Exosome Isolation: Is There an Optimal Method with Regard to Diagnosis or Treatment?

Extracellular vesicles (EV) gained considerable interest in recent years as both diagnostic tools and templates for therapeutic applications. EVs carry a number of cell-specific markers which gave researchers the opportunity of employing them as liquid biopsies causing no discomfort to patients. On the other hand, they are very exciting candidates for drug delivery due to their eobiotic origin, physicochemical and size characteristics. Isolation of EVs is performed by several strategies, having advantages and disatvantages over each other. As such, the method of EV isolation and in particular exosome isolation determines the quality and purity of obtained vesicles. In this chapter, extracellular vesicle isolation methods are evaluated with regard to their further use. Methods such as ultracentrifugation with different modifications, size exclusion chromatography, ultrafiltration, affinity and precipitation are compared with respect to the yield efficacy and purity of isolates. Furthermore, the advantages and disadvantages of different methods according to the purpose of use are revealed. Recent progress and remaining challenges in the isolation of EVs with regard to diagnosis and treatment is reviewed and discussed. In order to select the most suitable method researchers should clearly define purity, yield, quantity and quality requirements for exosomes, and consider disadvantages of distinct isolation methods

The relationship between Thiol/disulfide homeostasis and endometrial hyperplasia in patients with abnormal uterine bleeding/

Introduction: The role of oxidative stress and antioxidant capacity in the development of endometrial hyperplasia (EH) is controversial. Aim: The study aimed to evaluate Thiol/disulfide Homeostasis and ischemia modified albumin (IMA) levels in patients with EH without atypia. Materials and Methods: In this prospective case-control study, patients with EH without atypia (HP group) (n=28), patients with nonhyperplasia (proliferative/secretory/irregular proliferative/irregular secretory endometrium) (non-HP group) (n=28), and 28 healthy women (control group) were included. The patient's clinical characteristics, serum Thiol/disulfide parameters, and IMA levels were compared between groups. Results: A total of 84 patients were included in the study. Patients’ mean age, BMI, and mean native thiol (-SH-), total thiol (-SH-+-SS-), disulfide (-SS-), and IMA levels were similar among the three groups. The -SS- /-SH- ratio was higher in the HP group than the non-HP group. -SS- /-SH-+-SS- ratio was higher in the HP group vs. the other two groups. The -SS- /-SH-+-SS ratio was higher in the HP group vs. the non-HP group. -SH-/ -SH-+-SS- ratio was lower in the HP group than in the non-HP group. ET was greater in the HP group than in the non-HP and control groups. ET was also significantly greater in the non-HP group vs. in the control group. -SS-/-SH- ratio was found to be predictive with 64% sensitivity and 68% specificity for EH (area under curve = 0.672, p = 0.01). Conclusion and Suggestions: The dynamic thiol/disulfide balance shifted to the disulfide side in women with endometrial hyperplasia

Glioblastoma multiforme tedavisinde kullanılmak üzere siRNA taşıyıcı sistemlerin geliştirilmesi ve etkinliklerinin değerlendirilmesi

Amaç: Tez kapsamında Glioblastome Multiforme tedavisinde kullanılmak üzere beyine hedefleme özelliğine sahip siRNA taşıyıcı nanopartiküler sistemlerin geliştirilmesi amaçlandı.Gereç ve Yöntem: Mikroemülsiyon dilüsyon tekniği ile elde edilen fonksiyonel katı lipit nanopartiküllerinin dış yüzeyine spesifik olarak tümöre penetre olma özelliği bulunan iRGD peptidi bağlanarak hedeflenebilir nanopartiküller elde edildi. Elde elde edilen nanopartiküllerin karakterizasyonu gerçekleştirildi. Genetik materyal ile kompleks yapabilme, gerektiğinde kompleksten genetik materyali salabilme ve serum nükleazlarına karşı genetik materyali koruyabilme özellikleri incelendi. siRNA-EGFR ve siRNA-PDL1 ile kompleks hale getirilmiş nanopartiküllerin in vitro ve in vivo gen susturma etkinlikleri değerlendirildi. Yüzeyinde iRGD bağlı nanopartiküllerin hedefleme özellikleri in vitro ve in vivo olarak incelendi. Optimum formülasyonun sağ kalım üzerine etkisi intrakranial glioblastoma zenograft modelinde radyasyon varlığında ve yokluğunda değerlendirildi. Bulgular ve Sonuçlar: Tez kapsamında geliştirilen yöntem ile tümör hücrelerine iRGD bağımlı hedefleme özelliği kanıtlanmış, 50 nm’den küçük partikül boyutuna sahip, pozitif yüklü, monodispers nanopartikül sistemleri elde edildi. Optimum formülasyonun genetik materyal ile etkin bir şekilde kompleks yapabildiği ve onu serum nükleazlarından koruyabildiği tespit edildi. Nanopartikülün hedefleme özelliği in vitro ve in vivo olarak gösterildi. İn vivo olarak yapılan çalışmalar ile, geliştirilen genetik materyal taşıyıcı hedeflenebilir formülasyonun sağ kalımı anlamlı derecede uzattığı kanıtlandı (p<0,05).--------------------Aim: The aim of this thesis is to develop brain targeting siRNA delivery systems for the treatment of Glioblastoma Multiforme.Materials and Methods: The functional nanoparticles were obtained by microemulsion dilution method. The İRGD peptide that has spesific tumor penetration ability, was bound to the outer surfacer of nanoparticles. Characterization studies of the obtained nanoparticles were carried out. The complex formation ability of nanoparticles, the release feature of genetic material from the complex and the protection ability against serum nucelases were investigated. The in vitro and in vivo gene silencing efficiencies of siRNA-EGFR and siRNA-PDL1 carriying nanoparticles were evaluated. Targeting properties of iRGD-bound nanoparticles were investigated in vitro and in vivo. The survival benefit of the optimal formulation was assessed in the intracranial glioblastoma zenograft model, with or without radiation.Results and Conclusion: With the method developed in this thesis, positively charged, monodisperse nanoparticle systems with a particle size smaller than 50 nm were obtained. It has been found that the optimal formulation could efficiently complex with genetic material and protect it from serum nucleases. The-iRGD dependent targeted ability of nanoparticles were demonstrated in vitro and in vivo. Finally, in vivo studies which was carried on glioblastoma xenograft model have shown that the developed siRNA-EGFR and siRNA-PD-L1-carrying targetable nanopartarticles extends survival significantly (p <0.05)

