新型淀粉纳米囊人工红细胞有效性初步研究

目的:采用季铵盐阳离子淀粉为囊材包裹牛血红蛋白,制备成纳米级的新型季铵盐阳离子淀粉人工红细胞(Nanosized Cationic Amylose-encapsulated Hemoglobins,NCAHbs)并评价此新型人工红细胞的有效性.方法:应用大鼠重度失血性休克模型,将50只健康SD大鼠随机分成5组:休克对照组(Control组,n=10)、淀粉纳米囊人工红细胞组(NCAHbs组,n=10)、Ringer乳酸盐溶液组(Ringer's组,n=10)、羟乙基淀粉组(HES组,n=10)和全血组(Blood组,n=10);动态监测平均动脉血压(MAP),于休克前(T0)、休克末(T1)及复苏后1 h(T2)测定动脉血气指标,并记录24h内大鼠的存活情况.结果:Ringer's组、HES组复苏后大鼠MAP仍较低,NCAHbs组复苏后MAP接近Blood组,并能有效维持;复苏后1h,Blood组和NCAHbs组PCO2、PO2均得到改善,接近休克前基础水平;仅NCAHbs组及Blood组术后24h生存率为100％.结论:新型淀粉纳米囊人工红细胞能够有效的扩容、携氧,明显改善重度失血性休克时机体和组织的氧供,是较为理想的人工氧载体.     

壳聚糖-有机累托石/海藻酸钠复合纳米粒的制备

目的:以BSA作为模型药物,制备壳聚糖季铵盐-OREC复合物纳米微粒,建立一种安全有效的药物控释传递系统。方法:超声条件下,制备不同质量比的具有壳聚糖硅酸盐插层结构的复合物纳米微粒,观察其形态学特征、进行红外光谱分析。同时,测定OREC对BSA包封率和载药量的影响。结果:成功制备了不同质量比的OREC-HTCC纳米粒子。电镜结果显示纳米粒呈圆球形,均匀,平均粒径约为30nm。红外图谱分析证实,HTCC插入了OREC插层中,BSA成功地包裹入HTCC-ALG/OREC混合材料制备的纳米微粒。加入OREC后,纳米粒子的包封率及载药量均明显提高,但随着加入量的增加,包封率及载药量逐渐减少。结论:OREC-HTCC纳米粒子是良好的蛋白药物载体,具有粒径小、包封率高、缓释效果好等优点,为CS-OREC作为潜在的药物给药系统的进一步应用提供科学依据。     

牛血红蛋白PLGA纳米粒的制备及其体外功能的研究

目的:利用poly(lactide-co-glycolide)(PLGA)和poly(styrene-co-4-styrene-sulfonate)(PSS)制备带负电荷的牛血红蛋白纳米粒,并对其载氧性能和体外性能进行评价.方法:采用溶剂蒸发法制备出空白的PLGA-PSS的空白纳米粒,通过改变pH值,吸附牛血红蛋白,从而制备出牛血红蛋白PLGA纳米粒.通过TEM、粒径、Zeta电位、包裹率和载药量、体外释放及其携氧能力对该纳米粒进行了综合分析.结果:Hb-PLGA-PSS-NPsTEM电镜下呈类球形,平均粒径为226.8±23.4nm,Zeta电位为-68.62 mV,优化条件后最大包裹率约为99.3%,载药量约为28.6%,在37℃,pH7.4的PBS溶液中释放缓慢.通过对Hb结构的分析表明此工艺未对蛋白的结构造成影响,体外携氧实验测量了P50(29mmHg)和Hill系数(2.036),结果说明该Hb纳米微囊具有良好携氧功能.结论:成功制备了一种Hb-PLGA-PSS-NPs纳米粒,稳定性好,具有很好的携氧能力.     

