Acidic Phosphate-buffered Saline Research Articles

Investigation of the effect of solution pH value on rabbit corneal stroma biomechanics.

To facilitate the protection of corneal stability during corneal epithelium defects by determining the effect of solution pH on corneal stroma biomechanics. Thirty rabbit corneas were extracted, and the epithelium was scraped off. The samples were immediately subjected to inflation tests with pressures ranging from 0.3 to 6kPa at baseline and in three subsequent test cycles. During a 10-min interval between cycles, specimens were randomly divided into four groups; in three of these groups, phosphate-buffered saline (PBS) drops with pH values of 6.9, 7.4, or 7.9 were applied to the surface once per minute, whereas the fourth group did not receive drops. The corneal thickness significantly increased following the administration of PBS, while the corneal tangent modulus significantly decreased. At 2.5 and 4.5kPa, the modulus reduction was significantly smaller in the specimens treated with pH 6.9 PBS than in those treated with pH 7.4 or 7.9 PBS, adjusted for changes in corneal thickness. Linear fitting of the pressure-modulus plot revealed that the regression coefficient significantly decreased over time. The reduction in the coefficient was most prominent in the PBS-treated groups, and the administration of pH 6.9 PBS elicited the smallest reduction among those three groups, adjusted for corneal thickness changes. The study demonstrated that the administration of PBS drops with various pH values affected corneal biomechanics independent of corneal stromal swelling, and the impact of slightly acidic PBS was minimal. The effect became more prominent as posterior pressure increased. The research provides the basis for mediating the pH value of tear film and drops to maintain biomechanical stability of epithelium defects corneal stroma.

Ocular Effects of Glycyrrhizin at Acidic and Neutral pH.

PurposeTo test the effects of acidic vs. neutral pH glycyrrhizin (GLY) on the unwounded and wounded normal mouse cornea and after infection with Pseudomonas aeruginosa isolates KEI 1025 and multidrug-resistant MDR9.MethodsAcidic or neutral GLY vs. phosphate-buffered saline (PBS) was topically applied to normal or wounded corneas of C57BL/6 mice. In unwounded corneas, goblet cells and corneal nerves were stained and quantitated. After wounding, corneas were fluorescein stained and photographed using a slit lamp. Mice also were infected with KEI 1025 or MDR9 and the protective effects of GLY pH evaluated comparatively.ResultsIn the unwounded cornea, application of acidic or neutral GLY vs. PBS reduced the number of bulbar conjunctival goblet cells but did not alter corneal nerve density. Similar application of GLY to scarified corneas delayed wound closure. After KEI 1025 infection, none of the GLY vs. PBS-treated corneas perforated; GLY treatment also decreased plate count (neutral pH more effective) and reduced MPO and several cytokines. Similarly, for MDR9, GLY at either pH was protective and also enhanced the effects of moxifloxacin to which MDR9 is resistant.ConclusionAcidic or neutral pH GLY decreased goblet cell number but had no effect on nerve density. After corneal wounding, GLY at either pH (1) delayed wound closure and, (2) after infection, decreased keratitis when used alone or in combination with moxifloxacin. Neutral pH did not alter the therapeutic effect of GLY and would be preferred if used clinically.

Electrochemical behavior of open-cellular structured Ti-6Al-4V alloy fabricated by electron beam melting in simulated physiological fluid: the significance of pore characteristics

The cellular structured titanium alloys have attracted significant attention for implants because of their lower Youngʼs modulus, which is comparable to human bone and has the capability of providing space for bone tissue in-growth. However, there is a gap in the knowledge in regard to the relationship between the pore characteristics and the electrochemical performance of open-cellular structured titanium alloys. In this study, we elucidate the influence of pore characteristics on the electrochemical performance of open-cellular structured Ti-6Al-4V alloys produced by electron beam melting (EBM). Intriguingly, the passive film formed on cellular structured Ti-6Al-4V alloy with a larger pore size was more stable and protective, and the corrosion performance was superior compared to the samples with a smaller pore size in phosphate buffered saline (PBS), mainly because of relatively smaller exposed surface area and unlimited flow of electrolyte. However, in acidic PBS containing fluoride ions, the pore characteristics did not play an important role in the corrosion resistance. It was considered that the protective film breaks down such that the corrosion performance of cellular structured alloys was comparable to each other in this harsh environment.

