超顺磁性硫酸链霉素PLA-PEG微球的实验研究
|
摘要
儿童骨关节结核的治疗一直是儿童骨科医师面临的一大难题。目前骨关节结核的治疗是包括抗结核药物化疗、病灶清除术及营养支持等的综合疗法,而应用抗结核药物是治疗骨关节结核的中心环节。硫酸链霉素作为目前临床上的一线抗骨关节结核药物,其主要副作用是显著的第八对颅神经损害及肝肾毒性【1】,这极大地限制了其临床应用。近年来飞速发展的磁性微球靶向给药技术在提高药物对病灶的靶向性及降低药物的毒副作用方面显示出巨大的发展前景【2】。
普通磁性微球制剂进入体内后易被单核巨噬细胞系统作为异物认别而吞噬,显著降低了其对病灶的靶向性。目前重要的研究策略之一是对微球进行表面修饰,制备能逃避单核巨噬细胞系统识别和吞噬的隐形磁性微球系统【3】。本实验以能阻止单核巨噬系统细胞识别、吞噬的长链聚乙二醇修饰的聚乳酸共聚物(PLA-PEG)作为磁性微球载体,制备超顺磁性硫酸链霉素PLA-PEG微球。检测该微球的各项特征,研究该微球在振荡磁场作用下的释放规律,在不同温度下保存的稳定性,并同时考察了该微球的体外抗结核分支杆菌的效能。
本文首先采用目前较为成熟的化学共沉淀法制备超顺磁性壳聚糖Fe3O4纳米粒,通过透射电镜检测其粒径,振动样品磁强计测定纳米粒的磁感应性能。首次采用原子力显微镜(AFM)初步分析其微观构成方式。结果表明所制备的壳聚糖Fe3O4纳米粒粒径分布于20~30 nm,具有良好的超顺磁性,由AFM的检测结果推测Fe3O4中的Fe原子与壳聚糖中的游离氨基中的氮原子可能通过-NH-Fe-共价键结合。再采用复乳化-溶剂挥发法(W/O/W)制备超顺磁性硫酸链霉素PLA-PEG微球,所制得的微球平均粒径为2.467μm。本文同时建立了高效液相色谱法(HPLC)检测硫酸链霉素浓度以考察微球的包封率和载药量,外加静磁场考察微球磁感应。以微球的平均粒径、磁感应性及包封率等指标考察内外水相中PVA浓度、内水相与油相体积比、复乳与外水相体积比、超声乳化时间、均质机作用时间及均质机速度等因素对微球制备影响。确定了较佳的制备工艺为内外水相中PVA浓度分别为1.5%、0.5%,内水相与油相体积比为1∶4,复乳与外水相体积比为1∶20,超声乳化时间90 s,均质机作用时间90 s,均质机速度为24 kr/min。
本文研究了在自制大功率振荡磁场发生器产生的振动磁场干预下超顺磁性硫酸链霉素PLA-PEG微球药物释放规律。每隔24 h取样,取样前振荡磁场干预30 min,HPLC检测模拟体液中硫酸链霉素浓度,计算硫酸链霉素的释放率,并绘制各组微球的溶出曲线。结果表明,与未加磁场的超顺磁性硫酸链霉素PLA-PEG微球相比,各时间点的释放率都有提高。此外,未加磁场干预的超顺磁性硫酸链霉素PLA-PEG微球组与硫酸链霉素PLA-PEG微球组相比也有较高的释放率。
本实验同时考察了超顺磁性硫酸链霉素PLA-PEG微球在不同温度下保存的稳定性,结果表明该微球在-30℃条件下保存3月后微球的外观、载药量及磁感应性保持良好。
通过在改良罗氏培养基上采用绝对浓度法进行人型结核分支杆菌的硫酸链霉素药敏试验,以检验超顺磁性硫酸链霉素PLA-PEG微球的制备工艺及配方对微球中硫酸链霉素药效有无影响。用超顺磁性硫酸链霉素PLA-PEG微球的溶出液制备结核分支杆菌的药敏培养基,与硫酸链霉素培养基对照。药敏试验结果显示实验组、对照组中15株结核分支杆菌在高低浓度硫酸链霉素培养基上的生长均有抑制;分别比较实验组与对照组高、低浓度硫酸链霉素培养基对结核分支杆菌生长的抑制作用,采用M-H检验,P值均> 0.05,即实验组与对照组高、低浓度硫酸链霉素培养基对结核分支杆菌的抑菌效能差异无统计学意义,表明超顺磁性硫酸链霉素PLA-PEG微球的制备过程及配方对硫酸链霉素的药效无明显影响。实验组和对照组15株结核分支杆菌的耐药率分别为73.33%、80.00%,采用Fisher精确概率法比较,P>0.05,即实验组和对照组间耐药率比较差异无统计学意义。
本实验所制备的超顺磁性硫酸链霉素PLA-PEG微球外观完整、均匀,平均粒为2.467μm,在体外具有良好的磁响应性。在体外的溶出实验中振荡磁场可明显提高硫酸链霉素的释放。体外人型结核分支杆菌的药敏试验也表明该超顺磁性硫酸链霉素PLA-PEG微球具有良好的抗结核分支杆菌效能。
The treatment of tuberculosis of bone and joint in children has been a major problem to the children's orthopedic surgeons. At present the treatment of osteoarticular tuberculosis is including debridement surgery, drugs chemotherapy of anti-tuberculosis, nutritional support and so on. And the application of anti-tuberculosis drugs is critical to the treatment. The streptomycin sulfate is one of the clinical anti osteoarticular tuberculosis drugs, its main side effect is significant toxicity to the eighth cranial nerve, liver and kidney, and its clinical application has been greatly limited. In recent years, the targeted drug delivery system of the magnetic microspheres shows great prospect in improving the targeting on the lesion of drugs and reducing their toxic side effects.
