Effect of lamellarity and size on calorimetric phase transitions in single component phosphatidylcholine vesicles.

Nano-differential scanning calorimetry (nano-DSC) is a powerful tool in the investigation of unilamellar (small unilamellar, SUVs, or large unilamellar, LUVs) vesicles, as well as lipids on supported bilayers, since it measures the main gel-to-liquid phase transition temperature (Tm), enthalpies and entropies. In order to assign these transitions in single component systems, where Tm often occurred as a doublet, nano-DSC, dynamic light scattering and cryo-transmission electron microscopy (cryo-TEM) data were compared. The two Tms were not attributable to decoupled phase transitions between the two leaflets of the bilayer, i.e. nano-DSC measurements were not able to distinguish between the outer and inner leaflets of the vesicle bilayers. Instead, the two Tms were attributed to mixtures of oligolamellar and unilamellar vesicles, as confirmed by cryo-TEM images. Tm for the oligolamellar vesicles was assigned to the peak closest to that of the parent multilamellar vesicle (MLV) peak. The other transition was higher than that of the parent MLVs for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and increased in temperature as the vesicle size decreased, while it was lower in temperature than that of the parent MLVs for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and decreased as the vesicle size decreased. These subtle shifts arose due to small differences in the values of ΔH and ΔS, since Tm is determined by their ratio (ΔH/ΔS). It was not possible to completely eliminate oligolamellar structures for MLVs extruded with the 200 nm pore size filter, even after 120 passes, while these structures were eliminated for MLVs extruded through the 50 nm pore size filter.

Cationic gel-phase liposomes with "decorated" anionic SPIO nanoparticles: morphology, colloidal, and bilayer properties.

The assembly and complexation of oppositely charged colloids are important phenomena in many natural and synthetic processes. Liposome-nanoparticle assemblies (LNAs) represent an interesting hybrid system that combines "soft" and "hard" colloidal materials. This work describes the formation and characterization of gel-phase LNAs formed by the binding of anionic superparamagnetic iron oxide (SPIO) nanoparticles to cationic dipalmitoylphosphatidylcholine (DPPC)/dipalmitoyltrimethylammonium propane (DPTAP) liposomes. Particles were examined with hydrodynamic diameters below (16 nm) and above (30 nm) the cutoff reported for supported lipid bilayer formation. LNA formation with 16 nm particles was entropically driven and particles bound individually to yield "decorated" structures. In this case, increasing nanoparticle concentration yielded colloidal LNA aggregates and eventual charge inversion. In contrast, LNA formation with 30 nm particles was enthalpically driven, and the nanoparticles aggregated at the bilayer interface. These aggregates led to significant LNA aggregation and large bilayer sheets due to liposome rupture despite minimal charge screening of the liposome surface. In this case SLBs were present, but these structures were not dominant. Differences in LNA structure were also revealed through the lipid phase transition behavior. This work infers size-dependent nanoparticle binding and LNA formation mechanisms that can be used to tailor colloidal and bilayer properties. Analogies are made to polyelectrolyte patch charge heterogeneities and DNA complexation with cationic liposomes.

Multicomponent folate-targeted magnetoliposomes: design, characterization, and cellular uptake.

Folate-targeted cationic magnetoliposomes (FTMLs) have been prepared with coencapsulated doxorubicin (DOX) and anionic superparamagnetic iron oxide (SPIO) nanoparticles (NPs) with 5 nm γ-Fe(2)O(3) cores and 16 nm hydrodynamic diameters. NP encapsulation (89%) was confirmed by cryogenic transmission electron microscopy (TEM), and the presence of the oppositely charged NPs did not cause liposome aggregation. The FTMLs had an average diameter of 174 ± 53 nm and existed as unilamellar and cup-shaped liposomes, which was attributed to dissimilar lipid packing parameters and the presence of PEG-lipids. A 3-fold increase in DOX release was achieved over 2 hours when the encapsulated SPIO NPs were heated by an alternating current electromagnetic field operating at radio frequencies (RF). Results with human cervical cancer cells (HeLa), which have been shown to exhibit high folate receptor (FR) expression, confirmed FTML surface binding and cellular uptake. In contrast, no uptake was observed for lower FR-expressing human breast carcinoma cells (ZR-75-1). FROM THE CLINICAL EDITOR: This study discusses the design and cellular uptake of multifunctional folate-targeted cationic magnetoliposomes enabling doxorubicin delivery and SPIO labeling.

