Physicochemical characterization, rheological properties, and hypolipidemic and antioxidant activities of compound polysaccharides in Chinese herbal medicines by fractional precipitation.

This study aimed to investigate the effects of different compound polysaccharides (CPs) extracted from Folium nelumbinis, Fructus crataegi, Fagopyrum tataricum, Lycium barbarum, Semen cassiae, and Poria cocos (w/w, 2:4:2:1:1.5:1) by gradient ethanol precipitation on the physicochemical properties and biological activities. Three CPs (CP50, CP70, and CP80) were obtained and comprised rhamnose, arabinose, xylose, mannose, glucose, and galactose in different proportions. The CPs contained different amounts of total sugar, uronic acid, and proteins. These also exhibited different physical properties, including particle size, molecular weight, microstructure, and apparent viscosity. Scavenging abilities of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 1,1'-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and superoxide radicals of CP80 were more potent compared to those of the other two CPs. Furthermore, CP80 significantly increased serum levels of high-density lipoprotein cholesterol (HDL-C) and lipoprotein lipase (LPL), and hepatic lipase (HL) activity in the liver, while decreasing the serum levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C), along with LPS activity. Therefore, CP80 may serve as a natural novel lipid regulator in the field of medicinal and functional food.

Two-Step Purification of L-Asparaginase from Acrylaway® L

Abstract L-Asparaginase (L-ASNase) is a biopharmaceutical used for acute lymphoblastic leukaemia (ALL) treatment, dramatically increasing the patients' chance of cure. However, its production and distribution in developing countries were disrupted because of its low profitability, which caused great concern among patients. This study evaluates the feasibility of combining fractional precipitation and aqueous two-phase systems (ATPS) to purify L-ASNase from a low-grade product, commercially known as Acrylaway® L. The ATPS purification results were not particularly expressive compared to the two-step purification process composed of ethanol precipitation and gel filtration, which was able to recover the target molecule with a purification factor over 5 fold. Thus, we studied a purification process capable of manufacturing pharmaceutical grade L-ASNase from a commercially available low-grade raw material; however, improvements regarding its throughput must be achieved, and high purity is the first step to apply it as a new biopharmaceutical product. The proposed process could pose as a short-time solution to mitigate its shortage while a cost-effective production plant is being developed.

A simple 2-step purification process of α-amylase from Bacillus subtilis: Optimization by response surface methodology.

Purification of extracellular α-amylase from Bacillus subtilis was carried out via fractional precipitation by acetone and ion exchange chromatography. These steps provide fast precipitation as well as purification of α-amylase to improve enzyme purity, activity and stability. Compared with two-phase methods in which the yield was less than 1, this method resulted in a yield of more than 3. Moreover, 95% of acetone was recovered that enhanced the economy of the downstream process. Using the data provided by 2D electrophoresis, purification was done by a single step ion exchange chromatography. The enzyme exhibited a molecular mass (SDS-PAGE) of 50KD and the pI of 5. Maximum "yield" and "purification fold" were achieved through optimization of operation parameters such as volume and flowrate of loaded protein using response surface methodology (RSM). 0.5ml of loaded protein at a flow rate of 0.5 ml/min was purified as 48 folds and achieved a specific activity of 524 U/mg.

Simple, inexpensive and RNase-free purification of plasmid DNA by fractional precipitation with isopropanol.

We present a modified alkaline lysis method for purification of plasmid DNA (pDNA) from bacterial extract using fractional precipitation with isopropanol (FPI). This method includes two successive precipitations with 0.33 and 0.36 volumes of isopropanol and separates pDNA from total RNA and most of the lipopolysaccharides. Using different quality control tests, we demonstrate that plasmids purified with FPI show superior quality compared to plasmids prepared with commercial kits based on spin-column chromatography.

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin.

Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 µl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.

Isolation of Outer Membrane Vesicles from Helicobacter pylori.

