On the validity of fluorimetric intracellular calcium detection: Impact of lipid components.

We investigated the effects of different lipids on the activity of the angiotensin II type 1 receptor (AT1R). As calcium plays a key role in the signaling of the AT1R, we used the calcium-sensitive fluorescence indicators fura-2 to detect intracellular calcium release upon stimulation with the agonist angiotensin II. At first sight, cells preincubated with Very low-density lipoprotein (VLDL) showed a reduced calcium release triggered by angiontensin II compared to untreated control. However, on closer examination, this result seemed to be an artifact. Incubation with VLDL reduced also the amount of intracellular fura-2, as measured by fluorescence in the isosbestic point. Additionally, the maximal obtainable ratio, obtained after complete saturation with calcium ions, was reduced in cells preincubated with VLDL. These findings rendered our initial results questionable. We report the results of our work and our suggestions regarding the experimental setup to contribute to the understanding of the interpretation of fura-2 measurements and to avoid erroneous conclusions.

Reversible Shielding and Immobilization of Liposomes and Viral Vectors by Tailored Antibody-Ligand Interactions.

Controlling the time and dose of nanoparticulate drug delivery by administration of small molecule drugs holds promise for efficient and safer therapies. This study describes a versatile approach of exploiting antibody-ligand interactions for the design of small molecule-responsive nanocarrier and nanocomposite systems. For this purpose, antibody fragments (scFvs) specific for two distinct small molecule ligands are designed. Subsequently, the surface of nanoparticles (liposomes or adeno-associated viral vectors, AAVs) is modified with these ligands, serving as anchor points for scFv binding. By modifying the scFvs with polymer tails, they can act as a non-covalently bound shielding layer, which is recruited to the anchor points on the nanoparticle surface and prevents interactions with cultured mammalian cells. Administration of an excess of the respective ligand triggers competitive displacement of the shielding layer from the nanoparticle surface and restores nanoparticle-cell interactions. The same principle is applied for developing hydrogel depots that can release integrated AAVs or liposomes in response to small molecule ligands. The liberated nanoparticles subsequently deliver their cargoes to cells. In summary, the utilization of different antibody-ligand interactions, different nanoparticles, and different release systems validates the versatility of the design concept described herein.

Lipoprotein turnover and possible remnant accumulation in preeclampsia: insights from the Freiburg Preeclampsia H.E.L.P.-apheresis study.

BACKGROUND: Preeclampsia is a life-threatening disease in pregnancy, and its complex pathomechanisms are poorly understood. In preeclampsia, lipid metabolism is substantially altered. In late onset preeclampsia, remnant removal disease like lipoprotein profiles have been observed. Lipid apheresis is currently being explored as a possible therapeutic approach to prolong preeclamptic pregnancies. Here, apheresis-induced changes in serum lipid parameters are analyzed in detail and their implications for preeclamptic lipid metabolism are discussed. METHODS: In the Freiburg H.E.L.P.-Apheresis Study, 6 early onset preeclamptic patients underwent repeated apheresis treatments. Serum lipids pre- and post-apheresis and during lipid rebound were analyzed in depth via ultracentrifugation to yield lipoprotein subclasses. RESULTS: The net elimination of Apolipoprotein B and plasma lipids was lower than theoretically expected. Lipids returned to previous pre-apheresis levels before the next apheresis even though apheresis was repeated within 2.9 ± 1.2 days. Apparent fractional catabolic rates and synthetic rates were substantially elevated, with fractional catabolic rates for Apolipoprotein B / LDL-cholesterol being 0.7 ± 0.3 / 0.4 ± 0.2 [day- 1] and synthetic rates being 26 ± 8 / 17 ± 8 [mg*kg- 1*day- 1]. The distribution of LDL-subclasses after apheresis shifted to larger buoyant LDL, while intermediate-density lipoprotein-levels remained unaffected, supporting the notion of an underlying remnant removal disorder in preeclampsia. CONCLUSION: Lipid metabolism seems to be highly accelerated in preeclampsia, likely outbalancing remnant removal mechanisms. Since cholesterol-rich lipoprotein remnants are able to accumulate in the vessel wall, remnant lipoproteins may contribute to the severe endothelial dysfunction observed in preeclampsia. TRIAL REGISTRATION: ClinicalTrails.gov, NCT01967355 .