Calcium-siRNA Nanocomplexes Optimized by Bovine Serum Albumin Coating Can Achieve Convenient and Efficient siRNA Delivery for Periodontitis Therapy.

PurposeReducing toxicity, immunogenicity, and costs of small interfering RNAs (siRNA) carrier materials are key goals for RNA interference (RNAi) technology transition from bench to bed. Recently, calcium ions (Ca2+) have garnered attention as a novel, alternative material for delivering siRNA to cells. However, the tolerance for Ca2+ concentration varies in different cell types, which has limited its applications in vivo. Bovine serum albumin (BSA) can bind to Ca2+ through chelation. Moreover, BSA is a favorable coating material for nanoparticles owing to its excellent biocompatibility. Therefore, we hypothesized that coating Ca2+-siRNA with BSA helps buffer Ca2+ toxicity in vivo.MethodsBSA-Ca2+-siRNA nanoparticles were prepared, and the size, shape, encapsulation, and release efficiency were characterized using atomic force microscopy, scanning electronic microcopy, and gel electrophoresis. Binding nanoparticles were evaluated using attenuated total reflection-Fourier-transform infrared spectroscopy. The cellular uptake, intracellular release, cytotoxicity, and gene knockdown of nanoparticles were evaluated in periodontal ligament stem cells (PDLSCs) using laser-scanning confocal microscope, flow cytometry, and real-time quantitative polymerase chain reaction.ResultsBSA and Ca2+-siRNA could form a stable nano-scale complex (~140 nm in diameter). The nanocomplexes could maintain siRNA release for more than 1 week in neutral phosphate-buffered saline (PBS) and could induce accelerated degradation in acidic PBS (pH 5.0). The nanoparticles were taken up by the cells, primarily through macropinocytosis, and were then released intracellularly through the acidification of endosomes/lysosomes. Importantly, the BSA-Ca2+ carrier had high transfection efficiency and biocompatibility both in vitro and in vivo. To demonstrate the therapeutic potential of our BSA coating-optimized Ca2+-siRNA technology, we showed that BSA-Ca2+-siWWP1 complexes strongly enhanced the osteogenic differentiation of inflammatory PDLSCs.ConclusionBSA-Ca2+ could potentially be used for siRNA delivery, which is not only highly efficient and cost-effective but also biocompatible to host tissues owing to the BSA coating.

N-Terminal selective modification of peptides and proteins using 2-ethynylbenzaldehydes

Selective modification of the N-terminus of peptides and proteins is a promising strategy for single site modification methods. Here we report N-terminal selective modification of peptides and proteins by using 2-ethynylbenzaldehydes (2-EBA) for the production of well-defined bioconjugates. After reaction screening with a series of 2-EBA, excellent N-terminal selectivity is achieved by the reaction in slightly acidic phosphate-buffered saline using 2-EBA with electron-donating substituents. Selective modification of a library of peptides XSKFR (X = either one of 20 natural amino acids) by 2-ethynyl-4-hydroxy-5-methoxybenzaldehyde (2d) results in good-to-excellent N-terminal selectivity in peptides (up to >99:1). Lysozyme, ribonuclease A and a therapeutic recombinant Bacillus caldovelox arginase mutant (BCArg mutant) are N-terminally modified using alkyne- and fluorescein-linked 2-EBA. Alkyne-linked BCArg mutant is further modified by rhodamine azide via copper(I)-catalyzed [3 + 2] cycloaddition indicating that the reaction has high functional group compatibility. Moreover, the BCArg mutant modified by 2-ethynyl-5-methoxybenzaldehyde (2b) exhibits comparable activity in enzymatic and cytotoxic assays with the unmodified one.

