黄芪多糖纳米乳的制备及其免疫佐剂效应研究
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摘要
本研究以纳米乳给药系统为载体，对中药黄芪多糖进行剂型改造，以研制出可用于生产的纳米疫苗佐剂。本研究在空白纳米乳的基础上，利用伪三元相图，筛选出黄芪多糖纳米乳（APS-NE）给药系统，并对该系统的质量稳定性、安全性、药效学性能进行了研究。结果如下：
第一部分：旨在筛选出合适的油、表面活性剂和助表面活性剂及其配方，制备W/O型空白纳米乳。纳米乳是多组分的体系，其形成会受到多种因素的影响，如表面活性剂、助表面活性剂、油相、表面活性剂/助表面活性剂质量比（Km）以及药物的浓度等。本研究通过对形成的纳米乳外观和性质进行判断，结合各种因素对纳米乳伪三元相图的影响，筛选出W/O型空白纳米乳配方的最佳组成为：Tween80/Span80/无水乙醇/液体石蜡/水溶液，且Tween80∶Span80（m∶m）＝3∶1，Km=4:1。
第二部分：旨在最终确定W/O型APS-NE处方，并对纳米乳质量进行了评价。通过对APS-NE粒度、热力学稳定性、体外释药性等的研究，确定APS-NE处方为Tween80∶Span80∶无水乙醇∶液体石蜡∶水溶液=31∶11∶12∶22∶24（质量比），依此制备APS-NE样品，得到淡黄色透明APS-NE，证明为W/O型纳米乳。APS-NE液滴呈大小均一的球形，平均粒径为63.7nm，粒径分布范围窄。APS-NE的pH值为6.71，浊点74.1℃，粘度为168.5mPa s，APS含量经测定为9.6mg/mL。经光照试验、加速试验和长期稳定性试验，表明APS-NE均能稳定保持淡黄色透明态，没有出现分层、浑浊、絮凝和药物析出现象。
第三部分：旨在通过肌肉刺激性试验、热原试验、急性毒性和长期毒性试验以及细胞毒性试验研究，对研制的APS-NE的安全性进行评价。结果显示：空白纳米乳和APS-NE对肌肉平均刺激反应级别均低于1级；热原检查合格； LD_(50)分别为2.41mL和2.09mL（分别相当于24.1倍的空白纳米乳或20.9倍小鼠正常用药量），且95%可信区间分别为2.31～2.54mL和1.94～2.18mL；长期毒性试验显示空白纳米乳和APS-NE在20.27mL/Kg体重（按大鼠体重74g计算）范围长期连续使用对大鼠没有明显毒性伤害；一定浓度范围内（≤300μg/mL）对L929细胞无毒性作用。本研究表明，APS-NE的毒性极低，安全性好，满足药物临床试验的安全性要求。
第四部分：将APS-NE应用于雏鸡，观察其对血清新城疫抗体水平的影响。结果表明，第一次免疫接种14d后，APS和APS-NE两剂量组均显著提高雏鸡血清新城疫抗体滴度（P≤0.05）。与APSL组相比，APSH、APSL-NE和APSH-NE组雏鸡血清新城疫抗体滴度都有显着增加（P≤0.05）。而APSL-NE与APSH、APSH-NE与APSH和APSH-NE与APSL-NE各组雏鸡之间血清新城疫抗体滴度则差异不显著（P≥0.05）。此外，本研究发现，在新城疫抗体滴度随日龄增长变化曲线中，在14d到42d和42d到63d时间段内，APSH-NE和APSL-NE比对照组、APSL组以及APSH组有较高的抗体滴度。以上结果表明，APS和APS-NE，能够促使雏鸡较早产生特异性抗体并使之维持更长时间，提高对疫苗的免疫效果，其中以APS-NE表现尤为突出。本研究结果证实，在提高雏鸡血清ND抗体滴度方面，APS-NE比单一使用APS具有较好的效果，是一种理想的新型疫苗佐剂。
第五部分：为研究APS-NE对雏鸡免疫功能的调节作用机制，本章采用流式细胞技术检测了雏鸡外周血CD4~+和CD8~+T淋巴细胞数量的动态变化。结果发现APS-NE和APS均能够提高雏鸡外周血CD4~+和CD8~+T淋巴细胞的数量，降低由于注射疫苗引起的雏鸡外周血中CD4~+/CD8~+比值的升高。说明APS-NE和APS均能通过上调雏鸡外周血中CD4~+和CD8~+T淋巴细胞水平，增强机体对抗原的免疫应答，从而增强机体的免疫力和抵抗力。且因为纳米乳剂所具有的诸多特点，APS-NE对雏鸡的应用效果更为显著。
第六部分：本研究将APS-NE作用于体外培养的鸡脾脏淋巴细胞，以研究APS-NE对体外培养的鸡脾脏淋巴细胞增殖、合成NO以及分泌Th1类细胞因子(IFN-γ，IL-2等)和Th2类细胞因(IL-4)的影响。结果显示APS-NE和APS均能显著刺激淋巴细胞增殖(P≤0.05)，促进鸡脾脏淋巴细胞产生NO、NOS、iNOS和IL-2、IL-4、IFN-γ(P≤0.05)，且各检测指标中APS-NE作用均比APS更显著(P≤0.05)。另外，随着药物剂量的提高，本研究对体外培养鸡脾脏淋巴细胞的调节作用效果也变得更强，尤其是APS-NE两个剂量组之间的差异均达到了显著程度(P≤0.05)。结果表明APS-NE和APS均能通过增强鸡脾脏淋巴细胞NOS和iNOS活力，促进鸡脾脏淋巴细胞NO的产生，刺激T细胞不同亚群来调节Th细胞进一步分泌细胞因子，如IL-2、IL-4、IFN-γ等，进而发挥免疫调节作用，但APS-NE的作用更显著。另外，APS-NE对体外培养淋巴细胞分泌NO和细胞因子的作用具有一定的量效关系，在本研究中，均以300μg/mL（高剂量组）的效果最佳。
