Development of functional carrier systems for biotechnological enzyme drugs by using upconverting nanoparticles
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Tarih
2021
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İnönü Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
L-Asparaginaz (LASNaz) Akut Lenfeblast Lösemi (ALL) tedavisi için anahtar kemoterapötik ilaçdır. Polietilen glikol (PEG) ile modifiye edilmiş L-ASNaz formu, hastalarda bağışıklık tepkisini en aza indirmek için kullanılmaktadır. Bununla birlikte, bu enzimin kullanımı ve erişilebilirliği, kısa plazma yarı ömrü ve pahalı olması nedeniyle sınırlıdır. Ayrıca enzim immobilizasyonu sonrası meydana gelen enzim aktivitie kayıplarını ortadan kaldıran yeni taşıyıcı sistemlere ihtiyaç mevcuttur. Tez kapsamında klinikte kullanılan en önemli enzim ilaçlardan biri olan L-ASNaz için biyouyumlu ve fonksiyonel uç gruba sahip UCNP'lerin hazırlanması, PEG-L-ASNaz enziminin immobilizasyonu, NIR etkisi ile aktivitesinin tetiklenerek arttırılması ve mekanizmasına yönelik çalışmalar gerçekleştirilmiştir. Ayrıca hazırlanan immobilize sistemin in vitro biyouyumluluk özellikleri de belirlenmiştir. Çalışmada öncelikle 980 nm'de indüklenenen NaYF4:Yb3+, Er3+ ve 808 nm'de indüklenenen NaYF4:Nd3+, Yb3+, Er3+ UCNP'leri hidrotermal yöntemle sentezlenmiş ve yapısal ve termal karakterizasyonları sırasıyla XRD, DLS, Zeta-metre, TEM, FTIR, TGA, XPS ve floresans spektrometresi ile belirlenmiştir. PEG-L-ASNaz enziminin immobilizasyonu, PEI, GPTMS ve ICPTES ile modifiye nanopartiküller üzerinde fiziksel (elektrostatik) ve kimyasal (kovalent) yöntemler kullanılarak gerçekleştirilmiştir. Daha sonra, enzim içeren taşıyıcı platformlar için NIR tetikleme parametreleri olarak lazer yoğunluğu, maruz kalma süresi ve mesafe çalışmaları gerçekleştirilmiştir. İmmobilizasyon parametreleri (immobilizasyon verimliliği, optimum pH, sıcaklık, termal stabilite, yeniden kullanılabilirlik, in vitro yarılanma ömrü, depolama stabilitesi, tripsin sindirimi vb.) detaylı olarak incelenmiş ve serbest enzimle karşılaştırılmıştır. UCNP'ler ve UCNP-PEG-L-ASNazlar için in vitro toksisite çalışmaları, L-929 hücre hattı üzerinde gerçekleştirilmiştir. Sonuç olarak PEG-L-ASNaz, bu tez kapsamında ilk kez 980 ve 808 nm'de indüklenebilen UCNP'lere immobilize edilmiş ve enzimin NIR ile indüklenebileceği gösterilmiştir. NIR ile L-ASNaz enzim aktivitesinin indüksiyon oranlarında yaklaşık % 547'ye ulaşılarak mevcut sistemlerden daha iyi bir taşıyıcı sistem (NaYF4: Nd3+, Yb3+, Er3+/ICPTES) geliştirilmiştir. Ek olarak, bu taşıyıcı sistemin insanlar için toksik olmaması nedeniyle biyoteknolojik enzim ilaçları için umut verici bir sistem gibi görünmektedir.