Surface pH buffering to promote degradation of mesoporous silica nanoparticles under a physiological condition

Despite significant advancement of mesoporous silica nanoparticle (MSN)-based biomedical research, studies have not been done enough to understand biodegradability of functional MSNs for better clinical efficacy. Polyethyleneimine (PEI) is one of the mostly used surface functionalities of MSNs, owing to the amine-rich chemical composition and the well-known proton sponge effect. In this paper, we study degradation behaviors of PEI-coated MSNs (PEI-MSNs) under a neutral or acidic physiological condition in comparison to those of surface-uncoated or nonionic F-127-encapsulated MSNs. The results showed that the surface coating by PEI could promote particle degradation in both neutral and acidic phosphate buffered saline (PBS) solution (i.e., pH 7.4 and 5.0). Importantly, we demonstrated that the local pH buffering by the surface PEI could lead to a greater total degradation quantity of particles even in the acidic PBS solution. The PEI-induced pH buffering phenomenon was confirmed by using a fluorescent pH indicator dye, fluorescein, which was attached to the surface of PEI-MSNs (F-PEI-MSNs). The observed pH-insensitive fluorescing behavior of fluorescein attained by surface coating with PEI corroborates the buffering effect that minimizes the surface pH change regardless of the external pH. The presented results may offer a useful insight into the degradability of silica nanomaterials with PEI or related surface functionalities, especially in the acidic subcellular microenvironment.

Factors Affecting Urinary Myoglobin Stability In Vitro

Urine myoglobin concentrations are measured clinically to assess rhabdomyolysis and the related risk of renal damage. We studied urine myoglobin concentrations in vitro to explore the factors affecting stability. Myoglobin was very unstable in urine specimens, especially below pH 6.5, and its immunoreactivity deteriorated rapidly with increasing temperatures. The deterioration rate was influenced greatly by urine myoglobin concentration, suggesting rate-limiting kinetics. Myoglobin in acidic phosphate-buffered saline was significantly more stable than in acidic urine, indicating that urinary factors in addition to pH are involved in myoglobin instability. These unidentified urinary factors had a molecular weight of less than 10 kd. Our results provide additional insight into the mechanism involved in the instability of the urine myoglobin concentration. Understanding the stability of myoglobin in the preanalytic in vitro phase and its potential in vivo instability is essential in assuring the reliability and clinical usefulness of urine myoglobin measurements.

Factors Affecting Urinary Myoglobin Stability In Vitro

Urine myoglobin concentrations are measured clinically to assess rhabdomyolysis and the related risk of renal damage. We studied urine myoglobin concentrations in vitro to explore the factors affecting stability. Myoglobin was very unstable in urine specimens, especially below pH 6.5, and its immunoreactivity deteriorated rapidly with increasing temperatures. The deterioration rate was influenced greatly by urine myoglobin concentration, suggesting rate-limiting kinetics. Myoglobin in acidic phosphate-buffered saline was significantly more stable than in acidic urine, indicating that urinary factors in addition to pH are involved in myoglobin instability. These unidentified urinary factors had a molecular weight of less than 10 kd. Our results provide additional insight into the mechanism involved in the instability of the urine myoglobin concentration. Understanding the stability of myoglobin in the preanalytic in vitro phase and its potential in vivo instability is essential in assuring the reliability and clinical usefulness of urine myoglobin measurements.

Round window pH manipulation alters the ototoxicity of systemic cisplatin

The effect of manipulation of pH on the ototoxicity of systemic cisplatin was studied in Wistar rats. After control auditory brainstem responses (ABR) were performed, the auditory bullae were opened and acidic (pH 6.0), neutral (pH 7.4) or basic (pH 9.0) phosphate-buffered saline (PBS) was applied to fill the round window niche (RWN). After 30 min, 13 mg/kg cisplatin solution or saline was administered intraperitoneally. After 3 days, follow-up ABRs were performed and cochleae were processed for morphological analysis. Animals that received basic PBS on the RWN and cisplatin intraperitoneally had significantly smaller ABR threshold shifts compared to rats pretreated with neutral pH buffer ( P<0.05). Animals that received acidic PBS on the RWN and systemic cisplatin showed significantly greater ABR threshold shifts compared to those pretreated with neutral pH buffer ( P<0.05). No significant threshold changes were observed in animals that received buffer of any pH on the RWN, followed by saline intraperitoneally. Semiquantitative analysis of hair cell survival confirmed a protective effect by basic PBS against cisplatin and a synergistic effect by acidic PBS on cisplatin ototoxicity ( P<0.05). It appears that changes in cochlear pH can modulate the ototoxic effects of systemically applied cisplatin.