This study was used to reform the dosage formunation of the Chinese traditionalmedicine Astragalus Polysaccharide with nanoemulsion delivery system as the carrier. On thebase of the establishment of blank nanometer emulsion, through the Pseudo-ternary phasediagrams, the Astragalus polysaccharide nanoemulsion (APS-NE) delivery system wasprepared. The study had researched the quality stability, security and pharmacodynamicproperties of the APS-NE, and the effect on the adjuvant and its mechanism.
Part One: Nanoemulsion is a multicomponent system, which will be affected by manyfactors, such as surfactant, cosurfactant, oil phase, surfactant and cosurfactant mass ratio(Km) and drug concentration. This study observed the appearance and properties ofjudgment of the formunation of the nanoemulsion, combined with the Pseudo-ternary phasediagrams, screened out the formulation of the W/O astragalus polysaccharide nanoemulsion.The optimum composition was: Tween80/Span80/ethanol/liquid paraffin/water solution,and Tween80: Span80(M: m)=3:1, Km=4:1.
Part Two: Based on the APS-NE size, thermodynamic stability, drug release in vitro andso on, the APS-NE prescription was determined Tween80: Span80: ethanol: liquid paraffin:water=31:11:12:22:24(mass ratio). The APS-NE samples prepared with the mass ratiowere pale yellow transparent. The APS-NE was easy to be dyed red by oil soluble dyetonyred, and could maintain clear and transparent state, without flocculation and delamination,by liquid paraffin dilution or by high speed centrifugation. The APS-NE was proved a W/Onanoemulsion. APS-NE droplet was uniform size spherical, particle diameter less than100nm, no adhesion, good dispersibility, average particle diameter of63.7nm measured bylaser particle size instrument, and narrow range of particle size distribution. In addition,APS-NE was pH6.71, point74.1C, viscosity of168.5mPa s, APS content9.6mg/mL. Byillumination test, accelerated test and long term stability test, it showed that APS-NE couldkeep stable light yellow transparent state, no delamination, no turbidity, no flocculation andno drug precipitation phenomenon.
