Development of an Alcohol Dilution-Lyophilization Method for Preparing Lipid Nanoparticles Containing Encapsulated siRNA.

Systems for delivering nucleic acids are now fundamental technologies for realizing personalized medicine. Among the various nucleic acid delivery systems that are currently available, lipid-nanoparticles (LNPs) that contain short interfering RNA (siRNA) have been extensively investigated for clinical applications. LNPs are generally prepared by an alcohol dilution method. In this method, it is necessary to remove the alcohol and then concentrate the LNP sample before they can be used. In this study, we report on the development of an "alcohol dilution-lyophilization method" for preparing siRNA-encapsulating LNPs. This method involves the use of a freeze-drying (lyophilization) method to remove the residual alcohol and to simultaneously concentrate the preparation. At first, the compositions of cryoprotectants and polyethylene glycol (PEG)-lipids that were used were optimized from the point of view of particle stabilization. A combination of sucrose and 1-(monomethoxy polyethyleneglycol5000)-2,3-dimyristoylglycerol (DMG-PEG5000) was found to have the most efficient cryoprotective activity for the LNPs. The knockdown efficiency of the LNP prepared by the alcohol dilution-lyophilization method was comparable to that of an LNP prepared by the conventional ultrafiltration method.

Factor VII and protein C are phosphatidic acid-binding proteins.

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

Cell-derived vesicles exposing coagulant tissue factor in saliva.

On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is noncoagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism and physiologic relevance are unknown. Because saliva is known to contain TF, we hypothesized that this TF may also be associated with cell-derived vesicles to facilitate coagulation when saliva directly contacts blood. The saliva-induced shortening of the clotting time of autologous plasma and whole blood from healthy subjects (n = 10) proved TF-dependent. This TF was associated with various types of cell-derived vesicles, including microparticles and exosomes. The physiologic function was shown by adding saliva to human pericardial wound blood collected from patients undergoing cardiac surgery. Addition of saliva shortened the clotting time from 300 ± 96 to 186 ± 24 seconds (P = .03). Our results show that saliva triggers coagulation, thereby reducing blood loss and the risk of pathogens entering the blood. We postulate that our reflex to lick a wound may be a mechanism to enable TF-exposing vesicles, present in saliva, to aid in the coagulation process and thus protect the organism from entering pathogens. This unique compartmentalization may be highly conserved because also animals lick their wounds.

FVII dependent coagulation activation in citrated plasma by polymer hydrogels.

Polymer hydrogels containing positively charged functional groups were used to investigate the critical material and biological components of FVII activation and subsequent fibrin formation in citrated plasma. A FVIIa ELISA confirmed the ability of the polymer to induce FVII activation and provided insight into the material parameters which were influential in this activation. Experiments utilizing coagulation factor depleted and inhibited plasmas indicated that FVII, FX, FII, and FI are all vital to the process outlining the general mechanism of fibrin formation from the onset of FVII activation. Dynamic mechanical analysis and swelling experiments were used to establish a critical correlation between polymer microstructure and FVII activation.

Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates.

The effects on thrombosis and hemostasis of thrombin-induced activation of endogenous protein C (PC) were evaluated in baboons. Thrombosis was induced by placing into arteriovenous shunts a segment of Dacron vascular graft, which generated arterial platelet-rich thrombus, followed by an expansion region of low-shear blood flow, which in turn accumulated fibrin-rich venous-type thrombus. Thrombosis was quantified by 111In-platelet imaging and 125I-fibrinogen accumulation. Intravenous infusion of alpha-thrombin, 1-2 U/kg-min for 1 h, increased baseline activated PC levels (approximately 5 ng/ml) to 250-500 ng/ml (P < 0.01). The lower thrombin dose, which did not deplete circulating platelets, fibrinogen, or PC, reduced arterial graft platelet deposition by 48% (P < 0.05), and platelet and fibrin incorporation into venous-type thrombus by > 85% (P < 0.01). Thrombin infusion prolonged the activated partial thromboplastin clotting time, elevated fibrinopeptide A (FPA), thrombin-antithrombin III complex (T:AT III), and fibrin D-dimer plasma levels (P < 0.01), but did not affect bleeding times. Thrombin's antithrombotic effects were blocked by infusing a monoclonal antibody (HPC-4) which prevented PC activation in vivo, caused shunt occlusion, increased the consumption of platelets and fibrinogen, elevated plasma FPA and T:AT III levels, and reduced factor VIII (but not factor V) procoagulant activity (P < 0.05). We conclude that activated PC is a physiologic inhibitor of thrombosis, and that activation of endogenous PC may represent a novel and effective antithrombotic strategy.