Influence of pH on the ototoxicity of cisplatin: a round window application study

Cisplatin is an antineoplastic agent that produces a number of dose-limiting side effects, including ototoxicity. We investigated the effect of pH on cisplatin ototoxicity. Auditory brainstem responses (ABR) were recorded in chinchillas. Then the auditory bullae were opened and acidic (pH=6.5), neutral (pH=7.4) or alkaline (pH=10.2) phosphate-buffered saline (PBS) was applied to the round window membrane. After 30 min, any remaining solution was removed and cisplatin solution was applied to the round window membrane. After 3 days, follow-up ABRs were performed and the cochleae were processed for morphological analysis. Neutral PBS+cisplatin administration resulted in profound threshold changes at all frequencies. Acidic PBS+cisplatin administration showed had a trend of increased threshold changes, but the change did not reach statistical significance. However, the degree of hair cell loss was significantly higher than that of the neutral PBS-cisplatin group. Alkaline PBS significantly reduced cisplatin-induced threshold changes ( P<0.05) compared to the neutral PBS group. Because the pH of cisplatin solution was 6.0, pH 6.0 PBS was applied to round window membrane. This acidic PBS solution did not cause any hearing impairment. These results demonstrate that pH can modulate the ototoxic effects of cisplatin.

Incorporation of bovine serum albumin in calcium phosphate coating on titanium.

Calcium phosphate (Ca-P) and bovine serum albumin (BSA) were coprecipitated as a coating on commercially pure titanium (cpTi) with a high protein loading (15 wt %) by employing a recently developed wet-chemistry technique. It was observed that the incorporation of BSA significantly modified the morphology, composition, and crystallinity of the Ca-P coating. The Ca-P coating without BSA is a mixture of hydroxyapatite (HA) and octacalcium phosphate (OCP) with sharp-edged thin OCP crystal plates on the top layer, whereas only an HA phase was detected in the Ca-P/BSA coating. The crystal plates in the latter had a more rounded appearance. The Ca-P/BSA coatings were immersed respectively in neutral (pH 7.4) and acidic (starting pH 4.0) phosphate-buffered saline (PBS) at 37 degrees C over a 14-day period. No protein release was detected in the neutral PBS during the immersion; however, a continuous release of BSA was measured in the acidic PBS, subsequently leading to the formation of a very dense and well-adherent composite coating of BSA and Ca-P on cpTi. The present study provides the possibility to achieve a long-term effective release of biologically active proteins from a Ca-P-coated metallic implant.

Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.

Parathyroid hormone receptors are plasma membrane glycoproteins with asparagine-linked oligosaccharides.

In the preceding article, we described physicochemical and kinetic properties of parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8) using photoaffinity ligand labeling and showed that the physiologically relevant receptor-ligand complex has an apparent Mr = 80,000. In this study, the photoaffinity labeled Mr = 80,000 receptor was localized exclusively on the cell surface plasma membrane and its glycoprotein nature was demonstrated through the use of lectin affinity-chromatography and specific exo- and endoglycosidases. Rinsing ROS cells, preincubated in the dark with 125I-labeled [Nle8, N-epsilon-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NAP-NlePTH) (4 h, 15 degrees C, equilibrium conditions) with acidic phosphate-buffered saline (pH 2.5, 30 s, 4 degrees C) before photolysis resulted in selective and nearly total disappearance of the labeled Mr = 80,000 receptor. PTH receptor integrity to acid rinsing and photolysis was shown by relabeling the Mr = 80,000 receptor after a second incubation of these cells with 125I-labeled NAP-NlePTH, followed by photolysis. Adsorption of Triton X-100-solubilized, 125I-labeled NAP-NlePTH receptors to wheat germ agglutinin-agarose is nearly complete and highly selective, and elution with N-acetylglucosamine resulted in virtually total recovery of the labeled receptors from the column. The wheat germ agglutinin-retarded PTH receptors show increased electrophoretic mobility upon treatment with neuraminidase which was inhibited by simultaneous addition of 2,3-dehydro-3-desoxy-N-acetylneuraminic acid, a specific neuraminidase inhibitor. Endoglycosidase F treatment of the Mr = 80,000 receptors generated a single, labeled polypeptide with a Mr = 59,000 which migrated as a narrow band. PTH receptors on ROS 17/2.8 cells appear to be monomeric plasma membrane glycoproteins with an apparent Mr of 80,000 which contain a Mr = 59,000 polypeptide backbone and a polymeric arrangement of N-acetylglucosamine with N-acetylneuraminic acid as major terminal sugar residues.