盐酸洛拉曲克脂质体的制备及其质量考察

目的:制备盐酸洛拉曲克脂质体并考察其理化特性。方法:采用薄膜挤压-硫酸铵梯度法制备盐酸洛拉曲克脂质体,透射电镜及激光粒度分析仪分别观察和检测其粒径大小及分布,通过紫外分光光度法测定包封率及评估体外释药试验。结果:制备的盐酸洛拉曲克脂质体包封率达83.6%±2.37%,粒径103.5±26nm且分布均匀。24h体外释放实验结果提示约有66.5%的盐酸洛拉曲克从脂质体释放出来。结论:新制备的盐酸洛拉曲克脂质体粒径大小均匀,包封率尚有提高空间,具有体外缓慢释药的特性。     

自供氧光敏载体系统的制备工艺优化及释氧性能研究

目的:制备乙基纤维素(EC)载过氧化钙(CaO_2)和光敏剂血卟啉单甲醚(HMME)的自供氧微球,对其制备工艺进行优化,并考察其体外自释氧行为。方法:采用液中干燥法制备自供氧微球,设计单因素考察实验,以粒径和CaO_2/HMME包封率作为微球的质量评价指标,研究EC浓度、有机/水相体积比、水相温度、CaO_2投料量对微球性能的影响,从而筛选最优工艺,采用动态透析法测定微球中氧气的释放行为,以单线态氧荧光探针(SOSG)评价其促单线态氧生成能力。结果:通过单因素考察实验获得自供氧微球的最佳制备处方为:EC浓度为1%、有机/水相体积比1:3、水相温度30℃、CaO_2投料量40 mg;按照优化工艺制备的自供氧微球的粒径为(1198±147) nm、HMME包封率(91.83±3.48)%、CaO_2包封率(89.14±1.67)%、3 h内氧气累计释放量(99.87±4.32) m M、单线态氧释放量显著高于无CaO_2对照组。结论:优化的自供氧微球制备工艺合理可行,药物包封率高,且可增加单线态氧的生成。     

质粒纳米乳剂的制备与阴离子交换色谱测定方法

以纳米乳剂为载体包裹草原兔尾鼠卵透明带3DNA疫苗,制备获得了纳米乳剂DNA疫苗,并对其进行了质量评价。采用界面乳化法制备质粒纳米乳剂,用电子显微镜测定了其粒径及其分布,根据质粒的带电特性,利用强阴离子Q SepharoseTMXL色谱柱分离纳米乳剂和游离质粒,建立了强阴离子交换柱质粒纳米乳剂包封率的快速测定方法。结果表明:制备的纳米乳剂DNA疫苗的平均粒径为(23±10)nm,包封率为80.5%。选择0.05mol/L的Tris-HCl为平衡液,流速为0.7mL/min,紫外检测波长260nm,柱温30℃,进样量为2mL的实验条件,质粒的含量和峰面积的线性关系良好(r=0.9983),加样回收率在95%以上,该方法简便快速、灵敏度高,重复性好,可用于纳米乳剂DNA疫苗包封率的快速测定。     

盐酸洛拉曲克脂质体的制备及其质量考察

目的：制备盐酸洛拉曲克脂质体并考察其理化特性。方法：采用薄膜挤压-硫酸铵梯度法制备盐酸洛拉曲克脂质体,透射电镜及激光粒度分析仪分别观察和检测其粒径大小及分布,通过紫外分光光度法测定包封率及评估体外释药试验。结果：制备的盐酸洛拉曲克脂质体包封率达83.6%±2.37%,粒径103.5±26nm且分布均匀。24h体外释放实验结果提示约有66.5%的盐酸洛拉曲克从脂质体释放出来。结论：新制备的盐酸洛拉曲克脂质体粒径大小均匀,包封率尚有提高空间,具有体外缓慢释药的特性。     

万古霉素脂质体的制备及质量考察

目的:制备万古霉素脂质体并考察其质量.方法:采用薄膜分散冻干法制备万古霉素脂质体,透射电镜观察其粒径分布,通过紫外分光光度法测定包封率和体外释要特性.结果:透射电镜结果显示脂质体粒径大小均匀,测得平均包封率为79.23%±4.13%,体外释放度实验结果提示,振荡24h后,约有65%的万古霉素药物从脂质体释放出来,体外抑菌试验显示,6周后仍然有抑菌圈的形成.结论:薄膜分散冻干法适于制备万古霉素脂质体,该制剂稳定性好,粒径大小均匀,包封率高,具有体外缓慢释药的特性.     

