HEPES-buffered Saline Research Articles

Extracellular acidification increases uterine contraction in pregnant mouse by increasing intracellular calcium.

As uterine extracellular pH decreases during the ischemic conditions of labor, but its effects on myometrial contraction are largely unknown, there is a need to elucidate its physiological effects and mechanisms of action. Furthermore, it is not known if any of the effects of extracellular acidification are affected by pregnancy, thus we also determined how gestation affects the response to acidification. Nonpregnant, mid-, and term-pregnant myometrial strips were obtained from humanely killed mice. Contractions were recorded under spontaneous, depolarized, and oxytocin-stimulated conditions. The extracellular pH of the perfusate was changed from 7.4 to 6.9 or 7.9 in HEPES-buffered physiological saline. Intracellular pH was measured using SNARF, and intracellular calcium was measured using Indo-1. Statistical differences were tested using the appropriate t-test. Extracellular acidification significantly increased the frequency and amplitude of spontaneous contractions in pregnant, but not nonpregnant, myometrium, whereas alkalinization decreased contractions. Intracellular acidification, via Na-butyrate, transiently increased force in pregnant tissue. Intracellular pH was gradually acidified when extracellular pH was acidified, but extracellular acidification increased contractility before any significant change in intracellular pH. If myometrial force was driven by oxytocin or high-K depolarization, then extracellular pH did not further increase force. Intracellular calcium changes mirrored those of force in the spontaneously contracting pregnant myometrium, and if calcium entry was prevented by nifedipine, extracellular acidification could not induce a rise in force. Extracellular acidification increases excitability, calcium entry, and thus force in pregnant mouse myometrium, and this may contribute to increasing contractions during labor when ischemic conditions and acidemia occur.

Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy

We report the formulation, characterization, colloidal stability, and in vitro efficiency of Fisetin nanocrystals stabilized by poloxamer P407. Such nanocrystals present a nanometer scale (148.6 ± 1.1 nm) and a high homogeneity (polydispersity index of 0.17 ± 0.01), with a production yield of 97.0 ± 2.5%. The engineered formulations of nanocrystals suspension (pH of 7.4 ± 0.1), stabilized via steric repulsion, are stable for several days in aqueous environment (Milli Q water, NaCl 10 mM or mannitol 5% w/v), for few days in HEPES buffered saline (HBS) (20 / 150 mM) under sink conditions, and in culture medium. After freeze drying in 5% w/v mannitol, the nanocrystal formulations can be stored at −80 °C for at least 120 days. Drug release experiments displayed a 98.7 ± 5.1% cumulative release over 3 days in HBS. Compared to the free drug, the nanocrystal formulations showed an improved cytotoxicity highlighted by the decrease of the half maximal inhibitory concentration for both murine Lewis lung carcinoma (3LL) and human endothelial (EA.hy926) cell lines. In addition, after incubation with Fisetin nanosuspensions, significant changes in the cell morphology for both cell lines were observed, showing an improved anti-angiogenic effect of nanocrystals formulation compared to the free drug. Overall, Fisetin formulated as nanocrystals showed enhanced biopharmaceutical properties and in vitro activity, offering a wide range of indications for challenging applications in the clinic.

Reverse relation between cytotoxicity and Polyethylenimine/DNA ratio, the effect of using HEPES-buffered saline (HBS) medium in gene delivery

Polyethyleneimine (PEI) is considered a promising cationic polymer in non-viral gene delivery. DNA binding properties and other biochemical characteristics of PEI such as the proton sponge phenomenon, offered the branched 25 kDa PEI to be widely used for therapeutic DNA delivery, although the possible cytotoxic effects and the best conditions of PEI preparation are not still well recognized. While higher PEI/Plasmid ratios have increased transfection efficiencies, it induces more cell stress and toxicity. Considering that the PEI particle size and resulting cytotoxicity are affected by media ions, we used Neuro2A cells to assess the cell stress properties of PEI/Plasmid complexes prepared in a HEPES-buffered saline medium. Delivery of a plasmid containing EGFP happened in all increasing ratios of PEI/plasmid from 0.5, 2, 4, and 6, while higher ratios induced less unfolded protein response as evidenced by lower transcription of ER stress markers Grp78, Atf4, Chop, Xbp1, and induced Xbp1 splicing. These data were also supported by MTT cytotoxicity assay results. These findings indicate that preparing higher PEI/plasmid ratio complexes (using the equivalent of 200 ng DNA) in the HBS medium leads to less cytotoxicity.

