Antibody-conjugated soybean oil-filled calcium phosphate nanoshells for targetted delivery of hydrophobic molecules

Hollow calcium phosphate nanoparticles capable of encapsulating poorly water-soluble molecules were produced by self-assembly. Previously reported were solid calcium phosphate nanoparticles and water-filled calcium phosphate nanocapsules suited for encapsulating mostly hydrophilic, but not hydrophobic compounds. Here, calcium phosphate was deposited around 100?nm diameter, 1,2-dioleoyl-sn-glycero-3-phosphate stabilized soybean oil nanoemulsions using either calcium chloride or NaOH titrations to achieve shell thickness between 20–70?nm. The surface was functionalized with carboxylic acid via the addition of carboxyethylphosphonic acid to attach Molecular Probes AB-594C antibody using sulpho-n-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride with an efficiency of ～70%, while retaining near complete antibody function. Hydrophobic pyrene was encapsulated with an efficiency of 95%, at concentrations much higher than its water solubility limit, and exhibited spectral features characteristic of a hydrophobic environment. These materials can be used in the targeted delivery of many useful, yet poorly water-soluble pharmaceutical and nutraceutical compounds.     

Nasal mucosal administration of chitin microparticles boosts innate immunity against influenza A virus in the local pulmonary tissue

Influenza virus infection remains a major health concern due to morbidity and mortality associated with epidemics and occasional pandemics. The absence of acquired immunity to antigenically distinct, emerging virus strains stresses the need for a generic drug that protects independent of vaccination. Here, we demonstrate that prophylactic administration of chitin microparticles (CMP) via the intranasal route significantly reduced lung viral titres and clinical signs. Pre-treatment boosted the innate immune response to subsequent infection by recruiting innate cells, such as neutrophils, and increasing inflammatory cytokines. Although an increase in virus-specific T cells was observed, the memory phase was diminished. Our data demonstrate that in the absence of prior exposure to influenza virus, CMP reduce clinical signs by boosting innate immunity.     

Fetal corticotrophin-releasing hormone mRNA, but not phosphatidylserine-exposing microparticles, in maternal plasma are associated with factor VII activity in pre-eclampsia

Summary.  Background:  Pre-eclampsia is associated with increased placental debris circulating in maternal plasma. Objectives:  This study related placental debris to maternal markers of coagulation and endothelial activation in pre-eclampsia. Patients/methods:  Circulating fetal corticotrophin-releasing hormone (CRH) mRNA and phosphatidylserine (PS)-exposing microparticles were assayed in third trimester plasma from women with pre-eclampsia ( n  = 32) and controls ( n  = 32) matched for age, body mass index, parity, and gestational age at sampling. Markers of maternal hemostasis and endothelial function were assessed. Results:  Fetal CRH mRNA levels were higher in pre-eclampsia [mean 0.75 (SD 2.77) CRH/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio] than in control pregnancies [0.20 (0.74), P  = 0.014]. PS-exposing microparticle levels were not different between the groups. Women with pre-eclampsia had higher levels of tissue factor pathway inhibitor (TFPI), prothrombin F ₁₊₂ fragment ( F ₁₊₂), factor XIIa, soluble vascular cell adhesion molecule 1, von Willebrand factor and plasminogen activator inhibitor 1 than controls. Fetal CRH mRNA correlated with TFPI in pre-eclampsia and control groups ( r  = 0.38, P  = 0.031, and r  = 0.37, P  = 0.039, respectively). Fetal CRH mRNA correlated with FVII activity ( r  = 0.43, P  = 0.017) and PS-exposing microparticles correlated inversely with F ₁₊₂ ( r  = −0.64, P  < 0.001) in pre-eclampsia. Conclusions:  Placental debris, assessed by fetal CRH mRNA levels in maternal blood, is related to coagulation potential, i.e. FVII activity, but not to markers of coagulation or endothelial activation in pre-eclampsia.     