Anticoagulant mechanism of sulfonated polyisoprenes.

The influence of sulfonated polyisoprene (SPIP) on coagulation factors and human blood cells was investigated to elucidate and compare its anticoagulant mechanism with that of heparin. While the number of red cells was unaffected, the number of platelets decreased dramatically in the presence of SPIP due to aggregation. Using a synthetic peptide substrate to assay thrombin activity in the presence of its natural inhibitor, antithrombin (AT), we observed no stimulation by SPIP of AT-mediated inhibition. Nevertheless, thrombin cleavage of its natural substrate fibrinogen to fibrin peptide A was slightly inhibited. SPIP altered the electrophoretic mobility of fibrinogen and completely inhibited fibrinogen from clotting. We detected no significant influence of SPIP on factors II, VII, IX, and X, while factor XI and factors V and VIII were only slightly affected. Therefore, the main mechanism of SPIP's anticoagulant activity appears to be a strong interaction with fibrinogen and fibrin monomer, first, to prevent proteolytic conversion of the former to the latter and second, to inhibit polymerization of the fibrin monomer, once formed.

Phospholipase C mediated inhibition of factor VII requires triglyceride-rich lipoproteins.

The reduction of plasma factor VII (FVII) activity by phospholipase C (PLC), in vitro, has been proposed as a possible indication of a risk of cardiovascular disease. The ability of PLC to reduce FVII activity was found to require calcium ions and the presence of triglyceride-rich lipoproteins (e.g. chylomicra and very-low density lipoproteins) rather than high or low density lipoproteins. The PLC-mediated reduction of FVII activity was prevented by pre-incubation of PLC with chylomicra, before adding FVII, and this suggests that PLC may act on triglyceride-rich lipoproteins already bound to FVII in order to reduce FVII activity. At optimal PLC concentration, the extent of the reduction in FVII activity was proportional to the concentration of chylomicra. The detergent, Tween, prevented any loss of FVII activity, in both plasma and purified systems, if it was present at the beginning of the incubation with PLC. Addition of Tween, but not EDTA, after inhibition of FVII activity had occurred, caused a partial restoration of FVII activity. It is concluded that PLC reduces FVII activity by modifying triglyceride-rich lipoproteins to a form which binds to FVII, independently of calcium ions, and which inhibits procoagulant activity. The detection of PLC-sensitive procoagulant activity. The detection of PLC-sensitive FVII activity may therefore have no greater significance than the measurement of plasma triglyceride levels in predicting a risk of cardiovascular disease.

Analogous features of cold-promoted activation and contact system activation in human plasmas: the role of cryptic soluble surfaces in asthma, oral contraception and pregnancy.

PIP: Aspects of cold-promoted activation (CPA) and contact system activation (CSA) of human plasma were assayed in 13 normal subjects, 11 asthmatics and 15 patients with history of allergy to x-ray contrast media, to see whether the phenomena were correlated. Cold activation commonly occurs in stored plasma in 60% of women taking oral contraceptives, 93% of women in 3rd trimester of pregnancy, but only 15% of normal subjects. It is assayed by measuring kallikrein. CSA also occurs at low temperatures, but is dependent on soluble negatively-charged surfaces. It is assayed using dextran SO4 or polybrene. In this study, kallikrein amidolytic activity with dextran SO4-CDA and cold-dilution CDA were positively correlated in asthmatics. The allergic patients showing elevated B-CDA values also had shortened Thrombotest times with cold dilution, while those with normal B-CDA values had normal coagulation times. The significance of these results were discussed, including the theoretical possibility that cryptic surface phenomena may be involved in the heightened coagulability of plasma in women taking oral contraceptives.^ieng

Sphingosine-containing phospholipid vesicles support human factor VII autoactivation in the absence of tissue factor.

