCs for eradication of MDR biofilms. A biodegradable and clickable amphiphilic copolymer was used to stabilize carvacrol oil-in-water emulsion. Biofilm disruption ability of these NCs was evaluated on different MDR biofilms. In summary, potential bio-applications of various nano-constructs was evaluated.
Fabrication of degradable polymer coated nanoparticles
In recent years, interest in polymer drug conjugate delivery systems has increased. When these polymer drug conjugate systems are conjugated with magnetic nanoparticles, they can be used as delivery systems by creating magnetic field. In this thesis, firstly monodisperse oleic acid coated, iron oxide nanoparticles were synthesized by thermal decomposition method. By place exchange reaction, these nanoparticles were coated by chain transfer agents which are modified with dopamine and have disulfide bond in their structure. Then PEG based polymer were grafted from the surface of iron oxide nanoparticles by RAFT polymerization in order to make them hydrophilic. In this system, there are disulfide bonds between polymeric brushes and nanoparticles and these bonds can be cleaved by glutathione which is found in excess at diseased tissues. Importantly, it is difficult to characterize the molecular weights of the polymers which were grown from the surface of nanoparticles 'by grafting-from' approach. We envisioned that mild methods to cleave the polymers from nanoparticle surfaces will facilitate their characterization. We demonstrate that treatment with glutathione cleaves the polymeric brushes from nanoparticles under mild conditions. Thus, this study provides an excellent way to analyze molecular weight and molecular weight distributions of polymer brushes. In order to understand the cleavage kinetics, polymer brushes containing fluorescent dye were synthesized. Also, the fluorescent dye acts as a model hydrophobic drug. Incorporation of the dye into polymer brushes provides it with water solubility. Finally, we demonstrated that the trithiocarbonate end groups of polymers can be modified with azo initiators for purposes of attachment of any biomolecules.
Synthesis and application of thiol-reactive zwitterionic copolymers
In recent years, zwitterionic polymeric materials have gained attention thanks to their ultra-hydrophilicity due to the equimolar number of evenly distributed anionic and cationic moieties that they contain along their polymer chains while still preserving their overall charge neutrality. Moreover, zwitterionic polymers are typically regarded as an alternative to the popular poly(ethylene glycol) (PEG) polymers for anti-biofouling applications to prevent nonspecific protein adsorption and to reduce bacterial or mammalian cell adhesion. Due to its potential uses in interdisciplinary fields, including biomaterials, material science, and targeted delivery, the synthesis of zwitterionic polymers with reactive functional groups is a popular area of research. Polymers with reactive maleimide units as functional groups at the side chains can be obtained by a copolymerization technique using zwitterionic monomers and maleimide-containing monomers. In the thesis, the functionalization of zwitterionic structures was done by copolymerization with an acrylate-based 'latent-reactive' monomer in which maleimide is masked with furan by the Diels-Alder reaction to protect the double bond of the maleimide moiety. By using the advantage of the thermo-responsive behavior of the cycloadduct, the thiol-reactive maleimide unit was activated by the retro DA reaction. To show the effectiveness of the maleimide unit, the thiol-containing hydrophobic dye was conjugated to the linear polymeric structure by the 'click' reaction. Since the hydrophobic dye can become soluble in water when attached to the designed zwitterionic polymer, the methodology serves as a model of drug conjugation.
Reduced graphene oxide embedded polymeric nanofibers for thermo-responsive drug delivery systems
In recent years, nanofibers have become popular in biomedical applications because of their distinctive qualities, such as the large surface area to volume ratio, porosity, and appreciable mechanical strength. Electrospinning is the most widely utilized method to manufacture nanofibers due to its straightforward setup and cost-effective production. Fabricated nanofibers can be modified with different functional groups to impart characteristic properties and enable their application. In recent years, 'click' chemistry transformations have been preferred to functionalize nanofibers. In this thesis, clickable copolymers were synthesized to generate furan functionalized polymeric nanofibers with the help of electrospinning. Morphological features of the produced nanofibers were characterized with SEM. Reduced graphene oxide (r-GO) was embedded into the nanofiber during electrospinning to provide a photothermal property to the fibrous material. Irradiation of r-GO-containing materials with near-infrared (NIR) light results in the conversion of the light into heat and increases the temperature of the surface of the fibrous substrate. Doxorubicin (DOX) was modified with a maleimide functional group to enable Diels-Alder (DA) cycloaddition between furan-functionalized nanofibers and maleimide-modified drug molecules. After successfully conjugating the drug molecule, exposing drug conjugated nanofiber to NIR light leads to retro Diels-Alder (rDA) reaction resulting in drug release thanks to rapid light-to-heat conversion