After the ordinary magnetic microspheres injected into the blood circulation, the microspheres are easily identified and cleared as a foreign body by mononuclear phagocyte system (MPS), it will significantly reduce the concentration of drugs to their lesions. In current research, the main strategy is to prepare the stealh magnetic microsphere system via surface modification of microspheres to avoid mononuclear phagocyte cells. In this study, we use poly (d,l-lactic acid)-co-poly (ethylene glycol) (PLA-PEG) which can prevent the phagocytosis of MPS to prepare the magnetic microsphere carrier. Using streptomycin sulfate as the model drug, the superparamagnetic streptomycin sulfate PLA-PEG microspheres (spSPM) with the stealth capabilities were prepared, their featrues wre detcted, and the vitro susceptibility test of superparamagnetic streptomycin sulfate PLA-PEG microspheres were carried out.
In this paper, we used chemical coprecipitation which was a more mature method to prepare superparamagnetic chitosan Fe3O4 nanoparticles (spCFN), then using transmission electron microscopy to detect the particles size, vibration sample magnetometer to measure magnetic properties of the nanoparticles, atomic force microscope (AFM) to measure they composition preliminary. The size distribution of the spCFN was between 20 and 30 nm, which had perfect superparamagnetism. The results detected by the AFM showed that the Fe atoms in Fe3O4 and the nitrogen atoms of free aminos in chitosan may have adopted in -NH-Fe- covalent bond. Then spSPM were prepared by the method of multiple emulsion - solvent evaporation method (W / O / W), and the average particle size of these microspheres was 2.467μm. In this paper, we study the encapsulation rate and drug loading rate of these microspheres by the HPLC method which was established to detect the streptomycin sulfate. The magnetic inductions of these microspheres were studied by using the external static magnetic field magnetic. Using the average particle size of the microspheres, magnetic induction, and encapsulation rate of this microspheres to investigate the time of ultrasonic emulsification, the speed of homogenizer and homogenization time, the volume ratio of the aqueous phase to the oil phase, the concentration of PVA in internal and external aqueous phase, which were great factors in the preparation process of spSPM. In this research a better method was found as follows: the concentration of PVA in internal and external aqueous were 1.5% and 0.5%, the volume ratio of the aqueous phase to the oil phase was 1:4, the volume ratio of the multiple emulsion to the external aqueous was 1:20, the time of ultrasonic emulsification was 90 s, the time of homogenization was 90s and the speed of homogenizer was 24 kr/min.
In this paper, the release laws in vitro of were studied by using the oscillating magnetic field generator prepared by ourselves. Every 24hours sampled the solution, using the oscillating magnetic field generator to interfering 30 min, to detect the concentration of streptomycin sulfate in SBF by the HPLC, after calculating the release rate, stripping curves were prepared. The results show that the spSPM interfered by the oscillating magnetic field had significant higher release rate compared with spSPM without oscillating magnetic field at each time point. Besides, the release rate of spSPM without oscillating magnetic field was higher than the rate of streptomycin sulfate PLA-PEG microspheres (SPM).
This experiment also studied the stability of spSPM in different temperatures. The results showed that the appearance, the drug loading and the magnetic induction of these magnetic microspheres can be stable reserved at -30℃3 months later.
The drug susceptibility tests of streptomycin sulfate which were absolute concentration method based on L-J mediums in vitro against Mycobacterium tuberculosis were taken. It was studied whether the efficacy of streptomycin sulfate in the magnetic microspheres had been declined during the prescription and preparation process of this experiment. The drug susceptible culture mediums of mycobacterium tuberculosis were made by the streptomycin sulfate in the release liquid of spSPM, compared with the culture mediums made by streptomycin sulfate only. The results showed that the growth of 15 strains of Mycobacterium tuberculosis in high and low concentrations of streptomycin sulfate culture mediums of experimental group and the control group of of Mycobacterium tuberculosis had inhibited both. The growths of Mycobacterium tuberculosis in high and low concentrations of streptomycin sulfate culture mediums were compared by M-H test, P> 0.05. Compared the experimental group and control group of high and low concentrations of streptomycin sulfate medium on the antibacterial efficacy of Mycobacterium tuberculosis had no significant difference. The resistance rates of experimental group and control group of 15 strains Mycobacterium tuberculosis were 73.33% and 80.00%, compared by Fisher exact test, P> 0.05.And the efficacy of streptomycin sulfate in the magnetic microspheres had not been declined during the prescription and preparation process of this experiment.
In this study, the spSPM had complete and uniform appearance, the average partical size of these microspheres was 2.467μm, showed well superparamagnetic in vitro targeting test. The oscillating magnetic field could significantly increase the release of streptomycin sulfate in vitro dissolution experiments. Human type of Mycobacterium tuberculosis in vitro drug sensitivity tests also showed that the spSPM had well antibacterial activities to Mycobacterium tuberculosis.
