A microfluidic biosensor based on nucleic acid sequence recognition

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome–target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol L^–1 (10 pmol L^–1) . Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system.     

Formation of Giant Liposomes from Crystalline Complexes of Monoalkylammonium Surfactants and 4-Hydroxybiphenyl

A parallel rather than a perpendicular alignment of aromatic compounds with respect to surfactant molecules (see schematic representation) is preferred for the formation of a liposome structure, because the perpendicular alignment would reduce the hydrophilicity of the aggregate. This is the result of studies on crystalline complexes of monoalkylammonium halides and various aromatic compounds.     

Effect of delivery system on the pharmacokinetic and phototherapeutic properties of bis(methyloxyethyleneoxy)silicon-phthalocyanine in tumor-bearing mice

A Si(IV)-phthalocyanine bearing two methoxyethyleneglycol axial ligands bound to the central metal ion (SiPc) has been prepared by chemical synthesis and analyzed for its phototherapeutic activity after administration in a Cremophor or liposome formulation to C57B1/6 mice bearing a subcutaneously transplanted Lewis lung carcinoma (LLC). The maximum drug accumulation in the tumor is found at 24 h after intraperitoneal injection, independent of the delivery system. However, the tumor concentration of SiPc in the Cremophor formulation is about two-fold higher, while the drug concentration in liver and skin shows similar trends with the two delivery systems. The drug accumulation and retention in the brain is much larger when using Cremophor emulsion. Photodynamic therapy (672 nm, 370 mW m⁻², 360 J cm⁻²) at 24 h after the injection of Cremophor emulsion- or DPPC liposome-formulated SiPc causes a very efficient and similar response for the LLC (8 versus 22 mm mean tumor diameter for the control groups at 21 days after phototreatment). These very promising effects, obtained both at higher and lower tumor drug concentrations, clearly demonstrate the potential phototherapeutical activity of the newly synthesized SiPc.     

Bispidinone-based molecular switches for construction of stimulus-sensitive liposomal containers

It is demonstrated that new lipid-like amphiphilic compounds, derivatives of 3,7-diazabicyclo[3.3.1]nonan-9-one (bispidinone) with long alkyl substituents, can be integrated into liposomal membranes. They can serve as molecular switches changing the conformation from the chair–boat to chair–chair on addition of an aqueous solution of a bivalent copper salt, and thus enhancing the permeability of the lipid bilayer of liposomes and the release of encapsulated compounds.     

Systematic development and validation of RP-HPLC method for simultaneous estimation of tamoxifen and sulphoraphane with specific application for nanolipidic formulations

Tamoxifen (TAM) and Sulphoraphane (SFN) are well-known anti-estrogen drugs used for the treatment of breast cancer. Due to their synergistic therapeutic potential, their combination is preferred as it helps to minimize the drug-related toxicities and enhances therapeutic efficacy. A simple, robust and fast simultaneous reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed as well validated for the analysis of both the drugs based on their particular wavelength. The separation was performed on C₁₈ analytical column with dimensions of 4.6 × 250 mm, 5 μm using mobile phase methanol: water (pH 3.5) in the ratio 70:30 and flow rate of 0.8 min/mL. Box-Behnken experimental design was used to optimized these independent variables and analyze their effect on the response variables like retention time (RT), no. of theoretical plates and tailing factor of both analytes. Method validation was carried out for establishing the specificity, linearity range, accuracy, sensitivity, robustness, precision and ruggedness. The method applicability was evaluated on different nanoformulations, i.e., solid lipid nanoparticles (SLNs), liposomes (LIPO), nanostructured lipid carriers (NLCs). The peaks of the analyte were found to be well resolved and two distinct RT were recorded for TAM and SFN. Calibration curves were found to be linear for TAM and SFN over concentration range of 6–24 μg/mL. All method validation criteria were within the range of acceptance. Relative standard deviation (%RSD) was observed to be <2% for inter- and intra-day precision. The application of developed method for estimation of drugs from the nanoformulations was suitabile for in vitro as well as in vivo studies.     

Studying phase separation in confinement

Cells organize their interior through membrane-bound organelles and through membraneless condensates that are formed by liquid–liquid phase separation (LLPS). The complex process of coacervation that is involved in LLPS is challenging to study in living cells. Hence, studying coacervation in cell-mimicking synthetic containers can yield valuable insights. Here, we review recent progress with respect to studying LLPS (particularly coacervation) in artificial compartments, from water-in-oil droplets to membranous liposomes. We describe different strategies to form and control coacervates in microconfinements and to study their physicochemical and biological characteristics. We also describe how coacervation can itself be used in container formation. This review highlights the importance of in vitro coacervate studies for understanding cellular biology and for designing synthetic cells.     