Influence of short term storage conditions, concentration methods and excipients on extracellular vesicle recovery and function

Extracellular vesicles (EVs) are phospholipid bilayer enclosed vesicles which play an important role in intercellular communication. To date, many studies have focused on therapeutic application of EVs. However, to progress EV applications faster towards the clinic, more information about the physical stability and scalable production of EVs is needed. The goal of this study was to evaluate EV recovery and function after varying several conditions in the isolation process or during storage.Physical stability and recovery rates of EVs were evaluated by measuring EV size, particle and protein yields using nanoparticle tracking analysis, microBCA protein quantification assay and transmission electron microscopy. Western blot analyses of specific EV markers were performed to determine EV yields and purity. EV functionality was tested in an endothelial cell wound healing assay.Higher EV recovery rates were found when using HEPES buffered saline (HBS) as buffer compared to phosphate buffered saline (PBS) during EV isolation. When concentrating EVs, 15 ml spinfilters with a 10 kDa membrane cutoff gave the highest EV recovery. Next, EV storage in polypropylene tubes was shown to be superior compared to glass tubes. The use of protective excipients during EV storage, i.e. bovine serum albumin (BSA) and Tween 20, improved EV preservation without influencing their functionality. Finally, it was shown that both 4 °C and −80 °C are suitable for short term storage of EVs. Together, our results indicate that optimizing buffer compositions, concentrating steps, protective excipients and storage properties may collectively increase EV recovery rates significantly while preserving their functional properties, which accelerates translation of EV-based therapeutics towards clinical application.

Emizicumab Promotes Factor Xa Generation on Activated Endothelium in a Blood Cell-Independent Manner

IntroductionHemophilia A (HA) is an inherited bleeding disorder caused by the deficiency of coagulation factor VIII (FVIII) resulting in severe hemorrhage if untreated. Recombinant and plasma derived FVIII products have long been the standard of care in hemophilia. However, approximately 25-30% of patients with severe HA develop inhibitors, neutralizing alloantibodies to FVIII, a significant complication in the treatment of patients with HA that leads to bleeding despite factor therapy.First approved for bleed prophylaxis in HA with inhibitors in the US by the FDA in 2018, emicizumab (Genentech, USA) has initiated a new era of HA treatment. This drug is a bispecific, monoclonal antibody that binds to activated Factor IX (FIXa) and Factor X (FX), mimicking activated FVIII (FVIIIa) by bringing FIXa and FX into proximity to enable FX activation, even in the presence of inhibitors.Emicizumab prophylaxis drastically reduces bleed episodes. However, thromboses and thrombotic microangiopathy (TMA) were observed in trials, all associated with concomitant use of activated prothrombin complex concentrates (aPCC) (Callaghan et al., 2021). The mechanism of this devastating condition is uncertain, as emicizumab is not known to bind to phospholipid or vascular surfaces. We report that FX is more readily activated by FIXa and emicizumab on endothelium that has been activated by tumor necrosis factor alpha (TNF). This finding may partially explain the development of TMA in these patients.MethodsWe utilized novel, microfluidic devices that are inexpensive to manufacture and were modified from a technique previously described (Alapan et al. 2016). These devices are fabricated using a laser cut double-sided adhesive film sandwiched between a clear, gas-permeable polymer (Ibidi, Germany) and an acrylic top that is laser cut (Universal Laser Systems Inc., USA) (Figure 1).Human umbilical endothelial cells (HUVEC, Lonza, Switzerland) were harvested at passage 3 to 4 and seeded into the devices. These were then cultured under flow conditions using a non-peristaltic, air-driven pump (Ibidi GmbH, Germany) to achieve a confluent and quiescent endothelial surface. HUVEC are then activated by incubating with 5 nM TNF in serum-free growth medium for 4 hours. This treatment induced markers of endothelial activation without gross apoptosis. Non-activated HUVEC were incubated with endothelial cell growth medium (2% serum) until time of experiments.Factors IXa, X (Haemtech, USA), and/or emicizumab (discarded clinical material) were mixed in HEPES-buffered saline with 5 mM calcium chloride for all experimental conditions. Concentrations used of FIXa (30 nM), FX (170 nM), and emicizumab (55 ug/mL) were constant for all conditions. Combinations of factors and emicizumab were then incubated in the endothelialized device for 30 minutes at 37° C. The entire volume of the mixture was then aspirated (20 uL) and stored at -80° C.FXa activity was assayed on the effluent for 60 minutes using a chromogenic FXa substrate (Pefachrome, Pentapharm, Switzerland).ResultsNo significant generation of Xa was noted in the presence of healthy or activated endothelium with emicuzumab alone, emicizumab and FIXa, emicizumab and FX, or factors IXa and X. Median Xa generation observed with the combination of emicizumab, FIXa, and FX on healthy endothelium was 2 nM. Median Xa generation with the same combination on activated endothelium was 8.1 nM, a four-fold increase (P = 0.028, Mann-Whitney test) (Figure 2).DiscussionEmicizumab represents an evolving standard of care for hemophilia A. Considering data showing diminishing FVIII expression in the months to years after AAV gene therapy, (Pasi et al., 2020) it may well be the dominant treatment paradigm in HA for some time.However, much remains to be answered in the use of emicizumab, and the mechanism of thrombosis and TMA with concomitant aPCC use has resulted in the avoidance of aPCC use for breakthrough bleeding in patients on emicizumab therapy, even up to 6 months after cessation.Our data demonstrate that activated endothelial cells promote FX activation more readily than quiescent endothelial cells in the presence of FIXa and emicizumab. These findings demonstrate the potential of thrombotic angiopathy in an in vitro system. Further investigation of the interaction of endothelium with FIXa, FX, and FVIIIa-mimetic antibodies is warranted. [Display omitted] DisclosuresEllsworth: Takeda: Other: Salary supported as part of NHF-Takeda Clinical Fellowship Award. Monroe: Medexus Pharmaceuticals: Consultancy; Takeda: Consultancy; Otello Medical: Current equity holder in publicly-traded company. Hoffman: Takeda: Research Funding; CSL Behring: Consultancy; Sanofi: Consultancy; BPL (Bio Products Laboratory): Consultancy. Key: BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator; Sanofi: Consultancy.