Outer membrane vesicles (OMV) shed by pathogenic bacteria have multifunctional roles in disease initiation and progression. Further, their efficacy as novel vaccines has underscored their importance as potential therapeutics. Consequently, to advance allied research related to their immunogenicity and pathogenicity it is important to separate these vesicular structures from parental cells and demonstrate them to be free from cellular debris and other non-vesicle-related constituents such as protein aggregates. To do so represents a key step in initiating OMV-related studies and the techniques and strategies adopted by the H. pylori community to achieve this will be the focus of this chapter.The key methods used typically to obtain a heterogeneous mixture of OMV (size range: ~20-300 nm in diameter) include growth of bacteria in broth culture followed by differential centrifugation, filtration, and concentration to separate OMV from the intact organisms. Additional measures may be adopted to further size-fractionate the population of OMV including gel filtration or density gradient ultra-centrifugation in order to facilitate differentiation between the activities of small versus large OMV, as recent studies have demonstrated differential modes of entry into host cells as well as size-dependent differences in the OMV proteome (Turner et al., Front Immunol 9:1466, 2018). The OMV from H. pylori harbor many of the virulence factors associated with gastric disease including the CagA oncoprotein, the cytotoxin VacA, and the HtrA protease (Olofsson et al., mBio 5:e00979-14, 2014; Mullaney et al., Proteomics Clin Appl 3:785-96, 2009) and their close association with areas of cell-cell contact and efficient endocytosis supports a role for these complexes in gastric disease (Turkina et al., FEMS Microbiol Lett 362:fnv076, 2015).

Effect of Molecular Weight on Cloud Point of Aqueous Solution of Poly (ethylene oxide)-Poly (propylene oxide) Alternating Multiblock Copolymer.

A poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO) alternating multiblock (AMB) copolymer with various molecular weights was prepared via precipitation fractionation from an acetone/n-hexane mixture. The cloud point (Tc) of the aqueous solution of PEO-PPO AMB copolymer decreased as the number-average molecular weight of the sample increased. This phenomenon is generally observed for certain homopolymer systems having a lower critical solution temperature, such as PEO/water and poly(N,N-diethylacrylamide)/water systems. The relationship between the Tc of the solutions and the number of monomer units of the AMB copolymer suggests that the Shultz-Flory theory is applicable to this system.

Analytical Grade Purification of Phycocyanin from Cyanobacteria.

Phycocyanin is a blue-colored pigment-protein complex that exhibits numerous biofunctions such as anti-inflammation, antioxidation, antitumor, neuroprotective effect, and immunological enhancement. Purified phycocyanin has pharmaceutical and nutraceutical applications. In addition, as a nontoxic and non-carcinogenic natural coloring agent, phycocyanin has many applications in the food and cosmetic industries. This chapter describes a protocol for extraction and analytical grade purification of phycocyanin from cyanobacteria. The purification steps include (1) extraction of phycocyanin from biomass, (2) ammonium sulfate precipitation of phycocyanin and dialysis, and (3) purification of phycocyanin by gel filtration and ion-exchange chromatography.

Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix.

The preservation of nucleic acids from clinical samples is critical to facilitate accurate molecular diagnosis. The use of a paper matrix, Flinders Technology Associates (FTA) Elute cards, to archive DNA and viral RNA is well-documented. However, the feasibility of FTA Elute cards for archiving serum and serum exosomal microRNAs (miRNAs) remains unclear. Here, we performed a comprehensive evaluation of FTA Elute cards for miRNA storage and recovery in different pre-analytical conditions. The recovery of serum miRNA dry-spotted on FTA Elute cards by direct elution with water at high temperature was poor. However, serum miRNAs dry-spotted on the cards were isolated with about 40% yield when using QIAzol lysis reagent and recovery was improved remarkably (>80%) upon extraction from cards pre-treated with trehalose. miRNAs stored on the cards remained stable at room temperature and can be kept for prolonged periods. Furthermore, miRNAs could be similarly recovered from serum exosomes dry-spotted on the cards. Importantly, when using sera from gastric cancer (GC) patients, the miRNAs were efficiently recovered from trehalose pre-treated cards without affecting their representation. Collectively, we have demonstrated the potential of FTA Elute cards to archive serum and serum exosomal miRNAs, making it useful for biomarker discovery and diagnostics.