Part Three: the muscle average stimulus response levels of blank nanometer emulsionand APS-NE were all lower than1. pyrogen inspection was qualified. LD_(50)was2.41mL and 2.09mL (respectively equivalent to24.1times and20.9times of the normal dosage in mice),and the95%confidence intervals were2.31~2.54mL and1.94~2.18mL. long term toxicitytest showed that the blank nanoemulsion and APS-NE in20.27mL/Kg body weight (in ratbody weight74g calculation) range of long-term continuous use on rats were no obvioustoxic injury. A certain concentration range (less than300μg/mL) on L929cells was no toxiceffect. This study shows that APS-NE has very low toxicity, good security.
Part Four: This study was conducted to evaluate the effects of Astragalus polysaccharide(APS) nanoemulsion (NE) on the production of the serum Newcastle disease antibody invaccinated chickens. The results showed that APS and APS-NE at high (H) dosage and low(L) dosage all significantly improved the serum ND HI antibody titer of the chickenscompared with the control of medicine-free chickens after d14(P≤0.05). The serum ND HIantibody titers of APSH, APSL-NE and APSH-NE were significantly increased contrast to thatof APSL(P≤0.05). But, it was not significant between APSL-NE and APSH, APSH-NE andAPSH, APSH-NE and APSL-NE. In addition, we found that the curves of APSL-NE andAPSH-NE were higher from d14to d42and from d42to d63, than that of the control, APSL,even APSH. The results indicated that APS and APS-NE, especially APS-NE, could promotespecific antibody production earlier and maintain it longer, and thus improve the immuneeffect of the vaccine. Because of the similar composition of nanoemulsion to vaccine,APS-NE would be a possibility adjuvant of the vaccine. In summary, this research confirmsthat APS-NE displayed better effects on improvement the serum ND HI antibody titer inchickens and would be expected to be effective as component drugs of a novel vaccineadjuvant.
Part Five: To study the APS-NE on immune function of mechanism, this chapter usedflow cytometric to detect the population and dynamics of chickens peripheral blood CD4~+and CD8~+T lymphocyte. The results showed that APS-NE and APS were able to raise thepopulation of chickens peripheral blood CD4~+and CD8~+T lymphocyte, reduce the increasingdynamics of the peripheral blood CD4~+/CD8~+ratio caused by the vaccine in chicken.APS-NE and APS both enhanced the body of an immune response to an antigen, throughup-regulation the peripheral blood CD4~+and CD8~+T lymphocyte levels in chickens, thusenhanced the body's immunity and resistance. While the effect of APS-NE was more obviousbecause of the characteristics of nanoemulsion.
Part Six: The study observed the effects of APS-NE on the culture of chicken spleniclymphocytes, NO class, Th1cell factor (IFN-γ, IL-2) and Th2cell factor (IL-4) by culturedchicken splenic lymphocyte proliferation in vitro. The results showed that APS-NE and APScould significantly stimulate lymphocyte proliferation(P≤0.05), promote chicken splenic lymphocytes to produce NO, NOS, iNOS and IL-2, IL-4, IFN-γ(P≤0.05), and various indexesof APS-NE were more significant role than APS(P≤0.05). In addition, with the increasementof the drug dose, the regulate effect of the drugs became stronger on chicken spleniclymphocytes in vitro, especially two dose groups of APS-NE differences between bothreached a significant level (P≤0.05). The results showed that APS-NE promoted thepopulation of the chicken splenic lymphocytes on the generation of NO, by enhancement ofchicken splenic lymphocytes on NOS and iNOS activity, and stimulated further secretion ofcytokines of the different subsets T cells, such as IL-2, IL-4, IFN-γ, thus played betterimmune regulation effects than APS.In addition, the secrete of NO and cytokines on culturedlymphocytes had certain dose-effect relationship to the APS-NE, and in the study,300μg/mL(high dose group) was the best.