正交试验法优选PP1脂质体的制备工艺

目的:制备PP1脂质体,筛选最优处方。方法:用薄膜水化法制备PP1脂质体,以高效液相色谱法(HPLC)测定PP1脂质体的包封率,以包封率为主要指标,选取药脂比、胆固醇磷脂比、温度和水化时间为因素,用正交试验筛选最优配方。结果:用薄膜水法制备的PP1脂质体的最优处方的组成为:药脂比为1:10,胆固醇与二棕榈酰磷脂酰胆碱(DPPC)比为1:8,温度为40℃,水化时间为3 min。平均包封率为(63.27±3.32)%。PP1脂质体的平均粒径为(157.73±9.74)nm,Zeta电位为(-4.74±0.44)m V。结论:用薄膜水化法制备出的PP1脂质体包封率高,形态和粒径均匀,重现性好,为研究其在眼部的缓释作用奠定了基础。     

盐酸米托蒽醌聚乙二醇化脂质体的制备及包封率考察

目的:制备盐酸米托蒽醌聚乙二醇化(PEG化)脂质体,建立包封率测定方法.方法:采用乙醇注入结合高压均质法制备空白PEG化脂质体;以铵根离子梯度法进行主动载药制备盐酸米托蒽醌PEG化脂质体;采用G-25葡聚糖凝胶色谱分离脂质体和游离药物;使用紫外-可见分光光度法测定脂质体的包封率.结果:空白脂质体平均粒径为88.7nm,载药后粒径为95.3nm;在所建立色谱条件下,脂质体与游离米托蒽醌分离良好;盐酸米托蒽醌在0.5～10μg·ml-1范围内线性关系良好(R 2=0.9997),精密度高;脂质体的平均包封率大于96％.结论:乙醇注入-高压均质法结合铵根离子主动载药法适用于制备盐酸米托蒽醌PEG化脂质体;所建立分析方法简单快捷、准确可靠,可用于盐酸米托蒽醌长循环脂质体包封率的测定.     

复乳法制备红细胞代用品

以单甲氧基聚乙二醇聚乳酸共聚物 [methoxypoly(ethyleneglycol) poly DL lactide,PELA]为膜材 ,用W /O/W的复乳 溶剂扩散法制备了包埋牛血红蛋白 (bovinehemoglobin ,BHb)的微胶囊作为人造红细胞。研究发现复乳过程搅拌分散速率、有机溶剂种类和固化方法对BHb活性有明显影响。当搅拌分散速率小于 90 0 0r/min、以乙酸乙酯为有机相时 ,采用复乳 溶剂扩散法包埋BHb的过程对BHb活性无明显影响。当搅拌分散速率高于 12 0 0 0r/min时 ,BHb活性降低。复乳 溶剂扩散法制备微胶囊过程中固化液体积与微胶囊中BHb活性密切相关 ,通过增大固化液和复乳液的体积比可较好地保持BHb的活性。最后制得了粒径 10 μm左右、包埋率 93%、P50 和Hill系数均接近于天然BHb的微胶囊。     

血红蛋白携氧-释氧动力学研究

本文研究了鸡、家兔、鲤鱼、蟾蜍4种实验动物血红蛋白(hemoglobin,Hb)携氧-释氧动力学过程,初步建立Hb携氧-释氧动力学研究方法,并探讨Hb携氧-释氧动力学过程与动物生存环境之间的关系.结果显示:4种动物Hb携氧动力学曲线均呈"S"形曲线特征,与传统的Hb氧解离曲线(oxygen dissociation curve,ODC)相似;同时不同动物Hb携氧-释氧动力学曲线也有各自特点,如鸡Hb释氧时间长达(1 411±6)S;在Hb携氧.释氧曲线I阶段,鲤鱼上升斜率远大于家兔等.提示Hb携氧-释氧动力学曲线可反映不同动物Hb携氧效率的差异.与传统ODC参数P50相对应,由动力学曲线可得到Hb携氧动力学参数T50°T50是Hb达到50%氧饱和度所需时间,可直观反映Hb携氧效率的差异.4种实验动物Hb均有较稳定的T50,从大到小依次为:鸡、家兔、鲤鱼和蟾蜍.对Hb携氧动力学曲线与ODC综合分析,可得到Hb携氧效能参数E50,表示Hb达到50%氧饱和度所用时间与环境氧分压之间的关系,即E50(50% Sat,Xeo2,yr).E50有可能成为全面评价Hb携氧效能的综合指标.     

