Characterization and MRI detection of 19F-PLGA labelled human mesenchymal stem cells

Introduction 19F based stem-cell labelling is attractive, since it may enable long-term studies of cellular migration provided sufficient detection sensitivity can be achieved and has no background signal. Several 19F based compounds are based on a cocktail of approved pharmaceutical compounds, that facilitates translationonal studies. 19F-PLGA: poly(D,L-lactide-co-glycolide) has successfully been used for tracking of dendritic cells and has clinical research approval [1]. Methods 19F PLGA nanoparticles (19F PLGA NP) of various compositions, with and without surface modification were prepared by encapsulation of of perfluoro-15-crown-5-ether and by optical probes (carboxyfluorescein). The nanoparticle size was determined by DLS light scattering. Human mesenchymal stem cells (hMSC) were incubated in a solution containing a maximum of 4mg of the nanoparticles for a 1-3 days. The influence of 19F-PLGA-NP labelling on the following parameters was investigated: proliferation kinetics, colony generation, adhesion, surface migration, and presence of stem-cell markers. Fluorescence microscopy was used to verify cellular uptake of the compound, while cell loading was determined by MR spectroscopy and referencing to tri-fluoroacetic acid at 7T. The longitudinal relaxation time of the free nanoparticles and after hMSC labelling was determined at 7T. MRI sequence optimization was performed by simulation of the expected MR signal in Matlab [2] and labelled cells were measured either as a pellet or as an agarose suspension. Results Without surface modification, no uptake in hMSC occurred (0.01x1012 19F/cell). while the cell-load increased after surface modification and optimization of the loading protocol (0.29-1.16x1012 19F/cell). The size of the nanoparticles was 306nm, with a PDI of 0.16. No significant influence of 19F-PLGA-NPs on the cells was observed after achieving a cell loading of 1.16x1012 19F/cell. The T1 time of the 19F-PLGA-NP decreased from 1090 to 790ms after loading. In vitro, the T2 time was 590ms. The MRI detection limit with a cell load of 0.4x1012 19F/cell in a 2h scan was 20.000 cells (pellet) or 10.000 cells per microliter (suspension). Conclusions Besides optimization of the imaging protocol, a significant increase in MRI sensitivity can be achieved through improving the cell-load. The 19F-PLGA-NP did not significantly alter the properties of hMSC. In vivo studies of 19F-PLGA-NP labelled hMSC are under way to verify the utility of this technique in a pre-clinical setting

Magnetic retraction: A viable method for the purification of encapsulated islet grafts

Islet cell transplantation is a promising option for the restoration of normal glucose homeostatsis in patients with type-1 diabetes. But problems remain regarding the efficient use of donor cells and the prevention of graft rejection by the host immune system. Previously, we were able to show that xenotransplantation of microencapsulated rat and human islets cells can achieve life-long graft function in immunocompetent diabetic mice without the need for immunosuppression [1]. Since the microencapsulation of islets results in a significant amount of empty capsules, and since graft volume is a crucial issue, we developed a new method which uses magnetic labeling and separation of the microencapsulated islets witch supramagnetic iron particles (SPIO) in order to eliminate empty capsules. For this purpose, rat islets were isolated and labeled with two different concentrations of SPIO (3 and 30µl/ml Resovist®, respectively) before microencapsulation in alginate beads as described [1]. Before transplantation into diabetic mice, the magnetic capsules were separated from the empty capsules using a newly developed linear magnetic flow apparatus. Using this method we were able to reduce the ratio of empty capsules (EC) to islet containing capsules (IC) from 4:1 to at least 2:1 or 1:1 in the low (3µg/ml) and high (30µg/ml) SPIO concentration group, respectively. In vitro viability and functionality assessment using the insulin stimulation index did not show any differences between SPIO-labeted islets and freshly isolated unlabeled islets. For proof of in vivo function 3500 islet equivalents (Ieq) of SPIO-labeled islets from both concentrations were transplanted in the peritoneal cavity of streptozotozin-diabetic immunocompetent balb/c mice, resulting in long term (>30 weeks) normoglycemia. We conclude, that magnetic separation of SPIO-labelled encapsulated islets is a clinically safe and effective principle to significantly decrease the graft volume without impairing graft quality and function

Synthesis and Characterization of a series of Tetranuclear Heterometallic (Co/W and Ni/Mo)String Complexes with Tripyridyldiamino Ligand

