Effect of Cyclodextrins Formulated in Liposomes and Gold and Selenium Nanoparticles on siRNA Stability in Cell Culture Medium.

Background: Encapsulation of siRNA fragments inside liposome vesicles has emerged as an effective method for delivering siRNAs in vitro and in vivo. However, the liposome's fluid-phospholipid bilayer of liposomes allows siRNA fragments to diffuse out of the liposome, decreasing the dose concentration and therefore the effectiveness of the carrier. We have previously reported that β-cyclodextrins formulated in liposomes help increase the stability of siRNAs in cell culture medium.

A Fresh Look at the Potential of Cyclodextrins for Improving the Delivery of siRNA Encapsulated in Liposome Nanocarriers.

Liposomes are among the most effective vehicles to deliver siRNAs to cells, both and . However, despite numerous efforts to improve the potential of liposomes, siRNAs begin to leach out of liposomes as soon as they are formulated. This decreases the value of liposomes for drug delivery purposes significantly, masking their true potential.

Inhibitors of urokinase type plasminogen activator and cytostatic activity from crude plants extracts.

In view of the clear evidence that urokinase type plasminogen activator (uPA) plays an important role in the processes of tumor cell metastasis, aortic aneurysm, and multiple sclerosis, it has become a target of choice for pharmacological intervention. The goal of this study was thus to determine the presence of inhibitors of uPA in plants known traditionally for their anti-tumor properties. Crude methanol extracts were prepared from the leaves of plants (14) collected from the subtropical dry forest (Guanica, Puerto Rico), and tested for the presence of inhibitors of uPA using the fibrin plate assay.

Intracellular Delivery of siRNA by Polycationic Superparamagnetic Nanoparticles.

The siRNA transfection efficiency of nanoparticles (NPs), composed of a superparamagnetic iron oxide core modified with polycationic polymers (poly(hexamethylene biguanide) or branched polyethyleneimine), were studied in CHO-K1 and HeLa cell lines. Both NPs demonstrated to be good siRNA transfection vehicles, but unmodified branched polyethyleneimine (25 kD) was superior on both cell lines. However, application of an external magnetic field during transfection (magnetofection) increased the efficiency of the superparamagnetic NPs.

Low operational stability of enzymes in dry organic solvents: changes in the active site might affect catalysis.

The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme's initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results.

Immobilization of Thermoanaerobium brockii alcohol dehydrogenase on SBA-15.

The activity of Thermoanaerobium brockii alcohol dehydrogenase (TBADH) adsorbed on mesoporous silica SBA-15 was compared with that of the free enzyme in water and in biphasic system (water phase up to 50% v/v water). TBADH was active at a water concentration ≥10% v/v. In the reduction reaction of sulcatone to sulcatol carried out in biphasic systems, the yield obtained with SBA-15-adsorbed TBADH was up to 5.

Enantioselective Transesterification Catalysis by Nanosized Serine Protease Subtilisin Carlsberg Particles in Tetrahydrofuran.

Enzyme catalysis in organic solvents is a powerful tool for stereo-selective synthesis but the enantioselectivity is still hard to predict. To overcome this obstacle, we employed a nanoparticulate formulation of subtilisin Carlsberg (SC) and designed a series of 14 structurally related racemic alcohols. They were employed in the model transesterification reaction with vinyl butyrate and the enantioselectivities were determined.

Biguanide-, imine-, and guanidine- based networks as catalysts for transesterification of vegetable oil.

Polycationic systems based on poly(hexamethylene biguanide) (PHMBG), branched polyethyleneimine (PEI) and poly(N-vinylguanidine) (PVG) have been evaluated as heterogeneous catalysts for the transesterification of sunflower oil by methanol. Insoluble networks are synthesized via crosslinking of PHMBG by either 4,4'-methylenebis(N,N-diglycidylaniline) or polyisocyanate prepolymer, PEI with sebacoyl chloride, and PVG with 1,4-butanediol diglycidyl ether. PHMBG and its crosslinked networks appeared to be remarkably efficient catalysts, enabling 80-100% triglyceride conversion within 0.

Effect of prolonged exposure to organic solvents on the active site environment of subtilisin Carlsberg.