Interference of phosphatidylcholines with in-vitro cell proliferation - no flock without black sheep.

According to early experiments with natural extracts, phosphatidylcholines (PCs) are widely considered essentially non-toxic. In addition to these physiological mixed-chain PCs, many different synthetic diacyl-PCs are currently available, but they have never been systematically evaluated for any interference with cell proliferation. We thus investigated the cell proliferation of several cell lines in the presence of various liposomes consisting of a single PC component and cholesterol. Most of the PCs investigated did not interfere with cell proliferation, supporting the notion that most PCs are safe excipients. Significant IC50 values below 0.5mM were detected for PC(12:0/12:0), PC(14:1/14:1)trans and all diacyl-PCs containing two polyunsaturated fatty acids (PUFAs). The ω-3 PC(22:6/22:6) was the most toxic PC assessed, revealing IC50 values below 100 µM, but no rule concerning ω-3/6 configuration or acyl chain length could be observed. Physiological mixed-chain PCs containing PUFAs were much less toxic than respective non-physiological diacyl-PCs. All trans fatty acids in diacyl-PCs interfered more with proliferation than their respective cis-configured counterparts. Depending on the concentration, those diacyl-PCs not only inhibited proliferation but also induced cell death. Unlike the non-toxic PCs usually used for liposomal drug delivery, the elucidated diacyl-PCs may be worthy of further examination to eventually construct a toxic shell for toxic drugs, thereby enhancing anticancer drug delivery via lipid particles.

Systemic confounders affecting serum measurements of omega-3 and -6 polyunsaturated fatty acids in patients with retinal disease.

BACKGROUND: Omega-3 polyunsaturated fatty acids (PUFAs) have a highly anti-angiogenic effect in animal models. However, the clinical relevance of omega-3PUFAs in human retinal pathologies remains unclear. The ARED 2 study found no effect of omega-3 PUFA supplementation on progression of age related macular degeneration (AMD). The aim of this study was to compare serum levels of omega-3- and omega-6 PUFAs between patients with diabetic retinopathy (DR), AMD and retinal vein occlusion (RVO), and to identify potential confounders of serum level measurements. METHODS: Venous blood samples were collected from 44 patients with DR, 25 with AMD, 12 with RVO and 27 controls. The lipid phase was extracted and analyzed using mass spectrometry. Retinal disease staging was done by indirect funduscopy and FAG where appropriate. Patient demographics and medical history including current medication and fasting state were acquired. Tukey contrasts for multiple comparisons of the mean and linear regression analysis were used for statistical analysis. RESULTS: Our data revealed no significant differences in omega-6 PUFA serum levels between patients with AMD, DR, RVO and controls (p > 0.858). Uncorrected omega-3 PUFA levels were significantly higher in patients with AMD compared to DR but not compared to controls (p = 0.004). However, after correcting for possible confounders such as body mass index (BMI), age, sex, fasting and use of statins, no statistically significant difference remained for serum omega-3 PUFA levels. Fasting was identified as an independent confounder of total omega-6 PUFAs, three individual omega-6 PUFAs and one omega-3 PUFA(p < 0.0427). Statin use was identified as an independent confounder of α-linolenic acid (an omega-3PUFA; p = 0.0210). CONCLUSION: In this pilot study with relatively low patient numbers, we report significant differences in serum levels of omega-3PUFAs among patients with different types of retinal diseases. However, these differences were not robust for disease specificity after correction for possible confounders in our cohort. Our results demonstrate that serum lipid profiles need to be interpreted with caution since they are significantly altered by variables like fasting and medication use independent from the underlying disease. Correcting for respective confounders is thus necessary to compare serum lipid profiles in clinical studies.

