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Öğe Bioconjugated beta-cyclodextrin-perfluorohexane nanocone clusters as functional nanoparticles for nanoparticle-mediated histotripsy(American Chemical Society, 2022) Toydemir, Cemran; Hall, Sarah; Demirel, Erhan; Elmacı, Dilşah Nur; Göl, Deniz; Vlaisavljevich, Eli; Yüksel Durmaz, YaseminNanocone clusters (NCCs) are new-generation agents of nanoparticle-mediated histotripsy (NMH) recently developed to address the limitations of previously designed nanodroplets (NDs). NCCs can be obtained by simply mixing FDA-approved cyclodextrins (CD) and suitable perfluorocarbons (PFCs), which result in smaller size aggregates, detectable PFC amount, and more stable long-term storage since the obtained powder can be stored and redispersed as needed. Previous experimental and computational studies showed that NCCs consist of an organization of inclusion complexes of CD and PFC around free PFC droplets, and their aggregate behavior depends on the localization of PFC in the cavity and the water solubility of CD derivatives. It has been shown that beta-cyclodextrin (beta CD) and perfluorohexane (PFH) are ideal candidates for NCCs that can be isolated as a powder with high PFC content among various CD and PFC derivatives. This study focuses on the further development of the selected NCC composition to enhance the potential of NMH therapy while also enabling more detailed future experiments in vitro and in vivo. It is aimed to show the bioconjugation potential of NCCs through the example of the most commonly used functionalization methods such as targeting, PEGylation, and fluorescent labeling. For this purpose, beta CD as a building block was monofunctionalized with groups such as azide, alkyne, and amine groups that allow for effective coupling reactions such as the "click" reaction and N-(3-dimethylaminopropyl)-N '-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling. These monofunctional beta CDs were used as building blocks of NCCs in the presence of PFH to obtain functional NCCs as precursors of bioconjugation. EPPT1 as a synthetic peptide specific to uMUC1 and folic acid (FA) as the most commonly used targeting agent along with PEGylation were successfully shown as bioconjugation examples. Lastly, fluorescently labeled NCCs were obtained via fluorescein isothiocyanate (FITC) and alkyne functional NCC reaction through propargyl amine and isothiocyanate group reaction. The obtained bioconjugates were tested in vitro to validate the conjugation, and the ability to lower the histotripsy cavitation threshold, which is necessary for NMH, was demonstrated for all bioconjugates. Overall, the results showed that all obtained bioconjugates successfully lowered the cavitation threshold pressure while also fulfilling the desired bioconjugation metrics to serve as improved tools to enhance NMH as a targeted noninvasive ablation method.Öğe Development of acoustically active nanocones using the host-guest interaction as a new histotripsy agent(American Chemical Society, 2019) Rehman, Tanzeel Ur; Khirallah, Jennifer; Demirel, Erhan; Howell, Justin; Vlaisavljevich, Eli; Yüksel Durmaz, YaseminHistotripsy is a noninvasive and nonthermal ultrasound ablation technique, which mechanically ablates the tissues using very short, focused, high-pressured ultrasound pulses to generate dense cavitating bubble cloud. Histotripsy requires large negative pressures (>= 28 MPa) to generate cavitation in the target tissue, guided by real-time ultrasound imaging guidance. The high cavitation threshold and reliance on real-time image guidance are potential limitations of histotripsy, particularly for the treatment of multifocal or metastatic cancers. To address these potential limitations, we have recently developed nanoparticle-mediated histotripsy (NMH) where perfluorocarbon (PFC)-filled nanodroplets (NDs) with the size of similar to 200 nm were used as cavitation nuclei for histotripsy, as they are able to significantly lower the cavitation threshold. However, although NDs were shown to be an effective histotripsy agent, they pose several issues. Their generation requires multistep synthesis, they lack long-term stability, and determination of PFC concentration in the treatment dose is not possible. In this study, PFC-filled nanocones (NCs) were developed as a new generation of histotripsy agents to address the mentioned limitations of NDs. The developed NCs represent an inclusion complex of methylated beta-cyclodextrin as a water-soluble analog of beta-cyclodextrin and perfluorohexane (PFH) as more effective PFC derivatives for histotripsy. Results showed that NCs are easy to produce, biocompatible, have a size <50 nm, and have a quantitative complexation that allows us to directly calculate the PFH amount in the used NC dose. Results further demonstrated that NCs embedded into tissue-mimicking phantoms generated histotripsy cavitation "bubble clouds" at a significantly lower transducer amplitude compared to control phantoms, demonstrating the ability of NCs to function as effective histotripsy agents for NMH.