The potential of enzyme catalysis as a tool for organic synthesis is nowadays indisputable, as is the fact that organic solvents affect an enzyme's activity, selectivity and stability. Moreover, it was recently realized that an enzyme's initial activity is substantially decreased after prolonged exposure to organic media, an effect that further hampers their potential as catalysts for organic synthesis. Regrettably, the mechanistic reasons for these effects are still debatable.

Hydrogen/deuterium exchange study of subtilisin Carlsberg during prolonged exposure to organic solvents.

It has been previously reported that prolonged exposure of an enzyme to organic solvents leads to substantial decrease of activity. This effect was found to be unrelated to the catalysts' structure or their possible aggregation in organic solvents, and up to the present day the cause for activity loss remains unclear. In the present work, the structural dynamics of the serine protease subtilisin Carlsberg (SC) have been investigated during prolonged exposure to two organic solvents by following hydrogen/deuterium (H/D) exchange of mobile protons.

Stabilization of alpha-chymotrypsin upon PEGylation correlates with reduced structural dynamics.

Protein stability remains one of the main factors limiting the realization of the full potential of protein therapeutics. Poly(ethylene glycol) (PEG) conjugation to proteins has evolved into an important tool to overcome instability issues associated with proteins. The observed increase in thermodynamic stability of several proteins upon PEGylation has been hypothesized to arise from reduced protein structural dynamics, although experimental evidence for this hypothesis is currently missing.

Role of methoxypolyethylene glycol on the hydration, activity, conformation and dynamic properties of a lipase in a dry film.

A combined approach based on the use of ATR-FT/IR and steady-state fluorescence spectroscopy allowed to shed light on the effects of the additive methoxypolyethylene glycol (MePEG) on the hydration, conformation and dynamic properties of lipase from Burkholderia cepacia dehydrated to form a film. Spectroscopic data show that the additive has little effect on the structure of the protein; however, H/D exchange kinetic and fluorescence anisotropy suggest a more flexible enzyme molecule when in the presence of MePEG. By infrared spectroscopy, we estimated that, after conditioning the films at water activity of 1, the water content in the lipase dehydrated with MePEG is 5.

Effect of PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4-dioxane.

The employment of enzymes as catalysts within organic media has traditionally been hampered by the reduced enzymatic activities when compared to catalysis in aqueous solution. Although several complementary hypotheses have provided mechanistic insights into the causes of diminished activity, further development of biocatalysts would greatly benefit from effective chemical strategies (e.g.

On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg.

Background: Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours.

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18-crown-6 on lipase activity, enantioselectivity, and conformation.

Lipase from Burkholderia cepacia (lipase BC) and lipase B from Candida antarctica (CALB) show an increase of the transesterification activity in toluene (up to 2.4- and 1.7-fold, respectively), when lyophilized with 18-crown-6.

On the relationship between the activity and structure of PEG-alpha-chymotrypsin conjugates in organic solvents.

Enzymes are attractive catalysts for the production of optically active compounds in organic solvents. However, their often low catalytic activity in such applications hampers their practical use. To overcome this, we investigated the effectiveness of the covalent modification of alpha-chymotrypsin with methoxy poly(ethylene glycol) (PEG) with a Mw of 5,000 to enhance its activity.

High initial activity but low storage stability observed for several preparations of subtilisin Carslberg suspended in organic solvents.

Colyophilization with methyl-beta-cyclodextrin activates subtilisin Carlsberg by more than 200-fold in organic solvents, though this is a short-lived effect. About 93% of the enzyme's high initial activity observed in THF (at 45 degrees C) decreases exponentially with a t(1/2) of 1.8 h, until it reaches a residual activity (of 7%) that remains constant throughout the 4 days duration of the experiment.

Enzyme activation in organic solvents: co-lyophilization of subtilisin Carlsberg with methyl-beta-cyclodextrin renders an enzyme catalyst more active than the cross-linked enzyme crystals.

In this study we explored the efficiency of the additive methyl-beta-cyclodextrin (M beta CD) to enhance the activity and enantioselectivity of the serine protease subtilisin Carlsberg in organic solvents. These two parameters, measured for different transesterification reactions and in several solvents, are compared with results obtained by using two additional preparations of the same enzyme: lyophilized powder and cross-linked enzyme crystals (CLEC). The results suggest that co-lyophilization of subtilisin with M beta CD preserves the enzyme's active site tertiary structure rendering a highly active and enantioselective catalyst.