应用激光扫描共聚焦显微术观察血小板活化及其形态改变

目的建立应用激光扫描共聚焦显微术(laser scanning confocal microscopy,LSCM)观察血小板活化及其形态改变的方法。方法用不同浓度的胶原诱导血小板活化,通过LSCM观察与特异性荧光(FITC/PE)抗体结合的血小板膜糖蛋白Ⅱb/Ⅲa(GPⅡb/Ⅲa)荧光强度的变化,判断血小板微颗粒(platelet microparticle,PMP)的产生,同时观察正常人和糖尿病视网膜病变患者血小板形态的差异。结果加入诱导剂后血小板活化,镜下观察特异性抗体标记的血小板荧光强度呈规律性变化。在一定浓度时诱导剂胶原的加入可引起血小板GPⅡb/Ⅲa表达量的变化,且随着胶原浓度的增加,GPⅡb/Ⅲa表达量也有增加的趋势。并观察到糖尿病视网膜病变患者血小板有伪足伸出,末端附近有释放的微颗粒环绕,与正常人血小板形态有明显差异。结论应用LSCM可观察到诱导剂致血小板活化引起GPⅡb/Ⅲa表达量的变化以及血小板微颗粒的释放,同时可认为血小板形态改变的观察在血栓病血小板活化程度的判断上具有一定的价值。     

Novel Approaches to Vaccine Delivery

Although the currently available vaccines represent an outstanding success story in modern medicine and have had a dramatic effect on morbidity and mortality worldwide, it is clear that improvements are required in the current vaccine delivery technologies. Improvements are required to enable the successful development of vaccines against infectious diseases that have so far proven difficult to control with conventional approaches. Improvements may include the addition of novel injectable adjuvants or the use of novel routes of delivery, including mucosal immunization. Mucosal delivery may be required to provide protection against pathogens that infect at mucosal sites, including sexually transmitted diseases. Alternatively, novel approaches to delivery, including mucosal administration, may be used to improve compliance for existing vaccines. Of particular interest for safer mass immunization campaigns are needle-free delivery devices, which would avoid problems due to needle re-use in many parts of the world and would avoid needle-stick injuries.     

Phagocytosis and Phagosomal Fate of Surface-Modified Microparticles in Dendritic Cells and Macrophages

Purpose. We compared cationic, polyamine-coated microparticles (MPs) and anionic, protein-coated MPs with respect to their phagocytosis and phagosomal fate in dendritic cells (DCs) and macrophages (M). Methods. Polystyrene MPs were surface modified by covalent coupling with fluorescein isothiocyanate-labeled polyamines or proteins. Phagocytosis of MP and the pH of their intracellular microenvironment was assessed in human-derived DCs and M in a fluorescence plate reader. Visualization of MP phagocytosis in DCs was performed by transmission electron microscopy. Results. Phagocytosis of bovine serum albumin-coated MPs was low with significant differences between DC and M, whereas phagocytosis of IgG-coated MPs was significantly enhanced in both cell types. Phagocytosis of both particle types resulted in an acidified phagosomal microenvironment (pH 4.6-5.1). In contrast, cationic, polyamine-coated MPs were equally phagocytosed by DCs and M to a high extent and showed lower degrees of acidification (pH 6.0-6.8) in the phagosomal microenvironment. Transmission electron microscopy examination demonstrated all phagocytosed particles to be surrounded by a phagosomal membrane, which was more tightly apposed to the surface of cationic MPs and more loosely to bovine serum albumin-coated MPs. Conclusion. Phagocytosis of cationic, polyamine-coated MPs is suggested to lead to diminished phagosomal acidification. Thus, cationic MP are potential carriers that may display beneficial features for the intracellular delivery of immunomodulating therapeutics and their protection against lysosomal degradation.     

Receptor-mediated targeting of spray-dried lipid particles coformulated with immunoglobulin and loaded with a prototype vaccine

Purpose. Spray-dried lipid-based microparticles (SDLM) serve as a platform for delivery of a wide variety of compounds including peptides, proteins, and vaccines to the respiratory mucosa. In the present study, we assessed the impact of IgG-mediated targeting to phagocytic cells of inactivated influenza virus formulated in SDLM, on subsequent immune responses. Methods. SDLM were produced containing inactivated influenza virus strain A/WSN/32/H1N1 (WSN), with or without IgG. Using phagocytic antigen presenting cells (APC) and a T cell hybridoma (TcH) line specific for a dominant influenza virus epitope, we compared the in vitro responses elicited by ligand-formulated (SDLM-IgG-WSN) and non-ligand particles (SDLM-WSN). The effect of including the IgG ligand in the formulation was further characterized by measuring the immune responses of rodents vaccinated with SDLM. Results. SDLM-IgG-WSN were internalized in an Fc receptor (FcR)-dependent manner by phagocytic APC that were then able to effectively present a dominant, class II-restricted epitope to specific T cells. While SDLM-WSN elicited a lower response than administration of plain inactivated virus in saline, the level of the T cell response was restored both in vitro and in vivo by incorporating the APC FcR ligand, IgG, in the SDLM. Conclusions. Incorporation of FcR ligand (IgG) in SDLM restored the limited ability of formulated virus to elicit T-cell immunity, by receptor-mediated targeting to phagocytes.     

Microsphere translocation and immunopotentiation in systemic tissues following intranasal administration

With a view to developing improved mucosal immunisation strategies, we have quantitatively investigated the uptake of fluorescent polystyrene carboxylate microspheres (1.1 μm diameter), using histology and fluorescence-activated cell sorting, following intranasal delivery to BALB/c mice. To qualify these biodistribution data, antigen specific memory and effector responses in the spleens of mice immunised nasally with Yersinia pestis V antigen loaded poly(lactide) (PLA) microspheres (1.5 μm diameter) were assessed at 4, 7 and 11 days. Irrespective of administration vehicle volume (10 or 50 μl), appreciable numbers of fluorescent microspheres were detected within nasal associated lymphoid tissues (NALT) and draining cervical lymph nodes. Nasal administration of the particles suspended in 50 μl volumes of phosphate-buffered saline (PBS) served to deposit the fluorescent microspheres throughout the respiratory tract (P<0.05). In these animals, appreciable particle uptake into the mediastinal lymph node was noted (P<0.05). Also, spleens removed from mice 10 days after fluorescent particle application contained significantly more microspheres if the suspension had been nasally instilled using a 50 μl volume (P<0.05). Appreciable memory (and effector from day 7) responses were detected in mediastinal lymph nodes removed from mice immunised nasally with 50 μl volumes of microparticulated or soluble V antigen. Immunological responses in splenic tissue removed 7 days after intranasal immunisation corroborated the thesis that the spleen can act as an inductive site following bronchopulmonary deposition of particulated antigen: upon exposure to V in vitro, splenic T-cells from mice nasally immunised with 50 μl volumes of microspheres incorporated statistically greater (P<0.05) quantities of [³H]thymidine into newly synthesised DNA than did T-cells from cohorts nasally immunised with 50 μl volumes of V in solution. Similarly, significant numbers of anti-V IgG secreting cells were only detected in spleens from mice immunised intramuscularly or nasally with microparticles. These immunological and biodistribution data support the tenet that, following an appropriate method of mucosal delivery, microparticles can translocate to tissues in the systemic compartment of the immune system and thence provoke immunological reactions therein.     

In Vitro Evaluation of Microparticles and Polymer Gels for Use as Nasal Platforms for Protein Delivery

Purpose. Nasal delivery of protein therapeutics can be compromised by the brief residence time at this mucosal surface. Some bioadhesive polymers have been suggested to extend residence time and improve protein uptake across the nasal mucosa. We examined several potential polymer platforms for their in vitro protein release, relative bioadhesive properties and induction of cytokine release from respiratory epithelium. Methods. Starch, alginate, chitosan or Carbopol^® microparticles, containing the test protein bovine serum albumin (BSA), were prepared by spray-drying and characterized by laser diffraction and scanning electron microscopy. An open-membrane system was used to determine protein release profiles and confluent, polarized Calu-3 cell sheets were used to evaluate relative bioadhesion, enhancement of protein transport and induction of cytokine release in vitro. Results. All spray-dried microparticles averaged 2–4 m in diameter. Loaded BSA was not covalently aggregated or degraded. Starch and alginate microparticles released protein more rapidly but were less adhesive to polarized Calu-3 cells than chitosan and Carbopol^® microparticles. Protein transport across polarized Calu-3 cells was enhanced from Carbopol^® gels and chitosan microparticles. A mixture of chitosan microparticles with lysophosphatidylcholine increased protein transport further. Microparticles prepared from either chitosan or starch microparticles, applied apically, induced the basolateral release of IL-6 and IL-8 from polarized Calu-3 cells. Release of other cytokines, such as IL-l, TNF-, GM-CSF and TGF-, were not affected by an apical exposure to polymer formulations. Conclusions. We have described two systems for the in vitro assessment of potential nasal platforms for protein delivery. Based upon these assessments, Carbopol^® gels and chitosan microparticles provided the most desirable characteristics for protein therapeutic and protein antigen delivery, respectively, of the formulations examined.     