We studied the effects of lipid membrane composition on factor VII autoactivation and observed that factor VII was activated in the presence of phospholipid vesicles containing sphingosine. The time course for the factor VII activation was sigmoidal and the duration of the initial lag phase was decreased by the addition of exogenous factor VIIA. Kinetic studies revealed that factor VII activation in the presence of sphingosine-containing phospholipids can be defined by a second-order reaction mechanism with an apparent second-order rate constant of 1.1x10(4) M(-1)s(-1). The sphingosine-mediated factor VII autoactivation rate was dependent on the concentration of calcium ions and sphingosine content of the vesicles. Neither bovine serum albumin-conjugated sphingosine nor sphingosine analogues (ceramide, sphingomyelin) affected the factor VII autoactivation rate.

The activation of factor VII in citrated plasma by charged long-chain saturated fatty acids at the interface of large triglyceride-rich lipoproteins.

The prolonged incubation of dilute plasma on ice in the presence of added sulphatide vesicles or the long-chain saturated fatty acids (FA) stearic acid (C18:0) or behenic acid (C22:0) induced a concentration-dependent increase in factor VII coagulant activity (VIIc). The addition of FA at various ratios to human serum albumin showed the micellar non-bound pool to be responsible for this effect, FA bound to the high-affinity or low-affinity binding sites of albumin having no influence on VIIc. Plasma VIIc also increased following addition of behenate-enriched lipoprotein particles produced by incubation of the d < 1.006 g/ml lipoprotein fraction with this FA, or addition of lipoprotein remnants produced by pre-incubation of the d < 1.006 g/ml fraction with lipoprotein lipase. Long-chain saturated fatty acids in the interface of lipoprotein remnants, produced by the interaction of triglyceride-rich lipoprotein particles with lipoprotein lipase, appear to provide a surface that activates the contact system of coagulation and subsequently factor VII.

The effects of intravenous Triton WR-1339 on factor VII coagulant activity and plasma lipoproteins in normocholesterolaemic and hypercholesterolaemic rabbits.

The hypothesis that lipolysis of large lipoproteins by lipoprotein lipase (LPL) has an important influence on the activation of the contact system of coagulation and subsequently on factor VII activation was tested in rabbits rendered hyperlipidaemic by dietary means and/or by injection of Triton WR-1339. The dietary treatment involved a control diet and two isocaloric diets containing either a 0.5% cholesterol or 0.5% cholesterol and 7.5% safflower oil supplement. Other groups of rabbits were given either a standard diet or the standard diet supplemented with 1% cholesterol. All supplemented diets increased many-fold the concentrations of cholesterol associated with the chylomicron, very low-(VLDL), intermediate-(IDL) and low-density (LDL) lipoprotein fractions. Factor VII coagulant activity (FVIIc) increased significantly in all groups of rabbits fed the cholesterol supplement. The intravenous injection of Triton WR-1339 into rabbits fed either the standard or 1% cholesterol-supplemented diet resulted in increases of plasma cholesterol and triglyceride concentrations up to 36-48 h thereafter, followed by decreases up to completion of the experiment at 72 h. Most of these increases in plasma lipids were associated with the chylomicron and VLDL fractions. Following injection of Triton into rabbits fed either the standard or cholesterol-supplemented diet, changes in FVIIc were biphasic with a decrease in activity in the early intervals when rates of accumulation of plasma lipid were constant, and a progressive increase in activity at later intervals when rates of lipid accumulation declined and then reversed.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of reformulated 2-rod Norplant implant on hemostasis after three years.

Prolonged use of reformulated and the original 2-rod Norplant implants showed similar changes in most hemostatic parameters studied. Raised hemoglobin concentration and hematocrit values with not enhanced platelet activation or significant changes in platelet numbers were seen. Factor VII showed an increase from 18 months compared to the first 12 months of original Norplant implant use, while with reformulated Norplant implant, the level at 36 months was significantly higher than the first 24 months of implant use. Fibrinogen levels were significantly elevated by 36 months of both implant use. No evidence of enhanced activation of coagulation, fibrinolysis/inhibitor were observed during prolonged implant use. Overall, no significant changes in tissue plasminogen activator (t-PA) levels were observed but urokinase-like plasminogen activator (u-PA) levels were significantly reduced, indicating no enhancement of tissue breakdown. Plasminogen activation inhibitor (PAI-1) antigen levels were significantly reduced from 12 to 36 months with original Norplant implant use compared to the pre-insertion levels, while a nonsignificant decreased trend was seen with prolonged reformulated Norplant implant use. The increased levels of fibrinogen and FVII at the end of 36 months of implant use require further observation as these factors are known markers of hypercoagulation and associated with increased arteriosclerotic and cardiovascular risks. This study is on-going to evaluate the effects of levonorgestrel-containing subdermal reformulated Norplant implants on hemostasis after five years of use.

