Development and Characterization of Plant-derived Aristatoside C and Davisianoside B Saponin-loaded Phytosomes with Suppressed Hemolytic Activity.

Saponins are glycosides widely distributed in the plant kingdom and have many pharmacological activities. However, their tendency to bind to cell membranes can cause cell rupture, limiting their clinical use. In the previous study, aristatoside C and davisianoside B were isolated from Cephalaria species. Cytotoxicity assays showed that they are more active on A-549 cell lines than doxorubicin but caused hemolysis. In the current research, aristatoside C and davisianoside B were loaded to phytosomes called ALPs and DLPs respectively, and characterized for particle size, zeta potential, encapsulation efficiency, release kinetic, hemolytic activity, and cytotoxicity on A-549 cell line. DLPs maintained the cytotoxic activity of the free saponins against A-549 cells with IC50 of 9,64±0,02 µg/ml but dramatically reduced their hemolytic activity. Furthermore, temperature and time-dependent stability studies based on the size and zeta potential of ALPs and DLPs revealed that the phytosomes have sustained release properties over 2 weeks. Overall, DLPs displayed cytotoxicity against A-549 cells with minimal hemolysis and sustained release, highlighting their potential as nanotherapeutics for clinical applications.

Hydrophobically modified low molecular weight chitosans as efficient and nontoxic gene delivery vectors.

BACKGROUND: Chitosan derivatives are potential candidates for gene delivery because they are biocompatible and low toxic. However, their use has been limited by their moderate transfection efficiency and the rather large sizes of DNA complexes with high molecular weight chitosans. To circumvent these limitations, we used low molecular weight (approximately 5 kDa) chitosans grafted at 3 and 18 mol% with N-/2(3)-(dodec-2-enyl)succinoyl groups (HM-LMW-ch) that exhibit surfactant-like properties. METHODS: The physico-chemical properties of complexes of DNA with the two HM-LMW-ch derivatives and the nonmodified LMW-ch were compared by electrophoresis, dynamic light scattering, fluorescence spectroscopy and fluorescence correlation spectroscopy. Moreover, their transfection efficiencies and cytotoxicity were evaluated and their intracellular trafficking was monitored by confocal microscopy. Finally, their ability to deliver genes in mice kidneys after systemic administration was investigated. RESULTS: Complexes with HM(3%)-LMW-ch, but not with HM(18%)-LMW-ch and LMW-ch, efficiently delivered genes in mice kidneys. HM(3%)-LMW-ch formed small positively charged particles that were resistant to DNases and nucleases and marginally interact with serum components. Moreover, these particles were efficiently internalized in cells and low toxic. By contrast, HM(18%)-LMW-ch formed large and weakly charged aggregates with DNA that were highly susceptible to DNases and nucleases. CONCLUSIONS: HM(3%)-LMW-ch appears to be a promising nonviral vector with low cytotoxicity and efficient transfection properties.

Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging.

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.

Physicochemical properties of low molecular weight alkylated chitosans: a new class of potential nonviral vectors for gene delivery.

Low molecular weight chitosans grafted with N-/2(3)-(dodec-2-enyl)succinoyl groups (HM-LMW-chitosans) with a mean molecular mass of 5 kDa, a degree of acetylation of 3% and a degree of tetradecenoyl substitution (TDC) of 3-18 mol% have been synthesized. These molecules are monodisperse and soluble in water at neutral pH. Using tensiometry and Nile Red fluorescence, the HM-LMW-chitosans were found to form micelles through hydrophobic interactions involving their tetradecenoyl chains and nonprotonated glucosamine monomers. Their critical micelle concentration decreases with increasing TDC values but varies little with pH and salt. Interaction with large unilamellar vesicles taken as model membranes indicated that HM-LMW-chitosans interact mainly with vesicles mimicking the inner leaflet of biomembranes both through electrostatic and hydrophobic interactions. This preferential interaction may destabilize endosomal membranes and favor the DNA release into the cytoplasm in gene delivery applications. Moreover, since this interaction significantly decreased the membrane fluidity of these vesicles, the HM-LMC-chitosans are thought to exhibit limited lateral mobility and flip-flop ability, and thus, limited cytotoxicity. These properties suggest that the HM-LMW-chitosans may constitute a promising new class of nonviral vectors for gene therapy.

The binding of novel two-color fluorescence probe FA to serum albumins of different species.

The novel two-color ratiometric fluorescence probe FA belonging to a class of 3-hydroxychromone dyes was applied for characterization of binding sites in serum albumins obtained from seven species (bovine, dog, horse, human, pig, rabbit and sheep). On strong and highly specific FA binding to the same location in protein structure, the species-dependent differences were observed in positions of absorption maxima, positions of two fluorescence emission bands and the intensity ratios between them. They were analyzed by multiparametric algorithm that allowed a detailed characterization of probe-binding sites and were characterized by very low polarity, high electronic polarizability and different extent of intermolecular hydrogen bonding. The species-dependent differences were also observed in time-resolved fluorescence emission decays. Fluorescence competition experiments with the drug warfarin, suggested the location of FA binding site within or in proximity to Domain IIA.

Hemin-dependent induction and internalization of CD38 in K562 cells.

The cell surface antigen, CD38, is a bifunctional ecto-enzyme, which is predominantly expressed on hematopoietic cells during differentiation. In the present study, it is shown that hemin treatment of K562 cells gives rise to induction of enzymatic activities inherent to CD38. GDP-ribosyl cyclase activity, an indicator of CD38, increased initially in response to hemin in a time-dependent manner, reached a maximum level on the 5th day and, thereafter, declined sharply to the initial level. The increase in NAD(+) glycohydrolase and ADP-ribose uptake activities followed a similar time course. However, the decline in the latter activities after the 5th day of induction appeared to be rather slow in contrast to GDP-ribosyl cyclase activity. The time course of these changes was well correlated with the FACScan findings obtained by use of anti-CD38 monoclonal antibody. SDS-PAGE and Western blot analyses by use of the monoclonal antibody OKT10 revealed a transient hemin-dependent appearance of a 43 kDa membrane protein with maximum signal intensity on the first 4 days of incubation. There was subsequently a gradual decrease on the 5th day, concomitant with a reciprocal increase in activity of the internalized protein fraction. The results together indicated that hemin-induced expression of CD38 was followed by its down-regulation.

Novel two-color fluorescence probe with extreme specificity to bovine serum albumin.

We report on strong, highly specific and stochiometric binding to bovine serum albumin of novel fluorescence probe FA, 2-(6-diethylaminobenzo[b]furan-2-yl)-3-hydroxychromone, that exhibits a very characteristic two-band fluorescence spectrum. Both absorption band and two fluorescence bands of FA are very sensitive to non-covalent interactions in the immediate environment of the probe. Multiparametric analysis of this spectroscopic information allows us to conclude that the binding site is characterized by very low polarity, high extent of screening from aqueous environment and unusually high electronic polarizability. The latter suggests the proximal location of probe FA to the aromatic amino acid residues in the binding site. The new probe can be proposed for the study of interaction of ligands and drugs of different nature with serum albumins.