A circRNA-miRNA-mRNA network plays a role in the protective effect of diosgenin on alveolar bone loss in ovariectomized rats.

BACKGROUND: The present study aimed to assess the perturbation in circular RNA (circRNA)/mRNA expression profiles and a circRNA-miRNA-mRNA coexpression network involved in the potential protective effect of diosgenin (DIO) on alveolar bone loss in rats subjected to ovariectomy (OVX). METHODS: The Wistar rats (female) manipulated with sham operation were classified as the SHAM group and the grouping of OVX rats administered with DIO, estradiol valerate or vehicle for 12 weeks was DIO group, EV group and OVX group respectively. Following treatments, the plasmatic levels of osteocalcin and tumor necrosis factor-alpha and the microstructure of alveolar bone were assayed. Based on microarray analyses, we identified differentially expressed (DE) circRNAs and mRNAs in alveolar bone of rats in both OVX and DIO group. The DE circRNAs and DE mRNAs involved in the bone metabolism pathway validated by RT-qPCR were considered key circRNAs/mRNAs. On the basis of these key circRNAs/mRNAs, we predicted the overlapping relative miRNAs of key circRNAs/mRNAs, and a circRNA-miRNA-mRNA network was built. RESULTS: DIO showed an anti-osteopenic effect on the rat alveolar bone loss induced by OVX. In total, we found 10 DE circRNAs (6 downregulated and 4 upregulated) and 614 DE mRNAs (314 downregulated and 300 upregulated) in samples of the DIO group compared with those of the OVX group. However, only one circRNA (rno_circRNA_016717) and seven mRNAs (Sfrp1, Csf1, Il1rl1, Nfatc4, Tnfrsf1a, Pik3c2g, and Wnt9b) were validated by qRT-PCR and therefore considered key circRNA/mRNAs. According to these key circRNA/mRNAs and overlapping predicted miRNAs, a coexpression network was constructed. After network analysis, one circRNA-miRNA-mRNA axis (circRNA_016717/miR-501-5p/Sfrp1) was identified. CONCLUSION: The mechanism of DIO inhibiting alveolar bone loss after OVX is possibly relevant to the simultaneous inhibition of osteogenesis and osteoclastogenesis by mediating the expression of important molecules in the Wnt, PI3K, RANK/RANKL or osteoclastogenic cytokine pathways. The circRNA_016717/miR-501-5p/Sfrp1 axis may play important roles in these processes.

Protective effect of Rhizoma Dioscoreae extract against alveolar bone loss in ovariectomized rats via regulation of IL-6/STAT3 signaling.

The aim of the present study was to assess the effectiveness of Rhizoma Dioscoreae extract (RDE) on preventing rat alveolar bone loss induced by ovariectomy (OVX), and to determine the role of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in this effect. Female Wistar rats were subjected to OVX or sham surgery. The rats that had undergone OVX were treated with RDE (RDE group), vehicle (OVX group) or 17ß-estradiol subcutaneous injection (E2 group). Subsequently, bone metabolic activity was assessed by analyzing 3-D alveolar bone construction, bone mineral density, as well as the plasma biomarkers of bone turnover. The gene expression of alveolar bone in the OVX and RDE groups was evaluated by IL-6/STAT3 signaling pathway polymerase chain reaction (PCR) arrays, and differentially expressed genes were determined through reverse transcription-quantitative PCR. The inhibitory effect of RDE on alveolar bone loss in the OVX group was demonstrated in the study. In comparison with the OVX group, the RDE group exhibited 19 downregulated genes and 1 upregulated gene associated with the IL-6/STAT3 signaling pathway in alveolar bone. Thus, RDE was shown to relieve OVX-induced alveolar bone loss in rats, an effect which was likely associated with decreased abnormal bone remodeling via regulation of the IL-6/STAT3 signaling pathway.

