PH gradient techniques

Encapsulation of Drugs Within Liposomes by pH-Gradient Techniques... 

Over the past 20 years, our laboratory has played a major role in the development of liposomal systems optimized for the delivery of conventional drugs, almost all of which are encapsulated by pH-gradient techniques. Our initial studies led to the development of several liposomal drug delivery systems in which uptake was driven by the citrate method of generating pH gradients (15,21-23,27,54—58). This was followed by the development of new... 

Isoelectric Focusing. Isoelectric focusing is a technique used for protein separation, by driving proteins to a pH where they have no mobiUty. Resolution depends on the slope of a pH gradient that can be achieved in a gel. 

Apart from the passive encapsulation methods, different active entrapment techniques are described in the literature. Nichols and Deamer (1976) prepared liposomes with a pH gradient across the membrane (acidic interior with respect to the external buffer). These liposomes efficiently incorporated several catecholamines added to the external buffer. The same technique has been used to concentrate doxorubicin (DXR) in pH gradient liposomes (Mayer et al., 1986b). 

Fig. 3. Isoelectric focusing in ultrathin polyacrylamide layers (pH gradient 3 -10) of multiple forms of polygalacturonase produced by Candida boidinii under different cultivation conditions a - pectin, pH 3.51 b - pectin, pH 5.49 c -pectin, pH 7.01 d - 20% of D-galactopyranuronic acid in pectin e - pectate. A - Activity detection with print technique on colouress D-galacturonan DP 10 dyed additionally with ruthenium red ( both exo- and polygalacturonases) and B - activity detection with Ostazin Brilliant Red/D-galacturonan DP 10 agar print (polygalacturonases).

Because the pi of a protein is based on its amino acid sequence, this technique has good resolving power. The resolution can be adjusted further by changing the range of the pH gradient. The use of immobilized pH gradient (IPG) strips has enabled reproducible micropreparative fractionation of protein samples, which is not consistently possible when ampholytes are used in the first dimension (Gorg et al., 2000). 

Another limitation of 2D gels is that membrane proteins are underrepresented. Because membrane proteins account for approximately 30% of total proteins (Wallin and Von Heijne, 1998), this is a serious problem for characterization of the proteome. The relative lack of membrane proteins resolvable on 2D gels can be attributed to thee main factors (i) they are not abundant, and therefore are difficult to detect by standard staining techniques, (ii) they often possess alkaline pi values, which make them difficult to resolve on the pH gradients most often used for isolelectric focusing, and (iii) the most important reason for under representation may be that membrane proteins are poorly soluble in the aqueous media used for isoelectric focusing (Santoni et al., 2000). Membrane proteins are designed to be soluble in lipid bilayers and are therefore difficult to solubilize in water-based solutions. 

Regardless of the rotor speed and maximum velocity, sedimentation (or flotation) will not occur in a solution of equal density to the sample. Iso-density methods use this lack of movement in a manner comparable to a pH gradient in iso-electric focusing techniques. The methods are a combination of sedimentation and flotation, achieved by using a density gradient that straddles the density of the particles concerned. On centrifugation, the particles sediment until they reach a solvent zone with the same density. This results in the development of a zone for each type of particle present in the sample. 

It is impossible to recommend how far you should pursue the possibilities discussed in this chapter. Essentially, all analytical process developers routinely screen both anion and cation exchangers with salt gradients over a range of fixed pH values. A smaller subset routinely evaluates pH gradients as well. Exploration into more exotic territory is usually undertaken only when conventional approaches fail. This is probably as it should be. As powerful and versatile as IEC is, it is only one of a suite of proven chromatography techniques for protein analysis. If investigating the basics in IEC does not yield the results you seek, it... 

As in conventional IEF, the high resolving power of CIEF depends upon the focusing effect of the technique. At steady state, the ampholytes form a stable pH gradient within which proteins become focused at the positions where their net charges are zero, i.e., where pH = pi. Diffusion of a protein toward the anode (positive electrode) will result in the acquisition of positive charge, and the... 

Early studies in our laboratory on membrane potentials and the uptake of weak bases used for the measurement of ApH led to the recognition that a variety of chemotherapeutic drugs could be accumulated within LUVs exhibiting transmembrane pH gradients (59). This remote-loading technique, so named because drug is loaded into preformed vesicles, is based on the membrane permeability of the neutral form of weakly basic drugs such... 

---