Publication: SARS-CoV-2 Spike proteini içeren kitozan yapılı intranazal alt birim aşı formülasyonlarının geliştirilmesi ve in vitro-ın vivo değerlendirilmesi
Abstract
Amaç: Biyouyumlu, biyoparçalanabilir ve toksisite açısından güvenli olduğu bilinen kitozan polimerinden elde edilen nanopartiküller, bağışıklık sistemini uyarıcı ve mukoadezif özelliklerinin de etkisiyle aşı araştırmalarında dikkat çekmektedir. Bu çalışmada kitozan nanopartiküllerinin optimize edilmesi, intranazal yolla uygulanabilen spike proteini S1 ünitesini içeren kitozan yapılı nanopartiküler bir alt birim aşısı formüle edilmesi ve bu aşının in vitro ve in vivo çalışmalarla COVID-19 hastalığına karşı etkililiğinin ve güvenliğinin kanıtlanması amaçlanmıştır. Gereç ve Yöntem: Çalışmada, iyonik jelasyon yöntemiyle önce BSA içermeyen, ardından BSA içeren kitozan nanopartikül formülasyonları hazırlanmıştır. Optimum parametreler belirlenerek spike proteini S1 ünitesini içeren nanopartiküller üretilmiştir. Partikül büyüklüğü, zeta potansiyeli, morfoloji (SEM), liyofilizasyon etkisi, stabilite testleri, protein salım çalışmaları ve Franz hücresinde geçiş gibi in vitro analizler yapılmıştır. Toksisite, A549 hücrelerinde MTT yöntemiyle değerlendirilmiş, in vivo immün yanıt ise BALB/ c farelerde ELISA ile proteine spesifik IgG, IgA, total IFN-γ ve IL-6 seviyeleri ölçülerek incelenmiştir. Bulgular: pH 5,0 kitozan çözeltisi ve 3:1 kitozan:TPP oranıyla hazırlanan F3 formülasyonu ve aynı parametrelerle hazırlanan BSA yüklü formülasyon olan BNP7 formülasyonu, ön formülasyonlar arasında optimum formülasyonlar olarak seçilmiştir. Ardından aynı parametrelerle spike proteini S1 ünitesi içeren SNP formülasyonu elde edilmiştir. 249,5 nm boyut, 0,45 PDI ve +27,1 mV zeta potansiyeline sahip SNP nanopartikülleri, morfolojik olarak SEM ile, enkapsüle edilen S1 ünitesinin stabilitesi ise Western melezleme yöntemiyle doğrulanmıştır. Nanopartiküller, 4. günde S1 ünitesinin %80’ini pH 6,5 fosfat tamponu ortamına salarken, 48 saatte proteinin %50’den fazlası selüloz membranı geçmiştir. A549 hücrelerinde toksisite göstermeyen nanopartiküllerin, in vivo olarak IgG ve IgA seviyelerini artırdığı, antienflamatuvar etkisiyle IFN-γ ve IL-6 seviyelerini azalttığı kanıtlanmıştır. Sonuç: Bu çalışmada çeşitli optimizasyon ve karakterizasyon çalışmaları sonucunda SARS- CoV-2 proteini S1 ünitesi içeren SNP formülasyonu geliştirilmiş, karakterize edilmiş ve aşı olarak SARS-CoV-2 virüsüne karşı etkinliği in vivo çalışmalar ile kanıtlanmıştır.
Objective: Nanoparticles obtained from chitosan polymer, which is known to be biocompatible, biodegradable and safe in terms of toxicity, attract attention in vaccine research due to their immunostimulatory and mucoadhesive properties. This study aimed to optimize chitosan nanoparticles, formulate a chitosan-structured nanoparticle subunit vaccine containing the spike protein S1 unit that can be administered intranasally, and prove the efficacy and safety of this vaccine against COVID-19 disease with in vitro and in vivo studies. Materials and Methods: In the study, protein-free and then BSA-containing chitosan nanoparticle formulations were prepared by ionic gelation method. Accordingly, nanoparticles containing the spike protein S1 unit were produced with optimum parameters. In vitro analyses such as particle size, zeta potential, morphology (SEM), lyophilization effect, stability tests, protein release studies and permeability in Franz cells were performed. Toxicity was evaluated by MTT method in A549 cells. In vivo immune response was examined by measuring protein- specific IgG, IgA, total IFN-γ and IL-6 levels in BALB/ c mice using ELISA. Results: The F3 formulation prepared with pH 5.0 chitosan solution and 3:1 chitosan:TPP ratio and the BNP7 formulation, BSA-loaded formulation prepared with the same parameters, were selected as the optimum formulations. Then, the SNP formulation containing the spike protein S1 unit was obtained with the same parameters. SNP nanoparticles with a size of 249.5 nm, a PDI of 0.45 and a zeta potential of +27.1 mV were morphologically confirmed by SEM images and Western blotting for the stability of the S1 unit. While the nanoparticles released 80% of the S1 unit into the pH 6.5 phosphate buffer medium on day 4, more than 50% of the protein passed the cellulose membrane in 48 hours. Nanoparticles that did not show toxicity in A549 cells were also proven to increase protein specific IgG and IgA levels in vivo and reduce IFN- γ and IL-6 levels with their anti-inflammatory effects arising from chitosan. Conclusion: In this study, after various optimization and characterization studies, a SNP formulation containing the SARS-CoV-2 protein S1 unit was developed, characterized and its effectiveness against the SARS-CoV-2 virus as a vaccine was proven with in vivo studies.
Objective: Nanoparticles obtained from chitosan polymer, which is known to be biocompatible, biodegradable and safe in terms of toxicity, attract attention in vaccine research due to their immunostimulatory and mucoadhesive properties. This study aimed to optimize chitosan nanoparticles, formulate a chitosan-structured nanoparticle subunit vaccine containing the spike protein S1 unit that can be administered intranasally, and prove the efficacy and safety of this vaccine against COVID-19 disease with in vitro and in vivo studies. Materials and Methods: In the study, protein-free and then BSA-containing chitosan nanoparticle formulations were prepared by ionic gelation method. Accordingly, nanoparticles containing the spike protein S1 unit were produced with optimum parameters. In vitro analyses such as particle size, zeta potential, morphology (SEM), lyophilization effect, stability tests, protein release studies and permeability in Franz cells were performed. Toxicity was evaluated by MTT method in A549 cells. In vivo immune response was examined by measuring protein- specific IgG, IgA, total IFN-γ and IL-6 levels in BALB/ c mice using ELISA. Results: The F3 formulation prepared with pH 5.0 chitosan solution and 3:1 chitosan:TPP ratio and the BNP7 formulation, BSA-loaded formulation prepared with the same parameters, were selected as the optimum formulations. Then, the SNP formulation containing the spike protein S1 unit was obtained with the same parameters. SNP nanoparticles with a size of 249.5 nm, a PDI of 0.45 and a zeta potential of +27.1 mV were morphologically confirmed by SEM images and Western blotting for the stability of the S1 unit. While the nanoparticles released 80% of the S1 unit into the pH 6.5 phosphate buffer medium on day 4, more than 50% of the protein passed the cellulose membrane in 48 hours. Nanoparticles that did not show toxicity in A549 cells were also proven to increase protein specific IgG and IgA levels in vivo and reduce IFN- γ and IL-6 levels with their anti-inflammatory effects arising from chitosan. Conclusion: In this study, after various optimization and characterization studies, a SNP formulation containing the SARS-CoV-2 protein S1 unit was developed, characterized and its effectiveness against the SARS-CoV-2 virus as a vaccine was proven with in vivo studies.