Future prospects for artificial blood

Concern about potential infective agents in donated blood has stimulated the recent development of blood substitutes. Chemically cross-linked hemoglobins are already undergoing clinical trials and might soon be ready for routine use. New generations of modified hemoglobin are being prepared to modulate the effects of nitric oxide and oxygen radicals, and artificial red blood cells are also under development.     

Nanobiotechnological modification of hemoglobin and enzymes from this laboratory

Polyhemoglobin is formed by the nanobiotechnological assembling of hemoglobin molecules into soluble nanodimension complex. A further step involves the nanobiotechnological assembly of hemoglobin, catalase and superoxide dismutase into a soluble nanodimension complex. This acts both as oxygen carrier and antioxidant to prevent the oxidative effects of hemoglobin. A further step is the preparation of nanodimension artificial red blood cells that contain hemoglobin and all the enzymes present in red blood cells. Other approaches include a polyhemoglobin-fibrinogen that acts as an oxygen carrier with platelet-like activity, and a polyhemoglobin-tyrosinase to retard the growth of a fatal skin cancer, melanoma.     

修饰血红蛋白作为红细胞代用品的研究进展和挑战

血源紧缺和病毒污染问题推动了血液代用品的研究,以血红蛋白为代表的红细胞代用品成为研究的重点。为克服血红蛋白直接使用的毒副作用,各种修饰技术得到了迅速发展,其中包括双阿司匹林交联、戊二醛交联、棉子糖交联、聚乙二醇偶联、脂质体包埋、生物可降解高分子包埋等。其中一些技术已经形成规模化制备工艺,产品进入临床试验,有的已在个别国家上市。鉴于这项研究意义重大,我国有关研究已经起步并正在迅速发展,各国同行的研究有重要的参考价值,因此有必要对近年来血红蛋白作为红细胞的代用品研究状况进行分析,明确面临的挑战,这将有利于发展更全面和有效的研究方案,以期取得突破性进展。     

Effect of motor activity of different intensity upon the blood composition and respiratory function in domestic animals during their first months of life

Responses of the blood content, breathing function and gaseous metabolism to changes of motional activity level in calves in the first 30 days of a postnatal period were investigated. Keeping of calves in conditions natural for mature animals at different motor activity levels as compared with the locomotion limitation leads to a lesser reduction of hemoglobin content, haematocrit ratio, red cells and reticulocytes count in blood of calves during first 30 days of life. In comparison with calves with additional motor activity, 10-day old calves with natural motor activity and locomotion limitation showed an increase of the protein level and reduction of glucose and cholesterol in the blood. Locomotion limitation of calves during the first 30 days of postnatal period caused reduction of the blood oxygen carrying capacity (erythrocytes and reticulocytes count) with simultaneous decrease of breathing function and gaseous metabolism efficiency (breathing output, minute ventilation, carbon dioxide output and oxygen uptake).     

Determination of size distribution and encapsulation efficiency of liposome-encapsulated hemoglobin blood substitutes using asymmetric flow field-flow fractionation coupled with multi-angle static light scattering

In this study, we investigated the size distribution, encapsulation efficiency, and oxygen affinity of liposome-encapsulated tetrameric hemoglobin (LEHb) dispersions and correlated the data with the variation in extruder membrane pore size, ionic strength of the extrusion buffer, and hemoglobin (Hb) concentration. Asymmetric flow field-flow fractionation (AFFF) in series with multi-angle static light scattering (MASLS) was used to study the LEHb size distribution. We also introduced a novel method to measure the encapsulation efficiency using a differential interferometric refractive index (DIR) detector coupled to the AFFF-MASLS system. This technique was nondestructive toward the sample and easy to implement. LEHbs were prepared by extrusion using a lipid combination of dimyristoyl-phosphatidylcholine, cholesterol, and dimyristoyl-phosphatidylglycerol in a 10:9:1 molar ratio. Five initial Hb concentrations (50, 100, 150, 200, and 300 mg Hb per mL of buffer) extruded through five different membrane pore diameters (400, 200, 100, 80, and 50 nm) were studied. Phosphate buffered saline (PBS) and phosphate buffer (PB) both at pH 7.3 were used as extrusion buffers. Despite the variation, extrusion through 400-nm pore diameter membranes produced LEHbs smaller than the pore size, extrusion through 200-nm membranes produced LEHbs with diameters close to the pore diameter, and extrusion through 100-, 80-, and 50-nm membranes produced LEHbs larger than the pore sizes. We found that the choice of extrusion buffer had the greatest effect on the LEHb size distribution compared to either Hb concentration or extruder membrane pore size. Extrusion in PBS produced larger LEHbs and more monodisperse LEHb dispersions. However, LEHbs extruded in PB generally had higher Hb encapsulation efficiencies and lower methemoglobin (metHb) levels. The choice of extrusion buffer also affected how the encapsulation efficiency correlated with Hb concentration, extruder pore size, and the metHb level. The most optimum encapsulation efficiency and amount of Hb entrapped were achieved at the highest Hb concentration and the largest pore size for both extrusion buffers (62.38% and 187.14 mg Hb/mL of LEHb dispersion extruded in PBS, and 69.98% and 209.94 mg Hb/mL of LEHb dispersion extruded in PB). All LEHbs displayed good oxygen-carrying properties as indicated by their P(50) and cooperativity coefficients. LEHbs extruded in PB had an average P(50) of 23.04 mmHg and an average Hill number of 2.29, and those extruded in PBS had average values of 27.25 mmHg and 2.49. These oxygen-binding properties indicate that LEHbs possess strong potential as artificial blood substitutes. In addition, the metHb levels in PB-LEHb dispersions are significantly low even in the absence of antioxidants such as N-acetyl-L-cysteine.     