Comparison of electrochemical performance of various boron-doped diamond electrodes: Dopamine sensing in biomimicking media used for cell cultivation

Chemically inert and biocompatible boron-doped diamond (BDD) has been successfully used in neuroscience for sensitive neurochemicals sensing and/or as a growth substrate for neurons. In this study, several types of BDD differing in (i) fabrication route, i.e. conventional microwave plasma enhanced chemical vapour deposition (MW-PECVD) reactor vs. MW-PECVD with linear antenna delivery system, (ii) morphology, i.e. planar vs. porous BDD, and (iii) surface treatment, i.e. H-terminated (H-BDDs) vs. O-terminated (O-BDDs), were characterized from a morphological, structural, and electrochemical point of view. Further, planar and porous BDD-based electrodes were tested for sensing of dopamine in common biomimicking environments of pH 7.4, namely phosphate buffer (PB) and HEPES buffered saline (HBS). In HBS, potential windows are narrowed due to electrooxidation of its buffering component (i.e. HEPES), however, dopamine sensing in HBS is possible. H-BDDs (both planar and porous) outperformed O-BDDs as they provided clearer dopamine signals with higher peak currents. As expected, due to its enlarged surface area and increased sp2 content, the highest sensitivity and lowest detection limits of 8 × 10−8 mol L−1 and 6 × 10−8 mol L−1 in PB and HBS media, respectively, were achieved by square-wave voltammetry on porous H-BDD.

Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.

The aim of the present study was to improve the in vitro maturation (IVM) procedure using oocytes from surplus ovarian tissue after fertility preservation. Twenty-five patients aged 17-37years were included in the study. Maturation was compared between oocytes collected in HEPES-buffered medium or saline, and we determined whether transport on ice prior to oocyte collection affected maturation. Two different IVM media were used that were supplemented with and without recombinant human midkine. Mature oocytes were assessed for aneuploidy using next-generation sequencing (NGS). On average, 36 immature oocytes were collected from each patient (range 7-90, N = 895). Oocytes recovered from HEPES-buffered medium matured at a higher rate than oocytes recovered from saline (36% vs 26%, p < 0.01). Ovarian transportation on ice prior to the procedure negatively affected maturation compared with non-transported samples (42% vs 27%, p < 0.01). The addition of midkine improved maturation rate (34% vs 27%, p < 0.05). On average, 11 MII oocytes were obtained per patient (range 1-30). NGS of 53 MII oocytes and their first polar bodies indicated that 64% were euploid. The study demonstrated unexpectedly high number of immature oocytes collected from surplus ovarian tissue without any stimulation. The overall MII rate was one in three, resulting in a total number of MII oocytes that was similar to the number obtained after ovarian stimulation. If these MII oocytes prove suitable for IVF, they will provide a substantial improvement in fertility preservation for patients and advance IVM as an interesting platform for further improvements in assisted reproduction.