第一部分：旨在筛选出合适的油、表面活性剂和助表面活性剂及其配方，制备W/O型空白纳米乳。纳米乳是多组分的体系，其形成会受到多种因素的影响，如表面活性剂、助表面活性剂、油相、表面活性剂/助表面活性剂质量比（Km）以及药物的浓度等。本研究通过对形成的纳米乳外观和性质进行判断，结合各种因素对纳米乳伪三元相图的影响，筛选出W/O型空白纳米乳配方的最佳组成为：Tween80/Span80/无水乙醇/液体石蜡/水溶液，且Tween80∶Span80（m∶m）＝3∶1，Km=4:1。
第二部分：旨在最终确定W/O型APS-NE处方，并对纳米乳质量进行了评价。通过对APS-NE粒度、热力学稳定性、体外释药性等的研究，确定APS-NE处方为Tween80∶Span80∶无水乙醇∶液体石蜡∶水溶液=31∶11∶12∶22∶24（质量比），依此制备APS-NE样品，得到淡黄色透明APS-NE，证明为W/O型纳米乳。APS-NE液滴呈大小均一的球形，平均粒径为63.7nm，粒径分布范围窄。APS-NE的pH值为6.71，浊点74.1℃，粘度为168.5mPa s，APS含量经测定为9.6mg/mL。经光照试验、加速试验和长期稳定性试验，表明APS-NE均能稳定保持淡黄色透明态，没有出现分层、浑浊、絮凝和药物析出现象。
第三部分：旨在通过肌肉刺激性试验、热原试验、急性毒性和长期毒性试验以及细胞毒性试验研究，对研制的APS-NE的安全性进行评价。结果显示：空白纳米乳和APS-NE对肌肉平均刺激反应级别均低于1级；热原检查合格； LD_(50)分别为2.41mL和2.09mL（分别相当于24.1倍的空白纳米乳或20.9倍小鼠正常用药量），且95%可信区间分别为2.31～2.54mL和1.94～2.18mL；长期毒性试验显示空白纳米乳和APS-NE在20.27mL/Kg体重（按大鼠体重74g计算）范围长期连续使用对大鼠没有明显毒性伤害；一定浓度范围内（≤300μg/mL）对L929细胞无毒性作用。本研究表明，APS-NE的毒性极低，安全性好，满足药物临床试验的安全性要求。
第四部分：将APS-NE应用于雏鸡，观察其对血清新城疫抗体水平的影响。结果表明，第一次免疫接种14d后，APS和APS-NE两剂量组均显著提高雏鸡血清新城疫抗体滴度（P≤0.05）。与APSL组相比，APSH、APSL-NE和APSH-NE组雏鸡血清新城疫抗体滴度都有显着增加（P≤0.05）。而APSL-NE与APSH、APSH-NE与APSH和APSH-NE与APSL-NE各组雏鸡之间血清新城疫抗体滴度则差异不显著（P≥0.05）。此外，本研究发现，在新城疫抗体滴度随日龄增长变化曲线中，在14d到42d和42d到63d时间段内，APSH-NE和APSL-NE比对照组、APSL组以及APSH组有较高的抗体滴度。以上结果表明，APS和APS-NE，能够促使雏鸡较早产生特异性抗体并使之维持更长时间，提高对疫苗的免疫效果，其中以APS-NE表现尤为突出。本研究结果证实，在提高雏鸡血清ND抗体滴度方面，APS-NE比单一使用APS具有较好的效果，是一种理想的新型疫苗佐剂。
第五部分：为研究APS-NE对雏鸡免疫功能的调节作用机制，本章采用流式细胞技术检测了雏鸡外周血CD4~+和CD8~+T淋巴细胞数量的动态变化。结果发现APS-NE和APS均能够提高雏鸡外周血CD4~+和CD8~+T淋巴细胞的数量，降低由于注射疫苗引起的雏鸡外周血中CD4~+/CD8~+比值的升高。说明APS-NE和APS均能通过上调雏鸡外周血中CD4~+和CD8~+T淋巴细胞水平，增强机体对抗原的免疫应答，从而增强机体的免疫力和抵抗力。且因为纳米乳剂所具有的诸多特点，APS-NE对雏鸡的应用效果更为显著。
第六部分：本研究将APS-NE作用于体外培养的鸡脾脏淋巴细胞，以研究APS-NE对体外培养的鸡脾脏淋巴细胞增殖、合成NO以及分泌Th1类细胞因子(IFN-γ，IL-2等)和Th2类细胞因(IL-4)的影响。结果显示APS-NE和APS均能显著刺激淋巴细胞增殖(P≤0.05)，促进鸡脾脏淋巴细胞产生NO、NOS、iNOS和IL-2、IL-4、IFN-γ(P≤0.05)，且各检测指标中APS-NE作用均比APS更显著(P≤0.05)。另外，随着药物剂量的提高，本研究对体外培养鸡脾脏淋巴细胞的调节作用效果也变得更强，尤其是APS-NE两个剂量组之间的差异均达到了显著程度(P≤0.05)。结果表明APS-NE和APS均能通过增强鸡脾脏淋巴细胞NOS和iNOS活力，促进鸡脾脏淋巴细胞NO的产生，刺激T细胞不同亚群来调节Th细胞进一步分泌细胞因子，如IL-2、IL-4、IFN-γ等，进而发挥免疫调节作用，但APS-NE的作用更显著。另外，APS-NE对体外培养淋巴细胞分泌NO和细胞因子的作用具有一定的量效关系，在本研究中，均以300μg/mL（高剂量组）的效果最佳。