PIP: Researchers compared data on women using the 2-rod Norplant implant system formulated with the Silastic elastomer 4092 (original system) with those using the 2-rod Norplant made with a different elastomer (Q74910) (reformulated system) to examine their hemostatic effects during 36 months of use. All 33 women lived in Singapore. Both systems released the same amount of levonorgestrel at the same rate. In most hemostatic parameters, prolonged use of both 2-rod Norplant implant systems effected similar changes: increased hemoglobin concentration and hematocrit values, increased fibrinogen levels, no increased platelet activation, no increased activation of coagulation or fibrinolysis/inhibitor, no significant changes in platelet numbers, no significant changes in tissue plasminogen activator, and decreased urokinase-like plasminogen activator. Factor VII was higher after 18 months than it was during the first 12 months in original Norplant users (p 0.001). The factor VII level at 36 months during reformulated Norplant use was significantly higher than it was during the first 24 months of use (p 0.001). None of the changes common to both systems suggested tissue breakdown. Plasminogen activator inhibitor antigen levels were significantly lower during 12-36 months than preinsertion levels in original Norplant users, while they fell insignificantly in reformulated Norplant users. The increased levels of fibrinogen and factor VII at 36 months are areas of concern since they are markers of hypercoagulation and are linked to increased arteriosclerotic and cardiovascular risks. Researchers of this on-going study will later publish the two systems' effects on hemostasis after 5 years of use.

Efficacy of double-filtration plasmapheretic cross-circulation with a high-permeability membrane using canine harvested liver in porcine fulminant hepatic failure model.

INTRODUCTION: The use of bioartificial liver devices requires. A sufficient liver cell mass to provide adequate metabolic support, reduction of xenogeneic immune reactions, and avoidance of viral transmission. We have developed a plasmapheresis system using a semipermeable membrane combined with canine whole liver perfusion (PMCWLP). In this study, we investigated the efficacy of our system in a porcine fulminant hepatic failure (FHF) model. METHODS: The porcine FHF model was established by intraportal administration of alpha-amanitin (0.1 mg/kg) and lipopolysaccharide (1 microg/kg). Nine hours after drug injection, xenogenic perfusion treatment was performed twice within 6 hours (n = 5). As the plasmapheresis device, we used a hollow-fiber module with cellulose diacetate porous fibers (pore size, 0.05 microm, surface area, 2 m2). The canine whole liver was perfused with modified Krebs solution, which is commonly used in many laboratories, containing albumin (2 g/dL) and glucose (300 mg/dL). Control pigs (n = 10), had the circuit not connected to the whole canine liver. RESULTS: The survival of FHF pigs was significantly increased by the treatment (58.9 +/- 21.8 hour) compared with the controls (22.3 +/- 8.1 hour). Mean blood ammonia levels and intracranial pressure during treatment were significantly lower compared with control groups. CONCLUSION: Treatment of FHF pigs with the system significantly increased survival time, suggesting that this method may have applications as a clinical liver assist device.

Hematological and biochemical studies in calves living over 100 days with the polymethylmethacrylate total artificial heart TNS Brno II.

A polymethylmethacrylate total artificial heart (kinetic components made of polyetherurethane) of TNS Brno II type was implanted into seven calves (2-5 months of age) surviving for the average of 152.4 +/- 19.1 days after the implantation. During the entire post-operative period the animals received oral warfarin-sodium, acetylsalicylic acid, dipyridamole and alpha-tocopherol. Blood was taken for biochemical and hematological examinations twice a week from the jugular vein. During the experiments there were decreases in the number of red blood cells, hematocrit and hemoglobin levels. Plasma free hemoglobin and serum enzymes (alkaline phosphatase, AST, ALT, LDH) increased. Coagulation tests were abnormal because anticoagulation therapy was used. There were minimal changes in the number of white blood cells and platelets, fibrinogen, blood pH, blood glucose, serum electrolytes, bilirubin (total and direct), creatinine, blood urea, and lactate. Possible reasons for observed changes include the gradual rise in the central venous pressure and damaged function of the liver parenchyma. Other factors playing a possible role in inducing changes in laboratory findings are also discussed.