Facile synthesis of a ratiometric oxygen nanosensor for cellular imaging.

A new type of cell-penetrating ratiometric fluorescence oxygen sensing nanoparticle was prepared through a facile co-precipitation method. Amphiphilic polymer poly (styrene-co-maleic anhydride) (PSMA) was firstly cooperated with polystyrene (PS) to envelop the highly photostable phosphorescent oxygen indicator, platinum(II)-tetrakis(pentafluorophenyl)porphyrin (PtTFPP, emission at 648nm), and the reference fluorophore, poly(9, 9-dioctylfluorene) (PFO, emission at 440nm ), via hydrophobic interaction in aqueous solution. To improve the sensor biocompatibility, the biomacromolecule poly-l-lysine (PLL) was selected to act as a shell via electrostatic forces. The as-prepared PtTFPP doped core-shell nanoparticles (called PPMA/PLL NPs) exhibited an excellent ratiometric luminescence response to O2 content with high quenching efficiency and full reversibility in the oxygen sensing. More importantly, these oxygen nanosensors passed across the cell membrane after co-incubation without external force. Labeled cells exhibited high brightness in the matching blue and red channels of a digital camera. And most nanosensors were found locating in cytoplasm rather than being trapped in endosomes.

A sensitive impedimetric platform biosensing protein: Insoluble precipitates based on the biocatalysis of manganese(III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrinin in HCR-assisted dsDNA.

In this study, a sensitive biosensing interface for protein was reported based on nonconductive insoluble precipitates (IPs) by the biocatalysis of manganese(III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrin (MnTMPyP), which was intercalated into formed double-strand DNA (dsDNA) scaffold triggered by hybridization chain reaction (HCR). In the proposed impedimetric aptasensor, carcinoembryonic antigen (CEA) and its aptamer were used as testing model. PtPd nanowires (PtPdNWs) with large surface area and superior conductivity were employed as nanocarriers to greatly immobilize biomolecules (e.g. CEA aptamers). Then, two DNA hairpins H1 and H2 were introduced to trigger HCR with the assistance of DNA initiator, resulting in the formation of a long dsDNA scaffold. Meanwhile, mimicking enzyme MnTMPyP molecules were embedded into the resultant dsDNA, in situ generating the complex MnTMPyP-dsDNA with peroxidase-like activity. Under the biocatalysis of MnTMPyP-dsDNA, 3,3-diaminobenzidine (DAB) was oxidized to form nonconductive IPs. As a result, the electron transfer between electrode interface and redox probe was vastly hindered, leading to the significant amplification of electrochemical impedimetric signal. So, greatly improved analytical performances of the proposed aptasensor were achieved with a detection limit as low as 0.030pgmL(-1). And the successful assay of CEA in human serum samples enabled the developed biosensing platform to have promising potential in bioanalysis.

[Preliminary Investigation of the Amount, the Molecular Weight and the Activity of Polysaccharides from Chaenomeles Speciosa Fruits in Ethanol Fractional Precipitation].