This study was used to reform the dosage formunation of the Chinese traditionalmedicine Astragalus Polysaccharide with nanoemulsion delivery system as the carrier. On thebase of the establishment of blank nanometer emulsion, through the Pseudo-ternary phasediagrams, the Astragalus polysaccharide nanoemulsion (APS-NE) delivery system wasprepared. The study had researched the quality stability, security and pharmacodynamicproperties of the APS-NE, and the effect on the adjuvant and its mechanism.
Part One: Nanoemulsion is a multicomponent system, which will be affected by manyfactors, such as surfactant, cosurfactant, oil phase, surfactant and cosurfactant mass ratio(Km) and drug concentration. This study observed the appearance and properties ofjudgment of the formunation of the nanoemulsion, combined with the Pseudo-ternary phasediagrams, screened out the formulation of the W/O astragalus polysaccharide nanoemulsion.The optimum composition was: Tween80/Span80/ethanol/liquid paraffin/water solution,and Tween80: Span80(M: m)=3:1, Km=4:1.
Part Two: Based on the APS-NE size, thermodynamic stability, drug release in vitro andso on, the APS-NE prescription was determined Tween80: Span80: ethanol: liquid paraffin:water=31:11:12:22:24(mass ratio). The APS-NE samples prepared with the mass ratiowere pale yellow transparent. The APS-NE was easy to be dyed red by oil soluble dyetonyred, and could maintain clear and transparent state, without flocculation and delamination,by liquid paraffin dilution or by high speed centrifugation. The APS-NE was proved a W/Onanoemulsion. APS-NE droplet was uniform size spherical, particle diameter less than100nm, no adhesion, good dispersibility, average particle diameter of63.7nm measured bylaser particle size instrument, and narrow range of particle size distribution. In addition,APS-NE was pH6.71, point74.1C, viscosity of168.5mPa s, APS content9.6mg/mL. Byillumination test, accelerated test and long term stability test, it showed that APS-NE couldkeep stable light yellow transparent state, no delamination, no turbidity, no flocculation andno drug precipitation phenomenon.