Glycine Protects Muscle Cells From Wasting in vitro via mTORC1 Signaling.

Glycine supplementation can protect skeletal muscles of mice from cancer-induced wasting, but the mechanisms underlying this protection are not well-understood. The aim of this study was to determine whether exogenous glycine directly protects skeletal muscle cells from wasting. C2C12 muscle cells were exposed to non-inflammatory catabolic stimuli via two models: serum withdrawal (SF) for 48 h; or incubation in HEPES buffered saline (HBS) for up to 5 h. Cells were supplemented with glycine or equimolar concentrations of L-alanine. SF- and HBS-treated myotubes (with or without L-alanine) were ~20% and ~30% smaller than control myotubes. Glycine-treated myotubes were up to 20% larger (P < 0.01) compared to cells treated with L-alanine in both models of muscle cell atrophy. The mTORC1 inhibitor rapamycin prevented the glycine-stimulated protection of myotube diameter, and glycine-stimulated S6 phosphorylation, suggesting that mTORC1 signaling may be necessary for glycine's protective effects in vitro. Increasing glycine availability may be beneficial for muscle wasting conditions associated with inadequate nutrient intake.

Fibrinogen-Mediated Platelet Microaggregate Formation in Stored Whole Blood

Introduction: Whole blood is regaining popularity in resuscitation after hemorrhage. The platelet storage lesion that has been observed in apheresis platelets also exists in whole blood, where platelet count and aggregation response decline rapidly after collection and during storage in the blood bank. Platelet microaggregates form in unfiltered whole blood, resulting in relatively large but unstable thrombus formation in a flow model over collagen. We hypothesized that the presence of fibrinogen activates platelets within the whole blood unit and predisposes them toward premature aggregate formation. Methods: Fresh whole blood (WB) was collected in citrate tubes (4.3%, 10.9 mM) from healthy donors (n=4) according to an institutionally approved standard operating procedure. Blood was centrifuged at 200-g to collect the platelet-rich plasma (PRP). The remaining red blood cell (RBC) fraction was centrifuged at 2000-g to pellet RBCs which were washed with HEPES-buffered saline and repelleted; platelet-poor plasma (PPP) was saved. The PRP fraction was centrifuged with 1 µM prostacyclin at 700-g, and the platelet pellet was washed with Tyrode's buffer, repelleted, and resuspended in either fibrinogen-deficient plasma (Fg-DP) or PPP. Platelets were combined with washed RBCs to generate a reassembled whole blood sample containing 150,000 platelets/µl and a hematocrit of 40%. Samples were added to minibags and stored at 4 °C for 14 days. On days of testing (0, 3, 7, 14), 1 ml aliquots were stained with 1 µM calcein-AM for 30 min at 37 °C and run on the BioFlux 1000 microfluidics platform with arterial flow rates generating a shear rate of 980 s-1 over a collagen surface. Fluorescence microscopy images were captured and quantified over 10 min to observe platelet adhesion to the surface. Complete blood count (CBC) was measured on each aliquot. In a follow-up study, unmanipulated, stored whole blood was passed through a standard transfusion filter (200 µm pores) to determine the effects of removing microaggregates prior to microfluidics flow testing at each time point. Results: CBC results are in Figure 1, illustrating the decline of platelet count over storage as previously observed. Figure 2 shows representative images captured at the end of the 10 min flow period. In samples containing normal plasma, thrombus formation is impaired by day 3 of storage with aggregate structures appearing immediately in the microscopy images. These aggregates attach and release periodically over the course of flow, an effect that persisted through day 14. In samples reconstructed with Fg-DP, no microaggregates were observed during storage, although the density and size of thrombi diminished over time. In filtered whole blood samples, thrombus formation on days 3 and 7 more closely resembled the Day 0 fresh samples, but aggregates appeared on day 14 despite filtration. Discussion: WB contains all of the elements required for resuscitation after hemorrhage, however the platelet storage lesion may affect its function. The interaction of platelets and fibrinogen in a relatively static environment results in activation and aggregation of platelets. In apheresis units, studies have shown that using a platelet additive solution to dilute fibrinogen results in better platelet performance for longer periods of storage. Further study is required to determine the clinical significance of aggregate formation and whether mitigation strategies should be explored. Disclosures No relevant conflicts of interest to declare.