L-Asparaginase (LASNase) is the key chemotherapeutic drug for the treatment of Acute Lymphatic Leukemia (ALL). The L-ASNase form modified with polyethylene glycol (PEG) is used to minimize the immune response in patients. However, the use and accessibility of this enzyme are limited due to its short plasma half-life and expensive. In addition, there is a need for new carrier systems that eliminate the loss of enzyme activity after enzyme immobilization. Within the scope of the thesis, for L-ASNase, one of the most important enzyme drugs used in the clinic, the preparation of biocompatible and functional end group UCNPs immobilization of the PEG-L-ASNase enzyme, triggering its activity by NIR effect and its mechanism was carried out. In addition, the in vitro biocompatibility properties of the prepared immobilized system were determined. In this study, NaYF4: Yb3+, Er3+ induced at 980 nm and NaYF4: Nd3+ Yb3+, Er3+, induced at 808 nm UCNPs were firstly synthesized by hydrothermal method, and their structural and thermal characterizations were performed with XRD, DLS, Zeta-meter, TEM, FTIR, TGA, XPS and fluorescence spectrometry. Immobilization of PEG-L-ASNase on UCNP modified with PEI, GPTMS, and ICPTES was carried out using physical (electrostatic) and chemical (covalent) methods. Later intensity, exposure time, and laser distance studies were performed as NIR triggering parameters for carrier platforms containing enzymes. Immobilization parameters (immobilization efficiency, optimum pH, temperature, thermal stability, reusability, in vitro half-life, storage stability, trypsin digestion, etc.) were examined in detail and compared with the free enzyme. In vitro toxicity studies for UCNPs and UCNP-PEG-L-ASNases were performed on the L-929 cell line. In the conclusion, PEG-L-ASNaz was immobilized to UCNPs that can be induced at 980 and 808 nm for the first time within the scope of this thesis, and it has been shown that the enzyme can be induced by NIR. A better carrier system (NaYF4:Nd3+, Yb3+, Er3+/ICPTES) than existing systems has been developed by reaching approximately 547% in the induction rates of L-ASNase enzyme activity with NIR. In addition, the fact that this carrier system appears to be a promising system for biotechnological enzyme drugs due to no toxic to humans.
L-Asparaginase (LASNase) is the key chemotherapeutic drug for the treatment of Acute Lymphatic Leukemia (ALL). The L-ASNase form modified with polyethylene glycol (PEG) is used to minimize the immune response in patients. However, the use and accessibility of this enzyme are limited due to its short plasma half-life and expensive. In addition, there is a need for new carrier systems that eliminate the loss of enzyme activity after enzyme immobilization. Within the scope of the thesis, for L-ASNase, one of the most important enzyme drugs used in the clinic, the preparation of biocompatible and functional end group UCNPs immobilization of the PEG-L-ASNase enzyme, triggering its activity by NIR effect and its mechanism was carried out. In addition, the in vitro biocompatibility properties of the prepared immobilized system were determined. In this study, NaYF4: Yb3+, Er3+ induced at 980 nm and NaYF4: Nd3+ Yb3+, Er3+, induced at 808 nm UCNPs were firstly synthesized by hydrothermal method, and their structural and thermal characterizations were performed with XRD, DLS, Zeta-meter, TEM, FTIR, TGA, XPS and fluorescence spectrometry. Immobilization of PEG-L-ASNase on UCNP modified with PEI, GPTMS, and ICPTES was carried out using physical (electrostatic) and chemical (covalent) methods. Later intensity, exposure time, and laser distance studies were performed as NIR triggering parameters for carrier platforms containing enzymes. Immobilization parameters (immobilization efficiency, optimum pH, temperature, thermal stability, reusability, in vitro half-life, storage stability, trypsin digestion, etc.) were examined in detail and compared with the free enzyme. In vitro toxicity studies for UCNPs and UCNP-PEG-L-ASNases were performed on the L-929 cell line. In the conclusion, PEG-L-ASNaz was immobilized to UCNPs that can be induced at 980 and 808 nm for the first time within the scope of this thesis, and it has been shown that the enzyme can be induced by NIR. A better carrier system (NaYF4:Nd3+, Yb3+, Er3+/ICPTES) than existing systems has been developed by reaching approximately 547% in the induction rates of L-ASNase enzyme activity with NIR. In addition, the fact that this carrier system appears to be a promising system for biotechnological enzyme drugs due to no toxic to humans.
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ABBAS ALI NOMA, S.(2021). Development of functional carrier systems for biotechnological enzyme drugs by using upconverting nanoparticles,İnönü Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı.