Comparison of double-filtration plasmapheresis (DFPP) versus heparin-mediated extracorporeal LDL-precipitation (HELP)-apheresis in early-onset preeclampsia.

OBJECTIVES: Preeclampsia (PE) is a major cause of maternal and fetal mortality, and preterm birth. Previous studies indicate that lipid-apheresis may prolong pregnancy, namely heparin-mediated extracorporeal LDL-precipitation (HELP)- and dextran sulfate cellulose (DSC)-apheresis. We now report on double membrane plasmapheresis (DFPP) in early-onset preeclampsia (eoPE). STUDY DESIGN: Open pilot study assessing the prolongation of pregnancy in PE by lipoprotein-apheresis (DRKS00004527). Two women with eoPE were treated by DFPP and compared to a historical cohort of 6 patients with eoPE treated by HELP-apheresis (NCT01967355). MAIN OUTCOME MEASURES: Clinical outcome of mothers and babies and prolongation of pregnancies (time of admission to birth). RESULTS: Patient 1 (33y; 22 + 5/7GW) received 4 DFPP. Delivery day 19; birthweight 270 g; weight at discharge 2134 g on day 132. Patient 2 (35y; 21 + 4/7GW) received 2 DFPP. Delivery day 19; birthweight 465 g; weight at discharge 2540 g on day 104. DFPP was well tolerated by both patients. CONCLUSIONS: DFPP proved to be save and pregnancies remained stable as long as 19 days. Although babies were born very preterm both babies could finally be dismissed from hospital. No relevant clinical differences between DFPP and HELP-apheresis could be observed. Therefore, DFPP may extend the range of available apheresis techniques to prolong pregnancies in early-onset preeclampsia. However, further studies are necessary to gain more information. REGISTER: (DRKS00004527).

Kinetic targeting of pegylated liposomal doxorubicin: a new approach to reduce toxicity during chemotherapy (CARL-trial).

BACKGROUND: The therapeutic success of chemotherapeutic agents is often limited by severe adverse effects. To reduce toxicity of these drugs, nanoscale particle-based drug delivery systems (DDS) are used. DDS accumulate to some extent in tumor tissues, but only a very small portion of a given dose reaches this target. Accumulation of DDS in tumor tissues is supposed to be much faster than in certain other tissues in which side effects occur ("Kinetic Targeting"). Once saturation in tumor tissue is achieved, most of the administered DDS still circulate in the plasma. The extracorporeal elimination of these circulating nanoparticles would probably reduce toxicity. METHODS: For the CARL-trial (Controlled Application and Removal of Liposomal chemotherapeutics), pegylated liposomal doxorubicin (PLD) was used as chemotherapeutic agent and double filtration plasmapheresis (DFPP) was performed for extracorporeal elimination of liposomes. PLD was given as 40 mg/m2 every 3 weeks in combination with vinorelbine 2 × 25 mg/m2 (neoadjuvant treatment of breast cancer, 12 patients), or as 40 mg/m2 every 4 weeks (recurrent ovarian cancer, 3 patients). Primary endpoints were the efficiency and safety profile of DFPP, and secondary endpoints were side effects and tumor response. RESULTS: DFPP eliminated ~62% of circulating PLD, corresponding to ~45% of the total dose (n = 57 cycles). AUC of doxorubicin was reduced by 50%. No leakage of doxorubicin was detected during elimination, and no relevant DFPP-related side effects occurred. Reduction in tumor size > 30% occurred in 10/12 (neoadjuvant) and in 1/3 patients (recurrent). Only five grade 2 events and one grade 3 event (mucositis, neutropenia or leucopenia) and a single palmar-plantar erythrodysesthesia grade 2 were reported. CONCLUSION: Extracorporeal elimination of PLD by DFPP is safe and efficient. CARL can diminish the main dose-limiting side effects of PLD, and probably many different DDS alike. TRIAL REGISTRATION: DRKS00000163.