普通磁性微球制剂进入体内后易被单核巨噬细胞系统作为异物认别而吞噬,显著降低了其对病灶的靶向性。目前重要的研究策略之一是对微球进行表面修饰,制备能逃避单核巨噬细胞系统识别和吞噬的隐形磁性微球系统【3】。本实验以能阻止单核巨噬系统细胞识别、吞噬的长链聚乙二醇修饰的聚乳酸共聚物(PLA-PEG)作为磁性微球载体,制备超顺磁性硫酸链霉素PLA-PEG微球。检测该微球的各项特征,研究该微球在振荡磁场作用下的释放规律,在不同温度下保存的稳定性,并同时考察了该微球的体外抗结核分支杆菌的效能。
本文首先采用目前较为成熟的化学共沉淀法制备超顺磁性壳聚糖Fe3O4纳米粒,通过透射电镜检测其粒径,振动样品磁强计测定纳米粒的磁感应性能。首次采用原子力显微镜(AFM)初步分析其微观构成方式。结果表明所制备的壳聚糖Fe3O4纳米粒粒径分布于20~30 nm,具有良好的超顺磁性,由AFM的检测结果推测Fe3O4中的Fe原子与壳聚糖中的游离氨基中的氮原子可能通过-NH-Fe-共价键结合。再采用复乳化-溶剂挥发法(W/O/W)制备超顺磁性硫酸链霉素PLA-PEG微球,所制得的微球平均粒径为2.467μm。本文同时建立了高效液相色谱法(HPLC)检测硫酸链霉素浓度以考察微球的包封率和载药量,外加静磁场考察微球磁感应。以微球的平均粒径、磁感应性及包封率等指标考察内外水相中PVA浓度、内水相与油相体积比、复乳与外水相体积比、超声乳化时间、均质机作用时间及均质机速度等因素对微球制备影响。确定了较佳的制备工艺为内外水相中PVA浓度分别为1.5%、0.5%,内水相与油相体积比为1∶4,复乳与外水相体积比为1∶20,超声乳化时间90 s,均质机作用时间90 s,均质机速度为24 kr/min。
本文研究了在自制大功率振荡磁场发生器产生的振动磁场干预下超顺磁性硫酸链霉素PLA-PEG微球药物释放规律。每隔24 h取样,取样前振荡磁场干预30 min,HPLC检测模拟体液中硫酸链霉素浓度,计算硫酸链霉素的释放率,并绘制各组微球的溶出曲线。结果表明,与未加磁场的超顺磁性硫酸链霉素PLA-PEG微球相比,各时间点的释放率都有提高。此外,未加磁场干预的超顺磁性硫酸链霉素PLA-PEG微球组与硫酸链霉素PLA-PEG微球组相比也有较高的释放率。
本实验同时考察了超顺磁性硫酸链霉素PLA-PEG微球在不同温度下保存的稳定性,结果表明该微球在-30℃条件下保存3月后微球的外观、载药量及磁感应性保持良好。
通过在改良罗氏培养基上采用绝对浓度法进行人型结核分支杆菌的硫酸链霉素药敏试验,以检验超顺磁性硫酸链霉素PLA-PEG微球的制备工艺及配方对微球中硫酸链霉素药效有无影响。用超顺磁性硫酸链霉素PLA-PEG微球的溶出液制备结核分支杆菌的药敏培养基,与硫酸链霉素培养基对照。药敏试验结果显示实验组、对照组中15株结核分支杆菌在高低浓度硫酸链霉素培养基上的生长均有抑制;分别比较实验组与对照组高、低浓度硫酸链霉素培养基对结核分支杆菌生长的抑制作用,采用M-H检验,P值均> 0.05,即实验组与对照组高、低浓度硫酸链霉素培养基对结核分支杆菌的抑菌效能差异无统计学意义,表明超顺磁性硫酸链霉素PLA-PEG微球的制备过程及配方对硫酸链霉素的药效无明显影响。实验组和对照组15株结核分支杆菌的耐药率分别为73.33%、80.00%,采用Fisher精确概率法比较,P>0.05,即实验组和对照组间耐药率比较差异无统计学意义。
本实验所制备的超顺磁性硫酸链霉素PLA-PEG微球外观完整、均匀,平均粒为2.467μm,在体外具有良好的磁响应性。在体外的溶出实验中振荡磁场可明显提高硫酸链霉素的释放。体外人型结核分支杆菌的药敏试验也表明该超顺磁性硫酸链霉素PLA-PEG微球具有良好的抗结核分支杆菌效能。
The treatment of tuberculosis of bone and joint in children has been a major problem to the children's orthopedic surgeons. At present the treatment of osteoarticular tuberculosis is including debridement surgery, drugs chemotherapy of anti-tuberculosis, nutritional support and so on. And the application of anti-tuberculosis drugs is critical to the treatment. The streptomycin sulfate is one of the clinical anti osteoarticular tuberculosis drugs, its main side effect is significant toxicity to the eighth cranial nerve, liver and kidney, and its clinical application has been greatly limited. In recent years, the targeted drug delivery system of the magnetic microspheres shows great prospect in improving the targeting on the lesion of drugs and reducing their toxic side effects.