Hyperthermia-triggered release of hypoxic cell radiosensitizers from temperature-sensitive liposomes improves radiotherapy efficacy in vitro

Hypoxia is a characteristic feature of solid tumors and an important cause of resistance to radiotherapy. Hypoxic cell radiosensitizers have been shown to increase radiotherapy efficacy, but dose-limiting side effects prevent their widespread use in the clinic. We propose the encapsulation of hypoxic cell radiosensitizers in temperature-sensitive liposomes (TSL) to target the radiosensitizers specifically to tumors and to avoid unwanted accumulation in healthy tissues. The main objective of the present study is to develop and characterize TSL loaded with the radiosensitizer pimonidazole (PMZ) and to evaluate the in vitro efficacy of free PMZ and PMZ encapsulated in TSL in combination with hyperthermia and radiotherapy. PMZ was actively loaded into TSL at different drug/lipid ratios, and the physicochemical characteristics and the stability of the resulting TSL–PMZ were evaluated. PMZ release was determined at 37 °C and 42 °C in HEPES buffer saline and fetal bovine serum. The concentration-dependent radiosensitizing effect of PMZ was investigated by exposing FaDu cells to different PMZ concentrations under hypoxic conditions followed by exposure to ionizing irradiation. The efficacy of TSL–PMZ in combination with hyperthermia and radiotherapy was determined in vitro, assessing cell survival and DNA damage by means of the clonogenic assay and histone H2AX phosphorylation, respectively. All TSL–PMZ formulations showed high encapsulation efficiencies and were stable for 30 d upon storage at 4 °C and 20 °C. Fast PMZ release was observed at 42 °C, regardless of the drug/lipid ratio. Increasing the PMZ concentration significantly enhanced the effect of ionizing irradiation. Pre-heated TSL–PMZ in combination with radiotherapy caused a 14.3-fold increase in cell death as compared to radiotherapy treatment alone. In conclusion, our results indicate that TSL–PMZ in combination with hyperthermia can assist in improving the efficacy of radiotherapy under hypoxic conditions.

Aggregation stability of nanoparticles based on rare-earth elements in different microenvironment and biological media

Background: Development of new pharmacological forms able to increase the therapeutic effectiveness of already known drugs, to reduce side effects, and to increase the comfort of treatment for the patient is an actual task of modern medicine and pharmacy. To solve the problem, one of the most promising directions in this field is the use of various nanomaterials, among which in recent years the main attention was drawn to nanomaterials based on rare earth elements (REEs). At the same time, the question of the relation between the biological activity of nanomaterials and their physical and chemical properties, as well as the features of interaction with microenvironments in biosystems, remain controversial. Objectives: Estimation of the aggregation stability of REE-based nanoparticles (NPs) in incubation media of different compositions and of the role of certain factors in stabilizing NPs in the biological microenvironment. Materials and methods: Aggregation stability of GdYVO4:Eu3+, LaVO4: Eu3+, CeO2, GdVO4:Eu3+ NPs was studied using dynamic and electrophoretic light scattering techniques. NPs were incubated in 5% glucose or buffers: 50 mM Tris buffer (with different pH within of physiological values); Igla МЕМ medium; Krebs-Ringer buffer pH 7.4; HBSS-buffer (HEPES-buffered saline-solution) pH 7.4, in the absence or presence of 0.2% BSA, for 30 minutes and 24 hours. The effect of oxidized and reduced glutathione on the stability of solutions of NPs in Tris buffer at various pH values was also determined. Results: The results have shown that in contrast to the stabilizing effect of 5% glucose solution significant aggregation of NPs is observed in saline systems. The highest degree of aggregation was observed in the Igla МЕМ and Krebs-Ringer buffer environment. Addition of 0.2% of albumin to all media prevented aggregation. Interaction of immunoglobulin with NPs leads to increase in hydrodynamic diameter, especially for some types of NPs, already at the smallest of the used protein concentrations. Oxidized but not recovered glutathione promoted aggregation of all types of orthovanadate NPs in acidic medium (Tris buffer pH=6.7). Conclusions: The aggregative stability of NPs in the salt media increases significantly in the presence of serum albumin due to changes in the ratio of the electrostatic and steric components of the interaction of NPs with the microenvironment.