Rhizoma Dioscoreae extract protects against alveolar bone loss by regulating the cell cycle: A predictive study based on the protein­protein interaction network.

Rhizoma Dioscoreae extract (RDE) exhibits a protective effect on alveolar bone loss in ovariectomized (OVX) rats. The aim of this study was to predict the pathways or targets that are regulated by RDE, by re­assessing our previously reported data and conducting a protein­protein interaction (PPI) network analysis. In total, 383 differentially expressed genes (≥3­fold) between alveolar bone samples from the RDE and OVX group rats were identified, and a PPI network was constructed based on these genes. Furthermore, four molecular clusters (A­D) in the PPI network with the smallest P­values were detected by molecular complex detection (MCODE) algorithm. Using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA) tools, two molecular clusters (A and B) were enriched for biological process in Gene Ontology (GO). Only cluster A was associated with biological pathways in the IPA database. GO and pathway analysis results showed that cluster A, associated with cell cycle regulation, was the most important molecular cluster in the PPI network. In addition, cyclin­dependent kinase 1 (CDK1) may be a key molecule achieving the cell­cycle­regulatory function of cluster A. From the PPI network analysis, it was predicted that delayed cell cycle progression in excessive alveolar bone remodeling via downregulation of CDK1 may be another mechanism underling the anti­osteopenic effect of RDE on alveolar bone.

Rhizoma Dioscoreae extract protects against alveolar bone loss in ovariectomized rats via microRNAs regulation.

The aim of this study was to evaluate the osteoprotective effect of aqueous Rhizoma Dioscoreae extract (RDE) on the alveolar bone of rats with ovariectomy-induced bone loss. Female Wistar rats underwent either ovariectomy or sham operation (SHAM). The ovariectomized (OVX) rats were treated with vehicle (OVX), estradiol valerate (EV), or RDE. After treatments, the bone mineral density (BMD) and the three-dimensional microarchitecture of the alveolar bone were analyzed to assess bone mass. Microarrays were used to evaluate microRNA expression profiles in alveolar bone from RDE-treated and OVX rats. The differential expression of microRNAs was validated using real-time quantitative RT-PCR (qRT-PCR), and the target genes of validated microRNAs were predicted and further analyzed using Ingenuity Pathway Analysis (IPA). The key findings were verified using qRT-PCR. Our results show that RDE inhibits alveolar bone loss in OVX rats. Compared to the OVX rats, the RDE-treated rats showed upregulated expression levels of 8 microRNAs and downregulated expression levels of 8 microRNAs in the alveolar bone in the microarray analysis. qRT-PCR helped validate 13 of 16 differentially expressed microRNAs, and 114 putative target genes of the validated microRNAs were retrieved. The IPA showed that these putative target genes had the potential to code for proteins that were involved in the transforming growth factor (TGF)-ß/bone morphogenetic proteins (BMPs)/Smad signaling pathway (Tgfbr2/Bmpr2, Smad3/4/5, and Bcl-2) and interleukin (IL)-6/oncostatin M (OSM)/Jak1/STAT3 signaling pathway (Jak1, STAT3, and Il6r). These experiments revealed that RDE could inhibit ovariectomy-induced alveolar bone loss in rats. The mechanism of this anti-osteopenic effect in alveolar bone may involve the simultaneous inhibition of bone formation and bone resorption, which is associated with modulation of the TGF-ß/BMPs/Smad and the IL-6/OSM/Jak1/STAT3 signaling pathways via microRNA regulation.