After the ordinary magnetic microspheres injected into the blood circulation, the microspheres are easily identified and cleared as a foreign body by mononuclear phagocyte system (MPS), it will significantly reduce the concentration of drugs to their lesions. In current research, the main strategy is to prepare the stealh magnetic microsphere system via surface modification of microspheres to avoid mononuclear phagocyte cells. In this study, we use poly (d,l-lactic acid)-co-poly (ethylene glycol) (PLA-PEG) which can prevent the phagocytosis of MPS to prepare the magnetic microsphere carrier. Using streptomycin sulfate as the model drug, the superparamagnetic streptomycin sulfate PLA-PEG microspheres (spSPM) with the stealth capabilities were prepared, their featrues wre detcted, and the vitro susceptibility test of superparamagnetic streptomycin sulfate PLA-PEG microspheres were carried out.
In this paper, we used chemical coprecipitation which was a more mature method to prepare superparamagnetic chitosan Fe3O4 nanoparticles (spCFN), then using transmission electron microscopy to detect the particles size, vibration sample magnetometer to measure magnetic properties of the nanoparticles, atomic force microscope (AFM) to measure they composition preliminary. The size distribution of the spCFN was between 20 and 30 nm, which had perfect superparamagnetism. The results detected by the AFM showed that the Fe atoms in Fe3O4 and the nitrogen atoms of free aminos in chitosan may have adopted in -NH-Fe- covalent bond. Then spSPM were prepared by the method of multiple emulsion - solvent evaporation method (W / O / W), and the average particle size of these microspheres was 2.467μm. In this paper, we study the encapsulation rate and drug loading rate of these microspheres by the HPLC method which was established to detect the streptomycin sulfate. The magnetic inductions of these microspheres were studied by using the external static magnetic field magnetic. Using the average particle size of the microspheres, magnetic induction, and encapsulation rate of this microspheres to investigate the time of ultrasonic emulsification, the speed of homogenizer and homogenization time, the volume ratio of the aqueous phase to the oil phase, the concentration of PVA in internal and external aqueous phase, which were great factors in the preparation process of spSPM. In this research a better method was found as follows: the concentration of PVA in internal and external aqueous were 1.5% and 0.5%, the volume ratio of the aqueous phase to the oil phase was 1:4, the volume ratio of the multiple emulsion to the external aqueous was 1:20, the time of ultrasonic emulsification was 90 s, the time of homogenization was 90s and the speed of homogenizer was 24 kr/min.
In this paper, the release laws in vitro of were studied by using the oscillating magnetic field generator prepared by ourselves. Every 24hours sampled the solution, using the oscillating magnetic field generator to interfering 30 min, to detect the concentration of streptomycin sulfate in SBF by the HPLC, after calculating the release rate, stripping curves were prepared. The results show that the spSPM interfered by the oscillating magnetic field had significant higher release rate compared with spSPM without oscillating magnetic field at each time point. Besides, the release rate of spSPM without oscillating magnetic field was higher than the rate of streptomycin sulfate PLA-PEG microspheres (SPM).
This experiment also studied the stability of spSPM in different temperatures. The results showed that the appearance, the drug loading and the magnetic induction of these magnetic microspheres can be stable reserved at -30℃3 months later.
The drug susceptibility tests of streptomycin sulfate which were absolute concentration method based on L-J mediums in vitro against Mycobacterium tuberculosis were taken. It was studied whether the efficacy of streptomycin sulfate in the magnetic microspheres had been declined during the prescription and preparation process of this experiment. The drug susceptible culture mediums of mycobacterium tuberculosis were made by the streptomycin sulfate in the release liquid of spSPM, compared with the culture mediums made by streptomycin sulfate only. The results showed that the growth of 15 strains of Mycobacterium tuberculosis in high and low concentrations of streptomycin sulfate culture mediums of experimental group and the control group of of Mycobacterium tuberculosis had inhibited both. The growths of Mycobacterium tuberculosis in high and low concentrations of streptomycin sulfate culture mediums were compared by M-H test, P> 0.05. Compared the experimental group and control group of high and low concentrations of streptomycin sulfate medium on the antibacterial efficacy of Mycobacterium tuberculosis had no significant difference. The resistance rates of experimental group and control group of 15 strains Mycobacterium tuberculosis were 73.33% and 80.00%, compared by Fisher exact test, P> 0.05.And the efficacy of streptomycin sulfate in the magnetic microspheres had not been declined during the prescription and preparation process of this experiment.
In this study, the spSPM had complete and uniform appearance, the average partical size of these microspheres was 2.467μm, showed well superparamagnetic in vitro targeting test. The oscillating magnetic field could significantly increase the release of streptomycin sulfate in vitro dissolution experiments. Human type of Mycobacterium tuberculosis in vitro drug sensitivity tests also showed that the spSPM had well antibacterial activities to Mycobacterium tuberculosis.
引文
[1] Peloquin CA. Aminoglycoside toxicity: daily versus thrice-weekly dosing for treatment of mycobacterial diseases [J]. Clinical Infection Disease. 2004, 11(38):1538~44
[2] Theresa M. Drug Delivery Systems: Entering the Mainstream [J]. Science.2004, 303(5665):1818~22
[3] Chambers E. Long Circulating Nano-particles via Adhesion on Red Blood Cells: Mechanism and Extended Circulation [J]. Society for Experimental Biology and Medicine, 2007, 232:958~66
[4]David A. Tuberculosis of the Musculoskeletal System .Techniques in Orthopedics [J]. 2005,20(2):167~78.