Öğe Effective PEGylation method to improve biocompatibility of graphene derivatives(Elsevier, 2020) Demirel, Erhan; Karaca, Ezgi; Yüksel Durmaz, YaseminThe research on the use of graphene (G) in the biological applications has increased exponentially with emerging concerns regarding its biosafety and potential cytotoxicity. The modification of the surface with biocompatible polymers is a promising approach where poly(ethylene glycol) (PEG) have been extensively used. However, increasing water solubility may not be enough to make G biocompatible since it has different cytotoxicity mechanisms like absorbing cell nutrition on its wide surface area and creating reactive oxygen species through its functional groups. A PEGylation method that uses surface area of graphene oxide (GO) by taking advantage of its solubility in aqueous medium and simultaneously producing reduced GO (rGO) to eliminate the cytotoxicity that comes from functional groups might be a solution to improve biocompatibility and solubility of rGO which is an optimum graphene derivative for biological applications. To have surface PEGylated rGO instead of having edge PEGylated GO; P(PEGMA-co-MMA-co-PMA) copolymers were synthesized and coated on GO via ?-? interactions through multiple pyrene units in the copolymer. Healed conjugated surface of rGO was used as an advantage to increase the efficiency of PEGylation by in-situ reduction of GO to rGO in the presence of copolymer to obtain biocompatible, water dispersible, highly PEGylated rGO.Öğe Experimental design and characterization of dual-antibody-conjugated all-trans retinoic acid-loaded lipid nanoparticles as a potential cancer therapy(2024) İşlek, Zeynep; Sağıroğlu, Ali Asram; Üçışık, Mehmet Hikmet; Kırbaş, Oğuz Kaan; Demirel, Erhan; Yurdasiper, Aysu; Şahin, Fikrettin; Özer, ÖzgenAntibody-targeted immunotherapy has emerged in cancer therapies regarding checkpoint inhibition with monoclonal antibodies, such as anti-programmed death-ligand 1 (anti-PD-L1) either given alone or in combination. However, when given alone, it may fail to activate tumor-specific T cells. The combinational therapy of anti-PD-L1 with anti-4-1BB and all-trans retinoic acid (ATRA) has come into prominence due to disease heterogeneity, resulting in the synergistic effects associated with greater T-cell responses. This study introduces anti-PD-L1 and anti-4-1BB-conjugated ATRA-loaded solid lipid nanoparticles (SLNs), where the Design-Expert Program was applied for the optimization. Accordingly, antibody–conjugated ATRA-loaded SLNs had uniform dispersions with mean diameters of 179.6 ± 12.6 nm. The formulations achieved the encapsulation efficiency (EE %) of ATRA at 21.2 ± 1.4 %, regarding the three-dimensional response surface graph. The binding efficiency of anti-4-1BB and anti-PD-L1 antibodies were determined as 85.59 ± 7.3 % and 90.02 ± 5.4 %, respectively. The release profile of formulations indicated the biphasic release of ATRA (ie., 76 ± 4.4 %) from SLNs within 24 h via the Higuchi model. Particle size distributions of SLNs displayed a 7 % increase (i.e., 190.5 ± 7.63 nm) at 4 °C over 2 months. The experimental design of anti-PD-L1- and anti-4-1BB-conjugated- ATRA-loaded SLNs highlighted the promising strategy for the development of alternative formulations and the potential approach for further cancer therapies.Öğe Nanoparticle-mediated histotripsy (NMH) using perfluorohexane 'nanocones'(Institute of Physics Publishing, 2019) Khirallah, Jennifer; Schmieley, Rebecca; Demirel, Erhan; Ur Rehman, Tanzeel; Howell, Justin; Yüksel Durmaz, Yasemin; Vlaisavljevich, EliNanoparticle-mediated histotripsy (NMH) is an ultrasound treatment strategy that combines acoustically sensitive nanoparticles with histotripsy. Previous NMH studies using perfluorocarbon (PFC) nanodroplets (ND's), ?200 nm in diameter, demonstrated that NMH can selectively generate cavitation by reducing the cavitation threshold from ?25-30 MPa to ?10-15 MPa. Recent studies have also shown that cavitation nucleation in NMH is directly caused by the incident negative pressure (p-) exposed to the PFC, as predicted by classical nucleation theory (CNT), suggesting that the NMH cavitation threshold is dependent on the total volume of PFC present in the focal region. In this study, we investigate the use of a newly developed NMH nanoparticle synthesized using an inclusion complex of methylated ?