Chaenomeles speciosa fruits were extracted using water. The extracts were precipitated with 20%~95% (φ) ethanol, respectively. The amount of total polysaccharide was measured with phenol-sulfuric acid method. A method using high-performance size-exclusion chromatography (HPSEC) equipped with multiangle laser-light-scattering photometry (MALLS) and differential refractometry (RI) was presented for determining the molecular weight and molecular weigh distribution. RAW264.7 macrophage were cultured and stimulated with the polysaccharides in vitro and the production of nitric oxide in the cells was determined by the Griess assay. The aim of the study is to determine the amount and the molecular weight of the polysaccharides from Chaenomeles speciosa fruits, and preliminary investigate the immunomodulatory activity, The study provided the basis datas for the further research of Chaenomeles speciosa fruits. , and provided a simple and system method for the research of natural polysaccharide. The ethanol fractional precipitation showed that the order of total polysaccharide content was 95%>80%>40% ≥60%>20%. The results indicated that most polysaccharide from Chaenomeles speciosa fruits might be precipitated when ethanol concentration was up to 95% (T) and the crude polysaccharide purity had risen from 35. 1% to 45. 0% when the concentration of ethanol increased from 20% to 95%. HPSEC-MALLS-RI system showed that all the polysaccharide samples had the similar compositions. They appeared three chromatographic peaks and the retention time were not apparently different. The Mw were 6. 570 X 10(4) g . mol-1 and 1. 393 X 10(4) g . mol-1 respectively, and one less than 10 000 which was failure to obtain accurate values. The molecular weight of the first two polysaccharide distribution index(Mw/Mn)were 1. 336 and 1. 639 respectively. The polysaccharide samples had not exhibited immunomodulatory activity assessed on the basis of nitric oxide production by RAW264. 7 macrophage cells in the experiment.

Magnetic nanoparticles adapted for specific biomedical applications.

Magnetic nanoparticles (MNPs) are used in different biomedical applications, whereby each application requires specific particle properties. To fulfill these requirements, particle properties have to be optimized by means of variation of crystal structure, particle size, and size distribution. To this aim, improved aqueous precipitation procedures for magnetic iron oxide nanoparticle synthesis were developed. One procedure focused on the cyclic growth of MNPs without nucleation of new particle cores during precipitation. The second novel particle type are magnetic multicore nanoparticles, which consist of single cores of approximately 10 nm forming dense clusters in the size range from 40 to 80 nm. Their highest potential features these multicore particles in hyperthermia application. In our in vivo experiments, therapeutically suitable temperatures were reached after 20 s of heating for a particle concentration in the tumor of 1% and field parameters of H=24 kA/m and f=410 kHz. This review on our recent investigations for particle optimization demonstrates that tuning magnetic properties of MNPs can be obtained by the alteration of their structure, size, and size distribution. This can be realized by means of control of particle size during synthesis or subsequent size-dependent fractionation. The here-developed particles show high potential for biomedical applications.

Purification and characterization of a novel plantaricin, KL-1Y, from Lactobacillus plantarum KL-1.

Three bacteriocins from Lactobacillus plantarum KL-1 were successfully purified using ammonium sulfate precipitation, cation-exchange chromatography and reverse-phase HPLC. The bacteriocin peptides KL-1X, -1Y and -1Z had molecular masses of 3053.82, 3498.16 and 3533.16 Da, respectively. All three peptides were stable at pH 2-12 and 25 °C and at high temperatures of 80 and 100 °C for 30 min and 121 °C for 15 min. However, they differed in their susceptibility to proteolytic enzymes and their inhibition spectra. KL-1Y showed broad inhibitory activities against Gram-positive and Gram-negative bacteria, including Salmonella enterica serovar Enteritidis DMST 17368, Pseudomonas aeruginosa ATCC 15442, P. aeruginosa ATCC 9027, Escherichia coli O157:H7 and E. coli ATCC 8739. KL-1X and -1Z inhibited only Gram-positive bacteria. KL-1X, KL-1Y and KL-1Z exhibited synergistic activity. The successful amino acid sequencing of KL-1Y had a hydrophobicity of approximately 30 % and no cysteine residues suggested its novelty, and it was designated "plantaricin KL-1Y". Plantaricin KL-1Y exhibited bactericidal activity against Bacillus cereus JCM 2152(T). Compared to nisin, KL-1Y displayed broad inhibitory activities of 200, 800, 1600, 800, 400 and 400 AU/mL against the growth of Bacillus coagulans JCM 2257(T), B. cereus JCM 2152(T), Listeria innocua ATCC 33090(T), Staphylococcus aureus TISTR 118, E. coli O157:H7 and E. coli ATCC 8739, respectively, whereas nisin had similar activities against only B. coagulans JCM 2257(T) and B. cereus JCM 2152(T). Therefore, the novel plantaricin KL-1Y is a promising antimicrobial substance for food safety uses in the future.