Influence of cholesterol inclusion on the doxorubicin release characteristics of lysolipid-based thermosensitive liposomes

Fast hyperthermia (i.e. 39–42 °C) triggered doxorubicin release from lysolipid-containing thermosensitive liposomes (LTSL) in the tumor vasculature has been demonstrated to result in considerable enhancement of bioavailable drug levels in heated tumor tissue in preclinical tumor models. However, there is also significant leakage of doxorubicin already at 37 °C in the bloodstream, making these LTSL less efficient and increasing the risk for systemic toxicity. In conventional liposomes, cholesterol is incorporated in the bilayer to increase the stability of the liposomes. Here, we investigate the effect of cholesterol inclusion on the doxorubicin release characteristics of LTSL at 37 °C and hyperthermic temperatures.For this purpose, three LTSL formulations with 0, 5 and 10 mol% cholesterol were prepared. Inclusion of cholesterol reduced the undesired doxorubicin leakage at 37 °C in Hepes-buffered saline (HBS) as well as in fetal bovine serum (FBS). The incorporation of cholesterol in the LTSL bilayers did not influence the hyperthermia-triggered release property of the LTSL. These results were supported by DSC measurements.Therefore, in conclusion, our data indicate that cholesterol inclusion in LTSL offers a simple solution to the problem of significant leakage of doxorubicin from LTSL already at 37 °C in the bloodstream.

CA2+ SIGNALLING IN HUMAN BRAIN PERICYTES: INVESTIGATION OF EFFECTS OF AMYLOID BETA TREATMENT

Pericytes are contractile cells that regulate blood flow in the microvasculature. In the central nervous system (CNS), they are an important component of the gliovascular unit, a multicomponent cellular structure that is impacted by amyloid pathology. Here we test the effect of a synthetic human amyloid beta 1:42 peptide (hAβ 1–42) on resting Ca2+ concentrations and G-protein coupled receptor (GPCR)-mediated Ca2+ responses in human CNS pericytes. Human CNS pericytes and cell culture media were purchased from sciencellonline. Cells were grown in flasks according to the supplier's protocols. For Ca2+ imaging, cells were plated onto poly-D-lysine coated glass coverslips and pre-incubated for ∼24 hours with culture medium containing hAβ 1–42 (500 nM) or vehicle (DMSO 0.05%). The pericytes were then loaded with the Ca2+-sensitive ratiometric dye fura-2, before being placed on an inverted microscope equipped for single cell microfluorimetry. Imaging was performed in a standard HEPES-buffered saline solution (HBSS). HBSS variants containing three concentrations of glucose, 0.1, 2.5 and 6 mM, were individually examined. Pericytes exposed to hAβ1–42 displayed a lower basal Ca2+ concentration when experiments were performed in 2.5 and 6 mM glucose, although this was not observed in 0.1 mM glucose. We also tested Ca2+ responses to extracellular applications of a number of GPCR agonists relevant to pericyte function. Robust Ca2+ responses to endothelin1, bradykinin and histamine were noted; the latter producing the biggest overall population responses. Quite similar agonist responses were seen in bath solutions containing the different glucose concentrations. Furthermore, hAβ1–42 treatment produced no notable effect on agonist-induced Ca2+ responses. In summary, our initial data indicate that short term exposure of pericytes to hAβ1–42 can lower resting Ca2+ but has no discernible effect on GPCR-evoked pericyte Ca2+ signalling.

Characterizing release mechanisms of leuprolide acetate-loaded PLGA microspheres for IVIVC development I: In vitro evaluation

Release testing of parental controlled release microspheres is an essential step in controlling quality and predicting the duration of efficacy. In the first of a two-part study, we examined the effect of various incubation media on release from leuprolide-loaded PLGA microspheres to understand the influence of external pH, plasticization, and buffer type on mechanism of accelerated release. PLGA 50/50 microspheres encapsulating ~5% w/w leuprolide were prepared by the double emulsion-solvent evaporation method with or without gelatin or by the self-healing encapsulation method. The microspheres were incubated at 37°C up to 56days in various media: pH5.5, 6.5, and 7.4 phosphate buffered-saline (PBS) containing 0.02% Tween 80; pH7.4 PBS containing 1.0% triethyl citrate (PBStc); and pH7.4 HEPES buffered-saline containing 0.02% Tween 80 (all media contained 0.02% sodium azide). The recovered release media and microspheres were examined for released drug, polymer molecular weight (Mw), water uptake, mass loss, and BODIPY (green-fluorescent dye) diffusion coefficient in PLGA. After the initial burst release, release of leuprolide from acid-capped PLGA microspheres appeared to be controlled initially by erosion and then by a second mechanism after day 21, which likely consists of a combination of peptide desorption and/or water-mediated breakage of pore connections. PBStc and acidic buffers accelerated degradation of PLGA and pore-network development and increased BODIPY diffusion coefficient, resulting in faster release. Release of leuprolide from the end-capped PLGA showed similar trends as found with acid capped PLGA but with a longer lag time before release. These data provide a baseline mechanistic signature of in vitro release of leuprolide for future comparison with corresponding in vivo performance, and in turn could lead to future development of rational in vitro-in vivo correlations.