Effects of liposomal phophatidylserine on phagocytic uptake of liposomes by macrophage-like HL-60RG cells

The purpose of this work is to know the effect of surface properties of liposomes on their phagocytic uptake by macrophages. For this, liposomes were prepared by the Bangham technique from the mixture of phosphatidylcholine (PC) and cholesterol (Chol) incorporated either with phosphatidylserine (PS), phosphatidylethanolamine (PE) or phosphatidic acid (PA). The liposomes thus prepared had diameters in the range between 150 and 260 nm. Electric surface properties of the liposomes and the macrophages differentiated from HL-60RG cells were determined by measuring their electrophoretic mobilities. The phagocytic uptake of liposomes with different contents of PS, PE and PA by macrophage-like HL-60RG cells was investigated by measuring oxygen consumption associated with phagocytic uptake. The phagocytic activity was found to be the highest with the PC–Chol liposomes containing 7 mol% PS, but no significant effects were observed with PA- and PE-containing PC–Chol liposomes. As the uptake was independent of the electric surface property of liposomes, PS was concluded to be specifically important for phagocytic activity of macrophages.     

Use of liposomes to evaluate the role of membrane interactions on antioxidant activity

In this study the possibility of using liposomes as membrane mimetic systems was evaluated to estimate the antioxidant properties of oxicams and establish a relationship between the interactions of the drugs with the membrane and their consequent antioxidant activity. Different experiments were performed covering the study of the protective effect of oxicams in lipid peroxidation induced by the peroxyl radical (ROO) derived from 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and using two fluorescence probes with distinct lipophilic properties. Lipid peroxidation using the hydrophilic probe fluorescein was evaluated in lipid and aqueous media. Lipid systems labelled with the fluorescent probe diphenylhexatriene propionic acid (DPH-PA) were used to assess the effects of the drugs on membrane peroxidation simultaneously by fluorescence intensity decay and changes in membrane fluidity by steady-state anisotropy measurements. The use of different probes and liposomes as membrane mimetic systems allowed to conclude that membrane lipoperoxidation is related not only to the scavenging characteristics of the antioxidants but also to their ability to interact with the lipid bilayers.     

Capillary electrophoresis of liposomes functionalized for protein binding

CE enabled assessing the attachment of hexa-histidine-tagged proteins to functionalized phospholipid liposomes. The liposomes were made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, phosphatidyl-ethanolamine, cholesterol and distearoyl-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) in a molar ratio of 29:26:40:5. The unilamellar vesicles, which had an average diameter of 170 nm, were labelled by inclusion of FITC-dextran for fluorescence detection. CE was carried out in poly(vinyl alcohol) (PVA)-coated capillaries at 25 degrees C with a BGE consisting of Tris-HCl (50 mM, pH 8.0). For conjugation of the liposomes with the proteins (soluble synthetic receptor fragments with molecular mass of 60 and 70 kDa, respectively), Ni(2+) was implanted into the vesicle surface by an anchor lipid containing a nitrilotriacetate acid (NTA) group as complexation agent for the metal ions. The difference in surface charge enabled the separation of the different species of interest by CE: plain vesicles, vesicles functionalised with Ni-NTA, vesicle-protein complexes and the species formed upon removal of the Ni-ions by complexation with EDTA. Loss of the Ni-ions resulted in the release of the proteins and the reappearance of the plain Ni-free NTA-liposome species in the electropherograms.     

Intracellular enzyme-activatable prodrug for real-time monitoring of chlorambucil delivery and imaging

Carboxylesterase, a necessary enzyme in various mammalian cells, has been employed in various biological applications. Herein, we designed and synthesized a novel carboxylesterase-based prodrug, which can realize simultaneous drug-release imaging and cancer chemotherapy. This prodrug comprises three parts:coumarin as the fluorophore and the cleavable architecture, chlorambucil as the anticancer drug, and acetyl group as the enzyme-responsive unit. The presence of carboxylesterase leads to the activation of coumarin fluorescence, and this fluorescence serves as the reporting signal for assessing the enzyme level and drug release. Moreover, the prodrug was incorporated in liposome for monitoring drug release and chemotherapeutic effect in living cells. Upon internalization by HeLa cells, the prodrug can release chlorambucil and exhibit high cytotoxicity. This approach may provide some helpful insights for enhancing therapeutic effect and tracking the release of prodrug.