[5]罗卓荆.全国脊柱结核治疗专题座谈会纪要[N].中华骨科杂志.2007,27(9):669
[6] Tobias N. Superparamagnetic nanoparticles for biomedical applications: Possibilities and limitations of a new drug delivery system[J]. Journal of Magnetism and Magnetic Materials, 2005,293, 483~96
[7] Weissleder R, Stark D, Engelstad B, et al. Superparamaggnetic iron oxides:pharmacokinetics and toxicity[J].AM J Roent.1989,152(1)∶1 67~73
[8] Gref R. Development and characterization of CyA-loaded poly (lactic acid)-poly(ethylene glyco1) PEG micro -and nanoparticles comparison with conventional PLA particulate[J]. European Journal of Pharmace and Biopharm,2000,51(2):l11~8
[9] Diandra L. Advanced Magnetic Nanostructures [J].Springer US,2006, 15: 461~90
[10] Xingping Zhou,Siyu Ni, Xiaqin Wang,et al. Adsorption of Sodium Oleate on Nano-sized Fe3O4 Particles Prepared by Coprecipitation[J]. Current Nanoscience.2007, 3( 3) :259~63
[11] Gibaly E. Microencapsulation of ketoprofen using w/o/w complex emulsion technique [J].Journal of Microencapsulation. 1996 ,1(13):67~87
[12] Ravi Sheshala, Kok Khiang Peh, Yusrida Darwis. Preparation, characterization,and in vivo evaluation of insulin-loaded PLA–PEG microspheres for controlled parenteral drug delivery[J]. Drug Development and Industrial Pharmacy. 2009, 35(11): 1364~74
[13] Omar G. A direct HPLC method to estimate streptomycin and its putative ototoxic derivative, streptidine, in blood serum: Application to streptom–ycin treated humans [J]. Journal of Pharmaceutical and Biomedical Analysis, 2007, 43: 625~30.
[14] Gref R, Quellec P, Sanchez A, et al. Development and Characterization of CyA-loaded poly (actic acid)- poly(ethylene glycol) PEG micro-and nanoparticles Comparison with conventional PLA particulate carriers [J]. European Journal of Pharmaceutics. 2000, 51:111~8
[15] Lee Moorea, Macie Zborowskia, Masayuki Nakamur,et al. The use of magnetite doped polymeric microspheres in calibrating cell tracking velocimetry[J]. Journal of Biochemistry and Biophysicss. 2000, 44: 115~30
[16] Jia Zhi, Yujun Wang, Yangcheng Lu, et al. In situ preparation of magnetic chitosan/Fe3O4 composite nanoparticles in tiny pools of water-in-oil microemulsion [J].Reactive and Functional Polymers .2006, 66(12):1552~8
[17] Yasugi K., Kato M., Kataoka K., et al, Preparation and characterization of polymer micelles from poly(ethylene glycol)- poly (d,l-lactic acid) block copolymers as potential drug carrier[J]. Control Release .1999, 62:89~100
[18] Matsumoto J, Nakada Y, Sakurai K, et al. Preparation nanoparticles consisted of poly(L-lactide)-poly(ethylene giycol)-poly(Lactide) and their evaluation in vitro [J]. Internaional Journal of Pharmaceutics.1999,185:93~101
[19] Zambaux M, Bonneaux F, Gref R, et al. Protein C-loaded monomethoxypoly (ethylene oxide)-poly (lactic acid) nanoparticles [J]. International Journal of Pharmaceutics. 2001, 212: 1~9.
[20] Gecioni Neckel, Daiane Nemen, Ana Cristina, et al. Stealth and non-stealth nanocapsules containing camptothecin: in-vitro and in-vivo activity on B16-F10 melanoma[J]. Journal of Pharmacy and Pharmacology.2007, 59: 1359~64
[21] Elizabeth C, Samir M. Long Circulating Nanoparticles via Adhesion on Red Blood Cells: Mechanism and Extended Circulation [J].Society for Experimental Biology and Medicine, 2007, 232:958~66.
[22] Guosen H, Lwin L, Jie P, et al. ABA and BAB type triblock copolymers of PEG and PLA: A comparative study of drug release properties and“stealth”particle characteristics [J]. International Journal of Pharmaceutics. 2007, 334:48~55
[23] Zambaux M, Bonneaux F,Gref R, et al.MPEO-PLA nanoparticles;effect of MPEO cintent on some of their surface properties[J].J.Biomed.Mater.Res.1999,44:109~15
[24] Yi-You Huang, Tze-Wen Chung, Tzeng-Wen Tzeng. A method using biodegradable polylactides:polyethylene glycol for drug release with reduced initial burstInternational[J].Journal of Pharmaceutics. 1999, 182 (1999) 93~100
[25] Jie Ren, Xiao Yu, Tianbin Ren,et al. Preparation and characterization of fenofibrate-loaded PLA–PEG microspheres[J]. Journal of Materials Science Materials in Medicine.2007, 18:1481~7
[26] Shirui M, Jing X, Cuifang C, et al. Effect of WOW process parameters on morphology and burst release of FITC-dextran loaded PLGA microspheres[J]. International Journal of Pharmaceutics. 2007, 334:137~48
[27] Gang Ruana, Si-Shen Fenga.Preparation and characterization of poly(lactic acid)–poly(ethylene glycol)–poly(lactic acid) (PLA–PEG–PLA) microspheres for controlled release of paclitaxel[J].Biomaterials.2003, 24:5037~44
[28] Silva A, Silva E, Carri?o A, et al. Magnetic Carriers: A Promising Device for Targeting Drugs Into the human body [J]. Current Pharmaceutical Design.2007, 13, 1179~85
[29] Subbu S, Pan Jie, Feng Minb, et al. Micelle-like nanoparticles of PLA–PEG–PLA triblock copolymer as chemotherapeutic carrier[J]. International Journal of Pharmaceutics.2005, 298:219~32
[30] Noton Dutta, Kaushiki Mazumdar, Sujata Dastidar,et al. Activity of diclofenac used alone and in combination with streptomycin against Mycobacterium tuberculosis in mice[J]. International Journal of Antimicrobial Agents.2007,30 :336~40
[31] Thirumala Govender, Trevor Riley, Touraj Ehtezazi, et al.Defining the drug incorporation properties of PLA–PEG nanoparticles [J]. International Journal of Pharmaceutics 199 (2000) 95~110
[32]中国防痨协会.结核病诊断细菌学检验规程[J].中国防痨杂志.1996, 18(2):80~5
[33] Dutt M, Khuller GK. Therapeutic efficacy of poly(DL-lactide- co ethylene glycol)- encapsulated antitubercular drugs against Mycobacterium tuberculosis infection induced in mice. Antimicrob Agents Chemother. 2001; 45: 363~6.
