Browse

Synthesis and Characterization of 30Kc19-based Protein Nanoparticles for the Application to Drug Delivery
약물 전달에 응용하기 위한 30Kc19를 이용한 단백질 나노입자의 제조 및 특성 연구

DC Field Value Language
dc.contributor.advisor박태현-
dc.contributor.author이홍재-
dc.date.accessioned2017-07-13T08:38:44Z-
dc.date.available2017-07-13T08:38:44Z-
dc.date.issued2015-02-
dc.identifier.other000000024824-
dc.identifier.urihttps://hdl.handle.net/10371/119724-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2015. 2. 박태현.-
dc.description.abstract30Kc19 protein is a member of the 30K protein family from silkworm, having molecular weights of around 30 kDa. 30Kc19 protein is the most abundant among 30K proteins in the hemolymph. In previous studies, 30K proteins exhibited anti-apoptotic effect in various cells by gene expression or addition of 30K proteins in recombinant form produced from Escherichia coli. 30Kc19 also enhanced productivity and glycosylation by expression of a 30Kc19 gene or supplementation with a recombinant 30Kc19 protein. Additionally, 30Kc19 exhibited enzyme-stabilizing and cell-penetrating abilities in vitro.
In this study, it was hypothesized that supplemented 30Kc19 penetrated into the cell and enhanced the stability of the cellular enzyme, and investigated this using in vitro and cellular assessments. The activity of isolated mitochondrial complex I / III was enhanced with 30Kc19 in dose-dependent manner while initial reaction rate was unchanged, suggesting that 30Kc19 enhanced enzyme stability rather than specific activity. For intracellular enzyme activity assessment, mitochondrial complex II activity in HeLa cells increased more than 50% with 30Kc19. The enhanced mitochondrial complex activity increased mitochondrial membrane potential and ATP production in HeLa cells with 30Kc19, by over 50%.
Then cell penetrating and enzyme stabilizing effect of 30Kc19 was exploited to efficient drug delivery. 30Kc19 and HSA were used as building block of protein nanoparticles to exploit both beneficial effect of 30Kc19 protein stability of HSA nanoparticles. 30Kc19-HSA nanoparticles were successfully prepared using the desolvation method, with uniform spherical morphology and stable dispersion. 30Kc19-HSA nanoparticles showed negligible toxicity when treated to cells, and 30Kc19-HSA nanoparticles also exhibited increase in cellular uptake compared with HSA nanoparticles.
Because stable 30Kc19-HSA nanoparticles were successfully synthesized and characterized, nanoparticles loaded with model enzyme cargo to investigated effect of 30Kc19 on cargo enzyme. 30Kc19-HSA nanoparticles loaded with β-galactosidase were uniformly spherical in shape, dispersed evenly in phosphate buffered saline and cell culture media, and released β-galactosidase in a sustained manner. The 30Kc19-HSA nanoparticles had negligible toxicity to animal cells and exhibited enhanced cellular uptake and intracellular stability of β-galactosidase in HeLa and HEK293 cells when compared with those of HSA nanoparticles. These results suggest that 30Kc19-HSA protein nanoparticles could be used as a versatile tool for drug delivery to various cells.
Next, 30kc19-HSA nanoparticles were used to deliver actual therapeutic protein to cells. Fabry disease is a genetic lysosomal storage disease caused by deficiency of α-galactosidase, the enzyme that degrades neutral glycosphingolipids transported to lysosomes. Enzyme replacement therapy (ERT) using recombinant α-galactosidase is the only treatment available for Fabry disease. Because enhancing cellular delivery and enzyme stability is a challenge of ERT using α-galactosidase to maximize treatment efficacy, 30Kc19-HSA protein nanoparticles were used to enhance delivery and intracellular α-galactosidase stability. The 30Kc19-HSA nanoparticles had a uniform spherical shape and were well-dispersed. The 30Kc19-HSA nanoparticles had negligible toxicity to human cells. The nanoparticles exhibited enhanced cellular uptake and intracellular stability of the delivered α-galactosidase in human foreskin fibroblasts. Additionally, the nanoparticles enhanced globotriaosylceramide degradation in fibroblasts from a patient with Fabry disease. It is expected that 30Kc19-HSA protein nanoparticles will be used as an effective tool for efficient delivery and enhanced stability of drugs.
-
dc.description.tableofcontentsContents