本論文為利用三吡啶二胺為配基(Tripyridyldiamino ligand 簡稱H2tpda)與6B 以及9B ˴ 10B金屬在高溫萘燒法條件下進行反應，成功合成出不對稱四核混金屬串錯合物[CoWCo2(μ5-tpda)4(Cl)2](BF4) (1)、 [CoWCo2(μ5-tpda)4(Cl)2] (2)、[CoWCo2(μ5-tpda)4(NCS)2](PF6) (3)以及[Mo2Ni2(μ5-tpda)4(Cl)(O)](PF6) (4)。並對這類含八配位構型之金屬串錯合進行其結構、磁性及電化學進行探討。 由X-ray單晶繞射解析可知，所有產物皆是以四片tpda2- 配基以全順向式之模式螺旋纏繞在四個金屬離子外圍，中間的鉬˴鎢金屬離子是以四方反稜柱的配位模式與氮原子鍵結，其他的鈷˴鎳金屬離子則是平面四方以及四角的配位構型。中間的鉬、鎢與旁邊金屬的距離大於3.0 A，超過金屬單鍵的共價半徑，推斷其金屬間的作用力是非常弱的。 以超導量子干涉測量磁性，可知(1)、(3)在300K時的μeff為2.59及2.44 B.M ，推測(1) ˴(3)金屬電子自旋為 Co2+ (S = 1/2) ; W4+ (S = 0) ; Co25+ (S = 1/2)，接近於純自旋理論μeff = 2.45 B.M。錯合物(2)的還原的電子應是位於雙核鈷的單元，因此推測其只有一個磁性中心Co2+ (S = 1/2)，300 K時的μeff為2.01 B.M.，接近於純自旋理論的 1.73 B.M。錯合物(4)在300 K時的μeff為3.04 B.M ，推測其自旋組態為Mo4+(1) (S = 0) ; Mo4+ (2) (S = 0) ; Ni2+(1) (S = 0) ; Ni2+(2) (S = 1)，接近於純自旋理論μeff = 2.83 B.M.。A series of tetranuclear heterometallic string complexes, [CoWCo2 (μ5-tpda)4(Cl)2](BF4) (1) , [CoWCo2(μ5-tpda)4(Cl)2] (2) , [CoWCo2 (μ5-tpda)4(NCS)2](PF6) (3) and [Mo2Ni2(μ5-tpda)4(Cl)(O)](PF6) (4) (tpda = tripyridyldiamino anion) were synthesized and characterized. The X-ray structural studies showed that all of them are helically wrapped by four all syn type H2tpda ligand, and have one 6B metal ion with square anti-prismatic geometry chelated by one pyridine N atom and the amido N atom of each of tpda2-. Axial ligands Cl- and NCS- are bonded to two Co ions for (1), (2) and (3), Cl- and O2- are bonded to Ni and Mo for (4). The distance between the middle 6B and nearby metal is larger than 3.0 A , which is elongated beyond the single-bond covalent radii of the metal atoms. The magnetic susceptibility showed that the μeff values of (1), (2), (3) and (4) at 300K are 2.59 , 2.44 and 2.01 B.M. respectively. The spin state of (1), (3) are the same, with Co2+ (S = 1/2), W4+ (S = 0), Co25+ (S = 1/2) configuration, which is close to the spin-only value 2.45 B.M. (2) is Co2+ (S = 1/2), W4+ (S = 0), Co2+(2) = Co2+(3) (S = 0) ,which is close to (1.73 B.M.) of spin-only value. The μeff of (4) is 3.04 B.M. , corresponding to Mo4+(1) (S = 0) , Mo4+ (2) (S = 0) , Ni2+(1) (S = 0) ; Ni2+(2) (S = 1) spin state.目 錄 中文摘要 I 英文摘要 III 第一章 緒論 .1 1.1 前言 1 1.2 金屬-金屬鍵結理論 2 1.2.1 雙核及三核金屬錯合物 3 1.3 配基與多核金屬串錯合物 5 1.3.1 配基介紹 5 1.3.2 金屬串錯合物介紹 .. 8 1.4 研究方向 21 第二章 實驗部分 22 2.1 試藥與儀器 22 2.2 測量儀器 24 2.3 化合物之合成 25 2.3.1 配基的合成 25 2.3.2 金屬串錯合物的合成 26 2.4 晶體數據收集與處理 30 第三章 結果與討論 35 3.1 配基與金屬錯合物的合成探討 35 3.2 晶體結構解析 38 3.3 磁性分析 49 3.3 電化學分析 58 第四章 結論 61 4.1 總結 61 參考文獻 63 附錄 65 圖目錄 圖 1.1 含有橋接氮原子的雙合釕錯合物 2圖 1.2 [Re2Cl8]2- 的單晶結構 3圖 1.3 雙核金屬錯合物 d軌域混成示意圖 4圖 1.4 三核金屬串錯合物d軌域重疊圖及分子能階圖 4圖1.3.1 Hdpa的三種配位構型 5圖1.3.1 [Ni3(μ3-dpa)4Cl2] 之結構 6圖1.3.2 [Ni5(bna)4(Cl)2](PF6)2 之單晶結構 10圖1.3.3 [Ni11(tentra)4Cl2](PF6)2 之單晶結構 10圖1.3.4 [Ni5(ph2N5)4Cl2] 之單晶結構 10圖1.3.5 [Ni3(pta)4X2] 失序圖及修飾甲基之單晶結構 11圖1.3.6 [Ni5(phapaby)4Cl](PF)6之單晶結構 12 圖1.3.7 十一核鉻串九核鈷串及五核釕串之單晶結構圖 13圖1.3.8 [CoPdCo(dpa)4(Cl)2] 之晶體結構 14圖1.3.9 [CuPdCu(dpa)4(Cl)2] 及 [CuPtCu(dpa)4(Cl)2] 之單晶結構 14圖1.3.10 (a) [CuCuPd(npa)4Cl][PF6] 及 [Ru2Cu(dpa)4(Cl)2] 之單晶結構 15圖1.3.11 [NiCoRh(dpa)4Cl2] 的合成流程 16圖1.3.12 J. F. Berry團隊之異金屬錯合物合成流程 16圖1.3.13 [NiRu2Ni2(tpda)4(NCS)2] 及 [Ru2Co3(toda)4(NCS)2] 之單晶結構 17圖1.3.14 Ni3(H2pepta)2]2+ 之單晶結構 19圖1.3.15 [CoMoCo2(tpda)4(NCS)2] 之單晶結構 20圖1.3.16 [W2O(dpa)4]2+ 之合成流程及單晶結構圖 20 圖3.1.1 四氮配基副產物 35圖3.1.2 產物氧化前後之質譜圖 37圖3.2.1[CoWCo2(μ5-tpda)4(Cl)2](BF4) 之單晶結構圖 38 圖3.2.2 [CoWCo2(μ5-tpda)4(Cl)2](BF4) 之金屬失序情形 38圖3.2.3 [XeF8]2-及 W離子之配位構型 39圖3.2.4 [CoWCo2(μ5-tpda)4(Cl)2](BF4) 相關鍵長整理 40圖3.2.5 [W2O(dpa)4]2+ 的單晶結構及分子軌域示意圖 40圖3.2.6 [W2O(dpa)4]2+ 之相關鍵長整理 41圖3.2.7 [CoWCo2(μ5-tpda)4(Cl)2] 之單晶結構圖 42圖3.2.8 [CoWCo2(μ5-tpda)4(Cl)2] 的相關鍵長整理 43圖3.2.9 [CoWCo2(μ5-tpda)4(NCS)2](PF6) 之單晶結構圖 44圖3.2.10 [CoWCo2(μ5-tpda)4(NCS)2](PF6) 相關鍵長整理 45圖3.2.11 [Mo2Ni2(μ5-tpda)4(O)(Cl)](PF6) 之單晶結構圖 45圖3.2.12 [Mo2Ni2(μ5-tpda)4(O)(Cl)](PF6) 相關鍵長整理 46圖3.2.13 [Mo2O(dpa)4]2+ 相關鍵長整理 48圖3.2.14 [Mo2O(dpa)4]2+ 的分子軌域示意圖 50圖3.3.1 磁性示意圖 50圖3.3.2 莫耳磁化率及有效磁矩對溫度之關係圖 51圖3.3.3 [CoWCo2(μ5-tpda)4(Cl)2](BF4) 之磁性量測結果 52圖3.3.4 [CoWCo2(μ5-tpda)4(Cl)2](BF4) 之電子自旋圖 52圖3.3.5 [Co3(dpa)4(Cl)2](BF4) 有效磁矩對溫度的關係圖 54圖3.3.6 [Co3(dpa)4(Cl)2](BF4) 5E組態的分子軌域圖 54圖3.3.7 [CoWCo2(μ5-tpda)4(Cl)2之磁性量測結果 55圖3.3.8 [CoWCo2(μ5-tpda)4(Cl)2 之電子自旋圖 55圖3.3.9 [CoWCo2(μ5-tpda)4(NCS)2](PF6) 之磁性量測結果 56圖3.3.10 [CoWCo2(μ5-tpda)4(NCS)2](PF6) 之電子自旋圖 56 圖3.3.11 [Mo2Ni2(μ5-tpda)4(O)(Cl)](PF6) 之磁性量測結果 57圖3.3.12 [Mo2Ni2(μ5-tpda)4(O)(Cl)](PF6) 之電子自旋圖 57圖3.4.1 [CoWCo2(μ5-tpda)4(Cl)2](BF4) 之循環伏安圖 58圖3.4.2 [CoWCo2(μ5-tpda)4(NCS)2](PF6) 之循環伏安圖 59圖3.4.3 [Mo2Ni2(μ5-tpda)4(O)(Cl)](PF6) 之循環伏安圖 60 yo 表目錄 表1.3.1 使用三吡啶二胺配基所合成之異核金屬串 18 表 3.1 Pascal ̒s Constant 51表 3.2 Pascal ̒s Constant 5

Targeting the homing of stem cells via suppression of cell adhesion in the peripheral vasculature

The general approach of the so far developed technologies is the attempt to increase the homing rate of transplanted stem cells by modifying the molecules/receptors that interact directly with the chemokines/ligands in the damaged tissue. Our unique technology does not interact with the natural repertoire of specific molecules/receptors mediating homing of stem cells to the damaged tissue (SPECIFIC HOMING) but impairs the function of molecules/receptors that are responsible for the adhesion of stem cells throughout the non-damaged vasculature (NON-SPECIFIC CELL ADHESION). Moreover and most importantly, our highly effective technology is non-toxic, does not affect stem cell function and does not require genetic modifications thus making it a most promising candidate for clinical us