Elevation of circulating endothelial microparticles in patients with chronic renal failure

BACKGROUND: Chronic renal failure patients are at high risk of cardiovascular events and display endothelial dysfunction, a critical element in the pathogenesis of atherosclerosis. Upon activation, the endothelium sheds microparticles, considered as markers of endothelial dysfunction that also behave as vectors of bioactive molecules. AIM: To measure plasma levels of endothelial microparticles (EMPs) in chronic renal failure patients (CRF), either undialyzed or hemodialyzed (HD), and to investigate the ability of uremic toxins to induce EMP release in vitro. METHODS: Circulating EMPs were numerated by flow cytometry, after staining of platelet-free plasma with phycoerythrin (PE)-conjugated anti-CD144 (CD144+ EMP) or anti-CD146 (CD146+ EMP) monoclonal antibodies. Platelet MP (CD41+ PMP), leukocyte MP (CD45+ leukocyte microparticles (LMP)), and annexin-V+ MPs were also counted. In parallel, MPs were counted in supernatant of human umbilical vein endothelial cells incubated with uremic toxins [oxalate, indoxyl sulfate, p-cresol, and homocysteine (Hcy)], at concentrations found in patients. RESULTS AND CONCLUSIONS: CD144+ EMP and CD146+ EMP levels were significantly higher in CRF and HD patients than in healthy subjects. Furthermore, annexin-V+ MPs were elevated in both groups of uremic patients, and CD41+ PMP and CD45+ LMP were increased in CRF and HD patients, respectively. In vitro, p-cresol and indoxyl sulfate significantly increased both CD146+ and annexin-V+ EMP release. Increased levels of circulating EMP in CRF and HD patients represent a new marker of endothelial dysfunction in uremia. The ability of p-cresol and indoxyl sulfate to increase EMP release in vitro suggests that specific uremic factors may be involved in EMP elevation in patients.     

微颗粒研究进展

微颗粒是由多种细胞在活化、损伤或凋亡过程中产生的直径在0.1～1μm的胞外囊泡,主要形成机制是磷脂酰丝氨酸外翻及细胞骨架重构。微颗粒不仅含有脂质和膜蛋白,还含有其来源细胞的胞浆成分(蛋白质和核酸),具有来源细胞的功能。微颗粒存在于正常人体内,但在冠状动脉病、抗磷脂综合征、肿瘤、血栓等多种疾病患者中均增多,且不同疾病中增多的微颗粒种类不同。现已有多种方法检测微颗粒的大小、形态、来源及功能,但每种方法都有优势和局限性,可结合多种方法检测分析。微颗粒的检测有助于临床疾病的诊断和治疗。     

Preanalytical,analytical, and biological variation of blood plasma submicron particle levels measured with nanoparticle tracking analysis and tunable resistive pulse sensing

Background: Nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) enable measurement of extracellular vesicles (EVs) in blood plasma but also measure other particles present in plasma. Complete isolation of EVs from similarly sized particles with full EV recovery is currently not possible due to limitations in existing isolation techniques.

Aim: This study aimed to evaluate preanalytical, analytical, and biological variation of particle measurements with NTA and TRPS on blood plasma.

Methods: Blood from 20 healthy subjects was sampled in the fasting and postprandial state. Platelet free plasma (PFP) was analyzed immediately and after a freeze-thaw cycle. Additionally, the effect of prandial state and a freeze-thaw cycle on EV-enriched particle fractions obtained via size-exclusion chromatography (SEC) was examined.

Results: We observed analytical linearity in the range of 1.0–10.0?×?10⁸ particles/mL for NTA and 1.0?×?10⁸–1.8?×?10⁹ particles/mL for TRPS. The analytical variation was generally below 10%. A considerable intra- and inter-individual variation was demonstrated with estimated reference intervals of 1.4?×?10¹¹–1.2?×?10¹² particles/mL for NTA and 1.8?×?10⁸–1.6?×?10⁹ particles/mL for TRPS. Food intake and to a lesser extent a freeze-thaw cycle affected particle populations in PFP and, similarly, in EV-enriched fractions.

Conclusion: In this study NTA and TRPS enabled acceptably precise concentration and size measurement of submicron particles in PFP. An appreciable intra- and inter-individual biological variation was observed. In studies on particle populations in PFP or EV-enriched fractions, we recommend analysis of fresh, fasting samples.