Chapter 1. Research background and objectives 1
Chapter 2. Literature review 5
2.1 Nanoparticles for drug delivery 6
2.1.1 Terminology of nanoparticles 6
2.1.2 Classification of nanoparticles 7
2.2 Albumin nanoparticles 10
2.2.1 Albumins for nanoparticle production 10
2.2.2 Preparation of albumin nanoparticles 12
2.2.3 Surface modification of albumin nanoparticles 14
2.2.4 Uptake of albumin nanoparticles 18
Chapter 3. Experimental procedures 20
3.1 Production of recombinant 30Kc19 protein 21
3.2 Cell culture 21
3.3 Mitochondria isolation 22
3.4 Western blotting 22
3.5 Mitochondrial activity assay 22
3.6 Estimation of mitochondrial membrane potential and ATP generation 23
3.7 Preparation of nanoparticles 24
3.8 Size and zeta potential of the nanoparticles 24
3.9 Scanning electron microscopy (SEM) analysis of the nanoparticles 25
3.10 β-galactosidase loading efficiency of the nanoparticles 25
3.11 In vitro β-galactosidase release from nanoparticles 26
3.12 Cell viability assay 27
3.13 Cellular uptake of nanoparticles 27
3.14 Protein cargo activity of nanoparticle-treated cells 29
3.15 Globotriaosylceramide degradation 30
3.16 Statistical analysis 31
Chapter 4. Stabilization of cellular mitochondrial enzyme complex activity through supplementation of 30Kc19 protein 32
4.1 Introduction 33
4.2 Effect of 30Kc19 on in vitro mitochondrial enzyme stability 34
4.3 Intracellular and mitochondrial uptake of 30Kc19 35
4.4 Effect of 30Kc19 on intracellular mitochondrial enzyme stability 38
4.5 Effect of 30Kc19 on mitochondrial membrane potential and ATP generation 40
4.6 Conclusions 40
Chapter 5. Synthesis of protein nanoparticles using 30Kc19 protein and human serum albumin 42
5.1 Introduction 43
5.2 Preparation and characterization of 30K-HSA nanoparticles 45
5.3 Cellular toxicity of 30Kc19-HSA nanoparticles 49
5.4 Cellular uptake of 30Kc19-HSA nanoparticles 49
5.5 Conclusions 51
Chapter 6. Protein nanoparticles for protein cargo delivery using 30Kc19 protein and human serum albumin 53
6.1 Introduction 54
6.2 Characterization of β-gal-loaded 30Kc19-HSA nanoparticles 55
6.3 β-Galactosidase loading and in vitro release of 30Kc19-HSA nanoparticles 59
6.4 Cellular toxicity of 30Kc19-HSA nanoparticles loaded with β-Galactosidase 62
6.5 Cellular uptake of 30Kc19-HSA nanoparticles loaded with β-Galactosidase 64
6.6 Intracellular β-galactosidase activity delivered by nanoparticles 66
6.7 Conclusions 69
Chapter 7. Protein nanoparticles for therapeutic protein delivery using 30Kc19 protein and human serum albumin 70
7.1 Introduction 71
7.2 Production and characterization of 30Kc19-HSA nanoparticles 72
7.3 Cellular toxicity of 30Kc19-HSA nanoparticles 76
7.4 Cellular uptake of nanoparticles 76
7.5 Intracellular α-galactosidase activity delivered by nanoparticles 78
7.6 Globotriaosylceramide degradation activity in nanoparticle-treated Fabry disease fibroblasts 80
7.7 Conclusions 84
Chapter 8. Overall discussion and further suggestions 85
8.1 Overall discussion 86
8.2 Conclusion and further suggestions 92
References 96
국 문 초 록 110
-
dc.formatapplication/pdf-
dc.format.extent3222815 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subject약물 전달-
dc.subject.ddc660-
dc.titleSynthesis and Characterization of 30Kc19-based Protein Nanoparticles for the Application to Drug Delivery-
dc.title.alternative약물 전달에 응용하기 위한 30Kc19를 이용한 단백질 나노입자의 제조 및 특성 연구-
dc.typeThesis-
dc.contributor.AlternativeAuthorHong Jai Lee-
dc.description.degreeDoctor-
dc.citation.pagesviii, 112-
dc.contributor.affiliation공과대학 화학생물공학부-
dc.date.awarded2015-02-
Appears in Collections:
College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Theses (Ph.D. / Sc.D._화학생물공학부)
Files in This Item:
  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse