Calcium Phosphate Coprecipitation Research Articles

From classic to innovative: The continuation and modern application of calcium phosphate co-precipitation method in mRNA tumor delivery

From classic to innovative: The continuation and modern application of calcium phosphate co-precipitation method in mRNA tumor delivery

Hybrid functional membranes through layer-by-layer assembly of Ti3C2Tx MXene and gelatin-stabilized calcium phosphate nanospheres

MXene-based membranes exhibit promising properties for various applications; however, pure MXene membranes lack sufficient environmental stability and mechanical strength. This work demonstrates that incorporating gelatin-stabilized amorphous calcium phosphate (Gel-ACP) nanospheres into MXene membranes via layer-by-layer assembly significantly enhances flexibility and mechanical strength while retaining electrical conductivity. A pressure-assisted stacking technique was introduced to efficiently fabricate membranes with tunable thickness not achievable through single filtration steps. The Gel-ACP nanospheres, synthesized by co-precipitation of calcium phosphate within gelatin solution, provided a ductile and cytocompatible reinforcement that augmented the properties of the MXene matrix. Direct mixing of the components led to particle aggregation and non-uniform dispersion. In contrast, controlled layer-by-layer nanostructuring maintained membrane conductivity around 102 Scm−1 while dramatically improving mechanical integrity. The optimized hybrid membranes exhibited specific electromagnetic shielding effectiveness of ∼21,000 dBcm2g−1 and withstood over 90 kPa vacuum pressure without rupture, six times higher than pure MXene membranes. Cytocompatibility was confirmed by the proliferation of human mesenchymal stem cells on the membranes. Moreover, the layered membrane exhibited excellent adhesion to bone-mimicking structures, indicating potential utility for bone tissue engineering. Overall, this work provides new design principles for engineering hybrid membranes through controlled multicomponent assembly to overcome intrinsic limitations and impart multifunctionality.

Eco-friendly recycling of silicon−rich lye: Synthesis of hierarchically structured calcium silicate hydrate and its application for phosphorus removal

Silicon−rich lye (SRL), a byproduct generated from pre−treatment of coal−based solid waste (CSW), was considered as a preponderant silicon source to prepare hierarchically nanostructured calcium silicate hydrate (C-S-H). Through the novel mild−causticization synthesis strategy, C-S-H was prepared under optimal caustic process conditions at time of 3 h, temperature of 80 °C, Ca/Si of 1.25:1, and active CaO to obtain a conversion rate of Si up to 97.33 % during the high−value utilization of SRL. The synthesized C-S-H possesses abundant mesoporous structure and massive exchangeable active sites, whose formation is advanced through an appropriate elevation regulation of caustic temperature and time. The silicate chain depolymerization occurs to C-S-H prepared in the highly alkaline system at higher caustic temperature, longer caustic period, especially at existence of massive sodium ions, but it presents higher polymerization degree at more aluminum co-existing. The adsorption capacity up to 119.27 mg/g for C-S-H presents a valid removal performance toward phosphorus in the wastewater than massive present reports. The removal mechanism of phosphorus can be identified as the surface chemisorption and formation of calcium phosphate co-precipitation. This study can provide considerable and potential guidance to the coordinated disposal between industrial solid wastes and wastewater purification.

Exploration of mechanisms for calcium phosphate precipitation and accumulation in nitrifying granules by investigating the size effects of granules

Calcium phosphate could be accumulated in aerobic granules, which attracts attention recently for phosphorus removal and recovery from wastewater. In this study, partial nitrifying granules with high calcium precipitate content were sorted into different size groups for characterization and evaluation to reveal the dynamic balance of granules at stead state and relevant calcium phosphate precipitation and accumulation mechanism. It was found that light yellow small granules without calcium precipitates but high microbial activity co-existed with deep brown granules with calcium precipitate of around 91% and low microbial activity. Characterization with specific oxygen uptake rates, specific ammonium oxidation rates, calcium and phosphate removal rates from solution, EPS contents, elemental compositions by energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and scanning electron microscopy (SEM) for different size groups of granules enabled a proposal of a new hypothesized mechanism for calcium precipitation and accumulation. With this proposed mechanism, it is believed that sufficient granule retention time in reactors was critical for the accumulation of calcium precipitates followed by a slow microbial growth rate of biomass due to mass transfer resistance. The co-precipitation of calcium carbonate and calcium phosphate mainly occurred in granules with a size less than 710 µm while calcium phosphate dominant minerals were accumulated in granules larger than 710 µm. The results and conclusions in this study shed light on the mechanisms of calcium phosphate accumulation in granules, which could be used to better operate and control aerobic granular sludge with calcium phosphates for phosphorus removal and recovery.

Coprecipitation technique as a new approach of using solid-state scintillation

In this paper, the coprecipitation methods of selected radionuclides in connection with solid-state scintillation counting were explored. The calcium phosphate co-precipitation method exhibited best results from all investigated methods reaching almost absolute detection efficiencies for selected actinides and more than 60% for Sr/Y-90. The linear response of the measurement was in the range of 0.4 to 411.5 Bq for alpha/beta separation mode, respective 0.4 to 2000 Bq nuclear counting (high activity mode). Computed and optimized MDA values exhibited better results in comparison with previous works.

Expression of Thanatin in HEK293 Cells and Investigation of its Antibacterial Effects on Some Human Pathogens.

BackgroundThanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens.ObjectiveIn this study, the antimicrobial activity of this peptide against some species of human bacterial pathogens as well as its toxicity on NIH cells were evaluated.MethodsThanatin DNA sequence was cloned into pcDNA3.1+ vector and transformed into a DH5α bacterial strain. Then the recombinant plasmids were transfected into HEK-293 cells by calcium phosphate co-precipitation. After applying antibiotic treatment, the supernatant medium containing thanatin was collected. The peptide quantity was estimated by SDS-PAGE and GelQuant software. The antimicrobial activity of this peptide was performed with Minimum Inhibitory Concentration (MIC) method. In addition, its toxicity on NIH cells were evaluated by MTT assay.ResultsThe peptide quantity was estimated approximately 164.21 μmolL-1. The antibacterial activity of thanatin was estimated between 0.99 and 31.58 μmolL-1 using MIC method. The result of cytotoxicity test on NIH cell line showed that the peptide toxicity up to the concentration of 394.10 μmolL-1 and for 48 hours, was not statistically significant from negative control cells (P>0.05). The antimicrobial assay demonstrated that thanatin had an antibacterial effect on some tested microorganisms. The results obtained in this study also showed that thanatin had no toxicity on mammalian cell lines including HEK293 and NIH.ConclusionAntimicrobial peptides such as thanatin are considered to be appropriate alternatives to conventional antibiotics in treating various human pathological diseases bacteria.

Expression of Recombinant Factor IX Using the Transient Gene Expression Technique

Background: Pilot and large-scale production of recombinant proteins require the presence of stable clones, but the process of selecting stable clones is time consuming. Moreover, continuous clone culturing in large-scale production may cause loss of incoming plasmid and recombinant genes. Considering the advancements in Transient Gene Expression (TGE) technology, the large-scale expression of factor IX (FIX) was investigated in HEK cells by the TGE technique. Materials and Methods: HEK cells were seeded in a cell factory, and then transfected by pcDNA-hFIX plasmid using calcium phosphate co-precipitation method. Stable HEKhFIX cells were also seeded in a cell factory, separately. After adding vitamin K, recombinant FIX was quantified in conditioned media using an ELISA. Moreover, its functional activity was assayed using an aPTT test. Results: The results showed that the expression and activity of FIX by TGE technology was, respectively, 1.6 and 1.5 times higher than that obtained through stable HEK-FIX cells. Since calculating the specific activity revealed that for all time periods it is 0.2 mU/ng, so the increase in activity is due to the increase in the amount of FIX. Conclusions: HEK cells with higher transfectability seemed to be an appropriate alternative for transient expression for large-scale protein production. Furthermore, if rapid expression of recombinant proteins is intended, TGE can replace costly and lowyield methods.

Transfection of Difficult-to-Transfect Rat Primary Cortical Neurons with Magnetic Nanoparticles.

The efficient cell transfection method is vital for various biomedical applications, such as the CRISPR-Cas9 technique. Current cell transfection methods, including lipofectamine, calcium phosphate co-precipitation, nucleofection, and viral infection are not equally efficient for various cells and have their disadvantages. In this study, a magnetic nanoparticle (MNP)-based method was introduced for delivering both FITC dye and a functional EGFP gene into easy-to-transfect HEK cells and difficult-to-transfect rat primary cortical neurons. The transfection efficacy could be controlled in both time-dependent and magnetic strength-dependent manner. This cell transfection method could have substantial potential for targeted drug delivery.

Improving Fractionation of Human Milk Proteins through Calcium Phosphate Coprecipitation and Their Rapid Characterization by Capillary Electrophoresis.

This work describes a simple sample pretreatment method for the fractionation of human milk proteins (into their two main groups, whey and caseins) prior to their analysis. The protein-extraction protocol is based on the addition of calcium phosphate to nonadjusted pH human milk. The combination of calcium ions with phosphate results in an effective coprecipitation of caseins. To assess the suitability of this fractionation protocol, the protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), LC-MS/MS, and capillary electrophoresis (CE) analysis. The results evidence a significant decrease in contamination of casein fraction with whey proteins and vice versa compared with the conventional isoelectric precipitation of caseins. In addition, CE fraction collection coupled to LC-MS/MS (off-line coupling) has been successfully applied to the identification of minor proteins in this complex matrix. The methodology presented here constitutes a promising tool to enlarge the knowledge of human milk proteome.

A rapid method for the routine monitoring of 99Tc by liquid scintillation counting

A new simple routine method has been developed to rapidly determine technetium-99 (99Tc) in environmental waters using rhenium (Re) as a recovery tracer. A small water sample (40 mL) was first filtered. The potential interferents were then removed by a calcium phosphate co-precipitation followed by a TRU resin purification. 99Tc was measured by liquid scintillation counting and Re by UV–visible. The detection limit obtained (5 ± 2 Bq kg−1) is sufficiently low to determine if the water is safe for consumption.

Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene

The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction.Keywords: AAV, serotype DJ/8, GFP, Hepatocytes

In vitro mineralisation of grafted ePTFE membranes carrying carboxylate groups

In vitro mineralisation in simulated body fluid (SBF) of synthetic polymers continues to be an important area of research as the outcomes cannot be predicted. This study evaluates a series of ePTFE membranes grafted with carboxylate-containing copolymers, specifically using acrylic acid and itaconic acid for grafting. The samples differ with regards to graft density, carboxylate density and polymer topology. The type and amount of mineral produced in 1.5 × SBF was dependent on the sample characteristics as evident from XPS, SEM/EDX, and FTIR spectroscopy. It was found that the graft density affects the mineral phases that form and that low graft density appear to cause co-precipitation of calcium carbonate and calcium phosphate. Linear and branched graft copolymer topology led to hydroxyapatite mineralisation whereas crosslinked graft copolymers resulted in formation of a mixture of calcium-phosphate phases. This study demonstrates that in vitro mineralisation outcomes for carboxylate-containing graft copolymers are complex. The findings of this study have implications for the design of bioactive coatings and are important for understanding the bone-biomaterial interface.

Calcium phosphate nanoparticles-based systems for siRNA delivery.

Despite the enormous therapeutic potential of siRNA as a treatment strategy, the delivery is still a problem due to unfavorable biodistribution profiles and poor intracellular bioavailability. Calcium phosphate (CaP) co-precipitate has been used for nearly 40 years for in vitro transfection due to its non-toxic nature and simplicity of preparation. The surface charge of CaP will be tuned into positive by surface modification, which is important for siRNA loading and crossing cell membrane without enzymatic degradation. The new siRNA carrier system will also promote the siRNA escape from lysosome to achieve siRNA sustained delivery and high-efficiency silence. In this review, we focus on the current research activity in the development of CaP nanoparticles for siRNA delivery. These nanoparticles are mainly classified into lipid coated, polymer coated and various other types for discussion.

Facile Construction of Chloroquine Containing PLGA-Based pDNA Delivery System for Efficient Tumor and Pancreatitis Targeting in Vitro and in Vivo.

Chloroquine diphosphate (CQ) was ingeniously used to take place of phosphate salt in traditional calcium phosphate coprecipitation method for pDNA transfection. With multiple roles of CQ in the novel Ca-CQ-pDNA complex including pDNA compaction and assistance in lysosome escape, the transfection efficiency of the pDNA was significantly increased relative to the traditional method. CQ did not intercalate into the DNA double helix as free CQ did, which was probably ascribed to the prior mixing of the pDNA with high concentration of calcium chloride. In order to construct efficacious vector for in vivo gene delivery, Ca-CQ-pDNA-PLGA-NPs was designed and prepared. With entrapment efficiency, particle size and pDNA integrity as screening conditions, the optimal prescription was obtained and CaPi-pDNA-PLGA-NPs made with classic calcium phosphate coprecipitation method after optimization was also prepared as control to systematically study the role of CQ in the novel vector. Physical characters of the vectors were comprehensively studied using TEM, DSC, and XRD. The safety of the vector both in vitro and in vivo was evaluated using MTT, hemolysis test, and histological sections. The Ca-CQ-pDNA-PLGA-NPs dramatically enhanced the gene tranfection efficiency in Human Embryonic kidney HEK293 cells compared with the CaPi-pDNA-PLGA-NPs and presented an increasing gene transfection for up 144 h. The relative fast release of the CQ compared with pDNA from the nanoparticles was responsive for the increased transfection. The Did-labeled-Ca-CQ-pDNA-PLGA-NPs exhibited excellent tumor targeting efficiency and sustained circulation time in CT26 mouse model. The Ca-CQ-pDNA-PLGA-NP loaded with the plasmid pVITRO2 expressing mSurvivin-T34A protein gave 70% tumor inhibition rate, which was partially ascribed to CQ. The Ca-CQ-pDNA-PLGA-NPs showed high targeting efficiency in C57 acute pancreatitis model. In all, the Ca-CQ-pDNA-PLGA-NP was a promising candidate for targeted gene delivery to both tumor and pancreatitis.

EXTRACTION OF RADIUM ISOTOPES FROM SOME COASTAL SEAWATER SAMPLES IN EGYPT METHOD VALIDATION

A radiochemical separation of Ra isotopeswas carried out to determine the activity levels of radium (226Ra and 228Ra)in seawater samples, as well as providing a base-line data on background radiation in the investigated locations. Seawatersamples were collected from selected sites along the coasts. The water samples were analyzed for radium isotopes (Ra-226 & Ra-228) using alpha and liquid scintillation detectors.Development steps were applied to the radioanalysis procedure by using calcium phosphate co-precipitation and the addition of isopropanol. Quality assurance and method validation of the developed procedure was carried out to assure the results. The obtained results were within the uncertainty level ± 5%. Quality control steps were applied through the efficiency calibration of the detectors, the estimation of uncertainties, the use of blanks and the comparison with other laboratories.For comparison, selected samples were measured by gamma spectrometry using high purity germanium detector, after radiochemical separation of the isotopes with ion-exchange chromatography using a strong cation exchange resin, and the activity concentrations of 228Ra were comparable with those analyzed by the LSC. The average activity concentrations of 226Ra and228Ra in the Mediterranean seawater samples were found to be 166.8 and 500.6 mBq/L respectively, while in the Red seawater samples were 13.0 and 176.7 mBq/L respectively. The maximum value of radium activity was found at RasElBarr estuary, where the River Nile fresh water mixes with the coastal seawater. On the basis of the current results, we may conclude that the radium activities in the investigated seawater samples are well comparable to the corresponding reported values in literature.

Effect of polydopamine on the biomimetic mineralization of mussel-inspired calcium phosphate cement in vitro

Inspired by the excellent adhesive property of mussel adhesive protein, we added polydopamine (PDA) to calcium phosphate cement (PDA–CPC) to enhance its compressive strength previously. The mineralization and mechanism on PDA–CPC were investigated by soaking it in simulated body fluid in this study. The results indicated that PDA promoted the conversion of dicalcium phosphate dihydrate and α-tricalcium phosphate to hydroxyapatite (HA) in the early stage but inhibited this conversion subsequently. PDA promoted the rapid mineralization on PDA–CPC to form a layer of nanoscale calcium phosphate (CaP) whereas there was no CaP formation on the control-CPC after 1d of soaking. This layer of nanoscale CaP was similar to that of natural bone, which was always observed during soaking. X-ray photoelectron spectroscopy showed that the peak of CO of PDA existed in the newly formed CaP on PDA–CPC, indicating the co-precipitation of CaP with PDA. Furthermore, the newly formed CaP on PDA–CPC was HA confirmed by transmission electron microscopy, which the newly formed HA was in association with PDA. Therefore, PDA increased the capacity of mineralization of CPC and induced the formation of nanoscale bone-like apatite on PDA–CPC. Thus, this provides the feasible route for surface modification on CPC.

Improved gene transfer efficiency of a DNA-lipid-apatite composite layer by controlling the layer molecular composition

Surface-mediated nonviral gene transfer systems using biocompatible apatite-based composite layers have potential use in tissue engineering applications. Herein, we investigated a relatively efficient system based on a DNA-lipid-apatite composite layer (DLp-Ap layer): an apatite (Ap) layer with immobilized DNA and lipid (Lp) complexes (DLp complexes). DLp-Ap layers were fabricated on substrates using supersaturated calcium phosphate coprecipitation solutions supplemented with DLp complexes, and the molecular compositions of the DLp-Ap layers were controlled by varying the net DNA concentrations and Lp/DNA ratios in the coprecipitation solutions. Increases in both the DNA concentration and Lp/DNA ratio in the coprecipitation solution increased the DLp complex content of the resulting DLp-Ap layer. However, a higher DLp complex content did not always provide increased gene transfer efficiency to the CHO-K1 cells, because there was a threshold content of approximately 10μg/cm2. In addition, DLp-Ap layers with similar DLp complex contents exhibited different gene transfer efficiencies, most likely due to the different Lp/DNA ratios in the layers. Notably, the optimized Lp/DNA ratios in the coprecipitation solutions for maximizing the gene transfer efficiency were lower than those of the conventional particle-mediated lipofection systems. These findings will serve as a useful design guide for the preparation of DLp-Ap layers with high gene transfer efficiency.