Low-dose chemotherapy of hepatocellular carcinoma through triggered-release from bilayer-decorated magnetoliposomes.

Low-dose (LD) chemotherapy is a promising treatment strategy that may be improved by controlled delivery. Polyethylene glycol-stabilized bilayer-decorated magnetoliposomes (dMLs) have been designed as a stimuli-responsive LD chemotherapy drug delivery system and tested in vitro using Huh-7 hepatocellular carcinoma cell line. The dMLs contained hydrophobic superparamagnetic iron oxide nanoparticles within the lipid bilayer and doxorubicin hydrochloride (DOX, 2 µM) within the aqueous core. Structural analysis by cryogenic transmission electron microscopy and dynamic light scattering showed that the assemblies were approximately 120 nm in diameter. Furthermore, the samples consisted of a mixture of dMLs and bare liposomes (no nanoparticles), which provided dual burst and spontaneous DOX release profiles, respectively. Cell viability results show that the cytotoxicity of DOX-loaded dMLs was similar to that of bare dMLs (∼10%), which indicates that spontaneous DOX leakage had little cytotoxic effect. However, when subjected to a physiologically acceptable radiofrequency (RF) electromagnetic field, cell viability was reduced up to 40% after 8h and significant cell death (>90%) was observed after 24h. The therapeutic mechanism was intracellular RF-triggered DOX release from the dMLs and not intracellular hyperthermia due to nanoparticle heating via magnetic losses.

Structural and thermal analysis of lipid vesicles encapsulating hydrophobic gold nanoparticles.

The structure and stability of hybrid lipid vesicles containing bilayer-encapsulated hydrophobic nanoparticles is dependent upon lipid phase behavior. By embedding stearylamine-stabilized gold nanoparticles in dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol vesicles, we show that encapsulation at lipid to nanoparticle ratios from 10,000:1 to 5000:1 leads to bilayer thickening and hydrophobic mismatch, favoring nanoparticle inclusion in gel phase vesicles. High loadings lead to large increases in the gel to fluid melting temperature upon heating and significant hysteresis on cooling, which cannot be attributed solely to excess free ligand. This behavior is due to a cooperative effect of excess free SA ligand and nanoparticle embedment. Nanoparticle clustering was observed during lipid melting and could be reversed upon lipid freezing owing to lateral capillary forces within the bilayer. The impact of nanoparticle embedment on vesicle structure and properties at such low concentrations is reminiscent of hydrophobic proteins, suggesting that the underlying lipid biophysics between proteins and nanoparticle are similar and may provide a predictive design tool for therapeutic applications.

Controlled release from bilayer-decorated magnetoliposomes via electromagnetic heating.

Nanoscale assemblies that can be activated and controlled through external stimuli represent a next stage in multifunctional therapeutics. We report the formation, characterization, and release properties of bilayer-decorated magnetoliposomes (dMLs) that were prepared by embedding small hydrophobic SPIO nanoparticles at different lipid molecule to nanoparticle ratios within dipalmitoylphosphatidylcholine (DPPC) bilayers. The dML structure was examined by cryogenic transmission electron microscopy and differential scanning calorimetry, and release was examined by carboxyfluorescein leakage. Nanoparticle heating using alternating current electromagnetic fields (EMFs) operating at radio frequencies provided selective release of the encapsulated molecule at low nanoparticle concentrations and under physiologically acceptable EMF conditions. Without radio frequency heating, spontaneous leakage from the dMLs decreased with increasing nanoparticle loading, consistent with greater bilayer stability and a decrease in the effective dML surface area due to aggregation. With radio frequency heating, the initial rate and extent of leakage increased significantly as a function of nanoparticle loading and electromagnetic field strength. The mechanism of release is attributed to a combination of bilayer permeabilization and partial dML rupture.