-cyclodextrin and perfluorohexane (PFH). These 'nanocones' (NCs) have advantages compared to previously used ND's due to their smaller size (?50 nm), simple synthesis method, higher stability and information of definite PFH amount carried by the NC. To test the hypothesis that NCs can reduce the NMH cavitation threshold similar to ND's, and that the NMH cavitation threshold is dependent upon the total PFH concentration, tissue phantoms containing concentrations of NCs ranging from 10-5 to 10-10 (ml PFH/ml water) were exposed to single cycle ultrasound pulses using a 500 kHz focused transducer where high speed imaging captured cavitation data. Results showed that NCs significantly reduced the histotripsy cavitation threshold to 11.0 MPa for a concentration of 10-5 (ml PFH/ml water), with the threshold increasing at lower concentrations. Finally, the ability of NCs to be used for effective NMH ablation was demonstrated in tissue phantoms containing red blood cells (RBCs). Overall, the results of the study support our hypotheses that NCs can be used for effective NMH therapy and that NC concentration has a predictable threshold-reducing effect.Öğe PEGylated reduced graphene oxide as nanoplatform for targeted gene and drug delivery(Elsevier Ltd, 2023) Demirel, Erhan; Yüksel Durmaz, YaseminCombination therapies involving anticancer drugs and gene therapy agents have become a potential treatment for different cancer types reducing the side effects of anticancer drugs while enhancing their effectiveness. Here, we improved our previously published strategy that uses multiple pyrene groups of poly(ethylene glycol) (PEG) brushed graft copolymer to enhance the biological use of reduced graphene oxide (rGO) and introduced an azide containing monomer in the copolymer for further modifications to carry hydrophobic drugs like doxorubicin (Dox) along with oligonucleotides such as small interfering RNAs (siRNA). Azide functionality on the side chain of coated copolymer enables not only the conjugation of alkyne end group functional cationic copolymer but also allows the addition of targeting moieties like EPPT1 peptide that selectively binds MUC1, an early hallmark of tumorigenesis. To achieve the outlined carrier, first, a well-defined PEG, pyrene, azide, and methyl methacrylate containing poly[(poly(ethylene glycol) methyl ether methacrylate)-co-(3-azidopropyl methacrylate)-co-(methyl methacrylate)-co-(1-pyrenemethyl methacrylate)] (P(PEGMA-co-AzPMA-co-MMA-co-PMA), PAMP) copolymer was synthesized via ATRP and through its azide group another well-defined, cationic copolymer composed of hexyl methacrylate, 2-dimethyl amino ethyl methacrylate, and N,N,N-trimethylaminoethyl methacrylate iodide (P(HMA-co-DMAEMA-co-TMAEMA) was coupled via copper-catalyzed azide-alkyne cycloaddition (CuAAC). This cationic copolymer was synthesized using a unique ATRP initiator to allow click reaction as described as well as pH-dependent degradation through its hydrazone linkage. The obtained graft copolymer (PAMP-CP) was then coated on a graphene oxide (GO) surface and GO was in situ reduced to rGO (PAMP-CP-rGO) to have an rGO-based PEGylated nanoplatform that can be highly loaded with doxorubicin and successfully condensed with anti-GAPDH siRNA. Consequently, this dual drug delivery nanoplatform showed a controlled drug release that switches between release kinetics by the change of pH and high transfection efficiency regarding targeted GAPDH protein levels. Moreover, in vitro cytotoxicity tests showed that EPPT1-targeted Dox-loaded PAMP-CP-rGO shows concentration-dependent and selective cellular toxicity against cancer cells compared to its non-targeted versions. These results demonstrated the potential of a highly adaptable, multifunctional nanoplatform for safely and selectively transporting different cargoes to the tumor site.Öğe The development of multifunctional nanoplatforms for targeted dual drug delivery(İstanbul Medipol Üniversitesi, Fen Bilimleri Enstitüsü, 2022) Demirel, Erhan; Yüksel Durmaz, YaseminThe research on the use of graphene (G) in biological applications as a drug and gene carrier has increased exponentially simply due to its superior properties as a nanomaterial. On the other hand, many research studies have shown graphene and its derivatives graphene oxide (GO) and reduced graphene oxide (rGO) show cellular cytotoxicity with different mechanisms and emerge concerns regarding their use in biological applications. The modification of the surface with biocompatible polymers is a promising approach to overcome drawbacks that come from poor solubility or surface chemistry of graphene derivatives, where poly(ethylene glycol) (PEG) has been extensively used. In this PhD study, experimental studies were initiated by assessing the potential use of rGO as a platform for the use of 2D graphene-based nanomaterial for biomedical applications, with this goal poly[(poly(ethylene glycol) methyl ether methacrylate)-co-(methyl methacrylate)-co-(pyrene methacrylate)] (P(PEGMA-co-MMA-co-PMA), PMP) copolymer with different PEG length and polymer composition were prepared by using ATRP, introduced to GO surface via ?