Part Three: the muscle average stimulus response levels of blank nanometer emulsionand APS-NE were all lower than1. pyrogen inspection was qualified. LD_(50)was2.41mL and 2.09mL (respectively equivalent to24.1times and20.9times of the normal dosage in mice),and the95%confidence intervals were2.31~2.54mL and1.94~2.18mL. long term toxicitytest showed that the blank nanoemulsion and APS-NE in20.27mL/Kg body weight (in ratbody weight74g calculation) range of long-term continuous use on rats were no obvioustoxic injury. A certain concentration range (less than300μg/mL) on L929cells was no toxiceffect. This study shows that APS-NE has very low toxicity, good security.
Part Four: This study was conducted to evaluate the effects of Astragalus polysaccharide(APS) nanoemulsion (NE) on the production of the serum Newcastle disease antibody invaccinated chickens. The results showed that APS and APS-NE at high (H) dosage and low(L) dosage all significantly improved the serum ND HI antibody titer of the chickenscompared with the control of medicine-free chickens after d14(P≤0.05). The serum ND HIantibody titers of APSH, APSL-NE and APSH-NE were significantly increased contrast to thatof APSL(P≤0.05). But, it was not significant between APSL-NE and APSH, APSH-NE andAPSH, APSH-NE and APSL-NE. In addition, we found that the curves of APSL-NE andAPSH-NE were higher from d14to d42and from d42to d63, than that of the control, APSL,even APSH. The results indicated that APS and APS-NE, especially APS-NE, could promotespecific antibody production earlier and maintain it longer, and thus improve the immuneeffect of the vaccine. Because of the similar composition of nanoemulsion to vaccine,APS-NE would be a possibility adjuvant of the vaccine. In summary, this research confirmsthat APS-NE displayed better effects on improvement the serum ND HI antibody titer inchickens and would be expected to be effective as component drugs of a novel vaccineadjuvant.
Part Five: To study the APS-NE on immune function of mechanism, this chapter usedflow cytometric to detect the population and dynamics of chickens peripheral blood CD4~+and CD8~+T lymphocyte. The results showed that APS-NE and APS were able to raise thepopulation of chickens peripheral blood CD4~+and CD8~+T lymphocyte, reduce the increasingdynamics of the peripheral blood CD4~+/CD8~+ratio caused by the vaccine in chicken.APS-NE and APS both enhanced the body of an immune response to an antigen, throughup-regulation the peripheral blood CD4~+and CD8~+T lymphocyte levels in chickens, thusenhanced the body's immunity and resistance. While the effect of APS-NE was more obviousbecause of the characteristics of nanoemulsion.
Part Six: The study observed the effects of APS-NE on the culture of chicken spleniclymphocytes, NO class, Th1cell factor (IFN-γ, IL-2) and Th2cell factor (IL-4) by culturedchicken splenic lymphocyte proliferation in vitro. The results showed that APS-NE and APScould significantly stimulate lymphocyte proliferation(P≤0.05), promote chicken splenic lymphocytes to produce NO, NOS, iNOS and IL-2, IL-4, IFN-γ(P≤0.05), and various indexesof APS-NE were more significant role than APS(P≤0.05). In addition, with the increasementof the drug dose, the regulate effect of the drugs became stronger on chicken spleniclymphocytes in vitro, especially two dose groups of APS-NE differences between bothreached a significant level (P≤0.05). The results showed that APS-NE promoted thepopulation of the chicken splenic lymphocytes on the generation of NO, by enhancement ofchicken splenic lymphocytes on NOS and iNOS activity, and stimulated further secretion ofcytokines of the different subsets T cells, such as IL-2, IL-4, IFN-γ, thus played betterimmune regulation effects than APS.In addition, the secrete of NO and cytokines on culturedlymphocytes had certain dose-effect relationship to the APS-NE, and in the study,300μg/mL(high dose group) was the best.
引文
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