Heat-precipitation allows the efficient purification of a functional plant-derived malaria transmission-blocking vaccine candidate fusion protein.

Malaria is a vector-borne disease affecting more than two million people and accounting for more than 600,000 deaths each year, especially in developing countries. The most serious form of malaria is caused by Plasmodium falciparum. The complex life cycle of this parasite, involving pre-erythrocytic, asexual and sexual stages, makes vaccine development cumbersome but also offers a broad spectrum of vaccine candidates targeting exactly those stages. Vaccines targeting the sexual stage of P. falciparum are called transmission-blocking vaccines (TBVs). They do not confer protection for the vaccinated individual but aim to reduce or prevent the transmission of the parasite within a population and are therefore regarded as an essential tool in the fight against the disease. Malaria predominantly affects large populations in developing countries, so TBVs need to be produced in large quantities at low cost. Combining the advantages of eukaryotic expression with a virtually unlimited upscaling potential and a good product safety profile, plant-based expression systems represent a suitable alternative for the production of TBVs. We report here the high level (300 µg/g fresh leaf weight (FLW)) transient expression in Nicotiana benthamiana leaves of an effective TBV candidate based on a fusion protein F0 comprising Pfs25 and the C0-domain of Pfs230, and the implementation of a simple and cost-effective heat treatment step for purification that yields intact recombinant protein at >90% purity with a recovery rate of >70%. The immunization of mice clearly showed that antibodies raised against plant-derived F0 completely blocked the formation of oocysts in a malaria transmission-blocking assay (TBA) making F0 an interesting TBV candidate or a component of a multi-stage malaria vaccine cocktail.

Biosorption of cationic basic dye and cadmium by the novel biosorbent Bacillus catenulatus JB-022 strain.

Biosorption of heavy metals and dyes is a promising technology that involves the removal of toxic metals from industrial wastes. The present study aims to screen the bacterial strains isolated from soils and polluted pond for their potential biosorption of both cationic dye and cadmium. Bacillus catenulatus JB-022 strain removed 58% and 66% of cationic basic blue 3 (BB3) and cadmium (Cd(II)) at the respective concentrations of 2000 mg/L and 150 mg/L. The biosorption equilibrium data were well fitted by the Langmuir adsorption isotherm, and the kinetic studies indicated that the biosorption followed the pseudo-second-order model. The biosorption kinetics showed that the equilibrium was reached within 10 min and 5 min for BB3 and Cd(II), respectively. According to the Langmuir model, the maximum uptakes of BB3 and Cd(II) by the JB-022 biomass were estimated to be 139.74 and 64.28 mg/g, respectively. To confirm the surface morphology and functional groups, field emission scanning electron microscope, energy-dispersive X-ray spectrometer, X-ray diffraction, and Fourier transform infrared spectroscopy analyses were carried out, and the results revealed that the biomass of JB-022 has carboxyl and phosphonate groups as potential surface functional groups capable of binding to cationic pollutants. In conclusion, B. catenulatus JB-022 is proposed as an excellent biosorbent with potentially important applications in removal of cationic pollutants from wastewaters.

Targeted mass spectrometry analysis of the proteins IGF1, IGF2, IBP2, IBP3 and A2GL by blood protein precipitation.

Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extremely challenging due to the high protein concentration range, characterised by abundant proteins that suppress and mask other proteins of lower abundance. This situation is further aggravated when using fast high-throughput methods, which are necessary for analysis of hundreds and thousands of samples in clinical laboratories. The blood proteins IGF1, IGF2, IBP2, IBP3 and A2GL have been proposed as indirect biomarkers for detection of GH administration and as putative biomarkers for breast cancer diagnosis. We describe a sensitive and scalable method to quantify these 5 proteins of medium and low abundance by selected reaction monitoring (SRM) LC-MS/MS analysis in blood samples. Our method requires 7µL of plasma and reaches a throughput of up to ca. 80 analyses per day. It includes an initial protein precipitation protocol optimised for extraction of low mass proteins from blood samples for reduced signal suppression and increased sensitivity in LC-MS/MS. We benchmarked this method for the analysis of 40 individual blood samples including 20 patients diagnosed with breast cancer. BIOLOGICAL SIGNIFICANCE: The interest for MS-based biomarker analysis in body fluids is steadily increasing as proteomics methodology translates into clinical laboratories. We describe a method for detection of 5 distinct proteins of low mass and medium to low abundance, which are of interest in anti-doping and clinical analysis. The analytical setup is simple and robust and is suitable for high-throughput instrument configurations.