-? interactions through multiple pyrene units on the copolymer. PMP-rGO nanoplatforms were characterized chemically in many respects to understand the physicochemical properties of the composites, and they were compared to each other based on their stability in aqueous environments and their biocompatibility to select the most favorable outcome. In the first stage of the study, PEGylation method was perfected, produced nanomaterials were tested for their stability, hemolytic activity, cellular cytotoxicity and uptake, and the most favorable results were obtained from 3 pyrene units and 86% PEG containing copolymer coated rGO with a 71.2% coating efficiency. Taking into account the successful initiation, in the second phase of the study selected copolymer composition was enriched with the incorporation of an azide functional monomer on the side chain to enable easily conducted modifications by CuAAC reaction to introduce new functionalities to the system such as active targeting agents or versatile copolymers. A cationic charge carrying copolymer was synthesized by ATRP using a first-time reported ATRP initiator that has alkyne and hydrazone bond on its structure and grafted onto PAMP copolymer via CuAAC to produce poly[(poly(ethylene glycol) methyl ether methacrylate)-co-(3-azidopropyl methacrylate)-co-(methyl methacrylate)-co-(1-pyrenemethyl methacrylate)]-g-poly(hexyl methacrylate-co-2-(dimethylamino) ethyl methacrylate-co-N,N,N-trimethylaminoethyl methacrylate iodide) (PAMP-g-CP). Targeted, dual gene and drug delivery nano-platform was constructed by taking advantage of modular functionalization techniques that were utilized during this study, GO was coated with PAMP-g-CP, followed by EPPT1 conjugation, Dox loading, and siRNA complexation resulting in EPPT1-PAMP-CP-rGO-Dox-siRNA. The final nanoplatform and its derivative's physicochemical properties were thoroughly characterized, and its in vitro and in vivo applications and effectiveness were studied. The resulting graphene derivative could load high rates of doxorubicin and successfully conjugated anti-GAPDH and anti-Bcl-2 siRNA, consequently showing a controlled drug release that switches between release kinetics by the change of pH. Moreover, the in vitro cytotoxicity test of PAMP-CP-rGO's Dox loaded and targeted derivatives show concentration-dependent, selective cellular toxicity than non-targeted versions. Efficacy of EPPT1 targeting was tested by cellular uptake experiments using confocal microscopy and flow cytometric analysis and high uptake rates were detected, additionally, EPPT1-PAMP-CP-rGO-Dox-siRNA was located in the acidic compartments of MCF-7 cells by Lyso Tracker® Green DND-26 staining. The final nanoplatform induced high transfection and knockdown efficiency regarding targeted GAPDH protein and Bcl-2 protein levels as well as Bcl-2 coding mRNA levels. Anti-cancer effect of the nanoplatforms was studied by a xenograft model on athymic nude mice and significant tumor progress suppression was detected. In the light of these findings, PAMP-CP-rGO nanoparticles show high efficiency in transporting hydrophobic drug molecules and genetic material to the targeted tumor site, and the fact that they treat cancer cells with the combination of two different strategies has provided a modular approach to innovative cancer treatment methods.Öğe The effect of PEGylation components on drug release behavior of targeted reduced graphene oxide based drug delivery system(2024) Başavcı, Ezgi; Demirel, Erhan; Elhassan, Mohamad; Yüksel Durmaz, YaseminReduced graphene oxide (rGO) is a restored, defect-free version of graphene oxide (GO) with regained graphene-like properties but encounters challenges in biomedical applications due to its low solubility. Especially for drug release applications, rGO's stronger ?-? interaction of rGO compared to GO leads to low release, often demanding novel approaches for enhancement. The recently developed PEGylation method utilizes a copolymer with tunable PEG chain length and density for the PEGylation of GO simultaneously reduced to rGO, resulting in water-dispersible and biocompatible rGO-based nanoplatforms. This copolymer integrates functional monomers and enhances treatment options. Herein, we investigated how these PEGylation components and their incorporation order impact rGO's drug release behavior. A series of copolymers, (P(PEGMA-co-AzPMA-co-MMA-co-PMA), with different PEG brushes and azide groups, were synthesized via atom transfer radical polymerization. We explored the impact of ionic azide groups on drug release by comparing the azide-containing and azide-capped copolymers. Moreover, copolymers containing 500 or 2000 Da PEG brushes were compared to assess their role as a coating layer or diffusion barrier on drug release. Doxorubicin, a hydrophobic anticancer agent, was loaded onto the rGO surface before or after peptide-based targeting agent (EPPT1) conjugation. The results showed that the drug release behavior of 500 or 2000 Da PEG brushes containing rGO surfaces are different due to the density of PEG coating which makes the effect of azide groups not always visible. Incorporating the targeting peptide before drug loading was found to be optimal. The pH-dependent release profiles revealed 49 % and 44 % release from the rGO surface for the shorter and longer PEG brushes, respectively. Tuning the PEGylation components may further influence the release behavior.
