Biomod/2014/UANL/Background

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Background

A nanoreactor is a nanozised container for chemical reactions, in a range 1-100 nm, biological systems have always been an inspiration because of their complexity and diversity. Cell processes take place within constrained spaces and small volumes, and many are not yet fully understood. Advances in nanoscale fabrication have allowed us to mimic some of these spaces and features with other structures. The volumes that are present at this level allow molecules to collide more often, as opposed to an “open space”; simulations based on Brownian diffusion have shown that collision frequency between molecules strongly depend on vesicle size. Now, one of the main differences and reasons to use nanoreactors as opposed to “traditional” benchtop or micro reactors is that the reaction space at a nanoscale level strongly influences both movement and interactions of the regents. This may sound new, but nature uses this concept in many structures. Organelles support complex metabolic pathways and influence them due to their geometry and composition. The kinetics and mechanisms of chemical reactions in small-space restricted geometries has been studied in micelles and vesicles, polymer and zeolite structures and cells. The fluctuations of reactive species are larger, and may speed up or slow down reactions. Also, given the ratio between the container’s walls and the overall volume, the properties of the structure also have to be taken into account.

As the systems become smaller and smaller, the differences begin to grow larger. Enzyme kinetics are no exception, and are also modified, changing from the Michalis-Menten equations to a single-molecule perspective considering probabilities in each step of the reactions. As there is a (relative) small number of molecules within the nanoreactor, or a fixed number, depending on the type of container, the yield may be different than expected. A stochastic approach has been applied and used to model a system’s reaction kinetics. In general, inorganic structures were of greater interest because of their resilience to common industrial applications, mainly high temperature and pressure. However, self-assembled biological structures are of interest due to their possible application in vivo. One of the most widely studied containers is the liposome: a self-assembling structure formed by a lipid bilayer.

Liposomes

A liposome is a spherical soft-matter particle, composed by one or multiple lipid bilayers, which encapsulate an aqueous medium. This is possible because of their interactions with water: the “head” group, which is hydrophilic, and the “tail” group, which is hydrophobic. One example of a lipid bilayer is the cell membrane. Lipids as analogs of these membranes are generally assembled by spontaneous self-organization from pure lipids or lipid mixtures. The most commonly used lipids are phospholipids, particularly phosphatidylcholine for its neutral charge. Other compounds can be used in order to change the liposome’s electrical charge.

These structures were first described by Dr. Alec D. Bangham in 1961, and have since been widely studied. A liposome has the ability to encapsulate a solution within its membrane, preventing contact with the outer medium. Diffusion is dependant on a variety of factors, including, but not limited to, temperature, pH, ratio of saturated/insaturated phospholipids, size of the molecule, etc. Liposomes can be classified into several types according to their features, namely size and number of lamellae (bilayers).

Unimellar vesicles are of special interest to researchers due to their well-characterized membrane properties and facile preparation in a laboratory. Multimellar vesicles show a greater range of physical properties and general behavior when compared to unimellar vesicles, and are more used with industrial applications like drug delivery. Liposomes are not considered to be in a thermodynamic equilibrium because curvature energy is being confined in the vesicles as they are produced. The curvature free energy, also known as bending energy, is defined by the rigidity and curvature of the membrane, and is directly responsible for the large variety of sizes and shapes that liposomes can take.











































Lipids

Phosphatidylcholine

Phospholipid abundant in cells, consisting mainly of palmitic acid and unsaturated fatty acids. It contains an hydrophilic part and a hydrophobic moiety. The liposome is composed primarily of this component due to its structure, that confers permeability.

Cholesterol

Sterol with polar head consisting of hydroxyl group and an apolar portion formed by carbocycles. This lipid provides sttifness in body tissues, and is for this reason that is added to the liposome to acquire stability.

Encapsulation

The method used in this project to form the liposomes and encapsulate the enzymes uricase and catalase, is ethanol injection, we choose this method because lipids dissolved in ethanol are rapidly injected into a buffer solution where they spontaneously form small unilamellar vesicles. This procedure is simple, rapid, and gentle. At low concentrations of lipid (3 mM) 300 A diameter vesicles are formed, whereas at high concentrations (36 mM) 1 100 A diameter vesicles are formed. Uniform sizes of liposomes are formed in this method .

The injection of a small amount of an ethanolic solution of a bilayer-forming amphiphile (usually a PC) into an aqueous solution leads to the rapid formation of vesicles as the amphiphiles are exposed to water. The vesicles thus formed are abbreviated as VEI, ‘vesicles prepared by the ethanol injection method’ Instead of ethanol, methanol—which is not only more polar but also poisonous—in principle can also be used. Both alcohols are completely miscible with water. Unless specially removed (e.g. by dialysis), the alcohol remains in the vesicle preparation. If the aqueous solution contains enzyme molecules, they will be entrapped to some extent in the vesicles formed. This vesicles have a 250-50 nm diameter. Depending on the experimental conditions (e.g. lipid concentration, speed of adding the alcoholic solution, and stirring rate), VEI are more or less homogenous with respect to size andlamellarity Encapsulation efficiency is commonly measured by encapsulating a hydrophilic marker.

Enzymes

The reactions are carried out with biological catalysts. Application in biomedical and uricase catalase is used, while obtaining only renewable urease enzyme participates. The following are general characteristics of enzymes are presented.














References:

[1] http://en.wikibooks.org/wiki/Structural_Biochemistry/Liposomes

[2] http://books.google.com.mx/books?id=uLoA236Kwg0C

[3] http://www.structuralbiology.be/files/theses/2010%20Vocht.pdf

[4] http://lib.gen.in/3299714a9db912d26900bc8cc52b6519/jesorka2008.pdf

[5] Ostafin, A., & Landfester, K. (2009). Nanoreactor Engineering: For Life Sciences and Medicine. Norwood, Massachsetts: Artech House.

[6] Walde, P., & Ichikawa, S. (2001). Enzymes inside lipid vesicles: preparation, reactivity and applications. Biomol Eng , 143-177.

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