Recruitment of solid lipid nanoparticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing

Aim: The CRISPR/Cas9 system is a promising gene-editing tool for various anticancer therapies; however, development of a biocompatible, nonviral and efficient delivery of CRISPR/Cas9 expression systems remains a challenge. Materials & methods: Solid lipid nanoparticles (SLNs) were produced based on pseudo and 3D ternary plots. Obtained SLNs and their complexes with PX458 plasmid DNA were characterized and evaluated in terms of cytotoxicity and transfection efficiency. Results: SLNs were found to be nanosized, monodispersed, stable and nontoxic. Furthermore, they revealed similar transfection efficiency as the positive control. Conclusion: Overall, we have achieved a good SLN basis for CRISPR/Cas9 delivery and have the potential to produce SLNs with targeted anticancer properties by modifying production parameters and components to facilitate translating CRISPR/Cas9 into preclinical studies

Fabrication and Evaluation of Cationic Charged Magnetic NanopArticles for Enhanced Gene Delivery

Magnetofection; represents nucleic acid delivery by using magnetic nanopArticles (MNPs) under the influence of a magnetic field; gives promising results for gene delivery. However, pharmaceutical and biomedical studies in this area are very limited. To meet this need, we aimed to develop an effective magnetic gene delivery system in this study. The in-situ surface coating method was handled to develop cationic charged MNPs. Three different MNP formulations were obtained and investigated in terms of characterization, DNA binding, protection, and transfection ability. According to the results, the obtained MNPs have pArticles under 150 nm with a low PDI (0.3), and positive zeta potential with a spherical shape. The DNA binding and protecting ability from nucleases were shown by agarose gel studies. No significant cytotoxicity was observed on COS-7 cells in the concentration range of 4-20 µL/well. Moreover, transfection studies revealed that the optimal system (GMS-MNP-1) showed significantly higher transfection efficacy comparing the naked plasmid or non-magnetic version of nanopArticle under a magnetic field (p>0.05). Promising results have been obtained with the use of obtained GMS-MNPs in terms of magnetic gene delivery. This work can be extended to in vivo by using disease-specific therapeutic genetic materials

Design of Liposome Formulations for CRISPR/Cas9 Enzyme Immobilization: Evaluation of 5-Alpha-Reductase Enzyme Knockout for Androgenic Disorders

The enzyme steroid type II 5-alpha-reductase (SRD5 alpha 2) is responsible for the conversion of testosterone to dihydrotestosterone (DHT), which is involved in prostate cancer, benign prostatic hyperplasia, and androgenic alopecia. Inhibition of SRD5 alpha 2 activity has been explored and presented as a potential treatment for these conditions, but current drugs have side effects and alternative treatment approaches are needed. The CRISPR/Cas9 system, an innovative gene-editing tool, shows potential for targeting the SRD5 alpha 2 gene knockout as a therapeutic approach. Liposomes have been used for the immobilization and delivery of different proteins, and studies have shown that liposomes can enhance the stability and activity of enzymes. In this study, we provided the immobilization of Cas9 protein by encapsulating it in a novel cationic liposome formulation that carries sgRNA on its outer surface for gene delivery approaches. This novel delivery system has shown promising results in terms of physicochemical properties, stability, cytotoxicity, in vitro cellular uptake, and gene knockout efficiency, together with providing flexibility in sgRNA selection. The optimized final formulations showed an average diameter of 229.1 +/- 3.66 nm, a polydispersity index of 0.089 +/- 0.013, and a zeta potential value of 25.7 +/- 0.87 mV. The encapsulation efficiency of the developed formulations has been revealed as 80.60%. The cellular uptake efficiency was evaluated and measured as 45.6% for the final formulation. Furthermore, the Lipo/Cas9:sgRNA (1.5:1) formulation decreased the relative SRD5 alpha 2 mRNA expression by 29.7% compared to the control group. The results of this study reveal that the liposomal formulation based on enzyme immobilization of Cas9 protein using CRISPR technology, an innovative gene-editing tool for SRD5 alpha 2 suppression, might be an alternative treatment option for prostate cancer or BPH treatment without current drug side effects.This work was supported by Ege University Scientific Research Projects Coordination Unit (project no.: 22487) and The Scientific and Technological Research Council of Turkey (grant no.: SBAG-218S682).T?rkiye Bilimsel ve Teknolojik Arastirma Kurumu [22487]; Ege University Scientific Research Projects Coordination Unit [SBAG-218S682]; Scientific and Technological Research Council of Turke