Preparation and optimization of resveratrol nanosuspensions by antisolvent precipitation using Box-Behnken design.

Resveratrol, a natural polyphenolic component, has inspired considerable interest for its extensive physiological activities. However, the poor solubility of resveratrol circumscribes its therapeutic applications. The purpose of this study was to optimize and prepare resveratrol nanosuspensions using the antisolvent precipitation method. The effects of crucial formulation and process variables (drug concentration, stabilizer, and surfactant contents) on particle size were investigated by utilizing a three-factor three-level Box-Behnken design (BBD) to perform this experiment. Different mathematical polynomial models were used to identify the impact of selected parameters and to evaluate their interrelationship for predictive formulation purposes. The optimal formulation consisted of drug 29.2 (mg/ml), polyvinylpyrrolidone (PVP) K17 0.38%, and F188 3.63%, respectively. The morphology of nanosuspensions was found to be near-spherical shaped by scanning electron microscopy (SEM) observation. The X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) analysis confirmed that the nanoparticles were in the amorphous state. Furthermore, in comparison to raw material, resveratrol nanosuspensions showed significantly enhanced saturation solubility and accelerated dissolution rate resulting from the decrease in particle size and the amorphous status of nanoparticles. Meanwhile, resveratrol nanosuspensions exhibited the similar antioxidant potency to that of raw resveratrol. The in vivo pharmacokinetic study revealed that the C max and AUC0→∞ values of nanosuspension were approximately 3.35- and 1.27-fold greater than those of reference preparation, respectively. Taken together, these results suggest that this study provides a beneficial approach to address the poor solubility issue of the resveratrol and affords a rational strategy to widen the application range of this interesting substance.

Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection.

In situ precipitation: a novel approach for preparation of iron-oxide magnetoliposomes.

BACKGROUND: Conventional methods of preparing magnetoliposomes are complicated and inefficient. A novel approach for magnetoliposomes preparation was investigated in the study reported here. METHODS: FeCl3/FeCl2 solutions were hydrated with lipid films to obtain liposome-encapsulated iron ions by ultrasonic dispersion. Non-encapsulated iron ions were removed by dialysis. NH3·H2O was added to the system to adjust the pH to a critical value. Four different systems were prepared. Each was incubated at a different temperature for a different length of time to facilitate the permeation of NH3·H2O into the inner phase of the liposomes and the in situ formation of magnetic iron-oxide cores in the liposomes. Single-factor analysis and orthogonal-design experiments were applied to determinate the effects of alkalization pH, temperature, duration, and initial Fe concentration on encapsulation efficiency and drug loading. RESULTS: The magnetoliposomes prepared by in situ precipitation had an average particle size of 168±14 nm, zeta potential of -26.2±1.9 mV and polydispersity index of 0.23±0.06. The iron-oxide cores were confirmed as Fe3O4 by X-ray diffraction and demonstrated a superparamagnetic response. Encapsulation efficiency ranged from 3% to 22%, while drug loading ranged from 0.2 to 1.58 mol Fe/mol lipid. The optimal conditions for in situ precipitation were found to be an alkalization pH of 12, temperature of 60°C, time of 60 minutes, and initial Fe concentration of 100 mM Fe(3+) + 50 mM Fe(2+). CONCLUSION: In situ precipitation could be a simple and efficient approach for the preparation of iron-oxide magnetoliposomes.