不同抗凝剂种类、操作和保存方法对血浆微粒检测影响

目的探讨不同抗凝剂种类、标本处理方式、保存温度及时间对血浆微粒(MPs)检测效果的影响。方法采集2013年8月-2014年5月在苏州大学附属第二医院住院治疗的40例系统性红斑狼疮患者、41例类风湿性关节炎患者和52名健康对照者血浆标本;采用流式细胞术(FCM)计数不同抗凝剂[枸橼酸盐、乙二胺四乙酸二钾(EDTA-K₂)、肝素]、标本处理方式[洗涤贫血小板血浆(PPP)和直接PPP]、保存温度(-4、-20、-80 ℃)及时间(1、3、7、10、14 d)血浆中MPs的含量。结果采用枸橼酸盐、EDTA-K₂、肝素抗凝剂和无抗凝剂血浆中MPs含量分别为(1 527.0±620.4)、(981.4±247.9)、(877.7±176.2)和(480.8±112.2)μL, 差异有统计学意义(F=9.11, P<0.01);系统性红斑狼疮患者、类风湿性关节炎患者和健康对照者直接PPP MPs含量分别为(1 972.4±2 850.7)、(3 347.8±3431.5)和(2 157.7±1 901.1)μL, 均高于洗涤PPP的(1 406.4±2 205.7)、(2 375.6±2 500.2)和(1 502.8±1 337.1)μL(均P<0.01);与新鲜血浆标本MPs含量比较, -4 ℃、-20 ℃下血浆标本保存1、3、7、10 d 和-80 ℃下血浆标本保存3、7、10 d MPs含量均下降, 差异均有统计学意义(P<0.05);而在3个温度下保存14 d MPs含量开始升高, 与新鲜血浆标本MPs含量比较, 差异均无统计学意义(P>0.05);分别对3个温度下保存1、3、7、10、14 d血浆标本MPs含量比较, 差异均无统计学意义(P>0.05)。结论检测血浆MPs含量时宜选用新鲜EDTA-K₂抗凝标本且无需多次离心洗涤, 短期内标本保存在-4 ℃、-20 ℃、-80 ℃下均可。     

淫羊藿苷/载明胶纳米复合物-PLGA缓释系统的制备及工艺优化

目的：制备淫羊藿苷（ICA）@明胶纳米粒（GNPs）-聚乳酸-羟基乙酸共聚物（PLGA）（ICA@GNPs-PLGA）缓释系统,并对制备工艺进行优化。方法：采用二步去溶剂法和S/O/W乳化溶剂挥发法制备ICA@GNPs-PLGA缓释系统,检测投放的PLGA和纳米复合物的质量比和ICA的投入量等不同因素对微球包封率（EE）的影响以优化制备工艺。扫描电镜（SEM）观察纳米复合物和微球的表面特征;高效液相色谱法（HPLC）测量微球EE及其体外释放结果。结果：所制备的复合微球和纳米复合物均为白色粉末状;SEM下微球和纳米复合物表面光滑、圆整,粒径较为均一,粒径分布范围分别为4~12 μm和150~200 nm。当GNPs投入量为6 mg,PLGA在二氯甲烷（DCM）中的临界浓度为0.5%~1.0%;当GNPs的质量上升至12 mg时,PLGA在DCM中的临界浓度升至1.0%~2.0%;当PLGA的浓度低于0.25%时,无完全包封的复合微球可以形成。在临界浓度内,ICA@GNPs-PLGA微球的EE高于（62.00±1.25）%,且EE和ICA的投入量呈负相关关系（P<0.05）。24h时内微球累积释放率低于5.47%,40d时累积释放率为65.21%。结论：采用优化的制备工艺可以制备出粒径分布较窄、载药率较高、低突释和长期释放的ICA@GNPs-PLGA微球缓释系统。     

The Effect of Size on Uptake of Orally Administered Latex Microparticles in the Small Intestine and Transport to Mesenteric Lymph Nodes

Purpose. The present study examines the relationship between size and particle transit across the mucosal barrier of the gastrointestinal tract to other sites of the body. The extent of particle uptake with increasing size, the tissue distribution and cut-off points for 2–20µm particles is investigated. Methods. An established fluorescent latex particle-young adult rat model is used and particle numbers in small intestine and mesenteric lymph nodes, 0.5 h post administration, counted by fluorescence microscopy in bulk tissue specimens and cryosections. Results. Bulk tissue analysis provides evidence for the presence of particles of all sizes in the Peyer's patch regions, but only for 2 µm particles in the nodal tissues. Microscopy establishes uptake of both 2 and 6 µm particles in most intestinal and nodal tissue sites and compartments. By contrast, uptake of the larger particles is much reduced. Conclusions. Although more of the smaller (2 µm) particles are taken up, particularly by epithelial tissues, the 6µm size appears more efficient in terms of volume translocated to lymph nodes. This could have implications in the therapeutic use of particles for drug and vaccine delivery and for radiation safety.