[34] BurmanW J,Stone B L,Reves R R,et a1.The incidence of false positive cultures for Mycobacterium tuberculosis[J].Am J Respir Crit Care Med.1997,155(1):321-6
[35] Pablos M. Global surveillance for antituberculosis-drug resistance,1994–1997 [J]. The New England Journal of Medicine. 1998, 338:1641~9.
[36] J Dobson. Gene therapy progress and prospects:magnetic nanoparticle-based gene delivery[J]. Gene Therapy.(2006, (13): 283~7
[1] David A. Tuberculosis of the Musculoskeletal System [J] .Techniques in Orthopaedics.2005, 20 (2): 167~78
[2]罗卓荆.全国脊柱结核治疗专题座谈会纪要(N).中华骨科杂志.2007,27(9):669
[3] Global Tuberculosis Control: surveillance, planning, financing. World Health Organization.2007 (WHO/HTM/TB/2007.376)
[4] Tuli SM. General principles of osteoarticular tuberculosis [J]. Clinical Orthopedics Related Research. 2002, 398:11~9
[5] Pablos M. Global surveillance for antituberculosis-drug resistance, 1994–1997 [J]. The New England Journal of Medicine.1998, 338:1641~9
[6]朱莉贞.如何正确服用抗结核药物[J].中华结核和呼吸杂志. 2007, 3O (6): 476.
[7] Thomas R. The DOTS Strategy for Controlling the Global Tuberculosis Epidemic[J].Clinics in Chest Medicine.2005,26(2): 197~205.
[8]陈孝平.外科学(八年制[M].北京:人民卫生出版社.2005:1085.
[9] Zulia Wang .Treatment of spinal tuberculosis with ultrashort-course chemotherapy in conjunction with partial excision of pathologic vertebrae[J]. The Spine Journal. 2007, 7:671~81
[10] Umubyeyi A. Low levels of second-line drug resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Rwanda [J] .International Journal of Infectious Diseases. 2008, 12:152~6.
[11] Wolfart M. Mycobacterium tuberculosis resistance in HIV-infected patients from a tertiary care teaching hospital in Porto Alegre, southern Brazil [J]. Transactions of the Royal Society of Tropical Medicine and Hygiene.2008, 102:421~5
[12] Benator D. Rifapentine and isoniazid once a week versus rifampicin and isoniazid twice a week for treatment of drug-susceptible pulmonary tuberculosis in HIV-negative patients: a randomised clinical trial [J].The Lancet. 2002,360(9332): 528~34.
[13]王泓.常用抗结核药[J].中华综合临床医学杂志.2006, 8(4): 60~1.
[14]韩凤满,程宏,吴启秋,等.骨关节结核病灶结核菌L型状况的观察[J].中国防痨杂志.2004,04(26)233~7.
[15] Y. Bhusal.Determination of in vitro synergy when three antimicrobial agents arecombined against Mycobacterium tuberculosis[J].International Journal of Antimicrobial Agents. 2005, 26:292~7.
[16] Murugesan Dinakaran. Novel ofloxacin derivatives: Synthesis, antimycobacterial and toxicological evaluation[J].Bioorganic & Medicinal Chemistry Letters . 2008,18: 1229~36.
[17]谭守勇.氟喹诺酮类药物在耐多药结核病治疗中的应用[J].中国防痨杂志.2005, 04:262-7
[18] Maria V. Papadopoulou. NLCQ-1 and NLCQ-2, two new agents with activity against dormant Mycobacterium tuberculosis. International Journal of Antimicrobial Agents. 2007, 29:724~7.
[19]马光辉.高分子微球材料[M].北京:化学工业出版社.2005: 223~5
[20] Zahoor Ahmad1. Novel chemotherapy for tuberculosis: chemotherapeutic potential of econazole- and moxifloxacin-loaded PLG nanoparticles [J].International Journal of Antimicrobial Agents. 2008, 31: 142~6.
[2] Theresa M. Drug Delivery Systems: Entering the Mainstream [J]. Science.2004, 303(5665):1818~22
[3] Chambers E. Long Circulating Nano-particles via Adhesion on Red Blood Cells: Mechanism and Extended Circulation [J]. Society for Experimental Biology and Medicine, 2007, 232:958~66
[4]David A. Tuberculosis of the Musculoskeletal System .Techniques in Orthopedics [J]. 2005,20(2):167~78.