Comparison of sample preparation methods for reliable plutonium and neptunium urinalysis using automatic extraction chromatography

This paper describes improvement and comparison of analytical methods for simultaneous determination of trace-level plutonium and neptunium in urine samples by inductively coupled plasma mass spectrometry (ICP-MS). Four sample pre-concentration techniques, including calcium phosphate, iron hydroxide and manganese dioxide co-precipitation and evaporation were compared and the applicability of different techniques was discussed in order to evaluate and establish the optimal method for in vivo radioassay program. The analytical results indicate that the various sample pre-concentration approaches afford dissimilar method performances and care should be taken for specific experimental parameters for improving chemical yields. The best analytical performances in terms of turnaround time (6h) and chemical yields for plutonium (88.7±11.6%) and neptunium (94.2 ±2.0%) were achieved by manganese dioxide co-precipitation. The need of drying ashing (≥7h) for calcium phosphate co-precipitation and long-term aging (5d) for iron hydroxide co-precipitation, respectively, rendered time-consuming analytical protocols. Despite the fact that evaporation is also somewhat time-consuming (1.5d), it endows urinalysis methods with better reliability and repeatability compared with co-precipitation techniques. In view of the applicability of different pre-concentration techniques proposed previously in the literature, the main challenge behind relevant method development is pointed to be the release of plutonium and neptunium associated with organic compounds in real urine assays. In this work, different protocols for decomposing organic matter in urine were investigated, of which potassium persulfate (K2S2O8) treatment provided the highest chemical yield of neptunium in the iron hydroxide co-precipitation step, yet, the occurrence of sulfur compounds in the processed sample deteriorated the analytical performance of the ensuing extraction chromatographic separation with chemical yields of ≤50%.

Dopamine D2 Receptor Relies upon PPM/PP2C Protein Phosphatases to Dephosphorylate Huntingtin Protein

Striatal dopamine D2 receptor (D2R) relies upon G protein- and β-arrestin-dependent signaling pathways to convey its action on motor control and behavior. Considering that D2R activation inhibits Akt in the striatum and that huntingtin physiological functions are affected by Akt phosphorylation, we sought to investigate whether D2R-mediated signaling could regulate huntingtin phosphorylation. We demonstrate that D2R activation decreases huntingtin phosphorylation on its Akt site. This dephosphorylation event depends upon the Gαi-dependent engagement of specific members of the protein phosphatase metallo-dependent (PPM/PP2C) family and is independent of β-arrestin 2. These observations identify the PPM/PP2C family as a mediator of G protein-coupled receptor signaling and thereby suggest a novel mechanism of dopaminergic signaling.

A preliminary step of a novel strategy in suicide gene therapy with lentiviral vector

Background:One of the challenges in lentiviral vector–based suicide gene therapy by toxin or apoptosis-inducing genes is death of packaging cells. Therefore, the process of production of these lentiviral particles would be stopped in this step. We proposed that insertion of a reverse promoter between R and U5 regions of 5′ long terminal repeat (LTR) in transfer plasmid could be considered as a solution for this problem. But it is not known, whether the insertion of RΔU3 sequence between the promoter and target gene in proviral genome during the life-cycle of lentivirus may interfere whit gene expression in target cells.Materials and Methods:These following methods were performed in this study: insertion of RΔU3 sequence in pEGFP-N1 plasmid, evaluation of the expression of eGFP gene after calcium phosphate co-precipitation transfection of pCMV-RΔU3-GFP construction in 293T cells, and quantitative assay of eGFP gene by flow cytometry technique.Results:Our results from flow cytometry technique analysis showed that there was no significant difference between the expression of eGFP gene in transfected cells with pEGFP-N1 and pCMV-RΔU3-GFP plasmids (P > 0.05).Conclusion:In this step of our strategy, we demonstrated that modification of orientation and location of promoter may overcome some issues in lentiviral suicide gene therapy, especially when toxin or apoptosis-inducing genes are used.