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 in vitro and in vivo. 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. In this study, we examine the effect of ß-cyclodextrins on the retention time and transfection efficiency of siRNAs formulated in a liposome. Cyclodextrins have been widely studied as solvating agents and drug delivery vectors mainly because these cyclic nontoxic glucose structures can bind several molecules of different physicochemical characteristics, through H-bonding or by forming inclusion complexes. These properties, although beneficial for most applications, have resulted in some contradictory results published in the literature, whereas cyclodextrins have been found to destabilize a liposome's membrane. Here, we present a systematic study, which shows that ß-cyclodextrin binds, possibly via hydrogen bonding, with siRNA and DOPC liposomes, resulting in increased siRNA serum stability and in vitro siRNA's transfection efficiency when formulated together.

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. The extracts that tested positive (6) were then partitioned with petroleum ether, chloroform, ethyl acetate and n-butanol, in a sequential manner. The resulting fractions were then tested again using the fibrin plate assay. Extracts from leaves of Croton lucidus (C. lucidus) showed the presence of a strong uPA inhibitory activity. Serial dilutions of these C. lucidus partitions were performed to determine the uPA inhibition IC50 values. The chloroform extract showed the lowest IC50 value (3.52 µg/mL) and hence contained the most potent uPA inhibitor. Further investigations revealed that the crude methanol extract and its chloroform and n-butanol partitions did not significantly inhibit closely related proteases such as the tissue type plasminogen activator (tPA) and plasmin, indicating their selectivity for uPA, and hence superior potential for medicinal use with fewer side effects. In a further evaluation of their therapeutic potential for prevention of cancer metastasis, the C. lucidus extracts displayed cytostatic activity against human pancreatic carcinoma (PaCa-2) cells, as determined through an MTS assay. The cytostatic activities recorded for each of the partitions correlated with their relative uPA inhibitory activities. There are no existing reports of uPA inhibitors being present in any of the plants reported in this study.

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. Furthermore, our results reveal that these NPs are less toxic towards CHO-K1 cell lines than the unmodified polycationic-branched polyethyleneimine (PEI). In general, the external magnetic field did not alter the cell's viability nor it disrupted the cell membranes, except for the poly(hexamethylene biguanide)-modified NP, where it was observed that in CHO-K1 cells application of the external magnetic field promoted membrane damage. This paper presents new polycationic superparamagnetic NPs as promising transfection vehicles for siRNA and demonstrates the advantages of magnetofection.

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. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

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 5.5-fold higher than that with the free enzyme, which suggests a higher stability of the immobilized enzyme toward the organic solvent. The nature of the organic solvent substantially influenced the degree of conversion that, for example, was 7.4% in toluene and 31.6% in petroleum ether.

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. In general, short alcohol side chains led to low enantioselectivties, while larger and bulky side chains caused better discrimination of the enantiomers by the enzyme. With several bulky substrates high enantioselectivities with E>100 were obtained. Computational modeling highlighted that key to high enantioselectivity is the discrimination of the R and S substrates by the sole hydrophobic binding pocket based on their size and bulkiness. While bulky S enantiomer side chains could be accommodated within the binding pocket, bulky R enantiomer side chains could not. However, when also the S enantiomer side chain becomes too large and does not fit into the binding pocket anymore, enantioselectivity accordingly drops.

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 0.5 h at 70 degrees C. PEI-based networks catalyzed triglyceride transesterification with rates 8- to 12-fold slower than their PHMBG-based counterparts. The PVG-based networks, which were devoid of hydrophobic moieties, appeared to be inefficient catalysts due to limited accessibility of the basic guanidine groups to reactants. The PHMBG networks were shown to be recyclable by a simple centrifugal filtration. After 15 cycles of recovery and reuse, only 10-15% decline in performance was observed.

