PH lability

In 1961 it was reported that human leukocytes were capable of producing IFN in response to viral infections [8,9]. This viral stimulation of white blood cells was initially used to produce leukocyte IFN for clinical applications. Identification of a number of varied IFN inducers such as mycoplasma or other microorganisms in cell cultures, lipopolysaccharides (LPS, derived from bacteria membranes), tumor-derived or virus-transformed cells, and synthetic chemical compounds such as polyanions and poly I C (poly inosine-cytosine) suggested that different IFN mixtures could be derived from interaction of various inducing agents and appropriate target cells [10-16]. Another pH-labile, nonvirus-induced IFN termed immune-IFN (induced by immune effector cells) was discovered in 1965. It was produced by... 

Bowers WF, Fulton, Thompson J (1984) Ultrafiltration vs equilibrium dialysis for determination of free fraction. Clin Pharmacokinetic 9(Suppl l) 49-60 Brprs O, Jacobsen S (1985) pH lability in serum during equilibrium dialysis. Br J Clin Pharmacokinetic 20 85-88 Henricsson S (1987) Equilibrium Dialysis in a Stainless Steel Chamber Measurement of the Free Plasma Fraction of Cyclosporin. Pharmacy and Pharmacology Communications 6(10) 447-450... 

Is the product pH labile or do pH changes affect potency is such ways that are not evident in other assays, i.e., minimal degradation and/or unfolding ... 

Brooke, M. H. and Kaiser, K. K. (1970) Three myosin adenosine triphosphatase systems the nature of their pH lability and sulfhydryl dependence, J. Hlstochem. Cytochem. 18 670-2. 

Scheme 1. pH labile cationic lipids (2 and 4) and their stable analogs (1 and 3)... 

The size of the complexes was measured as a function of the cationic lipid/DNA charge ratio by dynamic light scattering with a Coulter N4+ particle sizer at 632 nm. The results, comparable to those obtained with the non-pH labile cationic lipids, are shown in Fig. la-c (see Note 10). 

Above 4-6 nmol cationic lipid/pg DNA, small particles were obtained containing compacted DNA. The formation of complexes between DNA and pH labile cationic lipids thus obeys similar rules as those observed with classical nonlabile cationic lipids. 

The degradation of lipoplexes obtained with pH labile cationic lipids 2 and 4 was evaluated by measuring the fluorescence percentage of EtBr intercalated with DNA. Acidic degradation of pH labile cationic lipids should result in the liberation of DNA and thus to an increase in the observed fluorescence, since EtBr will intercalate in uncompacted DNA. 

Liposomes containing either pH labile or stable cationic lipids were mixed with different amounts of DNA in order to obtain three different charge ratios 0.4 nmol cationic lipid/pg DNA corresponding to the uncompacted zone, 1.7 nmol cationic lipid/pg DNA corresponding to the aggregated zone, and 6 nmol cationic lipid/pg DNA corresponding to small lipo-plexes with compacted DNA. 

Fig. 2. Degradation kinetic of lipoplexes obtained with stable lipid 3 and pH labile lipids 2 and 4 at pH 5, and charge ratios 1.7 and 6. DNA release induced by degradation is measured by the % of fluorescence of intercalated ethidium bromide at 590 nm " 260 nm (100% refers to naked DNA)...

Lipoplexes containing pH labile cationic lipid 2 or 4 showed an increase of flnorescence at pH 5, after 4-6 h with a charge ratio of 1.7 and after 8-12 h with a charge ratio of 6 (Fig. 2) (see Note 10). 

In Vitro Transfection Experiments with pH Labile Lipoplexes... 

Formulations containing pH labile cationic lipids 2 and 4 were evaluated in vitro in transfection experiments with Hela cells with three different charge ratios (1.5, 6.0, and 10), using plasmid DNA containing the luciferase (Luc) reporter gene under the cytomegalovirus (CMV) promoter. 

The results were compared to those obtained with non-pH labile formulations. 

The results of transfection with pH labile lipid 4 and stable lipid 3 in the absence of serum, with or without bafilomycin A, are shown in Fig. 3. Formulations containing lipids 1 and 2 gave results similar to those obtained with 3 and 4 and for the sake of clarity are not represented in the figure. 

For a charge ratio = 10 which is the charge ratio commonly used in systemic injection, significant enhancement (xl2) of transfection was obtained with the pH labile formulation versus stable formulation. 

No significant differences were observed with bafilomycin-treated cells at ratios 1.5 and 6. However, at charge ratio = 10, the transfection level was decreased to one-fourth with bafilomycin-treated cells in the case of pH labile complexes compared to untreated cells, whereas no decrease was observed with the stable complexes in the same conditions. 

Serum decreased dramatically the transfection as expected, especially at high charge ratios. Nevertheless, a better transfection was still observed with the pH labile formulations (data not shown). 

In the present work, we have focused our studies on the pH sensitive strategy. This might involve the use of pH labile components into the formulation, such as pH degradable lipid (7-9) or polymer (10). The pH sensitivity might also be provided by a mixture of titrable anionic and cationic lipids in the formulation. Numerous works have been reported on such pH sensitive lipopiexes, mostly to increase gene delivery into the cells... 

Kaminskas, L.M. Kelly, B.D. McLeod, V.M. Sbema, G. Owen, D.J. Boyd, B.J. Porter, C.J. Characterisation and tumour targeting of PEGylated polylysine dendrimers bearing doxorubicin via a pH labile linker. J. Control. Release 2011,152 (2), 241-248. 

Hammond and coworkers reported tumor-specific intracellular delivery of poly-L-Lysine (PEL) coated quantum dots surface functionalized with PEG via a pH-labile iminobiotin-neutravidin bond (QD/PLLib/nav/PEG) for tumor acidosis triggered PEL positive charge exposure and cellular internalization [63], While the PEG layer allowed similar tumor biodistribution of QD/PEEib/nav/PEG and control particles prepared with a pH-stable biotin-neutravidin bond (QD/PEEb/nav/PEG) shortly after intravenous injection (30 min and 8 h), tumor-selective exposure of PEE enhanced tumor retention of QD/PEEib/nav/PEG at 48 h. Interestingly, QD/PEEib/nav/PEG nanoparticles selectively distributed to hypoxic regions of tumors, as expected from the correlation between hypoxia and acidosis [53]. 

Walker et al. [133] made use of the acidic milieu of the endosomes and introduced bioresponsive PEG-polycation conjugates with pH-labile hnkages (see Fig. 4). DNA particles shielded with these bio-reversible PEG conjugates... 

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