Controlled application and removal of liposomal therapeutics: effective elimination of pegylated liposomal doxorubicin by double-filtration plasmapheresis in vitro.

INTRODUCTION: Nanoscale particle-based drug delivery systems like long circulating liposomal doxorubicin show unique pharmacokinetic properties and improved toxicity profiles. Liposomal doxorubicin accumulates in tumor tissue due to the enhanced permeation and retention effect, but only a small fraction of a total dose reaches the tumor site. Accumulation of liposomal doxorubicin is much faster in tumor sites than in certain organs where dose limiting adverse effects occur. Finding a way to detoxify the predominant part of a given dose, circulating in the blood after accumulation is completed, will presumably reduce severe side effects during chemotherapy. METHODS: Elimination properties of therapeutic used pegylated liposomal doxorubicin (Doxil/Caelyx) and therapeutic used double-filtration plasmapheresis systems were evaluated in vitro and in reconstituted human blood. RESULTS: Liposomes can be filtered by appropriate membranes without leakage of doxorubicin up to a pressure of 1 bar. At higher pressures, liposomes ( approximately 85 nm) may squeeze through much smaller pores without significant leakage of doxorubicin, whereas decreasing pore size to approximately 8 nm leads to increased leakage of doxorubicin. With therapeutic used apheresis systems, liposomal doxorubicin can be efficiently eliminated out of buffer medium and reconstituted human blood. No leakage of doxorubicin was detected, even when liposomes were circulating for 48 h in human plasma before apheresis. CONCLUSIONS: Convenient apheresis techniques are capable of a safe and efficient elimination of therapeutic used liposomal doxorubicin in an experimental model system.

Controlled application and scheduled removal of nanoparticle based chemotherapeutics (CARL) will reduce dose limiting adverse events in anticancer chemotherapy.

Clinical success of cancer chemotherapy is impaired by dose limiting toxicities. Nanoscale particle based drug delivery systems (DDS) like long circulating liposomes show improved toxicity profiles. Nevertheless, their unique pharmacokinetic properties lead to new dose limiting adverse events such as elevated skin toxicity. Though DDS accumulate in tumor tissue, only a very small fraction of the total dose reaches the target site. The overwhelming amount of a given dose is needed only to build up a diffusion gradient for effective accumulation at the target site. Due to the altered endothelial barrier, accumulation of DDS in tumor tissue is much faster than accumulation in other tissues, where dose limiting side effects occur. On the basis of these pharmakinetic data we hypothesize, that once accumulation in the tumor tissue is completed, rapid elimination of the DDS fraction still circulating in the plasma may diminish otherwise dose limiting toxicities. Rapid elimination of circulating DDS might be performed by extracorporeal apheresis treatment. Within this paper the principle of kinetic targeting by scheduled extracorporeal elimination of long circulating DDS is presented in detail. Benefits for patients are as well discussed as possible criticisms and future developments. In conclusion, the combination of DDS and scheduled apheresis may allow the development of new chemotherapy regiments with higher impact and/or less toxicity.

Apheresis as emerging treatment option in severe early onset preeclampsia.

Based on an early suggestion by Winkler et al. 2003 and a subsequent successful study by Wang et al. 2006 using lipid apheresis (LA) in 9 patients with preeclampsia to prolong pregnancies, the use of apheresis as therapeutic option in severe early onset preeclampsia has received increasing attention. Further studies using different LA systems also prolonged pregnancy and have been published in the last few years. Albeit using different LA systems and relying on different working hypothesis, all studies demonstrated a promising stabilisation against the disease's progression. Overall time from hospitalisation to the need for mandatory delivery was longer for those patients receiving apheresis compared to historical or matched control patients not receiving apheresis. These data will be reviewed and different hypotheses about the beneficial mechanism of action of apheresis will be discussed. Since up to now there is no curative treatment for preeclampsia other than observation and delivery, future work shall be encouraged.