[Incidence of factor VII and C3 in vascular anastomosis]. / Wystepowanie czynnika VII krzepniecia krwi oraz czynnika C3 ukladu dopelniacza w zespoleniach naczyniowych.

In order to study pathogenesis of vascular prosthesis healing process the following experiment was designed. 16 dogs underwent implantation of unilateral straight aorto-femoral teflon (PTFE, polytetrafluoroethylene) by-pass. After 6 months all dogs were killed, dissected and vascular prostheses with margin of adjacent aorta and femoral artery were collected for further study. Areas of proximal and distal anastomosis were examined immunohistochemically. Presence of coagulation factor VII, and C3 complement factor were studied. The obtained results were analyzed statistically by means of t-Student test. Factor VII as well C3 were found in areas of both proximal and distal anastomosis. Concentration of all two substances in proximal and distal anastomosis was compared. No statistically valid differences in factor VII concentration in proximal and distal anastomosis were found, whereas amounts of C3 factor as well as degree of extracellular matrix infiltration were markedly higher in distal anastomosis.

Co-injection of a targeted, reversibly masked endosomolytic polymer dramatically improves the efficacy of cholesterol-conjugated small interfering RNAs in vivo.

Effective in vivo delivery of small interfering (siRNA) has been a major obstacle in the development of RNA interference therapeutics. One of the first attempts to overcome this obstacle utilized intravenous injection of cholesterol-conjugated siRNA (chol-siRNA). Although studies in mice revealed target gene knockdown in the liver, delivery was relatively inefficient, requiring 3 daily injections of 50 mg/kg of chol-siRNA to obtain measurable reduction in gene expression. Here we present a new delivery approach that increases the efficacy of the chol-siRNA over 500-fold and allows over 90% reduction in target gene expression in mice and, for the first time, high levels of gene knockdown in non-human primates. This improved efficacy is achieved by the co-injection of a hepatocyte-targeted and reversibly masked endosomolytic polymer. We show that knockdown is absolutely dependent on the presence of hepatocyte-targeting ligand on the polymer, the cognate hepatocyte receptor, and the cholesterol moiety of the siRNA. Importantly, we provide evidence that this increase in efficacy is not dependent on interactions between the chol-siRNA with the polymer prior to injection or in the bloodstream. The simplicity of the formulation and efficacy of this mode of siRNA delivery should prove beneficial in the use of siRNA as a therapeutic.

EGFP-EGF1 protein-conjugated PEG-PLA nanoparticles for tissue factor targeted drug delivery.

In a strategy for anti-thrombotic therapy, we have expressed EGFP-EGF1 fusion protein, in which EGF1 can bind with tissue factor (TF). EGFP has previously been widely used as a fluorescent protein marker. EGFP-EGF1 protein was thiolated and conjugated to the malemide covering on the pegylated nanoparticles (NP) to form the EGFP-EGF1-NP. The EGFP-EGF1-NP was characterized in terms of morphology, size and zeta potential. In vitro cell viability experiment confirmed that the biodegradable EGFP-EGF1-NP was safe. To evaluate the delivering ability of EGFP-EGF1-NP, a fluorochrome dye, Dir, was incorporated into the nanoparticle, and the loading capacity and release property of the particle were examined. In vitro results showed that the binding ability of EGFP-EGF1-NP with TF-expressing cells was significantly stronger than that of non-conjugated NP. In vivo multispectral fluorescent imaging demonstrated that EGFP-EGF1-NP had high specificity and sensitivity in targeting thrombi. Our study demonstrated that EGFP-EGF1-NP is a promising TF-targeting drug delivery system for thrombolytic treatment.

Effect of cirrhosis of the liver on the pharmacokinetics of chlormethiazole.

The pharmacokinetics of chlormethiazole were studied in eight patients with advanced cirrhosis of the liver and in six healthy volunteers after oral and intravenous administration of the drug. In the patients the systemic bioavailability of oral chlormethiazole was increased about tenfold, whereas its elimination was only slightly retarded. The increased bioavailability was clearly due to decreased first-pass metabolism of chlormethiazole in the cirrhotic liver. The results indicate that chlormethiazole should be used in reduced dosage when given by mouth to patients with cirrhosis of the liver.