Biochemical and rheodynamic properties of red blood cells crosslinked with glutaraldehyde.

New data on the properties of red blood cells (RBC) cross-linked with glutaraldehyde are presented (see also Biochem. Biophys. Res. Commun., 1988, 156, 970-977). Equilibrium and kinetic data show that by carrying out the fixation procedure in the absence of oxygen but in the presence of allosteric effectors (e.g., stabilizing the low-affinity (T) quaternary state of hemoglobin), it is possible to maintain, at least in part, the biochemical functions of the crosslinked hemoglobin inside the cell. Moreover, we show that the oxygen affinity of fixed red blood cells (RBC) is still modulated, even though to a smaller degree, by the allosteric effector bezafibrate (BZF), which is able to cross the fixed RBC membrane. Membrane filtration experiments indicate that the higher rigidity of fixed RBC alters significantly their rheodynamic properties and show that in order to exploit "engineered" RBC as "blood substitutes," more flexible cross-linking reagents may offer significant advantages.     

The binding of hemoglobin to membranes of normal and sickle erythrocytes.

The binding of hemoglobins A, S, and A2 to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with 14C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A2, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4-22 degrees C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied. An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites. These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.     

Targeted O2 delivery by low-P50 hemoglobin: a new basis for O2 therapeutics

To assess O2 delivery to tissue by a new surface-modified, polyethylene glycol-conjugated human hemoglobin [MP4; Po2 at 50% saturation of hemoglobin (P50); 5.4 mmHg], we studied microcirculatory hemodynamics and O2 release in golden Syrian hamsters hemodiluted with MP4 or polymerized bovine hemoglobin (PolyBvHb; P50 54.2 mmHg). Comparisons were made with the animals' hemodiluted blood with a non-O2 carrying plasma expander with similar solution properties (Dextran-70). Systemic hemodynamics (arterial blood pressure and heart rate) and acid-base parameters were not correlated with microhemodynamics (arteriolar and venular diameter, red blood cell velocity, and flow). Microscopic measurements of Po2 and the O2 equilibrium curves permitted analysis of O2 release in precapillary and capillary vessels by red blood cells and plasma hemoglobin separately. No significant differences between the groups of animals with respect to arteriolar diameter, flow, or flow velocity were observed, but the functional capillary density was significantly higher in the MP4-treated animals (67%) compared with PolyBvHb-treated animals (37%; P < 0.05) or dextran-treated animals (53%). In the PolyBvHb-treated animals, predominant O2 release (both red blood cells and plasma hemoglobin) occurred in precapillary vessels, whereas in MP4 animals most of the O2 was released from both red blood cells and plasma hemoglobin in capillaries. Base excess correlated directly with capillary O2 release but not systemic O2 content or total O2 release. Higher O2 extraction of both red blood cell and plasma hemoglobin in capillaries represents a new mechanism of action of cell-free hemoglobin. High O2 affinity appears to be an important property for cell-free hemoglobin solutions.