Optimizing Antitumor Efficacy and Adverse Effects of Pegylated Liposomal Doxorubicin by Scheduled Plasmapheresis: Impact of Timing and Dosing.

BACKGROUND: Nanoscale drug delivery systems accumulate in solid tumors preferentially by the enhanced permeation and retention effect (EPR-effect). Nevertheless, only a miniscule fraction of a given dosage reaches the tumor, while >90% of the given drug ends up in otherwise healthy tissues, leading to the severe toxic reactions observed during chemotherapy. Once accumulation in the tumor has reached its maximum, extracorporeal elimination of circulating nanoparticles by plasmapheresis can diminish toxicities. OBJECTIVE: In this study, we investigated the effect of dosing and plasmapheresis timing on adverse events and antitumor efficacy in a syngeneic rat tumor model. METHODS: MAT-B-III cells transfected with a luciferase reporter plasmid were inoculated into female Fisher rats, and pegylated liposomal doxorubicin (PLD) was used for treatment. Plasmapheresis was performed in a discontinuous manner via centrifugation and subsequent filtration of isolated plasma. RESULTS: Bioluminescence measurements of tumor growth could not substitute caliper measurements of tumor size. In the control group, raising the dosage above 9 mg PLD/kg body weight did not increase therapeutic efficacy in our fully immunocompetent animal model. Plasmapheresis was best done 36 h after injecting PLD, leading to similar antitumor efficacy with significantly less toxicity. Plasmapheresis 24 h after injection interfered with therapeutic efficacy, while plasmapheresis after 48 h led to fewer side effects but also to increased weight loss. CONCLUSION: Long-circulating nanoparticles offer the unique possibility to eliminate the excess of circulating particles after successful accumulation in tumors by EPR, thereby reducing toxicities and likely toxicity-related therapeutic limitations.

Accumulating nanoparticles by EPR: A route of no return.

Nanoparticle-based drug delivery to ease anticancer therapy relies primarily on the enhanced permeability and retention effect (EPR). The leaky vascular structure in tumors allows extravasation of nanoparticles, often termed passive targeting. Long term retention of nanoparticles is attributed to the lack of lymphatic drainage, and unidirectional extravasation has been implied. Fluorescent liposomes with a plasma half-life of 29h were injected into tumor-bearing rats, and biodistribution in tumor, skin, paws and ears was monitored via in vivo fluorescence measurements. To calculate tissue accumulation, an algorithm was developed to subtract the blood signal from the total fluorescence recorded. Accumulation in tumor tissue was much higher than that in other tissues monitored, initially exhibiting very rapid accumulation followed by a long plateau phase with little change. Discontinuous plasmapheresis was established that was as effective as highly sophisticated clinical plasmapheresis. We observed no difference in the tumor tissue's accumulation when plasmapheresis was performed 22h after liposome injection. In contrast, plasmapheresis led to a significant inhibition of further accumulation in other tissues. When the liposomes' blood concentration was rapidly lowered, we detected no drop in tumor fluorescence. Thus extravasation via EPR is most likely a route of no return. These data support the emerging view of a more dynamic model of EPR, where gaps or entire vessels may open and close over time, or accumulated liposomes become entangled within the pores, hampering further accumulation.

Molecular MRI in the Earth's Magnetic Field Using Continuous Hyperpolarization of a Biomolecule in Water.