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. In the present study we have made an attempt to explain the reasons behind the partial loss of enzyme activity on prolonged exposure to organic solvents. Fluorescence spectroscopic studies of the serine protease subtilisin Carlsberg chemically modified with polyethylene glycol (PEG-SC) and inhibited with a Dancyl fluorophore, and dissolved in two organic solvents (acetonitrile and 1,4-dioxane) indicate that when the enzyme is initially introduced into these solvents, the active site environment is similar to that in water; however prolonged exposure to the organic medium causes this environment to resemble that of the solvent in which the enzyme is dissolved. Furthermore, kinetic studies show a reduction on both V(max) and K(M) as a result of prolonged exposure to the solvents. One interpretation of these results is that during this prolonged exposure to organic solvents the active-site fluorescent label inhibitor adopts a different binding conformation. Extrapolating this to an enzymatic reaction we argue that substrates bind in a less catalytically favorable conformation after the enzyme has been exposed to organic media for several hours.

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. The enzyme, after lyophilization, was incubated in organic solvents at controlled deuteriated water activity for different times and the H/D exchange was allowed to take place. The amount of deuterium exchanged was evaluated by (2)H NMR, which in turn gave us a picture of the changing dynamics of our model enzyme during incubation and under different experimental conditions. Our results show that the flexibility of SC decreases during prolonged storage in 1,4-dioxane (Diox) and acetonitrile (ACN) as indicated by the observed 3- to 10-fold decrease in the apparent rate constants of exchange (k) of fast exchangeable protons (FEP) and slow exchangeable protons (SEP) in the protein. Our study also shows that SC is more flexible in ACN than in Diox (k 3-20 times higher in ACN for the FEP and SEP), suggesting that enzyme dynamics are affected by solvent physicochemical properties. Additionally, the enzyme dynamics are also affected by the method of preparation: decreased flexibility (k decreases 3- to 10-fold for FEP and SEP) is observed when the enzyme is chemically modified with poly ethylene glycol (PEGylated) or colyophilized with crown ethers. A possible relationship between activity, enantioselectivity (E), and structural dynamics is discussed, demonstrating that direct correlations, as have been attempted in the past, are hampered by the multi-variable nature and complexity of the system.

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. To test this hypothesis, the model protein alpha-chymotrypsin (alpha-CT) was covalently modified with PEGs with molecular weights (M(W)) of 700, 2,000 and 5,000 and the degree of modification was systematically varied. The procedure did not cause significant tertiary structure changes. Thermodynamic unfolding experiments revealed that PEGylation increased the thermal transition temperature (T(m)) of alpha-CT by up to 6 degrees C and the free energy of unfolding [DeltaG(U) (25 degrees C)] by up to 5 kcal/mol. The increase in stability was found to be independent of the PEG M(W) and it leveled off after an average of four PEG molecules were bound to alpha-CT. Fourier-transformed infrared (FTIR) H/D exchange experiments were conducted to characterize the conformational dynamics of the PEG-conjugates. It was found that the magnitude of thermodynamic stabilization correlates with a reduction in protein structural dynamics and was independent of the PEG M(W). Thus, the initial hypothesis proved positive. Similar to the thermodynamic stabilization of proteins by covalent modification with glycans, PEG thermodynamically stabilizes alpha-CT by reducing protein structural dynamics. These results provide guidance for the future development of stable protein formulations.

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 5.4- and 4.7-fold higher than in the absence of the additive and the additive alone, respectively. Additionally, our infrared data suggest that MePEG acts by hindering intermolecular protein-protein interactions and contributing to increase the accessibility and flexibility of the lipase in the dehydrated solid film. These factors also explain the enhancement of the enzyme catalytic activity (i.e., up to 3.7-fold in neat organic solvent) when in the presence of MePEG. The method and results presented might better address the use of additives for the preparation of enzymes employed in non-aqueous media or of proteins used in a dry form in different fields of biotechnology.

