臺灣博碩士論文加值系統

The basics material properties of nanodiamond (ND) are researched for many years. Nowadays, many groups want to apply this material in biomedical application using its advantages such as low cytotoxicity, intrinsic photoluminescence, sharp characteristic Raman signal, only carbon atoms composition and well-developed synthesis technologies. Due to these advantages, nanodiamond could be a potential material on cancer therapy and targeting drug delivery.
Studying the ND potential for drug delivery, the first problem we would face is nanodiamonds large aggregation. We can observe it in buffer solution (Phosphate buffered saline, PBS as well as cell growth mediums) or blood plasma. In this work we use blood protein albumin (rat serum albumin, RSA) adsorbed on nanodiamonds surface to prevent the ND aggregation. This effect has been analyzed using UV-visible spectrometer to measure RSA adsorption on nanodiamonds. Particle size and -potential are measured using dynamic light scattering analyzer (DLS analyzer) to evaluate the size of aggregates and the surface charge of cND and cND-RSA complexes. If this method to decrease the aggregation is feasible, in the future it could be used in pre-clinical preparation of ND to prevent the side effects which can arise due to large size aggregation.
In bio-medical applications nanodiamonds and their complexes have to be transported by blood circulation to reach the target tissues. If we want to apply any ND complexes in pre-clinical/ clinical studies, the second question we have to face is “does nanodiamonds affect the blood cells, first of all red blood cells (RBC) or not?” In this part of work, we used Raman spectroscopy to measure the oxygenation process of RBC. RBC’s function is to carry or release oxygen, which is bound by protein hemoglobin (Hb) contained in RBC. Hemoglobin conformation depends on oxygenation state and the structure and oxygenation state can be characterized by Raman spectroscopy. We use Raman spectroscopy to estimate the RBC function, using spontaneous oxygenation in the air oxygen-containing atmosphere and purging nitrogen for deoxygenation. Raman spectra were measured every five minutes and the changes of oxygen saturation was calculated using characteristic peaks of oxygenated or deoxygenated state to observe the effects of various treatments on the oxygenation/deoxygenation process.

The basics material properties of nanodiamond (ND) are researched for many years. Nowadays, many groups want to apply this material in biomedical application using its advantages such as low cytotoxicity, intrinsic photoluminescence, sharp characteristic Raman signal, only carbon atoms composition and well-developed synthesis technologies. Due to these advantages, nanodiamond could be a potential material on cancer therapy and targeting drug delivery.
Studying the ND potential for drug delivery, the first problem we would face is nanodiamonds large aggregation. We can observe it in buffer solution (Phosphate buffered saline, PBS as well as cell growth mediums) or blood plasma. In this work we use blood protein albumin (rat serum albumin, RSA) adsorbed on nanodiamonds surface to prevent the ND aggregation. This effect has been analyzed using UV-visible spectrometer to measure RSA adsorption on nanodiamonds. Particle size and -potential are measured using dynamic light scattering analyzer (DLS analyzer) to evaluate the size of aggregates and the surface charge of cND and cND-RSA complexes. If this method to decrease the aggregation is feasible, in the future it could be used in pre-clinical preparation of ND to prevent the side effects which can arise due to large size aggregation.
In bio-medical applications nanodiamonds and their complexes have to be transported by blood circulation to reach the target tissues. If we want to apply any ND complexes in pre-clinical/ clinical studies, the second question we have to face is “does nanodiamonds affect the blood cells, first of all red blood cells (RBC) or not?” In this part of work, we used Raman spectroscopy to measure the oxygenation process of RBC. RBC’s function is to carry or release oxygen, which is bound by protein hemoglobin (Hb) contained in RBC. Hemoglobin conformation depends on oxygenation state and the structure and oxygenation state can be characterized by Raman spectroscopy. We use Raman spectroscopy to estimate the RBC function, using spontaneous oxygenation in the air oxygen-containing atmosphere and purging nitrogen for deoxygenation. Raman spectra were measured every five minutes and the changes of oxygen saturation was calculated using characteristic peaks of oxygenated or deoxygenated state to observe the effects of various treatments on the oxygenation/deoxygenation process.
In the third part of work, we try to develop an oxygen carrier based on hemoglobin and cND-RSA, using rat hemoglobin. Using spectroscopic methods we analyzed its adsorption on ND and estimated the oxygenated and deoxygenated states of hemoglobin adsorbed on nanodiamonds surface. We show that 50 nm cND(RSA)-Hb can carry oxygen successfully, however, due to the complicate physiology condition, further use of this kind of oxygen carrier in-vivo needs more preliminary in-vitro investigations.

Contents
Acknowledgement II
Abstract III
Contents V
List of figure VII
List of table XI
Chapter 1 Introduction 1
1.1 Introduction of nanodiamond 1
1.2 Blood composition and rat serum albumin (RSA) 5
1.3 Red blood cell, hemoglobin and heme 6
1.4 Carboxylated Nanodiamond-RSA (cND-RSA) as Hemoglobin based Artificial Blood Substitutes 8
Chapter 2 Sample preparation 11
2.1 Nanodiamond carboxylation 11
2.2 Phosphate buffer saline (PBS) preparation 12
2.3 Hypotonic solution (20 mOsm) preparation 12
2.4 cND-RSA complex preparation 12
2.5 Red blood cells sample preparation 12
2.6 Preparation of cell free hemoglobin 13
2.7 Preparation of 50 nm cND(RSA)-Hb 13
Chapter 3 Experimental methods and set-up 15
3.1 UV-visible spectroscopy 15
3.2 Particle size and z-potential analysis 16
3.2.1 Particle size measurements 16
3.2.2 z-potential measurements 17
3.3 Introduction of Raman spectroscopy 18
3.4 Red blood cells Raman measurements 19
3.5 Introduction of laser confocal scanning fluorescence microscope 22
Chapter 4 Analysis of 50 nm cND-RSA and measurement of red blood cells (RBCs) oxygen saturation 27
4.1 Analyzing of 50 nm cND-RSA 27
4.2 Measurements of RBCs oxygen saturation 30
Chapter 5 Carboxylated Nanodiamond-RSA (cND-RSA) as Hemoglobin based Artificial Blood Substitutes 43
5.1 Analyzing 50 nm cND(RSA)-Hb 43
5.2 Hb function test using Raman spectroscopy 45
5.3 Stability test of cND(RSA)-Hb 47
5.4 Carboxylated ND(HSA)-Hb in human model 49
Chapter 6 Conclusion 53
Chapter 7 Reference 55

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