Mikrobiyal altın nanopartikül (AuNP) sentezinin optimizasyonu, karakterizasyonu ve adjuvan olarak kullanım potansiyelinin değerlendirilmesi
Küçük Resim Yok
Dosyalar
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Altın nanopartiküller (AuNP) yüzey plazmon rezonansları (SPR), kolay sentezlenebilmeleri, küçük parçacık boyutu ile geniş yüzey-hacim oranına sahip olmaları, yüzeylerine bağlanabilen fonksiyonel gruplar ile kolay modifikasyonları ve biyouyumlu yapıları sayesinde tıp, biyoteknoloji ve medikal alanda, görüntüleme, tanı ve tedavi uygulamalarında yaygın şekilde kullanılmakta ve araştırılmaktadır. Aşı teknolojisinde daha etkin ve güvenli adjuvan ve antijen taşıma sistemi konusundaki çalışmalarda nanopartiküllerin artan kullanımıyla beraber AuNP'ler de ilgi odağı haline gelmiştir. AuNP'ler fiziksel, kimyasal veya biyolojik sentez ile farklı boyut, şekil ve yüzey özelliklerinde üretilebilmektedir. Diğer yöntemlere göre iyi bir alternatif olan biyolojik sentezde (biyojenik) metabolitler kullanılarak Au+3 iyonlarının Au0'a indirgenmesi gerçekleşir.
Bu tez çalışmasında 30 aktinomiset (Actinobacteria) üyesi, AuNP sentezi açısından tarandı ve 5 aday izolat içerisinden M137-2 suşu ile en küçük boyutlarda AuNP (66,12±1,19 nm) sentezi gerçekleştirildi. Ardından, Streptomyces sp. M137-2 olarak tanımlanan izolat ile ekstrasellüler AuNP sentezinin pH, altın tuzu (HAuCl4) derişimi (M) ve inkübasyon süresi değişkenleri Merkezi Bileşen Tasarımı (CCD) ile optimize edildi. Sentezlenen AuNP'lerin karakterizasyonu [UV-Vis spektroskopisi, taramalı elektron mikroskobu (SEM), Dinamik Işık Saçılımı (DLS), X-Işını Kırınım Difraktometresi (XRD), X-Işını Fotoelektron Spektroskopisi (XPS), Fourier Dönüşümlü-Kızılötesi Spektroskopisi (FT-IR), stabilite] yapıldı ve L929 ile U937 hücreleri üzerinde sitotoksisitesi belirlendi. Daha sonra model antijen olan sığır serum albümin (BSA) ile konjüge edilen mikrobiyal AuNP'ler karakterize edildi ve adjuvan potansiyeli in vitro hücre kültürü denemeleri ile değerlendirildi. Bunun için karakterizasyonun ardından makrofaja farklılaştırılan U937 monosit hücreleri ile TNF-α ve IL-6 salınımı ve hücresel alım çalışmaları yapıldı. Kimyasal sentezlenen AuNP (tAuNP) ve aşı adjuvanı olan alüminyum tuzu (alum) kontrol olarak kullanıldı.
Sonuçta Yanıt yüzeyi metodolojisi (RSM) kullanılarak biyosentezin optimum pH 8, HAuCl4 10-3 M ve inkübasyon süresi 72 saat olarak belirlendi. Seçilen izolatın Streptomyces suşu olduğu tanımlandı ve yüzeyinde 20-25 nm protein koronaya sahip, 30-50 nm boyutunda, monodispers ve oldukça stabil formda küresel AuNP'ler sentezlendi. mAuNP'ler, XRD analizinde altına özgü karakteristik spektrum ve 541 nm'de ortalanmış SPR bandı ile doğrulandı. FT-IR sonuçları AuNP'lerin indirgenmesi ve stabilizasyonu için Streptomyces sp. M137-2 metabolitlerinin rolünü doğruladı. L929 hücreleri ile gerçekleştirilen sitotoksisite testi (MTT yöntemi) mAuNP'lerin tıp alanında güvenle kullanılabileceğini gösterdi. BSA konjugasyonu ile SPR bandında 7 nm kayma oldu, konjugat oluşumu toplam protein tayini ve FT-IR ile karakterize edildi. AuNP–BSA konjugatlarının SPR bantları ve zeta potansiyelleri stabil olduğunu doğruladı. Makrofaja farklılaşan U937 monosit hücreleri üzerinde AuNP-BSA ve alum-BSA konjugatlarının %70'in üzerinde canlılık gösterdiği belirlendi. AuNP-BSA ile indüklenen hücrelerin TNF-α ve IL-6 sitokin seviyeleri, alum-BSA ile indüklenenden sırasıyla 3 kat (p<0.05) ve 2 kat (p<0.05) daha yüksek olduğu belirlendi. AuNP'lerin hücresel alımı ve nükleusların çevresinde lokalize olduğu konfokal mikroskopi ile gösterildi.
Mikrobiyal AuNP'lerin adjuvan özelliği, muhtemelen güçlü inflamatuar yanıtı indüklemesinden ve antijenin makrofaj hücreleri tarafından alımını arttırmasından kaynaklanıyordu. Bu çalışma, mikrobiyal AuNP sentezinin boyut bağımlı istatistiksel optimizasyonunu ve mikrobiyal AuNP'lerin aşı adjuvanı olarak potansiyelini gösteren ilk rapordur.
Gold nanoparticles (AuNP) are widely used and researched in medicine, biotechnology and medical fields, imaging, diagnosis and treatment thanks to their surface plasmon resonance (SPR), easy synthesis, large surface-to-volume ratio due to small particle size and biocompatibility due to functional groups that can be attached to their surfaces. With the increasing use of nanoparticles in vaccine technology, studies on more effective and safe adjuvant and antigen delivery systems, AuNPs have also become the focus of attention. AuNPs can be produced in different sizes, shapes and surface properties by physical, chemical or biological synthesis. In biological synthesis (biogenic), which is a good alternative to other methods, the reduction of Au+3 ions to Au0 occurs by using metabolites. In this study, 30 actinomycete (Actinobacteria) members were screened for AuNP synthesis and the smallest AuNP (66,12±1,19 nm) synthesis was performed with M137-2 strain among 5 selected candidate isolates. Then, pH, gold salt (HAuCl4) concentration (M) and incubation time variables of extracellular AuNP synthesis with the isolate identified as Streptomyces sp. M137-2 were optimized by Central Component Design (CCD). The synthesized AuNPs were characterized [UV-Vis Spectroscopy, Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), X-Ray Diffraction (XRD) and X-Ray Photoelectron Spectroscopy (XPS), Fourier Transform-Infrared Spectroscopy (FT-IR), stability] and their cytotoxicity was determined on L929 and U937 cells. Next, microbial AuNPs conjugated with the model antigen, bovine serum albumin (BSA), were characterized and their adjuvant potential was evaluated by in vitro cell culture experiments. For this, TNF-α and IL-6 release and cellular uptake studies were performed with U937 monocyte cells differentiated into macrophages. Chemically synthesized AuNP (tAuNP) and the vaccine adjuvant aluminum salt (alum) were used as controls. As a result, the optimum pH of biosynthesis was determined as 8, HAuCl4 10-3 M and incubation time 72 hours using Response surface methodology (RSM).The selected isolate was identified as Streptomyces strain and spherical AuNPs were synthesized in monodisperse and highly stable form, 30-50 nm in size, with 20-25 nm protein corona on the surface. mAuNPs were confirmed by XRD analysis with gold-specific characteristic spectrum and SPR band centered at 541 nm. FT-IR results confirmed the role of Streptomyces sp. M137-2 metabolites for the reduction and stabilization of AuNPs. The cytotoxicity test (MTT method) performed with L929 cells showed that mAuNPs can be used safely in the medical field. After mAuNP-BSA conjugation, there was a 7 nm shift in the SPR band, and conjugate formation was characterized by total protein assay and FT-IR analysis. The SPR bands and zeta potentials of the mAuNP–BSA conjugates confirmed that they were stable. It was determined that AuNP-BSA and alum-BSA conjugates showed over 70% viability on U937 monocyte cells that differentiated into macrophages. TNF-α and IL-6 cytokine levels of cells induced by AuNP-BSA were determined to be 3-fold (p<0.05) and 2-fold (p<0.05) higher, respectively, than those induced by alum-BSA. Cellular uptake of AuNPs and localization around nuclei was demonstrated by confocal microscopy. The adjuvant property of mAuNPs was probably due to their induction of potent inflammatory response and increased uptake of antigen by macrophage cells. This study is the first to demonstrate the size-dependent statistical optimization of microbial AuNP synthesis and the potential of microbial AuNPs as vaccine adjuvants.
Gold nanoparticles (AuNP) are widely used and researched in medicine, biotechnology and medical fields, imaging, diagnosis and treatment thanks to their surface plasmon resonance (SPR), easy synthesis, large surface-to-volume ratio due to small particle size and biocompatibility due to functional groups that can be attached to their surfaces. With the increasing use of nanoparticles in vaccine technology, studies on more effective and safe adjuvant and antigen delivery systems, AuNPs have also become the focus of attention. AuNPs can be produced in different sizes, shapes and surface properties by physical, chemical or biological synthesis. In biological synthesis (biogenic), which is a good alternative to other methods, the reduction of Au+3 ions to Au0 occurs by using metabolites. In this study, 30 actinomycete (Actinobacteria) members were screened for AuNP synthesis and the smallest AuNP (66,12±1,19 nm) synthesis was performed with M137-2 strain among 5 selected candidate isolates. Then, pH, gold salt (HAuCl4) concentration (M) and incubation time variables of extracellular AuNP synthesis with the isolate identified as Streptomyces sp. M137-2 were optimized by Central Component Design (CCD). The synthesized AuNPs were characterized [UV-Vis Spectroscopy, Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), X-Ray Diffraction (XRD) and X-Ray Photoelectron Spectroscopy (XPS), Fourier Transform-Infrared Spectroscopy (FT-IR), stability] and their cytotoxicity was determined on L929 and U937 cells. Next, microbial AuNPs conjugated with the model antigen, bovine serum albumin (BSA), were characterized and their adjuvant potential was evaluated by in vitro cell culture experiments. For this, TNF-α and IL-6 release and cellular uptake studies were performed with U937 monocyte cells differentiated into macrophages. Chemically synthesized AuNP (tAuNP) and the vaccine adjuvant aluminum salt (alum) were used as controls. As a result, the optimum pH of biosynthesis was determined as 8, HAuCl4 10-3 M and incubation time 72 hours using Response surface methodology (RSM).The selected isolate was identified as Streptomyces strain and spherical AuNPs were synthesized in monodisperse and highly stable form, 30-50 nm in size, with 20-25 nm protein corona on the surface. mAuNPs were confirmed by XRD analysis with gold-specific characteristic spectrum and SPR band centered at 541 nm. FT-IR results confirmed the role of Streptomyces sp. M137-2 metabolites for the reduction and stabilization of AuNPs. The cytotoxicity test (MTT method) performed with L929 cells showed that mAuNPs can be used safely in the medical field. After mAuNP-BSA conjugation, there was a 7 nm shift in the SPR band, and conjugate formation was characterized by total protein assay and FT-IR analysis. The SPR bands and zeta potentials of the mAuNP–BSA conjugates confirmed that they were stable. It was determined that AuNP-BSA and alum-BSA conjugates showed over 70% viability on U937 monocyte cells that differentiated into macrophages. TNF-α and IL-6 cytokine levels of cells induced by AuNP-BSA were determined to be 3-fold (p<0.05) and 2-fold (p<0.05) higher, respectively, than those induced by alum-BSA. Cellular uptake of AuNPs and localization around nuclei was demonstrated by confocal microscopy. The adjuvant property of mAuNPs was probably due to their induction of potent inflammatory response and increased uptake of antigen by macrophage cells. This study is the first to demonstrate the size-dependent statistical optimization of microbial AuNP synthesis and the potential of microbial AuNPs as vaccine adjuvants.
Açıklama
Anahtar Kelimeler
Altın Nanopartikül, Aşı Adjuvanı, Yanıt Yüzeyi Metodolojisi, Actinobacteria, Streptomyces, Gold Nanoparticle (AuNP), Vaccine Adjuvant, Response Surface Method, Actinobacteria, Streptomyces