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., PEGylation) to ameliorate this event. Herein we explore the effects of altering the solvent composition from aqueous buffer to 1,4-dioxane on structural, dynamical, and catalytic properties of the model enzyme subtilisin Carlsberg (SBc). Furthermore, we also investigate the effects of dissolving the enzyme in 1,4-dioxane through chemical modification with poly(ethylene)-glycol (PEG, M(W) = 20 kDa) on these enzyme properties. In 1,4-dioxane a 10(4)-fold decrease in the enzyme's catalytic activity was observed for the hydrolysis reaction of vinyl butyrate with D(2)O and a 50% decrease in enzyme structural dynamics as evidenced by reduced amide H/D exchange kinetics occurred. Attaching increasing amounts of PEG to the enzyme reversed some of the activity loss. Evaluation of the structural dynamic behavior of the PEGylated enzyme within the organic solvent revealed an increase in structural dynamics at increased PEGylation. Correlation analysis between the catalytic and structural dynamic parameters revealed that the enzyme's catalytic activity and enantioselectivity depended on the changes in protein structural dynamics within 1,4-dioxane. These results demonstrate the importance of protein structural dynamics towards regulating the catalytic behavior of enzymes within organic media.

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. Nevertheless, the increase was observed only for low (less than 100) 18-crown-6/lipase molar ratio, while at higher ratios, the activity decreased for both enzymes to values lower than those obtained in the absence of the additive. In 1,4-dioxane, the activation is lower for lipase BC (1.7-fold) and for CALB (1.5-fold). Concerning enantioselectivity, tested in the kinetic resolution of 6-methyl-5-hepten-2-ol, only in the case of CALB, an effect of the additive (the E value varied from about 120 to 280) was observed. In water, 4% (w/w) of 18-crown-6 caused a loss of activity in the hydrolysis of p-nitrophenyl laurate of about 88 and 99.75%, compared to that observed in the absence of the crown ether for CALB and lipase BC, respectively. These data and the conformational analysis of both lipases, carried out by FT/IR spectroscopy indicate that the enzyme inactivation in water and in organic solvents at 18-crown-6/lipase molar ratios, higher than 100 might be due to conformational changes caused by the additive. Instead, at molar ratios lower than 100, 18-crown-6 might increase the activity - particularly, in toluene - thanks to the fact that in its presence, the enzyme has an hydrogen bonds pattern, more similar to that in water. This suggests that the additive would be able to provide the enzyme with more water.

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. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane. RESULTS: As model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-beta-cyclodextrin (MbetaCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM. CONCLUSION: These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.

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. The model transesterification reaction between sec-phenethyl alcohol and vinyl butyrate in various neat dry organic solvents and at a controlled water activity of 0.008 in two solvents was employed to measure the effect of PEGylation on activity and enantioselectivity. Synthesis conditions were varied to obtain various conjugates with average molar ratios of PEG-to-chymotrypsin ranging from ca. 1 to 7. While the enantioselectivity increased only modestly from ca. 4.4 to 6.1 when averaging results in all solvents, PEG was very efficient in increasing the activity of alpha-chymotrypsin up to more than 400-fold compared to that of the powder lyophilized from buffer alone. The activity increase was more pronounced in apolar than in polar organic solvents and also depended on the amount of PEG bound to the enzyme. For example, the activity of the modified enzyme towards the most reactive "S" enantiomer in octane increased 440-fold but increasing the molar ratio of PEG-to-enzyme from 1.1 to 7.1 resulted in a more than twofold decrease in enzyme activity. Controlling the water activity did not prevent the drop in activity. To investigate the possible origin of the activity changes, Fourier transform infrared (FTIR) spectroscopy experiments were conducted. It was found that PEGylation reduced lyophilization-induced structural perturbations, but exposure to the organic solvents caused structural perturbations. These perturbations were more pronounced in polar than in apolar solvents. The pronounced activity drop in polar solvents at increasing PEG-modification levels correlated with an increasing level of solvent-induced structural perturbations. This correlation was less pronounced in apolar solvents where both, activity drop and structural perturbations, were less pronounced at increasing PEGylation levels. In summary, PEG-modified alpha-chymotrypsin might be an interesting system to catalyze reactions, particularly in apolar organic solvents.

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. A further study of this enzyme reveals a general trend: the initial activities of the lyophilized powder and the cross-linked enzyme crystals are also greatly reduced upon incubation in this solvent, although these preparations retain 50% of their activity after about 20 h of incubation. All of the preparations studied retained some residual activity (which persisted throughout the duration of the experiments) after the initial exponential decay. The data here presented suggest that the mode of enzyme preparation is an important issue to consider when planning lengthy reactions.

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.