[5]罗卓荆.全国脊柱结核治疗专题座谈会纪要[N].中华骨科杂志.2007,27(9):669
[6] Tobias N. Superparamagnetic nanoparticles for biomedical applications: Possibilities and limitations of a new drug delivery system[J]. Journal of Magnetism and Magnetic Materials, 2005,293, 483~96
[7] Weissleder R, Stark D, Engelstad B, et al. Superparamaggnetic iron oxides:pharmacokinetics and toxicity[J].AM J Roent.1989,152(1)∶1 67~73
[8] Gref R. Development and characterization of CyA-loaded poly (lactic acid)-poly(ethylene glyco1) PEG micro -and nanoparticles comparison with conventional PLA particulate[J]. European Journal of Pharmace and Biopharm,2000,51(2):l11~8
[9] Diandra L. Advanced Magnetic Nanostructures [J].Springer US,2006, 15: 461~90
[10] Xingping Zhou,Siyu Ni, Xiaqin Wang,et al. Adsorption of Sodium Oleate on Nano-sized Fe3O4 Particles Prepared by Coprecipitation[J]. Current Nanoscience.2007, 3( 3) :259~63
[11] Gibaly E. Microencapsulation of ketoprofen using w/o/w complex emulsion technique [J].Journal of Microencapsulation. 1996 ,1(13):67~87
[12] Ravi Sheshala, Kok Khiang Peh, Yusrida Darwis. Preparation, characterization,and in vivo evaluation of insulin-loaded PLA–PEG microspheres for controlled parenteral drug delivery[J]. Drug Development and Industrial Pharmacy. 2009, 35(11): 1364~74
[13] Omar G. A direct HPLC method to estimate streptomycin and its putative ototoxic derivative, streptidine, in blood serum: Application to streptom–ycin treated humans [J]. Journal of Pharmaceutical and Biomedical Analysis, 2007, 43: 625~30.
[14] Gref R, Quellec P, Sanchez A, et al. Development and Characterization of CyA-loaded poly (actic acid)- poly(ethylene glycol) PEG micro-and nanoparticles Comparison with conventional PLA particulate carriers [J]. European Journal of Pharmaceutics. 2000, 51:111~8
[15] Lee Moorea, Macie Zborowskia, Masayuki Nakamur,et al. The use of magnetite doped polymeric microspheres in calibrating cell tracking velocimetry[J]. Journal of Biochemistry and Biophysicss. 2000, 44: 115~30
[16] Jia Zhi, Yujun Wang, Yangcheng Lu, et al. In situ preparation of magnetic chitosan/Fe3O4 composite nanoparticles in tiny pools of water-in-oil microemulsion [J].Reactive and Functional Polymers .2006, 66(12):1552~8
[17] Yasugi K., Kato M., Kataoka K., et al, Preparation and characterization of polymer micelles from poly(ethylene glycol)- poly (d,l-lactic acid) block copolymers as potential drug carrier[J]. Control Release .1999, 62:89~100
[18] Matsumoto J, Nakada Y, Sakurai K, et al. Preparation nanoparticles consisted of poly(L-lactide)-poly(ethylene giycol)-poly(Lactide) and their evaluation in vitro [J]. Internaional Journal of Pharmaceutics.1999,185:93~101
[19] Zambaux M, Bonneaux F, Gref R, et al. Protein C-loaded monomethoxypoly (ethylene oxide)-poly (lactic acid) nanoparticles [J]. International Journal of Pharmaceutics. 2001, 212: 1~9.
[20] Gecioni Neckel, Daiane Nemen, Ana Cristina, et al. Stealth and non-stealth nanocapsules containing camptothecin: in-vitro and in-vivo activity on B16-F10 melanoma[J]. Journal of Pharmacy and Pharmacology.2007, 59: 1359~64
[21] Elizabeth C, Samir M. Long Circulating Nanoparticles via Adhesion on Red Blood Cells: Mechanism and Extended Circulation [J].Society for Experimental Biology and Medicine, 2007, 232:958~66.