In this work, we illustrate a method to continuously hyperpolarize a biomolecule, nicotinamide, in water using parahydrogen and signal amplification by reversible exchange (SABRE). Building on the preparation procedure described recently by Truong et al. [ J. Phys. Chem. B , 2014 , 118 , 13882 - 13889 ], aqueous solutions of nicotinamide and an Ir-IMes catalyst were prepared for low-field NMR and MRI. The (1)H-polarization was continuously renewed and monitored by NMR experiments at 5.9 mT for more than 1000 s. The polarization achieved corresponds to that induced by a 46 T magnet (P = 1.6 × 10(-4)) or an enhancement of 10(4). The polarization persisted, although reduced, if cell culture medium (DPBS with Ca(2+) and Mg(2+)) or human cells (HL-60) were added, but was no longer observable after the addition of human blood. Using a portable MRI unit, fast (1)H-MRI was enabled by cycling the magnetic field between 5 mT and the Earth's field for hyperpolarization and imaging, respectively. A model describing the underlying spin physics was developed that revealed a polarization pattern depending on both contact time and magnetic field. Furthermore, the model predicts an opposite phase of the dihydrogen and substrate signal after one exchange, which is likely to result in the cancelation of some signal at low field.

Elimination of liposomes by different separation principles used in low-density lipoprotein apheresis.

Clinical success of many therapies is impaired by dose limiting toxicities. Nanoscale particle-based drug delivery systems such as liposomes show unique pharmacokinetic properties and improved toxicity profiles. Liposomes accumulate in tumor tissue, but only a small fraction of a total dose reaches the target site. The overwhelming amount of a given dose is needed only to build up a diffusion gradient for effective accumulation at the target site. In order to find a way to detoxify this predominant fraction after accumulation is completed, the different separation principles used for the apheresis of lipoproteins were evaluated for the extracorporeal elimination of liposomes. Appropriate radiolabeled model liposomes were prepared by extrusion. Separation efficacy, leakage of liposomal content and influence of plasma contact were measured. Membranes with pore sizes between 25 and 400 nm were used to investigate filtration properties of liposomes. Liposomes were precipitated by adding heparin and Ca(2+). Adsorption chromatography was investigated using dextran sulfate, heparin sepharose and functionalized polyacrylamide beads. Membrane filtration allowed the elimination of various liposomes, while precipitation and adsorption were only useful for positively charged liposomes. Leakage of liposomal content was not induced by adsorption, but precipitation induced leakage. Leakage during filtration was dependent on liposomal membrane lipids. Plasma contact reduced precipitation and adsorption efficacy of positively charged liposomes, while filtration properties of liposomes remained unchanged. For extracorporeal elimination of liposomal drug delivery systems, filtration-based techniques are presumably more convenient and versatile than precipitation- or adsorption-based apheresis technologies.

Central role of a serine phosphorylation site within duck hepatitis B virus core protein for capsid trafficking and genome release.

Viral nucleocapsids compartmentalize and protect viral genomes during assembly while they mediate targeted genome release during viral infection. This dual role of the capsid in the viral life cycle must be tightly regulated to ensure efficient virus spread. Here, we used the duck hepatitis B virus (DHBV) infection model to analyze the effects of capsid phosphorylation and hydrogen bond formation. The potential key phosphorylation site at serine 245 within the core protein, the building block of DHBV capsids, was substituted by alanine (S245A), aspartic acid (S245D) and asparagine (S245N), respectively. Mutant capsids were analyzed for replication competence, stability, nuclear transport, and infectivity. All mutants formed DHBV DNA-containing nucleocapsids. Wild-type and S245N but not S245A and S245D fully protected capsid-associated mature viral DNA from nuclease action. A negative ionic charge as contributed by phosphorylated serine or aspartic acid-supported nuclear localization of the viral capsid and generation of nuclear superhelical DNA. Finally, wild-type and S245D but not S245N virions were infectious in primary duck hepatocytes. These results suggest that hydrogen bonds formed by non-phosphorylated serine 245 stabilize the quarterny structure of DHBV nucleocapsids during viral assembly, while serine phosphorylation plays an important role in nuclear targeting and DNA release from capsids during viral infection.