HEPES-Buffered Saline (HBS) Precipitation Buffer (2×)

HEPES-Buffered Saline (HBS) Precipitation Buffer (2×)

Plasma Constitutive Serum Amyloid A4 Is Procoagulant and Is Elevated in Venous Thrombosis Patients

Regulation of thrombin generation by the factors (F) Xa:Va complex is key for achieving hemostasis and avoiding thrombosis, i.e., for the hemostatic balance. Plasma lipoproteins and minor abundance lipids can influence the hemostatic balance. For example, oxidized LDL is procoagulant, HDL is anticoagulant as an APC:protein S cofactor, certain glycosphingolipids are anticoagulant cofactors, and acyl carnitines and lysosulfatides inhibit the FXa;Va complex. To identify novel lipoprotein factors that affect FXa:Va activity (prothrombinase (IIase) activity), lipoprotein fractions from ten adult varied subjects were made by ultracentrifugation and, following dialysis into Hepes-buffered saline, screened for their procoagulant activity in purified IIase assays. Each lipoprotein fraction, except that from one subject (#939) with only 22% activity, similarly enhanced thrombin generation by IIase. SDS page analysis of these lipoprotein fractions showed a very low level of one protein band (14 kDa) for subject #939. Proteomics analysis of that band from normal lipoprotein fractions implied this 14 kDa band was constitutive serum amyloid A4 (SAA4). The biologic functions of SAA4 are unclear. To assess its procoagulant activity, recombinant 14 kDa SAA4 was studied in various coagulation assays. When SAA4 was added to IIase assays, it caused a robust procoagulant activity in the presence of FVa but not in the absence of FVa. Plasma-based coagulation assays, done without phospholipids added, were also used, including FXIa-induced thrombin generation, and FXa-1-stage clotting assays. Recombinant SAA4 was mildly or strongly procoagulant in these plasma-based assays. SAA4 dose-dependently shortened the FXa-induced clotting time from 320 sec to 55 sec; and SAA4 increased thrombin generation by 15 % in normal plasma and by 85 % in plasma from subject #939. Direct interactions between recombinant SAA4 and purified FXa were studied using SPR (Octet Red™) with immobilized-FXa subjected to titrations of SAA4. Kinetic constants gave a value for Kd apparent of 25 nM for SAA4 binding to FXa. Thus, SAA4 binds FXa and is procoagulant by enhancing IIase activity. We then assessed potential clinical relevance of SAA4's procoagulant activity. SAA4 is a plasma apolipoprotein, mainly made in the liver, with a constant plasma level of 4 µM. It is not an acute phase protein, unlike SAA1 and SAA2. SAA4 contains 112 residues and plasma contains two monomeric isoforms (14 kDa and 19 kDa) due to the absence or presence of N-glycosylation at Asn-76. The plasma levels of monomeric SAA4 (combined 14 and 19 kDa isoforms) were determined by quantitative IR immunoblotting (non-reduced SDS PAGE) using the Odyssey IR imaging system (Li-Cor Biosciences) for 110 venous thrombosis (VTE) subjects and 110 matched controls from the Scripps Venous Thrombosis Registry. The Registry samples were from male and female adults without cancer or lipid altering drugs who were < 55 yrs old and gave fasting blood samples > 3 months after the clinical event (see H. Deguchi et al, BLOOD 2015). When the relative fluorescence unit (RFU) values for SAA4 14 and 19 kDa monomer bands for 110 normal subjects were normalized to a median of 100 %, patients with VTE had significantly higher plasma levels of monomeric SAA4 (median 116.4 RFU vs median 100 RFU, p=0.0005) (see median values in Figure). The odds ratio (OR) for the 75%-ile cut-off was 2.1 (95% CI = 1.2 - 3.8, p=0.01) (see Figure). Thus, SAA4 monomers are elevated in VTE patients compared to matched controls for the Scripps Venous Thrombosis Registry. Further studies are needed to replicate this finding and to gather more data towards deciphering whether the link between elevated SAA4 and VTE is causal, consequential or coincidental. However, the fact that recombinant 14 kDa SAA4 binds FXa and is procoagulant in purified IIase assays and in plasma-based clotting assays provides biological plausibility for a causal prothrombotic role for SAA4. These findings also raise the possibility that SAA4 procoagulant activity might contribute to normal hemostasis. Further studies are needed to clarify the details for the procoagulant effects of SAA4 on coagulation pathways. In summary, these results show that elevated monomeric plasma levels of SAA4 are strongly linked to VTE in adults (< 55 yrs old) and that SAA4 itself is a potential enhancer of thrombin generation in plasma. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Coefficient of Friction of Human Corneal Tissue.