[22] Guosen H, Lwin L, Jie P, et al. ABA and BAB type triblock copolymers of PEG and PLA: A comparative study of drug release properties and“stealth”particle characteristics [J]. International Journal of Pharmaceutics. 2007, 334:48~55
[23] Zambaux M, Bonneaux F,Gref R, et al.MPEO-PLA nanoparticles;effect of MPEO cintent on some of their surface properties[J].J.Biomed.Mater.Res.1999,44:109~15
[24] Yi-You Huang, Tze-Wen Chung, Tzeng-Wen Tzeng. A method using biodegradable polylactides:polyethylene glycol for drug release with reduced initial burstInternational[J].Journal of Pharmaceutics. 1999, 182 (1999) 93~100
[25] Jie Ren, Xiao Yu, Tianbin Ren,et al. Preparation and characterization of fenofibrate-loaded PLA–PEG microspheres[J]. Journal of Materials Science Materials in Medicine.2007, 18:1481~7
[26] Shirui M, Jing X, Cuifang C, et al. Effect of WOW process parameters on morphology and burst release of FITC-dextran loaded PLGA microspheres[J]. International Journal of Pharmaceutics. 2007, 334:137~48
[27] Gang Ruana, Si-Shen Fenga.Preparation and characterization of poly(lactic acid)–poly(ethylene glycol)–poly(lactic acid) (PLA–PEG–PLA) microspheres for controlled release of paclitaxel[J].Biomaterials.2003, 24:5037~44
[28] Silva A, Silva E, Carri?o A, et al. Magnetic Carriers: A Promising Device for Targeting Drugs Into the human body [J]. Current Pharmaceutical Design.2007, 13, 1179~85
[29] Subbu S, Pan Jie, Feng Minb, et al. Micelle-like nanoparticles of PLA–PEG–PLA triblock copolymer as chemotherapeutic carrier[J]. International Journal of Pharmaceutics.2005, 298:219~32
[30] Noton Dutta, Kaushiki Mazumdar, Sujata Dastidar,et al. Activity of diclofenac used alone and in combination with streptomycin against Mycobacterium tuberculosis in mice[J]. International Journal of Antimicrobial Agents.2007,30 :336~40
[31] Thirumala Govender, Trevor Riley, Touraj Ehtezazi, et al.Defining the drug incorporation properties of PLA–PEG nanoparticles [J]. International Journal of Pharmaceutics 199 (2000) 95~110
[32]中国防痨协会.结核病诊断细菌学检验规程[J].中国防痨杂志.1996, 18(2):80~5
[33] Dutt M, Khuller GK. Therapeutic efficacy of poly(DL-lactide- co ethylene glycol)- encapsulated antitubercular drugs against Mycobacterium tuberculosis infection induced in mice. Antimicrob Agents Chemother. 2001; 45: 363~6.
[34] BurmanW J,Stone B L,Reves R R,et a1.The incidence of false positive cultures for Mycobacterium tuberculosis[J].Am J Respir Crit Care Med.1997,155(1):321-6
[35] Pablos M. Global surveillance for antituberculosis-drug resistance,1994–1997 [J]. The New England Journal of Medicine. 1998, 338:1641~9.
[36] J Dobson. Gene therapy progress and prospects:magnetic nanoparticle-based gene delivery[J]. Gene Therapy.(2006, (13): 283~7
[1] David A. Tuberculosis of the Musculoskeletal System [J] .Techniques in Orthopaedics.2005, 20 (2): 167~78
[2]罗卓荆.全国脊柱结核治疗专题座谈会纪要(N).中华骨科杂志.2007,27(9):669
[3] Global Tuberculosis Control: surveillance, planning, financing. World Health Organization.2007 (WHO/HTM/TB/2007.376)
[4] Tuli SM. General principles of osteoarticular tuberculosis [J]. Clinical Orthopedics Related Research. 2002, 398:11~9
[5] Pablos M. Global surveillance for antituberculosis-drug resistance, 1994–1997 [J]. The New England Journal of Medicine.1998, 338:1641~9
[6]朱莉贞.如何正确服用抗结核药物[J].中华结核和呼吸杂志. 2007, 3O (6): 476.
[7] Thomas R. The DOTS Strategy for Controlling the Global Tuberculosis Epidemic[J].Clinics in Chest Medicine.2005,26(2): 197~205.
[8]陈孝平.外科学(八年制[M].北京:人民卫生出版社.2005:1085.
[9] Zulia Wang .Treatment of spinal tuberculosis with ultrashort-course chemotherapy in conjunction with partial excision of pathologic vertebrae[J]. The Spine Journal. 2007, 7:671~81
[10] Umubyeyi A. Low levels of second-line drug resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Rwanda [J] .International Journal of Infectious Diseases. 2008, 12:152~6.
[11] Wolfart M. Mycobacterium tuberculosis resistance in HIV-infected patients from a tertiary care teaching hospital in Porto Alegre, southern Brazil [J]. Transactions of the Royal Society of Tropical Medicine and Hygiene.2008, 102:421~5
[12] Benator D. Rifapentine and isoniazid once a week versus rifampicin and isoniazid twice a week for treatment of drug-susceptible pulmonary tuberculosis in HIV-negative patients: a randomised clinical trial [J].The Lancet. 2002,360(9332): 528~34.
[13]王泓.常用抗结核药[J].中华综合临床医学杂志.2006, 8(4): 60~1.
[14]韩凤满,程宏,吴启秋,等.骨关节结核病灶结核菌L型状况的观察[J].中国防痨杂志.2004,04(26)233~7.
[15] Y. Bhusal.Determination of in vitro synergy when three antimicrobial agents arecombined against Mycobacterium tuberculosis[J].International Journal of Antimicrobial Agents. 2005, 26:292~7.
[16] Murugesan Dinakaran. Novel ofloxacin derivatives: Synthesis, antimycobacterial and toxicological evaluation[J].Bioorganic & Medicinal Chemistry Letters . 2008,18: 1229~36.
[17]谭守勇.氟喹诺酮类药物在耐多药结核病治疗中的应用[J].中国防痨杂志.2005, 04:262-7
[18] Maria V. Papadopoulou. NLCQ-1 and NLCQ-2, two new agents with activity against dormant Mycobacterium tuberculosis. International Journal of Antimicrobial Agents. 2007, 29:724~7.
[19]马光辉.高分子微球材料[M].北京:化学工业出版社.2005: 223~5
[20] Zahoor Ahmad1. Novel chemotherapy for tuberculosis: chemotherapeutic potential of econazole- and moxifloxacin-loaded PLG nanoparticles [J].International Journal of Antimicrobial Agents. 2008, 31: 142~6.