A novel property evaluation methodology was used to determine the elusive value for the human corneal coefficient of friction (CoF). Using a microtribometer on 28 fresh human donor corneas with intact epithelia, the CoF was determined in 4 test solutions (≥5 corneas/solution): tear-mimicking solution (TMS) in borate-buffered saline (TMS-PS), TMS in phosphate-buffered saline (TMS-PBS), TMS with HEPES-buffered saline (TMS-HEPES), and tear-like fluid in PBS (TLF-PBS). Mean (SD) CoF values ranged from 0.006 to 0.015 and were 0.013 (0.010) in TMS-PS, 0.006 (0.003) in TMS-PBS, 0.014 (0.005) in TMS-HEPES, and 0.015 (0.009) in TLF-PBS. Statistically significant differences were shown for TMS-PBS versus TLF (P = 0.0424) and TMS-PBS versus TMS-HEPES (P = 0.0179), but not for TMS-PBS versus TMS-PS (P = 0.2389). Successful measurement of the fresh human corneal tissue CoF was demonstrated, with values differing in the evaluated buffer solutions, within this limited sample size.

A pH and redox dual responsive 4-arm poly(ethylene glycol)-block-poly(disulfide histamine) copolymer for non-viral gene transfection in vitro and in vivo.

A novel 4-arm poly(ethylene glycol)-b-poly(disulfide histamine) copolymer was synthesized by Michael addition reaction of poly(ethylene glycol) (PEG) vinyl sulfone and amine-capped poly(disulfide histamine) oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average diameter) with almost neutral surface charge (+(5–10) mV). Besides, these polyplexes were colloidal stable within 4 h in HEPES buffer saline at pH 7.4 (physiological environment), but rapidly dissociated to liberate DNA in the presence of 10 mM glutathione (intracellular reducing environment). The polyplexes also revealed pH-responsive surface charges which markedly increased with reducing pH values from 7.4–6.3 (tumor microenvironment). In vitro transfection experiments showed that polyplexes of 4-arm PEG-SSPHIS were capable of exerting enhanced transfection efficacy in MCF-7 and HepG2 cancer cells under acidic conditions (pH 6.3–7.0). Moreover, intravenous administration of the polyplexes to nude mice bearing HepG2-tumor yielded high transgene expression largely in tumor rather other normal organs. Importantly, this copolymer and its polyplexes had low cytotoxicity against the cells in vitro and caused no death of the mice. The results of this study indicate that 4-arm PEG-SSPHIS has high potential as a dual responsive gene delivery vector for cancer gene therapy.

The Electrogenic Sodium Bicarbonate Cotransporter NBCe1 Is a High-Affinity Bicarbonate Carrier in Cortical Astrocytes

The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) is a robust regulator of intracellular H(+) and a significant base carrier in many cell types. Using wild-type (WT) and NBCe1-deficient (NBC-KO) mice, we have studied the role of NBCe1 in cortical astrocytes in culture and in situ by monitoring intracellular H(+) using the H(+)-sensitive dye BCECF [2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein] in wide-field and confocal microscopy. Adding 0.1-3 mm HCO3(-) to an O2-gassed, HEPES-buffered saline solution lowered the intracellular H(+) concentration with a Km of 0.65 mm HCO3(-) in WT astrocytes, but slowly raised [H(+)]i in NBCe1-KO astrocytes. Human NBCe1 heterologously expressed in Xenopus oocytes could be activated by adding 1-3 mm HCO3(-), and even by residual HCO3(-) in a nominally CO2/HCO3(-)-free saline solution. Our results demonstrate a surprisingly high apparent bicarbonate sensitivity mediated by NBCe1 in cortical astrocytes, suggesting that NBCe1 may operate over a wide bicarbonate concentration in these cells.

A Surface Plasmon Resonance Method to Study HCV NS5B Inhibitors

Surface Plasmon Resonance (SPR) technology is a well-established platform used to evaluate the kinetic parameters of protein-small molecule interactions. Below, we describe the use of the ProteOn XPR36 biosensor from Bio-Rad (Hercules, CA) to evaluate the binding of small molecule inhibitors to recombinant NS5B protein. The high pI (&gt; 9) of this construct allows for chemical immobilization using HEPES-buffered saline at pH 7.5. This is in contrast to traditional biosensor protocols that use both low pH and ionic strength. The use of a more physiological buffer to immobilize this enzyme leads to improved surface activity.